Gonadotrophin-inhibitory hormone and its mammalian orthologue RFamide-related peptide-3: Discovery and functional implications for reproduction and stress.

At the turn of the millennium, a neuropeptide with pronounced inhibitory actions on avian pituitary gonadotrophin secretion was identified and named gonadotrophin-inhibitory hormone (GnIH). Across bird species, GnIH acts at the level of the pituitary and the gonadotrophin-releasing hormone (GnRH) neuronal system to inhibit reproduction. Subsequent to this initial discovery, orthologues of GnIH have been identified and characterised across a broad range of species. In many vertebrates, the actions of GnIH and its orthologues serve functional roles analogous to those seen in birds. In other cases, GnIH and its orthologues exhibit more diverse actions dependent on sex, species, season and reproductive condition. The present review highlights the discovery and functional implications of GnIH across species, focusing on research domains in which the significance of this neuropeptide has been explored most.

The roles of RFamide-related peptide-3 in mammalian reproductive function and behaviour.

To maximise reproductive success, organisms restrict breeding to optimal times of the day or year, when internal physiology and external environmental conditions are suitable for the survival of both parent and offspring. To appropriately coordinate reproductive activity, internal and external standing is communicated to the hypothalamic-pituitary-gonadal axis via a coordinated balance of stimulatory and inhibitory neurochemical systems. The cumulative balance of these mediators ultimately drives the pattern of gonadotrophin-releasing hormone secretion, a neurohormone that stimulates pituitary gonadotrophin secretion. Until 2000, a complementary inhibitor of pituitary gonadotrophin secretion had not been identified. At this time, a novel, avian hypothalamic peptide capable of inhibiting gonadotrophin secretion in cultured quail pituitary cells was uncovered and named gonadotrophin-inhibitory hormone (GnIH). Subsequently, the presence and functional role for the mammalian orthologue of GnIH, RFamide-related peptide, (RFRP-3), was examined, confirming a conserved role for this peptide across several rodent species. To date, a similar distribution and functional role for RFRP-3 have been observed across all mammals investigated, including humans. This overview summarises the role that RFRP-3 plays in mammals and considers the implications and opportunities for further study with respect to reproductive physiology and the neural control of sexual behaviour and motivation.

Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction.

Gonadotrophin-releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin-inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic-pituitary-gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS-1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS-1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS-1, designated KiSS-2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.

Gonadotrophin-inhibitory hormone: a multifunctional neuropeptide.

Gonadotrophin-inhibitory hormone (GnIH) was discovered 8 years ago in birds. Its identification raised the possibility that gonadotrophin-releasing hormone (GnRH) is not the sole hypothalamic neuropeptide that directly influences pituitary gonadotrophin release. Initial studies on GnIH focused on the avian anterior pituitary as comprising the only physiological target of GnIH. There are now several lines of evidence indicating that GnIH directly inhibits pituitary gonadotrophin synthesis and release in birds and mammals. Histological studies on projections from hypothalamic GnIH neurones subsequently implied direct actions of GnIH within the brain and in the periphery. In addition to actions on the pars distalis via the median eminence, GnIH axons and terminals are present in multiple brain areas in birds, and the GnIH receptor is expressed on GnRH-I and -II neurones. Furthermore, we have demonstrated the presence of GnIH and its receptor in avian and mammalian gonads. Thus, GnIH can act directly at multiple levels: within the brain, on the pituitary and in the gonads. In sum, our data indicate that GnIH and its related peptides are important modulators of reproductive function at the level of the GnRH neurone, the gonadotroph and the gonads. Here, we provide an overview of the known levels of GnIH action in birds and mammals. In addition, environmental and physiological factors that are involved in GnIH regulation are reviewed.

Photoperiod and testosterone interact to drive seasonal changes in kisspeptin expression in Siberian hamsters (Phodopus sungorus).

Kisspeptin, a neuropeptide product of the KiSS-1 gene, has recently been implicated in the regulation of seasonal breeding in a number of species, including Siberian hamsters. In this species, kisspeptin expression is reduced in the anteroventral periventricular nucleus (AVPV) following exposure to inhibitory day lengths, and exogenous kisspeptin activates the reproductive neuroendocrine axis of reproductively quiescent animals. Because sex steroids can impact kisspeptin expression, it is unclear whether changes in kisspeptin occur in direct response to photoperiodic cues or secondarily in response to changes in sex steroid concentrations resulting from the transition to reproductive quiescence. The present study aimed to assess the relative contributions of photoperiod and testosterone in regulating kisspeptin expression in Siberian hamsters. Animals housed in long or short day lengths for 8 weeks were either castrated or received sham surgeries. Half of the hamsters in each photoperiod were given testosterone to mimic long-day sex steroid concentrations. The results obtained indicate that kisspeptin neurones in the AVPV and arcuate nuclei were influenced by both photoperiod and testosterone. In the AVPV, removal of testosterone or exposure to inhibitory day lengths led to a marked reduction in kisspeptin-immunoreactive cells, and testosterone treatment increased cell numbers across conditions. Importantly, long-day castrates exhibited significantly more kisspeptin cells than short-day castrates or intact short-day animals with empty capsules, suggesting the influences of photoperiod, independent of gonadal steroids. In general, the opposite pattern emerged for the arcuate nuclei. Collectively, these data suggest a role for both gonadal-dependent and independent (i.e. photoperiodic) mechanisms regulating seasonal changes in kisspeptin expression in Siberian hamsters.

Vasoactive intestinal polypeptide contacts on gonadotropin-releasing hormone neurones increase following puberty in female rats.

Successful reproduction requires precise temporal coordination among various endocrine and behavioural events. The circadian system regulates daily temporal organization in behaviour and physiology, including neuroendocrine rhythms. The main circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sends direct efferents to the reproductive axis via monosynaptic projections to gonadotropin-releasing hormone (GnRH) neurones. This communication generates circadian endocrine rhythms as well as the preovulatory luteinizing hormone (LH) surge necessary for successful ovulation. One SCN peptide thought to be important for the regulation of oestrous cycles is vasoactive intestinal polypeptide (VIP). VIP neurones from the SCN contact GnRH cells, and these cells are preferentially activated during an LH surge in rats. Unlike adult rats, prepubertal females do not exhibit oestrous cycles, nor do they exhibit an LH surge in response to oestradiol positive-feedback. The present study was undertaken to determine the extent to which the development of a 'mature' reproductive axis in female rats is associated with modifications in VIP contacts on GnRH neurones. The brains of diestrus adult (approximately 60 days of age) and prepubertal (21 days of age) female rats were examined using double-label fluorescence immunohistochemistry for VIP and GnRH, with light and confocal microscopy. Although the total number of GnRH-immunoreactive neurones did not differ between adult and prepubertal females, adults had a significant increase in the percentage of GnRH cells receiving VIP contacts compared to juveniles. These data suggest that the development of reproductive hormone rhythms and oestrous cyclicity may be, in part, due to modifications of VIP input to the GnRH system.

Calbindin-D(28K) cells selectively contact intra-SCN neurons.

Calbindin-D(28K)-immunoreactive cells are tightly packed within a discrete region of the caudal aspect of the suprachiasmatic nuclei of hamsters. These cells receive direct retinal input and are Fos-positive in response to a light pulse. Knowledge of their afferent and efferent connections is necessary to understand suprachiasmatic nucleus organization. The first aim of the present study is to identify interconnections between calbindin and other peptidergic cells of the suprachiasmatic nuclei, using epi- and confocal microscopy and intra-suprachiasmatic nucleus tract tracing. The results indicate that essentially all calbindin cells receive numerous appositions from vasoactive intestinal polypeptide (VIP), neuropeptide Y and serotonin fibers and that most receive appositions from gastrin releasing peptide (GRP) and cholecystokinin (CCK) fibers. Reciprocal connections are seen from VIP, GRP and CCK cells but surprisingly, not from dorsomedial vasopressin cells. Injection of biotinylated dextran amine into the suprachiasmatic nucleus indicates that the ventrolateral suprachiasmatic nucleus projects to the entire nucleus, while the dorsal and medial regions of the suprachiasmatic nucleus project densely to most of the nucleus, except to the calbindin region. Analysis of colocalization of the peptides in the calbindin cell region shows that 91% of the substance P cells, 42% of the GRP cells and 60% of the VIP cells in the calbindin subnucleus coexpress calbindin-D(28K). Our results reveal a highly specialized topographical organization of connections among suprachiasmatic nucleus cells.

Food restriction affects the gonadotropin releasing hormone neuronal system of male prairie voles (Microtus ochrogaster).

Individuals of species inhabiting temperate and boreal latitudes optimize the timing of energetically costly processes by curtailing nonessential energetically demanding processes when environmental conditions are not favourable. One proximate environmental variable used to fine-tune moment-to-moment changes in reproductive physiology and behaviour is food intake. The neuroendocrine mechanisms by which food restriction leads to the cessation of reproduction in seasonally breeding rodent species remain largely unspecified. The present study sought to determine the effects of extended food restriction on the gonadotropin releasing hormone (GnRH) neuronal system. Male prairie voles (Microtus ochrogaster) were either fed ad libitum or were exposed to either 1, 2 or 3 weeks of moderate (70% of daily mean) food restriction. In accordance with previous studies of food restriction, gross reproductive organ masses and body mass were unaffected by food deprivation. Although 1 week of food restriction did not result in alterations in the GnRH neuronal system, food restriction for 2 weeks was associated with increased GnRH-immunoreactive (GnRH-ir) neurone soma size. Three weeks of food restriction resulted in a pronounced increase in GnRH-ir neurone numbers, as well as an increase in fibre intensity in the main fibre pathway to the median eminence. Taken together, these findings suggest that extended food restriction leads to modifications in the GnRH neuronal system, providing a means for temporary cessation of reproduction without gross alterations in reproductive physiology. This transient change in the hypothalmo-pituitary-gonadal axis, without pronounced changes in reproductive organ morphology, likely provides a mechanism for the rapid reinitiation of breeding in nature when local conditions provide adequate food availability.

Photoperiodic polyphenisms in rodents: neuroendocrine mechanisms, costs, and functions.

Annual changes in daylength figure prominently in the generation of seasonal rhythms in reproduction, and a wide variety of mammals use ambient photoperiod as a proximate cue to time critical reproductive events. Nevertheless, within many reproductively photoperiodic mammalian species, there exist individuals--termed "photoperiod nonresponders"--that fail to adopt a seasonal breeding strategy and instead exhibit reproductive competence at a time of year when their conspecifics are reproductively quiescent. Photoperiod nonresponsiveness has been principally characterized by laboratory observations--over half of the species known to be reproductively photoperiodic contain a proportion of nonresponsive individuals. The study of nonresponders has generated basic insights regarding photic regulation of reproduction in mammals. The neuroendocrine mechanisms by which the short-day photoperiodic signal is degraded or lost in nonresponders varies between species: differences in features of the circadian pacemaker, which provides photoperiodic input to the reproductive neuroendocrine system, have been identified in hamsters; changes in the responsiveness of hypothalamic gonadotrophs to melatonin and as-yet-unspecified inhibitory signals have been implicated in voles and mice. Individuals that continue to breed when their conspecifics refrain might enjoy higher fitness under certain circumstances. Statements regarding the adaptive function of reproductive nonresponsiveness to photoperiod require additional information on the costs (metabolic and fitness) of sustaining reproductive function during the winter months and how these costs vary as a function of environmental conditions. Reproductive nonresponders thus continue to represent a challenge to theories that extol the adaptive function of seasonality. Several nonexclusive hypotheses are proposed to account for the maintenance of nonresponsive individuals in wild rodent populations.

In vitro melatonin treatment enhances cell-mediated immune function in male prairie voles (Microtus ochrogaster).

The present study was designed (1) to determine the extent to which male prairie voles (Microtus ochrogaster) alter immune status in response to short-day lengths, (2) to evaluate the role of melatonin in coordinating these alterations in immune function, and (3) to assess the association between alterations in immune function and reproductive responsiveness to photoperiod. Male voles were housed in either long- or short-day lengths for 10 wk; voles in short days were subdivided into reproductive "responders" (R) or "non-responders" (NR) based on testicular mass at autopsy. After 10 wk of exposure to photoperiodic conditions, cell-mediated immune function was evaluated using an in vitro splenocyte proliferation assay. The direct effects of melatonin on immune cells were evaluated by adding melatonin to one-half of the cultures in each experimental condition. Melatonin treatment led to enhanced splenocyte proliferation for all experimental groups. Neither photoperiodic condition nor reproductive status was associated with alterations in immune function or the degree of immuno-enhancing effects of melatonin. Taken together, the results of the present study suggest that melatonin is capable of enhancing immune function in male voles potentially by acting directly on immune cells.

Circadian organization in male mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-).

Circadian (approximately 24 h) rhythms in physiology and behavior are generated by the bilateral suprachiasmatic nucleus (SCN) of the anterior hypothalamus. For these endogenous rhythms to be synchronized with the external environment, light information must be transmitted to pacemaker cells within the SCN. This transmission of light information is accomplished via a direct retino-hypothalamic tract (RHT). Nitric oxide (NO), an endogenous gas that functions as a neurotransmitter, has been implicated as a messenger necessary for photic entrainment. Three isoforms of the enzyme that form NO, NO synthase, have been identified (a) in neurons (nNOS), (b) in the endothelial lining of blood vessels (eNOS), and (c) as an inducible form in macrophages (iNOS). The present study was undertaken to determine the specific role of eNOS in circadian organization and photic entrainment. Wild-type (WT) and eNOS-/- mice were initially entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was phase advanced. After an additional 3 weeks, animals were held in constant darkness (DD). eNOS-/- animals did not exhibit a deficit in the ability to entrain to the LD cycle, phase-shift locomotor activity, or free-run in constant conditions. Animals held in DD were killed after light exposure during either the subjective day or the subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos protein expression in the SCN of both WT and eNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that endothelial isoform of NOS may not be necessary for photic entrainment in mice.

Photoperiod and temperature interact to affect the GnRH neuronal system of male prairie voles (Microtus ochrogaster).

Individuals of numerous species limit energy expenditure during winter by inhibiting reproduction and other nonessential functions. To time these adaptations appropriately with the annual cycle, animals rely on environmental cues that predict, well in advance, the onset of winter. The most commonly studied environmental factor that animals use to time reproduction is photoperiod. Rodents housed in short photoperiods in the laboratory or in naturally declining day lengths exhibit pronounced alterations in reproductive function concomitant with alterations in the hypothalamic gonadotropin-releasing hormone neuronal system. Because animals in their natural environment use factors in addition to photoperiod to time reproduction, the present study sought to determine the independent effects of photoperiod and temperature, as well as the interaction between these factors, on reproductive parameters and the GnRH neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 10 weeks. Animals in each photoperiod were further subdivided into groups housed in either mild (i.e., 20 degrees C) or low (i.e., 8 degrees C) temperatures. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods and long-day voles housed in low temperatures (and maintained large gonads) exhibit higher GnRH-immunoreactive (GnRH-ir) neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to all other groups. In addition, voles that underwent gonadal regression in response to both short days and low temperatures did not exhibit an increase in GnRH-ir neuron numbers compared to long-day, mild-temperature controls. These data suggest that photoperiod and temperature interact to influence reproductive function potentially by alterations of the GnRH neuronal system.

Temperature and photoperiod interact to affect reproduction and GnRH synthesis in male prairie voles.

Prairie voles (Microtus ochrogaster), like most rodent species, exhibit a phenotypic polymorphism in reproductive response to winter conditions or to short day lengths in the laboratory. Laboratory studies on seasonally breeding species have traditionally focused on the role of photoperiod in modulating reproduction and other seasonal adaptions. However, because animals use proximate environmental factors in addition to photoperiod to phase seasonal adaptions with the appropriate time of year, the present study investigated the interaction of photoperiod and temperature on reproductive function and the gonadotropin-releasing hormone (GnRH) neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) photoperiods. Voles in each photoperiodic condition were also exposed to either mild (20 degrees C) or low (8 degrees C) temperatures. After 10 weeks, voles were killed and their brains were processed using in situ hybridization for mRNA for proGnRH. The results suggest that GnRH synthesis is not affected by exposure to a single inhibitory proximate factor (i.e. short days or low temperatures alone), even when reproduction is inhibited, whereas a combination of inhibitory proximate factors leads to a decrease in GnRH synthesis (i.e. fewer neurones staining for mRNA for proGnRH). These data suggest that the neuroendocrine mechanisms regulating seasonal alterations in reproductive function are likely to differ between harsh and mild winters.

Leptin, but not immune function, is linked to reproductive responsiveness to photoperiod.

Energetic demands are high while energy availability is minimum during winter. To cope with this energetic bottleneck, animals exhibit numerous energy-conserving adaptations during winter, including changes in immune and reproductive functions. A majority of individual rodents within a population inhibits reproductive function (responders) as winter approaches. A substantial proportion of small rodents within a species, however, fails to inhibit reproduction (nonresponders) during winter in the field or in the laboratory when maintained in winter-simulated day lengths. In contrast, immune function is bolstered by short day lengths in some species. The specific mechanisms that link reproductive and immune functions remain unspecified. Leptin is a hormone produced by adipose tissue, and several studies suggest that leptin modulates reproductive and immune functions. The present study sought to determine if photoperiodic alterations in reproductive function and leptin concentrations are linked to photoperiod-modulated changes in immune function. Siberian hamsters (Phodopus sungorus) were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 9 wk. After 9 wk, blood samples were collected during the middle of the light and dark phase to assess leptin concentrations. One week later, animals were injected with keyhole limpet hemocyanin to evaluate humoral immunity. Body mass, body fat content, and serum leptin concentrations were correlated with reproductive responsiveness to photoperiod; short-day animals with regressed gonads exhibited a reduction in these measures, whereas short-day nonresponders resembled long-day animals. In contrast, immune function was influenced by photoperiod but not reproductive status. Taken together, these data suggest that humoral immune function in Siberian hamsters is independent of photoperiod-mediated changes in leptin concentrations.

Ejaculatory abnormalities in mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-).

Nitric oxide (NO) has been established as a neurotransmitter in both the central and peripheral nervous systems. Three isoforms of its synthetic enzyme, NO synthase (NOS), have been identified: 1) in the endothelial lining of blood vessels (eNOS), 2) an inducible form found in macrophages (iNOS), and 3) in neurons (nNOS). Previous studies using pharmacological agents that block all three isoforms of NOS have revealed that NO mediates several aspects of reproductive physiology and behavior, including anomalies in male sexual behavior and erectile function. To determine the specific contribution of the endothelial isoform of NOS in male reproductive behavior, we studied mice missing the gene for only eNOS (eNOS-/-). Wild-type (WT) and eNOS-/- animals were placed with an estrous WT female and observed for 45 min. Both WT and eNOS-/- mice displayed equivalent motivation to mount the stimulus female. However, eNOS-/- mice exhibited striking anomalies in ejaculatory function. A higher percentage of eNOS-/- than WT mice ejaculated during the testing period (p < 0.001). This increased propensity to ejaculate was apparently due to reduced stimulation required to elicit ejaculation; eNOS-/- mice required significantly fewer mounts (p < 0.003) and intromissions (p < 0.001) to ejaculate compared to WT mice. Taken together, these results suggest that NO synthesized by eNOS may be involved in ejaculatory physiology, but not sexual motivation.

Effects of photoperiod and reproductive responsiveness on pituitary sensitivity to GnRH in male prairie voles (Microtus ochrogaster).

In order to promote survival and reproductive success, many nontropical rodents inhibit reproduction well in advance of winter in response to decreasing day lengths. Male prairie voles (Microtus ochrogaster), small temperate zone rodents, vary in their reproductive response to photoperiod. Some male voles undergo complete gonadal regression when housed in short days (responders) whereas others fail to inhibit reproduction when exposed to short (i. e., <12 h light/day) day lengths (nonresponders). Previous research has shown that phenotypic variation in reproductive response is reflected at the level of the hypothalamic gonadotropin-releasing hormone (GnRH) neuronal system. The present study sought to determine if photoperiod or reproductive condition alters pituitary responsiveness to a GnRH challenge. Animals were housed in either long (LD 16:8) or short (LD 8:16) photoperiods for 10 weeks. Subsequently, short-day voles were separated into responders and nonresponders based on testicular size. To reduce the influence of endogenous testosterone on luteinizing hormone concentrations, half of the animals in each group were castrated. All animals were injected (i.p.) with either 100 or 50 ng of GnRH and a blood sample was collected after 15 min. Although castration resulted in a significant increase in LH concentrations (P < 0.05), neither photoperiod nor reproductive condition affected LH concentrations in response to a GnRH challenge (P > 0.05). Taken together, these data support the hypothesis that short photoperiods lead to reproductive inhibition by acting at the level of the hypothalamus rather than the pituitary.

Elimination of aggressive behavior in male mice lacking endothelial nitric oxide synthase.

Male mice with targeted deletion of the gene encoding the neuronal isoform of nitric oxide synthase (nNOS(-/-)) display increased aggressive behavior compared with wild-type (WT) mice. Specific pharmacological inhibition of nNOS with 7-nitroindazole also augments aggressive behavior. We report here that male mice with targeted deletion of the gene encoding endothelial NOS (eNOS(-/-)) display dramatic reductions in aggression. The effects are selective, because an extensive battery of behavioral tests reveals no other deficits. In the resident-intruder model of aggression, resident eNOS(-/-) males show virtually no aggression. Latency for aggression onset is 25-30 times longer in eNOS(-/-) males compared with WT males in the rare instances of aggressive behaviors. Similarly, a striking lack of aggression is noted in tests of aggression among groups of four mice monitored in neutral cages. Although eNOS(-/-) mice are hypertensive ( approximately 14 mmHg blood pressure elevation), hypertension does not appear responsible for the diminished aggression. Reduction of hypertension with hydralazine does not change the prevalence of aggression in eNOS(-/-) mice. Extensive examination of brains from eNOS(-/-) male mice reveals no obvious neural damage from chronic hypertension. In situ hybridization in WT animals reveals eNOS mRNA in the brain associated exclusively with blood vessels and no neuronal localizations. Accordingly, vascular eNOS in the brain appears capable of influencing behavior with considerable selectivity.

Photoperiod affects the gonadotropin-releasing hormone neuronal system of male prairie voles (Microtus ochrogaster).

In order to maximize survival, animals inhabiting temperate and boreal latitudes exhibit numerous adaptations to changing seasons. Central among this suite of coping strategies is the cessation of breeding during the suboptimal conditions of winter. Many nontropical rodents inhibit reproduction well in advance of winter in response to short day lengths. Male prairie voles (Microtus ochrogaster) are small temperate-zone rodents that vary in their reproductive response to photoperiod. Some male voles undergo complete gonadal regression during short days (responders) while others fail to inhibit reproduction when exposed to short day lengths (nonresponders). The neuroendocrine mechanisms regulating this differential response to photoperiod have not been investigated in this species. Presumably, photoperiod can act at any or all levels of the hypothalamo-pituitary-gonadal (HPG) axis to regulate reproduction. The present study sought to determine the contribution of the GnRH system to this variable reproductive response to photoperiod. Male prairie voles were housed in either long or short day lengths for 10 weeks. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods exhibited increased GnRH neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to both long-day animals and short-day voles that maintained reproductive function. Mean optical density of staining and cell size did not differ among groups. These data suggest that the differential reproductive response to photoperiod in male voles is mediated, in part, by alterations in the GnRH neuronal system.

Nocturnal motor coordination deficits in neuronal nitric oxide synthase knock-out mice.

Nitric oxide is formed in the brain primarily by neurons containing neuronal nitric oxide synthase (nNOS), though some neurons may express endothelial NOS (eNOS), and inducible NOS (iNOS) only occurs in neurons following toxic stimuli. Mice with targeted disruption of nNOS (nNOS-) display distended stomachs with hypertrophied pyloric sphincters reflecting loss of nNOS in myenteric plexus neurons. nNOS- animals resist brain damage following middle cerebral artery occlusions consistent with evidence that excess release of nitric oxide mediates neurotoxicity in ischemic stroke. Neuronal NOS- mice have no grossly evident defects in locomotor activity, breeding long-term depression in the cerebellum, long-term potentiation in the hippocampus, and overall sensorimotor function. However, nNOS- animals display excessive, inappropriate sexual behavior and dramatic increases in aggression. Because the cerebellum possesses the greatest levels of nNOS neurons in the brain, it was surprising that presumed cerebellar functions such as balance and coordination were grossly normal in nNOS- mice. These previous studies were all conducted during the day (between 1400 and 1600, lights on at 0700). We now report striking, discrete abnormalities in balance and motor coordination in nNOS-mice reflected selectively at night.

Circadian locomotor analysis of male mice lacking the gene for neuronal nitric oxide synthase (nNOS-/-)

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.