Biomedical subjects

F J Ebling

Publications and source records attributed to F J Ebling.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1995

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

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↗

PubMed1995

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-[125I]-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗

PubMed1995

Effects of N-methyl-D-aspartate (NMDA) on seasonal cycles of reproduction, body weight and pelage colour in the male Siberian hamster.

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 of 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗

PubMed1994

Photoperiodic differences during development in the dwarf hamsters Phodopus sungorus and Phodopus campbelli.

The dwarf hamsters Phodopus sungorus and to a lesser extent Phodopus campbelli are widely used to study pineal physiology and seasonal reproduction because of their marked responses to photoperiod in adulthood. These species are frequently confused, partly because the common names "Siberian hamster" and "Djungarian hamster" are applied to both, often interchangeably. The current study examines whether the effects of photoperiod on reproductive maturation and prolactin secretion differ between these closely related species. Both male and female P. sungorus and P. campbelli were gestated and raised in long days (LD) of 16 hr light: 8 hr dark or in short days (SD) of 8 hr dark: 16 hr light. They were raised in pairs of the same sex after weaning at 21 days of age. Reproductive organ weights and serum prolactin concentrations were studied at 35 and 70 days of age. Whereas reproductive development proceeded rapidly in LD in both male and female P. sungorus, SD photoperiod completely inhibited growth of the gonads during this study. There were also marked differences in serum prolactin concentrations in both sexes of P. sungorus at all ages: levels were approximately 10-fold higher in LD than in SD. In P. sungorus of both sexes, SD also significantly decreased body weight relative to conspecifics raised in LD. In P. campbelli, reproductive maturation in LD occurred even more rapidly than in P. sungorus. Although significant effects of photoperiod were still apparent, the magnitude of its effect was less marked than in P. sungorus, and a substantial degree of reproductive development was observed in SD-reared males and females.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Photoperiodically induced changes in glutamatergic stimulation of LH secretion in male Syrian hamsters: role of circulating testosterone and endogenous opioids.

In seasonally breeding mammals, the sensitivity of LH secretion to stimulation by the glutamate agonist N-methyl-D-aspartic acid (NMDA) is lower in the sexually active condition than in animals with testicular regression. One experiment determined if this increased sensitivity to NMDA in the reproductively inactive animal reflects reduced circulating testosterone. Responses to NMDA were determined during long days (LD) and short days (SD) in castrated hamsters bearing silastic testosterone implants, designed to maintain constant serum testosterone concentrations throughout a photoperiodically induced seasonal cycle. As expected, no significant effect of NMDA (50 mg/kg BW, sc) on secretion of LH occurred in testosterone-implanted castrate or intact control hamsters when challenged in LD. In contrast, both groups of hamsters responded to this dose of NMDA after 8 weeks exposure to SD, despite the maintenance of high serum testosterone concentrations in the castrate group. Moreover, the increased response to NMDA was not a reflection of lower LH concentrations associated with this photoperiod, because a response to NMDA persisted after removal of implants when endogenous secretion of LH had increased. Thus, the low circulating concentrations of testosterone in male hamsters exposed to an inhibitory SD photoperiod cannot explain the increased response to glutamatergic stimulation in the sexually inactive state. Photoperiod, acting centrally, is the major determinant of the response to activation of NMDA receptors. Other experiments investigated whether the lack of response to glutamatergic stimulation in the LD sexually active state results from endogenous opioid (EOP) tone that inhibits further increases in LH secretion at this stage of the reproductive cycle. If this is so, then pretreatment with an opioid antagonist would reveal or increase the stimulatory effect of NMDA on secretion of LH. Hamsters in LD were pretreated with the opioid antagonist naloxone (NAL; 5 mg/kg BW sc) before NMDA treatment (50 mg/kg BW sc), and blood samples collected 15 min later. Compared with controls, serum LH was significantly elevated in hamsters pretreated with NAL, but NMDA alone did not elevate LH. Surprisingly, LH concentrations in hamsters pretreated with NAL and then injected with NMDA were significantly lower than in hamsters receiving NAL only. Treatment with a submaximal dose of NAL (0.1 mg/kg) did not increase serum LH, nor did it reveal a stimulatory effect of subsequent NMDA treatment. The results demonstrate that the decreased sensitivity to glutamatergic agonists in the sexually active state is not a reflection of masking by inhibitory EOP mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity.

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Non-photic circadian entrainment in the Syrian hamster is not associated with phosphorylation of the transcriptional regulator CREB within the suprachiasmatic nucleus, but is associated with adrenocortical activation.

The mechanisms which mediate non-photic entrainment of the circadian clock are not known. The free-running circadian rhythm of the wheel-running activity of Syrian hamsters was phase advanced by subcutaneous injection of saline and the associated handling at circadian time (CT) 10 (2 h before activity onset, CT 12). Over several experiments, approximately 80% of the animals were sensitive to this non-photic cue and were, therefore, designated as 'shifters'. Injection at CT 6 had no phase-shifting effect in any animals. A series of six injections presented to shifters at CT 10 over 14 circadian cycles produced a cumulative phase advance to the activity rhythm which free ran from the phase established by the final injection, demonstrating that the non-photic stimulus caused true entrainment of the circadian clock, rather than simply masking its expression. Entrainment by light is associated with the rapid phosphorylation of the transcription regulatory factor, cyclic adenosine monophosphate response element binding protein (CREB), in the suprachiasmatic nuclei (SCN), the principal circadian clock. The induction of immunoreactivity to the phosphorylated form of CREB (P-CREB-ir) was used to determine whether phosphorylation is also activated by a non-photic phase-shifting cue. No significant P-CREB-ir was found in shifters maintained under constant dim red light and sampled at either CT 10.05 or 14.05, i.e., times when animals are sensitive to non-photic stimuli and light pulses, respectively. Exposure of animals to light for 5 min at CT 14 caused a dramatic increase in P-CREB-ir within the SCN at CT 14.05. However, despite being sensitive to the phase-shifting effects of handling and injection at CT 10, none of the shifters sampled 5 min after injection of saline at CT 10 exhibited P-CREB-ir within the SCN. These data demonstrate that non-photic cues do not activate a cellular pathway (phosphorylation of CREB) known to be activated within the SCN by light. To assess the importance of arousal in non-photic entrainment, the effect of injection of saline upon activation of the adrenocortical axis, a peripheral index of arousal, was investigated. As anticipated, there was a marked circadian variation in serum cortisol concentrations. In animals identified as shifters, the levels were low during mid-subjective day (CT 6) and rose in advance of activity onset (CT 10). Injection and handling at CT 6 did not have a did not have a significant effect upon serum cortisol levels. In contrast, injection of shifters at CT 10 was associated with a pronounced rise in serum cortisol levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗

PubMed1993

Distribution of N-methyl D-aspartate (NMDA) receptor mRNAs in the rat suprachiasmatic nucleus.

Photic entrainment of the circadian oscillator located in the hypothalamic suprachiasmatic nucleus (SCN) is considered to be mediated at least partly by release of glutamate from the retinal presynaptic nerve terminals acting via a NMDA receptor. Several NMDA receptor subtypes have been cloned and expressed in model systems. The NMDA-R1 subtype is essential for the function of the NMDA receptor, and the multiple NMDA-R2(-A, -B, or -C) subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDA-R1. The aim of this study was to use in situ hybridization histochemistry with oligonucleotide sequences (42-48-mer) labeled with 35S to detect whether NMDA receptor mRNA is present in the rat SCN, and if so, to characterize which receptor subtypes occur. In order to identify the precise location of NMDA receptor mRNAs within the SCN, sections were dipped in emulsion and cellular resolution was achieved. The hybridization revealed a high abundance of NMDA-R1 mRNA in the SCN as well as in many other forebrain areas. The NMDA-R1 expressing cells were distributed throughout the SCN. NMDA-R2A and NMDA-R2B mRNAs were found in the hippocampus, but not in the SCN. In contrast, NMDA-R2C mRNA was found in relative high amounts in the rat SCN, but not in other hypothalamic areas. In dipped sections, it was evident that the localization of NMDA-2RC was mostly confined to the dorsomedial part of the SCN. Thus, the rat SCN contains a specific combination of NMDA receptor mRNA subtypes not found in other forebrain structures. These observations are consistent with the hypothesis that glutamate mediates the effect of light on entrainment of the circadian oscillator.

Animals↗

PubMed1993

Testicular regression in pinealectomized Syrian hamsters following infusions of melatonin delivered on non-circadian schedules.

The aims of this study were to validate the use of a timed infusion protocol for restoring physiologically appropriate rhythms of melatonin in the circulation of pinealectomized hamsters and, using such infusions, to compare the relative importance of the parameters of the nocturnal melatonin signal--frequency, phase, and duration of the interval between signals--in the photoperiodic control of testicular function in male Syrian hamsters. Hamsters were pinealectomized and fitted with a chronic s.c. cannula enabling them to receive timed infusions of melatonin (50 ng/h) or saline vehicle (50 microliters/h). In experiment 1, RIA of serum samples confirmed that s.c. infusions produced a pattern of melatonin in the blood equivalent in amplitude and duration to that observed previously in pineal-intact animals exposed to a short photoperiod. In experiment 2, we investigated the relative importance of the frequency of the melatonin signal and the duration of the interval between signals. Pinealectomized animals that received infusions of saline for 6 wk had large testes and high concentrations of LH in the serum. Animals that received a series of short-day-like infusions of melatonin of 14-h duration, separated by an interval of either 10 h or 6 h, underwent gonadal regression and had low serum concentrations of LH. Animals that received infusions of melatonin of 8-h duration, separated by intervals of 12 h, also exhibited full gonadal collapse. However, animals that received the same 8-h infusions separated by intervals of 8 h (i.e., once every 16 h) did not undergo gonadal regression and their circulating levels of LH remained high.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

19-Aldehydo-4-androstene-3,17-dione: an estrogen precursor that inhibits sebaceous secretion in a rat model.

19-aldehydo-4-androstene-3,17-dione (A; R = CHO), a biogenetic precursor of estrone in the body, has been found to suppress sebum secretion in the ovariectomized testosterone-treated Wistar rat at 1/2000 times the dose of cyproterone acetate required to produce an equivalent effect. The action of this steroid must therefore be analogous to that of an estrogen even though, in striking contrast to estradiol, it is without effect on uterine weight or vaginal cornification. It is postulated that 19-aldehydo-4-androstene-3,17-dione (A; R = CHO) is converted locally into estrone (B) by aromatase present in skin but only in low concentrations in vagina and uterus. The potential of biogenetic precursors (I) of estrogens for therapy of acne or alleviation of the worst effects of skin aging is worth investigation.

Androstenedione↗

PubMed1993

Photoperiod regulates the LH response to central glutamatergic stimulation in the male Syrian hamster.

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 reproductively 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 alpha-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 (POA) 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 regulated 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.

Animals↗

PubMed1993

Hypothalamic versus pituitary stimulation of luteinizing hormone secretion in the prepubertal female lamb.

Glutamate and aspartate have been hypothesized to function as neurotransmitters in the regulation of the gonadotropin-releasing hormone (GnRH) neurosecretory system. We, therefore, determined if hypothalamic stimulation of luteinizing hormone (LH) secretion in the intact prepubertal female lamb could be achieved by intravenous injection of N-methyl-D,L-aspartate (NMA), a glutamate agonist. A pilot study determined a dose of NMA that would induce physiologic pulses of LH (GnRH). Subsequently, we compared the ability of NMA with exogenous GnRH to induce ovulation in the prepubertal lamb when administered chronically. Eighteen prepubertal lambs (21 weeks of age, 34.2 +/- 1.5 kg body weight) were treated intravenously with either NMA (2 mg/kg, n = 6) or GnRH (68 ng/injection or approximately 2 ng/kg, n = 6) for 3 days, every 2 h on day 1 and every 1 h on days 2 and 3, or received no treatment (controls, n = 6). Gonadotropin surges were detected only in GnRH-treated lambs (5/6 lambs, onset = 54.0 +/- 4.5 h from the start of study, mean +/- SE). Compared to 83% of GnRH injections inducing LH pulses, only 47% of NMA injections induced LH pulses. Because each injection of NMA did not induce a pulse of LH, a second experiment was performed in an attempt to optimize the LH response to NMA. Ten prepubertal lambs (25 weeks of age) were injected every 2 h for 24 h with higher doses of NMA, either 4 mg/kg (n = 5) or 16 mg/kg (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Distribution of estrogen receptor-immunoreactive cells in the sheep brain.

The distribution of immunoreactive (IR) estrogen receptor (ER)-containing cells was studied in the brains of adult Suffolk ewes using a rat monoclonal antibody (H222) which recognizes the human estrogen receptor. IR cells were characterized by dense nuclear reaction product, and in some instances, cytoplasmic immunostaining which filled dendrite-like processes. The greatest densities of ER-IR cells were found in the medial preoptic area, the mediobasal hypothalamus, and in a number of limbic system structures (amygdala, bed nucleus of the stria terminalis, lateral septum). ER-IR cells were found at lower densities in several other subregions of the hypothalamus and limbic system, and in the periaqueductal gray of the caudal midbrain. Cytoplasmic ER immunoreactivity was most prominent among ER-IR cells in the ventrolateral-ventromedial nucleus, the bed nucleus of the stria terminalis, the midbrain periaqueductal gray, and some ER-IR cells in the substantia innominata. The distribution of ER-containing cells in the sheep brain closely parallels that seen in other mammals. ER-IR cells are found in sites such as the medial preoptic area and ventrolateral-ventromedial hypothalamus which have been implicated as targets in this species and others for the influence of estradiol on sexual behavior and reproductive neuroendocrine function.

Animals↗

PubMed1992

Non-photic phase shifting of the circadian activity rhythm of Syrian hamsters: the relative potency of arousal and melatonin.

This study investigated the relative potency of melatonin and arousal as Zeitgebers in the non-photic phase shifting of circadian rhythmicity in the adult Syrian hamster. Animals held under dim red light (DD) exhibited robust free-running rhythms of wheel-running activity. Melatonin (1 mg/kg) or ethanolic saline vehicle, delivered manually by subcutaneous injection after removing the animal from its cage, resulted in phase advances of the activity rhythm. This effect was phase dependent, injections at CT 8 and 10 being effective (CT 12 = anticipated activity onset), whereas injection at CT 2, 6, 14 and 20 did not cause a shift. There was no significant difference between the magnitude or timing of phase shifts in response to injections of saline or melatonin. To determine whether the observed shifts were related to arousal of the animals induced by handling, a second group held under DD were fitted with chronic s.c. cannulae so that melatonin solution or vehicle could be delivered remotely at projected CT 10. Neither solution had any effect upon the free-running rhythm. However, when these animals received manual s.c. injection of saline or melatonin solution, they exhibited phase advances similar to those observed in Expt. 1. These results fail to support the hypothesis that melatonin can exert a chemically specific, acute phase-shifting action in the adult Syrian hamster. They do, however, demonstrate the potent effect of arousing stimuli upon the circadian clock in this species.

Animals↗

PubMed1992

Effect of nutritional repletion on pituitary and serum follicle-stimulating hormone isoform distribution in growth-retarded lambs.

Using nutritionally restricted ovariectomized lambs, we tested the hypothesis that nutritionally regulated endogenous increases in GnRH secretion (as assessed by LH pulsatility) not only alter the quantity of FSH present in the pituitary and serum, but also alter the pituitary and serum FSH isoform distribution. Eleven lambs were nutritionally restricted from weaning and ovariectomized at 12 wk of age. Beginning at 56 wk, 6 were fed ad libitum for 14 days, and the other 5 were continued on the restricted diet. Jugular blood samples were collected frequently (12-min interval) for 4 h prior to pituitary removal. Immunoreactive ovine LH (I-oLH) and immunoreactive ovine FSH (I-oFSH) concentrations were measured in sera and pituitary extracts. Bioactive (B) oFSH and I-oFSH isoform distribution patterns were determined in serum pools and pituitary extracts. Ad libitum feeding increased I-oLH pulsatility and mean concentrations of pituitary and serum I-oFSH and B-oFSH. The I-oFSH isoform distribution patterns in the pituitaries from the nutritionally restricted animals were not different from those of repleted lambs; in both, the predominant FSH peak eluted in the pH range of 3.5-5.6. A similar predominance of I-oFSH isoforms was also evident in the serum of ad libitum-fed animals. This predominance was not demonstrable in 3 of the restricted-fed animals due to low circulating concentrations of FSH (less than 2.5 ng/ml). Subsequent studies, utilizing serum from 4 additional restricted-fed lambs with circulating I-oFSH concentrations in the range of 4-14 ng/ml (but no detectable LH pulses) revealed similar predominance of oFSH isoforms in the pH 3.5-5.6 range.(ABSTRACT TRUNCATED AT 250 WORDS)

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