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Notes: 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.

Notes: This study investigated central glutamatergic function in relation to photoperiodically-induced changes in the secretion of luteinizing hormone (LH). The experimental approach was to compare the central effects of glutamate agonists on LH secretion in reproduc-tively active hamsters kept in long days (LD) with those in photoinhibited hamsters kept in short days (SD) for 6 weeks and having regressed testes. Agonists were delivered via a cannula into the III ventricle of freely moving hamsters, and blood samples collected 10 to 15 min after the start of the infusion. A high dose (3.0 nmole) of N-methyl-D-L-aspartate (NMDA) induced significant (P〈0.01) increases in serum concentrations of LH in hamsters in both photoperiods, though the NMDA-induced increase relative to endogenous LH concentrations was greater in SD than in LD. However, a lower dose of NMDA (0.3 nmole) revealed a difference in sensitivity. This dose significantly increased serum LH (P〈0.05) in hamsters in SD but had no effect in those in LD.The seasonal difference in response to NMDA was compared with the response to an equimolar dose of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA agonist. This dose of AMPA (0.3 nmole) induced a two-fold increase (P〈0.05) in serum concentrations of LH in hamsters in both photoperiods, relative to vehicle-treated controls. In a third experiment the dose-response effects of central AMPA on LH secretion were examined more closely. The sensitivity of LH secretion to stimulation with AMPA did not differ between SD- and LD-housed hamsters. Thus the photoperiod-related difference in sensitivity to stimulation with glutamate agonists is specific for NMDA receptor-mediated activation, rather than a passive reflection of differences in the capacity to secrete GnRH/LH in SD and LD photoperiods.To investigate the site of action of NMDA, the expression of the c-fos immediate-early gene, as assessed by immunocytochemistry for its protein product Fos, was used as a marker of neuronal activation, because previous studies in rodents indicate that a high proportion of GnRH neurons express c-fos at the time of the mid-cycle LH surge. NMDA induced widespread expression of c-fos in many periventricular regions including the medial preoptic area (PDA) and ventromedial hypothalamic nucleus. However, dual ICC revealed that in neither photoperiod was Fos present in GnRH-positive neurons 1 h after infusion of 3 nmole of NMDA, despite the increases in LH secretion induced by the infusion. AMPA injected icv at doses which released LH did not enhance expression of c-fos in the hypothalamus. Thus, in the male, enhanced expression of c-fos cannot be detected in GnRH neurons at the time of increased secretion of this hormone induced by glutamate agonists.In conclusion, these results show that both NMDA and non-NMDA glutamatergic pathways potentially regulate LH secretion in the Syrian hamster. The increased sensitivity to NMDA but unaltered sensitivity to AMPA in photoinhibited hamsters in SD is consistent with the view that changes in photoperiod might induce specific alterations in NMDA-mediated pathways that ultimately regulate GnRH neurosecretory activity.

Notes: Siberian hamsters (Phodopus sungorus) transferred from stimulatory photoperiods (long days: LD) to inhibitory photoperiods (short days: SD) undergo testicular regression within 8 weeks. This reproductive response to photoperiod was blocked by systemic daily treatment with the glutamatergic agonist N-methyl-D-aspartate (NMDA: 20 mg/kg BW, sc). This powerful effect of NMDA demonstrates the potential for endogenous glutamate to regulate reproductive function.The overall aim of the subsequent studies was to investigate the site and mechanism of action of this glutamatergic agonist in order to identify potential mechanisms through which endogenous glutamate might act. To investigate whether the effect of systemic NMDA was via an effect on the circadian timing system, alterations in gonadal regression and recrudescence, seasonal coat changes (pelage) and body weight (BW) were examined. It would be predicted that long-term cycles of all these seasonal parameters would be affected if the action of NMDA were to perturb the transduction of photoperiodic information. Daily treatments with NMDA, which initially maintained reproductive function in hamsters exposed to SD, did not influence the time course of subsequent testicular recrudescence, nor did they influence long-term cycles of pelage and BW. Moreover, treatment with NMDA induced a dose-dependent increase in serum concentrations of LH within 15 min of systemic injection. These data are consistent with the hypothesis that systemic NMDA exerts it reproductive effects not via an action on the circadian system, but via an action on secretion of GnRH.To investigate potential central sites of action of glutamate, induction of the immediate early gene c-fos, an acute marker of cellular response, was evaluated immunocytochemically (ICC) in brain areas after treatment with NMDA. Although dual-label ICC studies revealed that NMDA did not induce c-fos within GnRH neurons, NMDA did induce c-fos in many cells in the region of the organum vasculosum of the lamina terminalis (OVLT), an area containing a large number of GnRH perikarya, and in the arcuate nucleus, a region close to GnRH secretory terminals in the median eminence. The lack of c-fos induction in GnRH cells argues against a direct effect of NMDA on GnRH neurons. Thus, we examined immunocytochemically the distribution of the common NMDAR1 glutamate receptor subunit to evaluate further the potential sites of glutamatergic action. As expected, NMDAR1-ir was widespread in perikarya throughout the brain, including the region of the OVLT and the arcuate nucleus. However, NMDAR1-ir was also observed in cell processes in hypothalamic areas, including the median eminence, demonstrating the potential for actions of glutamate on neurosecretion in this structure. Collectively, these pharmacological, endocrine and neuroanatomical studies suggest that NMDA acts to release GnRH when delivered systemically, though not necessarily via a direct action on GnRH neurons. These findings are consistent with the view that glutamate is an important regulator of seasonal changes in reproductive function.

Notes: The circadian clock of the suprachiasmatic nuclei (SCN) of perinatal rodents is entrained by maternally derived cues. The SCN of neonatal Syrian hamsters express high-affinity D1 dopamine receptors, and the circadian activity–rest cycle of pups can be entrained by maternal injection of dopaminergic agonists. The present study sought to characterize the intracellular pathways mediating dopaminergic signalling in neonatal rodent SCN. Both dopamine and the D1 agonist SKF81297 caused a dose-dependent increase in phosphorylation of the transcriptional regulator Ca2+/cyclic AMP response element (CRE) binding protein (CREB) in suprachiasmatic GABA-immunoreactive (-IR) neurons held in primary culture. The D1 antagonist SCH23390 blocked this effect. Dopaminergic induction of pCREB-IR in GABA-IR neurons was also blocked by a protein kinase A (PKA) inhibitor, 5–24, and by the MAPK inhibitor, PD98059, whereas KN-62, an inhibitor of Ca2+/calmodulin-dependent (CAM) kinase II/IV was ineffective. Treatment with NMDA increased the level of intracellular Ca2+ in the cultured primary SCN neurons in Mg2+-free medium, but SKF81297 did not. Blockade of CaM kinase II/IV with KN-62 inhibited glutamatergic induction of pCREB-IR in GABA-IR neurons, whereas 5–24 was ineffective, confirming the independent action of Ca2+- and cAMP-mediated inputs on pCREB. SKF81297 caused an increase in pERK-IR in SCN cells, and this was blocked by 5–24, indicative of activation of MAPK via D1/cAMP. These results demonstrate that dopaminergic signalling in the neonatal SCN is mediated via the D1-dependent activation of PKA and MAPK, and that this is independent of the glutamatergic regulation via Ca2+ and CaM kinase II/IV responsible for entrainment to the light/dark cycle.

Notes: The circadian clockwork of the hypothalamic suprachiasmatic nuclei (SCN) is synchronized by light and by nonphotic cues. The core timing mechanism is cell-autonomous, based on an autoregulatory transcriptional/translational feedback loop of circadian genes and their products. This study investigated the effects of neuropeptide Y (NPY), a potent nonphotic resetting cue, and its interaction with light in regulating clock gene expression in the SCN in vivo. Injection of NPY adjacent to the SCN and transfer to darkness 7 h before scheduled lights out, shifted the circadian activity–rest cycle. Exposure to light for 1 h immediately after NPY infusion blocked this behavioural response. NPY-induced shifts were accompanied by suppression of both mPer1 and mPer2 mRNA in the SCN, assessed 3 h after infusion. mPer mRNAs were not altered 1 h after infusion. Levels of mClock mRNA or mCLOCK immunoreactivity in the SCN were not affected by NPY at either time point. In parallel to the behavioural response, the NPY-induced suppression of mPer genes in the SCN was attenuated when a light pulse was delivered immediately after the infusion. These results identify mPer1 and mPer2 as molecular targets for both photic and nonphotic (NPY-induced) resetting of the clockwork, and support a synthetic model of circadian entrainment based upon convergent up- and downregulation of mPer expression.

Notes: The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights-on. These rhythms are circadian in nature as they continue approximately under free-running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.

Notes: Secretion of gonadotropin-releasing hormone (GnRH) at the median eminence is the essential activator of the reproductive axis. The mechanisms by which embryonic GnRH neurons migrate from the olfactory placode to the preoptic area and then elaborate neurites that course through the hypothalamus to terminate at the median eminence are largely unknown. We investigated the hypothesis that GnRH neurite outgrowth is promoted by brain-derived neurotrophic factor (BDNF) because GnRH neurites course through BDNF-rich areas of the forebrain during their development. Confocal microscopy revealed that most (86%) cultured embryonic GnRH cells tagged with a green fluorescent protein reporter were immunoreactive for TrkB. In primary cultures of E12.5 olfactory tissue, treatment with BDNF induced a dose-dependent increase in neurite outgrowth, but had no discernible effect on branching. BDNF induced phosphorylation of Ca2+/cAMP response element-binding protein (pCREB) in both GnRH and non-GnRH cells in these cultures. This was not associated with phosphorylation of ERK in GnRH-immunoreactive cells, though BDNF treatment did stimulate pERK in neighbouring non-GnRH cells. Promotion of neurite outgrowth is unlikely therefore to result from activation of the Ras-MAPK/ERK pathway. We conclude that the developing GnRH secretory system is directly sensitive to BDNF and that this polypeptide functions as a neurotrophic factor for GnRH neurons.

Notes: 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.

Notes: Precise temporal regulation of transcription is pivotal to the role of the mammalian pineal gland as a transducer of circadian and seasonal information. The circadian clock genes Per1 and Per2 encode factors implicated in temporally gated transcriptional programmes in brain and pituitary. Here we show that the nocturnal circadian expression of Per1 and Per2 in the rat pineal gland parallels that of serotonin N-acetyltransferase (NAT) mRNA, which encodes the rate-limiting enzyme of melatonin biosynthesis. This rhythm is dependent upon an intact sympathetic innervation. Increases in rPer1 (r indicates rat) and rPer2, as well as rNAT, expression during subjective night were blocked completely by superior cervical ganglionectomy (SCGX). In SCGX rats, the β-adrenergic receptor agonist isoproterenol rapidly induced the rPer1 mRNA with dynamics very similar to its effect on rNAT mRNA. In contrast, isoproterenol was without effect on expression of rPer2 mRNA. These findings demonstrate that circadian pineal expression of both rPer1 and rPer2 is controlled by sympathetic afferent innervation, but whereas β-adrenergic signalling regulates rPer1 and rNAT, an alternative route mediates sympathetic regulation over rPer2 expression.

Notes: Abstract: Two experiments investigated the response of the pituitary-gonadal axis of pinealectomized male Syrian hamsters to programmed systemic administration of melatonin. In the first experiment, castrated male Syrian hamsters were housed in a short photoperiod (8L:16D) and maintained on subcutaneous testosterone implants for 7 weeks. These males were then pinealectomized or sham-pinealectomized and their testosterone capsules removed. Daily infusions of melatonin 250 ng/infusion) or its vehicle were administered for 3 weeks; infusion duration was long (11 or 12 hr) or short (6 hr). Measurement of serum luteinizing hormone (LH) following this 3-week period indicated that long-duration melatonin infusions mimicked short-day conditions (LH levels were low), but short-duration infusions did not (LH levels were significantly elevated). In the second experiment, pinealectomized, gonadally intact males were housed in a 12L:12D photoperiod and injected once daily with melatonin or its vehicle, either 3 or 5 hr after dark onset for 11 weeks. These times were chosen to coincide with the light: dark cycle phase that according to published reports is optimally responsive to exogenous melatonin for the induction of short-photoperiodic effects. Melatonin injections did not induce gonadal regression in pinealectomized hamsters. Melatonin and vehicle-treated males responded similarly; their testis widths and serum testosterone levels were not significantly different at the end of the experiment. These results support the hypothesis that the duration of melatonin secretion each night is an important variable in conveying photoperiodic information, but that the circadian phase during which melatonin is present is not.