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Handbook of
Mouse
Auditory
Research
From Behavior
to Molecular Biology
James F. Willott, Ph.D.

University of South Florida
Tampa, Florida
and
The Jackson Laboratory
Bar Harbor, Maine
CRC Press
Boca Raton London New York Washington, D.C.
2328cFM/frame Page 2 Monday, April 2, 2001 1:44 PM
Library of Congress Cataloging-in-Publication Data
Handbook of mouse auditory research : from behavior to molecular biology / edited by

James F. Willcott.
p. cm.
Includes bibliographical references.
ISBN 0-8493-2328-2 (alk. paper)
1. Hearing--Research--Metholdogy--Handbooks, manuals, etc. 2. Mice as laboratory
animals. I. Willott, James F.
QP461 .H26 2001

573.8′91935—dc21 00-068879
This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with
permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish
reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials
or for the consequences of their use.
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No claim to original U.S. Government works

International Standard Book Number 0-8493-2328-2
Library of Congress Card Number 00-068879
Printed in the United States of America 1 2 3 4 5 6 7 8 9 0
Printed on acid-free paper
Dedication
This book is dedicated to the students and colleagues who worked

in my laboratory over the past 24 years.
Preface
It has been almost two decades since I edited The Auditory Psychobiology of the Mouse (Charles
Thomas, Springfield, Illinois, 1983). That book covered the field of mouse auditory research quite
adequately with only a third as many chapters as this one. Things have changed dramatically during
the last two decades, and truly exciting developments have emerged in areas of molecular biology,
genetics, and mouse models of human hearing disorders. Many of the topics and techniques
reviewed here were not even a gleam in the eye of the authors writing in the early 1980s. But make
no mistake; familiar areas of research such as auditory behavior and psychophysics, development,
physiology, and anatomy have not been standing still, and equally exciting, cutting edge work is
being done in these areas as well. There is a growing interest in mouse auditory phenotypes for
their own sake, as well as to inform the molecular and genetic research. Fortuitously, the various
disciplines fuel activity in one another. Behavioral and psychophysical research identifies the mouse
models that are worthy of study at more molecular levels, whereas molecular biology helps to
explain the properties of interesting auditory phenotypes. This mutual, integrative aspect of mouse
auditory research is evident throughout the chapters of this book.
This book brings together as much information about these areas as is physically possible
within the covers of a reasonably sized volume. The authors have done a marvelous job of presenting
general background along with the current mouse research to present the state of the art of many
diverse disciplines. There are always omissions in a handbook, and for those I am sorry. Every
topic cannot be covered, but not many have been excluded here.
The list of contributors is close to my dream team: a mix of seasoned leaders in the field who
have been well known for decades, as well as a number of rising young stars. Some of the authors
were also involved with The Auditory Psychobiology of the Mouse, including John Nyby, Jim
Saunders, and David Ryugo. Despite my begging, two other original contributors, Karen Steel and
Guenter Ehret, were unable to write chapters this time because of other commitments. Their fine
work on mice has continued over the last two decades and is well represented in various chapters.
It is hoped that the reader will come away from this handbook with an appreciation for the
value and power of mouse auditory research, while being brought up to date on many topics for
which mice are important subjects. We have tried to provide a mixture of reviews of the literature,
current research (much of it not previously published), along with insights into the various meth-
odologies being used. Several “focus” chapters deal with specific topics as well.
MICE AS RESEARCH ANIMALS

Of course, the use of mice in research has a long history. As reviewed recently by Pennisi (2000),
it essentially began in 1664, when Robert Hooke made the first known scientific observations on
mice. In 1909 Clarence Little (who would later found The Jackson Laboratory in 1929) began to
develop the first inbred strain, DBA (dilute, brown, non-agouti), followed by BALB/c (Bagg albino)
from 1913 to 1916, and the C57BL strain (BL = black) in 1921. Interestingly, all three of these
now widely used strains possess a gene that results in progressive hearing loss, making them
valuable animal models, as evidenced throughout this book. Whereas the availability of these inbred
strains as models was fortunate, it has become possible to intentionally generate a variety of new
models with the development of techniques to produce transgenic mice in the 1980s and knockout
mice in 1987. Indeed, progress in genetic engineering of mice continues to accelerate in the 21st
century, with the development of techniques that were unheard of only a few years ago.
As Davisson (1999) notes, mice have several unique advantages as animal models. First, they
possess the best characterized genome of any experimental vertebrate, soon to be fully sequenced.
As of 1999 about 4000 human gene homologies had already been identified and hundreds of targeted
mutations had been made. Second, the variety and power of genetic techniques are unrivaled among
vertebrates. Recombinant and congenic strains of various types have been produced; genes can be
transferred from one genetic background to another to identify modifying genes; it is relatively
easy to find polymorphic markers and phenotypic variability; sophisticated computer analysis
programs are available; and others. Third, mice are practical; they are cost-efficient, easy to handle
(excepting a few strains that are well known to exasperated researchers), and they reproduce rapidly.
Added to this is the substantial support for mouse research provided by the NIH and other funding
agencies.
The backbone of mouse research is the use of inbred strains. Many inbred strains of mice have
interesting and useful auditory phenotypes themselves, but they also serve as hosts for myriad
mutations and genetically engineered traits that are invaluable for auditory research. The occurrence
of spontaneous mutations, and nowadays, induced-mutagenesis, provide congenic mutations that
can be studied within the homogeneous inbred background. Pure-inbred strains of mice can be
studied and reproducibly compared for virtually any characteristics of development, anatomy,
physiology, or behavior.
Given any documented differences among the inbred strains, those strains can be crossed to
produce F1 hybrids which are also genetically homogenous; that is, genetically identical except
for the sex differences. All truly F1 hybrids will be either homozygous (AA or bb) or heterozygous
(Cc, Dd), according to the parental inbreds. In fact, the F1 hybrids often exhibit “hybrid vigor,” or
advantage(s) over their respective inbred parents.
As Dr. Larry Erway (one of our contributors) notes, the greatest advantage of inbred and F1
hybrid strains of mice is that the F1 hybrids can be backcrossed to either of the parental strains
for detection of: (1) equal segregation of alleles, (2) independent assortment among loci, and (3)
linkage-recombination between genes. In the current age of genetic mapping, this includes some
ten thousand microsatellite DNA markers closely linked among the genetic loci. Given any F1
hybrids, they can be backcrossed to either of the parent inbreds, thus the possibility of detecting
equal segregation or recombination among linked genes. By crossing F1 males and females derived
from the same inbred stains, the F2 progeny also provide the advantages of comparing the expression
of all three genotypes (AA, Aa, aa). Potentially such comparisons can be extended to the interaction
of two loci, including all nine genetic combinations: e.g., AA;BB;cc, Aa;bb;cc, and aa;Bb;cc.
As many as 25 million mice may be raised worldwide in the year 2000, accounting for more
than 90% of mammals used in research (Malakooff, 2000). Whereas this is twice as many mice as
were used a decade ago, the rate of use is expected to continue growing by 10 to 20% annually.
Only a relatively small portion of these animals are used as subjects in research on the auditory
system. However, our share of the pie is becoming larger all the time. Auditory research on mice
will only continue to grow and, in all likelihood, the best is yet to come.
James F. Willott
2328c01/frame Page 1 Wednesday, April 4, 2001 11:52 AM
Section I
Audiology, Auditory Behavior,
and Psychophysics
This section presents what is currently known about mouse auditory behaviors and abilities, and
how to measure these in the laboratory. This is the best way to begin a book on the mouse auditory
system, because one needs to know what mice hear and how they use auditory information, before
one can address biological mechanisms and processes. In addition to reviewing past and current
literature, methodological issues are discussed when appropriate.
Chapter 1 (Nyby) makes it clear that (unlike some other laboratory species) mice are highly
vocal animals, for which auditory communication is of great importance; mice make great use of
their auditory system for social and other purposes. The various ways researchers can assess hearing
behaviorally in mice are reviewed by Drs. Heffner in Chapter 2, with a focus on auditory localization
as well (Chapter 3, Heffner, Koay, and Heffner).
The method of choice for measuring hearing thresholds is the auditory brainstem response
(ABR), while otoacoustic emissions are finding increasing use for evaluation of the mouse cochlea.
The literature and methodology are the topic of Chapter 4 (Parham, Sun, and Kim).
With the growing interest in evaluation of mouse auditory phenotypes in mutagensis and other
programs of research, the acoustic startle response and its modification by preceding sounds
(prepulse inhibition) are showing up in most phenotype screening protocols. One suspects that
many researchers who use these methods do not have a thorough appreciation of the rich history
of research on these topics or the complexity of these “simple” behaviors. Such deficiencies should
be remedied by Chapter 5, written by James Ison, one of the pioneers in the field. Chapter 6 (Carlson
and Willott) provides additional information on variables that influence the startle response in mice.
Chapter 7 (Falls and Pistell) takes a different turn, focusing on the relationship between the auditory
system and fear conditioning.
2328cFM/frame Page 7 Wednesday, April 4, 2001 4:43 PM
Contributors
Stuart Apfel Donald M. Caspary
Kennedy Center Department of Pharmacology
Albert Einstein College of Medicine Southern Illinois University School of
Bronx, New York Medicine
Springfield, Illinois
James F. Battey, Jr.
NIDCD E. Bryan Crenshaw III
NIH Department of Neuroscience
Bethesda, Maryland University of Pennsylvania
Philadelphia, Pennsylvania
James Bell
Departments of Psychiatry and Pharmacology Mark A. Crumling
SUNY at Stony Brook Department of Otorhinolaryngology HNS
Stony Brook, New York University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania
Lynne M. Bianchi
Neuroscience Program
Thomas M. Daly
Oberlin College
Department of Pathology
Oberlin, Ohio
Washington University School of Medicine
St. Louis, Missouri
Nenad Bogdanovic
Karolinska Institute
Stockholm, Sweden Rickie R. Davis
Hearing Loss Prevention Section, Engineering
Barbara A. Bohne and Physical Hazards Branch
Washington University School of Medicine Division of Applied Research and Technology
Department of Otolaryngology National Institute for Occupational Safety and
Head and Neck Surgery Health
St. Louis, Missouri Cincinnati, Ohio
Robert Burkard Dalian Ding

Center for Hearing and Deafness Center for Hearing and Deafness
SUNY at Buffalo SUNY at Buffalo
Buffalo, New York Buffalo, New York
Barbara Canlon Blanca Durand

Department of Physiology II Center for Hearing and Deafness
Karolinska Institute SUNY at Buffalo
Stockhom, Sweden Buffalo, New York
Stephanie Carlson Lawrence C. Erway

Department of Psychology Department of Biological Science
Bethel College University of Cincinnati
Mishawaka, Indiana Cincinnati, Ohio
William Falls Kenneth R. Johnson

Department of Psychology The Jackson Laboratory
The University of Vermont Bar Harbor, Maine
Burlington, Vermont
Matthew Kelley
Michael J. Ferragamo Unit on Developmental Neuroscience
Department of Biology National Institute on Deafness and Other
Gustavus Adolphus College Communication Disorders
Saint Peter, Minnesota National Institutes of Health
Rockville, Maryland
Robert D. Frisina
Otolaryngology Division Duck O. Kim
University of Rochester Medical Center Department of Neuroscience
Rochester, New York University of Connecticut Health Center
Farmington, Connecticut
Gary W. Harding
Washington University School of Medicine Gimseong Koay
Department of Otolaryngology — Head and Department of Psychology
Neck Surgery University of Toledo
St. Louis, Missouri Toledo, Ohio
Henry E. Heffner Verity A. Letts

Department of Psychology The Jackson Laboratory
University of Toledo Bar Harbor, Maine
Toledo, Ohio
Charles J. Limb
Rickye S. Heffner Center for Hearing Sciences
Department of Psychology Johns Hopkins University School of Medicine
University of Toledo Baltimore, Maryland
Toledo, Ohio
James McCaughran, Jr.
Robert Hitzemann Department of Psychiatry
Department of Behavioral Neuroscience SUNY at Stony Brook
Oregon Health Sciences University Stony Brook, New York
Portland, Oregon
Sandra L. McFadden
Esma Idrizbegovic Center for Hearing and Deafness
Department of Audiology SUNY at Buffalo
Huddinge University Hospital Buffalo, New York
Huddinge, Sweden
JoAnn McGee
James R. Ison Developmental Auditory Physiology Laboratory
Department of Brain and Cognitive Sciences Boys Town National Research Hospital
University of Rochester Omaha, Nebraska
Rochester, New York
D. Kent Morest
Jennifer Jeskey Department of Neuroscience
Department of Psychology Center for Neurological Sciences
Northern Illinois University University of Connecticut Health Center
DeKalb, Illinois Farmington, Connecticut
Lina M. Mullen Erik Rasmussen

Department of Surgery Department of Psychiatry
University of California San Diego SUNY at Stony Brook
La Jolla, California Stony Brook, New York
Alfred L. Nuttall Allen F. Ryan

Oregon Hearing Research Center Division of Otolaryngology
Department of Otolaryngology University of California San Diego
Head and Neck Surgery LaJolla, California
Oregon Health Sciences University
Portland, Oregon David K. Ryugo
John G. Nyby Center for Hearing Sciences
Department of Biological Sciences Johns Hopkins University School of Medicine
Lehigh University Baltimore, Maryland
Bethlehem, Pennsylvania
Richard J. Salvi
Donata Oertel Center for Hearing and Deafness
Department of Physiology SUNY at Buffalo
University of Wisconsin — Madison Medical Buffalo, New York
School,
Madison, Wisconsin Mark S. Sands
Departments of Internal Medicine and Genetics
Kevin K. Ohlemiller Washington University School of Medicine
Research Department St. Louis Missouri
Central Institute for the Deaf
St. Louis Missouri James C. Saunders
William E. O’Neill Department of Otorhinolaryngology HNS
Department of Neurobiology and Anatomy University of Pennsylvania School of Medicine
University of Rochester Medical Center Philadelphia, Pennsylvania
Rochester, New York
Carrie Secor
Henry C. Ou Center for Hearing and Deafness
Department of Otolaryngology SUNY at Buffalo
University of Washington Buffalo, New York
Seattle, Washington
Hanna M. Sobkowicz
Kourosh Parham Department of Neurology
Division of Otolaryngology University of Wisconsin School of Medicine
Department of Surgery Madison, Wisconsin
University of Connecticut Health Center
Farmington, Connecticut Hinrich Staecker
De-Ann M. Pillers University of Maryland School of Medicine
Department of Pediatrics Division of Otolaryngology
Oregon Health Sciences University Head and Neck Surgery
Portland, Oregon Baltimore, Maryland
Paul Pistell Xiao-Ming Sun

Department of Psychology Department of Speech Pathology and Audiology
The University of Vermont University of South Alabama
Burlington, Vermont Mobile, Alabama
Victoria Sundin Carole A. Vogler

Department of Psychology Department of Pathology
Northern Illinois University St. Louis University School of Medicine
DeKalb, Illinois St. Louis, Missouri
Joseph Trettel Edward Walsh

Department of Neuroscience Developmental Auditory Physiology Lab
Center for Neurological Sciences Boys Town National Research Hospital
University of Connecticut Health Center Omaha, Nebraska
Farmington, Connecticut
Dennis R. Trune Joseph P. Walton

Oregon Hearing Research Center Otolaryngology Division
Department of Otolaryngology University of Rochester Medical Center
Head and Neck Surgery Rochester, New York
Oregon Health Sciences University
Portland, Oregon James F. Willott
Department of Psychology
Thomas R. Van De Water University of South Florida
Kennedy Center Tampa, Florida
Albert Einstein College of Medicine and The Jackson Laboratory
Bronx, New York Bar Harbor, Maine
Qing Yin Zheng

The Jackson Laboratory
Bar Harbor, Maine
Contents
SECTION I
AUDIOLOGY, AUDITORY BEHAVIOR, AND PSYCHOPHYSICS .......................................1
Chapter 1
Auditory Communication among Adults...........................................................................................3
John G. Nyby
Chapter 2
Behavioral Assessment of Hearing in Mice ....................................................................................19
Henry E. Heffner and Rickye S. Heffner
Chapter 3
Focus: Sound-Localization Acuity Changes with Age in C57BL/6J Mice ....................................31
Rickye S. Heffner, Gimseong Koay, and Henry E. Heffner
Chapter 4
Noninvasive Assessment of Auditory Function in Mice: Auditory Brainstem Response and
Distortion Product Otoacoustic Emissions ......................................................................................37
Kourosh Parham, Xiao-Ming Sun, and Duck O. Kim
Chapter 5
The Acoustic Startle Response: Reflex Elicitation and Reflex Modification by Preliminary
Stimuli ..............................................................................................................................................59
James R. Ison
Chapter 6
Modulation of the Acoustic Startle Response by Background Sound in C57BL/6J Mice ............83
Stephanie Carlson and James F. Willott
Chapter 7
Focus: Learning and the Auditory System — Fear-Potentiated Startle Studies ............................91
William A. Falls and Paul J. Pistell
SECTION II
PERIPHERAL AUDITORY SYSTEM .......................................................................................97
Chapter 8
The Outer and Middle Ear...............................................................................................................99
James C. Saunders and Mark A. Crumling
Chapter 9
The Development of the GABAergic Innervation in the Organ of Corti of the Mouse..............117
Hanna M. Sobkowicz
Chapter 10
Development and Neuronal Innervation of the Organ of Corti....................................................137
Matthew W. Kelley and Lynne M. Bianchi
Chapter 11
The Role of Neurotrophic Factors in the Development and Maintenance of Innervation
in the Mouse Inner Ear ..................................................................................................................157
Hinrich Staecker, Stuart Apfel, and Thomas R. Van De Water
Chapter 12
Preparation and Evaluation of the Mouse Temporal Bone ...........................................................171
Barbara A. Bohne, Gary W. Harding, and Henry C. Ou
Chapter 13
Cochlear Hair Cell Densities and Inner-Ear Staining Techniques................................................189
Dalian Ding, Sandra L. McFadden, and Richard J. Salvi
Chapter 14
Effects of Exposure to an Augmented Acoustic Environment on the Mouse Auditory System .....205
James F. Willott, Victoria Sundin, and Jennifer Jeskey
Chapter 15
Cochlear Blood Flow .....................................................................................................................215
Alfred L. Nuttall
Chapter 16
Development of the Endbulbs of Held ..........................................................................................225
Charles J. Limb and David K. Ryugo
SECTION III
THE CENTRAL AUDITORY SYSTEM ..................................................................................237
Chapter 17
Focus: Diversity of the Mouse Central Auditory System .............................................................239
James F. Willott
Chapter 18
Neuroanatomy of the Central Auditory System............................................................................243
Robert D. Frisina and Joseph P. Walton
Chapter 19
Cytoarchitectonic Atlas of the Cochlear Nucleus of the Mouse ..................................................279
Joseph Trettel and D. Kent Morest
Chapter 20
Functional Circuitry of the Cochlear Nucleus: In Vitro Studies in Slices....................................297
Michael J. Ferragamo and Donata Oertel
Chapter 21
Focus: GABA and Glycine Neurotransmission in Mouse Auditory Brainstem Structures .........317
Donald M. Caspary
Chapter 22
Calcium Binding Proteins in the Central Auditory System: Modulation by Noise Exposure
and Aging .......................................................................................................................................321
Esma Idrizbegovic, Nenad Bogdanovic, and Barbara Canlon
Chapter 23
Auditory Neurons in the Reticular Formation of C57BL/6J Mice...............................................331
Stephanie Carlson
Chapter 24
Aging of the Mouse Central Auditory System..............................................................................339
Robert D. Frisina and Joseph P. Walton
Chapter 25
Focus: Elicitation and Inhibition of the Startle Reflex by Acoustic Transients: Studies of
Age-Related Changes in Temporal Processing .............................................................................381
James R. Ison, Joseph P. Walton, Robert D. Frisina, and William E. O’Neill
SECTION IV
GENETICS AND THE MOUSE AUDITORY SYSTEM ......................................................389
Chapter 26
Human Hereditary Hearing Impairment: Research Progress Fueled by the Human Genome
Project and Mouse Models ............................................................................................................391
James F. Battey, Jr.
Chapter 27
Genetic Analyses of Non-Transgenic Mouse Mutations Affecting Ear Morphology or
Function..........................................................................................................................................401
Kenneth R. Johnson, Qing Yin Zheng, and Verity A. Letts
Chapter 28
Inbred Strains of Mice for Genetics of Hearing in Mammals: Searching for Genes for
Hearing Loss ..................................................................................................................................429
L.C. Erway, Q.Y. Zheng, and K.R. Johnson
Chapter 29
Mapping the Genes for the Acoustic Startle Response (ASR) and Prepulse Inhibition of
the ASR in the BXD Recombinant Inbred Series: Effect of High-Frequency Hearing
Loss and Cochlear Pathology ........................................................................................................441
Robert Hitzemann, James Bell, Erik Rasmussen, and James McCaughran, Jr.
Chapter 30
Transgenic Mice: Genome Manipulation and Induced Mutations ...............................................457
Lina M. Mullen and Allen F. Ryan
SECTION V
MURINE MODELS OF DISEASES OR CONDITIONS AFFECTING THE
AUDITORY SYSTEM.................................................................................................................475
Chapter 31
Noise-Induced Hearing Loss .........................................................................................................477
Rickie R. Davis
Chapter 32
The Role of Superoxide Dismutase in Age-Related and Noise-Induced Hearing Loss:
Clues from SOD1 Knockout Mice ................................................................................................489
Sandra L. McFadden, Dalian Ding, Kevin Ohlemiller, and Richard J. Salvi
Chapter 33
Mouse Models for Immunologic Diseases of the Auditory System.............................................505
Dennis R. Trune
Chapter 34
Focus: Muscular Dystrophy Mouse Models and Hearing Deficits...............................................533
De-Ann M. Pillers and Dennis R. Trune
Chapter 35
Hypothyroidism in the TSHR Mutant Mouse...............................................................................537
Edward J. Walsh and JoAnn McGee
Chapter 36
Mouse Models for Usher and Alport Syndromes .........................................................................557
JoAnn McGee and Edward J. Walsh
Chapter 37
Preventing Sensory Loss in a Mouse Model of Lysosomal Storage Disease ..............................581
Kevin K. Ohlemiller, Carole A. Vogler, Thomas M. Daly, and Mark S. Sands
Chapter 38
Auditory Brainstem Responses in CBA Mice and in Mice with Deletion of the RAB3A
Gene ...............................................................................................................................................603
Robert Burkard, Blanca Durand, Carrie Secor, and Sandra L. McFadden
Chapter 39
Focus: Mutations of the Brn4/Pou3f4 Locus ................................................................................617
E. Bryan Crenshaw, III
References .....................................................................................................................................621
Index ..............................................................................................................................................717
1 Auditory Communication
among Adults
John G. Nyby
INTRODUCTION
Any comprehensive understanding of mouse hearing should be informed by an understanding of
the sounds that the mouse auditory system has evolved to detect. All sensory systems, including
the auditory system, have evolved because they allow an organism to process sensory inputs
important for survival and reproduction. In the case of the mouse, the sounds that mice produce
as part of their social behavior have likely contributed to the evolution of the mouse auditory
system. This conclusion would seem warranted by auditory sensitivity in normal-hearing house
mice roughly corresponding to frequencies at which mice emit audible and ultrasonic sounds. This
chapter examines the auditory communication of adult mice.
While mice can certainly vocalize at frequencies audible to the human ear, a great deal of
mouse vocalizing occurs at ultrasonic frequencies. This chapter examines both the ultrasonic and
audible signals produced by mice, the factors that regulate and modulate their production, and what
is known of their role in mouse communication. Because much of the seminal research describing
infant rodent vocalizations was performed some time ago and has been ably reviewed by others
(rodents in general [Brown, 1976; Noirot, 1972; Okon, 1972; Sales and Pye, 1974] and mice in
particular [Haack, Markl, and Ehret, 1983]), the present review concentrates on communication in
adult mice.
MECHANISM OF RODENT VOCALIZING

A great deal of evidence (reviewed by Nyby and Whitney, 1978) supports the hypothesis that both
audible and ultrasonic vocalizations in rodents are produced in the larynx by air passing over the
vocal cords. While audible vocalizations are produced by vibrating vocal cords (Roberts, 1975),
ultrasonic vocalizations are produced when the vocal cords are constricted so tightly that they can
no longer vibrate. In so doing, the vocal cords are thought to provide the plates and aperture of a
whistle mechanism. In fact, all of the sonagraphic features of rodent ultrasounds can be duplicated
by passing air through a bird whistle at physiological pressures (Roberts, 1975). A laryngeal
mechanism for both audible and ultrasonic vocalizations would also account for the occasional
instantaneous transitions from audible to ultrasonic calls and vice versa seen in rodents (Roberts,
1972).
As expected, mouse ultrasonic vocalizations are severely disrupted by transecting the nerves
that control the laryngeal musculature. One group of investigators (Nunez, Pomerantz, Bean, and
Youngstrom, 1985) found that the best strategy for devocalizing mice was to transect unilaterally
the inferior laryngeal nerve. However, such males remained devocalized for less than a week. Thus,
a relatively short window of opportunity exists for using such devocalized mice experimentally.
On the other hand, bilaterally cutting the inferior laryngeal nerve was usually fatal because of
breathing difficulties, while bilateral superior laryngeal nerve cuts had only minor effects in reducing
ultrasounds.
0-8493-2328-2/01/$0.00+$.50
© 2001 by CRC Press LLC 3
4 Handbook of Mouse Auditory Research
ULTRASONIC VOCALIZATIONS
Adult mice, like virtually all myomorph rodents, emit ultrasonic vocalizations during adult social
interactions. Among rodents in general, such vocalizations are emitted mainly in reproductive or
agonistic situations. Anderson (1954), the discoverer of rodent ultrasounds, speculated that such
vocalizations also might be used for echolocation although little evidence has since been found to
support this hypothesis.
For earlier reviews of adult rodent ultrasonic vocalizations, see Sales and Pye (1974), Brown
(1976), and Nyby and Whitney (1978). The reader is also directed to earlier reviews of mouse
ultrasonic communication (Whitney and Nyby, 1983) and of the stimuli that elicit mouse ultrasonic
vocalizations (Whitney and Nyby, 1979).
MALE ULTRASONIC VOCALIZATIONS

Much research has focused on the ultrasonic vocalizations emitted by male mice in response to
females. While females have the capability to vocalize ultrasonically (Sales, 1972b), and under
certain circumstances vocalize quite a lot (Maggio and Whitney, 1985), they rarely vocalize in
male/female pairs. Thus, males are responsible for most of the vocalizations when a male and
female are placed together. This conclusion has been supported by anesthetizing (Whitney, Coble,
Stockton, and Tilson, 1973) or by devocalizing (Nunez et al., 1985; Warburton, Sales, and Milligan,
1989; White, Prasad, Barfield, and Nyby, 1998) one member of a male/female pair. If the male
was either anesthetized or devocalized prior to pairing, few or no ultrasounds were detected.
However, if the female was devocalized or anesthetized prior to pairing, the resulting high level of
ultrasound was similar to that in unmanipulated male/female pairs.
In contrast to the results of Whitney et al. (1973), Warburton et al. (1989) reported that males
emitted few vocalizations to anesthetized females. The likely explanation for this difference is that
the act of anesthetizing a mouse can induce variable degrees of stress. In Warburton’s experiments,
the stress may have been sufficient to cause the release of an alarm pheromone (Abel, 1991; 1992;
1994)) that stimulated male responses incompatible with ultrasonic vocalizations. Consistent with the
findings of both groups, this author has observed considerable variability in the effectiveness of
anesthetized females to elicit vocalizations from one experiment to the next (unpublished observations).
Mice are different from rats (White and Barfield, 1987) and hamsters (Floody and Pfaff, 1977b),
where both the male and the female vocalize during reproductive behavior. Mice are also unusual
among rodents in that they do not normally emit ultrasounds during agonistic situations (Sales,
1972b). In mice, the only social context that involves male ultrasonic vocalizations is reproduction.
Sales (1972a) detected ultrasounds on occasion in male/male pairs but only when one male was
investigating or mounting the other. Thus, such ultrasounds would appear misguided and usually
disappear at the first sign of intermale agonistic behavior (Whitney and Nyby, 1983).
Sonagraphic Analysis of Male Mouse Ultrasounds
Adult mice emit ultrasonic vocalizations as short pulses, with each exhalation typically containing
one pulse. Each individual pulse is typically from 50 to 300 ms in duration, with approximately
200 ms between pulses. A train of ultrasonic pulses may contain from 3 to 80 such pulses in quick
succession, with the total train of pulses lasting from about 0.5 s up to about 30 s in duration. These
trains of pulses are most frequent just after a male and female are placed together (Sales, 1972b).
Although substantial individual variability exists, up to 450 of these pulses have been detected in
the 3-min period immediately after pairing (Nyby and Whitney, 1978). Male mice clearly expend
more effort emitting ultrasonic vocalizations during reproductive behavior than do male rats (Sales,
1972b) or hamsters (Floody and Pfaff, 1977a).
While audible mouse squeals typically have a major frequency plus harmonically related minor
frequencies (as would be expected from vibrating vocal cords), ultrasonic vocalizations are quite
Auditory Communication among Adults 5
often pure tones with no harmonics. Most of these ultrasonic calls can be detected from adults with
an ultrasonic receiver tuned to 70 kHz (with a bandwidth of ±10 kHz). Hence, these calls are
sometimes referred to as 70-kHz calls. However, sonagraphic analysis reveals frequency modulation
with frequency drifts, rapid frequency changes, and instantaneous frequency steps up to 60 kHz
(Sales, 1972b). Sales’ sonograms also provide evidence for some ultrasounds at frequencies that
do not include 70 kHz.
A more recent study (White et al., 1998) systematically followed sonagrams of ultrasonic
vocalizations across a mating bout. This work confirmed Sales’ observations of the relationship
between 70-kHz vocalizations and male-typical courtship and sexual behavior. However, once the
male began mounting the female, a second ultrasonic vocalization of about 40-kHz call was
interspersed among the 70-kHz calls. Both types of vocalizations declined across intromissions.
The 40-kHz calls were louder, more variable in length, and less variable in frequency.
The suggestion was made that perhaps the 40-kHz and 70-kHz calls are serving different
functions (White et al., 1998). Because the 70-kHz calls occurred at their highest rates during
investigatory behavior and early in the mating sequence, these calls may be involved in communi-
cating information during courtship and the early stages of copulation. On the other hand, the 40-kHz
calls occurred mainly during mounting and were suggested to play a role during copulation only.
Ethographic Analysis of Male Mouse Ultrasounds
One way of obtaining a first approximation of the functional significance of male vocalizations has
been to observe exactly what the male is doing when his ultrasonic vocalizations are detected.
Sales (1972b) found that in male/female pairs of C3H, T.O. Swiss, and E.N. mice, ultrasounds
correlated better with the behaviors of the male than with those of the female. The ultrasounds
were most common immediately after the male and female were placed together when the male
was engaging in olfactory investigation of the female. However, the ultrasounds continued, although
at a lower level, after investigatory behavior had ceased and sexual behavior had begun. Once
mounting began, the ultrasounds occurred mainly during the mounting attempts. Relatively few
ultrasounds were detected between mounts when the male and female were separated and not
interacting. Sales also reported that for some male/female pairs, the ultrasounds correlated with
the pelvic thrusts of the male during mounts both with and without intromissions. The ultrasounds
declined across intromissions, and in males that ejaculated, no ultrasounds were detected during
and immediately after ejaculation.
In general, the ultrasounds of male mice correlated well with other male behaviors indicative
of sexual arousal. These observations led Sales to conclude that ultrasounds themselves can serve
as a good index of male sexual arousal. On the other hand, audible squeals, when they occurred,
correlated best with the nonreceptive behaviors of the female, suggesting that audible sounds were
most often female-produced.
Ejaculation by male mice is usually followed by a postejaculatory refractory period of about
15 to 30 min in duration (Mosig and Dewsbury, 1976; Nyby, 1983; but see also McGill, 1962). At
the end of this refractory period, many males reinitiate another mating sequence leading to a second
ejaculation, which then generally leads to sexual exhaustion (Mosig and Dewsbury, 1976). In
rodents, such a postejaculatory refractory period is further subdivided into an absolute refractory
period when the male is physiologically incapable of mating and a relative refractory period when
mating is physiologically possible but normally does not occur (Barfield and Geyer, 1972).
Nyby (1983) quantified levels of 70-kHz ultrasonic vocalizations through the first ejaculation,
the postejaculatory refractory period, and into a second mating bout. The cessation of vocalizations
at the time of ejaculation continued for about the first 75% of the refractory period. The vocalizations
began again and continued for about the last 25% of the refractory period, generally coincident
with male investigation of the female. Once the refractory period ended and the male resumed
mounting, the patterning of male mouse vocalizations was similar to that preceding ejaculation
with an even stronger temporal relationship to mounting. The period of ultrasonic silence following
ejaculation was hypothesized to demarcate the absolute refractory period, while the resumption of
ultrasounds during the latter portion of the refractory period was hypothesized to index that the
male had entered the relative refractory period. The period of ultrasonic silence during the poste-
jaculatory refractory period appears analogous to the emission of 22-kHz postejaculatory ultra-
sounds by male rats which similarly demarcate the absolute refractory period in that species
(Barfield and Geyer, 1975).
In summary, male mice emit substantial amounts of 70-kHz ultrasound immediately preceding
and during the time that they are engaging in either olfactory investigation or mounting of the
female. On the other hand, males typically emit fewer ultrasounds when disengaged from the female
between investigatory or sexual encounters and emit no ultrasounds at all for much of the poste-
jaculatory refractory period. These findings support the hypothesis that male ultrasounds index
immediate sexual motivation on the part of the male.
Genetic Basis for Motivation to Vocalize
Early work (described in Nyby and Whitney (1978)) examined a number of inbred, hybrid, and
outbred strains of mice for their latency to emit 70-kHz vocalizations in response to a female.
Although strain differences were evident, males of all strains emitted at least some ultrasound,
indicating that such vocalizing is likely a general phenomenon among male mice. Males from a
“low-vocalizing” strain (A/J) in which less than 10% of the males vocalized during a 3-min test
were later found to reliably emit ultrasound when tested over a longer time period (Nyby, 1983).
The low levels of ultrasound originally attributed to this strain simply reflected that this strain was
slower to begin vocalizing. Thus, conclusions about vocalization levels obtained from short time-
sampling periods can sometimes be misleading, as has also been pointed out by others (Warburton,
Stoughton, Demaine, Sales, and Milligan, 1988).
The latency of male mice to vocalize to a female does exhibit the genetic property of heterosis.
That is, hybrid mice are generally quicker to begin vocalizing than males of their progenitor inbred
strains. Such a relationship is indicative of directional dominance. In addition, in an unpublished
doctoral dissertation, Coble (1972) found that the realized heritability for latency to vocalize from
one generation of bi-directional selection in a genetically heterogeneous population was not sig-
nificantly different from zero. Directional dominance and a low heritability represent the genetic
architecture of a trait that has undergone strong directional selection and is closely related to
reproductive fitness (Falconer, 1960). Thus, genetic work is certainly consistent with male vocal-
izations subserving some important biological function.
Androgenic Regulation of Male Mouse Ultrasounds
Androgenic hormones are generally hypothesized to influence behavior by acting upon the brain
in two fashions. Androgens can exert organizational effects early in development during sensitive
periods where they permanently and irreversibly masculinize target tissues (particularly the brain).
On the other hand, androgens can also exert activational effects, mainly in adulthood, that produce
reversible effects. While this dichotomy is not absolute (Arnold and Breedlove, 1985), it has
nonetheless proven a useful heuristic for guiding a great deal of research examining hormone effects
upon the brain and behavior (Breedlove, 1992). While considerable evidence supports the idea that
testosterone exerts activational effects upon the production of male vocalizations, the evidence that
testosterone organizes the neural substrate underlying this behavior is not very compelling.
Activational Effects
Several early observations indirectly suggested that male vocalizations might be activated by male-
typical sex hormones. For example, infant mice emit ultrasonic distress calls that stimulate retrieval
and reduce maternal aggression (Noirot, 1972). By 14 to 16 days of age, these ultrasounds cease.
However, ultrasounds, similar in frequency to the infant ultrasounds, reappear in C57BL/6J x
BALB/cJ male mice sometime after 35 days of age, at roughly the time of puberty (Whitney et al.,
1973). In a more systematic study (Warburton et al., 1989), the time of reappearance of vocalizations
in male T.O. Swiss mice was between 30 and 40 days of age, the approximate age of puberty. The
exact age at which the ultrasounds reappeared depended on the amount of previous experience that
young males had with females. Despite this variation, the reappearance of vocalizations in young
males clearly correlated with the endogenous increase in androgenic hormones at puberty.
Another observation suggesting hormonal control of male vocalizations was that dominant
DBA/2J males were quicker to begin emitting ultrasound and emitted more ultrasound in response
to females than did subordinates (Nyby, Dizinno, and Whitney, 1976). Because dominant male
mice generally have higher reproductive fitness than subordinates (Horn, 1974), this finding again
raised the possibility that ultrasounds might be regulated by androgenic hormones.
Dizinno and Whitney (1977) provided the first direct evidence that androgens regulate male
ultrasounds. In that work, castrated hybrid males (C57BL/6J x DBA/2J) emitted substantially fewer
ultrasounds to females than gonadally intact males. However, ultrasounds were restored in castrates
by treatments with testosterone propionate. The positive role of testosterone in activating male
ultrasounds has since been confirmed in DBA/2J (Nunez, Nyby, and Whitney, 1978), Swiss-Webster
(Nunez and Tan, 1984), and Tucks Swiss T.O. (Warburton et al., 1989) strains and thus appears as
a general phenomenon among mice.
A widely used approach to determine the receptor system by which testosterone (T) regulates
male-typical behaviors involves assessing the behavior-restorative properties of estradiol (E2) and
dihydrotestosterone (DHT), the major CNS metabolites of T (Larsson, 1979; Luttge, 1979). Estro-
gen receptors are assumed to mediate the effectiveness of E2 while DHT (a nonaromatizable
androgen) is thought to be specific for androgen receptor activation.
Initial work with DBA/2J mice (Nunez et al., 1978) was consistent with testosterone activating
male-typical reproductive behavior following aromatization to estradiol and then binding to estrogen
receptors in the brain. In contrast, subsequent work with Swiss-Webster mice (Nunez and Tan,
1984) and with C57BL/6J x AKR/J mice (Bean, Nyby, Kerchner, and Dahinden, 1986b) demon-
strated that both E2 and DHT were effective in restoring ultrasonic vocalizations in males of these
strains. This work suggests that mice possess redundant neural receptor mechanisms for androgenic
responsiveness, both of which are present in some genetic strains but not in others. Work on
copulatory behavior in mice and rats similarly finds that all strains uniformly show estrogenic
responsiveness but show much more variation in their degree of androgenic responsiveness (Nyby
and Simon, 1987).
A later study (Nyby and Simon, 1987) demonstrated that ultrasounds could be restored in
castrated DBA/2J males (the DHT insensitive strain) with high (900 µg) but not lower doses (600 µg
and 300 µg) of methyltrienolone (R1881). R1881 is an artificial nonaromatizable androgen thought
to be even more specific for androgen receptors than DHT (Doering and Leyra, 1984). However,
in contrast to expectations, the 900-µg dosage that restored behavior also showed significant binding
with estrogen receptors in the brain (Nyby and Simon, 1987). This finding raised the possibility
that the restorative effects of R1881 in this study, and perhaps DHT in the previous studies (Bean,
Nunez, and Wysocki, 1986a; Nunez and Tan, 1984) were mediated by these androgens binding
estrogen receptors. Because estrogens restored ultrasounds in all studies at much lower dosages
than androgens, the estrogen receptor system is clearly important in regulating sex hormone
activation of ultrasonic vocalizations. Establishing unequivocally that the androgen receptor system
can also mediate responsiveness clearly requires further work.
Female mice also respond to androgen treatment with increased levels of ultrasonic vocaliza-
tions (Nyby, Dizinno, and Whitney, 1977a). In fact, ovariectomized DBA/2J females were quite
similar to castrated males of this genotype in their responsiveness to testosterone. In contrast,
C57BL/6J x AKR/J females did not respond quite as quickly to repeated testosterone injections as
castrated males of their own strain. However, with extended treatment over several weeks, their
amount of ultrasound eventually became indistinguishable from that of males. Such androgenic
treatments also increased the females’ incidence of male-typical mounting of other females in both
strains (Nyby et al., 1977a). Thus, females appear to possess the same potential as males for showing
male-typical courtship and mounting behavior and will quite reliably express this potential following
extended androgenic stimulation.
Organizational Effects
One approach to demonstrate organizational effects is simply to administer male hormones to

females in adulthood. If parts of the female brain have been organized differently from those of
males, then females should respond differently to androgens. As noted above (Nyby et al., 1977a),
females were either similar to or slightly slower in responding to androgenic treatment. Whether
the delayed female response reflects an organizational difference or whether females were simply
more refractory because of lower initial androgen levels is not clear. However, if a sex difference
in hormone responsiveness exists, it is not profound.
Another strategy to demonstrate organizational effects examines whether phenotypic differences
between animals of the same sex can be accounted for by their intrauterine position. Specifically,
individuals developing in utero between two males (2M mice) are exposed to more androgen early
in development than individuals developing between two females (0M mice) (Clemens, Gladue,
and Coniglio, 1978; vom Saal, 1981). As a result, 2M female mice should be more masculinized
in adulthood than 0M females for traits organized by the early effects of androgens. Consistent
with this hypothesis, adult 2M female mice were more aggressive (Gandelman, Vom Saal, and
Reinisch, 1977; Kinsley, Miele, Konen, Ghiraldi, and Svare, 1986; Quadagno et al., 1987; Rines
and vom Saal, 1984) and more likely to engage in male-typical copulatory behavior (Quadagno
et al., 1987; Rines and vom Saal, 1984) than 0M females.
However, Jubilan and Nyby (1992) found intrauterine position to have little influence on the
ultrasonic vocalizations of either adult females or adult males to either a female mouse or her urine.
Thus, this approach also did not support an early organizational influence of androgens upon
ultrasonic vocalizations. In conclusion, females have much the same potential to vocalize as males,
and the sex difference in adulthood is likely due to a sex difference in androgen titers.
Neural Regulation of Male Mouse Ultrasounds
Because 70-kHz ultrasounds appear to be a male-typical courtship behavior, research examining

their neural regulation has focused on areas of the brain known to be important in male reproductive
behavior. One such area is the medial preoptic area (MPOA). Bilateral lesions of the MPOA cause
severe impairment or abolishment of male-typical copulatory behavior in other mammals (Hart and
Leedy, 1985). Bean, Nunez, and Conner (1981) demonstrated that mouse copulatory behavior was
similarly impaired by MPOA lesions but that ultrasonic vocalizations, either to adult females or to
female bedding, were relatively unaffected. These results suggested that while male copulatory
behavior is regulated by the MPOA, ultrasonic vocalizations are regulated by other parts of the brain.
In contrast, other studies have consistently implicated the MPOA as the important site at which
androgens activate male vocalizations (Matochik, Sipos, Nyby, and Barfield, 1994; Nyby, Matochik,
and Barfield, 1992; Sipos and Nyby, 1996; 1998). In these studies, cannula implants of either
testosterone or testosterone propionate into the MPOA were highly effective in restoring ultrasonic
vocalizations in castrated male C57BL/6J x AKR mice. However, implants into the anterior hypo-
thalamus (Nyby et al., 1992), ventromedial hypothalamus (Nyby et al., 1992), septum (Nyby, 1992;
Matochik, 1994), medial amygdala (Matochik et al., 1994; Nyby et al., 1992), and ventral tegmental
area (Sipos and Nyby, 1996) were without effect.
Perhaps the reason that MPOA lesions did not eliminate ultrasounds (Bean et al., 1981) was
that not enough of the MPOA was lesioned. While plausible, this hypothesis may be difficult to
test because large bilateral lesions of the MPOA are often lethal. Nonetheless, androgenic implant
work clearly implicates the MPOA as an important site for the neural regulation of male ultrasonic
vocalizations.
It seems likely that other areas of the brain also participate in the regulation of male mouse
ultrasonic vocalizations. Newman (1999) hypothesized that all social behaviors (including sexual,
aggressive, and parental behaviors) may be regulated by a common neural network (the medial
extended amygdala) that includes the corticomedial amygdala, lateral septum, ventromedial hypo-
thalamus, midbrain central gray and tegmentum, anterior hypothalamus, as well as the medial
preoptic area. While testosterone implants demonstrated the involvement of the medial preoptic
area, other strategies will be necessary to examine the importance of these other areas, since most
of them are androgenically unresponsive for the activation of ultrasounds (Matochik et al., 1994;
Nyby et al., 1992; Sipos and Nyby, 1996; 1998). In addition, the parts of the nervous system that
control motor output to the larynx should also be involved.
At the same time, estradiol implants restored ultrasounds in the MPOA, ventromedial hypo-
thalamus, anterior hypothalamus, and lateral septum (Nyby et al., 1992). These results could reflect
the greater activity of this treatment, more diffusion of hormone from the implant site, or that the
distribution of estrogen-responsive neurons was not identical to that of androgen-responsive neu-
rons. Further research is necessary to choose among these alternatives.
Pheromonal Elicitation of Male Mouse Ultrasounds
Not only are female mice good stimuli for eliciting ultrasonic vocalizations from males, the odors
of females are also quite effective. Early work showed, for example, that the soiled cage shavings
of females were good stimuli (Whitney, Alpern, Dizinno, and Horowitz, 1974). Subsequent work
revealed that female urine (Nyby, Wysocki, Whitney, and Dizinno, 1977b), female saliva (Byatt
and Nyby, 1986), and female vaginal fluids (Nyby et al., 1977b) were also effective. Whether these
different body fluids all contain the same ultrasound-eliciting chemosignal or whether they contain
different female chemosignals, all of which elicit ultrasounds, has not been determined.
However, most of the research on the chemosensory elicitation of male ultrasounds has focused
on female urine. One reason was to keep the work as comparable as possible to a much larger body
of research examining pheromonal communication in mice. For example, in addition to eliciting
ultrasonic vocalizations, female urine causes a reflexive release of luteinizing hormone in male
mice (Maruniak and Bronson, 1976), promotes male copulatory behavior (Dixon and Mackintosh,
1971), and reduces intermale aggression (Mugford and Nowell, 1971). In recent unpublished work,
we (Sipos, Nyby, Snyder, and Kerchner, 1999) present evidence that all of these different effects
of female urine upon males, including ultrasound elicitation, may be mediated by the same urinary
chemosignals. In addition, work in my laboratory provided evidence that two different urinary
chemosignals of female mice elicit ultrasounds from male mice. In what follows, some of our
earlier work on pheromonal elicitation of ultrasounds is reinterpreted in light of these newer
findings.
Freshly voided urine of female mice contains a potent ultrasound-eliciting chemosignal that in
most ways fits the most rigorous definitions of a pheromone (Beauchamp, Doty, Moulton, and
Mugford, 1976). This pheromone is salient even to sexually naïve adult males (Sipos, Kerchner,
and Nyby, 1992), remains a potent stimulus for eliciting vocalizations with repeated testing (Sipos
et al., 1992), and can even serve as an unconditioned stimulus for causing neutral stimuli to acquire
ultrasound-eliciting properties (Sipos et al., 1992). At the same time, this chemosignal is ephemeral;
its activity is normally destroyed by oxidation within 15 to 18 h after voiding (Sipos, Nyby, and
Serran, 1993). This chemosignal is not present in urine collected overnight (12 h) in a metabolic
cage, indicating that this method of urine collection likely hastens the oxidation that destroys the
pheromone.
Because urine aged well beyond 24 h (up to 30 days) also elicits ultrasounds under certain
circumstances (Nyby and Zakeski, 1980), a second ultrasound-eliciting chemosignal must exist as
well. However, aged urine elicits ultrasounds only under limited circumstances. First, males do not
vocalize to aged urine unless they are sexually experienced (Dizinno, Whitney, and Nyby, 1978;
Sipos et al., 1992). Although such males initially vocalize at high levels to aged urine, their
vocalizations rapidly habituate following three to four exposures (Dizinno et al., 1978; Sipos et al.,
1992). Furthermore, repeatedly pairing a neutral stimulus with aged urine does not result in
vocalizations to the neutral stimulus (Sipos et al., 1992).
Although the chemosignal in aged urine elicits ultrasounds in certain circumstances, its prop-
erties do not fit the original narrow definition of a pheromone (Beauchamp et al., 1976). However,
some workers subscribe to a broader definition to include chemosignals where the roles of learning
and contextual cues are important in establishing the signal value of the chemosignal (reviewed by
Albone, 1984). As such, we (Sipos et al., 1992) have referred to these two chemosignals as the
“ephemeral pheromone” and the “stable pheromone,” respectively. We believe that both chemosig-
nals are probably present in freshly voided urine and that, when a male encounters such urine, their
association permits aged urine to acquire the property to elicit male vocalizations. Moreover, the
relative concentrations of these two chemosignals in a female scent mark may also provide important
information about how recently a female has been in the area. However, attempts to chemically
characterize these chemosignals have not been successful, and it is not known whether these
chemosignals are single molecules or chemical mixtures. We have hypothesized, however, that
oxidation may turn the ephemeral pheromone into the stable pheromone.
Other work has demonstrated that males can learn to vocalize not only to the stable pheromone
in aged female urine, but also to other odors that normally do not elicit ultrasounds. For example,
by pairing the following with repeated exposures to a female, males acquired the capacity to vocalize
to some degree to: urine from female rats (Kerchner, Vatza, and Nyby, 1986), urine from hypo-
physectomized females (Maggio, Maggio, and Whitney, 1983), perfume (Nyby, Whitney, Schmitz,
and Dizinno, 1978), foenugreek (Kerchner et al., 1986), clean cotton swabs (Sipos, Wysocki, Nyby,
Wysocki, and Nemura, 1995), and plastic bags (Nyby, Wysocki, Whitney, Dizinno, and Schneider,
1979). In fact, some males will vocalize to the entry of a human experimenter into the colony room
if the male has been paired repeatedly with females by an experimenter (unpublished). Almost any
stimulus that allows a male to anticipate an encounter with a female would appear to acquire the
property of eliciting ultrasounds. However, some constraints must exist on the cues that can elicit
ultrasounds because attempts to train males to vocalize to the urine of other males were not
successful (unpublished).
How male mice learn to vocalize to stimuli that normally do not elicit vocalizations has been
the subject of some research. Although previous work suggested that encountering a novel odor
during a socio/sexual encounter with a female provided the best learning, others (Marr and Gardner,
1965; Müller-Schwarze and Müller-Schwarze, 1971) have hypothesized that the salience of sex
odors is acquired as a result of neonatal imprinting on the mother. Using a perfume that normally
does not elicit vocalizations, we systematically explored whether male experience with such per-
fume during infancy (associated with the dam) or during adulthood (associated with a female during
sexual encounters) was most important in causing the perfume to acquire ultrasound-eliciting
properties (Nyby et al., 1978). Very briefly, adult experience with an odorized female was far more
important than infant experience with an odorized dam. However, some synergy appeared to exist
between the two experiences, suggesting that imprinting might enhance the learning of novel signals
in adulthood. At the same time, males that had seen only perfumed females at these two develop-
mental periods nonetheless exhibited high levels of ultrasonic responsiveness to normal female
urine (Nyby et al., 1978). Thus, responsiveness to perfume, when it occurred, was superimposed
upon, and did not replace, normal ultrasonic responsiveness to female urine.
Whether male vocalizations to the ephemeral chemosignal in freshly voided urine are “learned”
as a result of neonatal imprinting or are simply an innate response is not known.
Sensory Detection of Ultrasound-Eliciting Pheromones

Potential nasal chemosensory systems for mediating responsiveness to ultrasound-eliciting
chemosignals include the main olfactory system, the accessory olfactory or vomeronasal system
(Jacobson’s organ), the terminal nerve system, and the trigeminal nerve system (Wysocki, 1979;
Wysocki and Lepri, 1991). In addition, some investigators classify the septal organ system as an
additional chemosensory system separate from the main olfactory system. However, work on
ultrasound elicitation in mice has centered on the relative contributions of the main olfactory system
and the vomeronasal system.
Early work (Bean, 1982; Wysocki, Nyby, Whitney, Beauchamp, and Katz, 1982), which
involved deafferenting all of the nasal chemosensory systems of male mice by olfactory bulbectomy,
greatly reduced ultrasonic vocalizations to female mice. However, vocalizations to the female herself
were not totally eliminated in all bulbectomized males, particularly if the male was sexually
experienced prior to olfactory bulbectomy (Wysocki et al., 1982). This finding was consistent with
sexually inexperienced males relying more on chemosensory input for vocalization elicitation, while
experienced males were better able to use non-chemosensory cues in making this determination.
On the other hand, vocalizations of sexually experienced male mice to the soiled cage shavings of
females (perceived mainly by chemosensory input) were eliminated by bulbectomy (Wysocki et al.,
1982). Thus, olfactory bulbectomy did not eliminate the motivation to vocalize, but rather eliminated
the detection of pheromonal cues that normally elicit vocalizations.
Earlier work (Wysocki et al., 1982) indicated that sexually inexperienced males required a
functioning vomeronasal organ to learn to vocalize to the stable chemosignal in aged urine.
However, sexually experienced males vocalized to the stable chemosignal after vomeronasalectomy.
Thus, the ability of a male to recognize the stable chemosignal based upon non-vomeronasal
chemoreception required previous experience with such cues in the presence of a functioning
vomeronasal organ (Wysocki et al., 1982). Again, the deficits in vocalizing following vomeronasal
removal were sensory rather than motivational deficits.
Another line of research (Sipos et al., 1995) examined the relative roles of the main and accessory
olfactory systems in vocalizing to the potent but ephemeral pheromone in recently voided urine. In
one experiment, selectively deafferenting the accessory system (by surgically removing the vomer-
onasal organ) or selectively deafferenting the main olfactory system (by intranasal irrigation with
ZnSO4) each had only minor effects in reducing ultrasounds to freshly collected urine. However, if
both chemosensory systems were simultaneously deafferented, ultrasounds to fresh urine were
eliminated. Thus, for this pheromone, both chemosensory systems were approximately equally
important and appeared redundant for pheromone detection. For a chemosignal carrying biologically
important information, it is not surprising that redundant sensory systems exist for its detection.
Function of Male Mouse Ultrasounds
Most hypotheses on the function of male mouse ultrasounds have focused on the 70-kHz calls.
Sales [nee Sewell (Sewell, 1967; 1968)] first suggested that male vocalizations serve to signal
females that the male is sexually and not aggressively motivated. Sales further suggested that
ultrasounds also serve both to reduce female aggression and to promote female sexual motivation.
Whitney (1973) developed this hypothesis further by suggesting that ultrasounds may, in fact, be
a ritualized courtship display in which the male is mimicking ultrasounds emitted by infant mice
of both sexes. Because infant ultrasounds are attractive to the dam and reduce rough parental
handling, adult male vocalizations may similarly attract adult females and reduce their aggression.
Such ritualized use of infantile behaviors in adult male courtship occurs in a variety of birds and
mammals (Eibl-Eibesfeldt, 1970).
The hypothesis that male ultrasounds serve to reduce female aggression becomes more plausible
in light of the fact that most mouse matings in nature occur during the female’s postpartum estrus
(Whitney et al., 1973). At this time, the female is very protective of her pups and very aggressive
toward strange males. Perhaps male ultrasounds are particularly important in reducing female
aggression at this time.
Various indirect lines of evidence are certainly consistent with a courtship/reproductive function
for 70-kHz ultrasounds. As described earlier, ultrasound production is hormonally and neurologi-
cally regulated very similar to other components of male-typical reproductive behavior. This
phenotype also exhibits the genetic architecture (directional dominance and low heritability) of a
trait closely related to reproductive fitness. At the same time, some strains of laboratory mice are
deaf in adulthood and yet clearly mate without obvious difficulty. While ultrasonic vocalizations
likely promote reproductive behavior, such vocalizations are clearly not essential. The effects of
ultrasounds may be subtle. For example, in hamsters, male ultrasonic vocalizations promote female
receptivity through the prolongation of lordosis (Floody and Pfaff, 1977c); and in rats, male
ultrasounds increase the female proceptive behaviors of darting and hopping (Thomas, Howard,
and Barfield, 1982).
However, attempts to experimentally demonstrate functional significance for male mouse ultra-
sounds have been mixed. One approach (Pomerantz, Nunez, and Bean, 1983) gave females access
to tethered males in adjoining compartments that were either vocalizing or not and observing which
male they preferred. The rate of vocalizing was manipulated by either devocalizing only one of the
males or by devocalizing both males and using an ultrasound generator to associate artificial
vocalizations with only one of the males. While neither natural nor artificial ultrasounds caused
more entries into a compartment, once the female entered a compartment containing ultrasounds,
she stayed in that compartment longer. Thus, one possible function of 70-kHz ultrasounds may be
to keep the female in close proximity.
Although performed in the context of mother/infant communication, it is also quite interesting
that female mice are more attracted to infant ultrasounds if they can hear them with their right ear
than their left ear (Ehret, 1987). Such a finding suggests that the decoding of ultrasonic vocalizations
is a left-hemisphere phenomenon, providing certain parallels to human speech. If female processing
of male-produced ultrasounds is similarly lateralized, indirect evidence would certainly be provided
for functional significance.
If male vocalizations serve a courtship function, one might predict that devocalized males would
be less successful in initiating reproductive behavior. However, attempts to demonstrate such a
relationship were not successful (Nunez et al., 1985). Devocalized males readily mated and did not
show an impairment in their latency to begin mounting. Thus, while male ultrasounds may play a
role in mating, they clearly are not required for successful mating.
Recognizing that mating with an ovariectomized female in hormone-induced receptivity may
not entirely reflect mating situations in nature, Bean et al. (1986a) looked at the ability of male
vocalizations to reduce aggression in lactating females (when estrus occurs most often in natural
situations). In contrast to expectations, the lactating females attacked vocalizing males even more
quickly than devocalized males. At the same time, these lactating females were likely not experi-
encing postpartum estrus, which may have been necessary to show an effect of male ultrasounds
in reducing female aggression.
An alternative hypothesis for why it has been difficult to experimentally demonstrate function-
ality for male ultrasounds is that perhaps ultrasonic vocalizations have no communicatory signifi-
cance. Such an explanation was postulated for Mongolian gerbils by Thiessen and colleagues
(Thiessen and Kittrell, 1979; Thiessen, Kittrell, and Graham, 1980), who were unable to find any
evidence that ultrasonic vocalizations serve as a form of communication in this species. These
workers further observed that about 90% of gerbil ultrasonic vocalizations occur during hopping
movements when the forepaws hit the ground. Other situations that involved ultrasounds included
turning, stretching, compression of the upper body, and hind-foot thumping. What all of these
behaviors had in common was the physical compression of the thorax and lungs, which forced air
out of the lungs, through the larynx, and out the nose. Thiessen suggested that the ultrasonic
vocalizations produced by gerbils are simply an incidental by-product of bodily movements and
have no communicatory significance.
Blumberg (1992) critically evaluated this hypothesis for rodents in general and concluded that
house mice, rats, woodrats, hamsters, and gerbils are indeed often engaging in behaviors that
promote thoracic compression while emitting ultrasounds. In mice, the correlation between ultra-
sound production and pelvic thrusting during mounting was seen as particularly consistent with
this idea.
However, Blumberg (1992) also makes additional important points in favor of ultrasonic
vocalizations possessing communicatory significance. For example, even if an ultrasound is pro-
duced as an incidental by-product of thoracic compression, it could still acquire communicatory
significance during evolution if it reliably predicts a particular behavioral or physiological state.
As already noted, male mouse ultrasounds clearly correlate with male sexual arousal and thus could
clearly communicate the male’s motivational state to other nearby mice. Blumberg (1992) also
points out that not all rodent ultrasounds can be related to biomechanical strain alone. For example,
training rats to hop (and thereby compress their thorax) did not cause them to emit ultrasonic
vocalizations (Blumberg, 1992).
With regard to mice, I have observed high levels of 70-kHz ultrasounds when a male is stationary
and have even noted repeated vocalizations from males hanging by their front paws from the top
of a mouse cage. While thoracic compression may accompany and perhaps even aid in the emission
of some mouse ultrasonic vocalizations, thoracic compression is not essential. Whether the 40-kHz
calls of male mice better fit the thoracic compression hypothesis is not clear. More work on the
40-kHz call is clearly necessary.
Two additional caveats are necessary for investigators interested in experimentally examining
the functional effects of male mouse ultrasounds on female behavior. The first is that while male
ultrasounds may play a communicatory role in wild mice, such a role may have been altered in
domesticated mice by years of selective breeding. Clearly, domesticated mice have been bred, either
on purpose or inadvertently, to readily engage in reproductive behavior. As a result, perhaps female
reproductive behavior in domesticated mice does not require the same degree of ultrasonic facili-
tation as it does for wild mice (possibly accounting for the difficulty that experimenters have had
in demonstrating function). The second caveat is that many domesticated mouse strains (particularly
inbred strains) have high-frequency hearing loss during development and in some cases become
deaf in adulthood (Nyby and Whitney, 1978) (see Chapters 5, 6, 13, 24, 28). For either of these
reasons, studying the functional aspects of male mouse vocalizations in domesticated mice can
present problems of interpretation. On the other hand, studying the function of ultrasonic vocal-
izations using wild mice, which in many ways would be more desirable, has its own attendant
difficulties.
ULTRASONIC VOCALIZATIONS BY FEMALE MICE

Although most of the work on adult mouse ultrasonic vocalizations has concentrated on male-
produced vocalizations, normal females clearly have the capability to vocalize. For example, Sales
in an early paper (Sales, 1972b) reported two cases in which 70-kHz ultrasounds were detected
from female/female pairs. Maggio and Whitney (1985) subsequently confirmed that the best stim-
ulus for eliciting ultrasounds from female mice was indeed another female.
Similar to findings from males, females vocalized not only to awake females but also to
anesthetized females (Maggio and Whitney, 1985). Anesthetized males, however, were not good
stimuli for ultrasound elicitation. Such findings might suggest that the females were vocalizing to
odor cues from other females. However, attempts to verify the importance of odors were not
successful: very few females vocalized either to female-soiled cage shavings or to female urine
(Maggio and Whitney, 1985). Moreover, giving the females increased experience with females
(which promotes male vocalizations to female odors) was without effect in promoting female
vocalizations to female odors (Maggio and Whitney, 1985). Maggio and Whitney (1985) also found
that a brief exposure to a male inhibited vocalizing to other females for at least 48 hours.
Genetics of Female Vocalizing
Two strain surveys indicated that while females of all inbred and hybrid strains emitted ultrasonic
vocalizations, strain differences clearly existed (Maggio and Whitney, 1985). In fact, the propensity
of females of a given genotype to vocalize and their levels of vocalizations were similar to that of
males of the same genotype. Moreover, female vocalizing exhibited the genetic property of het-
erosis, with hybrids generally vocalizing more than their inbred progenitors.
Function of Female Ultrasounds
Maggio and Whitney (1985) saw little evidence that females were also engaging in male-typical
behaviors while vocalizing to other females, suggesting that these vocalizations were not simply
an inappropriate male-typical behavior. As a result, these authors hypothesized that female vocal-
izations may contribute to the establishment of social dominance hierarchies among females within
demes (i.e., breeding groups).
However, a result that complicates this interpretation was that brief exposure to an anesthetized
male greatly inhibited female vocalizations to other females for at least several days (Maggio and
Whitney, 1985). The social structure of mice usually involves females living in a deme that includes
one or more males (Bronson, 1979). Thus, if the presence of a male inhibits female vocalizing,
females would be unlikely to vocalize under naturalistic conditions. This finding raises the possi-
bility that female vocalizations are just a laboratory artifact that arises when females are kept
isolated from males. Clearly, more work is necessary on vocalizations by females to determine
whether these vocalizations are functionally important or simply an epiphenonmenon.
Alternatively, perhaps female ultrasonic vocalizations are an adaptive facultative response to
isolation from males. If the males in a deme leave or die, it may be necessary for a female to
assume some role normally performed by males. In the absence of a male, perhaps one of the
females in the deme (perhaps the most dominant female) would take over this function. This line
of reasoning is highly speculative and further research certainly is necessary to test this and other
hypotheses about female vocalizations.
AUDIBLE SOUNDS OF ADULT MICE

Audible mouse sounds during social behavior have not received nearly the attention of ultrasonic
vocalizations. However, audible sounds are common and often index a distinct emotional state in
the vocalizer. As such, they clearly convey information that could potentially be useful to another
mouse. Two audible sounds that could have communicatory significance during social behavior
include squealing (also referred to as squeaking) and tail rattling (also called tail lashing).
SQUEALING
Both adult male and female mice squeal during normal social/sexual and agonistic encounters. For
example, males often squeal during intermale aggression, while females are likely to squeal during
reproductive behavior in response to the sexual advances of a male. However, in response to human
handling, male and female mice squeal with about equal probability (Whitney, 1969). Thus, it
seems likely that this vocalization is indexing the same emotional state in both sexes. Mice of both
sexes also squeal in response to a predatory attack (Blanchard et al., 1998).
Sonagrams of Mouse Squealing
Houseknect (1968) examined the fundamental frequencies and durations of squeals produced by
adult house mice during aggressive interactions (both intraspecific and interspecific) and when
being held by the tail. During aggressive interactions, the subordinate mouse always appeared to
be the vocalizer. Squeals were much more common in male/male interactions than in female/female
interactions. However, the sonagrams of mouse squealing were similar regardless of sex and context,
with a fundamental frequency of about 4 kHz, a secondary frequency of about 8 kHz, and a duration
of about 125 ms.
Female Squealing
In male/female pairs, squealing is observed most often when a sexually motivated male is interacting
with a nonreceptive female. I have observed that nonreceptive females often squeal not only to the
mounting attempts of a male, but also, in some cases, to mere tactile contact by the male. Thus,
the squeals index not only painful interactions, but also the anticipation of such interactions.
Whether female squealing has any impact on the male’s sexual behavior has not been rigorously
tested. I have observed that males often reduce their sexual advances to nonreceptive squealing
females. Whether the reduction in male sexual behavior is caused by the squealing itself or by
other aspects of the female’s nonreceptivity (not presenting the genital region to male and pushing
the male away) is not clear.
Note in this regard that female mice are more difficult to bring into reliable behavioral estrus
through exogenous hormone treatment than female rats or hamsters. Thus, to ensure the availability
of sufficient receptive females in an experiment, it is often desirable to screen females for receptivity
with stud males prior to their use. In this context, repeated squealing by a female in response to
the male’s advances is a clear indication of nonreceptivity. In fact, McGill (1962) has used amount
of squealing as a major component in defining a five-point scale of female sexual receptivity.
Male Squealing
In male/male encounters, squeals are often emitted by the subordinate member of the pair when
attacked by the dominant member and are usually associated with defensive, escape, or submissive
behaviors of the subordinate (Scott, 1966; Scott and Fredericson, 1951). Squealing is occasionally
seen in a subordinate simply in response to being sniffed or approached by a dominant (Clark and
Schein, 1966; Scott, 1966). Thus, males squeal not only during painful interactions but also in
anticipation of such interactions. Whether the squeals reduce aggression is not clear. Many dominant
males are relentless in their aggression toward a subordinate and do not appear affected by the
subordinate’s squeals. However, the types of experimental studies that would be necessary to
definitively test this hypothesis have not been performed.
Squealing has been used to index the occurrence of agonistic behavior among male mice
(Morgret, 1973; Morgret and Dengerink, 1972). Using an electronic monitoring device, Morgret
and Dengerink (1972) found that the number of squeals during an encounter between two males
correlated with time fighting (r = 0.76), and number of attacks (r = 0.72). Moreover, the conditional
probability of a squeal occurring during a fight was p = 0.83, while during periods of no fighting
the conditional probability of a squeal was p = 0.03. The authors concluded that although squealing
directly measures one mouse’s painful stimulation during an agonistic encounter, it indirectly
provides a good measure of the occurrence of aggression. Another group of investigators (Brain,
Benton, Cole, and Prowse, 1980) built a similar device to monitor mouse squealing over extended
periods of time and presented evidence of similar impressive relationships between the amount of
squealing and various indices of aggression.
By monitoring squealing as an index of fighting, some variables that influence fighting in mice
have been examined. Male mice showed an increase in fighting around the time of puberty and
more fighting occurred at night than during the day (Morgret, 1973). Morgret also found evidence
of fighting between female mice although the incidence was much lower than for males. The rate
of aggression between male mice (as indexed by squealing) was also reduced by alcohol adminis-
tration (Bertilson, Mead, Morgret, and Dengerink, 1977).
Genetics of Squealing
As noted earlier, male and female mice are equally likely to squeal when picked up by the tail by
a human experimenter. Whitney (Whitney, 1969; 1970; 1973; Whitney and Nyby, 1983) provided
evidence for a single gene accounting for much of the strain variation observed. Picking up a
domesticated mouse by the tail is a relatively mild stressor and, accordingly, less than 5% of mice
from most strains squeal under this circumstance. However, in mice of one strain (IsBi), approxi-
mately 50% of males and females squealed when picked up by the tail. Whitney examined this
phenotypic difference using a variety of different genetic approaches and concluded that a single
dominant gene with 50% penetrance best accounted for high levels of squealing. While work by
St. John (1978) was consistent with much of Whitney’s interpretation, an unexpected age effect
suggested that the genetics might be somewhat more complicated than originally hypothesized.
Nonetheless, squealing by domesticated mice may be a naturally occurring model system for
examining the effects of single genes upon behavior.
Squealing as an Index of Nociception
Because squealing usually occurs in response to painful stimulation, pharmacologists have used this
behavior to screen analgesic drugs (Winter, 1965). The general paradigm involves testing whether
various drugs can affect the latency to begin squealing as increasing shock levels are delivered
through a metal grid floor. This “grid-shock analgesia test” has been validated as a reliable test for
antinociception and agrees well with drug data obtained by other methods (Swedberg, 1994).
Moreover, by using freely moving animals, this test has the advantage of eliminating the confounding
effects of restraint-induced analgesia present in several other analgesic testing methods.
Function of Squealing
Although squealing is usually caused by either a painful experience or the anticipation of such an
experience, only anecdotal evidence bears on whether such squeals affect the behavior of other
animals. As mentioned, squeals may serve to inhibit agonistic and sexual advances. Note in this
regard that the squeals are very disorienting to a novice human experimenter trying to inject a
mouse with a drug or anesthetic. Perhaps the squeals function to temporarily disorient the listener,
thus allowing the squeaking mouse to escape. If true, the response to squealing would likely depend
on past experience with squealing mice. Again, the types of experimental studies required to test
whether other animals are behaviorally affected by squeals have not been done.
TAIL RATTLING
Another mouse sound with potential communicatory significance is tail rattling (also called tail
lashing). In this situation, the mouse rapidly lashes its tail back and forth. As the tail rubs the
substrate, a distinctive sound is produced that can easily be heard by a human observer and
presumably by other mice as well. While this sound has audible components, ultrasonic components,
detectable by an ultrasonic receiver, exist as well (unpublished observations). However, to my
knowledge, no one has ever produced a sonagram of tail rattling.
Situations where Tail Rattling is Observed
Tail rattling is seen most often during agonistic behavior, although conflicting observations exist
about whether a dominant or subordinate male is most likely to perform the behavior. Clark and
Schein (1966) observed tail rattling more often in dominant mice just after dominance had been
established. Tail rattling was typically seen by the dominant just before attacking a subordinate.
However, tail rattling was also observed in both males prior to the establishment of dominance, by
a mouse approaching another mouse, by a mouse after grooming another, and occasionally by a
subordinate animal when being attacked or chased. Beilharz and Beilharz (1975) similarly observed
tail rattling most often after initial aggressive interactions. However, according to these investigators,
the male performing the tail rattling was usually tending toward submission. Once the mouse had
clearly become submissive, tail rattling was usually replaced by squealing in intermale encounters.
Whether tail rattling normally occurs most often in dominant or subordinate males may simply
reflect that mice of different strains possess different thresholds for the activation of this behavior.
Beilharz and Beilharz (1975) also observed tail rattling more often in domesticated mice than
in wild mice. Wild mice were more likely to flee agonistic situations before either tail rattling or
squealing could occur. Tail rattling can also be elicited by electric shock (St. John, 1973).
Genetics of Tail Rattling
St. John (1973) examined a number of inbred strains as well as some of their hybrid crosses for
amount of tail rattling in response to shock. Clear strain differences were apparent and, in general,
hybrid strains were more likely to tail rattle than their progenitor inbred strains. Such heterosis is
indicative of directional dominance and is reflective of a trait that has been directionally selected
by natural selection. Thus, genetic evidence favors the hypothesis that tail rattling serves some
adaptive function.
Function of Tail Rattling
R.Z. Brown (1953) interpreted tail rattling as a manifestation of extreme excitement in mice. Scott
and Fredericson (1951) interpreted tail rattling as a threat produced in agonistic situations in which
the mouse is considering attacking an opponent. In a later paper, Scott (1966) analogized tail rattling
in mice to growling in dogs. Clark and Schein (1966) similarly believe it to be a threat behavior,
particularly when the mouse is undecided about whether to attack or retreat. However, no strong
evidence exists that other mice respond to tail rattling as a threat. While tail rattling may be an
attempt to threaten other mice, experimental research to test this hypothesis is necessary to reach
definitive conclusions.
CONCLUDING REMARKS
Much research has examined the mechanisms by which mice produce sounds, the physical char-
acteristics of the sounds, the eliciting stimuli and contexts in which the sounds are produced, and
in the case of ultrasounds, the physiology and neuroscience related to their production. While much
remains to be learned in these different domains, one area of inquiry that has been much less
successful than others is in experimentally establishing the functional significance of mouse sounds.
While many in this field feel confident that mouse sounds play an important role in mouse social
communication, work on the functional significance of mouse sounds will ultimately be necessary
to validate much of the other work that has been performed. Such functional work, while fraught
with difficulties, should be a high priority for future research in this field.
However, independent of their communicatory significance, mouse ultrasounds have been
validated as an excellent index of male sexual motivation with a number of advantages over using
copulatory behavior. In addition to the greater speed and ease of obtaining data, it is also possible
to observe this behavior without the confounding presence of a sexual partner. Thus, this behavior
may provide a purer measure of sexual motivation than copulatory behavior itself. In addition, it
is a naturally occurring behavior that is part of the normal species repertoire that provides certain
advantages over operant measures of sexual motivation.
ACKNOWLEDGMENTS
The author thanks Murray Itzkowitz and Peter James for reading and commenting on an earlier
version of this manuscript.
2 Behavioral Assessment
of Hearing in Mice
Henry E. Heffner and Rickye S. Heffner
INTRODUCTION
The domestic house mouse (Mus musculus) has become an important animal model for the study
of genetic hearing loss. Not only are there strains with naturally occurring hearing impairments,
but genetically engineered mice can be used to identify the specific genes involved in hearing
disorders. To fully understand the effects of genetic mutations on hearing, however, it is necessary
to determine with some precision the hearing abilities of these mice. This includes not only absolute
thresholds, but other aspects of hearing such as frequency and intensity discrimination, the effect
of masking, and sound localization acuity.
There are two general approaches to assessing hearing in mice: electrophysiological and
behavioral. A popular way of assessing hearing in mice is to use an electrophysiological measure;
specifically, the auditory brainstem response or ABR (e.g., Q.Y. Zheng et al., 1999b). The popularity
of this technique stems from the fact that it is relatively easy to learn and can provide rapid results,
with measurements usually taking no more than an hour or so. However, the ABR is actually a
measure of neural synchrony — not auditory sensitivity — and at best can only be used to infer
absolute sensitivity (Hood, 1998). In addition, it cannot provide information regarding an animal’s
ability to discriminate sounds. In the clinic, the ABR is useful in diagnosing auditory disorders,
but has not supplanted behavioral tests of hearing. Indeed, it is known on occasion to significantly
overestimate and underestimate the effect of auditory malfunctions on sensory thresholds (Hood
et al., 1994; Starr et al., 1996). Thus, although the ABR is a useful tool in assessing hearing
disorders, it is necessary to employ behavioral procedures to obtain valid measures of auditory
function. The ABR method is discussed in detail in Chapter 4.
The behavioral procedures available for assessing hearing in animals can be divided into two
types: those that train an animal to respond to sound using conditioning procedures, and those that
make use of unconditioned or reflexive responses to sound. Conditioning procedures have been
considered to be more sensitive than reflexive measures, because the animals are carefully trained
to be reliable observers. Moreover, these procedures are easily adapted to testing the ability of
animals to discriminate between, as well as to detect sounds. However, conditioning procedures
can be difficult to use, and an animal may require lengthy training before it is ready for testing.
This has led to the development of procedures involving unconditioned reflexes that are simpler to
administer and involve no training of the animal. Although most tests that make use of unconditioned
reflexes are limited to determining an animal’s ability to hear loud sounds, one procedure, prepulse
inhibition, is able to determine both an animal’s ability to hear low-level sounds and to discriminate
between sounds (see Chapter 5 for a detailed discussion).
The essential feature of any auditory test is that the animal makes a clearly defined response
when one stimulus is presented and a different response when either no stimulus or a different
stimulus is presented. Although it is possible to determine an animal’s response through direct
observation, it is preferable to use an automated recording system to rule out the possibility of
observer bias. The animal should respond reliably to obviously suprathreshold stimuli, and decreasing
0-8493-2328-2/01/$0.00+$.50
the stimulus level or the difference between the stimuli must eventually result in chance perfor-
mance, indicating that the animal is not using some extraneous cue to perform the task. In addition,
it is commonly observed in conditioning procedures that the thresholds of naïve animals improve
as the animals become experienced observers, suggesting that it may be necessary for animals to
learn to listen for sounds near threshold (Stebbins, 1970). Finally, the ideal test is one that is easy
to use, works with all individuals, and provides reliable and valid results in a short period of time.
The acoustic environment is also important. Test procedures should be capable of fixing an
animal’s head within the sound field so that the sound reaching the ears can be precisely specified.
Reflections from the walls of a cage should be avoided by constructing the test cage of sound-
transparent material (e.g., wire mesh) and any necessary response or reward mechanisms kept small
and out of the path of the sound. For example, a water reward can be delivered through a thin
vertical water spout that comes up through the cage floor and is thus well below the level of the
animal’s ears (Heffner and Heffner, 1995). Sounds to be detected or discriminated should be
carefully checked for distortion as well as for onset and offset artifacts that may occur when sounds
are turned on and off abruptly.
There are a number of behavioral procedures available for assessing animal hearing (cf., Klump
et al., 1995), and it is the purpose of this chapter to describe and evaluate those that have been, or
could be, used with mice. It is not our intention to provide a manual on how to conduct these tests,
but to describe them in sufficient detail that one might choose between them. Anyone interested in
using a particular procedure should contact investigators who use them because no written descrip-
tion can cover all details, and procedures are constantly being updated and refined.
PROCEDURES USING CONDITIONED RESPONSES

It is not uncommon to divide conditioning procedures into (1) those using operant (or instrumental)
conditioning, in which an animal emits a response to obtain a reward or avoid a punisher, and
(2) those using classical conditioning, in which a stimulus elicits a response. However, whether or
not a particular procedure is true classical conditioning is a technical issue that does not affect its
use in sensory testing, and the main issue in this chapter is the ease of use and validity of the results.
All conditioning procedures involve either a reward, a punisher, or both. Rewards used for mice
have included sweetened water and milk (Birch et al., 1968; Sidman et al., 1966). However, water
by itself works well and can be reliably delivered with a commercial syringe pump or water dipper
(e.g., Markl and Ehret, 1973; Chapter 3). Electric shock is commonly used in both avoidance and
classical conditioning with the levels kept relatively low, because high levels can interfere with
performance by causing the animals to develop a fear of the test apparatus (Heffner and Heffner,
1995).
CONDITIONED SUPPRESSION/AVOIDANCE
In devising a psychophysical procedure for use with animals, it is helpful to choose a task that
utilizes an animal’s natural responses, thus making the task easier to learn. One response common
to all mammals is to suppress ongoing behavior (i.e., freeze) upon detection of a stimulus that
might signal danger. The suppression of behavior as a procedure for testing hearing was originally
developed for mice and has since been adapted for auditory testing in other animals (Heffner and
Heffner, 1998; Ray, 1970; Sidman et al., 1966). The current procedure consists of allowing an
animal to make steady contact with a water spout to obtain water, and then training it to momentarily
break contact whenever it hears a sound that signals impending shock. Because this procedure
involves avoidable shock, we have previously referred to it as “conditioned avoidance” to distinguish
it from earlier conditioned suppression procedures that used unavoidable shock (cf., Heffner and
Heffner, 1995; Sidman et al., 1966). However, because the suppression of ongoing activity is the
key feature, we refer to it here as “conditioned suppression/avoidance.”
Behavioral Assessment of Hearing in Mice 21
To determine absolute thresholds, a thirsty mouse is placed in a test cage where it drinks from
a water spout that delivers water as long as the animal is in contact with the spout. Next, a tone is
presented for 2 s, after which a mild electric shock is delivered through the water spout. The animal
quickly learns to associate the tone with the shock and breaks contact with the spout whenever it
detects the tone in order to avoid the shock. The use of avoidable, as opposed to unavoidable shock,
significantly increases the number of trials that can be given.
Test sessions are divided into 2-s trials with 1.5-s intertrial intervals. Tone or “warning” trials
are presented on a random schedule with approximately 22% of the trials containing a tone. The
response of an animal is scored as a “hit” if the animal breaks contact during the last 150 ms of a
trial that contains a tone. Breaking contact during a trial that does not contain a tone (a “safe” trial)
is scored as a “false alarm” (FA). The hit rate is then corrected for the false alarm rate; one common
correction is: Corrected Hit Rate = Hit rate – (Hit rate × FA rate). Another is: Corrected hit rate =
Hit rate – FA rate. The intensity of the tone is lowered until the animal’s performance falls to
statistical chance (i.e., the hit and false alarm rates are not significantly different), and threshold is
defined as the intensity yielding a corrected hit rate of 0.50. Sessions last approximately 20 min,
during which 25 to 30 warning trials can be given.
There are four features of the conditioned suppression/avoidance procedure that give it an
advantage over other conditioning procedures. First, the animal’s head is held in a fixed position
by requiring it to make contact with the water spout. This not only allows the sound pressure level
at the animal’s ears to be specified with precision (e.g., ±1 dB), but is essential for sound localization
tests in which the azimuth of a sound source relative to an animal’s head must be specified. Second,
the procedure incorporates a “ready” or “observing” response in that trials are not presented unless
the animal is in contact with the spout during the preceding intertrial interval. This requirement
avoids presenting trials when an animal is grooming or otherwise not attending to the task. Third,
the false alarm rate is easily controlled by changing the shock level and/or the reward rate. Thus,
a high false alarm rate can be quickly lowered by reducing the level of the shock and/or changing
the water flow rate. Finally, the procedure is easily adapted for testing any auditory discrimination,
such as frequency discrimination, intensity discrimination, or sound localization acuity, and can
even be used to assess an animal’s ability to categorize sounds (e.g., Heffner and Heffner, 1986;
Chapter 3). Tests of auditory discrimination are conducted by presenting one stimulus during safe
trials (e.g., a sound from the right) and a different stimulus during warning trials (e.g., a sound
from the left).
In summary, conditioned suppression/avoidance is a simple procedure for an animal to learn
and has been used to test hearing in more species than any other method, including wild house
mice (Heffner and Heffner, 1998; Heffner and Masterton, 1980). Given a water spout of appropriate
size and height for mice and a reliable syringe pump to deliver the 0.5 to 2 ml of water that a
domestic mouse will drink in one session, it takes 5 to 10 sessions for a naive mouse to become
accustomed to the test cage and learn to reliably respond to suprathreshold stimuli. The time required
for testing depends on the number of data points to be collected and thresholds may be most
efficiently obtained by using a tracking procedure (Ray, 1970; Sidman et al., 1966).
GO/NO-GO
The standard procedure for assessing hearing in humans is to ask subjects to raise a hand when
they hear a tone. The equivalent approach in animals is a go/no-go procedure that requires an
animal to wait patiently until a stimulus is presented and then respond within a fixed amount of time.
The first go/no-go procedure to obtain a complete audiogram for mice employed a test cage
that was divided in half by a barrier (Birch et al., 1968). The animals were required to wait
underneath a loudspeaker on one side of the barrier (a “listening” compartment) and to cross over
to the other side and press a lever within 10 s of tone onset to receive a sweetened water reward.
Trials were presented at random intervals and the intensity of the tone was reduced to obtain a
threshold. Requiring the animals to wait under the loudspeaker positioned them in the sound field,
although the intensity of the sound at the animal’s ears varied depending on where the mouse stood
and whether or not its ears were directed up at the loudspeaker. The false alarm rate was monitored,
and thresholds were retested if the rate was too high. The animals learned to respond reliably to a
suprathreshold tone in an average of 20 sessions consisting of 10 to 20 trials per session.
Although a complete audiogram was successfully obtained using this procedure, there was
considerable variability in the animals’ thresholds. One factor contributing to this was the variation
in the intensity of the sound reaching an animal, which depended both on where the animal was
standing and whether its head was oriented toward or away from the loudspeaker. Another factor
was the lack of an effective observing or ready response, and an animal would often fail to respond
to a tone if it was grooming or otherwise engaged, although it was in the listening compartment.
A simpler version of the go/no-go procedure, one that dispenses with the listening compartment,
has been used successfully to assess the ability of mice to perform a number of auditory discrim-
inations (Ehret, 1975a; Markl and Ehret, 1973). In this procedure, a mouse is placed in a small
test cage with a water spout and loudspeaker located in one corner. Variation in the sound pressure
level is minimized by confining the animal to a small area, in this case a 10 × 10-cm platform, and
presenting tones when the animal is facing the loudspeaker. Tones are presented at random intervals,
and the animal is required to maintain contact with the spout for 3 s while a tone is on in order to
obtain a water reward. Because the animals reportedly do not lick the water spout for more than
2 s in the absence of a tone, false positives are not a problem. Sessions last approximately 10 min,
during which 20 tone presentations are made.
This procedure has been used to assess absolute thresholds, frequency and intensity difference
limens, temporal integration, and masking (Ehret, 1983). Because it is not necessary to train the
animal to enter a listening compartment, initial conditioning is accomplished in 8 days. However,
the procedure works best with tame mice; that is, animals that have been handled so they are not
overly shy or nervous. As a result, not all animals can be successfully trained with this procedure,
especially when the task involves discriminating between two different sounds rather than simply
detecting a sound (Ehret, 1975b; 1976b).
Because an animal’s head is not fixed, this procedure cannot be used for sound-localization
testing. However, this could be corrected by adding a second water spout to serve as an observing
response. For example, an animal could be trained to place its mouth on one water spout while
waiting for a tone and to contact a second water spout located directly below it when a tone is
presented (a similar procedure has been used with other species; e.g., Heffner and Heffner, 1983).
This modification would both fix an animal’s head within the sound field and allow for the automated
presentation of trials and the recording of responses. In addition, the inclusion of an observing
response might increase the success rate, although the amount of time needed to train an animal
would increase.
Recently, a modification of the procedure used by Birch and colleagues has appeared that
contains several new features (Prosen et al., 2000). Photocells are used to determine the location
of an animal, and trials start automatically once it has entered the listening side of the test cage.
Instead of requiring the animal to press a lever when it hears a tone, a response is counted as soon
the animal crosses over to the side of the cage containing the water reward. Errors — both false
alarms and failure to respond to a tone (misses) — are punished by a time-out, which requires the
animal to wait an additional 5 s before testing resumes. Finally, the detection rate is corrected for
false alarms. Unfortunately, these modifications do not seem to have solved the problems of the
earlier version, as the animal’s head is still not fixed within the sound field and the procedure results
in false alarm rates of 20% and higher, well above the rate generally considered acceptable
(Stebbins, 1970). Moreover, this version involves a complicated training procedure that requires a
month or more before an animal is ready to test, making it the slowest procedure of all.
CONDITIONED EYEBLINK
Auditory thresholds have been obtained in mice by conditioning them to close their eyes when a
sound signaling impending shock is presented (Ehret, 1976a). It is important to note, however, that
this is not the standard eyeblink reflex used by others as the aversive stimulus is delivered to an
animal’s feet, not to one of its eyes (cf, Martin et al., 1980). Thus, the response of closing the eyes
is most likely part of a larger pattern of behavior in which the animal reacts to the anticipated shock.
For testing, the animal is placed in a small cage with a floor constructed of metal bars. Tones
are presented at random intervals for a duration of 1 s and are followed immediately by a brief
electric shock delivered through the cage floor. Sessions last about 10 min, during which 20 trials
are given. The interval between trials varies from 3 to 60 s, depending on the behavior of the
animal, because good responses can only be obtained when the animal is not moving. This procedure
is a simple method for assessing hearing, and those mice that are able to learn it respond reliably
after eight training sessions.
The eyeblink procedure can give good results, and audiograms generated by it are virtually
identical to those obtained using the go/no-go procedure (Markl and Ehret, 1973). In addition, it
has the advantage that it is not necessary to deprive an animal of food or water. However, it requires
the animal to sit motionless during testing, and only about 50% of mice can be trained to respond
reliably (e.g., Ehret, 1976a). Moreover, the response is determined subjectively by the experimenter
and does not lend itself to automation, opening the possibility of observer bias. Finally, the procedure
has not been used to test auditory discriminations, most likely because discrimination tests tend to
have high false positive rates and this procedure has no means to control them.
AVOIDANCE CONDITIONING
Two procedures have made use of avoidance conditioning to obtain auditory thresholds in mice.
The first procedure used a shuttle box, also known as a double grill box, to obtain absolute thresholds
(Schleidt and Kickert-Magg, 1979). This procedure consists of placing an animal in a test cage
consisting of two compartments (the shuttle box). The animal is then required to cross from one
compartment to the other whenever it hears a tone in order to avoid an electric shock applied to
the bars of the cage floor. This task is basically a go/no-go procedure in which the animal makes
a response to avoid shock, as opposed to obtaining a positive reward as in the go/no-go procedures
described above.
The second procedure trained mice to jump onto a platform to avoid electric shock in response
to a change in the frequency of a tone (Kulig and Willott, 1984). The animals were placed in a
box with a grid floor and trained to jump onto a small platform whenever an ongoing train of tone
pips of the same frequency was replaced by a train of tone pips that alternated in frequency. The
animals were given 5 s to respond to the change in frequency, after which electric shock was
delivered through the grid floor. Jumping onto the platform when the tone pips were all the same
frequency (false alarms) was punished by allowing the platform to collapse.
Although the shock avoidance procedure has an advantage in that it is not necessary to deprive
an animal of food or water, it does not appear to work well with mice. The animals tested in the
shuttle box required 3 months of training, and the investigators were only able to train 6 out of the
15 animals. Furthermore, an animal’s head is not fixed in the sound field and the animal can
continuously cross back and forth between the two compartments to avoid the shock, thus rendering
it untestable. Similar problems were encountered in the test in which the animals jumped onto a
platform to avoid shock. Although only 1 of the 12 animals failed to reach criterion, the false
positive rates were high (usually exceeding 20%) and performances even at large frequencies
differences tended to be poor. As better procedures are now available for testing mice, there is no
longer any reason to consider using shock avoidance.
APPROACH THE SOURCE OF A SOUND

An attempt was made to assess the ability of mice to localize sound by training them to approach
the source of a sound (Ehret and Dreyer, 1984). In this procedure, the mice were placed in a circular
test cage and trained to initiate trials by contacting a water spout located in the center of the cage,
which turned on a sound from one of three loudspeakers placed at the edge of the test cage 120°
apart. The animals were then required to approach the active loudspeaker and contact the water
spout located in front of it. However, the animals never performed the task well and failed to
respond if the sound was turned off before they reached the water spout. Even if the sound was
left on until they completed their response, they often did not directly approach the loudspeaker.
It is not clear why this procedure did not work well with mice, as it has been used successfully
to assess sound localization in other species, including rats and gerbils (e.g., Heffner and Heffner,
1988b; Masterton et al., 1975). As used by others, the procedure ensures that an animal’s head is
precisely oriented with respect to the loudspeakers by requiring the animal to stand on a platform
to reach the center spout. A contact switch then detects when the animal is in the proper position
and turns on the sound (Thompson et al., 1974). One possible explanation for the inability of mice
to do well in this task is that small prey animals may have a general reluctance to approach the
source of a sound, especially if they have to cross a large open area. If so, then mice might perform
better in a smaller test cage than the 1.55 m-diameter cage used by Ehret and Dreyer (1984).
It should be noted that sound localization ability can also be assessed using the conditioned
suppression/avoidance procedure in which an animal ceases to drink when a sound comes from
one direction, but not from another (Chapter 3). Indeed, the two procedures (approaching the source
of a sound and suppressing to a change in the location of a sound source) have been shown in
other mammals to give similar results (Heffner and Heffner, 1988a; 1992b). However, the suppres-
sion technique, which allows an animal to respond immediately, may be a more accurate measure
of sensory ability because an animal that is required to approach the source of a sound may become
distracted before it can complete its response.
GALVANIC SKIN RESPONSE (GSR) AUDIOMETRY

The galvanic skin response (GSR) is a measure of skin conductance and is a popular measure of
autonomic arousal (e.g., Woodworth and Schlosberg, 1965). It occurs as an unconditioned response
to a sudden loud sound that habituates with repeated presentation. However, a GSR can be obtained
to low-intensity sounds by pairing the sounds with electric shock. In this way, the GSR has been
used to obtain absolute thresholds in mice (Berlin, 1963).
For testing, a mouse is sedated to reduce extraneous movements and to allow it to be restrained.
Shock electrodes are attached to the animal’s front paws and recording electrodes to its hind paws.
For conditioning, 1-s tones are presented at random intervals accompanied by an electric shock
that is turned on 0.5 s after tone onset. Because the GSR occurs 0.5 to 3.5 s after tone onset, a
GSR response to the tone itself can only be observed for tone presentations not followed by shock.
Therefore, tones are followed by shock 40% of the time to maintain conditioning, with the tone-
only trials analyzed to determine if a response occurred. Control trials are used in which no tone
or shock is presented so as to obtain a measure of the animal’s false alarm rate. The tones are
attenuated to obtain the lowest intensity that yields a detectable response.
An audiogram for mice, derived by taking the lowest 10% of the thresholds obtained from a
group of 50 mice, showed reasonable sensitivity at low and middle frequencies, although they
appeared to be less sensitive to high frequencies than indicated by another audiogram that used the
same strain of mice (CBA/J). This difference raises the possibility that GSR conditioning may not
give accurate results at high frequencies (cf., Berlin, 1963; Birch et al., 1968). In addition, the
animals showed extreme variation, differing by as much as 80 dB at some frequencies. Moreover,
the animals failed to condition to a tone approximately 25% of the time and pregnant and estrous
females proved too variable to be used. Thus, as noted by the investigator, although GSR condi-
tioning may be of interest in its own right, it does not appear to be a good method for assessing
auditory sensitivity in individual animals (Berlin, 1963).
SUMMARY OF PROCEDURES USING CONDITIONED RESPONSES

Of the six behavioral conditioning procedures reviewed here, the conditioned suppression/avoidance
procedure appears to be the most effective for the following reasons: it has the shortest training
time; it allows the most trials to be given; it holds the animal’s head fixed in the sound field, thus
permitting accurate specification of the auditory stimulus reaching the animal; and it works with
animals considered difficult to test. Although go/no-go and eyeblink procedures can also give good
results, these two procedures do not fix an animal’s head within the sound field and will not work
with all animals, especially those that are overly active.
UNCONDITIONED RESPONSE PROCEDURES

The simplest methods for assessing hearing in mammals take advantage of an animal’s uncondi-
tioned responses to sudden loud sounds. As a result, it is possible to show that an animal can detect
a sound without having to engage in lengthy training. In most cases, such tests are limited in that
animals show an unconditioned response only to sounds that are very loud, the response habituates,
and the procedures cannot be used to measure an animal’s ability to discriminate between sounds.
However, there is one unconditioned procedure that may have overcome these limitations —
prepulse inhibition — which takes advantage of the fact that the acoustic startle reflex to a loud
sound can be modified by preceding it with another sound.
ACOUSTIC STARTLE REFLEX

Mice, like other mammals, show an unconditioned motor reaction to sudden loud sounds, a response
referred to as the acoustic startle reflex (e.g., Hoffman and Ison, 1980). The sound used to produce
the startle reflex must be loud (e.g., 100 dB re 20 µPa) and have a near instantaneous onset time
because sounds with onset times much greater than 10 ms may not elicit a startle. In addition, the
startle reflex is best elicited when the animal is sitting quietly, because a moving animal may not
show a startle response (see Chapter 5 for a detailed review).
The reflex is measured by placing an animal in a cage and presenting a startle sound at random
intervals when the animal is not moving (e.g., Parham and Willott, 1988). The response of the
animal to the startle sound is detected with an accelerometer attached to the test cage. A variety
of startle sounds have been used, including noise (e.g., 10 to 25 ms noise burst, 100 to 115 dB,
1 to 5 ms rise/fall time) and tones (e.g., 4 to 24 kHz tone burst, 70 to 110 dB, 10 ms duration,
1 ms rise/fall time). Although habituation of the startle reflex does occur, it is generally possible
to obtain a large number of trials (e.g., 60) within a single session.
Because the startle reflex occurs only to relatively loud sounds, it cannot be used to determine
absolute sensitivity. However, it can provide information that may be used to supplement threshold
measurements (e.g., Parham and Willott, 1988). For example, a normal startle to loud sounds in
an animal with a hearing loss might indicate the occurrence of recruitment, a phenomenon in which
absolute thresholds are elevated, but the apparent loudness of sounds at suprathreshold levels is
unchanged (e.g., Moore, 1997). Thus, the acoustic startle reflex can provide additional information
about an animal’s hearing ability.
PREPULSE INHIBITION
Although the acoustic startle reflex itself can only determine an animal’s ability to respond to loud
sounds, the latency or amplitude of the response can be modified by a less intense sound that
precedes the startle sound, but which does not itself cause a startle (e.g., Hoffman and Ison, 1980).
Thus, the ability of an animal to detect a particular sound can be investigated by determining if
presenting that sound before the startle sound modifies the resulting reflex, a procedure referred to
as reflex modification or prepulse inhibition (see Chapter 5 for a detailed review).
As in the startle reflex test described above, a mouse is placed in a cage that has an accelerometer
attached to it to detect the animal’s movements (e.g., Ison et al., 1998; Willott and Turner, 1999).
A startle sound, such as a 25-ms noise burst at 115 dB with a near-instantaneous rise/fall time, is
presented at random intervals when the animal is sitting quietly. The startle stimulus is preceded
on most trials (e.g., 75%) by a prepulse stimulus, such as a 40-ms tone, with the startle stimulus
presented alone on the remaining trials for comparison. A typical interval between the two stimuli
is 100 ms, although the optimal interval must be determined empirically. The effect of the prepulse
stimulus is expressed as a percent reduction of the unmodified startle response. Test sessions may
last 1 h, during which about 50 to 100 trials are presented.
Prepulse inhibition can be used to measure both absolute thresholds and the ability to discrim-
inate between different sounds. Absolute thresholds are determined by reducing the intensity of
the prepulse stimulus until it no longer has an effect on the startle reflex. The ability to discriminate
between stimuli is determined using a change in an ongoing sound as the prepulse stimulus. For
example, the ability to discriminate continuous noise from noise containing a gap is determined
by presenting ongoing noise interrupted by a gap that occurs just before the presentation of the
startle stimulus, that is, the gap serves as the prepulse stimulus. This procedure has produced
reasonable gap detection thresholds in mice (Ison et al., 1998), and there appears to be no reason
that it cannot be used for other auditory discriminations. That is, frequency- and intensity-difference
thresholds, as well as sound-localization thresholds, could be determined by using a prepulse
stimulus that consists of a change in the frequency, intensity, or location of an ongoing sound.
Although prepulse inhibition appears to be ideal for sensory testing, the question arises as to
whether it is as sensitive as conditioned response procedures. First, experience with conditioning
procedures suggests that animals must learn to listen before they becomes reliable observers; that
is, initial thresholds are generally higher than those obtained in later sessions (e.g., Stebbins, 1970).
Because prepulse inhibition does not involve any training for vigilance, it might not reflect an
animal’s best sensitivity. Second, the current method for fixing an animal’s head in the sound field
involves tranquilizing the animal and holding its head in place with a wooden Q-tip glued to the
skin of its head (e.g., Ison and Agrawal, 1998), raising the question of whether sedation would
affect thresholds.
At present, there is reason to believe that prepulse inhibition can yield sensitive thresholds.
Specifically, a reflex modification study using rats obtained thresholds as low as those found using
conditioned response procedures, at least for the frequencies in the midrange of the audiogram
(Fechter et al., 1988a). However, the issue of validity is best settled by comparing thresholds
obtained using this procedure to those obtained for the same animals in the same acoustic envi-
ronment using a conditioned response procedure. Moreover, the entire audiogram should be deter-
mined to ensure that the procedure is equally sensitive at all frequencies because at least one
procedure — the conditioned GSR — appears to underestimate high-frequency hearing (see above).
In summary, prepulse inhibition appears to be a simple and rapid method for assessing hearing.
Because it uses a natural reflex, an animal requires no training and usable results can be obtained
in the first session. However, it should be noted that tests are sometimes carried out with 2 to 4 days
between sessions to avoid habituation, with the result that some tests require as much as 2 months
to conduct (e.g., Ison and Agrawal, 1998). Moreover, the optimal parameters may vary with the
particular task, making it necessary to conduct pilot studies before detailed testing can begin. For
example, it is helpful to know the optimal startle stimulus and interval between the prepulse and
startle stimuli that yield the best results, as well as the maximum number of trials that can be given
without causing excessive habituation.
PINNA MOVEMENTS
Because mammals with mobile ears will move their pinnae when presented with an unexpected
sound, one of the first tests of animal hearing was to look for pinna movements in response to
sound. One type of pinna movement is the Preyer reflex, which is a movement of the pinna in
response to loud sounds (Ehret, 1983; Francis, 1979). This response is considered to be a startle
reflex because it occurs only in response to loud sounds, and thresholds obtained with it are about
60 to 90 dB above those obtained with conditioning procedures (e.g., Hack, 1968; Markl and Ehret,
1973).
Another type of response is the pinna movements that occur in reaction to sounds as low as
25 dB (re 20 µPa), much lower than the sounds that elicit a Preyer reflex (Ehret, 1976a; 1983). In
this case, the animal appears to be making use of the directional filtering properties of its pinnae
to search its auditory environment. Although the response rapidly habituates, it can be reinstated
by briefly handling the animals, which probably has the effect of sensitizing them (Ehret, 1976a).
Although both of these responses are of interest in their own right, the Preyer reflex occurs
only in response to very loud sound, and it is unknown whether pinna movements to less intense
sounds are a measure of absolute threshold. In other words, the absence of a response does not
indicate that the animal cannot hear the sound, only that it is not responding to it. The best
demonstration of the usefulness of pinna movements elicited by less intense sounds has been in
studies of the development of hearing in young mice (Ehret, 1976a; 1977). However, it may now
be possible to study hearing, even in young mice, using other techniques such as the acoustic startle
reflex modification procedure and even, perhaps, the conditioned suppression/avoidance procedure.
FREEZING RESPONSE
An animal that is moving about may stop or freeze when it hears an unexpected sound. This reaction
has been used to demonstrate hearing in mice under 12 days of age, at which time the pinna detaches
from the scalp and pinna movements can be observed (Ehret, 1976a; 1977). As with the pinna
movements discussed above, the freezing response is probably not a measure of absolute sensitivity,
although it can provide useful information in the absence of other measures. More sensitive
procedures, such as the acoustic startle reflex modification procedure or conditioned suppres-
sion/avoidance procedure, should be tried first before using this procedure.
GALVANIC SKIN RESPONSE (GSR)

As previously noted, galvanic skin response (GSR) occurs as an unconditioned response to loud
sounds. The unconditioned GSR has been used to study the relative response of mice to tones from
2 to 40 kHz delivered at a constant sound pressure level of 100 dB (Berlin et al., 1968). As in the
conditioned GSR procedure, the mouse is sedated to reduce extraneous movements and GSR
recording electrodes attached to its hind feet. The magnitude of the GSR appears to parallel its
audiogram, suggesting that the unconditioned GSR might provide an equal loudness contour.
However, given the uncertainty as to whether the GSR is an accurate indicator of high-frequency
hearing (see above), other estimates of loudness (e.g., the acoustic startle reflex) might provide
more accurate results.
SUMMARY OF UNCONDITIONED RESPONSE PROCEDURES

Of the five unconditioned procedures reviewed here, prepulse inhibition appears to hold the most
potential for auditory testing. Not only is this procedure capable of demonstrating an animal’s
ability to detect sounds of low intensity, but it can also be used to determine the ability to
discriminate between sounds. All that is required is to verify that the thresholds obtained with this
procedure are as sensitive as those obtained with procedures using conditioned responses. In
addition, the acoustic startle reflex itself is a useful indicator of an animal’s responsiveness to loud
sounds and is therefore an important adjunct to tests of absolute threshold. Both prepulse inhibition
and the acoustic startle reflex have been the subject of numerous studies, with the result that much
is known about them (see Chapter 5). Moreover, the acoustic startle response is measured objec-
tively, whereas most other unconditioned procedures rely on the subjective report of the experi-
menter. Therefore, these two procedures are to be preferred when assessing hearing with uncondi-
tioned procedures.
CONCLUSION
Because mice play a key role in the study of the genetics of hearing, it is important to develop
procedures that can quickly and accurately measure their hearing. Not only should a procedure be
capable of assessing absolute sensitivity, but it also should be able to determine an animal’s ability
to discriminate between sounds.
Of the procedures reviewed here, two hold the most promise for testing mice. The first,
conditioned suppression/avoidance, is a relatively simple procedure because the animals need only
stop drinking when they hear a sound that signals impending shock. As a result, it is possible to
train animals in a minimum amount of time. In addition, requiring an animal to drink from a water
spout fixes its head within a sound field, which allows for precise measurement of the sound
reaching the animal and makes it possible to test sound localization. Indeed, this is one of the few
procedures that can be used to test any auditory discrimination.
However, the current conditioned suppression/avoidance procedure can be improved by increas-
ing the speed with which thresholds are assessed. For example, current practice for assessing
absolute thresholds is to present tones of a particular intensity in blocks of five or more trials,
calculate the animal’s performance, and then change to a new intensity. Because mice require little
water and thus work for only 15 to 20 min, only one threshold can be obtained per daily session
using this procedure. However, the speed with which thresholds are determined could be increased
using an automated tracking procedure in which the intensity of the tone is changed from trial to
trial, as originally recommended by Sidman and co-workers (1966), instead of collecting informa-
tion in blocks of trials. Other changes that might also increase the speed of testing would be to
optimize the test apparatus by determining the size and shape of the water spout and water reward
rate that work best for mice.
The other behavioral procedure that holds great promise is prepulse inhibition. Because this
procedure makes use of an unconditioned reflex, it is potentially the fastest of all procedures for
assessing hearing in mice because the animals need not be trained. However, it remains to be
determined whether absolute thresholds obtained with this procedure are as sensitive as those
obtained with conditioned response procedures. In addition, it is important to find a good way to
keep an animal’s head fixed within the sound field because there is the possibility that the current
technique, which involves sedation, might affect thresholds. Should prepulse inhibition prove to
provide valid thresholds, it could easily supplant conditioned response procedures for assessing
hearing in mice and other animals. Despite the fact that it may be necessary to space test sessions
several days apart to reduce habituation, prepulse inhibition is simpler to use and would be able
to test more animals in the same amount of time than any of the conditioned response procedures,
including conditioned suppression/avoidance.
SUMMARY
Six conditioned and five unconditioned response procedures for assessing hearing in mice are
reviewed. The six conditioned response procedures include conditioned suppression/avoidance,
go/no-go, eyeblink conditioning, avoidance conditioning, approaching the source of a sound, and
GSR conditioning. Of these, the conditioned suppression/avoidance procedure is the most effective
because it has the shortest training time, allows the most trials to be given, holds the animal’s head
fixed in the sound field, and is successful with animals considered difficult to test. The five
unconditioned procedures are the acoustic startle reflex, prepulse inhibition, pinna movements,
freezing response, and GSR. Of these, the prepulse inhibition procedure holds the most promise
for auditory testing; unlike other unconditioned response procedures, it is it capable of demonstrat-
ing an animal’s ability to detect low-intensity sounds as well as discriminate between sounds.
ACKNOWLEDGMENTS
We thank G. Koay and I. Harrington for their comments on an earlier draft of this chapter. Many
of the ideas expressed here were developed in work supported by NIH grants NS 30539, DC 02960,
and DC 03258.
3 Focus: Sound-Localization
Acuity Changes with Age
in C57BL/6J Mice
Rickye S. Heffner, Gimseong Koay, and Henry E. Heffner
INTRODUCTION
C57BL/6J mice show a progressive high-frequency hearing loss beginning about 2 to 3 months of
age (Chapters 13, 24, 28; Mikaelian, 1979). Because mammalian high-frequency hearing (i.e., above
the10-kHz limit of other vertebrates) evolved under strong selective pressure for sound localization,
such a hearing loss should affect not only the ability of mice to detect sound, but also to localize it
(R. Heffner and Heffner, 1992a; H. Heffner and Heffner, 1998). Specifically, high frequencies are
necessary for two of the three basic locus cues: the binaural intensity difference of a sound at the two
ears and the monaural pinna cues that arise from the directionality of the pinnae (the third cue being
the binaural time difference, primarily a low-frequency cue). The two high-frequency cues require
sounds that are effectively shadowed by the head and/or pinnae because low frequencies bend around
small obstacles with little attenuation. Just how high an animal must hear to be able to use these cues
depends on the size of its head and pinnae — the smaller the animal, the higher it must hear, which
is why mice can hear above 80 kHz (H. Heffner and Masterton, 1980; Markl and Ehret, 1973). Thus,
the loss of high-frequency hearing should have a detrimental effect on the ability of mice to localize
sound, especially because they may not be able to compensate by relying on binaural time cues
because the maximum interaural delay their small heads generate is so small (about 60 µs).
We performed a study to observe the effect of age-related, high-frequency hearing loss in
C57BL/6J mice by determining their sound-localization acuity at two different ages. An additional
goal was to assess the ability of mice to localize brief sounds because previous research had only
been able to demonstrate an ability to home in on sounds that were continuously repeated (Ehret
and Dryer, 1984). Sound-localization thresholds and the ability to localize filtered noise bursts were
determined for three C57BL/6J mice using an avoidance procedure involving suppression of
drinking (for details, see H. Heffner and Heffner, 1995). The animals were then retested later, at
which time their absolute thresholds for 16- and 32-kHz tones were also determined. The animals
had free access to food in their home cages and received water during daily test sessions.
The mice were tested in a small, sound-transparent, wire mesh cage (15 × 8 × 10 cm) mounted
on a camera tripod in the center of a carpeted, double-walled acoustic chamber, the walls and
ceiling of which were lined with eggcrate foam to reduce sound reflections. The animals were
trained to make steady contact with a water spout that came up through the floor of the cage in
order to receive a slow but steady trickle of water dispensed via a syringe pump located outside
the chamber. Drinking from the spout served to fix the animal’s head in the center of a perimeter
bar (1-m radius) on which loudspeakers were mounted. A typical test session lasted approximately
20 min, during which a mouse consumed 0.5 to 1.5 mL of water.
The mice were trained to drink in the presence of a 100-ms noise burst emitted from a
loudspeaker to their right, but to break contact with the water spout whenever a sound was emitted
0-8493-2328-2/01/$0.00+$.50
from a loudspeaker to their left in order to avoid a mild electric shock delivered through the spout.
A 100-ms noise burst was presented once every 2.3 s from the left or right side on a quasi-random
schedule in which about 22% of signals were from the left. An animal’s response on each trial was
determined by recording whether it was in contact with the spout during the last 200 ms of a trial
(which was 1.8 s in duration). Breaking contact following a sound from the left was recorded as
a hit, whereas breaking contact following a sound from the right was recorded as a false alarm.
Performance was quantified by determining hit and false alarm rates for a particular stimulus
or angle in blocks of trials (typically 6 left and 25 right sounds). A performance measure was
determined by correcting the hit rate according to the following formula: Performance = Hit rate –
(Hit rate × False alarm rate). Threshold, defined as the angle at which performance equaled 0.50,
was determined by progressively reducing the separation between the loudspeakers until perfor-
mance fell to chance (i.e., the hit and false alarm rates did not differ significantly).
Sound-localization thresholds were determined using a 100-ms broadband noise burst containing
measurable frequencies from 3 to 80 kHz (6 to 48 kHz ± 3dB), covering most of the hearing range of
the domestic mouse (which, for sounds of 60 dB sound pressure level (SPL) ranges from 900 Hz to
79 kHz; Markl and Ehret, 1973). The intensity of the noise was varied randomly from 63 to 70 dB
SPL to prevent the animals from using possible intensity differences between the loudspeakers as a cue.
A second test investigated the importance of high frequencies for localization by determining
average performance for localizing low-pass filtered noise (48 dB/octave) at a fixed angle of 60°
(±30° left and right of midline). The five low-pass filter settings used (and the signal intensities re
20 µPa) were: 80 kHz (70 dB), 60 kHz (70 dB), 40 kHz (69 dB), 20 kHz (71.5 dB), and 10 kHz
(67 dB). The filtered noise was presented with a 50-ms rise/fall time to avoid generating high-
frequency onset and offset artifacts. (For the sound production and measurement equipment, see
R. Heffner, Heffner, and Koay, 1995). Blocks of six left trials (and the associated right trials) were
given at four different filter settings each day until 36 left trials had been given for each noise band.
Finally, absolute thresholds for 16- and 32-kHz pure tones were determined at the end of testing
to verify that the animals had a high-frequency hearing loss. In this case, the animals were trained
to break contact with the spout whenever they heard a tone presented from a loudspeaker located
in front of the cage (3 pulses, 400 ms on, 100 ms off, 10 ms rise/decay). The intensity of the tone
was reduced in 5-dB increments until performance fell to chance and threshold was defined as the
level at which performance equalled 0.50.
LOCALIZATION ACUITY
The mice had little difficulty learning to localize single, brief noise bursts. Initial training and
detailed testing for localizing the 100-ms broadband noise required 21 and 25 sessions, respectively.
During final threshold determination at 2.4 to 2.5 months of age, all three mice were performing
well at angles of 90° or larger, and their mean threshold was 33° (Figure 3.1). Retraining and testing
after more than a 3-month break required only 14 sessions, as the mice were now experienced with
the test. Each animal’s threshold, determined between 6.9 and 7.1 months of age, had increased
on average by 13° to a new average of 46° (Figure 3.1). The increased thresholds cannot be attributed
to an inability to hear the noise burst, nor to any deterioration in motivation, intellect, or general
localization ability because performance at large angles remained normal.
LOCALIZATION OF LOW-PASS NOISE

Determination of the ability of the mice to localize low-pass noise bursts (60° separation) required
six to eight sessions. Initial performance was determined at 2.5 to 2.7 months of age, and retesting
took place at 7.2 to 7.4 months (Figure 3.2). Performance declined progressively as the high
frequencies were removed, demonstrating the importance of high frequencies for azimuthal local-
ization in mice. Animals A and B fell to chance when the filter was set at 20 kHz low-pass, and
Focus: Sound-Localization Acuity Changes with Age in C57BL/6J Mice 33
FIGURE 3.1 Sound-localization performances of the three mice at 2.4 to 2.5 months of age (solid lines)
and 6.9 to 7.1 months (dashed lines). Thresholds increased with age from 37° to 51° for mouse A, 38° to 59°
for mouse B, and 23.5° to 37° for mouse C.
all three mice were unable to localize above chance when the filter was set at 10 kHz low-pass
although the noise burst was clearly audible. The animals showed a mild performance decrement
as they aged, even when the signal contained high frequencies, presumably because a high-
frequency hearing loss prevented the mice from making full use of them.
PURE-TONE THRESHOLDS
Detection thresholds at 16 and 32 kHz were completed when the mice were 7.7 months of age.
Compared with NMRI mice (Markl and Ehret, 1973), the C57BL/6J mice showed hearing losses
at 16 kHz of 16 to 54 dB and at 32 kHz of 52 to 59 dB (Table 3.1). Thus, these animals had an
obvious hearing loss by 7.7 months of age.
DISCUSSION
TEST PROCEDURE
The use of an avoidance procedure in which an animal ceases responding when it detects a sound
is a natural response that all three of the mice easily learned. Moreover, the animals could easily
shift from one task (discriminating locus) to another (detecting tones) with little difficulty. Indeed,
FIGURE 3.2 Sound localization performances of the three mice at 2.5 to 2.7 months of age (solid lines),
and 7.2 to 7.4 months (dashed lines) with 100-ms low-pass filtered noise bursts presented 30° to the left or
right of midline.
TABLE 3.1
Detection Thresholds (dB SPL) at 16 and 32 kHz
for Three C57BL/6J Mice at the End of Sound
Localization Testing (7.7 months old)
Frequency
Strain 16 kHz 32 kHz
NMRI (average)a 4 dB 22 dB
C57BL/6J (7.7 mo)
Mouse A 20 dB 74 dB
Mouse B 41 dB 80 dB
Mouse C 58 dB 81 dB
a Source: From Markl and Ehret, 1973. With permission.
Focus: Sound-Localization Acuity Changes with Age in C57BL/6J Mice 35
this procedure has proven successful in assessing hearing in a wide variety of animals (e.g.,
H. Heffner and Heffner, 1995; 1998), and it probably accounts for the success in demonstrating
that mice can localize brief sounds when previous studies have failed (Ehret and Dryer, 1984).
Moreover, the combination of this task with the response of eating or drinking from a spout, which
fixes an animal’s head in the sound field, makes it ideal for auditory testing.
SOUND LOCALIZATION CHANGES ACCOMPANYING AGING

From 2.7 to 7.4 months of age, the average localization thresholds of the mice increased from 33°
to 46°. Because this strain of mice is known to show a progressive high-frequency hearing loss
beginning at 2 to 3 months of age, and because the mice tested here demonstrated a large high-
frequency hearing loss at 7.7 months, it is probable that the increased locus thresholds were due
to their hearing loss. That the mice did not suffer from any general deterioration in localization
ability is suggested by the observation that their localization performance at large angles was
essentially normal (Figure 3.1).
The loss of high frequencies does not affect left-right sound localization acuity in all species.
For example, humans with high-frequency sensorineural hearing loss retain good left-right acuity,
presumably because binaural time differences, which use low frequencies, are sufficient to maintain
good localization (Colburn, Zurek, and Durlach, 1987; Noble, Byrne, and Lepage, 1994). Similarly,
the left-right localization acuity of chinchillas is not degraded by filtering out high frequencies, as
it is in mice. However, it should be noted that both humans and chinchillas require high frequencies
to discriminate the elevation of sound and to prevent front-back reversals (Belendiuk and Butler,
1975; R. Heffner et al., 1994; 1995). The fact that mice require high frequencies for left-right
localization, however, suggests that they may not use the binaural time-difference cue for locus.
This would not be unique because at least one other small mammal, the big brown bat, appears to
have relinquished the use of the binaural time cue, relying solely on interaural intensity differences
and monaural pinna cues for passive localization (Koay et al., 1997).
COMPARISON OF MICE TO OTHER MAMMALS

The 33° mean threshold for these mice at 2.5 to 2.7 months of age is larger than that of most
mammals, but comparable to that of gerbils, kangaroo rats, horses, and cattle (H. Heffner and
Heffner, 1998). Although some investigators have suggested that there may be small high-frequency
losses in C57BL/6J mice at 2 months of age (Li and Borg, 1991), it is likely that the explanation
for the comparatively poor locus acuity in these mice lies in the relationship between hearing and
vision — the primary function of sound localization is to direct the eyes to the source of a sound
for visual examination (R. Heffner and Heffner, 1992c; Koay et al., 1998). Just how accurately the
ears must direct the eyes depends on the width of an animal’s field of best vision. Specifically,
animals with narrow fields of best vision (e.g., humans and elephants) require better locus acuity
than animals with broad fields of best vision (e.g., kangaroo rats and cattle). Because the density
of the ganglion cells in the mouse retina indicates that it has a relatively broad field of best vision
of 114° (based on retinal ganglion-cell density gradients, R. Heffner, unpublished), its comparatively
poor localization acuity is not unexpected.
ACKNOWLEDGMENTS
This research was supported by NIH Grant R01 DC 02960. We thank J. Willott for suggesting this
experiment and for providing the mice. In addition, we thank Terry Donnel for help in testing the
animals.
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These were substantials, and there were jocose extravagances to
chorus them. One heard of a pair of cuff-links, diamond and emerald
studded, purchased to order at the price of a king’s ransom; of a
sybaritic Fortunatus who reveled luxuriously in night-shirts at three
thousand dollars a dozen; of men who scorned the humble “chip” in
the crowded gaming rooms and played with twenty-dollar gold
pieces for counters.
Philip saw and heard and was conscious of penetrating inward
stirrings. Was the totting-up of figures all he was good for? True,
there were money-making opportunities even at the railroad desk;
chances to lend his thrifty savings at usurious interest to potential
prospectors; chances to make quick turn-overs on small margins,
and with certain profits, in real-estate; invitations to get in on
“ground floors” in many promising enterprises, not excepting the
carefully guarded inside stock pool of the railroad company he was
working for. But the inward stirrings were not for these ventures in
the commonplace; they were even scornful of them. Money-lending,
trading, stockjobbing—these were for the timid. For the venturer
unafraid there was a braver and a richer field.
“How much experience does a fellow have to have to go
prospecting?” he asked of Middleton, one day when the figure-
adding had grown to be an anæsthetizing monotony hard to be
borne.
Middleton grinned mockingly. “Hello!” he said. “It’s got you at last,
has it?”
“I asked a plain question. What’s the answer?—if you know it.”
“Experience? Nine-tenths of the fools who are chasing into the hills
don’t know free gold from iron pyrites, or carbonates from any other
kind of black sand. It’s mostly bull luck when they find anything.”
“Yet they are finding it,” Philip put in.
“You hear of those who find it. The Lord knows, they make racket
enough spending the proceeds. But you never hear much about the
ninety-nine in a hundred who don’t find it.”
“Just the same, according to your tell, one man’s chance seems to
be about as good as another’s. I believe I’d like to have a try at it,
Middleton. Want to go along?”
“Not in a thousand years!” was the laughing refusal. “I’ll take mine
straight, and in the peopled cities. I’ve got a girl back in Ohio, and
I’m going after her one of these days—after this wild town settles
down and quits being so rude and boisterous.”
Philip looked his desk-mate accusingly in the eye.
“It’s an even bet that you don’t,” he said calmly.
“Why won’t I?”
“I saw you last night-down at the corner of Holladay and
Seventeenth.”
Middleton, lately a country-town bank clerk in his native Ohio, but
who was now beginning to answer the invitation of a pair of rather
moist eyes and lips that were a trifle too full, tried to laugh it off.
“You mean the ‘chippy’ I was with? I’m no monk, Philly; never set up
to be. Besides, I’m willing to admit that I may have had one too
many whiskey sours last night. Cheese it, and tell me what’s on your
mind.”
“I’ve already told you. I think I’ll try my luck in the hills, if I can find
a partner.”
“Good-by,” grunted Middleton, turning back to his tonnage sheet. “As
for the partner part, all you have to do is to chase down to the
station and shoot your invitation at the first likely looking fellow who
gets off the next incoming train. He’ll be a rank tenderfoot, of
course, but that won’t make any difference: there’ll be a pair of you
—both innocents. Why, say, Philly; I’ll bet you’ve still got your first
drink of red liquor waiting for you! Come, now—own up; haven’t
you?”
“I should hope so,” said Philip austerely. “I didn’t come all the way
out here to make a fool of myself.”
This time Middleton’s grin was openly derisive.
“My, my!” he jeered. “The spirit moves me to prophesy. I know your
kind, Philly—up one side and down the other. When you let go, I
hope I’ll be there to see. It’ll be better than a three-ring circus.
Wine, women and song, and all the rest of it. Speaking of women
——”
“You needn’t,” Philip cut in shortly; and he got up to answer the
auditor’s desk bell.
The process of securing a partner for a prospecting trip was scarcely
the simple matter that Middleton’s gibing suggestion had made it.
Though there were many haphazard matings achieved hastily at the
outfitting moment, a goodly proportion of the treasure hunters were
coming in pairs and trios hailing from a common starting point in the
east. In spite of the free-and-easy levelling of the conventions, Philip
found it difficult to make acquaintances, his shell of provincial
reserve remaining unchipped, though he tried hard enough to break
it. Besides, he felt that he was justified in trying to choose
judiciously. He could conceive of no experience more devastating
than to be isolated in the wilderness for weeks and perhaps months
with an ill-chosen partner for his only companion. The very intimacy
of such an association would make it unbearable.
It was while he was still hesitating that a small duty urged itself
upon him. It concerned the Mississippi family with the death-
threatened husband and father. In a city where all were strangers he
had fully intended keeping in touch with the Dabneys, if only to be
ready to offer what small help a passing acquaintance might in case
the threatened catastrophe should climax. Since he would shortly be
leaving Denver, the duty pressed again, and he set apart an evening
for the tracing of the Mississippians, going first to the American
House to make inquiries.
Fortunately for his purpose, one of the hotel clerks, himself a
Southerner, remembered the Dabneys. They had remained but a few
days in the hotel; were now, so the clerk believed, camping in one of
the tent colonies out on California or Stout Streets somewhere
between Twentieth and Twenty-third. Yes, Captain Dabney had been
in pretty bad shape, but it was to be assumed that he was still living.
The clerk had been sufficiently interested to keep track of the
obituary notices in the newspapers, and the Dabney name had not
appeared in any of them. Inquiry among the tenters would probably
enable Philip to find them.
Reproaching himself for his prolonged negligence, Philip set out to
extend his search to the tent colonies. It was after he had reached
the more sparsely built-up district, and was crossing a vacant square
beyond the better-lighted streets, that a slender figure, seemingly
materializing out of the ground at his feet, rose up to confront him,
a pistol was thrust into his face, and he heard the familiar formula:
“Hands up—and be quick about it!”
It is probably a fact that the element of shocked surprise, no less
than the natural instinct of self-preservation, accounts for the easy
success of the majority of hold-ups. Sudden impulse automatically
prompts obedience, and the chance of making any resistance is lost.
But impulsiveness was an inconsequent part of Philip’s equipment.
Quite coolly measuring his chances, and well assured that he had a
considerable advantage in avoirdupois, he knocked the threatening
weapon aside and closed in a quick grapple with the highwayman.
He was not greatly surprised when he found that his antagonist,
though slightly built, was as wiry and supple as a trained acrobat;
but in the clinching struggle it was weight that counted, and when
the brief wrestling match ended in a fall, the hold-up man was
disarmed and spread-eagled on the ground and Philip was sitting on
him.
When he could get his breath the vanquished one laughed.
“Made a complete, beautiful and finished fizzle of it, d-didn’t I?” he
gasped. “Let me tell you, my friend, it isn’t half so easy as it is made
to appear in the yellow-back novels.”
“What the devil do you mean—trying to hold me up with a gun?”
Philip demanded angrily.
“Why—if you must know, I meant to rob you; to take and
appropriate to my own base uses that which I have not, and which
you presumably have. Not having had the practice which makes
perfect, I seem to have fallen down. Would you mind sitting a little
farther back on me? I could breathe much better if you would.”
Philip got up and picked up the dropped weapon.
“I suppose I ought to shoot you with your own gun,” he snapped;
and the reply to that was another chuckling laugh.
“You couldn’t, you know,” said the highwayman, sitting up. “It’s
perfectly harmless—empty, as you may see for yourself if you’ll
break it. You were quite safe in ignoring it.”
Philip regarded him curiously.
“What kind of a hold-up are you, anyway?” he asked.
“The rottenest of amateurs, as you have just proved upon my poor
body. I thank you for the demonstration. It decides a nice question
for me. I hesitated quite some time before I could tip the balance
between this, and going into a restaurant, ordering and consuming a
full meal, and being kicked out ignominiously for non-payment
afterward. This seemed the more decent thing to do, but it is pretty
evident that I lack something in the way of technique. Wouldn’t you
say so?”
“I should say that you are either a fool or crazy,” said Philip bluntly.
“Wrong, both ways from the middle,” was the jocular retort. “At the
present moment I am merely an empty stomach; and empty
stomachs, as you may have observed, are notoriously lacking in any
moral sense. May I get up?”
“Yes,” said Philip; and when the man was afoot: “Now walk ahead of
me to that street lamp on the corner. I want to have a look at you.”
What the street lamp revealed was what he was rather expecting to
see; a handsome, boyish face a trifle thin and haggard, eyes that
were sunken a little, but with an unextinguished smile in them, a
fairly good chin and jaw, a mouth just now wreathing itself in an
impish grin under his captor’s frowning scrutiny.
“Umph!—you don’t look like a very hard case,” Philip decided.
“Oh, but I am, I assure you. I’ve been kicked out of two respectable
colleges, dropped from the home club for conduct unbecoming a
gentleman, and finally turned out of house and home by a justifiably
irate father. Can I say more?”
“What are you doing in Denver?”
“You saw what I was trying to do a few minutes ago. The outcome
dovetails accurately with everything else I have attempted since I
parted with the final dollar of the even thousand with which I was
disinherited. Failure seems to be my baptismal name.”
“What is your name?”
“Henry Wigglesworth Bromley. Please don’t smile at the middle third
of it. That is a family heirloom—worse luck. But to the matter in
hand: I’m afraid I’m detaining you. Shall I—‘mog,’ is the proper
frontier word, I believe—shall I mog along down-town and surrender
myself to the police?”
“Would you do that if I should tell you to?”
“Why not, if you require it? You are the victor, and to the victors
belong the spoils—such as they are. If you hunger for vengeance,
you shall have it. Only I warn you in advance that it won’t be
complete. If the police lock me up, they will probably feed me, so
you won’t be punishing me very savagely.”
For once in a way Philip yielded to an impulse, a prompting that he
was never afterward able to trace to any satisfactory source.
Dropping the captured revolver into his coat pocket, he pressed a
gold piece into the hand of the amateur hold-up.
“Say that I’ve bought your gun and go get you a square meal,” he
said, trying to say it gruffly. “Afterward, if you feel like it, go and sit
in the lobby of the American House for your after-dinner smoke. I’m
not making it mandatory. If you’re not there when I get back, it will
be all right.”
“Thank you; while I’m eating I’ll think about that potential
appointment. If I can sufficiently forget the Wigglesworth in my
name I may keep the tryst, but don’t bank on it. I may—with a full-
fed stomach—have a resurgence of the Wigglesworth family pride,
and in that case——”
“Good-night,” said Philip abruptly, and went his way toward the tent
colony in the next open square, wondering again where the impulse
to brother this impish but curiously engaging highwayman came
from.
III
In circumstances in which it would have been easy enough to fail,
Philip found the family he was looking for almost at once; and it was
the young woman with the dark eyes and hair and the enticing
Southland voice and accent who slipped between the flaps of the
lighted tent when he made his presence known.
“Oh, Mr. Trask—is it you?” she exclaimed, as the dim lamplight
filtering through the canvas enabled her to recognize him. “This is
kind of you, I’m sure. We’ve been wondering if we should ever see
you again. I can’t imagine how you were ever able to find us.”
Philip, rejoicing in the softly smothered “r’s” of her speech, explained
soberly. It was not so difficult. He had gone first to the hotel; and
once in the tented square, a few inquiries had sufficed.
“I’m sorry I can’t ask you in,” she hastened to say. “You see, there
isn’t so awfully much room in a tent, and—and the children are just
going to bed. Would you mind sitting out here?”
There was a bench on the board platform that served as a dooryard
for the tent, and they sat together on that. For the first few minutes
Philip had an attack of self-consciousness that made him boil
inwardly with suppressed rage. His human contacts for the past few
weeks—or months, for that matter—had been strictly masculine, and
he had never been wholly at ease with women, save those of his
own family. Stilted inquiries as to how the sick Captain was getting
along, and how they all liked Denver and the West, and how they
thought the climate and the high altitude were going to agree with
them, were as far as he got before a low laugh, with enough
mockery in it to prick him sharply, interrupted him.
“Excuse me,” she murmured, “but it’s so deliciously funny to see you
make such hard work of it. Are there no girls in the part of
Yankeeland you come from?”
“Plenty of them,” he admitted; “but they are not like you.”
“Oh!” she laughed. “Is that a compliment, or the other thing?”
“It is just the plain truth. But the trouble isn’t with girls—it’s with
me. I guess I’m not much of a ladies’ man.”
“I’m glad you’re not; I can’t imagine anything more deadly. Are you
still working for the railroad?”
“Just at present, yes. But I’ll be quitting in a few days. I’m going to
try my luck in the mountains—prospecting.”
“But—I thought you said you wouldn’t!”
“I did; but I was younger then than I am now.”
She laughed again. “All of six weeks younger. But I’m glad you are
going. If the Captain could get well, and I were a man, I’d go, too.”
Philip was on the point of saying that he wished she were a man and
would go with him; but upon second thought he concluded he didn’t
wish it. Before he could straighten out the tangle of the first and
second thoughts she was asking him if he knew anything about
minerals and mining.
“Nothing at all. But others who don’t know any more than I do are
going, and some of them are finding what they hoped to find.”
“It’s in the air,” she said. “You hear nothing but ‘strikes’ and ‘leads’
and ‘mother veins’ and ‘bonanzas.’ Lots of the people in these tents
around us are here because some member of the family is sick, but
they all talk excitedly about the big fortunes that are being made,
and how Tom or Dick or Harry has just come in with a haversack full
of ‘the pure quill,’ whatever that may mean.”
“You haven’t been hearing any more of it than I have,” said Philip.
“Not as much, I think. The town is mad with the mining fever. I’m
only waiting until I can find a suitable partner.”
“That ought not to be very hard—with everybody wanting to go.”
Philip shook his head. “I guess I’m not built right for mixing with
people. I can’t seem to chum in with just anybody that comes
along.”
“You oughtn’t to,” she returned decisively. Then, again with the
mocking note in her voice: “They say the prospectors often have to
fight to hold their claims after they have found them: you ought to
pick out some big, strong fighting man for a partner, don’t you
think?”
Philip was glad the canvas-filtered lamplight was too dim to let her
see the flush her words evoked.
“You are thinking of that day on the train, and how I let the husky
miner take your part when I should have done it myself? I’m not
such a coward as that. I was trying to get out of my seat when the
miner man got ahead of me. I want you to believe that.”
“Of course I’ll believe it.” Then, quite penitently: “You must forgive
me for being rude again: I simply can’t help saying the meanest
things, sometimes. Still, you know, I can’t imagine you as a fighter,
really.”
“Can’t you?” said Philip; and then he boasted: “I had a fight with a
hold-up on the way out here this evening—and got the best of him,
too.” Whereupon he described with dry humor Henry Wigglesworth
Bromley’s attempt to raise the price of a square meal; the brief
battle and its outcome.
“You haven’t told all of it,” she suggested, when he paused with his
refusal to accept Bromley’s offer to arrest himself on a charge of
attempted highway robbery.
“What part have I left out?”
“Just the last of it, I think. You gave the robber some money to buy
the square meal—I’m sure you did.”
“You are a witch!” Philip laughed. “That is exactly what I did do. I
don’t know why I did it, but I did.”
“I know why,” was the prompt reply. “It was because you couldn’t
help it. The poor boy’s desperation appealed to you—it appeals to
me just in your telling of it. He isn’t bad; he is merely good stock
gone to seed. Couldn’t you see that?”
“Not as clearly as you seem to. But he did appear to be worth
helping a bit.”
“Ah; that is the chord I was trying to touch. You ought to help him
some more, Mr. Trask. Don’t you reckon so?”
“‘Mr. Trask’ wouldn’t, but perhaps ‘Philip’ would,” he suggested
mildly.
“Well, ‘Philip,’ then. Don’t you see how brave he is?—to laugh at
himself and all his misfortunes, the hardships his wildness has
brought upon him? You say you are looking for a prospecting
partner; why don’t you take him?”
“Do you really mean that?”
“Certainly I mean it. It might result in two good things. If you could
get him off in the mountains by himself, and live with him, and make
him work hard, you might make a real man of him.”
“Yes?” said Philip. “That is one of the two good things. What is the
other?”
“The other is what it might do for you. Or am I wrong about that?”
“No,” he said, after a little pause. “I still think you are a witch. You’ve
found out that I live in a shell, and it’s so. I guess I was born that
way. You think the shell would crack if I should take hold of a man
like this Bromley and try to brother him?”
“I am sure it would,” she replied gravely. “It couldn’t help cracking.”
Then, as a low-toned call came from the inside of the tent: “Yes,
mummie, dear,—I’m coming.”
Philip got up and held out his hand.
“I am sorry I’m going away, because I’d like to be within call if you
should need me. If you should move into a house, or leave Denver,
will you let me know? A note addressed to me in care of the railroad
office will be either forwarded or held until I come back.”
“I’ll write,” she promised, and the quick veiling of the dark eyes told
him that she knew very well what he meant by her possible need.
“And about this young scapegrace who tried to hold me up: what
you have suggested never occurred to me until you spoke of it. But
if you think I ought to offer to take him along into the mountains, I
don’t know but I’ll do it. It wouldn’t be any crazier than the things a
lot of other people are doing in this mining-mad corner of the world
just now.”
“Oh, you mustn’t take me too seriously. I have no right to tell you
what you should do. But I did have a glimpse of what it might mean.
I’m going to wish you good luck—the very best of luck. If you really
want to be rich, I hope you’ll find one of these beautiful ‘bonanzas’
people are talking about; find it and live happily ever after. Good-by.”
“Good-by,” said Philip; and when she disappeared behind the tent
flap, he picked his way out of the campers’ square and turned his
steps townward.
It was after he had walked the five squares westward on Champa
and the six northward down Seventeenth, and was turning into
Blake, with the American House only a block distant, that a girlish
figure slipped out of a doorway shadow, caught step with him, and
slid a caressing arm under his with a murmured, “You look
lonesome, baby, and I’m lonesome, too. Take me around to Min’s
and stake me to a bottle of wine. I’m so thirsty I don’t know where
I’m going to sleep to-night.”
Philip freed himself with a twist that had in it all the fierce virtue of
his Puritan ancestry. Being fresh from a very human contact with a
young woman of another sort, this appeal of the street-girl was like
a stumbling plunge into muddy water. Backing away from the
temptation which, he told himself hotly, was no temptation at all, he
walked on quickly, and had scarcely recovered his balance when he
entered the lobby of the hotel. Almost immediately he found
Bromley, sprawled in one of the lounging chairs, deep in the
enjoyment of a cigar which he waved airily as he caught sight of
Philip.
“Benedicite, good wrestler! Pull up a chair and rest your face and
hands,” he invited. Then, with a cheerful smile: “Why the pallid
countenance? You look as though you’d just seen a ghost. Did some
other fellow try to hold you up?”
Philip’s answering smile was a twisted grimace. “No; it was a
woman, this time.”
“Worse and more of it. Lots of little devils in skirts chivvying around
this town. Too many men fools roaming the streets with money in
their pockets. ‘Wheresoever the body is, there will the eagles be
gathered together’—only they’re not exactly eagles; they are birds of
another feather. I know, because they clawed me a bit before my
wallet went dry.”
“You were one of the fools?” said Philip sourly.
“You’d know it without my telling you. But to the law and the
testimony. You see, the Wigglesworth family pride didn’t prevent me
from keeping your kindly hint in mind—and I’ve obeyed it. Where do
we begin?”
“Suppose we begin where we left off,” said Philip guardedly. “Is there
any decent ambition left in you?”
Bromley took time to consider, and when he replied he was shaking
his head doubtfully.
“To be perfectly frank about it, I’m not sure there has ever been
anything worthy the high sounding name of ambition. You see, there
is quite a lot of Bromley property scattered about in my home town
—which is Philadelphia, if you care to know—and the income from it
has heretofore proved fatal to anything like decent ambition on the
part of a play-boy.”
“Your property?” Philip queried.
“Oh, dear, no; the governor’s. But he hasn’t kept too tight a hand on
the purse strings; not tight enough, if we are to judge from the
effects—the present horrible example being the most disastrous of
the same. As I intimated on the scene of my latest fiasco, I
stretched the rubber band once too often and it snapped back at me
with a disinheriting thousand-dollar check attached. That, my dear
benefactor, is my poor tale, poorly told. You see before you what
might have been a man, but what probably—most probably—never
will be a man.”
“Of course, if you are willing to let it go at that——” said Philip,
leaving the sentence unfinished.
“You mean that I ought to pitch in and do something useful? My
dear Mr. Good-wrestler——”
“My name is Trask,” Philip cut in shortly.
“Well, then, my dear Trask, I have never learned how to do useful
things. One has to learn, I believe, if it’s only washing dishes in a
cheap restaurant, or chopping wood. I should inevitably break the
dishes, or let the axe slip and chop my foot.”
Philip made a gesture of impatience.
“I had a proposal to make to you, but it seems that it’s no use. I am
about to strike out for the mountains, to try my luck prospecting. A
friend of yours, whom you have never seen or even heard of,
suggested that you might want to go along—as my partner.”
Bromley straightened himself in his chair and the mocking smile died
out of his boyish eyes.
“A friend of mine, you say? I had some friends while my thousand
lasted, but I haven’t any now.”
“Yes, you have at least one; though, as I have said, you have never
seen or heard of her.”
The play-boy sank back into the depths of his chair.
“Ah, I see; a woman, and you told her about me. Am I such an
object of pity as that, Trask?”
Philip forgot his New England insularity for a moment and put his
hand on the play-boy’s knee.
“It was angelic pity, Bromley. Surely that needn’t hurt your pride.”
“Angels,” was the half-musing reply. “They can rise so much, so
infinitely much, higher than a man when they hold on, and sink so
much lower when they let go. This angel you speak of—is she yours,
Trask?”
“No; only an acquaintance. I have met her only twice. But you
haven’t said what you think of my proposal. It is made in good
faith.”
“Don’t you see how impossible it is?”
“Why is it impossible?”
“A partnership presupposes mutual contributions. I have nothing to
contribute; not even skill with a miner’s pick.”
“You have yourself and your two hands—which are probably not
more unskilled than mine, for the kind of work we’d have to do.”
“But the outfit—the grub-stake?”
“I have money enough to carry the two of us through the summer. If
we strike something, you can pay me back out of your half of the
stake.”
For quite a long time Bromley sat with his head thrown back, and
with the half-burned cigar, which had gone out and was cold and
dead, clamped between his teeth. And his answer, when he made it,
was strictly in character.
“What a hellish pity it is that I didn’t find you and try to hold you up
weeks ago, Trask—while I had some few ravellings of the thousand
left. Will you take a beggar with you on your quest of the golden
fleece? Because, if you will, the beggar is yours. We mustn’t
disappoint the angel.”
IV
The August sun had dropped behind a high-pitched horizon of saw-
tooth peaks and broken ranges, leaving the upper air still shot
through with a golden glow that was like the dome lighting of a vast
celestial theater, by the time two young men, whose burro packs of
camp equipment, supplies and digging tools marked them as
prospectors, had picked their way down the last precipitous rock
slide into a valley hemmed in by the broken ranges. At the close of a
hard day’s march the straggling procession was heading for running
water and a camp site; the water being a clear mountain stream
brawling over its rocky bed in the valley bottom. Reaching the
stream before the upper-air effulgencies had quite faded into the
smoke-gray of twilight, a halt was made and preparations for a night
camp briskly begun.
Two full months had elapsed since the partnership bargain had been
struck in the lobby of the American House in Denver, and during the
greater part of that interval Philip and the play-boy had prospected
diligently in the foot-hills and eastern spurs of the Continental
Divide, combing the gulches in the vicinity of Fair Play and Alma, and
finding nothing more significant than an occasional abandoned
tunnel or shaft, mute evidences that others had anticipated their
own disappointment in this particular field. Drifting southwestward,
past Mount Princeton, they had ascended Chalk Creek, crossed the
range over a high pass into Taylor Park, and were now in new
ground on the western side of the Divide.
“This side of the world looks better; or at least a little less
shopworn,” Bromley remarked, after they had cooked and eaten
their supper and were smoking bed-time pipes before the camp fire.
“I think we have outrun the crowd, at last, and that is something to
be thankful for.”
Philip opened his pocket knife and dug with the blade into the bowl
of his pipe to make it draw better. The two months of outdoor life
and hard manual labor had done for him what the treasure search
was doing for many who had never before known what it was to lack
a roof over their heads at night, or to live on a diet of pan-bread and
bacon cooked over a camp fire. With the shedding of the white collar
and its accompaniments and the donning of flannel shirt, belted
trousers and top boots had come a gradual change in habits and
outlook, and—surest distinguishing mark of the tenderfoot—a more
or less unconscious aping of the “old-timer.” Since his razor had
grown dull after the first week or two, he had let his beard grow;
and for the single clerkly cigar smoked leisurely after the evening
meal, he had substituted a manly pipe filled with shavings from a
chewing plug.
Bromley had changed, too, though in a different way. Two months’
abstention from the hectic lights and their debilitating effects had
put more flesh and better on his bones, a clearer light in his eyes
and a springy alertness in his carriage; and though his clothes were
as workmanlike as Philip’s, he contrived to wear them with a certain
easy grace and freedom, and to look fit and trim in them. Also,
though his razor was much duller than Philip’s, and their one scrap
of looking-glass was broken, he continued to shave every second
day.
“I’ve been wondering if a later crowd, with more ‘savvy’ than we
have, perhaps, won’t go over the same ground that we have gone
over and find a lot of stuff that we’ve missed,” said Philip, after the
pipe-clearing pause.
“‘Savvy,’” Bromley chuckled. “When we started out I was moved to
speculate upon what the wilderness might do to you, Phil; whether it
would carve a lot of new hieroglyphs on you, or leave you unscarred
in the security of your solid old Puritan shell. ‘Savvy’ is the answer.”
“Oh, go and hire a hall!” Philip grumbled good-naturedly. “Your
vaporings make my back ache. Give us a rest!”
“There it is again,” laughed the play-boy. “Set the clock back six
months or so and imagine yourself saying, ‘Go hire a hall,’ and ‘Give
us a rest!’ to a group of the New England Trasks.”
“Humph! If it comes to that, you’ve changed some, too, in a couple
of months,” Philip countered.
“Don’t I know it? Attrition—rubbing up against the right sort of thing
—will occasionally work the miracle of making something out of
nothing. You’ve rubbed off some of your New England virtues on
me; I’m coming to be fairly plastered with them. There are even
times when I can almost begin to look back with horror upon my
young life wasted.”
“Keep it up, if you feel like it and it amuses you,” was the grunted
comment. “I believe if you were dying, you’d joke about it.”
“Life, and death, too, are a joke, Philip, if you can get the right
perspective on them. Have you ever, in an idle moment, observed
the activities of the humble ant, whose ways we are so solemnly
advised to consider for the acquiring of wisdom? Granting that the
ant may know well enough what she is about, according to her
lights, you must admit that her apparently aimless and futile
chasings to and fro—up one side of a blade of grass and down the
other, over a pebble and then under it—don’t impress the human
beholder as evidences of anything more than mere restlessness, a
frantic urge to keep moving. I’ve often wondered if we human ants
may not be giving the same impression to any Being intelligent
enough to philosophize about us.”
“This feverish mineral hunt, you mean?”
“Oh, that, and pretty nearly everything else we do. ‘Life’s fitful fever,’
Elizabethan Billy calls it—and he knew. But in one way we have the
advantage of the ant; we can realize that our successive blades of
grass and pebbles are all different.”
“How, different?”
“We put the day that is past behind us and step into another which
is never the same. Or, if the day is the same, we are not. You’ll
never be able to go back to the peace and quiet of a railroad desk,
for example.”
“Maybe not. And you?”
“God knows. As I have said, you’ve rubbed off some of your virtues
on me—suffering some little loss of them yourself, I fancy. We’ll see
what they will do to me. It will be something interesting to look
forward to.”
“Umph!” Philip snorted; “you’re getting grubby again—maggoty, I
mean. Which proves that it’s time to hit the blankets. If you’ll look
after the jacks and hobble them, I’ll gather wood for the fire.”
Bromley sat up and finished freeing his mind.
“Philip, if anybody had told me a year ago that within a short twelve-
month I’d be out here in the Colorado mountains, picking,
shovelling, driving jack-asses, cooking at least half of my own meals,
and liking it all ... well, ‘liar’ would have been the mildest epithet I
should have chucked at him. Comical, isn’t it?” And with that he
went to valet the burros.
The first day after their arrival in the western valley was spent in
exploring, and they finally settled upon a gulch not far from their
camp of the night before as the most promising place in which to
dig. Though they had as yet mastered only the bare rudiments of a
trained prospector’s education, the two summer months had given
them a modicum of experience; enough to enable them to know
roughly what to look for, and how to recognize it when they found it.
The gulch in which they began operations was a miniature canyon,
and the favored site was indicated by the half-hidden outcropping of
a vein of brownish material which they could trace for some distance
up the steep slope of the canyon wall. During the day’s explorations
they had frequently tested the sands of the stream bed for gold
“colors,” washing the sand miner-fashion in their frying pan, and it
was upon the hint given by the “colors” that they had pitched upon
the gulch location. Below the gulch mouth microscopic flakes of gold
appeared now and again in the washings. But the sands above were
barren.
“It looks as if we may have found something worth while, this time,”
Philip hazarded, after they had cleared the rock face to reveal the
extent of the vein. “The ‘colors’ we’ve been finding in the creek sand
come from a lode somewhere, and this may be the mother vein.
We’ll put the drills and powder to it to-morrow and see what
happens.”
Accordingly, for a toilsome fortnight they drilled and blasted in the
gulch, and by the end of that time the prospect had developed into a
well-defined vein of quartz wide enough to admit the opening of a
working tunnel. Having no equipment for making field tests, they
could only guess at the value of their discovery, but the indications
were favorable. The magnifying glass showed flecks and dustings of
yellow metal in selected specimens of the quartz; and, in addition,
the ore body was of the character they had learned to distinguish as
“free milling”—vein-matter from which the gold can be extracted by
the simplest and cheapest of the crushing processes.
Taking it all in all, they had good reason to be hopeful; and on the
final day of the two weeks of drilling and blasting they skipped the
noonday meal to save time and were thus enabled to fire the
evening round of shots in the shallow tunnel just before sunset. A
hasty examination of the spoil blown out removed all doubt as to the
character of the material in which they were driving. The vein was
gold-bearing quartz, beyond question; how rich, they had no means
of determining; but there were tiny pockets—lenses—in which the
free metal was plainly visible without the aid of the magnifier.
That night, before their camp fire, they held a council of war.
Though it seemed more than likely that the lode was a rich one,
they were now brought face to face with the disheartening fact that
the mere ownership of a potential gold mine is only the first step in
a long and uphill road to fortune. In the mining regions it is a
common saying that the owner of a silver prospect needs a gold
mine at his back to enable him to develop it, and the converse is
equally true.
“Well,” Bromley began, after the pipes had been lighted, “it seems
we’ve got something, at last. What do we do with it?”
“I wish I knew, Harry,” was the sober reply. “If the thing turns out to
be as good as it looks, we’ve got the world by the tail—or we would
have if we could only figure out some way to hold on. But we can’t
hold on and develop it; that is out of the question. We have no
capital, and we are a good many mountain miles from a stamp-mill.
Unless the lode is richer than anything we’ve ever heard of, the ore
wouldn’t stand the cost of jack-freighting to a mill.”
“That says itself,” Bromley agreed. “But if we can’t develop the thing,
what is the alternative?”
“There is only one. If our map is any good, and if we have figured
out our location with any degree of accuracy, we are about thirty
miles, as the crow flies, from Leadville—which will probably mean
forty or fifty the way we’d have to go to find a pass over the range.
We have provisions enough to stake us on the way out, but not very
much more than enough. I cut into the last piece of bacon to-night
for supper.”
“All right; say we head for Leadville. We’d have to do that anyway, to
record our discovery in the land office. What next?”
“Assays,” said Philip. “We’ll take a couple of sacks of the quartz along
and find out what we’ve got. If the assays make a good showing,
we’ll have something to sell, and it will go hard with us if we can’t
find some speculator in the big camp who will take a chance and
buy our claim.”
“What?—sell out, lock, stock and barrel for what we can get, and
then stand aside and see somebody come in here and make a
million or so that ought to be ours?” Bromley burst out. “Say, Philip
—that would be death by slow torture!”
“I know,” Philip admitted. “It is what the poor prospector gets in nine
cases out of ten because, being poor, he has to take it. If we had a
mine, instead of a mere prospect hole, we might hope to be able to
capitalize it; but as it is—well, you know what’s in the common
purse. My savings are about used up; we came in on a shoe-string,
in the beginning.”
“Yes, but, land of love, Philip!—to have a thing, like this may turn
out to be, right in our hands, and then have to sell it, most likely for
a mere song! ... why, we’d never live long enough to get over it,
neither one of us!”
Philip shook his head. “It’s tough luck, I’ll admit; but what else is
there to do?”
Bromley got up and kicked a half burnt log into the heart of the fire.
“How nearly broke are we, Phil?” he asked.
The financing partner named the sum still remaining in the
partnership purse, which, as he had intimated, was pitifully small.
“You said, just now, if we had a mine to sell, instead of a bare
prospect,” Bromley went on.... “We’ve got nerve, and two pairs of
hands. Suppose we stay with it and make it a mine? I know good
and well what that will mean: a freezing winter in the mountains,
hardships till you can’t rest, half starvation, maybe. Just the same,
I’m game for it, if you are.”
Philip rapped the ashes from his pipe and refilled it. From the very
beginning of the summer Bromley had been offering a series of
grateful surprises: dogged endurance, cheerfulness under privations,
willingness to share hard labor—a loyal partner in all that the word
implied. Slow to admit any one to the inner intimacies of friendship,
as his Puritan heritage constrained him to be, Philip had weighed
and measured the play-boy coldly, impartially, and before they had
been many weeks together he was honest enough to admit that
Bromley was as tempered steel to his own roughly forged iron; that
it had been merely a lack of an adequate object in life that had
made him a spendthrift and a derelict.
“You’d tackle a winter here in these mountains rather than let go?”
he said, after the refilled pipe was alight. “It will be hell, Harry. You
remember what those fellows in Chalk Creek told us about the
snows on this side of the range.”
“I’m discounting everything but the kind of hell that will be ours if
we should let go and see somebody else come in and reap where
we’ve sown.”
“All right; let’s see what we’ve got to buck up against. First, we’ll
have to go out for the recording, the assays, and the winter’s
provisions. We’d have to buy at least one more burro to freight the
grub-stake in; and then one of us will have to take the jacks out for
the winter. They’d starve to death here. All this is going to take time,
and the summer is already gone. And that isn’t all; we’ll have to
build a cabin and cut the winter’s wood. It will be a fierce race
against time to get holed in before we’re snowed under.”
“Still I’m game,” declared Bromley stoutly. “If it turns out that we
have something worth fighting for—and the assays will say yes or no
to that—I’m for the fight.”
Philip scowled amiably at the transformed play-boy. “You nervy little
rat!” he exclaimed in gruff affection. “Think you can back me down
on a fighting proposition? I’ll call your bluff. We’ll put in one more
day setting things to rights, and then we’ll pull out for Leadville and
that starvation winter grub-stake.”
“Setting things to rights,” as Philip phrased it, did not ask for an
entire day. By noon they had cached their tools and what remained
of the stock of provisions after enough had been reserved to supply
them on the journey; had filled a couple of ore sacks with samples
for the assay; and had paced off and re-staked their claim, posting it
with the proper notice and christening it the “Little Jean,”—this at
Philip’s suggestion, though he did not tell Bromley why he chose this
particular name.
With nothing more to be done, Philip was impatiently eager to break
camp at once, but Bromley pleaded for a few hours’ rest.
“It’s Sunday,” he protested. “Can’t you possess your soul in patience
for one little afternoon? This bonanza of ours—which may not be a
bonanza, after all—won’t run away. I’d like to sleep up a bit before
we strike out to climb any more mountains.”
The impatient one consented reluctantly to the delay; and while
Bromley, wearier than he cared to admit, slept for the better part of
the afternoon, Philip dumped the sacked ore and spent the time
raking over the pile of broken rock and vein-matter blasted out of
the shallow opening, selecting other samples which he thought
might yield a fairer average of values. Beside the camp fire that
evening he stretched himself out with the two sacks of ore for a
back rest; and Bromley, awake now and fully refreshed, noted the
back rest and smiled.
“Like the feel of it, even in the rough, don’t you, Phil?” he jested.
Then: “I’m wondering if this treasure hunt hasn’t got under your skin
in more ways than one. At first, you were out for the pure
excitement of the chase; but now you are past all that; you are plain
money-hungry.”
“Well, who isn’t?” Philip demanded, frowning into the heart of the
fire. “Still, you’re wrong. It isn’t the money so much, as what it will
buy.”
“What will it buy—more than you’ve always had? You won’t be able
to eat any more or any better food, or wear any more clothes, or get
more than one tight roof to shelter you at a time,—needs you have
always had supplied, or have been able to supply for yourself.”
Getting no reply to this, he went on. “Suppose this strike of ours
should pan out a million or so—which is perhaps as unlikely as
anything in the world—what would you do with the money?”
For the moment Philip became a conventional, traditional worshipper
at the altar of thrift.
“I think I should emulate the example of the careful dog with a
bone; go and dig a safe hole and bury most of it.”
Bromley’s laugh came back in cachinnating echoes from the gulch
cliffs.
“Not on your life you wouldn’t, Philip. I can read your horoscope
better than that. If it does happen to happen that we’ve really made
a ten-strike, I can see you making the good old welkin ring till the
neighbors won’t be able to hear themselves think, for the noise
you’ll make.”
“I don’t know why you should say anything like that,” Philip objected
morosely.
“Of course you don’t. You’d have to have eyes like a snail’s to be
able to see yourself. But just wait, and hold my little prophecy in
mind.”
Philip, still staring into the heart of the fire, remembered a similar
prediction made by his desk-mate in the Denver railroad office. “I
know your kind....” Middleton had said. What was there about his
kind that made other people so sure that the good thread of self-
control had been left out in his weaving?
“I’ll wait,” he said; and then: “You haven’t said what you’d do in case
it should turn out that we’ve made the improbable ten-strike.”
“I?” queried the play-boy. “Everybody who has ever known me could
answer that, off-hand. You know my sweet and kindly disposition. I
wouldn’t want to disappoint all the old ladies in Philadelphia. And
they’d be horribly disappointed if I didn’t proceed to paint everything
within reach a bright, bright shade of vermilion.”
Philip looked at his watch.
“Nine o’clock,” he announced, “and we start at daybreak, sharp. I’m
turning in.”
In strict accordance with the programme of impatience, the start
was made at dawn on the Monday morning. Their map, though
rather uncertain as to the smaller streams, seemed to enable them
to locate their valley and its small river, and their nearest practicable
route to Leadville appeared to be by way of the stream to its
junction with a larger river, and then eastward up the valley of the
main stream, which the map showed as heading in the gulches
gashing the western shoulder of Mount Massive.
A day’s tramping behind the two diminutive pack beasts brought
them to the larger stream, and the third evening found them
zigzagging up the slopes of the great chain which forms the
watershed backbone of the continent. Philip had been hastening the
slow march of the burros all day, hoping to reach the pass over the
range before night. But darkness overtook them when they were
approaching timber line and they were forced to camp. It was at this
high camp that they had the unique experience of melting snow
from a year-old snowbank at the end of summer to water the burros
and to make coffee over their camp fire. And even with double
blankets and the tarpaulins from the packs, they slept cold.
Pushing on in the first graying light of the Thursday dawn, they
came to the most difficult stretch of mountain climbing they had yet
encountered: a bare, boulder-strewn steep, gullied by rifts and
gulches in which the old snow was still lying. At the summit of the
rugged pass, which they reached, after many breathing halts, a little
before noon, there was a deep drift, sand-covered and treacherous,
and through the crust of this the animals broke and floundered, and
finally did what over-driven burros will always do—got down and
tried to roll their packs off. It was then that Philip flew into a rage
and swore savagely at the jacks; at which Bromley laughed.
“You’re coming along nicely, Phil,” he chuckled. “A few more weeks
of this, and you’ll be able to qualify for a post-graduate course in the
higher profanities. Not but what you are fairly fluent, as it is.”
Philip made no reply; he was silent through the scarcely less difficult
descent into a wide basin on the eastern front of the range. On the
lower level the going was easier, and in the latter half of the
afternoon they came to the farther lip of the high-pitched basin from
which they could look down into the valley of the Arkansas; into the
valley and across it to a distant, shack-built camp city spreading
upward from a series of gulch heads over swelling hills with mighty
mountains for a background—the great carbonate camp whose fame
was by this time penetrating to the remotest hamlet in the land. A
yellow streak winding up one of the swelling hills marked the course
of the stage road, and on it, in a cloud of golden dust, one of the
rail-head stages drawn by six horses was worming its way upward
from the river valley.
“Think we can make it before dark?” Bromley asked.
“We’ve got to make it,” Philip declared doggedly; adding: “I’m not
going to wait another day before I find out what we’ve got in that
hole we’ve been digging. Come on.”
The slogging march was resumed, but distances are marvelously
deceptive in the clear air of the altitudes, and darkness was upon
them before the lights of the big camp came in sight over the last of
the hills. Bromley, thoroughly outworn by the three-days’ forced
march coming upon the heels of two weeks of drilling and blasting
and shovelling, had no curiosity sharp enough to keep him going,
after the burros had been stabled and lodgings had been secured in
the least crowded of the hotels; but Philip bolted his supper hastily
and announced his intention of proceeding at once in search of an
assay office.
“You won’t find one open at this time of night,” the play-boy yawned.
“There’s another day coming, or if there isn’t, it won’t matter for any
of us.”
“I tell you, I’m not going to wait!” Philip snapped impatiently; and he
departed, leaving Bromley to smoke and doze in the crowded and ill-
smelling hotel office which also served as the bar-room.
It was perhaps an hour later when Bromley, who, in spite of the
noise and confusion of the place, had been sleeping the sleep of
utter exhaustion in his chair in the corner of the smoke-befogged
bar-room, was awakened by a shot, a crash of glass and a strident
voice bellowing, “Yippee! That’s the kind of a hellion I am! Walk up,
gen’lemen, an’ le’s irrigate; the drinks’re on me. Th’ li’l’ ol’ prospect
hole’s gone an’ turned an ace an’ I’m paintin’ the town. Yippee! Line
up, gen’lemen, an’ name yer pizen. Big Ike’s buyin’ fer th’ crowd!”
Vaguely, through the smoke fog, Bromley saw a burly miner, bearded
like a fictional pirate, beckoning the bar-room crowd up to the bar,
weaving pistol in hand. Possibly, if he had been fully awake, he
would have understood that the easy way to avoid trouble with a
manifestly drunken roisterer was by the road of quietly following the
example of the others. But before he could gather his faculties the
big man had marked him down.
“Hey, there—yuh li’l’ black-haired runt in th’ corner! Tail in yere afore
I make yuh git up an’ dance fer th’ crowd!” he shouted. “I’m a rip-
snortin’ hell-roarer fr’m ol’ Mizzoo, an’ this is my night fer flappin’ my
wings—yippee!”
Bromley was awake now and was foolish enough to laugh and wave
the invitation aside airily. Instantly there was a flash and crash, and
the window at his elbow was shattered.
“L-laugh at me, will yuh!” stuttered the half-crazed celebrator. “Git up
an’ come yere! I’m goin’ to make yuh drink a whole durn’ quart o’
red-eye fer that! Come a-runnin’, I say, afore I——”
The door opened and Philip came in. He had heard the shot, but was
wholly unprepared for what he saw; Bromley, his partner, white as a
sheet and staggering to his feet at the menace of the revolver in the
drunken miner’s fist; the shattered window and bar mirror; the
group of card players and loungers crowding against the bar, and the
barkeeper ducking to safety behind it.
In the drawing of a breath a curious transformation came over him.
Gone in an instant were all the inhibitions of a restrained and
conventional childhood and youth, and in their room there was only
a mad prompting to kill. At a bound he was upon the big man, and
the very fierceness and suddenness of the barehanded attack made
it successful. With his victim down on the sawdust-covered floor, and
the pistol wrested out of his grasp, he swung the clubbed weapon to
beat the fallen man over the head with it and would doubtless have
had a human life to answer for if the bystanders had not rushed in
to pull him off with cries of “Let up, stranger—let up! Can’t you see
he’s drunk?”
Philip stood aside, half-dazed, with the clubbed revolver still grasped
by its barrel. He was gasping, not so much from the violence of his
exertions as at the appalling glimpse he had been given of the
potentialities within himself; of the purely primitive and savage
underman that had so suddenly risen up to sweep away the last
vestiges of the traditions, to make his tongue like a dry stick in his
mouth with a mad thirst for blood.
It was Bromley who drew him away, and nothing was said until they
had climbed the rough board stair and Bromley was lighting the
lamp in the room they were to share. Then, in an attempt to lessen
the strain under which he knew his companion was laboring, he
said: “It’s lucky for me that you didn’t have your real fighting clothes
on, that night when I tried to hold you up, Philip. There wouldn’t
have been anything left of me if you had really meant business. Did
you find an assay shop?”
Philip dropped into a chair and nodded. “A sampling works that runs
night and day. We’ll get the results in the morning.”
“For richer?—or poorer?”
“I wish to God I knew! I showed the assayer some of the quartz, but
he wouldn’t commit himself; he talked off; said you could never tell
from the looks of the stuff; that the bright specks we’ve been
banking on might not be metal at all. God, Harry!—if it were only
morning!” he finished, and his eyes were burning.
“Easy,” said Bromley soothingly. “You mustn’t let it mean so much to
you, old man. You’ve worked yourself pretty well up to the breaking
point. There are plenty of other gulches if ours shouldn’t happen to
pan out. Get your clothes off and turn in. That’s the best thing to do
now.”
Philip sprang up and began to walk the floor of the small bed-room.

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Handbook of Computational Molecular Biology 1st Edition

Handbook of Molecular Chaperones Roles Structures and

Molecular Cell Biology 6th Edition Harvey F. Lodish

The Mouse in Biomedical Research Volume 3 Second

The Microbial Models of Molecular Biology From Genes to

Blood Brain Barrier Biology and Research Protocols

Molecular Beam Epitaxy From Research to Mass Production

Functional Family Therapy for Adolescent Behavior

James F. Willott, Ph.D.

Library of Congress Cataloging-in-Publication Data

Handbook of mouse auditory research : from behavior to molecular biology / edited by

QP461 .H26 2001

Visit the CRC Press Web site at www.crcpress.com

© 2001 by CRC Press LLC

No claim to original U.S. Government works

This book is dedicated to the students and colleagues who worked

MICE AS RESEARCH ANIMALS

Robert Burkard Dalian Ding

Barbara Canlon Blanca Durand

Stephanie Carlson Lawrence C. Erway

William Falls Kenneth R. Johnson

Henry E. Heffner Verity A. Letts

Lina M. Mullen Erik Rasmussen

Alfred L. Nuttall Allen F. Ryan

Paul Pistell Xiao-Ming Sun

Victoria Sundin Carole A. Vogler

Joseph Trettel Edward Walsh

Dennis R. Trune Joseph P. Walton

Qing Yin Zheng

MECHANISM OF RODENT VOCALIZING

4 Handbook of Mouse Auditory Research

MALE ULTRASONIC VOCALIZATIONS

Sonagraphic Analysis of Male Mouse Ultrasounds

Auditory Communication among Adults 5

Ethographic Analysis of Male Mouse Ultrasounds

6 Handbook of Mouse Auditory Research

Genetic Basis for Motivation to Vocalize

Androgenic Regulation of Male Mouse Ultrasounds

Auditory Communication among Adults 7

8 Handbook of Mouse Auditory Research

One approach to demonstrate organizational effects is simply to administer male hormones to

Neural Regulation of Male Mouse Ultrasounds

Because 70-kHz ultrasounds appear to be a male-typical courtship behavior, research examining

Auditory Communication among Adults 9

Pheromonal Elicitation of Male Mouse Ultrasounds

10 Handbook of Mouse Auditory Research

Auditory Communication among Adults 11

Sensory Detection of Ultrasound-Eliciting Pheromones

Function of Male Mouse Ultrasounds

12 Handbook of Mouse Auditory Research

Auditory Communication among Adults 13

ULTRASONIC VOCALIZATIONS BY FEMALE MICE

14 Handbook of Mouse Auditory Research

Genetics of Female Vocalizing

Function of Female Ultrasounds

AUDIBLE SOUNDS OF ADULT MICE