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M H Hastings

Publications and source records attributed to M H Hastings.

At least 19 recordsLinked to original sources
PubMed1998

Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro.

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at approximately 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-D-aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 microM], or native glutamate (1 microM) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser133. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 microM) or dopamine (1 microM) had no effect on total CREB-ir, but increased pCREB-ir in approximately 50 and 30% of cells, respectively, whereas forskolin (1 microM) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 microM) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.

Animals↗
PubMed1997

Central clocking.

The main questions in circadian neurobiology are: how many oscillators are involved; how are their daily oscillations generated and synchronized to the external world; and how do they signal time of day to the organism. The suprachiasmatic nuclei of the hypothalamus (SCN) are well established as the principal circadian oscillator of mammals. Their 10,000 or so 'clock' neurones drive our overt rhythms-the daily patterning we observe in our physiology and behaviour being mirrored perfectly by their spontaneous cycle of neuronal activity. However, they are not our only circadian oscillator, their molecular timekeeping is not understood and they ways in which they communicate with other parts of the brain are more unusual than was previously assumed.

Animals↗
PubMed1997

Non-photic signalling in the suprachiasmatic nucleus.

Scheduled arousal by handling and sub-cutaneous saline injection entrains the free-running clock of the adult Syrian hamster and outbred (ID(ICR)) but not inbred (C57B16) mice. Syrian hamsters bearing lesions of the intergeniculate leaflet of the thalamus remain able to entrain and phase-shift to light, but the lesions block completely entrainment by serial arousal, even though lesioned animals continue to respond acutely to the arousing cue. This suggests that the innervation from the IGL to the SCN is a necessary component of the pathways which signal an aroused state to the clock. Siberian hamsters do not entrain to serial arousal but they do entrain to serial injections of melatonin, whereas in adult Syrian hamster, systemic treatment with melatonin has no effect above that of arousal. In contrast to the adult, the foetal and neonatal Syrian hamster can be entrained by melatonin. These variations in sensitivity correlate with inter-specific and developmental differences in the pattern and level of expression of melatonin receptors in the SCN. The perinatal hamster can also be entrained by dopaminergic agonists. SCN tissue from neonatal Syrian hamsters was used to characterise the biochemical actions of dopamine and melatonin. In primary culture and tissue explants, forskolin, dopamine and glutamatergic agonists all stimulated the phosphorylation of the transcription factor CREB. This probably occurred via convergent actions through Ca2+ (glutamate) and cyclic AMP-dependent (forskolin, dopamine) signalling pathways. Dopamine induced phospho-CREB-ir exclusively in GABA-ir neurons and melatonin reversed this effect of dopamine, indicative of an inhibitory Gi protein linking via the Mel1a receptor to adenylyl cyclase. The regulation of phospho-CREB by multiple entraining cues in the SCN highlights its position as a point of convergence for regulators of the clock, and indicates a possible role in entrainment.

Afferent Pathways↗
PubMed1997

A thalamic contribution to arousal-induced, non-photic entrainment of the circadian clock of the Syrian hamster.

It is well established that the circadian clock of the suprachiasmatic nuclei (SCN) is entrained by light. More recently, the potent effects of arousing, non-photic cues on the clock have been recognized. The neural mediators of non-photic entrainment are yet to be identified. To examine the contribution of the thalamic intergeniculate leaflet (IGL) and its NPY-immunopositive projection, the geniculo-hypothalamic tract to non-photic entrainment by arousal, male Syrian hamsters received lesions of the IGL (IGLX) which ablated NPY-immunoreactivity in the SCN. Their circadian responses to both photic and non-photic cues were then tested. Lesions resulted in a delay in the timing of activity onset following lights out, but had no effect on the behavioural or cellular circadian responses to phase-advancing light pulses presented at circadian time (CT) CT19 (where CT12 represents the time of activity onset). Injection with a benzodiazepine (chlordiazepoxide, 100 mg/kg) at CT6 suppressed wheel-running, increased general locomotion of intact controls and induced large phase advances of the circadian rhythm of wheel-running. Chlordiazepoxide also inhibited wheel-running in lesioned animals, but there was no significant increase in general locomotion and the lesioned animals did not phase advance. Serial arousal by injection of saline at intervals of 23.5 h for 6 days entrained the circadian rhythm of wheel-running of intact hamsters and was associated with an increase in general locomotor activity. Entrainment by serial arousal was abolished by IGLX. However, the lesioned animals did show a clear behavioural response to every presentation of the non-photic cue. These results show that the IGL is a necessary component of the neural pathways mediating both arousal- and benzodiazepine-induced non-photic entrainment.

Animals↗
PubMed1997

Melatonin-sensitive, serum-stimulated signalling in ovine pars tuberalis.

In primary cultures of ovine pars tuberalis (oPT), serum acts through melatonin-sensitive mechanisms independent of cyclic AMP to increase the phosphorylation of the Ca2+/cyclic AMP response element binding protein (CREB). Immunocytochemical and biochemical assays were used to characterize the active components of serum and the signalling pathways through which they and melatonin function in oPT. The stimulatory effect of serum was heat-labile, sensitive to precipitation by methanol, and required components with a mass greater than 10 KDa implicating peptide or protein factors as the active agent. Serum increased the cytosolic free Ca2+ concentration ([Ca2+]i) of oPT cells. Serum also enhanced the release of [3H]-choline and [3H]-arachidonic acid from prelabeled cells, demonstrating that factors present in serum increase the breakdown of cellular phospholipids. This effect, however, was not blocked by melatonin (1 microM). Serum also caused a dose-dependent increase in levels of immediate early gene immunoreactivity, confirming that factors in serum have the ability to control transcription in the oPT. Down-regulation of protein kinase C (PKC) by treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA, 100 nM) or treatment with a specific PKC inhibitor (RO-31-8220, 1 microM), did not affect protein kinase A-mediated stimulation of CREB phosphorylation. However, down-regulation of PKC blocked the acute stimulatory effects of TPA (100 nM) and of serum (1%). Moreover, RO-31-8220 abolished the stimulatory effect of TPA (100 nM) and strongly attenuated that of serum (1%). These results demonstrate that serum increases the phosphorylation of CREB by stimulating cyclic AMP-independent, PKC-dependent, signalling pathways within the oPT. PKC may be activated through increased phospholipid catabolism and/or raised [Ca2+]i.

Animals↗
PubMed1996

Are nuclear receptors involved in pituitary responsiveness to melatonin?

Evidence suggests that the pineal hormone melatonin modulates prolactin (PRL) secretion in part through direct effects on the anterior pituitary. However, high-affinity membrane receptors for melatonin are only found in the pars tuberalis (PT) of the anterior pituitary, whereas lactotrophs are confined to the pars distalis (PD). This study therefore sought to determine whether melatonin might have direct effects on the PD, through alternate pathways. Such a possibility had been suggested by recent reports of melatonin binding to members of the retinoid-related orphan nuclear receptor family (ROR/RZR). Expression of ROR(alpha)/RZR(alpha) isoforms was observed by reverse transcription PCR in the ovine PT and PD. Correspondingly unidentified nuclear proteins from these tissues showed binding to consensus DNA response elements for members of the ROR/RZR family. In contrast nuclear 2-[125I]iodomelatonin (IMEL) binding was not detectable in PD or PT extracts even at high ligand and tissue concentrations. Nevertheless, the conditions used allowed membrane and nuclear IMEL binding to be observed in PT and liver extracts, respectively. Overall these findings do not support the possibility of direct effects of melatonin on the adult PD, and by implication they reinforce the view that the melatonin-responsive PT is an intermediary in the control of PRL secretion.

Animals↗
PubMed1996

Serotonergic antagonists impair arousal-induced phase shifts of the circadian system of the syrian hamster.

Single episodes of arousal of Syrian hamsters 2 h before projected activity onset (i.e., CT 10) phase-advanced their free-running circadian rhythm of wheel-running. Serial arousal once every 23 h or once every 23.5 h for 7 days caused large composite phase-advances to the wheel-running rhythm, the latter period being more effective in supporting an interval of stable entrainment. Pre-treatment of hamsters at CT 6 with the serotonergic antagonist ritanserin (1-5 mg/kg, which acts at both 5-HT2 and the putative 5-HT7 receptor, impaired the phase-advancing response to arousal at CT 10 but the drug was without effect on phase advances induced by exposure to light. Pre-treatment with a second serotonergic antagonist, ketanserin (1-5 mg/kg), which is without effect at 5-HT7 but has high affinity for 5-HT2 receptors, was also effective in attenuating the phase advancing effect of arousal at CT 10. However, neither agent was able to achieve complete blockade of the phase advances. These results are discussed in relation to in vitro and in vivo studies in the rat which have identified a role for 5-HT7 receptors in serotonin-mediated circadian entrainment.

Animals↗
PubMed1996

FosB in the suprachiasmatic nucleus of the Syrian and Siberian hamster.

The suprachiasmatic nucleus (SCN) generates circadian rhythms of behavior and hormone secretion in mammals, and integrates responses to light and nonphotic stimuli to synchronize such rhythms with the external environment. Previous studies have demonstrated a close association between the induction of the immediate early gene (IEG) c-fos in the SCN by light and phase shifts of circadian rhythms induced by light, but nonphotic stimuli (e.g., arousal), which also cause phase shifts, do not increase c-fos expression in the SCN. Because c-fos is now known to be a member of a large family of IEGs which can regulate transcription and thus cellular function, the aim of the current study was to determine whether induction of another member of this immediate early gene family, fosB, is associated with photic and nonphotic phase shifts. An antiserum that recognizes a unique peptide sequence derived from FosB was produced so that the expression of fosB could be investigated in cells within the SCN by immunocytochemical detection of its protein product. The regional distribution of FosB-immunoreactive (ir) cells in the SCN of Syrian and Siberian hamsters was broadly similar to that for c-Fos-ir cells. However, whereas c-fos expression in the SCN was constitutively low, but could be massively induced by light at particular circadian phases, FosB-ir cells were present at all circadian phases studied, irrespective of photic stimulation, and light only produced marginal increases in the number of FosB-ir cells compared with nonstimulated controls. Moreover, blockade of glutamatergic neurotransmission by pretreatment of hamsters with the NMDA receptor antagonist MK801 significantly reduced photic induction of c-Fos-ir cells, but did not influence the number of FosB-ir cells in the SCN. Finally, an arousing nonphotic stimulus known to cause phase advances in wheel-running behavior in Syrian hamsters did not alter significantly the number of FosB-ir cells in the SCN. These observations indicate that light and nonphotic stimuli are not potent regulators of fosB expression in the SCN. However, because fosB and c-fos can be present in the SCN at the same time after a light pulse, these studies indicate the potential for interactions with each other and with members of the Jun family in the regulation of the circadian timing system.

Animals↗
PubMed1996

Production of a prolactin releasing factor by the ovine pars tuberalis.

The anterior pituitary hormone prolactin is involved in the regulation of a wide variety of processes including mammary growth, lactation, reproduction and pelage growth (1). Correspondingly, in seasonal breeders, pronounced photoperiod-driven, seasonal changes occur in the levels of prolactin secretion (2). These effects are thought to be mediated by the pineal hormone melatonin, which acts as a humoral indicator of photoperiod (3). Melatonin is thought to act through hypothalamic sites to control the gonadotrophic axis (4-6), but the sites through which melatonin modulates prolactin remain to be established. One possibility is that melatonin acts at the level of the hypothalamus to modulate the release of the hypothalamic prolactin inhibitory factor, dopamine (7). However, recent evidence from hypothalamo-pituitary disconnection experiments performed in the ram suggests that the photoperiodic modulation of prolactin secretion can occur independently of the hypothalamus, presumably due to direct effects of melatonin on the anterior pituitary (8). The only identified site of melatonin receptor expression within the ovine pituitary is the pars tuberalis (PT) (9), the cells of which are almost completely non-lactotrophic (10). Thus the possibility exists that factors released by the PT regulate the activity of lactotrophs in the pars distalis (PD). We have investigated this hypothesis using a range of co-culture and medium-conditioning experiments on primary cultures of ovine PT and PD cells, and here we report that PT cells secrete an unidentified factor that is a potent stimulus of prolactin secretion by PD cells.

Animals↗
PubMed1996

Lesions of the melatonin- and androgen-responsive tissue of the dorsomedial nucleus of the hypothalamus block the gonadal response of male Syrian hamsters to programmed infusions of melatonin.

The objective of this study was to characterize a site at which it is likely that melatonin mediates photoperiodic control of reproduction in the male Syrian hamster. The first experiment was a comparison of the distributions of iodomelatonin (IMEL)-binding sites and cells immunoreactive to androgen receptors (AR-ir) in the medio-basal hypothalamus (MBH). AR-ir cells extended throughout the MBH, whereas IMEL binding was restricted to the dorsomedial nucleus (DMN). Comparisons between IMEL binding and AR-ir on adjacent cryostat sections revealed a clear overlap between the IMEL-binding sites and a distinct subpopulation of AR-ir cells within the DMN. The second experiment examined whether lesions of these IMEL- and androgen-responsive cells affected the response of the hamsters to short-day (SD)-like infusions of melatonin. Animals received sham or bilateral electrolytic lesions of the IMEL-binding sites within the DMN of the hypothalamus (MBH-X). Animals were pinealectomized and 4 wk later fitted with an s.c. cannula for the daily infusion of either melatonin (50 ng/h) or saline (500 microliters/10 h). After 6 wk the animals with sham lesions showed gonadal atrophy and lower serum concentrations of LH and prolactin (PRL) after infusions with melatonin. In contrast, MBH-X animals given melatonin had large testes and long-day (LD)-like serum LH concentrations. Infusions of melatonin did, however, cause a significant decline in serum PRL level. This study shows that an intact MBH is essential for the expression of gonadotrophic but not lactotrophic responses to melatonin and/or photoperiod. It also suggests that cells responsive to both gondal steroids and melatonin may be involved in the seasonal variation in GnRH release, and indicates a site at which melatonin might influence sensitivity to steroid feedback, a hypothalamic function known to be regulated by photoperiod.

Androgens↗
PubMed1996

A role for the circadian clock of the suprachiasmatic nuclei in the interpretation of serial melatonin signals in the Syrian hamster.

Seasonal rhythms of reproduction in the Syrian hamster are triggered by the pineal hormone melatonin. By varying the parameters of systemic infusions of exogenous melatonin delivered to pinealectomized hamsters, it has been shown that the hypothalamus is sensitive to the duration of individual signals, which serve as an inverse coding of day length. It also has been shown that animals are sensitive to the temporal structure of a series of signals insofar as a series of melatonin infusions of appropriate number and duration may fail to invoke a gonadal response if they are presented at inappropriate frequencies. Although the endogenous circadian pacemaker of the suprachiasmatic nucleus (SCN) is not thought to be involved in the measurement of or response to melatonin signal duration, its contribution to the interpretation of a series of melatonin signals remains to be determined. Syrian hamsters are able to show a short-day-like gonadal response to a series of melatonin signals delivered on a variety of noncircadian schedules, including one in which a "random" pattern of infusions is employed. This study investigated the role of the SCN in the interpretation of such infusion paradigms. Adult male Syrian hamsters received electrolytic lesions of the SCN. Pinealectomized, lesioned, and intact hamsters then were infused with melatonin or saline at one of three different phases of the day in a random pattern such that no signal was predictive of the timing of the next. Other lesioned and intact animals received melatonin or saline at the same time daily. After 6 weeks, control saline-infused animals in both lesioned and intact groups had large testes. However, sham animals receiving melatonin in the random infusion pattern had regressed testes, as did the lesioned animals receiving melatonin at the same phase every day. By contrast, lesioned animals that received melatonin in the random pattern of infusion did not show a short-day gonadal response. These results suggest that although the SCN is not necessary for measurement of the duration of individual signals, it may play a role in the interpretation of a series of melatonin signals in which the number of melatonin signals and the period of time over which they are encountered need to be compared.

Animals↗
PubMed1996

Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin.

The pars tuberalis (PT) of the anterior pituitary is notable for the expression of levels of melatonin receptors that consistently exceed those in all other tissues in mammals. For this reason and because of its anatomical position, it has been suggested that the PT may play a role in seasonal reproductive responsiveness. However, no data have been forthcoming on the nature of the melatonin-responsive cells in this tissue or on the interaction of melatonin with other hormonal signals in the control of PT cells. A number of recent studies have reported that the tubero-infundibular region of the pituitary in several species contains binding sites for insulin-like growth factor-1 (IGF-1). The present study, therefore, sought to address the question of whether functional receptors for IGF-1 exist in the ovine PT (oPT). Primary cultures of cells from the oPT contained a widespread distribution of cells staining positively with a monoclonal antibody to the human IGF-1 receptor, with the strongest staining occurring over the small phase-bright cells that predominate in this culture system and are thought to constitute the melatonin-responsive cell type. As a functional assay for responsiveness to IGF-1, primary cultures of oPT cells were assayed for activation of mitogen-activated protein kinase (MAPK) using a previously validated phosphotransferase assay. Cytosolic extracts from PT cells treated with IGF-1 (100 pM-10 nM) caused a dose-dependent increase in the rate of phosphorylation of myelin basic protein; in contrast, treatment with melatonin had no significant effect on myelin basic protein phosphorylation. Immunostaining of Western blots of PT cell extracts with a pan-extracellular regulated kinase antibody demonstrated that both p42 and p44 MAPK are strongly expressed in this tissue. To confirm that the effects observed in the cytosol assay were indeed attributable to increased activation of p42/p44, gel renaturation assays of protein kinase activity were performed. These experiments revealed that IGF-1 (10 nM) and forskolin (1 microM) were both potent activators of 42- and 44-kDa moeities; however, neither of these agents had any significant effect on the phosphotransferase activity associated with several other higher molecular weight kinases also detected by the gel-renaturation assay procedure. Melatonin (10 nm) was consistently found to be a highly potent inhibitor of the activation of MAPK induced by forskolin; in contrast, melatonin did not inhibit the activation of MAPK induced by IGF-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

The tau mutation in the Syrian hamster alters the photoperiodic responsiveness of the gonadal axis to melatonin signal frequency.

This study investigated the role of the circadian timing system (CTS) in photoperiodic time measurement by examining the response of the tau mutant hamster to programmed infusions of melatonin. The mutation is a single Mendelian gene defect which accelerates circadian period from 24 h in the wild-type (WT) to 20 h in the homozygote. If the CTS does not contribute to the photoperiodic interpretation of the melatonin signal, then the tau mutation would not influence photoperiodic responses of pinealectomised (PX) animals to systemic infusions of melatonin (10 h) once every 20, 24 or 25 h, mimics short-daylengths and causes gonadal involution. More ( < 18 h) or less ( > 25 h) frequent signals are ineffective. In this study, taus which received melatonin (10 h) once every 16 or 20 h exhibited significant gonadal atrophy relative to saline controls, whereas infusions of melatonin every 24 or 28 h were ineffective. Serum concentrations of LH and PRL were also significantly reduced in both the 16 and 20 h, but not 24 and 28 h groups. The tau mutant hamster may therefore respond to a different and higher ranger of melatonin signal frequencies than those reported for WTs. The 4 h shift in the frequency-response function correlates with the altered circadian period and suggests that the CTS contributes to the photoperiodic interpretation of a series of melatonin signals.

Animals↗
PubMed1996

Phosphorylation of CREB in ovine pars tuberalis is regulated both by cyclic AMP-dependent and cyclic AMP-independent mechanisms.

This study used a combination of Western blotting and immunocytochemistry to test whether signalling pathways independent of cyclic AMP have the potential to induce phospho-CREB (pCREB)-like immunoreactivity (-ir) in the oPT. Western blot analysis of extracts of primary cultures of oPT using an antiserum against CREB, revealed a major band of CREB-ir at 44 KDa. The intensity of this band did not vary systematically with treatment. In extracts from untreated cells, Western blot analysis revealed a major band of pCREB-ir at 42 KDa which was not sensitive to agonist treatment. Treatment of cells with forskolin (10(-6) M) increased the intensity of a number of other pCREB-ir bands at between ca. 38 and 44 KDa. The band at 44 KDa probably represented native pCREB whilst the other bands induced by forskolin probably represented pCREB-like proteins. Melatonin (10(-6) M) alone had no effect on pCREB-ir, but it did inhibit the effect of forskolin on the ca. 38 and 44 KDa pCREB-ir bands. Treatment with lamb serum (1%) consistently increased the intensity of the ca. 38 and 44 KDa pCREB-ir bands relative to control cells, as assessed by Western blot. However, Western blot analysis did not reveal a consistent effect of melatonin on the pCREB-ir response to serum. The effect of serum on pCREB-ir in oPT cells was characterized further by immunocytochemical analysis. In contrast to experiments utilizing Western blotting, untreated cells did not possess detectable pCREB-ir. In serum-starved oPT and oPD cultures, treatment with serum induced exclusively nuclear pCREB-ir. A large majority of oPT cells (> or = 90%) were sensitive to serum (1%), and serum caused a time- and dose-dependent increase of nuclear pCREB-ir. Melatonin attenuated the response to serum in the oPT. This inhibition of the response to serum was not apparent in the oPD, demonstrating that the effect of melatonin was specific for a tissue known to express melatonin receptors. In oPT cultures, physiological concentrations of melatonin (10(-9) M) partially reversed (ca. 70%) the inductive effect of 0.1% serum on nuclear pCREB-ir. However, in contrast to studies applying forskolin, the induction of pCREB-ir by serum occurred in the absence of measurable changes in the concentration of cyclic AMP, indicating that components of serum are able to stimulate the phosphorylation of CREB in the oPT through mechanisms independent of cyclic AMP. Both adenosine and prostaglandin E2 (PGE2) also induced nuclear pCREB-ir in the absence of increased levels of cyclic AMP. These results demonstrate that transcriptional activities in the oPT which are under the control of CREB may be modulated by convergent cyclic AMP-dependent and cyclic AMP-independent pathways. Regulation of these pathways by melatonin and other factors present in serum may be an important control-point in the generation of seasonal neuroendocrine cycles.

Adenosine↗
PubMed1995

Immediate-early genes and the neural bases of photic and non-photic entrainment.

The expression of immediate-early genes (IEGs) within the mammalian suprachiasmatic nucleus (SCN) identifies individual light-responsive cells of the circadian system. Cells immunoreactive for products of IEGs form a neurochemically heterogeneous population, of which a few are VIP (vasoactive intestinal peptide)-immunoreactive or GRP (gastrin-releasing peptide)-immunoreactive, although the phenotypes of most of the others have yet to be determined. Dual-labelling experiments with anatomical tracers reveal that only a minority of efferent projection neurons of the SCN are immunoreactive for IEG products, and it is likely that the majority of the immunoreactive cells are interneurons or glia. Photic induction of IEGs is mediated via NMDA (N-methyl-D-aspartate) and non-NMDA glutamatergic receptors, the SCN expressing a topographically specific complement of subtypes of the NMDA receptor. Non-photic cues (arousal) can shift the clock but this is not associated with expression of IEGs, demonstrating that the proteins encoded by IEGs are probably involved in transducing photic cues, rather than shifting the clock per se. Their induction provides an anatomically explicit marker for circadian phase and photic sensitivity and so is useful in analyses of circadian function, for example, in the tau mutant hamster. Non-photic phase shifts are accompanied by adrenocortical activation, confirming the importance of arousal in shifting of the clock. The phase-shifting effect of arousal can be blocked by treatment with the serotonin receptor antagonist ketanserin, suggesting that ascending serotonergic input to the forebrain, possibly directly to the SCN, is an important mediator of entrainment by arousal.

Animals↗
PubMed1995

Gating of retinal inputs through the suprachiasmatic nucleus: role of excitatory neurotransmission.

The mammalian circadian clock, located in the hypothalamic suprachiasmatic nucleus (SCN) is important in the regulation of many circadian rhythms, including regulation of pineal gland metabolism and melatonin secretion. Transsection of the optic nerves, disrupting the retinohypothalamic pathway, lesion of the SCN, or lesion of the hypothalamic paraventricular nucleus (PVN) abolish the regulation of pineal serotonin N-acetyltransferase activity by light. Therefore, the pathways linking the retina and the pineal gland must be channelled from the retina through the SCN and the PVN. Many lines of evidence indicate that the major neurotransmitter in the retinal afferents is glutamate. The first aim was therefore to study the retinal target neurons by localising glutamate receptors in the rodent SCN. Using in situ hybridisation, we detected NMDA-R1 and NMDA-R2C mRNA subunits in the SCN. Using immunocytochemistry, immunoreactivity for the AMPA type receptors GluR1, GluR2,3 and GluR4 was also detected in the SCN. Presentation of a short light pulse during the subjective night [i.e. circadian time (CT) 14 or 19], when light induced phase-shifting of activity-rest cycles can be accomplished, also induces expression of the immediate early-genes c-fos and junB in the rodent SCN. The second aim was to use this cellular correlate of behavioural function to determine the location of potential retinal target neurons in the SCN, and to investigate the hypothesis that glutamatergic neurotransmission mediates the effects of light on the circadian system. Thus, the ability of the NMDA receptor antagonist MK-801 to block light-induced c-fos expression in the SCN was studied. In the rat, this antagonist blocked c-fos mRNA expression in a subpopulation of cells in the ventral SCN at doses of 6, but not 2 mg/kg. In contrast, in the hamster both doses blocked light-induced c-fos expression in the ventral SCN. These data provide support for the hypothesis that glutamate mediates effects of light in the SCN, although it appers that the complexes of NMDA receptor subunits, which are involved in light-induced expression of c-fos after light, are relatively insensitive to MK-801. The diversity, heterogeneous distribution, and complexity of glutamate receptor subunits in the SCN suggest that processing of light pulses in the SCN is mediated by several cell types in the SCN. Via an integration process in the clock, the transmission of photic information takes place to other brain structures.

Animals↗