Research ReportCircadian changes in the expression of vasoactive intestinal peptide 2 receptor mRNA in the rat suprachiasmatic nuclei
Introduction
Daily rhythms in physiological and behavioural processes of animals and humans have pervasive influences on their adaptation to the environment. These rhythms are dependent upon an endogenous circadian clock located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The photic entrainment of this clock's activity involves pathways to the SCN that originate in the retina 11, 13. Several peptidergic neurotransmitters have been identified in the SCN; one of the most abundant of these peptides is the 28-amino acid peptide vasoactive intestinal peptide (VIP) 3, 4, 26. Neurons that synthesize VIP are found in the ventral portion of the SCN; these neurons receive photic information directly from the retina [9]and also indirectly from the intergeniculate leaflet of the lateral geniculate body [8]. In vitro studies on the circadian rhythm of electrical activity in rat SCN slices have indicated that VIP has phase-resetting properties [20]. Furthermore, in vivo investigations have shown that VIP, alone or in combination with other peptides, can reproduce the phase-advancing and -delaying effects of light pulses on wheel-running rhythms when injected into the region of the hamster SCN 1, 15. The mechanisms underlying the phase-dependent actions of VIP on the circadian pacemaker are unknown and the ways in which this peptide may contribute to the expression of circadian rhythms [17]remains unclear.
Autoradiographic studies have shown widespread distribution of VIP binding sites in the rat central nervous system 2, 7, 18. Three receptors for the pituitary adenylate cyclase activating polypeptide (PACAP)–VIP group of peptides have been identified [5]. The PACAP type I receptor appears to bind PACAP with a thousand times greater affinity than VIP 6, 14, 16. There are also two PACAP type II receptors (denoted as VIP1 and VIP2) that show approximately equal affinity for PACAP and VIP 10, 12, 22. In situ hybridization studies have shown that mRNAs for the PACAP-type I and VIP2 receptor but not for the VIP1 receptor are expressed in the rodent SCN 6, 10, 19, 25; they did not, however, address the possibility that the expression of these receptors varies across the 24 h cycle.
Previous studies have reported that although VIP and its mRNA display a diurnal rhythm in the SCN 21, 27, no such variation occurs in the absence of a light–dark cycle 21, 24. We cannot assume that absence of an autonomous rhythm in the content of VIP indicates absence of a rhythm in the activity of the local VIP neurons; nevertheless, it seems possible that changes in VIP receptor expression may introduce a functionally significant rhythm into the system. We have therefore examined the expression of the mRNA for the VIP2 receptor in the SCN across the light–dark cycle and also in the absence of photic cues.
Section snippets
Animals and experimental procedures
Adult male Wistar rats (250 g; Charles River, UK) were used. Animals were maintained in a 12:12 h light–dark (LD) schedule (lights on at 0700 h and designated as Zeitgeber time [ZT] 0; temperature at 21±2°C); `darkness' consisted of constant dim red light providing <2 lux at cage level. The emission spectrum of this light source was between 600 and 700 nM, a range within the red part of the spectrum and outside the spectral sensitivity curve for the circadian phase-shifting response [23]. Food
Results
The film autoradiographs for the VIP2 receptor mRNA exhibit a marked local density of the hybridization signal in the SCN (Fig. 1). They also show expression of the receptor mRNA in the cerebral cortex, hippocampus and thalamus (Fig. 1). The regional pattern of expression detected in the coronal sections is consistent with the distribution we have previously observed [19]. In RNase-treated sections, no signals were generated in these areas of the brain (results not shown).
Quantitative analysis
Discussion
This study demonstrates that the mRNA for the VIP2 receptor is abundantly expressed in the SCN under either the LD or DD condition. Furthermore, under each of these conditions its expression in these nuclei varies across the 24 h cycle. In the LD animals there is a biphasic variation in the expression which peaks at mid-day and during the latter part of the night. Two peaks also occur in DD; one during the latter part of the subjective day and the other during the latter part of the subjective
Conclusion
In summary, the present results demonstrate that the mRNA for VIP2 receptors is differentially expressed in the rat SCN across the 24 h cycle under both LD and DD conditions. The differential expression of VIP2 receptor mRNA may underlie the temporal sensitivity of the circadian pacemaker contained in the SCN to VIP. Further studies are required to determine the mechanisms through which VIP resets the rodent clock in a phase-dependent manner.
Acknowledgements
We are grateful to Dr N.W.S. Chong and Mr. T. Kalamatianos for their technical assistance. This work was supported by a grant from the BBSRC.
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