Neurobiology of Aging 33 (2012) 383–392

www.elsevier.com/locate/neuaging

Impact of age and retinal degeneration on the light input to circadian

brain structures
Daniela Lupia, Ma’ayan Semob, Russell G. Fostera,*
a
University of Oxford, Circadian and Visual Neuroscience Group, Nuffield Laboratory of Ophthalmology, Level 5 and 6, West Wing, The John Radcliffe
Hospital, Headley Way, Headington, Oxford OX3 9DU, UK
b
Ocular Biology and Therapeutics, Institute of Ophthalmology, University College London, 11– 43 Bath Street, London EC1V 9EL, UK

Abstract

Aging causes anatomical and functional changes in visual and circadian systems. In wild type mice rods, cones, and photosensitive retinal
ganglion cells (pRGCs) decline with age. In rd/rd cl mice, the early loss of rods and cones is followed by protracted transneuronal loss of
inner retinal neurons as well as the pRGCs. Here we use Fos induction to study the light input pathway to the suprachiasmatic nuclei (SCN),
the intergeniculate leaflets (IGL) and ventral lateral geniculate nuclei (vLGN) of old (⬃700 days) and young (⬃150 days) wild type and
rd/rd cl mice. Cholera toxin tracing was used in parallel to study the anatomy of this pathway. We find that aging rather than retinal
degeneration is a more important factor in reducing light input to the SCN, causing both a reduction in Fos expression and retinal afferents.
Furthermore, we show light-induced Fos within the vLGN and IGL is predominantly subserved by rods and cones, and once again aging
reduces the amplitude of this response.
© 2012 Elsevier Inc. All rights reserved.

Keywords: Circadian rhythms; Immediate early gene; Melanopsin; Neuronal tracing; Photoreceptors; Retinohypothalamic tract

Photoreception in rodents is mediated by rods, cones, in the rods, cones, and retinal ganglion cells (RGCs) (Cav-
and melanopsin-based photosensitive retinal ganglion cells allotti et al., 2001; Danias et al., 2003; Gao and Hollyfield,
(pRGCs) (Hattar et al., 2003; Lucas et al., 2003; Panda et 1992; Katz and Robison, 1986). In the circadian system,
al., 2003; Sekaran et al., 2003). The pRGCs alone are both the amplitude of the pacemaker and its responses to
capable of mediating circadian photoentrainment and the light have been shown to be reduced in humans (Czeisler
phase shifting effect of single pulses of light on activity et al., 1991; Mirmiran et al., 1992), hamsters (Davis and
onset delivered to animals maintained under constant dark- Viswanathan, 1998; Penev et al., 1997; Zee et al., 1992;
ness (Barnard et al., 2004; Freedman et al., 1999), and Zhang et al., 1996), rats (Sutin et al., 1993) and mice
multiple other irradiance detection tasks including the sup- (Aujard et al., 2001; Benloucif et al., 1997; Valentinuzzi et
pression of pineal melatonin (Lucas et al., 1999), pupil al., 1997; Welsh et al., 1986). Here we use a mouse model
constriction (Lucas et al., 2001), negative masking behavior (rd/rd cl) (Freedman et al., 1999; Lucas et al., 1999) to
(Thompson et al., 2008) and the modulation of sleep (Alti- assess the relative importance of age versus retinal degen-
mus et al., 2008; Lupi et al., 2008). eration on the responsiveness of the circadian system to
Aging affects both the retina and circadian system. In the light. In old rd/rd cl mice the complete loss of the outer
retina there are age related neuronal reductions, particularly retina is followed by a more protracted decline of the inner
retina that starts at approximately 9 months of age. By
18 –24 months the inner nuclear layer is considerably thin-
* Corresponding author at: University of Oxford, Circadian and Visual ner and in many regions totally absent. Our previous studies
Neuroscience Group, Nuffield Laboratory of Ophthalmology, Level 5 and
6, West Wing, The John Radcliffe Hospital, Headley Way, Headington,
demonstrated that there is an age related decline of mel-
Oxford OX3 9DU, UK. Tel.: ⫹44(0)1865 234792. anopsin and Thy-1 expression in wild type and rd/rd cl mice
E-mail address: [email protected]. (Semo et al., 2003b), both genotypes showing an ⬃40%

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.neurobiolaging.2010.03.006
384 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392

reduction in melanopsin pRGCs (Semo et al., 2003a). Here 1.2. Fos induction
we study the impact of the rd/rd cl lesion on the light-
Quantitative analysis of Fos induction in brain nuclei
induced gene expression (c-fos) within, and the projections
was undertaken in both old mice (610 – 847 d old; wild type
to, the master circadian pacemaker (suprachiasmatic nuclei/
pulsed n ⫽ 6; wild type sham n ⫽ 4; rd/rd cl pulsed n ⫽ 7;
SCN) in aging congenic rd/rd cl and wild type mice. The
rd/rd cl sham n ⫽ 6) and in young mice (100 –200 d; wild
immediate early gene c-Fos provides a powerful marker of
type pulsed n ⫽ 5; wild type sham n ⫽ 5; rd/rd cl pulsed
photic signaling to the SCN. In rodents, pulses of light
n ⫽ 3; rd/rd cl sham n ⫽ 3). Mice were entrained for at least
during the night results in Fos induction in the retinorecipi-
10 d to a 12 : 12 LD, lights on at 4:00 and off 16:00. On the
ent region of the SCN (Chambille et al., 1993; Earnest et al.,
day of the light pulse the animals remained in darkness. At
1990; Rea, 1989; Rusak et al., 1990; Schwartz et al., 2000). 20:00 (4 h after the lights would normally have been turned
Moreover, the numbers of Fos positive neurons (Beaule and off) mice were transferred to the light pulse equipment under
Amir, 1999), and the overall levels of c-fos and Fos expres- infrared illumination and exposed to a 15 min pulse of light
sion in the SCN is broadly correlated with the effects of (␭max at 505 nm at an irradiance of 8.0 ␮W/cm2 measured
light on circadian behavior (Dkhissi-Benyahya et al., 2000; using an optical power meter, Macam Photometrics). Sham
Kornhauser et al., 1990; Lupi et al., 1999). pulsed mice were handled in a similar manner but no light
In addition to the SCN, we have used light-induced Fos pulse was given. Following the light pulse mice were returned
and tract-tracing to assess the impact of age and retinal to their cages. Ninety min after the beginning of light treatment
degeneration on two other retinorecipient structures that animals were deeply anesthetized with sodium pentobarbitone
receive afferents from pRGCs; the intergeniculate leaflets (60 mg/kg), perfused transcardially with warm (32 °C) 0.9%
(IGL) and the ventral lateral geniculate nuclei (vLGN) NaCl, followed by 300 mL of cold 4% paraformaldehyde in
(Gooley et al., 2001; Gooley et al., 2003; Hattar et al., 2002; 0.1-M phosphate buffer, pH 7.4. The brain was removed and
Hattar et al., 2006; Morin et al., 2003). The IGL of the processed as described in Section 1.4.
lateral geniculate complex receives direct bilateral retinal
input from RGCs (Hickey and Spear, 1976; Ling et al., 1.3. Neuronal tracing
1998; Morin and Blanchard, 1997; Morin et al., 1992;
Neuronal tracing was carried out in old and young wild
Muscat et al., 2003; Pickard, 1982) that are collateral to
type and rd/rd cl mice (n ⫽ 3 young wild type, n ⫽ 3 young
those that project to the SCN (Pickard, 1985). In contrast to
rd/rd cl, n ⫽ 2 old wild type and n ⫽ 2 old rd/rd cl). Mice
the SCN, the relationship between light and Fos induction
were anaesthetized with ketamine hydrochloride, 60 mg
within the IGL has received relatively little attention and
kg⫺1 and xylazine, 7 mg kg⫺1 and a topical analgesic
remains poorly understood (Lupi et al., 1999; Peters et al.,
ophthalmic solution (Proparacaine parachloride). Cholera
1996). Through its projections to the SCN, the IGL is a
toxin Subunit B tracer (CTB, List Biological, Campbell,
functional contributor to circadian rhythmicity by its mod-
CA) (1% diluted in sterile distilled water) was injected into
ification of circadian responses to both photic and nonphotic the vitreous chamber of one eye with the aid of a micropi-
cues (Harrington and Rusak, 1986; Janik and Mrosovsky, pette (50 mm tip) sealed to the needle of a 5 ␮L Hamilton
1994; Johnson et al., 1989; Morin and Pace, 2002; Mros- syringe. Twenty-four hours later, the mice were perfused
ovsky, 1996; Pickard et al., 1987; Reebs and Mrosovsky, transcardially as described in Section 1.2.
1989). The vLGN is another component of the visual system
that has been shown to project bilaterally via the optic tracts 1.4. Tissue preparation and immunohistochemistry
to the SCN (Legg, 1979; Pickard, 1982; Ribak and Peters,
1975; Swanson et al., 1974). While the vLGN shows up- Following perfusion, brains were removed, postfixed
regulation of Fos in response to light, any role it may have overnight in the same fixative at 4 °C, and cryo-protected by
in circadian organization remains unresolved (Prichard et immersion in a solution of 30% sucrose in PBS overnight at
al., 2002; Rusak et al., 1990). 4 °C, then stored in PBS-A (0.01 M PB, 0.9% NaCl, 0.1%
NaN3 0.1%). Following cryo-protection in 30% sucrose,
serial coronal brain sections (40 ␮m) containing the SCN,
IGL, and vLGN were cut from each brain on a freezing
1. Methods microtome. Sections were processed for Fos or CTB immu-
1.1. Animals nohistochemistry depending on previous treatment (light
induction or intraocular injection). They were incubated in
Congenic wild type and rd/rd cl C3H/He mice (described 50% ethanol, 0.9% NaCl, and 0.05% H2O2 to block endog-
in Lucas et al., 1999) were maintained at 22 °C, 50% enous peroxidase at 4 °C for 1 h. Then they were washed in
humidity in a 12 : 12 h light/dark (12 : 12 LD) cycle. Food PBS (0.01 M PB, 0.9% NaCl) and blocked in 1% normal
and water available ad libitum. All procedures were con- serum (goat or rabbit according to the primary antibody
ducted according to the Home Office (UK) regulations, host) in PBST-A (0.1 M PB, 0.9% NaCl, 0.3% Triton-X
under the Animals (Scientific Procedures) Act of 1986. 100, NaN3 0.1%) for 60 min at 4 °C followed by incubation
 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392 385

in primary antibody rabbit anti-Fos (Ab-5 Oncogene Re- SCN by three way ANOVA (factors: pulsing, genotype and
search Products Calbiochem) at a final dilution of 1 : 20,000 age) shows: (1) there is a significant effect of light (F1,29 ⫽
or goat anti-CTB (List Biological, Campbell, CA) at a 108.3, p ⬍ 0.0001), a light pulse causes a substantial in-
dilution of 1 : 10,000 at 4 °C for 72 h. The secondary crease in Fos immunoreactivity; (2) a significant effect of
antibody binding and avidin biotin amplification was carried age (F1,29 ⫽ 20.15, p ⬍ 0.0001), older animals show atten-
out using the Vectastain ABC Elite kit (PK-6101 for Fos or uated Fos levels and (3) an interaction between light pulsing
PK-6105 for CTB). Brain sections were then washed in Tris and age (F1,29 ⫽ 21.58, p ⬍ 0.0001) the amplitude of light
buffer 0.05M, pH 7.4 twice for 10 min and then transferred induced Fos is reduced in old animals. In both young wild
to chilled 4 °C Tris buffer containing 0.02% 3, -3= Diamino- type and rd/rd cl mice there is a ⬃25 fold induction of Fos
benzidine, 0.5% nickel ammonium sulfate (DAB-Ni) and after a light pulse. Fos is also induced by light in old wild
0.001% H2O2. The development of the Chromagen was type and rd/rd cl mice but the magnitude of induction is
kept the same for all brain slices. The slices were thoroughly attenuated to ⬃9 fold (for post hoc comparisons see Table 1).
washed with Tris buffer (two times and left overnight at Interestingly there is no significant difference in the ampli-
4 °C). Then they were mounted on chrome alum and gela- tude of Fos induction between genotypes (Fig. 2).
tin-coated slides, air dried, dehydrated in a series of alco- We have also investigated the extent of retinal projec-
hols, cleared in xylene, and coverslipped with DePeX. Con- tions to the SCN in young versus old rd/rd cl and wild type
trol slices where the primary or secondary antibodies were mice. Representative sections are shown in Fig. 1I–L. Ret-
replaced with normal serum did not show any label. inal projections to the SCN show a similar symmetrical
1.5. Image analysis bilateral distribution for both rd/rd cl and wild type mice.
Significantly, labeling appears reduced in old animals, and
All the brain sections through the SCN, IGL, and vLGN again is similar for both genotypes. Densitometry values
were analyzed using a computerized image analysis system. indicate that the projection is similar between young wild
They were examined under a Zeiss Axioplan 2 microscope type (average CTB IOD 413,905 ⫾ SD 35,474) and young
and images captured with a Spot Digital Camera (Diagnos- rd/rd cl (average CTB IOD 319,483 ⫾ SD 11,938). In old
tic Instruments). In our study, the computerized image anal- wild type and rd/rd cl these values are reduced (wild type
ysis software Image-Pro Plus (Media Cybernetics) was used 159,408 ⫾ SD 30,873 and rd/rd cl 269,764 ⫾ SD 8,313).
to determine the integral optical density (IOD) of the la-
beled brain sections. The IOD represents the integral sum of 2.2. IGL: light activation and retinal afferents
the surface area of single pixels multiplied by their corre-
spondent optical density values. This method has been used Analysis of the Fos IOD in the paired IGL nuclei by three
widely to measure levels of Fos in the SCN (Barnard et al., way ANOVA shows: (1) a significant effect of light (F1,32 ⫽
2004; Dkhissi-Benyahya et al., 2000; Lupi et al., 1999; Lupi 18.01, p ⬍ 0.0002) resulting in an increase in Fos; (2) a
et al., 2006; Sekaran et al., 2005) and takes into account not significant effect of age (F1,32 ⫽ 10.52 p ⬍ 0.003) with
only the total area of Fos positive nuclei but also the density of attenuated Fos levels in old animals and (3) a significant
the label (Rieux et al., 2002). For the purposes of standardiza- effect of genotype (F1,32 ⫽ 4.50, p ⬍ 0.04), the rd/rd cl
tion each brain area was processed in parallel. The IOD values show slightly lower levels of Fos (Figs 3 and 4A). Post hoc
were summed for paired nuclei and in all sections, rostral to tests (Table 1) indicate that there is significant light induc-
caudal in which the nuclei could be identified. tion of Fos in the IGL of young wild types (p ⬍ 0.05), and
1.6. Statistics that this is reduced in old animals (p ⬍ 0.05) such that there
is not a significant light induction of Fos in the IGL of old
For the Fos induction studies we used three way light pulsed wild type animals compared with old sham
ANOVA for three factors (pulse, genotype, and age), car- pulsed wild types. Although the level of Fos in the IGL is
ried out using StatView SE⫹ Graphics, v1.0.3 (SAS Insti- higher in the rd/rd cl following light administration (in both
tute, Inc, Cary, USA), followed by Bonferroni’s multiple young and old) this is not to a significant level.
comparison test for post hoc comparisons. For neuronal The retinal projections to the IGL have an asymmetrical
tracing the average and standard deviations for densitometry distribution with a different contralateral/ipsilateral compo-
measurements are quoted in the results, the number of nent. In both rd/rd cl and wild type young and old there is
animals precluded statistical analysis of these data. a higher contralateral component than ipsilateral (Fig. 5).
The sum of the IOD values for CTB from both the ipsi- and
2. Results contralateral contribution to the IGL shows a marked reduc-
tion with aging in the wild type animals (wild type young
2.1. SCN: light activation and retinal afferents
175,232 ⫾ 22,559 vs. wild type old 80,462 ⫾ 17,899). The
Light-induced Fos occurs in the SCN of both wild type values from the rd/rd cl IGL also show a slight reduction
and rd/rd cl mice at both young and old ages (Fig. 1). with aging (rd/rd cl young 156,044 ⫾ 33,280 vs. rd/rd cl
Analysis of all the IOD data of Fos immunoreactivity in the old 150,201 ⫾ 28,666).
386 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392

Fig. 1. Light induced Fos and cholera toxin B (CTB) immunoreactivity in the suprachiasmatic nuclei (SCN) of young and old, wild type, and rd/rd cl mice.
Representative images of Fos immunoreactivity are shown in A–H. Images A, C, E, and G are from animals that received no light (sham pulse) while those
in B, D, F, and H received a pulse of light. SCN from young wild type mice are shown in (A) and (B), and young rd/rd cl in (C) and (D), those from old
wild type mice are shown in (E) and (F), and old rd/rd cl in (G) and (H). Examples of regions of interest analyzed for integrated optical density of Fos in
the SCN are shown in (B). I–L representative images of CTB immunoreactivity following unilateral intravitreal injection of CTB. SCN from (I) wild type
young, (J) rd/rd cl young, (K) wild type old and (L) rd/rd cl old mice. Scale bar for all images in (A) 100 ␮m.

2.3. vLGN: light activation and retinal afferents (Figs 3 and 4B). Post hoc testing (Table 1) indicates that
Three way ANOVA on IOD data from the vLGN shows there is a significant light induction of Fos in the young wild
a significant effect of light only (F1,27 ⫽ 27.76, p ⬍ type animals (p ⬍ 0.001) and that the magnitude of this
0.0001), with no significant effects of either age or genotype induction is reduced in old wild types (p ⬍ 0.05). Three way
 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392 387

Table 1 light input to the SCN in rd/rd cl mice is unaffected by outer

Post hoc Bonferroni comparisons for IOD of Fos in the SCN, IGL retinal cell loss. Thus, melanopsin-based pRGCs alone are
and vLGN
able to induce normal levels of Fos. These findings are
Post hoc comparison SCN IGL vLGN consistent with our previous results showing that the circa-
p-values p-values p-values
dian behavior of mice lacking rods and cones and that of
WT young light v. WT young p ⬍ 0.001 p ⬍ 0.05 p ⬍ 0.001 wild type mice is broadly similar (Barnard et al., 2004;
sham
RC young light v. RC young sham p⬍ 0.001 NS NS Freedman et al., 1999; Lucas et al., 1999; Semo et al.,
WT old light v. WT old sham p⬍ 0.05 NS NS 2003b). Indeed the magnitudes of phase shifts to a light
RC old light v. RC old sham p⬍ 0.05 NS NS pulse of 505-nm wavelength (as used here) are not signifi-
WT young light v. WT old light p⬍ 0.01 p ⬍ 0.05 p ⬍ 0.05 cantly different between wild type and rd/rd cl mice, sug-
RC young light v. RC old light p⬍ 0.001 NS NS
WT young light v. RC young light NS NS NS gesting compensatory mechanisms of the pRGC system in
WT old light v. RC old light NS NS NS the absence of outer retinal photoreception (Semo et al.,
Abbreviations: IOD, integrated optical density; IGL, intergeniculate leaf- 2003b). The rods and cones, however, do provide light
lets; NS, not significant; SCN, suprachiasmatic nuclei; RC, rd/rd cl; vLGN, information to the SCN. For example, in the absence of
ventral lateral geniculate nuclei; WT, wild type. melanopsin (Opn4⫺/⫺ mice) rods and cones can partially
compensate for the loss of functional pRGCs, showing
ANOVA does not describe any genotype differences, how- attenuated phase shifts and Fos induction of ⬃40% (Hattar
ever at the post hoc level we have been unable to detect a et al., 2003; Panda et al., 2002; Ruby et al., 2002). In view
significant light induction of Fos in the vLGN of rd/rd cl, of this input from the rods and cones it is perhaps surprising
the IOD levels do indicate a subtle light induced increase in that their loss does not appear to attenuate light-induced Fos
Fos compared with sham pulsed rd/rd cl (Fig. 4B). in the SCN of rd/rd cl mice.
Retinal projections to the vLGN again have an assy- In aged wild type and rd/rd cl mice, SCN neurons still
metrical distribution with a higher contralateral than ipsilat- show significant light-induced Fos, but there is a marked
eral component (Fig. 5). The IOD values for CTB in the reduction in levels compared with younger animals of both
vLGN (ipsi- and contralateral) are markedly reduced with genotypes. We have shown previously that aged rd/rd cl and
age in the wild type animals (wild type young 761,795 ⫾ wild type mice have significantly fewer melanopsin positive
114,706 vs. wild type old 468,464 ⫾ 98,007). There is a RGCs than young mice, but that the numbers of melanopsin
slight reduction in the IOD values for the rd/rd cl projection cells are not significantly different between the two genotypes
but again this is not as marked as for the wild types (rd/rd cl (Semo et al., 2003a; Semo et al., 2003b). This attenuation in
young 798,531 ⫾ 118,799 vs. rd/rd cl old 668,448 ⫾
49,229).

3. Discussion
In the present study we have used a mouse model (rd/rd cl)
to assess the relative importance of age versus retinal
degeneration on light-induced Fos within specific retino-
recipient areas (SCN, IGL, and vLGN) of the brain. Fos
expression has been used widely as a marker for neuronal
activation in response to various stimuli including light
(Dkhissi-Benyahya et al., 2000; Kornhauser et al., 1992). In
parallel, we have employed CTB as an anterograde tracer to
label these retinal target areas (Angelucci et al., 1996; Mik-
kelsen, 1992; Reiner et al., 1996).
3.1. Light activation in the SCN following retinal
degeneration and aging
Previously, levels of Fos expression in the SCN have
been shown to correlate with light intensity and the magni-
tude of circadian phase shifts (Dkhissi-Benyahya et al.,
2000; Kornhauser et al., 1990; Lupi et al., 1999). In the Fig. 2. The total integrated optical density (IOD) for Fos immunoreactivity
present study, we show light induction of Fos in both young measured in the paired nuclei of the suprachiasmatic nuclei (SCN) from
sham and light pulsed, old and young, wild type and rd/rd cl mice. Fos is
rd/rd cl and wild type animals and we also show that the significantly induced by light in both the rd/rd cl and wild type (post hoc
levels of Fos in the SCN of both genotypes are statistically p ⬍ 0.05). The magnitude of this induction is equivalent in both genotypes
indistinguishable, indicating that at the cellular level, the and is reduced in the old mice by ⬃58%.
388 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392

Fig. 3. Light induced Fos immunoreactivity in the intergeniculate leaflets (IGL) and ventral lateral geniculate nuclei (vLGN) of young and old wild
type and rd/rd cl mice. The images A, C, E, and G are from animals that received no light (sham pulse) while those in B, D, F, and H received a pulse
of light. IGL/vLGN from young wild type mice are shown in (A) and (B), and young rd/rd cl in (C) and (D), while those from old wild type mice
are shown in (E) and (F), and old rd/rd cl in (G) and (H). The smaller dashed area in (H) marks an example of the region of interest that would be
anlaysed for Fos in the IGL, while the larger area defines the region of interest for the vLGN. Scale bar 100 ␮m.
 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392 389

have been reported within melanopsin RGCs of aging indi-

viduals (Vugler et al., 2007). Although loss of melanopsin
pRGCs seems to be the most parsimonious explanation for
the reduction in Fos within the SCN, we cannot entirely
exclude the existence of other causes. For example, a re-
duction in light transmission through the lens has been
reported in old hamsters (Zhang et al., 1998) and humans
(Cuthbertson et al., 2009). It is also possible that there might
be age related impairments in the physiological responses of
the SCN neurons perhaps by changes in neurotransmitter
release and/or a reduction in neurotransmitter receptors
(Aujard et al., 2001; Tamaru et al., 1991).

3.2. Light activation in the IGL and vLGN following

retinal degeneration and aging
In the IGL and vLGN, in contrast to the SCN, we observe
differences in Fos expression between young rd/rd cl and
wild type mice. Wild type animals show a marked induction
of Fos in the IGL and vLGN in response to light, while rd/rd cl
mice show only a slight elevation of Fos in response to
light and this fails to reach statistical significance when
compared with sham treated controls. This suggests
strongly that rod and cone photoreceptors play a more
dominant role in light activation of the IGL and vLGN. In
view of the different sensory tasks mediated by rods/cones
and pRGCs, these results are not too surprising (Hankins et
al., 2008), reflecting their respective roles as image and
irradiance detectors. These findings are similar to those in
another visually impaired mouse (Rho⫺/⫺ Cnga3⫺/⫺) which
lacks functional rods and cones, where light stimuli failed to
elicit Fos induction in the IGL (Barnard et al., 2004). Old
rd/rd cl and wild type mice fail to show any significant
light-induced Fos within the IGL and vLGN. We suggest
this lack of Fos expression in the old wild type, compared
with young animals, is related to a loss of rod/cone input.
Our neuronal tracing suggests a reduction of fiber density of
retinal afferents to the IGL/vLGN in aged wild type mice
and correlates well with previously reported results showing
a reduction of rods and cones in the aged human and rodent
Fig. 4. The total integrated optical density (IOD) for Fos immunoreactivity retina (Gao and Hollyfield, 1992; Katz and Robison, 1986).
measured in the paired nuclei of the (A) intergeniculate leaflets (IGL) and
(B) ventral lateral geniculate nuclei (vLGN) from sham and light pulsed,
old and young, wild type and rd/rd cl mice. In the IGL and vLGN there is 3.3. Overall summary and conclusions
a significant light induction of Fos only in wild type animals and the
magnitude of this induction is reduced in old animals. There is no statis- We show a generalized loss of retinal afferents (⬃40% to
tically significant light induction of Fos in the rd/rd cl. the SCN) and a reduction in light activation in both normal
and retinally degenerate mice (rd/rd cl) (20 –27 months of
RGCs may well account for the reduction in Fos expression age). Previous studies in aging wild type mice have shown
we observe within the SCN of aged mice and is consistent a steady decrease in RGC numbers, declining by ⬃41% at
with our observation of an ⬃40% decrease in retinal affer- 18 months of age (Neufeld and Gachie, 2003). Outer retinal
ents in these mice (Fig. 1). Since we did not find differences degeneration appears to accelerate RGC loss in rd/rd mice
in melanopsin numbers between the genotypes, our result as they show ⬃20% fewer RGCs at 11–12 months com-
showing that the aged rd/rd cl and wild type have a similar pared to wild types (Wang et al., 2000). What is perhaps
magnitude of Fos induction within the SCN is also consis- surprising in the present study is that retinal afferent loss is
tent. Studies in humans also suggest age-related changes in not enhanced by rod and cone loss in the aged rd/rd cl mouse.
the pRGC system. In a recent paper, lipofuscin deposits Indeed, at this advanced age it appears that the afferents are
390 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392

Fig. 5. Cholera toxin B (CTB) immunoreactivity in the intergeniculate leaflets (IGL) and ventral lateral geniculate nuclei (vLGN) of young and old wild type
and rd/rd cl mice after unilateral intravitreal injection of CTB. The images A, C, E, and G are contralateral to the injected eye while those in B, D, F, and
H are ipsilateral. Projections to the IGL/vLGN in young wild type mice are shown in (A) and (B), and young rd/rd cl in (C) and (D), while those from old
wild type mice are shown in (E) and (F), and old rd/rd cl in (G) and (H). Black arrows indicate the vLGN and white arrows the IGL. Scale bar 200 ␮m.

preserved to the same level as wild types. Our data indicate Acknowledgements
that the extensive loss of RGCs over the life span of the
The first two authors contributed equally to this work.
mouse is not related to the loss of outer retinal photorecep-
This work was supported by the Biotechnology and Biolog-
tors and other factors must be involved, such as nutritional
ical Sciences Research Council (BBSRC), the Wellcome
intake, light levels, and/or strain specific diseases (Danias et
Trust, and a BBSRC studentship to MS. The authors would
al., 2003; Neufeld and Gachie, 2003). Our results might also like to thank Prof. Robert J. Lucas for technical assistance
reflect the fact that we have focused on regions that receive and Stuart N. Peirson for constructive comments during
inputs from melanopsin pRGCs, and these neurons may be preparation of this manuscript.
somewhat more resistant to axotomy induced cell death
(Robinson and Madison, 2004), that may be occurring in the
dystrophic retina (Wang et al., 2000). References
In summary, our data allow us to conclude that advanced
age is a more important factor than retinal degeneration in Altimus, C.M., Guler, A.D., Villa, K.L., McNeill, D.S., Legates, T.A.,
reducing the level of light activation in the SCN. Our study Hattar, S., 2008. Rods-cones and melanopsin detect light and dark to
modulate sleep independent of image formation. Proc. Natl. Acad. Sci.
provides important anatomical correlates to recent studies USA 105, 19998 –20003.
showing the therapeutic benefit of providing increased light Angelucci, A., Clasca, F., Sur, M., 1996. Anterograde axonal tracing with
to elderly people for the improvement of circadian rhythm the subunit B of cholera toxin: a highly sensitive immunohistochemical
deficits (Dowling et al., 2008; Lieverse et al., 2008; Riem- protocol for revealing fine axonal morphology in adult and neonatal
brains. J. Neurosci. Methods 65, 101–112.
ersma-van der Lek et al., 2008).
Aujard, F., Herzog, E.D., Block, G.D., 2001. Circadian rhythms in firing
rate of individual suprachiasmatic nucleus neurons from adult and
middle-aged mice. Neuroscience 106, 255–261.
Barnard, A.R., Appleford, J.M., Sekaran, S., Chinthapalli, K., Jenkins, A.,
Disclosure statement Seeliger, M., Biel, M., Humphries, P., Douglas, R.H., Wenzel, A.,
Foster, R.G., Hankins, M.W., Lucas, R.J., 2004. Residual photosensi-
The authors report no actual or potential conflicts of tivity in mice lacking both rod opsin and cone photoreceptor cyclic
interest. nucleotide gated channel 3 alpha subunit. Vis. Neurosci. 21, 675– 683.
 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392 391

Beaule, C., Amir, S., 1999. Photic entrainment and induction of immediate- Johnson, R.F., Moore, R.Y., Morin, L.P., 1989. Lateral geniculate lesions
early genes within the rat circadian system. Brain Res. 821, 95–100. alter circadian activity rhythms in the hamster. Brain Res. Bull. 22,
Benloucif, S., Masana, M.I., Dubocovich, M.L., 1997. Responsiveness to 411– 422.
melatonin and its receptor expression in the aging circadian clock of Katz, M.L., Robison, W.G., Jr, 1986. Evidence of cell loss from the rat
mice. Am. J. Physiol. 273, R1855–R1860. retina during senescence. Exp. Eye Res. 42, 293–304.
Cavallotti, C., Artico, M., Pescosolido, N., Feher, J., 2001. Age-related Kornhauser, J.M., Nelson, D.E., Mayo, K.E., Takahashi, J.S., 1990. Photic
changes in rat retina. Jpn. J. Ophthalmol. 45, 68 –75. and circadian regulation of c-fos gene expression in the hamster su-
Chambille, I., Doyle, S., Serviere, J., 1993. Photic induction and circadian prachiasmatic nucleus. Neuron 5, 127–134.
expression of Fos-like protein. Immunohistochemical study in the ret- Kornhauser, J.M., Nelson, D.E., Mayo, K.E., Takahashi, J.S., 1992. Reg-
ina and suprachiasmatic nuclei of hamster. Brain Res. 612, 138 –150. ulation of June-B messenger RNA and AP-1 activity by light and a
Cuthbertson, F.M., Peirson, S.N., Wulff, K., Foster, R.G., Downes, S.M., circadian clock. Science 255, 1581–1584.
2009. Blue light-filtering intraocular lenses: review of potential benefits Legg, C.R., 1979. An autoradiographic study of the efferent projections of
and side effects. J. Cataract Refract. Surg. 35, 1281–1297. the ventral lateral geniculate nucleus of the hooded rat. Brain Res. 170,
Czeisler, C.A., Chiasera, A.J., Duffy, J.F., 1991. Research on sleep, circa- 349 –352.
dian rhythms and aging: applications to manned spaceflight. Exp. Lieverse, R., Nielen, M.M., Veltman, D.J., Uitdehaag, B.M., van Someren,
Gerontol. 26, 217–232. E.J., Smit, J.H., Hoogendijk, W.J., 2008. Bright light in elderly subjects
Danias, J., Lee, K.C., Zamora, M.F., Chen, B., Shen, F., Filippopoulos, T., with nonseasonal major depressive disorder: a double blind randomised
Su, Y., Goldblum, D., Podos, S.M., Mittag, T., 2003. Quantitative clinical trial using early morning bright blue light comparing dim red
analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia light treatment. Trials 9, 48.
glaucomatous mice: comparison with RGC loss in aging C57/BL6 Ling, C., Schneider, G.E., Jhaveri, S., 1998. Target-specific morphology of
mice. Invest. Ophthalmol. Vis. Sci. 44, 5151–5162. retinal axon arbors in the adult hamster. Vis. Neurosci. 15, 559 –579.
Davis, F.C., Viswanathan, N., 1998. Stability of circadian timing with age Lucas, R.J., Douglas, R.H., Foster, R.G., 2001. Characterization of an
in Syrian hamsters. Am. J. Physiol. 275, R960 –R968. ocular photopigment capable of driving pupillary constriction in mice.
Dkhissi-Benyahya, O., Sicard, B., Cooper, H.M., 2000. Effects of irradi- Nat. Neurosci. 4, 621– 626.
ance and stimulus duration on early gene expression (Fos) in the Lucas, R.J., Freedman, M.S., Munoz, M., Garcia-Fernandez, J.M., Foster,
suprachiasmatic nucleus: temporal summation and reciprocity. J. Neu- R.G., 1999. Regulation of the mammalian pineal by non-rod, non-cone,
rosci. 20, 7790 –7797. ocular photoreceptors. Science 284, 505–507.
Dowling, G.A., Burr, R.L., Van Someren, E.J., Hubbard, E.M., Luxenberg, Lucas, R.J., Hattar, S., Takao, M., Berson, D.M., Foster, R.G., Yau, K.W.,
J.S., Mastick, J., Cooper, B.A., 2008. Melatonin and bright-light treat- 2003. Diminished pupillary light reflex at high irradiances in melanop-
ment for rest-activity disruption in institutionalized patients with Alz- sin-knockout mice. Science 299, 245–247.
heimer’s disease. J. Am. Geriatr. Soc. 56, 239 –246. Lupi, D., Cooper, H.M., Froehlich, A., Standford, L., McCall, M.A.,
Earnest, D.J., Iadarola, M., Yeh, H.H., Olschowka, J.A., 1990. Photic Foster, R.G., 1999. Transgenic ablation of rod photoreceptors alters the
regulation of c-fos expression in neural components governing the circadian phenotype of mice. Neuroscience 89, 363–374.
entrainment of circadian rhythms. Exp. Neurol. 109, 353–361. Lupi, D., Oster, H., Thompson, S., Foster, R.G., 2008. The acute light-
Freedman, M.S., Lucas, R.J., Soni, B., von Schantz, M., Munoz, M., induction of sleep is mediated by OPN4-based photoreception. Nat.
David-Gray, Z., Foster, R., 1999. Regulation of mammalian circadian Neurosci. 11, 1068 –1073.
behavior by non-rod, non-cone, ocular photoreceptors. Science 284, Lupi, D., Sekaran, S., Jones, S.L., Hankins, M.W., Foster, R.G., 2006.
502–504. Light-evoked FOS induction within the suprachiasmatic nuclei (SCN)
Gao, H., Hollyfield, J.G., 1992. Aging of the human retina. Differential loss of melanopsin knockout (Opn4⫺/⫺) mice: a developmental study.
of neurons and retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Chronobiol. Int. 23, 167–179.
Sci. 33, 1–17. Mikkelsen, J.D., 1992. Visualization of efferent retinal projections by
Gooley, J.J., Lu, J., Chou, T.C., Scammell, T.E., Saper, C.B., 2001. immunohistochemical identification of cholera toxin subunit B. Brain
Melanopsin in cells of origin of the retinohypothalamic tract. Nat. Res. Bulletin 28, 619 – 623.
Neurosci Dec;4(12):1165. Mirmiran, M., Swaab, D.F., Kok, J.H., Hofman, M.A., Witting, W., Van
Gooley, J.J., Lu, J., Fischer, D., Saper, C.B., 2003. A broad role for Gool, W.A., 1992. Circadian rhythms and the suprachiasmatic nucleus
melanopsin in nonvisual photoreception. J. Neurosci. 23, 7093–7106. in perinatal development, aging and Alzheimer’s disease. Prog. Brain
Hankins, M.W., Peirson, S.N., Foster, R.G., 2008. Melanopsin: an exciting Res. 93, 151–163.
photopigment. Trends Neurosci. 31, 27–36. Morin, L.P., Blanchard, J., Moore, R.Y., 1992. Intergeniculate leaflet and
Harrington, M.E., Rusak, B., 1986. Lesions of the thalamic intergeniculate suprachiasmatic nucleus organization and connections in the golden
leaflet alter hamster circadian rhythms. J. Biol. Rhythms 1, 309 –325. hamster. Vis. Neurosci. 8, 219 –230.
Hattar, S., Kumar, M., Park, A., Tong, P., Tung, J., Yau, K.W., Berson, Morin, L.P., Blanchard, J.H., 1997. Neuropeptide Y and enkephalin im-
D.M., 2006. Central projections of melanopsin-expressing retinal gan- munoreactivity in retinorecipient nuclei of the hamster pretectum and
glion cells in the mouse. J. Comp. Neurol. 497, 326 –349. thalamus. Vis. Neurosci. 14, 765–777.
Hattar, S., Liao, H.W., Takao, M., Berson, D.M., Yau, K.W., 2002. Mel- Morin, L.P., Blanchard, J.H., Provencio, I., 2003. Retinal ganglion cell
anopsin-containing retinal ganglion cells: architecture, projections, and projections to the hamster suprachiasmatic nucleus, intergeniculate
intrinsic photosensitivity. Science 295, 1065–1070. leaflet, and visual midbrain: bifurcation and melanopsin immunoreac-
Hattar, S., Lucas, R.J., Mrosovsky, N., Thompson, S., Douglas, R.H., tivity. J. Comp. Neurol. 465, 401– 416.
Hankins, M.W., Lem, J., Biel, M., Hofmann, F., Foster, R.G., Yau, Morin, L.P., Pace, L., 2002. The intergeniculate leaflet, but not the visual
K.W., 2003. Melanopsin and rod-cone photoreceptive systems account midbrain, mediates hamster circadian rhythm response to constant
for all major accessory visual functions in mice. Nature 424, 75– 81. light. J. Biol. Rhythms 17, 217–226.
Hickey, T.L., Spear, P.D., 1976. Retinogeniculate projections in hooded Mrosovsky, N., 1996. Locomotor activity and non-photic influences on
and albino rats: an autoradiographic study. Exp. Brain Res. 24, 523– circadian clocks. Biol. Rev. Camb. Philos. Soc. 71, 343–372.
529. Muscat, L., Huberman, A.D., Jordan, C.L., Morin, L.P., 2003. Crossed and
Janik, D., Mrosovsky, N., 1994. Intergeniculate leaflet lesions and behav- uncrossed retinal projections to the hamster circadian system. J. Comp.
iorally-induced shifts of circadian rhythms. Brain Res. 651, 174 –182. Neurol. 466, 513–524.
392 D. Lupi et al. / Neurobiology of Aging 33 (2012) 383–392

Neufeld, A.H., Gachie, E.N., 2003. The inherent, age-dependent loss of Schwartz, W.J., Carpino A., Jr, de la Iglesia, H.O., Baler, R., Klein, D.C.,
retinal ganglion cells is related to the lifespan of the species. Neurobiol. Nakabeppu, Y., Aronin, N., 2000. Differential regulation of fos family
Aging 24, 167–172. genes in the ventrolateral and dorsomedial subdivisions of the rat
Panda, S., Provencio, I., Tu, D.C., Pires, S.S., Rollag, M.D., Castrucci, suprachiasmatic nucleus. Neuroscience 98, 535–547.
A.M., Pletcher, M.T., Sato, T.K., Wiltshire, T., Andahazy, M., Kay, Sekaran, S., Foster, R.G., Lucas, R.J., Hankins, M.W., 2003. Calcium
S.A., Van Gelder, R.N., Hogenesch, J.B., 2003. Melanopsin is required imaging reveals a network of intrinsically light-sensitive inner-retinal
for non-image-forming photic responses in blind mice. Science 301, neurons. Curr. Biol. 13, 1290 –1298.
525–527. Sekaran, S., Lupi, D., Jones, S.L., Sheely, C.J., Hattar, S., Yau, K.W.,
Panda, S., Sato, T.K., Castrucci, A.M., Rollag, M.D., DeGrip, W.J., Ho- Lucas, R.J., Foster, R.G., Hankins, M.W., 2005. Melanopsin-dependent
genesch, J.B., Provencio, I., Kay, S.A., 2002. Melanopsin (Opn4) photoreception provides earliest light detection in the Mammalian
Requirement for Normal Light-Induced Circadian Phase Shifting. Sci- retina. Curr. Biol. 15, 1099 –1107.
ence 298, 2213–2216.
Semo, M., Lupi, D., Peirson, S.N., Butler, J.N., Foster, R.G., 2003a.
Penev, P.D., Turek, F.W., Wallen, E.P., Zee, P.C., 1997. Aging alters the
Light-induced c-fos in melanopsin retinal ganglion cells of young and
serotonergic modulation of light-induced phase advances in golden
aged rodless/coneless (rd/rd cl) mice. Eur. J. Neurosci. 18, 3007–3017.
hamsters. Am. J. Physiol. 272, R509 –R513.
Semo, M., Peirson, S., Lupi, D., Lucas, R.J., Jeffery, G., Foster, R.G.,
Peters, R.V., Aronin, N., Schwartz, W.J., 1996. c-Fos expression in the rat
2003b. Melanopsin retinal ganglion cells and the maintenance of cir-
intergeniculate leaflet: photic regulation, co-localization with Fos-B,
cadian and pupillary responses to light in aged rodless/coneless (rd/rd
and cellular identification. Brain Res. 728, 231–241.
Pickard, G.E., 1985. Bifurcating axons of retinal ganglion cells terminate cl) mice. Eur. J. Neurosci. 17, 1793–1801.
in the hypothalamic suprachiasmatic nucleus and the intergeniculate Sutin, E.L., Dement, W.C., Heller, H.C., Kilduff, T.S., 1993. Light-induced
leaflet of the thalamus. Neurosci. Lett. 55, 211–217. gene expression in the suprachiasmatic nucleus of young and aging
Pickard, G.E., 1982. The afferent connections of the suprachiasmatic nu- rats. Neurobiol. Aging 14(5), 441– 446.
cleus of the golden hamster with emphasis on the retinohypothalamic Swanson, L.W., Cowan, W.M., Jones, E.G., 1974. An autoradiographic
projection. J. Comp. Neurol. 211, 65– 83. study of the efferent connections of the ventral lateral geniculate
Pickard, G.E., Ralph, M.R., Menaker, M., 1987. The intergeniculate leaflet nucleus in the albino rat and the cat. J. Comp. Neurol. 156, 143–163.
partially mediates effects of light on circadian rhythms. J. Biol. Tamaru, M., Yoneda, Y., Ogita, K., Shimizu, J., Nagata, Y., 1991. Age-
Rhythms 2, 35–56. related decreases of the N-methyl-D-aspartate receptor complex in the
Prichard, J.R., Stoffel, R.T., Quimby, D.L., Obermeyer, W.H., Benca, rat cerebral cortex and hippocampus. Brain Res. 542, 83–90.
R.M., Behan, M., 2002. Fos immunoreactivity in rat subcortical visual Thompson, S., Foster, R.G., Stone, E.M., Sheffield, V.C., Mrosovsky, N.,
shell in response to illuminance changes. Neuroscience 114, 781–793. 2008. Classical and melanopsin photoreception in irradiance detection:
Rea, M.A., 1989. Light increases Fos-related protein immunoreactivity in negative masking of locomotor activity by light. Eur. J. Neurosci. 27,
the rat suprachiasmatic nuclei. Brain Res. Bull. 23, 577–581. 1973–1979.
Reebs, S.G., Mrosovsky, N., 1989. Large phase-shifts of circadian rhythms Valentinuzzi, V.S., Scarbrough, K., Takahashi, J.S., Turek, F.W., 1997.
caused by induced running in a re-entrainment paradigm: the role of Effects of aging on the circadian rhythm of wheel-running activity in
pulse duration and light. J. Comp. Physiol. [A] 165(6), 819 – 825. C57BL/6 mice. Am. J. Physiol. 273, R1957–R1964.
Reiner, A., Zhang, D., Eldred, W.D., 1996. Use of the sensitive anterograde Vugler, A.A., Redgrave, P., Semo, M., Lawrence, J., Greenwood, J.,
tracer cholera toxin fragment B reveals new details of the central retinal Coffey, P.J., 2007. Dopamine neurones form a discrete plexus with
projections in turtles. Brain Behav. Evol. 48, 307–337. melanopsin cells in normal and degenerating retina. Exp. Neurol. 205,
Ribak, C.E., Peters, A., 1975. An autoradiographic study of the projections 26 –35.
from the lateral geniculate body of the rat. Brain Res. 92, 341–368. Wang, S.M., Villegas-Pérez, M.P., Vidal-Sanz, M., Lund, R.D., 2000.
Riemersma-van der Lek, R.F., Swaab, D.F., Twisk, J., Hol, E.M.,
Progressive optic axon dystrophy and vascular changes in rd mice.
Hoogendijk, W.J., Van Someren, E.J., 2008. Effect of bright light and
Invest. Ophthalmol. Vis. Sci. 41, 537–545.
melatonin on cognitive and noncognitive function in elderly residents
Welsh, D.K., Richardson, G.S., Dement, W.C., 1986. Effect of age on the
of group care facilities: a randomized controlled trial. J. Am. Med.
circadian pattern of sleep and wakefulness in the mouse. J. Gerontol.
Assoc. 299, 2642–2655.
41, 579 –586.
Rieux, C., Carney, R., Lupi, D., Dkhissi-Benyahya, O., Jansen, K., Chounla-
mountri, N., Foster, R.G., Cooper, H.M., 2002. Analysis of immunohis- Zee, P.C., Rosenberg, R.S., Turek, F.W., 1992. Effects of aging on en-
tochemical label of Fos protein in the suprachiasmatic nucleus: comparison trainment and rate of resynchronization of circadian locomotor activity.
of different methods of quantification. J. Biol. Rhythms 17, 121–136. Am. J. Physiol. 263, R1099 –R1103.
Robinson, G.A., Madison, R.D., 2004. Axotomized mouse retinal ganglion Zhang, Y., Brainard, G.C., Zee, P.C., Pinto, L.H., Takahashi, J.S., Turek,
cells containing melanopsin show enhanced survival, but not enhanced F.W., 1998. Effects of aging on lens transmittance and retinal input to
axon regrowth into a peripheral nerve graft. Vis. Res. 44, 2667–2674. the suprachiasmatic nucleus in golden hamsters. Neurosci. Lett. 258,
Ruby, N.F., Brennan, T.J., Xie, X., Cao, V., Franken, P., Heller, H.C., 167–170.
O’Hara, B.F., 2002. Role of Melanopsin in Circadian Responses to Zhang, Y., Kornhauser, J.M., Zee, P.C., Mayo, K.E., Takahashi, J.S.,
Light. Science 298, 2211–2213. Turek, F.W., 1996. Effects of aging on light-induced phase-shifting of
Rusak, B., Robertson, H.A., Wisden, W., Hunt, S.P., 1990. Light pulses circadian behavioral rhythms, fos expression and CREB phosphoryla-
that shift rhythms induce gene expression in the suprachiasmatic nu- tion in the hamster suprachiasmatic nucleus. Neuroscience 70, 951–
cleus. Science 248, 1237–1240. 961.