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Department of Biology, Imperial College of Science Technology and Medicine, London, United Kingdom SW7 2AZ
Address all correspondence and requests for reprints to: Dr. Robert Lucas, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, Imperial College of Science Technology and Medicine, London, United Kingdom SW7 2AZ. E-mail: r.j.lucas{at}ic.ac.uk
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Abstract |
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max 509 nm) in C3H mice
bearing either of two mutations affecting the retina: retinal
degeneration (rd), a disruption of rod
phototransduction, and retinal degeneration slow (rds),
an ablation of photoreceptor outer segments. Diurnal profiles of pineal
melatonin content were similar in both mutant genotypes and in
wild-type mice; melatonin peaked between 3–5 h before lights on. All
three genotypes exhibited irradiance dependent inhibition of pineal
melatonin content; 2.6 x 10-2
microwatts/cm2 509 nm light induced complete suppression in
all three genotypes, whereas lower irradiances were ineffective in all
cases. Bilateral enucleation abolished responses even to 6
microwatts/cm2 509 nm light. These results demonstrate that
the process of irradiance detection for pineal melatonin inhibition is
buffered against considerable loss of photoreceptive capacity and that
neither rod photoreceptors nor rod or cone outer segments are required
for mediating this response in mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous descriptions of the spectral sensitivity of melatonin suppression in rats (4, 5) and Syrian hamsters (6) have suggested the involvement of rod photoreceptors in mediating these responses. However, definitive evidence associating any specific retinal photoreceptor with the task of regulating the mammalian pineal is absent. Here, we set out to examine the involvement of rod and cone photoreceptors in this process. To this end, we assessed the effect of two retinal degeneration mouse models, the retinal degeneration (rd) and retinal degeneration slow (rds) mutations, on the ability of light to acutely suppress pineal melatonin. rd is a mutation of the ß-subunit of the rod-specific phosphodiesterase (7, 8). The absence of a functional phosphodiesterase in homozygous rd/rd mice leads to a constitutive elevation of intracellular cGMP in rod photoreceptors, thus destroying their ability to respond to photic stimulation with appropriate changes in membrane potential. This primary ablation of rod phototransduction is accompanied by an attrition initially of rod and subsequently of cone photoreceptors. By 85–90 days (the age at which the current experiments were carried out), rod cell bodies are absent, and cone cell bodies are reduced by at least 50% (9). rds is an insertion mutation of the peripherin gene that encodes a key structural component of photoreceptor outer segments (10, 11, 12). Homozygous rds/rds mice never develop photoreceptor outer segments and show a gradual degeneration of both rod and cone cell bodies. By 85–90 days of age, the outer nuclear layer of the rds/rds retina is reduced by more than 50% (13).
In a previous examination of melatonin suppression in rd/rd mice, Goto and Ebihara (14) reported that although rd/rd mice were capable of exhibiting photic melatonin suppression, their sensitivity to light was significantly reduced when compared to wild-type animals. The ability of rd/rd mice to show melatonin suppression indicates that photoreceptors other than rods are capable of mediating this response. However, the reduction in sensitivity was interpreted as strong evidence that, when present, rod photoreceptors contribute to this pathway. On this basis, Goto and Ebihara (14) concluded that regulation of the mammalian pineal is mediated by both rod and non-rod photoreceptors. However, this interpretation is complicated by strain differences between the rd/rd (C3H/He strain) and wild-type (CBA/Ms) mice. Thus, it is possible that the differences reported were related to strain background rather than retinal phenotype. The present study set out to resolve this issue and to independently examine the requirement for rod and cone outer segments by comparing photic sensitivity in C3H mice homozygous for either the rd or rds mutation with that in mice wild-type at both loci. Our results indicate that despite the large reductions in photoreceptive capacity induced by rd and rds mutations, there is no concomitant reduction in the sensitivity of pineal melatonin production to monochromatic (509-nm) light exposure in either genotype. These findings demonstrate that neither rod photoreceptors nor rod/cone outer segments are required for the acute inhibition of pineal melatonin in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experimental animals were stably entrained to a 12-h light, 12-h dark cycle for at least 2 weeks before sampling. At around 85–90 days of age (mean ± SEM; rd/rd, 82 ± 0.5; rds/rds, 89 ± 2.1; wild-type, 91 ± 1.6) animals were sampled according to one of the following protocols.
Diurnal profile
To describe diurnal profiles of pineal melatonin content for
each of the genotypes, pineals were collected from between six and
eight animals of each genotype at 2-h intervals through the dark phase
of the light-dark cycle and at one time point during the light phase,
zeitgeber time (ZT) 8, 13, 15, 17, 19, 21, or 23 (where ZT12 is the
time of lights off). Animals were killed by cervical dislocation and
bilaterally enucleated under infrared illumination. Subsequently, the
pineal was quickly removed under white fluorescent light and
snap-frozen on dry ice. Pineals were stored at -80 C until assayed for
melatonin content.
Exposure to monochromatic 509-nm light
Mice of all three genotypes were individually exposed to 15 min
of defined irradiance monochromatic 509 nm light timed to start between
ZT20 and ZT21 of the light-dark cycle. A remote source light-pulsing
apparatus was employed, as previously described (15). Importantly, this
apparatus employs a fiber optic cable to ensure that the mice are not
exposed to any heat output of the light source during pulsing. The
spectral transmission of this light source was controlled using a
monochromatic filter (Oriel Corp., Stratford, CT; max, 509
nm; half-band width, 10 nm). Irradiance was controlled by the use of a
series of neutral density impedance filters (Oriel Corp.) and was
measured using an optical power meter (Graseby Optronics, Orlando,
FL). Between six and eight animals from each genotype were
exposed to monochromatic green light at irradiances of 1.2 x
10-4, 1.2 x 10-3, and 2.6 x
10-2 microwatts (µW)/cm2. In addition, six
to eight animals from each genotype were placed in the pulsing
apparatus for 15 min without exposure to light to act as experimental
controls. At the end of the 15-min pulses, mice were removed from the
pulsing apparatus under infrared illumination, their eyes were removed,
and pineals were collected as described above.
Enucleated mice
Young adult mice from each of the three genotypes (five
wild-type, three rd/rd, and two rds/rds) were
bilaterally enucleated under halothane anesthesia. After recovering
from the surgery, these animals were exposed to 12-h light, 12-h dark
cycles. To assess circadian phase in these animals they were singly
housed with free access to a running wheel from which circadian rhythms
of wheel-running activity were monitored using a DataQuest system
(Minimitter Co. Inc., Sunriver, OR). Enucleated mice did not entrain to
the light-dark cycle, but after some transient arrhythmicity exhibited
free running rhythms. When at least 14 days of stable circadian
activity rhythm had been observed, mice were exposed to a 15-min 509-nm
light pulse of 6 µW/cm2 using the apparatus described
above. These pulses were timed with respect to the free running
activity rhythm to start around circadian time (CT) 20, where CT12 is
the time of activity onset. At the end of the pulse they were killed by
cervical dislocation under infrared light, and pineals were
collected.
Melatonin assay
A direct RIA was employed for the detection of melatonin
concentrations in pineal homogenates (after Ref. 16). Briefly,
single pineal glands were rapidly homogenized in assay buffer using
ultrasound. These samples were then incubated in duplicate with a
specific antiserum for melatonin raised in sheep (Stockgrand Ltd.,
Guildford, UK), and [3H]melatonin (Amersham,
Aylesbury, UK) was added. Free and antibody-bound fractions of
melatonin were separated using dextran (Sigma Chemical Co., Poole, UK)-coated activated charcoal (Sigma Chemical Co.), and the amount of bound
[3H]melatonin was estimated using a scintillation counter
(Fluoransafe, Fisher Life Science, Loughborough, UK;
RackBeta, Wallac AC, Turku, Finland). The concentration of
melatonin in the sample was estimated by comparison with standards of
known melatonin (Sigma Chemical Co.) concentration.
Quality control samples were included at 73 and 159 pg/ml; the
intraassay coefficients of variation were 4.6% and 4.2%, and the
interassay coefficients of variation were 10.5% and 7.6% for the
lower and higher quality controls, respectively. The minimum
detectable dose was 10 pg/ml. The assay was validated for use with
mouse pineal homogenates by demonstrating parallelism over the range
10–200 pg/ml.
Statistical analysis
The effect of increasing irradiances of 509 nm light on pineal
melatonin content was tested in each genotype using a one-way ANOVA;
post-hoc t tests were made against the unpulsed
control group employing Bonferroni’s correction. The t test
comparisons were made between pineal melatonin content in enucleated
mice exposed to 6 µW/cm2 of light and intact animals with
either peak (not light pulsed) or completely suppressed (exposed to
2.6 x 10-2 µW/cm2 light) melatonin.
Because of the differences in estimating circadian phase from the
external light cycle compared with the running wheel activity rhythm,
pineal samples from intact animals at ZT19, -20, and -21 were compared
with the enucleated animals exposed to 6 µW/cm2 at CT20.
To check for differences between genotypes in peak melatonin production
or the response to enucleation, one-way ANOVA tests were employed on
the pineal melatonin contents of the nonlight-pulsed and enucleated
mice. Statistical significance was defined as P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In each case melatonin
content was basal during the light phase and reached a peak during the
second half of the dark phase before falling again preceding lights on.
Pineal melatonin content was stable between ZT19 and ZT21 in all three
genotypes.
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).
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; by one-way ANOVA,
P < 0.01). There was no indication that either
rd/rd or rds/rds mice showed a decrease in
sensitivity compared with wild-type animals, with all three genotypes
showing suppression (P < 0.01, by post-hoc
t tests with Bonferroni’s correction against unpulsed
animals) in response to 2.6 x 10-2
µW/cm2.
Enucleation effectively abolished pineal melatonin suppression in
response to light exposure. Animals of all three genotypes showed a
similar response (by one-way ANOVA, P > 0.05) to 6
µW/cm2 light after enucleation (Fig. 3). Pineal melatonin content was
significantly (by t test, P < 0.0001)
elevated compared with that in intact animals exposed to 2.6 x
10-2 µW/cm2 light. There was a modest
reduction compared with intact animals not exposed to a light pulse,
but this was not statistically significant (by t test,
P > 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of bilateral enucleation reported here confirm previous reports in mammals that the photoreceptors regulating the mammalian pineal are located in the eyes (1, 17). There has been a recent report suggesting that humans are capable of showing circadian phase shifts through an as yet undefined extraocular photoreceptor (18; for review, see Ref. 19). However, that study did not extend to an examination of pineal melatonin suppression, and here we report that after bilateral enucleation, melatonin was not significantly suppressed even by an irradiance more than 2 orders of magnitude higher than that capable of complete suppression in intact animals. Although not statistically significant, pineal melatonin content did appear moderately lower in enucleated animals exposed to bright light compared with that in intact, unpulsed mice. However, we do not consider that this supports the hypothesis of extraocular photoreception in mammals. It is most likely that this effect is accounted for by three difficulties associated with ensuring that pineals were collected at the same phase of the melatonin rhythm in free running (enucleated) and entrained (intact) animals. Firstly, different phase reference points (ZT12, lights off, and CT12, activity onset) were employed for intact and enucleated animals which may not be wholly comparable in terms of the circadian phase they represent or the accuracy with which they do this. Secondly, free running (enucleated) mice typically exhibited a period significantly different from 24 h. Finally, the phasing of melatonin production in mammals varies according to night length (for review, see Ref. 20) and may well have been altered during the several weeks following blinding.
Although it seems clear that the photoreceptors regulating the
mammalian pineal are exclusively ocular, their precise nature remains
unknown. The murine retina contains three known photoreceptor cell
types: rods with a maximal absorbance (max) around 498 nm (21) and
two populations of cone photoreceptors absorbing green (max =
508 nm) (22) and UV (max = 359 nm) (23) light. The
rd mutation blocks functional phototransduction in rods (7, 8), and by 80 days of age, rd/rd mice lack rod
photoreceptors (9). Consequently, the demonstration of melatonin
suppression in rd/rd mice indicates that functional rod
photoreceptors are not required for mediating this response. This
finding implicates the green cones in regulation of the pineal, as
these are the sole remaining photoreceptors in the rd/rd
retina sensitive to 509 nm light. This hypothesis is currently under
examination in our laboratory using mice lacking green cone
photoreceptors. In addition, the involvement of some as yet
unidentified nonrod-, non-cone-based photopigment cannot be
excluded.
The evidence suggests that rod photoreceptors are not required for the acute suppression of pineal melatonin. However, it remains possible that, where present in the wild-type mouse retina, they do contribute to this response. Although several investigators have reported significant suppression in rats or Syrian hamsters by light outside the normal sensitivity of rodopsin (24, 25, 26), the overall spectral sensitivity of pineal melatonin inhibition in these species is consistent with the involvement of a rod-like opsin photopigment (4, 5, 6). Similarly, action spectra for light-induced changes in electrical activity within the mammalian pineal suggest input from both rod and cone photoreceptors (27). These findings suggest that multiple photoreceptor types mediate pineal responses to light. Previously, Goto and Ebihara (14) have presented data from rd/rd mice that seem to support this hypothesis. They reported that although rd/rd mice are capable of showing pineal responses to white light, their sensitivity was greatly reduced compared with that of wild-type animals. However, that study compared C3H/He rd/rd mice with CBA/Ms wild types. Here we have demonstrated that when wild-type and rd/rd mice from the same (C3H) strain are compared, no decrease in sensitivity is evident. This finding indicates that a complete loss of rod phototransduction has no demonstrable effect on the sensitivity of pineal suppression. Consequently, our data suggest either that rod photoreceptors are not involved in regulating this response or that in their absence some other photoreceptor can completely compensate for their loss.
Pineal melatonin production is under photic control via two independent mechanisms. In this report we have examined the acute effects of light on melatonin production. However, light also effects melatonin production by entraining the circadian clock in the SCN that drives the activity of the pineal. To date, both anatomical and experimental evidence supports the hypothesis that the same photoreceptors mediate these parallel irradiance-dependent processes. Both processes are intimately associated with the SCN (2, 28), which receive a direct retinal projection via the retinohypothalamic tract (29, 30) and, through variations in stimulatory input, regulate the activity of the pineal gland according both to circadian phase and environmental illumination (3). In Syrian hamsters, the spectral sensitivity of both pineal melatonin suppression (6, 24) and circadian phase shifting (31) has been examined. In each case, maximal responses were observed to light of around 500 nm, suggesting that these two processes are mediated by photoreceptors with a similar absorbance spectrum. However, although the spectral sensitivity of phase shifting and melatonin suppression responses in this species are similar, the absolute sensitivities of these two tasks are significantly different, with melatonin suppression sensitive to irradiances 1.4 log units lower than those required for phase shifts (32). Whether these differences in sensitivity are caused by differential processing of output from the same photoreceptors or the use of different photoreceptors is unknown. Here, we have shown that both rd/rd and rds/rds mice show unattenuated photic inhibition of pineal melatonin. Previous reports confirm that these genotypes also exhibit unattenuated phase shifts in response to appropriate light pulses (15, 33). Together these data suggest that whatever the ocular elements mediating these two irradiance-dependent responses, both are spared by the massive photoreceptor degenerations caused by the rd and rds mutations.
Although it seems likely that different irradiance detection tasks employ the same ocular photoreceptors, the hypothesis that these are the same photoreceptors known to mediate vision (i.e. rods and cones) is currently unproven. The presence of a dedicated retinohypothalamic tract in mammals indicates that at some structural level, visual and irradiance detection functions are separated. Whether this separation extends to the use of different photoreceptor cells and pigments remains unknown. A previous study using a shuttle-box classical conditioning paradigm has suggested that aged rd/rd mice are visually blind (34). Consequently, the demonstration shown here and reported previously (15, 34) that irradiance detection is not significantly impaired in rd/rd mice suggests that visual blindness is not necessarily associated with impaired irradiance detection. This conclusion is supported by reports of visually blind humans showing responses consistent with the presence of functioning irradiance detection (35). It seems clear from these various reports that irradiance detection functions are buffered against a loss of photoreceptive capacity that is sufficient to induce visual blindness. Whether the basis of this buffering is the use of a dedicated irradiance detection photoreceptor or an up-regulation of input from the remaining conventional photoreceptors remains to be determined.
In summary, neither rd nor rds mutations induced a significant decrease in the sensitivity of pineal melatonin production to photic inhibition. By contrast, enucleation abolished this response. Thus, our results confirm that the photoreceptors mediating the acute suppression of pineal melatonin are located in the eye. In addition, they demonstrate that at least in mice, neither rod photoreception nor rod or cone outer segments are required for photic regulation of the pineal.
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Footnotes |
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Received August 20, 1998.
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