DOPAMINERGIC CONTROL OF OXYTOCIN RELEASE IN

LACTATING RATS

G. CLARKE, D. W. LINCOLN AND LYNDA P. MERRICK

Department of Anatomy, University of Bristol Medical School, University Walk,
Bristol, BS8 1TD
(Revised manuscript received 23 March 1979)
SUMMARY

During suckling, anaesthetized lactating rats release regular (about every 7 min) but brief
)ulses of oxytocin (0\m=.\5\p=n-\1\m=.\0mu.) which produce single transient increases in intramammary
[unk]ressure. Drugs which selectively impair synaptic transmission were used to determine the
[unk]oleof dopamine and noradrenaline in regulating this natural reflex. Diethyldithiocarbamate
100\p=n-\200mg/kg, i.v.) and \g=a\-methylparatyrosine (100\p=n-\400mg/kg, i.v.) which inhibit the
[unk]ynthesisof catecholamines both blocked the suckling-induced release of oxytocin. The milk-
[unk]jectionreflex was also inhibited in a dose-dependent manner by the intravenous administra-
ion of the dopamine antagonists, fluphenazine (0\m=.\7mg/kg), pimozide (1\m=.\4mg/kg), cis\x=req-\
lupenthixol (4\m=.\5mg/kg) and metoclopramide (6\m=.\0mg/kg), and caused a significant inhibition
P < 0\m=.\01)of the reflex in 50% of the rats tested. The \g=a\-adrenoceptorantagonist phenoxy-
[unk]enzamine(1\m=.\4mg/kg) was similarly effective. Dopamine (40 \g=m\g),bromocriptine (10 \g=m\g),
[unk]pomorphine(100 \g=m\g),noradrenaline (10 \g=m\g)and phenylephrine (2 \g=m\g)injected into the
;erebral ventricles evoked a sustained release of oxytocin which produced multiple increases
n intramammary pressure ; isoprenaline (4 \g=m\g)was ineffective. The release of oxytocin evoked
[unk]ydopamine and noradrenaline was prevented by cis-flupenthixol and phenoxybenzamine
respectively. None of the drugs used affected the mammary sensitivity to exogenous oxytocin
lor were their actions modified by pretreatment with propranolol (1 mg/kg). The results
[unk]uggestthat the neural pathway for the reflex release of oxytocin during suckling in the rat
[unk]ontainsboth dopaminergic and noradrenergic synapses, the latter acting through \g=a\\x=req-\
[unk]drenoceptorsand being distal in the pathway to the dopaminergic component.

INTRODUCTION
The magnocellular neurones of the paraventricular and supraoptic nuclei, which synthesize
ind release the peptide hormones oxytocin and vasopressin, may be regulated by aminergic
nechanisms. Both these hypothalamic nuclei are rich in noradrenaline and dopamine (Fuxe,
1965; Palkovits, Brownstein, Saavedra & Axelrod, 1974), many of the synapses impinging
jn the cells of these nuclei contain dense-cored vesicles characteristic of aminergic terminals
'Morris, 1974), and the injection of these amines releases both oxytocin and vasopressin
¡Konstantinova, 1967; Olsson, 1970; Kuhn, 1974; Bridges, Hillhouse & Jones, 1976).
However, such amines may exhibit both excitatory and inhibitory postsynaptic functions.
Evidence from studies using selective adrenoceptor antagonists indicates that noradrenaline
nay promote vasopressin release via an -receptor mechanism and inhibit release by a ß-
-eceptor mechanism (Bhargava, Kulshrestha & Srivastava, 1972), and a similar dual system
would appear to apply to the control of oxytocin (Tribollet, Clarke, Dreifuss & Lincoln,
1978). Phenoxybenzamine and other a-adrenoceptor antagonists, for example, cause a
reversible and dose-dependent inhibition of oxytocin release in the lactating rat, whilst
propranolol, a ß-adrenoceptor antagonist, facilitates the suckling-evoked release of oxytocin
(Tribollet et al. 1978). Alternatively dopamine rather than noradrenaline could be the more
important catecholamine for the stimulation of neurohypophysial hormone release. It is
notable that the turnover of dopamine in the hypothalamus increases during suckling in the
rat, whereas noradrenaline turnover is barely affected (Voogt & Carr, 1974).
The involvement of dopamine in the release of oxytocin, and the possible interaction of
noradrenaline with a dopamine-dependent mechanism, was investigated in lactating rats by
the administration of (1) catecholamine synthesis inhibitors and (2) selective receptor
antagonists. Oxytocin release was evoked by the natural stimulus of the sucking of the young
on the nipples and/or by the intraventricular injection of amines. The release of oxytocin was
determined by the measurement of intramammary pressure using procedures described
previously (Clarke, Fall, Lincoln & Merrick, 1978).
MATERIALS AND METHODS
All experiments were performed on rats between days 7 and 11 of lactation. These animals
were laboratory bred from Wistar stock and weighed 250-400 g. The environmental condi¬
tions of the animal rooms were controlled for temperature (22 CC) and photoperiod
(14 h light : 10 h darkness), and food and water were provided ad libitum.
The young were separated from their mothers and placed in adjacent cages for 12-16 h
before the experiments. The accumulation of milk within the mammary tissue which resulted
from this period of separation facilitated the measurement of intramammary pressure.
Furthermore, hungry young were more willing to remain attached to the nipples of the
mother throughout the experimental period than were their well-fed counterparts. Between
09.00 and 10.00 h on the morning following separation, the mothers were anaesthetized with
1-25 g ethyl carbamate/kg, i.p. (as a 25% solution, w/v). One of the saphenous veins and the
teat ducts of two of the mammary glands in the inguinal region were cannulated for the
administration of drugs and the measurement of intramammary pressure respectively. Ten
hungry pups were then placed on the nipples for a trial period of about 10 min.
Suckling-induced release of oxytocin
Three hours after the injection of the anaesthetic, and while the animals were still deeply
anaesthetized, ten hungry pups were applied to the uncannulated nipples of the mother for
a second time, and left to feed for 3 h or more. Milk ejection commenced within 30 min
in most animals, and thereafter milk ejections recurred at relatively regular intervals of
5-10 min. Each milk ejection was characterized by an abrupt but transient rise in intra¬
mammary pressure (Lincoln, Hill & Wakerley, 1973).
In all experiments using antagonists, and in some experiments using synthesis inhibitors,
drugs were injected intravenously after at least six milk ejections. The milk-ejection reflex
was considered to have been inhibited if, in the 30 min following drug treatment, one of the
intervals between successive milk ejections was longer (P 0-01) than the intervals observed
=

in the pretreatment period (for further details see Clarke et al. 1978). The degree of inhibition
was quantified by dividing the duration of the longest milk-ejection interval following drug
treatment by the mean interval before treatment (milk-ejection ratio). The sensitivity of the
myoepithelial cells of the mammary glands to oxytocin was checked at various intervals
before and after drug treatment by the intravenous injection of synthetic oxytocin. The
introduction of such additional milk ejections does not reset the endogenous pattern of milk
ejections displayed by the nursing mother (Lincoln, 1974; Wakerley & Deverson, 1975).
Control animals were given injections of sodium chloride at an osmolarity approximately
equal to that of the test solution.
Release of oxytocin by intraventricular injection of amines
For the injection of materials into the lateral ventricles the rats were mounted in a stereotaxic
frame and small holes drilled in the skull to permit a 27 gauge needle to be lowered into one
or other of the cerebral ventricles (co-ordinates of A 5-0 mm, L 2-5 mm, according to De
Groot (1959) and to a depth of 5-5 mm from the surface of the skull). In some of these animals
a common carotid artery was cannulated for the measurement of arterial blood pressure.
Further details have been given previously (Clarke et al. 1978).

Drugs
The following substances were used: atropine sulphate (B.D.H.), bromocriptine mesylate
(Parlodel ; Sandoz), carbamylcholine chloride (Carbachol ; Sigma), chlorpheniramine maléate
(Piriton; Allen and Hanbury), diazepam (Valium; Roche), diethyldithiocarbamate (Sigma),
3-hydroxytyramine hydrochloride (dopamine, Sigma), ethyl carbamate (urethane, B.D.H.),
c/s-flupenthixol hydrochloride (a-flupenthixol, Lundbeck), rrawi-flupenthixol hydrochloride
(ß-flupenthixol, Lundbeck), fluphenazine hydrochloride (Moditen; Squibb), hyoscine hydro-
bromide (scopolamine, Sigma), DL-isoproterenol hydrochloride (isoprenaline, Sigma),
metoclopramide hydrochloride (Primperan; Berk), mecamylamine hydrochloride (Sigma),
-methyl paratyrosine methyl ester (Sigma), naloxone hydrochloride (Endo), L-noradrenaline
hydrochloride (Sigma), oxytocin (Syntocinon; Sandoz), phenoxybenzamine hydrochloride
(Dibenyline; Smith, Kline & French), L-phenylephrine hydrochloride (Sigma), picrotoxin
(Sigma), pimozide (Orap; Janssen), promazine hydrochloride (Sparine; Wyeth), prometha-
zine hydrochloride (Phenergan; May and Baker), propranolol hydrochloride (Inderai;
I.C.I.) and vasopressin (Pitressin; Parke-Davis).
All the substances injected intravenously were made up to 0-3-0-6 ml with distilled water.
Drug doses are expressed in terms of the salts used. Pimozide was initially dissolved in a few
drops of glacial acetic acid, and phenoxybenzamine was dissolved in a few drops of ethyl
alcohol (70%) acidified with hydrochloric acid, and made up to volume with distilled water.
Substances for intraventricular injection were dissolved in distilled water, up to 1-2 µ .
When necessary, solutions containing catecholamines were adjusted to pH 4 by the addition
of ascorbic acid. Bromocriptine was initially dissolved in a few drops of ethyl alcohol
acidified with tartaric acid. Control injections were prepared in the same way as the drug
solutions except that the active drug was omitted and replaced by sodium chloride to
approximately the same concentration.
RESULTS
The patterns of reflex milk ejection observed during the suckling of the young were in every
way similar to those reported previously (Lincoln et al. 1973 ; Clarke et al. 1978). During the
3 h nursing period, milk ejections were both regular in their recurrence and uniform in
character (Fig. 1). The individual pressure responses recorded from the mammary gland
during the expression of the suckling-induced reflex were very similar in form to the responses
obtained by a bolus i.v. injection of 0-5-1-0 mu. oxytocin.

Effect of amine synthesis inhibitors in reflex milk ejection

a-Methylparatyrosine methyl ester (AMPT), which inhibits the synthesis of both nora¬
drenaline and dopamine, and diethyldithiocarbamate (DDC), which inhibits the synthesis of
noradrenaline alone, both blocked the milk-ejection reflex of the rat. Fifteen rats were given
AMPT at doses of 50^400 mg/kg and 19 rats received DDC at doses of 50-200 mg/kg, i.v.
At the highest doses both drugs blocked the milk-ejection reflex for the entire 2-3 h observa¬
tion period. With doses of 100-300 mg AMPT/kg and 100-150 mg DDC/kg occasional
milk ejections were seen during the remainder of the recording period, but they were always
very infrequent.
The effect of the synthesis inhibitors was extremely rapid. When the synthesis inhibitors
were administered after the occurrence of at least six regularly spaced milk ejections, the
subsequent milk ejection was invariably delayed, though it should have occurred within
5-10 min of the drug injection. By contrast, different results were obtained when DDC
(200 mg/kg, i.v.) was administered 15 min before the pups were allowed access to the nipples.
Of the nine rats treated in this way, four started to eject milk at the predicted time following
the application of the pups, but only two to four milk ejections were observed. The remain¬
ing five animals failed to display a single milk ejection. The eight control rats given saline
before suckling displayed between them 105 milk ejections during the 2 h of observation.

L
RME7
K. K.

<2>
L·
AMPT
200 mg/kg

L
L
5 min
Fig. 1. Continuous intramammary pressure recording from an anaesthetized lactating rat which
began to milk-eject 40 min after the onset of suckling, the trace begins 45 min later just before the
seventh milk-ejection response (RME 7). a-Methylparatyrosine (AMPT) was injected intravenously
2 min after the 11th response which resulted in the subsequent two milk-ejection responses being
delayed and attenuated compared with the previous responses. During the long periods without
milk-ejection responses small and distorted increases in pressure occurred (black arrow heads) but
were undetected by the sucking pups.

In experiments notable for the stability of the intramammary pressure recording, small
deflections in the pressure trace were sometimes seen at times when full responses might
have been predicted (Fig. 1). Furthermore, the few pressure responses which occurred
following the application of the synthesis inhibitors were not as large in amplitude as those
observed before treatment. Neither AMPT nor DDC decreased the sensitivity of the
mammary glands to exogenous oxytocin, and the inhibition of reflex milk ejection by AMPT
and DDC was not modified by i.v. pretreatment with 1 mg propranolol/kg (18 animals).

Effect of catecholamine antagonists on reflex milk ejection

Fluphenazine (0-5-4 mg/kg), ris-flupenthixol (4-8 mg/kg), metoclopramide (5-10 mg/kg)
and pimozide (0-5-6 mg/kg), all drugs with the property of antagonizing dopamine receptors,
abolished the milk-ejection reflex, and at the doses used the effects were reversible. The
inhibition of the reflex was dose-dependent, though there were considerable differences in
the slope of the dose-response lines (Fig. 2). Fluphenazine was the most potent drug followed
by pimozide, ci's-flupenthixol and metoclopramide. A similar rank order of potency was
obtained when the dose required to produce a statistically significant (P= 0-01) increase in
the milk-ejection interval in half of the rats tested (ED50) was calculated for each drug
(Table 1).
Whilst c/i-flupenthixol effectively blocked the reflex the inactive isomer rrawi-flupenthixol
was generally without effect (Fig. 3), regardless of which isomer was injected first. Although
 13
-

1 7"

Dose of antagonist (mg/kg)

Fig. 2. Dose-dependent effects of dopamine antagonists upon reflexly milk-ejecting rats. Graph of the
milk-ejection ratio (the longest interval between milk ejections after administration of the antagonist
divided by the mean interval between responses before treatment) plotted against the dose of
antagonist used. Each point is the mean value from at least three experiments and the lines are re¬
gression lines calculated from the individual data points. A, Fluphenazine (r 0-74); , pimozide
=

(r 0-80); ·, c/s-flupenthixol (/ 0·63); , metoclopramide (r 0-65); D, ira/w-flupenthixol.

= = =

The correlation coefficients were all significant at < 0-05.

Table 1. Efficacy of catecholamine antagonists at blocking reflex milk-ejection during suckling

Mean dose (mg/kg) for
significant (<0-01)
No. of inhibition of the milk-
rats Dose range ejection reflex in 50%
Drug treatment tested (mg/kg, i.v.) of rats (ED60)
Fluphenazine 18 0-5-40 0-7
Pimozide 22 0-5-60 1-4
cí'í-Flupenthixol 20 2-8 4-5
Metoclopramide 11 5-10 60
ira/ii-Flupenthixol 23 2-20 18-5 (see text)
Phenoxybenzamine 22 0-5-4 1-4
ED50 values were obtained graphically by plotting the number of rats (%) showing a significant (P<0-01)
inhibition of the milk-ejection reflex against dose of antagonist for the range of doses shown.

rrani-flupenthixol at doses greater than 16 mg/kg caused an inhibition of the reflex in some
animals the dose-response line was very shallow and the ED50 of 18-5 mg/kg was close to
the lethal dose of 20-25 mg/kg.
Tribollet et al. (1978) reported that the a-adrenoceptor antagonist, phenoxybenzamine
(0-5-4-0 mg/kg, i.v.), abolishes the milk-ejection reflex of the rat. Results obtained in the
present studies indicate that phenoxybenzamine has a similar potency to pimozide (Table 1).
The dose-response line for phenoxybenzamine, if plotted in Fig. 2, would have fallen between
the lines for fluphenazine and pimozide with a milk-ejection ratio of 2-9 at 2 mg/kg and 9-4
at 4 mg/kg.
None of the antagonists used in the preceding studies caused any marked change in the
sensitivity of the mammary glands to oxytocin, and their actions were unaffected by the
 8c

8t 8c

8t 16t

Saline

0 12 3 4
Time (h)
Fig. 3. Effect of the two isomers of fiupenthixol on the milk-ejection reflex of the urethane-
anaesthetized rat. The four rows show the position of milk ejection in individual rats suckling
ten pups each; the milk-ejection responses are represented by black vertical bars. The drugs were
injected intravenously at the times indicated (black arrows), the figures denote the dose (mg/kg)
and the letters indicate whether the eis or trans isomer of fiupenthixol was used. The control rat
received 0-3 ml 0-9% saline.

pretreatment of the animals with the ß-adrenoeeptor antagonist, propranolol. Furthermore,

it is unlikely that these
antagonists acted by causing a general depression of the central
nervous system for of the following centrally active drugs influenced the milk-ejection
none
reflex when given by intravenous injection : atropine (1-200 mg/kg, 40 rats), chlorpheniramine
(1-4 mg/kg, 5 rats), diazepam (1-5 mg/kg, 10 rats), hyoscine (2-90 mg/kg, 11 rats), naloxone
(5-20 mg/kg, 13 rats), picrotoxin (1-5 mg/kg, 9 rats), promazine (5 mg/kg, 3 rats) and prome-
thazine (1-5 mg/kg, 7 rats).
Intraventricular injection of catecholamines
An injection of 40 µg dopamine into the lateral cerebral ventricles caused a large and
sustained series of mammary contractions in all seven rats treated (Fig. 4), but with 20 µg
a response was only obtained in two out of four rats. Similarly, an increase in intramammary
pressure occurred in six out of seven rats given 10 µg bromocriptine, and all five rats given
100 µg apomorphine. Injections of the vehicle alone were without effect.
Both noradrenaline (1-10 µg) and the a-adrenoceptor agonist phenylephrine (1-2 µg)
evoked prolonged mammary contractions in all animals when administered into the cerebral
ventricles of 14 and six rats respectively. By contrast the ß-adrenoeeptor agonist isoprenaline
failed to elicit a mammary contraction when given in doses up to 4 µg.
Whilst the initial response to dopamine or noradrenaline was very consistent, the response
was not readily repeatable. Thus of the seven rats responding to an injection of 40 µg
dopamine only three displayed a response to a second injection, though 2 h had elapsed in
some experiments. Similarly, of 14 rats displaying a response to an intraventricular injection
of noradrenaline only three displayed a response to a second injection. However, animals
refractory to a second injection of dopamine were found to respond to an injection of
noradrenaline, and vice versa.
 Dopamine and noradrenaline both failed to elicit a mammary response when injected
intravenously at the dose which was effective intraventricularly, indicating a site of action
within the central nervous system. Blood pressure was recorded in six rats given 10 µg
noradrenaline into the cerebral ventricles. Whilst an increase in intramammary pressure was
observed in all animals, an increase in blood pressure was only observed in two animals. In
the same six animals, vasopressin injected intravenously only caused a rise in intramammary
pressure at a dose (2-4 mu.) which also produced a rise in arterial pressure. The intravenous
administration of 1 mu. oxytocin had no effect upon blood pressure (Fig. Ab).

-> 0

5 min

-1 8
(b)

i__i A>^—^_L
130
M
X

: 90
O V N
5 min
Fig. 4. Intramammary pressure responses evoked by the intraventricular injection of catecholamines
( ) and the intravenous injection of neurohypophysial hormones (A), (a) Continuous pressure
record from one rat showing the increase in intramammary pressure produced by the intravenous
injection of 0-5 mu. oxytocin (O) and by the intraventricular injection of 40 µg dopamine (D) and
10 µg noradrenaline (N). (6) Combined intramammary pressure and arterial blood pressure records.
Intravenous 0-5 mu. oxytocin (O) produced only a mammary pressure response whereas 2 mu.
vasopressin (V) also caused a sustained rise in blood pressure. The intraventricular injection of 10 µg
noradrenaline (N) resulted in a multiple contraction of the mammary gland but did not affect the
arterial blood pressure.

Effect of antagonists on amine evoked release of neurohypophysial hormones

As it was not possible to obtain repeated responses to the intraventricular injection of dopa¬
mine and noradrenaline, it was necessary to administer the antagonists before the administra¬
tion of the amines and compare the responses observed with those obtained following the
injection of the amines alone. To check on the accurate placement of the microsyringe, 0-2 µg
carbachol was injected into the cerebral ventricles, for previous studies had shown that this
would produce a consistent and reproducible release of oxytocin (Clarke et al. 1978).
The dopamine antagonist m-flupenthixol (6 mg/kg) abolished the predicted response to
dopamine in all four rats treated, but was totally ineffective against noradrenaline in four
rats. The inactive isomer rrans-flupenthixol was ineffective against both dopamine and
noradrenaline (Fig. 5 and Table 2). The a-adrenoceptor antagonist, phenoxybenzamine
(4 mg/kg), eliminated the responses expected to both noradrenaline and dopamine in most
animals (Table 2).
 (a)

O c

c
10

\_L
O D
5 min

10

5 min
Fig. 5. Effect of the two isomers of fiupenthixol on the release of oxytocin evoked by the intra¬
ventricular injection ( ) of dopamine and carbachol. (a) Intramammary pressure record showing
in row 1 the pressure response resulting from the intravenous injection of 0-5 mu. oxytocin (O) and
from the intraventricular injection of carbachol (C). Five minutes before row 2, m-flupenthixol
was injected intravenously and then the responses were repeated as shown. When 40 µg dopamine
(D) were injected intraventricularly (row 3) there was no effect, even though the mammary gland
was still responsive to oxytocin. (Ten minutes of the record have been deleted between each row.) (6)
As in (a) except that ira/w-flupenthixol was injected between rows 1 and 2, which did not prevent the
release of oxytocin evoked by intraventricular dopamine (row 3).

Table 2. Effect of various neurotransmitter antagonists injected intravenously (i.v.) on the

release of oxytocin evoked by dopamine (40 µ#) or noradrenaline (10 ¡ig) injected intra¬
ventricularly (i.c.v.) into rats
Dopamine evoked Noradrenaline evoked
release of oxytocin
release of oxytocin
/no. responding\ no. responding^
Drug
pretreatment no. tested ) ( no. tested Ì
No drug 7/7 14/14
ci'j-Flupenthixol 0/4 4/4
(6 mg/kg, i.v.)
/rani-Flupenthixol 5/5 3/3
(6 mg/kg, i.v.)
Phenoxybenzamine 0/4 2/7
(4 mg/kg, i.v.)
Mecamylamine 3/3 4/4
(2 mg/kg, i.v.)
Atropine 3/3
(1 mg/kg, i.v.)
 The nicotinic cholinoceptor antagonist mecamylamine (2 mg/kg), which blocks the
reflex release of oxytocin during suckling (Clarke et al. 1978), and atropine (1 mg/kg),
which blocks the response to carbachol injected into the cerebral ventricles (Clarke et al.
1978), both failed to prevent the release of neurohypophysial hormones following the intra¬
ventricular injection of catecholamines.

DISCUSSION
Bridges al. (1976) have provided good evidence to implicate catecholamines in the central
et
regulation of oxytocin release. The present results are entirely complementary to these
studies, and further suggest that both dopamine and noradrenaline have a functional role in
the reflex release of oxytocin during suckling.
The enzyme inhibitors a-methylparatyrosine and diethyldithiocarbamate, which prevent
the synthesis of dopamine and noradrenaline (Carlsson, Lindqvist, Fuxe & Hokfelt, 1966;
Spencer & Turner, 1969; Krulich, Giachetti, Marchlewska-Koj, Hefco & Jameson, 1977)
caused a prolonged abolition of the milk-ejection reflex. As the sensitivity of the mammary
glands to exogenous oxytocin was not reduced by these synthesis inhibitors the lack of a
milk-ejection response was in all probability due to a failure ofthe animals to release oxytocin.
Although the brains of the rats were not assayed to determine the levels of catecholamines,
other studies on rats treated with comparable doses of inhibitors have recorded a substantial
reduction in amine levels (Carlsson et al. 1966; Spencer & Turner, 1969; Krulich et al. 1977).
It was noteworthy that when the milk-ejection reflex was well established the synthesis
inhibitors caused an immediate cessation of the reflex, whereas a few rudimentary milk
ejections were sometimes observed when the inhibitors were given before the young were
applied to the nipples. This could be taken to indicate that in the non-suckling animal there
is sufficient transmitter stored in the nerve-terminals to elaborate a few releases of oxytocin.
Conversely, the abrupt cessation of the reflex when the inhibitors were administered during
suckling suggests that the synthesis of these amines is critical to the successful operation of
the milk-ejection reflex.
Although the results obtained with the synthesis inhibitors indicate the involvement of
catecholamines they do not indicate the exact identity of the neurotransmitters involved. The
milk-ejection reflex was effectively abolished, however, by a variety of drugs which were
known from numerous other studies to function as dopamine-receptor antagonists. As
most of these neuroleptic drugs have other central actions apart from their effects on
dopaminergic systems, several different classes of antagonists were used such that their
potencies could be compared with those observed in other neurochemical and psycho-
pharmacological studies. Of particular importance were the observations that c/'j-flupenthixol
eliminated the reflex whilst the inactive isomer, /ra/ii-flupenthixol was without effect, and
that metoclopramide, an antiemetic drug with dopamine-receptor antagonist properties
but unrelated to the neuroleptic drugs (Peringer, Jenner, Donaldson & Marsden 1976) was
also effective. The rank order of potency of the neuroleptic drugs at eliminating the release
of oxytocin during suckling compared favourably with that obtained in experiments utilizing
binding to dopamine receptors (Peroutka, U'Prichard, Greenberg & Snyder, 1977), in the
measurement of behavioural changes in conscious animals (Costali & Naylor, 1974, 1976;
Pycock, Tarsy & Marsden, 1975), and in the inhibition of release of prolactin (Langer,
Sachar, Gruen & Halpern, 1977). These neuroleptic drugs have a wide range of actions on the
central nervous system and thus it is possible, though unlikely, that a neurotransmitter other
than dopamine was responsible for the effects observed. An antimuscarinic action of the
neuroleptic drugs (Miller & Hiley, 1974), implicating an involvement of acetylcholine, is
unlikely, for atropine and hyoscine are without effect on the reflex at very high doses
(Clarke et al. 1978). Similarly, the involvement of gamma aminobutyric acid and the opiate
 peptides can be largely eliminated since picrotoxin and naloxone fail to block the milk-
ejection reflex. Furthermore, the two isomers of fiupenthixol do not show stereospecificity
for muscarinic, opiate or neutral amino acid receptors, whereas they do for dopamine
receptors (Enna, Bennett, Burt, Creese & Snyder, 1976). Histamine has been implicated in
the release of vasopressin (Dogterom, van Wimersma Greidanus & De Weid, 1976) but the
histamine antagonists chlorpheniramine and promethazine were both without effect upon
the milk-ejection reflex. Finally, it is unlikely that the neuroleptic drugs were preventing
oxytocin release by antagonizing serotonin for this amine inhibits the release of oxytocin
(Mizuno, Talwalker & Meites, 1967).
It is possible that some of the antagonists used in this study were not sufficiently selective
to distinguish between receptors for noradrenaline and receptors for dopamine. For example,
some dopamine antagonists influence the binding of ligands to a-adrenoceptors (Peroutka
et al. 1977) and antagonize the noradrenaline sensitive adenylate cyclase system (Bockaert,
Tassin, Thierry, Glowinski & Premont, 1977). Milk ejection is prevented by the a-adreno-
ceptor antagonists (Moos & Richard, 1975; Tribollet et al. 1978), whilst noradrenaline and
the a-adrenoceptor agonist phenylephrine are very powerful stimulants for the release of
oxytocin. On the other hand, the prevention of the milk-ejection reflex by m-flupenthixol
and pimozide strongly suggests a role for dopamine in the reflex release of oxytocin because
c/s-flupenthixol selectively prevented the release of oxytocin evoked by the intraventricular
injection of dopamine. Pimozide was shown to be selective in a similar way by Bridges et al.
(1976). They stimulated the release of oxytocin with the intraventricular injection of dopa¬
mine, apomorphine and noradrenaline, and using pimozide selectively blocked the dopamine-
and apomorphine-evoked release. More recently it has been reported that haloperidol,
another dopamine antagonist, blocks oxytocin release during suckling in both anaesthetized
and conscious rats when injected into the ventricles (Moos & Richard, 1979). We therefore
endorse the views expressed by Bridges et al. (1976) that both dopamine- and noradrenaline-
containing neurones form part of the reflex pathway for oxytocin release, and that the nora-
drenergic component is closer to the paraventricular and supraoptic nuclei of the hypo¬
thalamus than the dopaminergic component. The exact location of these two synaptic
mechanisms in the reflex pathway for oxytocin release remains to be determined.
Mecamylamine, a nicotinic cholinergic antagonist, blocks the milk-ejection reflex of the
anaesthetized rat (Clarke et al. 1978) but this drug had no effect on either the dopamine or
the noradrenaline evoked release of oxytocin. Thus, it would appear that both the aminergic
components of the reflex are closer to the oxytocin producing cells of the hypothalamus than
the cholinergic link, and adds further evidence to the view we have expressed elsewhere
(Clarke et al. 1978) that the final excitatory transmitter in the reflex arc is not acetylcholine
as might be inferred from studies with the iontophoresis of acetylcholine (Dreifuss & Kelly,
1972; Moss, Urban & Cross, 1972). The cholinergic component of the milk-ejection reflex
could be presynaptic to the dopamine terminals. In the striatum of the brain the dopaminergic
neurones possess nicotinic receptors on their axon terminals, which when activated facilitate
the release of dopamine (Giorguieff, Le Floc'h, Westfall, Glowinski & Besson, 1976).
Furthermore, it has been suggested that stimulation of these nicotinic receptors to release
dopamine is important at times of high neurotransmitter output (Ahtee & Kaakkola, 1978)
precisely the conditions which might be needed to cause the explosive activation of the
-

oxytocinergic neurones associated with each pulse of oxytocin release (Lincoln & Wakerley,
1974, 1975).
We should like to acknowledge the gifts of drugs from Sandoz, Lundbeck, Squibb, Endo,
Janssen, I.C.I, and Smith, Kline & French.
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