Substance P receptor regulates the photic induction of Fos-like protein in the suprachiasmatic nucleus of Syrian hamsters

Substance P (SP) is a candidate neurotransmitter or neuromodulator for conveying light information from the retina to the hypothalamic suprachiasmatic nucleus (SCN) where a circadian oscillator(s) is located in mammals. Immediate early gene c-fos has been demonstrated to be induced in the SCN with a brief light exposure at the subjective night, and suggested to play an important role in the photic entrainment of the oscillator. To clarify the possibility of an involvement of the SP receptor in the photic-induction of c-fos in the SCN, we examined effects of a SP receptor antagonist, spantide, on the light-induced Fos-like protein immunoreactivity (Fos-lir) in the SCN of Syrian hamster. The light-induced Fos-lir was inhibited with the pretreatment of spantide in a dose-related manner and in an anatomically distinctive way. The higher dose of spantide (8 nmol) blocked light-induced Fos-lir substantially in the rostral and central areas of the SCN, and in the dorsal portion of the caudal SCN. However, it blocked Fos-lir only slightly in the ventral portion of the caudal SCN. These results suggest that the SP is involved in conveying light information to induce Fos protein in the hamster SCN, and that different neurotransmitter systems are involved in the light-induced Fos-lir in the different portions of hamster SCN.     

Distribution of substance P and neurokinin-1 receptor immunoreactivity in the suprachiasmatic nuclei and intergeniculate leaflet of hamster, mouse, and rat

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied.     

Regional Distribution of Iodomelatonin Binding Sites within the Suprachiasmatic Nucleus of the Syrian Hamster and the Siberian Hamster

The pineal hormone melatonin is a potent regulator of seasonal and circadian rhythms in vertebrates. In order to characterize potential target tissues of melatonin, the distribution of iodomelatonin (IMEL)-binding sites was examined within neurochemically and anatomically defined subdivisions of the suprachiasmatic nucleus (SCN), a structure necessary for seasonal and circadian rhythms in mammals. Studies were carried out in both the adult Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamster. The retinoreceptive zone of the SCN was identified anatomically by immunocytochemical (ICC) visualization of cholera toxin B subunit tracer (ChTB-ir) following its intra-ocular injection. Photically-responsive SCN cells were identified by immunostaining for the protein product of the immediate-early gene c-fos (Fos-ir) following exposure of the animal to light. The non-photoresponsive zone of the SCN was identified using in situ hybridization (ISH) for arginine vasopressin (AVP) mRNA, whilst sites of IMEL-binding in the SCN were identified by in vitro film autoradiography using the specific ligand 2-[¹²⁵l]-iodomelatonin. To compare directly the distribution of IMEL-binding sites and one of the functional zones of the nucleus, alternate serial coronal sections through the SCN were processed for autoradiography for IMEL and one of the following: ICC for ChTB-ir or Fos-ir, or ISH for AVP mRNA. Overall, the regional distribution of the various markers within the SCN was comparable in the two species. The retinorecipient (ChTB-ir) and photically-responsive (Fos-ir) zones of the SCN mapped together to the middle and caudal thirds of the nucleus, predominantly in its ventro-lateral division. IMEL-binding was present throughout the full rostro-caudal extent of the SCN, but by far the most extensive area of IMEL-binding was in the rostral half of the nucleus, leading to a clear dissociation along the rostro-caudal axis of the principal zone of IMEL-binding and the retinorecipient zone of the nucleus. In the Syrian hamster, in coronal sections of the caudal SCN which did contain significant amounts of both IMEL-binding and Fos-ir, IMEL-binding was confined to the medial zone, distinct from the Fos-ir region of the ventro-lateral SCN. The segregation was less clear-cut in the Siberian hamster where the area of IMEL-binding was more extensive. The dissociation of IMEL-binding and photically-responsive cells in the Syrian hamster was confirmed in a series of sagittal sections which were processed alternately for Fos-ir and IMEL-binding. Whereas Fos-ir was confined to the ventro-lateral SCN, IMEL-binding was concentrated in the medial zone of the nucleus. In both species, mRNA for AVP was found throughout the rostro-caudal extent of the SCN, but the peak area was located in the rostral half, and so was segregated from the principal retinorecipient zone. The distribution of mRNA for AVP along the rostro-caudal and medio-lateral axes was in direct register with the IMEL-binding in both species. These studies suggest that melatonin acts upon pathways within the SCN different to those addressed by light, and that it may influence directly the efferent activity of the nucleus, possibly via an effect on vasopressinergic cells.     

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.     

Ontogeny of a Photic Response in the Suprachiasmatic Nucleus in the Siberian Hamster (Phodopus sungorus)

The ontogeny of photic responsiveness in the suprachiasmatic nucleus of the Siberian hamster (Phodopus sungorus) was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localization of its protein product. Hamsters were kept on a 16 h light:8 h dark photocycle. The adult Siberian hamster showed a marked increase in the number of c-fos-immunoreactive (c-fos-ir) cells within the suprachiasmatic nuclei (SCN) in response to a 1 h light pulse delivered 1–3 h after lights off, in comparison to controls kept in the dark. This is consistent with previous studies in the Syrian hamster and rat. The development of the photic response was examined. The first study investigated the effects of a light pulse on c-fos induction in pups at 5, 9, 12 and 24 postnatal days of age (PD). The suprachiasmatic region was identified by immunocytochemical localization of peptide-histidine-isoleucine in adjacent sections, a peptide expressed early in the development of the rodent SCN. The distribution of c-fos-ir cells was also compared with the location of retinal efferents, as determined by intraocular injection of the tract tracer cholera toxin B subunit 24 h previously. At PD 9, 12 and 24, significant increases in the number of c-fos-ir cells occurred in the light pulsed animals in comparison to age-matched control animals which were moved within the non-illuminated room to provide a ‘dark’ pulse. Induction of c-fos mainly occurred in the ventrolateral region of the medial and caudal SCN, the region which receives the greatest density of retinal innervation. The second study focused on the earliest age at which light could induce c-fos expression in the SCN by investigating the effects of light on c-fos-ir cells in pups at PD 2–4. Animals at PD 3 and 4 showed enhanced c-fos expression in the ventrolateral region, whilst PD 2 animals showed no response. The number of c-fos-ir cells in the SCN region in light-pulsed animals increased from PD 3 to adulthood. These results indicate that retinal input can activate cells in the SCN even before the eyelids open on PD 11, and the SCN can potentially be entrained by photic inputs as early as day 3 after birth.     

Distribution of Ca-dependent protein kinase C isoforms in the suprachiasmatic nucleus of the diurnal murid rodent, Arvicanthis niloticus

The suprachiasmatic nuclei (SCN) contain the major ‘biological clock' in mammals that controls most circadian rhythms expressed by these animals. The functional importance of protein phosphorylation and intracellular Ca²⁺ in the mammalian circadian pacemaker is becoming increasingly apparent. Here we report the immunocytochemical localization of the four Ca²⁺-dependent protein kinase C (PKC) isoforms (α, βI, βII, γ) within the SCN of the diurnal murid rodent, Arvicanthis niloticus, and the nocturnal golden hamster. In the SCN of A. niloticus, PKCα was the most abundant of the four isoforms. Cells containing PKCα were homogeneously distributed throughout the SCN. PKCβI cells were sparsely distributed in the perimeter of the SCN and were absent in its central area. PKCβII and -γ were not found in the SCN of A. niloticus. In the SCN of the golden hamster, PKCα cells were most heavily concentrated in the dorsomedial region, though some were also present laterally and ventrally. The distribution of arginine–vasopressin (AVP) cells in the SCN overlapped with that of PKC in both species. Species differences in the location of the Ca²⁺-dependent PKC isoforms suggest differences in function such as the relaying of photic or non-photic information to the clock mechanism, or the synchronization of AVP neurons and their subsequent output signals.     

Neurogenesis of the hamster suprachiasmatic nucleus

Neurogenesis of the hypothalamic suprachiasmatic nucleus (SCN) was described in the Syrian hamster (Mesocricetus auratus) using tritiated [3H]thymidine autoradiography. Pregnant hamsters were given single intraperitoneal injections of [3H]thymidine at different times during prenatal development, and labeled cells were analyzed in the offspring of 4-5 weeks of age. Cells of the hamster SCN became postmitotic (were 'born') over two and a half days from 10.5 to 13.0 days postfertilization (dpf) with a peak around 11.5 dpf, 4 days before birth. Two gradients in SCN neurogenesis were observed. Posterior cells were produced somewhat earlier than anterior cells and ventrolateral cells were produced before dorsomedial cells. An exception to the second gradient was a small population of ventrolateral cells produced near the end of SCN neurogenesis. The pattern of SCN neurogenesis in the hamster was similar to that described in the rat, including a predominant ventrolateral to dorsomedial gradient and the presence of ventral or ventrolateral cells produced relatively late, contrary to the predominant gradient.     

Effects of Microinjections of Substance P Into the Suprachiasmatic Nucleus Region on Hamster Wheel-Running Rhythms

The suprachiasmatic nucleus (SCN) receives a direct retinal projection, which in rats includes substance P (SP)-immunoreactive retinal ganglion cells. While SP has been shown to have neurophysiological effects on SCN cells in Syrian hamsters and rats, it is not known what effects SP in the SCN has on circadian rhythms in hamsters. We examined this question using male Syrian hamsters that were implanted with cannulas aimed at the SCN region and maintained in constant dim red lighting conditions. Hamsters received 0.5

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microinjections of saline or SP (500 pmol in saline) at a variety of circadian times (CT). Saline injections had little or no phase-shifting effects at any phase tested. SP had no significant effects at CT4–8, 16–20, or 20–24, but did cause small phase delays of −23.7 ± 7 min (mean ± sem) at CT12–16. In order to examine the dose-response relations of this effect, hamsters were also microinjected with 50 and 2500 pmol of SP at CT12–16. Both the 50 and 2500 pmol doses induced very small phase delays (−14.2 ± 7 min and −18.2 ± 5 min, respectively), indicating no obvious dose dependence within this range. These results do not suggest that SP alone in the SCN mimics light effects on circadian rhythms or is a key neurotransmitter involved in photic entrainment. It remains to be determined whether SP interacts with other transmitters in the SCN to modulate their effects on rhythm phase.     

5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons

Serotonin (5-HT) has been shown to phase shift circadian rhythms in mammals and to affect responses of the circadian system to light, but it is not clear which receptors are involved in these actions. We found that drugs which act as 5-HT_1A receptor agonists suppressed photic responses of hamster SCN cells, but these drugs also exhibit high affinity for the recently cloned 5-HT₇ receptor. We therefore studied the effects of 5-HT agonists and antagonists with differential affinities for 5-HT₇ and 5-HT_1A receptors on responses of hamster SCN cells to retinal illumination. We confirmed that the 5-HT receptor agonists 5-HT, 8-OH-DPAT and 5-CT, dose-dependently reduced photic activation of SCN cells. These effects could be blocked by co-application of antagonists with high affinities for 5-HT₇ receptors: ritanserin or clozapine. The 5-HT_1A/B/D antagonist, cyanopindolol, which is inactive at 5-HT₇ receptors, did not antagonize the actions of 8-OH-DPAT. Selective 5-HT_1A antagonists, WAY100635 and p-MPPI, had weak or no antagonist effects on the responses to 8-OH-DPAT in the SCN, but they effectively antagonized the actions of 8-OH-DPAT in the hippocampus. In the cerebellar cortex where few 5-HT₇ receptors are present, ritanserin failed to antagonize the effects of 8-OH-DPAT. Our results indicate that the 5-HT₇ receptor subtype plays a major role in mediating the effects of 5-HT on photic responses of SCN cells in the hamster.     

Physiological mechanisms regulating photic induction of Fos-like protein in hamster suprachiasmatic nucleus

Immediate early genes including c-fos are selectively induced in cells of the hamster suprachiasmatic nucleus (SCN) by nocturnal light stimulation, suggesting that the Fos protein may play a role in the photic entrainment of circadian rhythms. To examine the physiological regulation of the induction of c-fos in the SCN, we studied the effects of antagonists of excitatory amino acids (EAA) receptors on photic induction of Fos-like immunoreactivity (Fos-lir) in the hamster SCN. We also examined the effects of electrical stimulation of the intergeniculate leaflet (IGL) to see whether neural input from IGL to SCN is involved in the induction of Fos protein in SCN cells. The results indicate that for most SCN cells EAA receptors mediate photic input involved in Fos induction but that another mechanism affects cells in restricted area of the caudal SCN. The neurochemical mechanisms and pathways by which these cells are activated by light remain undetermined.     

Luminance coding in a circadian pacemaker: the suprachiasmatic nucleus of the rat and the hamster

The hypothalamic suprachiasmatic nuclei (SCN) of mammals function as a pacemaker driving circadian rhythms. This pacemaker is entrained to the daily light-dark cycle in the environment via the retina and central retinal projections to the anterior hypothalamus. We carried out a comparative study of the visual properties of rat and hamster SCN neurons. Extracellular single cell activity was recorded in the SCN of urethane-anaesthetized animals. In both species, visual SCN neurons responded to retinal illumination with a sustained increase or a sustained decrease in electrical discharge. The majority (75%) of these cells were activated by light. In both the rat and the hamster SCN, visually responsive cells altered their discharge rate as a monotonic function of luminance. The intensity-response curve could be described by a Michaelis function with a small working range between threshold and saturation (2-3 log units) and a relatively high threshold. Intensity-response curves in both species were occasionally different for increasing as opposed to decreasing luminance. Thus, hysteresis effects of illumination may occur in the SCN. The spontaneous firing rates as well as the responsiveness of visual SCN cells were subject to marked variations between and within cells. The overall photic responsiveness of SCN neurons, however, indicated that they are specialized for luminance coding in the range of light intensities naturally occurring at dawn and dusk. This property makes these cells suitable to mediate photic entrainment of circadian rhythms as well as the measurement of photoperiod.     

Distribution of delta opioid receptor immunoreactivity in the hamster suprachiasmatic nucleus and intergeniculate leaflet

The hamster suprachiasmatic nucleus (SCN) is innervated by a dense plexus of enkephalin-containing axons originating from cells in the intergeniculate leaflet (IGL) of the thalamus. However, the distribution of opioid receptors within the hamster SCN has not been reported. Opioid receptors consist of three primary subtypes: mu, delta and kappa opioid receptors. Enkephalins have the highest affinity for delta opioid receptors. Therefore, in the present study, we examined the distribution of delta opioid receptor immunoreactivity in the hamster SCN and the IGL of the thalamus. Coronal sections of the hamster hypothalamus inclusive of the SCN or thalamic regions containing the IGL were prepared at specific times of the day and labeled with anti-delta opioid receptor polyclonal antisera using standard immunohistochemical techniques. delta opioid receptors were heavily distributed within rostral-caudal regions of the SCN, with the densest labeling located in the ventral and medial regions of the mid-SCN. Similar patterns of labeling were observed for tissue prepared during mid-day or mid-night times. In contrast, delta opioid receptor immunoreactivity only sparsely labeled cells in the IGL. Cellular staining in all regions appeared as dark punctate labeling surrounding cells, indicative of terminal boutons. Therefore, it is suggested that delta opioid receptors are located presynaptically on axon terminals within the hamster SCN and IGL. These results suggest that delta opioid receptors may play a role in modulating circadian rhythms generated within the SCN, possibly by regulating transmitter release within the nucleus.     

[I]Vasoactive intestinal peptide binding in rodent suprachiasmatic nucleus: Developmental and circadian studies

The suprachiasmatic nucleus (SCN) of rat and hamster have been studied extensively and shown to play critical roles in circadian rhythmicity. [¹²⁵I]Vasoactive intestinal peptide (VIP) binding levels are high in the rat SCN, suggesting that VIP receptors may be an important component of SCN function. In contrast to previously demonstrated diurnal variations in VIP immunoreactivity and VIP mRNA, the present study found [¹²⁵I]VIP binding to be stable across the light-dark cycle in both rat and hamster SCN. High [¹²⁵I]VIP labeling appeared to be coextensive with the rat SCN but extended somewhat beyond the cytoarchitectonic boundaries of the hamster SCN. Binding density in hamster SCN was slightly higher than in rat. In the developing rat SCN, [¹²⁵I]VIP binding levels distinguished the SCN on embryonic day 18, and appeared to increase to postnatal day 10 before declining to adult levels. The early presence of [¹²⁵I]VIP binding suggests possible involvement of VIP receptors in fetal entrainment of circadian rhythms.     

Neuromodulator content of hamster intergeniculate leaflet neurons and their projection to the suprachiasmatic nucleus or visual midbrain

The intergeniculate leaflet (IGL) of the lateral geniculate complex has widespread, bilateral, and reciprocal connections with nuclei in the subcortical visual shell. Its function is poorly understood with respect to its role in visual processing. The most well-known IGL projection, and the only one with a clear function, is the geniculohypothalamic tract (GHT) that terminates in the suprachiasmatic nucleus (SCN), site of the primary circadian clock. The hamster GHT is derived, in part, from IGL neurons containing neuropeptide Y and enkephalin. IGL neurons containing these peptides also project to the pretectal region. The present studies used a combination of immunohistochemical, lesion, and retrograde tracing techniques to study neuron types in the IGL and their projections to hamster SCN and pretectum. Two additional neuromodulators, gamma-aminobutyric acid (GABA) and neurotensin, are shown to be present in IGL neurons. The GABA- and neurotensin-immunoreactive neurons project to the SCN with terminal field patterns very similar to those for neuropeptide Y and enkephalin. IGL neurons of all four types also send projections to the pretectum, but rarely do individual cells project to both the SCN and the pretectum. Nearly all neurotensin is colocalized with neuropeptide Y in IGL neurons, although about half of the neuropeptide Y cells do not contain neurotensin. Otherwise, the extent to which the four neuromodulators are colocalized varies from 6% to 54%. Nearly every SCN neuron appears to contain GABA. In the IGL, the majority of cells studied are not identifiable by GABA immunoreactivity. Putative functions of the various neuromodulator projections from the IGL to pretectum or SCN are discussed.     

Anatomical and functional characterisation of a dopaminergic system in the suprachiasmatic nucleus of the neonatal Siberian hamster.

In altricial rodents, maternal influences entrain the developing circadian system in the perinatal period before the capacity to respond directly to photic cues develops. The aim of these studies was to investigate the potential role of dopamine in this process in the Siberian hamster. An initial study investigated the ontogeny of retinal innervation of the suprachiasmatic nuclei (SCN) by using cholera toxin B subunit as a tracer. This revealed that retinal fibres first innervate the SCN on postnatal day 3 (PD3), and ingrowth of fibres is extensive by PD6. In situ hybridisation studies revealed the presence of D1-dopamine receptor (D1-R) mRNA in the SCN on PD2, and levels of expression were similar in PD6 pups and adult hamsters. Immunocytochemical staining for tyrosine hydroxylase revealed abundant catecholaminergic fibres within the ventromedial zone of the SCN from the day of birth through PD20; however, in contrast, few fibres were present in adult SCN. Dopamine-beta-hydroxylase-immunoreactive fibres were absent from the neonatal and adult SCN, suggesting that the fibres in the SCN are dopaminergic. The function of this dopaminergic system was investigated by determining the effects of D1-R agonists on the expression of the immediate-early gene c-fos in the SCN. This was assessed in pups ages PD1- PD5 by in situ hybridisation and immunocytochemical localisation of its protein product. No induction was seen in the SCN, in marked contrast to studies in the developing rat. A final series of studies investigated dopaminergic function by determining whether a D1-agonist could induce phosphorylation of Ca2+/cyclic AMP response element-binding protein (CREB) on Ser133. Hypothalamic slices containing SCN taken from PD1 and PD2 hamsters were treated with D1-R agonists, and levels of phosphorylated CREB were assayed by Western blots. Phosphorylation of CREB was stimulated by D1-R agonists in both Syrian and Siberian hamster hypothalamus, but the response was far greater in Syrian hamster tissue (+138%+/-28%) than in Siberian hamster tissue (+43%+/-11%). Although the anatomical studies demonstrate the existence of a dopaminergic system in the SCN of the early postnatal Siberian hamster, the unresponsiveness of c-fos expression and the relative lack of phosphorylation of CREB after D1-R activation suggests a diminished role for dopamine in the regulation of circadian events during the postnatal period in this species.     

Circadian rhythm of spontaneous neuronal activity in the suprachiasmatic nucleus of old hamster in vitro

The effects of aging on neuronal activity in the suprachiasmatic nucleus (SCN) were examined in hamsters kept under light-dark (LD) or constant light (LL) conditions. The free-running period in wheel-running rhythm of 24-month-old hamsters (24.2 ± 0.04) was shorter than that of the 2-month-old hamsters (24.4 ± 0.057). There was a significant difference in the mean firing rates of SCN neuron activity between old and young hamster during subjective day (6.58 ± 0.36 spikes/s in young and 5.63 ± 0.24 in old hamsters), but not during subjective night (4.33 ± 0.47 in young and 4.05 ± 0.39 in old). Similar to LL condition, the firing activity during zeitgeber time 3–11 (4.33 ± 0.27) in old hamsters kept under LD condition, was significantly lower than that of young hamster (6.22 ± 0.32). These results suggest that deterioration of SCN neuronal activity in old hamsters assessed as reduction of daytime activity may reflect changes in the interaction between SCN clocks and the overt behaviors and/or pacemaking properties of SCN cells.     

Does the intergeniculate leaflet play a role in the integration of the photoperiod by the suprachiasmatic nucleus?

The circadian clock located in the suprachiasmatic nuclei (SCN) is influenced by the photoperiod. After the transfer from a long (LP 14:10) to a short photoperiod (SP 10:14), the adjustment of the light sensitivity of the SCN, in terms of Fos expression, takes 25 nights. To examine the contribution of the thalamic intergeniculate leaflet (IGL) and its NPY-immunoreactive projection in the extension of the duration of the photosensitive phase of the SCN, male Syrian hamsters received electrolytic lesions of the IGL. We showed a lower number of Fos-ir cells in the SCN of IGLx hamsters following a light pulse applied 13 h after dark onset, 25 nights after the transfer from LP to SP compared to sham operated hamsters. The present study shows that the integrity of the IGL is necessary to have a complete integration of photoperiodic changes by the SCN. This demonstrates the involvement of the IGL in the integration of photoperiodic information by the SCN.     

Single unit response of neurons within the hamster suprachiasmatic nucleus to GABA and low chloride perfusate during the day and night

Using the in vitro hamster hypothalamic slice preparation, the effects of GABA and 80% chloride (Cl-) reduced medium on the single unit activity of SCN neurons was investigated. GABA 10(-4) M produced inhibitory responses in 55% of the 69 SCN neurons examined. No statistically significant day-night difference was observed in either the percentage of SCN units responding to GABA, or in their threshold response. During the day 80% Cl- reduced medium had an excitatory effect on SCN neurons; however, following the return to normal Cl- concentrations a transient, but significant inhibition was observed. During the night, 80% Cl- reduced medium produced an excitatory response similar to that observed during the day, but no inhibition following return to the medium containing normal Cl- concentrations. Only during the night was Cl- reduced medium found to initiate activity in a dose-dependent manner in some silent cells. No significant day-night difference in response to 80% Cl- reduced medium occurred in neurons of the paraventricular nucleus of the hypothalamus. These results indicate that SCN neurons whose activity is mediated by Cl- may be involved in the control of circadian rhythms.     

Neurophysiological responses to melatonin in the SCN of short-day sensitive and refractory hamsters

The pineal hormone melatonin plays a central role in the regulation of seasonal reproductive cycles in mammals and several studies have implicated the suprachiasmatic nucleus (SCN) as a target on which melatonin acts. The Syrian hamster is a long-day breeder which exhibits gonadal regression when housed in short (less than 12.5 h) daily photoperiods or injected daily with melatonin in long photoperiods. In the present paper we address the question whether melatonin affects firing rates of SCN neurones and whether these effects change as the animals become refractory to short photoperiods. In long-day (LD14:10) hamsters SCN neurones were suppressed (31%), activated (15%) or unaffected (54%) by melatonin. In contrast, there was an increased proportion of melatonin insensitive cells (88%) in short-day (refractory) hamsters. Melatonin-responsive cells were found primarily during the late projected day and early projected night in both long-day and short-day animals. This reduced responsiveness of SCN neurones to melatonin in hamsters refractory to short-day exposure may represent part of the mechanism underlying the development of gonadal refractoriness and the onset of gonadal growth in anticipation of long spring photoperiods.