Targeted mutation of the calbindin D28K gene disrupts circadian rhythmicity and entrainment - PubMed (original) (raw)
Targeted mutation of the calbindin D28K gene disrupts circadian rhythmicity and entrainment
Lance J Kriegsfeld et al. Eur J Neurosci. 2008 Jun.
Erratum in
- Eur J Neurosci. 2008 Sep;28(5):1030
Abstract
The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function.
Figures
Figure 1. CalB-/- mice exhibit pronounced abnormalities in circadian rhythms and entrainment
Activity records from animals initially held in a 12:12 light:dark (LD) cycle then transferred to constant darkness (DD). Wild-type animals demonstrate entrainment in LD as indicated by the ability to predict activity onset when placed into DD (A). Approximately 60% of CalB-/- mice exhibit low-amplitude rhythms when transferred to DD. In these animals, although the majority of activity was confined to the dark period in LD, activity onset in DD was variable relative to the phase of LD behavior (B). Approximately 40% of CalB-/- become arrhythmic soon after exposure to DD (C). Red arrow points to onset of activity after transfer to DD. Red boxes in A and B outline areas enlarged in D that show activity onset in DD relative to the previous LD cycle. Panel E provides further examples of the random nature of activity onset following transition from LD to DD in CalB-/- mice. The onset of activity after transfer to DD, relative to the previous LD cycle, is shown quantified in Panel F. Shaded regions indicate periods of darkness mirrored in LD bars above the actograms.
Figure 2. CalB-/- mice exhibit abnormal entrainment to a skeleton photoperiod
Representative activity records from one WT (A) and three CalB-/- animals (B, C, D) held in a skeleton photoperiod. WT mice synchronize their behavior to the two one hour light pulses as if they were maintained in a complete LD cycle. Rhythmic CalB-/- mice exhibited more activity throughout both dark periods compared to WT mice. Animals in B and D show signs of entrainment as manifested by the production of a 24h rhythm and entrainment with an abnormal phase angle. Arrhythmic CalB-/- mice exhibit equal amounts of activity throughout both dark periods with making during brief periods of light. Shaded regions of the activity records indicate periods of darkness.
Figure 3. Phase shifting is unaffected in CalB mutants
CalB-/- mice phase shift normally in response to light pulses during the subjective day (CT4) and night (CT16, CT22). Both WT and CalB-/- mice do not phase shift at CT4 and exhibit characteristic phase delays at CT16 and phase advances at CT22.
Figure 4. Developmental changes in CalB expression in mouse SCN
Calbindin protein is expressed in SCN core early during postnatal development, with scattered expression in the adult mouse SCN. Photomicrographs depict CalB protein expression in the caudal SCN of wild-type mice at either P10 (top), P16 (middle), or in adulthood (bottom). Dashed lines outline the SCN. Magnification bar = 100 μm
Figure 5. Colocalization of Calbindin and Melanopsin
(a) Melanopsin (red); (b) Calbindin (green); (c) merged image from retinal whole mount focused on ganglion cell layer. Single arrow: melanopsin ganglion cell in which calbindin is confined to the nucleus. Double arrow: melanopsin ganglion cell in which calbindin is found in both nucleus and perikaryal cytoplasm. Asterisk: melanopsin ganglion cell lacking calbindin. d. vertical section of wt retina showing layers: gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer. Multiple retinal cell types show calbindin-IR. A melanopsin ganglion cell with a calbindin signal is seen in gcl. Arrow indicates calbindin-IR ganglion cell lacking melanopsin-IR. e. vertical section of calbindin ko retina. No calbindin-IR is seen but non-specific label is noted in blood vessels (bv). f. Whole mount preparation of CalB-/- retina. Note both bright and dim melanopsin-IR ganglion cells. 10 μm marker bar in (e) applies to layers a-f. g-i. Anti-melanopsin immunoreactivity is present in control retina (g), but is lacking in preadsorption control (h) and when the primary anti-melanopsin antibody was omitted (i). 20 μm marker bar in (h) applies to g-i.
Figure 6. Rhythmic Per2 expression
Pattern of PER2 protein expression across the day in animals held in a 12:12 light:dark (LD) cycle (Zeitgeber time; ZT) or in constant darkness (Circadian time; CT). Photomicrographs depict PER2 expression in the SCN that is quantified in the graphs. Both in LD and DD, pronounced disruptions in the amplitude of clock gene expression are noted. *=significantly greater than CalB-/- animals at the same time-point. Magnification bar = 100μm
Figure 7. Vasopressin expression is affected by deletion of CalB
Photomicrographs depicting the rhythm of vasopressin peptide expression in WT and CalB-/- animals held in DD. Images are shown for the middle region of the SCN. Quantified cell counts indicate a blunted vasopressin expression in CalB-/- mice relative to WT controls (bottom). *=significantly less than WT animals at the same time-point. Magnification bar = 100μm
Figure 8. VIP expression in the SCN of WT and CalB-/- mice
Representive photomicrographs of VIP-ir staining in the SCN of WT (top) and CalB-/- (bottom) mice. Unilateral counts of VIP-ir-labeled cells did not differ between genotypes (bottom).
Figure 9. Light-induced mPer1 expression in not affected in CalB-/- mice
Medium-power photomicrographs depict the pattern of mPer1 expression in animal exposed to a light pulse (light +) at CT4, CT16, or CT22, or control animals sacrificed at the same time that were not exposed to a light pulse (light -) (top). Graphs depict quantified mPer1 expression in the SCN core (left) and shell (right) of WT and CalB-/- mice. Animals did not differ on any of the parameters investigated. Magnification bar = 100μm
Figure 10. Light-induced FOS expression is minimally reduced in CalB-/- mice
Medium-power photomicrographs depict the pattern of FOS expression in animals exposed to a light pulse (light+) at CT4, 16, or 22, or control animals sacrificed at the same time that were not exposed to a light pulse (light-) (top). Graphs depict quantified FOS expression in the SCN core (left) and shell (right) of WT and CalB-/- mice. Magnification bar = 100μm *=significantly greater than light- controls from the same genotype; +=significantly less than light+ WT animals.
References
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