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The Journal of Neuroscience, December 1, 2002, 22(23):10427-10433
Loss of Photic Entrainment and Altered Free-Running Circadian
Rhythms in math5 / Mice
Raymond
Wee1,
Ana Maria
Castrucci3,
Ignacio
Provencio3,
Lin
Gan4, and
Russell N.
Van
Gelder1, 2
Departments of 1 Ophthalmology and Visual Sciences and
2 Molecular Biology and Pharmacology, Washington University
Medical School, St. Louis, Missouri 63110, 3 Department of
Anatomy, Physiology, and Genetics, Uniformed Services University of
Health Sciences, Bethesda, Maryland 20814, and 4 Department
of Neurobiology and Anatomy, University of Rochester Medical School,
Rochester, New York 14642
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ABSTRACT |
Mammalian free-running circadian rhythms are entrained to the
external light/dark cycle by photic signaling to the suprachiasmatic nuclei via the retinohypothalamic tract (RHT). We investigated the
circadian entrainment and clock properties of
math5 / mutant mice.
math5 is a critical regulator of retinal ganglion cell
development; math5/ mice show
severe optic nerve hypoplasia. By anterograde cholera toxin B tracing,
we find that math5/ mice do not
develop an identifiable RHT pathway. This appears to be attributable to
agenesis or dysgenesis of the majority of RHT-projecting retinal
ganglion cells. math5/ mice
display free-running circadian rhythms with a period ~1 hr longer
than B6/129 controls (24.43 ± 0.10 vs 23.62 ± 0.19 hr; p < 0.00001). The free-running period of
heterozygote mice is indistinguishable from that of controls.
math5/ mice show no entrainment
to light/dark cycles, whereas heterozygote mice show normal entrainment
to both 12 hr light/dark cycles and to a 1 hr skeletal photoperiod.
math5/ mice show reduced ability
to entrain their rhythms to the nonphotic time cue of restricted
running wheel access but demonstrate both free-running behavior and
entrained anticipation of wheel unlocking in these conditions,
suggesting the presence of a second diurnal oscillatory system in
math5/ animals. These results
demonstrate that retinal ganglion cell input is not necessary for the
development of a free-running circadian timekeeping system in the
suprachiasmatic nucleus but is important for both photic entrainment
and determination of the free-running period.
Key words:
circadian rhythm; math5; photic entrainment; retinohypothalamic tract; nonphotic entrainment; phase shifting
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INTRODUCTION |
In the absence of external time
cues, mammalian behavior remains temporally organized in nearly
day-long cycles of activity and rest. These circadian rhythms have a
discrete genetic and anatomic basis (Herzog, 2001 ; Reppert and Weaver,
2001). The suprachiasmatic nuclei (SCN) of the hypothalamus contain the
central circadian pacemaker. Lesions of the suprachiasmatic nucleus
result in behavioral arrhythmicity, whereas transplantation of fetal
SCN tissue can restore rhythmic behavior with the clock period
dependent on the period of the donor tissue (Ralph et al., 1990;
Weaver, 1998). In the absence of external time cues (i.e., free-running
conditions), the period of the circadian clock differs significantly
from 24 hr in most mammals. Free-running rhythms thus require continual synchronization with the exogenous 24 hr light/dark cycle.
The eyes are necessary for photic entrainment in most mammals, but the
classical photoreceptors (rods and cones) are dispensable (Foster et
al., 1991; Lucas and Foster, 1999). The identity of the photopigment
underlying circadian entrainment has not been definitively
demonstrated, but its localization appears to be in the inner retina
(Berson et al., 2002). The retinohypothalamic tract (RHT) connects a
small subset of retinal ganglion cells with the SCN (Hendrickson et
al., 1972; Moore, 1973, 1995). This tract is thought to play an
essential role in photic entrainment. In hamsters, resection of the RHT
eliminates photic entrainment (Johnson et al., 1988). Conversely,
resection of the optic tract with preservation of the RHT tract in
hamsters results in a blind animal still able to entrain to light
(Klein and Moore, 1979). Recently, a candidate photopigment,
melanopsin, has been found to be expressed in RHT and other
non-visual-projecting retinal ganglion cells (Gooley et al., 2001;
Hannibal et al., 2002; Hattar et al., 2002; Provencio et al., 2002).
math5, a murine atonal homolog, is a
helix-loop-helix proneural gene necessary for retinal ganglion cell
differentiation in mice (Brown et al., 2001; Wang et al., 2001). This
gene is expressed nearly exclusively in developing retinal ganglion
cells (RGCs) (Brown et al., 1998). Homozygous
math5/ mice lack >80% of
retinal ganglion cells, including nearly all Brn3b-positive cells
(Brown et al., 2001; Liu et al., 2001; Wang et al., 2001). Heterozygous
mice are anatomically normal. The current study was designed to
determine whether the RHT-projecting retinal ganglion cells are also
dependent on math5 for their development or whether these
cells are among the math5-independent RGCs seen in
math5/ mice. We find that >90%
of melanopsin-positive cells fail to develop in
math5/ mice, leading to loss of
an identifiable retinohypothalamic tract. The
math5/ mouse thus has a
genetically deafferented circadian clock. We demonstrate that this
deafferented clock cannot entrain to light/dark cycles, has an abnormal
free-running circadian period, and has abnormal responses to nonphotic stimuli.
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MATERIALS AND METHODS |
Animals.
math5/ mice were generated as
described previously (Wang et al., 2001). All analysis was performed on
a mixed C57BL/6J and 129/SvEv background (hereafter referred to as
B6/129). Mice ranged in age from 12 to 16 weeks at the start of the
experiment. Each mouse was individually housed in a polycarbonate cage
containing a running wheel. All animal work was performed under animal
care guidelines from the Association for Research in Vision and Ophthalmology.
Genotyping of mice was performed by PCR of DNA extracted from distal
tail biopsy. Primers for wild-type math5 were 5'-TAC GCG AAA
GGT CAG AGG TCA C-3' and 5'-TGA GCC ACG AAC AGA TGA AAG C-3', and
primers for disrupted math5 were 5'-AGG GCC GCA AGA AAA CTA
TCC-3' and 5'-ACT TCG GCA CCT TAC GCT TCT TCT-3'. After 2 min of
denaturation at 94°C, 35 cycles of denaturation at 94°C for 30 sec,
annealing at 64°C for 30 sec, and extension at 72°C for 50 sec were
performed. Products were analyzed by agarose gel electrophoresis with
ethidium bromide staining.
Cholera toxin subunit B immunohistochemistry. Cholera toxin
subunit B (Sigma, St. Louis, MO) was pressure-injected into the posterior chamber of the right eye in adult
math5/ and B6/129 control mice
as described previously (Mikkelsen, 1992; Mikkelsen and Serviere,
1992). Cholera toxin subunit B antiserum raised in goat was purchased
from List Biologic (Campbell, CA; product 703, lot 7032G). Mice were
anesthetized with ketamine and xylazine and injected with 5 µl (50 µg) of cholera toxin subunit B using a Hamilton (Reno, NV) syringe
(gauge 33) into the vitreous humor of the right eye. After 24 hr, the mice were anesthetized, killed, and fixed by perfusion. Animals
were perfused initially with 30 ml of PBS containing 15 IU/l
heparin for 5 min and then with 400 ml 4% paraformaldehyde dissolved
in 0.1 M phosphate buffer, pH 7.4, for 15 min.
Brains were removed, immersed in the same fixative for 4 hr at room
temperature, cryoprotected in 30% sucrose and PBS for 2 d at
4°C, immersed in optimal cutting temperature compound for 4 hr, frozen, cut into 40-µm-thick sections in a cryostat, and
collected in PBS as free-floating sections. Immunohistochemistry was
performed using the avidin-biotin procedure as described previously (Hsu et al., 1981).
Melanopsin immunohistochemistry. Adult
math5/ and B6/129 control mice
were anesthetized with ketamine and xylazine, killed, and fixed by
transcardiac perfusion with 30 ml of 15 IU/l heparin followed by 30 ml
of 4% paraformaldehyde. The cornea and lens were removed. A fiducial
mark was made by burning the dorsal aspect of the eye with a heated 21 gauge needle before the eyes were removed. A cut was made from the
fiducial mark to the optic nerve to maintain orientation. The eyes were
postfixed for 48 hr in 4% paraformaldehyde. Flat-mounted retina
preparations were subjected to a 72 hr incubation at 4°C with a
1:2500 dilution of anti-melanopsin antiserum. The primary antiserum was
raised in a rabbit against a peptide representing the 15 N-terminal
amino acids of mouse melanopsin with an appended C-terminal cysteine to
facilitate conjugation to keyhole limpet hemocyanin. Immunopositive
cells were visualized by incubating the retina in a 1:500 dilution of a
Cy3-conjugated anti-rabbit IgG secondary antibody (1:500;
Jackson ImmunoResearch, West Grove, PA) for 1 hr at room temperature. Processed retinas were mounted and coverslipped under Vectashield (Vector Laboratories, Burlingame, CA) and viewed under epifluorescence.
Photic entrainment. Wheel-running activity was recorded
through continuous computer sampling of magnetic switches triggered by
running wheel rotation (Actimetrics, Evanson, IL). Mice were kept in
total darkness (DD) and allowed ad libitum access to the running wheel, food, and water. Free-running periods were calculated by
periodogram analysis using ClockLab software (Actimetrics). To assay
photic entrainment, mice were placed in a 12 hr light/dark (LD 12:12)
lighting cycle with broad-spectrum fluorescent lighting. Luminance was
measured as 100 lux at the cage surface. Mice were monitored in LD
12:12 for 14 d, after which the mice were again placed in DD. To
test heterozygous mice for subtle defects in photic entrainment,
math5 heterozygote and B6/129 control sibling mice were
placed into a skeletal photoperiod of 1 hr of light/d (LD 1:23).
Phase response measurement. Six
math5+/ mice and 4 B6/129 sibling
controls were singly housed and placed in LD 12:12 for 7 d to
coordinate entrainment among the mice. The mice were then released into
DD for 4 d. On the fifth day, the mice were subjected to ~100
lux of light for 1 hr; this corresponded to early subjective night
[mean circadian time (CD) ± SD, 14.9 ± 1.4].
Free-running rhythms were again monitored for 7 d, and phase shift
was assayed by determination of wheel-running onsets. For the phase
advance portion of the curve, the mice were entrained with 10 d of
LD 12:12 before being released into darkness for 4 d to
reestablish a free-running rhythm. On the fifth day, the mice received
1 hr of ~100 lux of light in the late subjective night (mean CT,
22.0 ± 1.1). Phase shift was measured as for the phase delay
curve above.
Wheel lock with activity monitor. After 11 d of
free-running activity, seven
math5/ mice and seven B6/129
sibling controls were kept in constant darkness with restricted access
to the running wheel for 2 hr/d (10 A.M. to 12 P.M.). All mice
ran vigorously of their own volition; no special measures had to be
taken to induce activity. Activity was monitored through the use of a
plastic platform placed adjacent to the water bottle spout. When
drinking or incidentally stepping on this platform, the mouse triggered
a microswitch that sent a signal to the computer recording activity
data. Scheduled running wheel access occurred for 46 d, after
which mice were allowed unlimited access.
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RESULTS |
math5/ mice lack a well developed
retinohypothalamic tract
The murine RHT consists of ~500 retinal ganglion cell axons that
project directly to the SCN (Moore et al., 1995). Intravitreal injection of cholera toxin subunit B is an effective method to trace
efferent projections from the retina (Mikkelsen, 1992). To determine
whether the RHT is spared or lost in homozygous
math5/ mutant mice, four
math5/ mice and sibling B6/129
control mice underwent intravitreal injection with cholera toxin
subunit B. Cholera toxin staining confirmed substantial loss of the
optic chiasm in math5/ mice. In
all math5/ mice, no significant
staining of the SCN was observed, whereas the SCN of B6/129 mice
exhibited robust staining (Fig. 1).
Hematoxylin staining of math5/ mouse
brain sections revealed anatomically normal-appearing SCN.
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Figure 1.
Anterograde cholera toxin B staining for the
retinohypothalamic tract in control (B6/129; left) and
math5/ (right)
mice. Note the minimal optic chiasm in
math5/mice. No staining was seen
in any math5/ SCN.
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math5/ retinas show markedly reduced
numbers of melanopsin-positive retinal ganglion cells
Loss of anterograde cholera toxin staining of the SCN from the
retina could be attributable either to loss of the melanopsin-positive, light-sensitive retinal ganglion cells (Gooley et al., 2001; Hannibal et al., 2002; Hattar et al., 2002; Provencio et al., 2002) or to
misrouting of the axons of these cells. We stained the retinas of
math5/ and B6/129 mice for
melanopsin immunoreactivity (Provencio et al., 2002). The number of
melanopsin-expressing cells was markedly reduced in
math5/ retinas, which
showed only a few melanopsin-positive cells (Fig. 2). Cell counts of
math5/ retinas revealed
60.3 ± 10.5 melanopsin-positive cells per retina (mean ± SE; n = 4). This corresponds to ~6% of the normal
complement of melanopsin-positive cells (A. M. Castrucci and I. Provencio, unpublished observations). The few melanopsin-positive cells
tended to be found in the peripheral retina and had normal-appearing morphology. Loss of the retinohypothalamic tract is therefore most
likely attributable to agenesis or dysgenesis of the majority of
RHT-projecting cells rather than misrouting of their axons.
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Figure 2.
Representative whole-mount retinal
immunohistochemical specimens for melanopsin in control B6/129 and
math5/ retinas. One retinal
quadrant is shown in each image.
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Loss of photic entrainment and lengthened free-running period
in math5/ mice
If photic entrainment of circadian activity requires an intact
retinohypothalamic tract, one would expect that mice genetically lacking this pathway would be unable to synchronize their circadian rhythms to external lighting cues. All
math5/ mice recorded in LD 12:12
conditions failed to entrain their circadian rhythms to external time
cues and showed free-running behavior (Fig.
3). In contrast, all
math5+/ heterozygotes and control
siblings showed normal entrainment to 100 lux light/dark cycles.
Unexpectedly, math5/ mice
uniformly exhibited free-running periods significantly >24 hr
(24.43 ± 0.10 hr; n = 7; Table
1). There was no difference in
free-running periods between
math5/ mice kept in a light/dark
cycle and those in total darkness, suggesting that the circadian clock
of these animals was oblivious to external lighting conditions.
math5+/ heterozygote mice showed
free-running periods (23.52 ± 0.13 hr) indistinguishable from
those of B6/129 control animals (23.62 ± 0.19 hr). The period
difference between math5/ and
math5+/ mice (and control mice)
was highly statistically significant (p < 0.00001 by two-tailed pair-wise Student's t test).
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Figure 3.
math5/ mice
do not entrain to a light/dark cycle. Top panels, Mice
were allowed to run freely for 1 week and were then subjected to LD
12:12 (lights on represented by gray bars) for 14 d, followed by 1 week of DD conditions. All data are shown as
double-plotted wheel-running raster plots. Bottom panel,
Comparison of math5/ and
math5+/ mice for entrainment during
50 d of LD 12:12. No entrainment was seen in any of seven
math5/ mice tested.
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math5+/ mice show normal entrainment to
the skeletal photoperiod and normal phase response characteristics to
light
The prolonged free-running period of
math5/ mice could be
attributable to either loss of an influence of the
retinohypothalamic-projecting ganglion cells on the free-running
period of SCN pacemaking cells or to a direct effect of
math5 within the circadian clock mechanism. All murine clock
mechanism genes studied to date (including mPer1, mPer2, Bmal, Clock, mCry1,
and mCry2) have shown at least subtle free-running or
entrained circadian abnormalities in heterozygotes and behave as
semidominant alleles (Antoch et al., 1997; Thresher et al., 1998; van
der Horst et al., 1999; Vitaterna et al., 1999; Bunger, 2000; Bae et
al., 2001; Zheng et al., 2001). We therefore further analyzed the
circadian entrainment of heterozygous
math5+/ mice. We evaluated these
mice for a subtle entrainment phenotype by testing the ability of
heterozygous and control mice to entrain to a minimal (skeleton)
photoperiod of LD 1:23 (n = 4 of each genotype). No
difference in the ability to entrain to a skeletal photoperiod was seen
between math5+/ and control B6/129
animals (Fig. 4, top
panel). All heterozygotes and control mice were able to
entrain to the skeletal photoperiod with no qualitative difference in
entrainment angle or stability.
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Figure 4.
math5+/ mice
entrain to a skeletal photoperiod and show normal photic phase shifting
responses. Top, Representative double-plotted wheel
running actograms for math5+/
(left) and control (right) mice kept in
LD 1:23 (lights on represented by gray bars).
Bottom, Phase responses for photic phase shifting in
math5+/ and control B6/129 mice.
Phase responses were calculated on the basis of the change in the
free-running wheel activity phase after a 1 hr 100 lux light stimulus
given at the indicated phase. n = 7 for
math5+/ mice; n = 4 for control mice. Differences between genotypes were not
statistically significant (p > 0.4 for
each).
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We additionally asked whether differences in the phase-shifting
response of the free-running clock to a brief light pulse could be
observed between the math5+/ and
B6/129 mice. Free-running mice were subjected to light pulses in the
early and late subjective night (CT, 15 and 22), and subsequent phase
shifts were measured. As shown in Figure 4, bottom panel, there was no qualitative difference in the photic phase response of
math5+/ mice in either the advance
or delay portion of the phase-response curve.
math5/ mice do not entrain activity to
running wheel availability
Regularly scheduled exercise can entrain free-running circadian
rhythms in mice (Edgar et al., 1991). To determine whether the
math5/ mouse's circadian clock
could entrain to this nonphotic stimulus, we subjected
math5/ and B6/129 control mice
to scheduled wheel-running availability. Mice were allowed access to
their running wheels for 2 hr/d. During this window of activity, mice
spontaneously ran vigorously on their wheels (Fig.
5). At all other times, the wheels were
mechanically locked. Drinking activity was monitored to assess
behavioral rhythmicity. Six of seven control mice demonstrated
entrainment to running wheel availability within 46 d of wheel
lock (Fig. 5). The single mouse that failed to entrain did not run
vigorously during times of wheel availability. Only one of seven
math5/ mice showed entrainment
to wheel availability after 46 d of wheel lock. It is unlikely
that this failure to entrain to an external stimulus reflects the wheel
availability falling in the "dead zone" of the phase-response
curve, because the long duration of wheel locking ensured that wheel
availability was presented at all circadian phases.
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Figure 5.
math5/ mice
fail to synchronize activity to limited wheel availability.
Double-plotted drinking activity actograms are shown for three
math5+/ (left) and
three control (right) mice. Mice were housed in DD
conditions. Wheel access was limited to 2 hr/d (gray
bars) for 46 d.
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Free-running math5/ mice anticipate a
24 hr wheel availability rhythm
We analyzed the daily drinking activity of the
math5/ mice during activity
restriction by plotting this activity summated for each 1 hr of the 24 hr day for the six nonentraining
math5/ mice and 46 d of
recording (total, 276 recording days) (Fig. 6). These data demonstrated that the
math5/ mice anticipated the
release of wheel lock by at least ~2 hr despite not having overall
activity entrained by the limited wheel availability.
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Figure 6.
math5/ mice
show 24 hr anticipatory behavior during limited wheel availability.
Mean drinking monitor activity per hour was summated over the six
nonentraining math5/ mice during
46 d of limited wheel availability (n = 276 d for each point). Data are shown as mean ± SE. The
gray bar represents the period of wheel availability.
Points represent succeeding hours (i.e., the
point at 0 hr represents time
0-1).
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DISCUSSION |
In this study, we have demonstrated that
math5/ mutant mice do not
develop a detectable retinohypothalamic tract, resulting in a
genetically deafferented circadian clock. This clock cannot entrain to
external light/dark stimuli. Two properties of this "blind"
circadian clock are unexpectedly altered: the free-running period of
activity is increased by ~1 hr, and this clock is not readily
entrained by timed running wheel availability.
Photic entrainment is the primary process by which most animals
synchronize their circadian rhythms with the external environment (Lowrey and Takahashi, 2000). Previous experiments in genetically blind
mice, including rd/rd (Foster et al., 1991),
rds/rds (Argamaso et al., 1995), and rd/rd; cl
(Freedman et al., 1999), have demonstrated that outer retinal
photoreceptors (rods and cones) are not necessary for photic
entrainment. These mice all entrain normally to external light/dark
cycles. However, enucleated mice do not entrain their rhythms to light
(Halberg et al., 1954; Freedman et al., 1999), demonstrating that the
circadian photoreceptive system in rodents is ocular. In
math5/ mice, differentiation of
retinal ganglion cells is disrupted, and <20% of the normal
complement of retinal ganglion cells develop (Brown et al., 2001; Wang
et al., 2001). The retinas of
math5/ mice maintain their inner
laminar structure, and no loss of photoreceptor cells in the outer
nuclear layer is observed. The inability of math5/ mice to entrain to light
thus demonstrates the necessity of retinal ganglion cells for circadian
entrainment. Recent work has demonstrated that the subset of
RHT-projecting, melanopsin-expressing retinal ganglion cells is
directly photoresponsive (Berson et al., 2002; Hattar et al., 2002). In
the present work, we demonstrate that math5 is necessary for
the development of most melanopsin-expressing retinal ganglion cells
and the retinohypothalamic tract. The loss of photic entrainment in
these mice demonstrates that retinal ganglion cells are essential for
this process and is consistent with the hypothesis that the
retinohypothalamic tract is necessary for photic entrainment (Moore,
1995).
To our knowledge, the math5/
mouse is the second genetically blind strain to show the absence of
photic circadian entrainment. The more severe anophthalmic mouse
(ZRDCT-AN) carries a mutation that results in abortive embryonic eye
development (Tucker et al., 2001). Several of the features of circadian
rhythmicity found in the math5/
mice were also seen in anophthalmic mice. Anophthalmic mice fail to
entrain to light/dark cycles (Silver, 1977; Faradji et al., 1980;
Laemle and Ottenweller, 1998). Free-running periods of anophthalmic mice are also consistently >24 hr (although they are ~15 min shorter than observed in math5/ mice;
Laemle and Ottenweller, 1998). However, unlike
math5/ mutants, anophthalmic
mice show significant variability in the presence, stability, and
nonphotic entrainment of their rhythms (Laemle and Ottenweller, 1998).
Anophthalmic mice also show variable morphologic defects in the SCN
(Silver, 1977), which were not observed in our studies of the
math5/ mice. The nature of the
anophthalmic mutation in mice is not definitively known, but related
anophthalmic mutants in hamsters show pleiotropic phenotypes with both
ocular and hypothalamic dysgenesis (Asher, 1981). It is likely that the
phenotype of the anophthalmic mouse is an amalgam of an ocular
developmental phenotype and associated (and variable) ventral
hypothalamic dysmorphogenesis.
The cause of the lengthened free-running period in
math5/ mice is unclear. It is
doubtful that math5 plays a direct role in the inner clock
mechanism, because it is not expressed in the brains of adult animals
(Brown et al., 1998; Wang et al., 2001). Furthermore, math5
heterozygotes show no abnormalities in the free-running period,
entrainment to the skeletal photoperiod, or phase-shifting response to
light as has been observed for heterozygous animals of most central
oscillator genes, including both cryptochromes (Thresher et
al., 1998; van der Horst et al., 1999; Vitaterna et al., 1999),
period 1 and 2 (Zheng et al., 1999; Albrecht et al., 2001; Bae et al., 2001; Zheng et al., 2001), Clock
(Vitaterna et al., 1994), and Bmal/MOP3 (Bunger,
2000). The apparent absence of a retinohypothalamic tract may result in
a developmental abnormality in the SCN; a decrease in the number of
GFAP-positive astrocytes has been noted in rats enucleated at birth,
for example (Munekawa et al., 2000). Early enucleation similarly causes
changes in the anatomy and location of the SCN (Holtzman et al.,
1989; Nagai et al., 1992). Alternatively, the RHT could provide a tonic
input that in part determines the period in the mouse. Input from the RHT can clearly affect the free-running period, as seen in the period
lengthening observed in mice in constant lighting conditions (i.e.,
Aschoff's rule; for review, see Daan, 2000). However, no consistent
lengthening of the free-running period has been reported in mice
enucleated as adults, so it is unlikely that the RHT provides a "dark
current" of tonic input to the SCN that helps determine the
free-running period.
Six of seven tested math5/ mice
did not entrain to a nonphotic stimulus. A similar failure to entrain
to wheel lock conditions has been noted in a subset of anophthalmic
mice (Laemle and Ottenweller, 1999). The mechanism of nonphotic phase
shifting is not as well understood as that of photic phase shifts.
Although serotonergic pathways have been implicated (Penev et al.,
1995), depletion of serotonin in hamsters does not affect phase shifts
in a novelty wheel-running paradigm (Bobrzynska et al., 1996). However,
ablations of the intergeniculate leaflet do attenuate the
phase-shifting effects of nonphotic stimuli (Janik and Mrosovsky,
1994). It is possible that the failure of retinogeniculate connections
to form in the math5/ mutant
similarly disrupts the IGL pathway and thereby affects nonphotic as
well as photic phase shifting. Alternatively, the failure of these mice
to entrain to a 24 hr period of wheel lock may reflect the altered
relationship between a ~24.5 hr free-running circadian clock and the
nonphotic phase-response curve. It is possible that these mice might
entrain more easily to a non-24 hr nonphotic stimulus (i.e., wheel
locking on a 25 hr time cycle).
The wheel-anticipatory behavior of
math5/ animals reveals the
presence of a second circadian timekeeping system in these animals. This oscillator is desynchronized from the general behavioral oscillator. A similar second oscillator has been seen with experiments on food availability (the so-called food-entrainable oscillator; Stephan et al., 1979a,b; Rosenwasser et al., 1984; Abe et al., 1989).
This oscillator is not dependent on the suprachiasmatic nucleus
(Stephan et al., 1979b; Stephan, 1983) but is attenuated by hindbrain
lesions in the parabrachial region (Davidson et al., 2000). It remains
to be seen whether the wheel-anticipatory oscillator seen in the
math5/ mice is the same as the
described food-entrainable oscillator. The
math5/mouse may be a very useful
model for studying secondary oscillators, because these animals
spontaneously display both anticipatory and free-running rhythm in
internal desynchronization.
The findings in the present study may have clinical ramifications for
the relationship between human eye disease and circadian synchronization. It is known that a subset of blind humans can still
entrain their rhythms to light, whereas others cannot (Sack et al.,
1992); similarly, some blind human subjects show suppression of their
melatonin levels by light, whereas others do not (Czeisler et al.,
1995). By analogy to the circadian entrainment phenotypes shown by the
rodless and coneless rd/rd; cl mice (which show
normal circadian entrainment; Freedman et al., 1999; Lucas et al.,
1999) and the math5/ mice
described in the present study, one might expect that human patients
with outer retinal disease (such as retinitis pigmentosa) would show
normal entrainment to external light/dark cycles, but individuals with
inner retinal disease (i.e., end-stage glaucoma or optic nerve
hypoplasia) would have difficulty. This might be manifested by an
increased incidence of sleep disorders. In one study of the
self-reported sleep problems in blind individuals (Tabandeh et al.,
1998), patients with glaucoma and inner retinal disease showed a higher
incidence of self-reported poor sleep compared with patients with outer
retinal dysfunction. If the inability to entrain to nonphotic stimuli
is also a feature of severe or developmental inner retinal disease,
this would compound poor photic phase entrainment and might explain why
social cues fail to entrain subsets of blind patients (Klerman et al.,
1998).
Together, math5/ mice and outer
retinal degenerate mice form a complementary model demonstrating that
the phototransduction pathway required for vision is mechanistically
distinct from the phototransduction pathway required for resetting the
circadian clock. Both types of mice are behaviorally blind, yet mice
that lack classical photoreceptors maintain photic entrainment, whereas mice that lack retinal ganglion cells lose this ability. Results from
this study support the model of separate pathways for vision and
circadian entrainment, implicating the retinal ganglion cell as a
necessary component for signaling of photic information to the
circadian clock.
| |
FOOTNOTES |
Received May 6, 2002; revised Aug. 22, 2002; accepted Aug 29, 2002.
R.W. was supported by a Doris Duke Fellowship. I.P. was supported by
National Institutes of Health (NIH) Grant R01MH062405. L.G. was
supported by NIH Grant EY013426. R.N.V.G. was supported by a career
development award from Research to Prevent Blindness (RPB), the
Becker/Association of University Professors of Ophthalmology/RPB clinician-scientist award, and NIH Grant K08EY00403.
Correspondence should be addressed to Dr. Russell N. Van Gelder,
Department of Ophthalmology and Visual Sciences, Campus Box 8096, Washington University School of Medicine, 660 South Euclid Avenue, St.
Louis, MO 63110. E-mail: vangelder{at}vision.wustl.edu.
| |
REFERENCES |
-
Abe H,
Kida M,
Tsuji K,
Mano T
(1989)
Feeding cycles entrain circadian rhythms of locomotor activity in CS mice but not in C57BL/6J mice.
Physiol Behav
45:397-401[Medline].
-
Albrecht U,
Zheng B,
Larkin D,
Sun ZS,
Lee CC
(2001)
MPer1 and mper2 are essential for normal resetting of the circadian clock.
J Biol Rhythms
16:100-104[Abstract].
-
Antoch MP,
Song EJ,
Chang AM,
Vitaterna MH,
Zhao Y,
Wilsbacher LD,
Sangoram AM,
King DP,
Pinto LH,
Takahashi JS
(1997)
Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.
Cell
89:655-667[ISI][Medline].
-
Argamaso SM,
Froehlich AC,
McCall MA,
Nevo E,
Provencio I,
Foster RG
(1995)
Photopigments and circadian systems of vertebrates.
Biophys Chem
56:3-11[ISI][Medline].
-
Asher Jr JH
(1981)
Concerning the primary defect leading to the pleiotropic effects caused by anophthalmic white (Wh) in the Syrian hamster Mesocricetus auratus.
J Exp Zool
217:159-169[Medline].
-
Bae K,
Jin X,
Maywood ES,
Hastings MH,
Reppert SM,
Weaver DR
(2001)
Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock.
Neuron
30:525-536[ISI][Medline].
-
Berson DM,
Dunn FA,
Takao M
(2002)
Phototransduction by retinal ganglion cells that set the circadian clock.
Science
295:1070-1073[Abstract/Free Full Text].
-
Bobrzynska KJ,
Vrang N,
Mrosovsky N
(1996)
Persistence of nonphotic phase shifts in hamsters after serotonin depletion in the suprachiasmatic nucleus.
Brain Res
741:205-214[ISI][Medline].
-
Brown NL,
Kanekar S,
Vetter ML,
Tucker PK,
Gemza DL,
Glaser T
(1998)
Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis.
Development [Suppl]
125:4821-4833.
-
Brown NL,
Patel S,
Brzezinski J,
Glaser T
(2001)
Math5 is required for retinal ganglion cell and optic nerve formation.
Development [Suppl]
128:2497-2508.
-
Bunger MK
(2000)
Mop3 is an essential component of the master circadian pacemaker in mammals.
Cell
103:1009-1017[ISI][Medline].
-
Czeisler CA,
Shanahan TL,
Klerman EB,
Martens H,
Brotman DJ,
Emens JS,
Klein T,
Rizzo JF
(1995)
Suppression of melatonin secretion in some blind patients by exposure to bright light.
N Engl J Med
332:6-11[Abstract/Free Full Text].
-
Daan S
(2000)
The Colin S. Pittendrigh Lecture: Colin Pittendrigh, Jurgen Aschoff, and the natural entrainment of circadian systems.
J Biol Rhythms
15:195-207[ISI][Medline].
-
Davidson AJ,
Cappendijk SL,
Stephan FK
(2000)
Feeding-entrained circadian rhythms are attenuated by lesions of the parabrachial region in rats.
Am J Physiol
278:R1296-R1304[Abstract/Free Full Text].
-
Edgar DM,
Kilduff TS,
Martin CE,
Dement WC
(1991)
Influence of running wheel activity on free-running sleep/wake and drinking circadian rhythms in mice.
Physiol Behav
50:373-378[Medline].
-
Faradji H,
Cespuglio R,
Rondot G,
Paut L,
Jouvet M
(1980)
Absence of light-dark entrainment on the sleep-waking cycle in mice with intact visual perception.
Brain Res
202:41-49[ISI][Medline].
-
Foster RG,
Provencio I,
Hudson D,
Fiske S,
De Grip W,
Menaker M
(1991)
Circadian photoreception in the retinally degenerate mouse (rd/rd).
J Comp Physiol [A]
169:39-50[Medline].
-
Freedman MS,
Lucas RJ,
Soni B,
von Schantz M,
Munoz M,
David-Gray Z,
Foster R
(1999)
Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors.
Science
284:502-504[Abstract/Free Full Text].
-
Gooley JJ,
Lu J,
Chou TC,
Scammell TE,
Saper CB
(2001)
Melanopsin in cells of origin of the retinohypothalamic tract.
Nat Neurosci
4:1165[ISI][Medline].
-
Halberg F,
Visscher MB,
Bittner JJ
(1954)
Relation of visual factors to eosinophil rhythm in mice.
Am J Physiol
179:229-235[Free Full Text].
-
Hannibal J,
Hindersson P,
Knudsen SM,
Georg B,
Fahrenkrug J
(2002)
The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract.
J Neurosci
22:RC191[Abstract/Free Full Text](1-7).
-
Hattar S,
Liao HW,
Takao M,
Berson DM,
Yau KW
(2002)
Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity.
Science
295:1065-1070[Abstract/Free Full Text].
-
Hendrickson AE,
Wagoner N,
Cowan WM
(1972)
An autoradiographic and electron microscopic study of retino-hypothalamic connections.
Z Zellforsch Mikrosk Anat
135:1-26[ISI][Medline].
-
Herzog ED
(2001)
The mammalian circadian clock shop.
Semin Cell Dev Biol
12:295-303[ISI][Medline].
-
Holtzman RL,
Malach R,
Gozes I
(1989)
Disruption of the optic pathway during development affects vasoactive intestinal peptide mRNA expression.
New Biol
1:215-221[Medline].
-
Hsu SM,
Raine L,
Fanger H
(1981)
The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics.
Am J Clin Pathol
75:816-821[ISI][Medline].
-
Janik D,
Mrosovsky N
(1994)
Intergeniculate leaflet lesions and behaviorally-induced shifts of circadian rhythms.
Brain Res
651:174-182[ISI][Medline].
-
Johnson RF,
Moore RY,
Morin LP
(1988)
Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract.
Brain Res
460:297-313[ISI][Medline].
-
Klein DC,
Moore RY
(1979)
Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinohypothalamic tract and the suprachiasmatic nucleus.
Brain Res
174:245-262[ISI][Medline].
-
Klerman EB,
Rimmer DW,
Dijk DJ,
Kronauer RE,
Rizzo III JF,
Czeisler CA
(1998)
Nonphotic entrainment of the human circadian pacemaker.
Am J Physiol
274:R991-R996.
-
Laemle LK,
Ottenweller JE
(1998)
Daily patterns of running wheel activity in male anophthalmic mice.
Physiol Behav
64:165-171[Medline].
-
Laemle LK,
Ottenweller JE
(1999)
Nonphotic entrainment of activity and temperature rhythms in anophthalmic mice.
Physiol Behav
66:461-471[Medline].
-
Liu W,
Mo Z,
Xiang M
(2001)
The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development.
Proc Natl Acad Sci USA
98:1649-1654[Abstract/Free Full Text].
-
Lowrey PL,
Takahashi JS
(2000)
Genetics of the mammalian circadian system: photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation.
Annu Rev Genet
34:533-562[ISI][Medline].
-
Lucas RJ,
Foster RG
(1999)
Neither functional rod photoreceptors nor rod or cone outer segments are required for the photic inhibition of pineal melatonin.
Endocrinology
140:1520-1524[Abstract/Free Full Text].
-
Lucas RJ,
Freedman MS,
Munoz M,
Garcia-Fernandez JM,
Foster RG
(1999)
Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors.
Science
284:505-507[Abstract/Free Full Text].
-
Mikkelsen JD
(1992)
Visualization of efferent retinal projections by immunohistochemical identification of cholera toxin subunit B.
Brain Res Bull
28:619-623[ISI][Medline].
-
Mikkelsen JD,
Serviere J
(1992)
Demonstration of a direct projection from the retina to the hypothalamic supraoptic nucleus of the hamster.
Neurosci Lett
139:149-152[Medline].
-
Moore RY
(1973)
Retinohypothalamic projection in mammals: a comparative study.
Brain Res
49:403-409[ISI][Medline].
-
Moore RY
(1995)
Organization of the mammalian circadian system.
Ciba Found Symp
183:88-106[Medline].
-
Moore RY,
Speh JC,
Card JP
(1995)
The retinohypothalamic tract originates from a distinct subset of retinal ganglion cells.
J Comp Neurol
352:351-366[ISI][Medline].
-
Munekawa K,
Tamada Y,
Iijima N,
Hayashi S,
Ishihara A,
Inoue K,
Tanaka M,
Ibata Y
(2000)
Development of astroglial elements in the suprachiasmatic nucleus of the rat: with special reference to the involvement of the optic nerve.
Exp Neurol
166:44-51[Medline].
-
Nagai N,
Nagai K,
Nakagawa H
(1992)
Effect of orbital enucleation on glucose homeostasis and morphology of the suprachiasmatic nucleus.
Brain Res
589:243-252[Medline].
-
Penev PD,
Turek FW,
Zee PC
(1995)
A serotonin neurotoxin attenuates the phase-shifting effects of triazolam on the circadian clock in hamsters.
Brain Res
669:207-216[ISI][Medline].
-
Provencio I,
Rollag MD,
Castrucci AM
(2002)
Photoreceptive net in the mammalian retina: this mesh of cells may explain how some blind mice can still tell day from night.
Nature
415:493[Medline].
-
Ralph MR,
Foster RG,
Davis FC,
Menaker M
(1990)
Transplanted suprachiasmatic nucleus determines circadian period.
Science
247:975-978[Abstract/Free Full Text].
-
Reppert SM,
Weaver DR
(2001)
Molecular analysis of mammalian circadian rhythms.
Annu Rev Physiol
63:647-676[ISI][Medline].
-
Rosenwasser AM,
Pelchat RJ,
Adler NT
(1984)
Memory for feeding time: possible dependence on coupled circadian oscillators.
Physiol Behav
32:25-30[Medline].
-
Sack RL,
Lewy AJ,
Blood ML,
Keith LD,
Nakagawa H
(1992)
Circadian rhythm abnormalities in totally blind people: incidence and clinical significance.
J Clin Endocrinol Metab
75:127-134[Abstract].
-
Silver J
(1977)
Abnormal development of the suprachiasmatic nuclei of the hypothalamus in a strain of genetically anophthalmic mice.
J Comp Neurol
176:589-606[ISI][Medline].
-
Stephan FK
(1983)
Circadian rhythm dissociation induced by periodic feeding in rats with suprachiasmatic lesions.
Behav Brain Res
7:81-98[ISI][Medline].
-
Stephan FK,
Swann JM,
Sisk CL
(1979a)
Anticipation of 24-hr feeding schedules in rats with lesions of the suprachiasmatic nucleus.
Behav Neural Biol
25:346-363[ISI][Medline].
-
Stephan FK,
Swann JM,
Sisk CL
(1979b)
Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic lesions.
Behav Neural Biol
25:545-554[ISI][Medline].
-
Tabandeh H,
Lockley S,
Buttery R,
Skene D,
Defrance R,
Arendt J,
Bird A
(1998)
Disturbance of sleep in blindness.
Am J Ophthalmol
126:707-712[Medline].
-
Thresher RJ,
Vitaterna MH,
Miyamoto Y,
Kazantsev A,
Hsu DS,
Petit C,
Selby CP,
Dawut L,
Smithies O,
Takahashi JS,
Sancar A
(1998)
Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses.
Science
282:1490-1494[Abstract/Free Full Text].
-
Tucker P,
Laemle L,
Munson A,
Kanekar S,
Oliver ER,
Brown N,
Schlecht H,
Vetter M,
Glaser T
(2001)
The eyeless mouse mutation (ey1) removes an alternative start codon from the Rx/rax homeobox gene.
Genesis
31:43-53[ISI][Medline].
-
van der Horst GT,
Muijtjens M,
Kobayashi K,
Takano R,
Kanno S,
Takao M,
de Wit J,
Verkerk A,
Eker AP,
van Leenen D,
Buijs R,
Bootsma D,
Hoeijmakers JH,
Yasui A
(1999)
Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms.
Nature
398:627-630[Medline].
-
Vitaterna MH,
King DP,
Chang AM,
Kornhauser JM,
Lowrey PL,
McDonald JD,
Dove WF,
Pinto LH,
Turek FW,
Takahashi JS
(1994)
Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.
Science
264:719-725[Abstract/Free Full Text].
-
Vitaterna MH,
Selby CP,
Todo T,
Niwa H,
Thompson C,
Fruechte EM,
Hitomi K,
Thresher RJ,
Ishikawa T,
Miyazaki J,
Takahashi JS,
Sancar A
(1999)
Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.
Proc Natl Acad Sci USA
96:12114-12119[Abstract/Free Full Text].
-
Wang SW,
Kim BS,
Ding K,
Wang H,
Sun D,
Johnson RL,
Klein WH,
Gan L
(2001)
Requirement for math5 in the development of retinal ganglion cells.
Genes Dev
15:24-29[Abstract/Free Full Text].
-
Weaver DR
(1998)
The suprachiasmatic nucleus: a 25-year retrospective.
J Biol Rhythms
13:100-112[Abstract].
-
Zheng B,
Larkin DW,
Albrecht U,
Sun ZS,
Sage M,
Eichele G,
Lee CC,
Bradley A
(1999)
The mPer2 gene encodes a functional component of the mammalian circadian clock.
Nature
400:169-173[Medline].
-
Zheng B,
Albrecht U,
Kaasik K,
Sage M,
Lu W,
Vaishnav S,
Li Q,
Sun ZS,
Eichele G,
Bradley A,
Lee CC
(2001)
Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock.
Cell
105:683-694[ISI][Medline].
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INTRODUCTION
