Neural production of 17β-oestradiol via aromatisation of testosterone may play a critical role in rapid, non-genomic regulation of physiological and behavioural processes. In brain nuclei implicated in the control of sexual behaviour, sexual or stressfull stimuli induce respectively a rapid inhibition or increase in preoptic aromatase activity (AA). Here, we tested quail that were either non-stressed or acutely stressed (15 min restraint) immediately prior to sexual interaction (5 min) with stressed or non-stressed partners. We measured nuclei-specific AA changes, corresponding behavioural output, fertilisation rates and corticosterone (CORT) concentrations. In males, sexual interaction rapidly reversed stress-induced increases of AA in the medial preoptic nucleus (POM). This time scale (<5min) highlights the dynamic potential of the aromatase system to integrate input from stimuli that drive AA in opposing directions. Moreover, acute stress had minimal effects on male behaviour suggesting that the input from the sexual stimuli on POM AA may actively preserve sexual behaviour despite stress exposure. We also found distinct sex differences in contextual physiological responses: while males did not show any effect of partner status, females responded to both their stress exposure and the male partner’s stress exposure at the level of circulating CORT and AA. In addition, fertilisation rates and female CORT correlated with the male partner’s exhibition of sexually aggressive behaviour suggesting that female perception of the male can affect their physiology as much as direct stress. Overall, male reproduction appears relatively simple – sexual stimuli, irrespective of stress, drives major neural changes including rapid reversal of stress-induced changes of AA. In contrast, female reproduction appears more nuanced and context specific, with subjects responding physiologically and behaviourally to stress, the male partner’s stress exposure, and female-directed male behaviour.
Oestrogens; acute stress; sexual behavior; sex differences; medial preoptic nucleus
Oestrogen receptor (ER)α and ERβ are members of the ligand-activated superfamily of nuclear receptors and mediate most facets of oestrogen signalling. Several naturally occurring splice variants of each ER have been identified in the human brain, yet the biological significance of these splice variants in the brain remains unknown. In the present study, we exploit the unique structural differences of the human ERβ splice variants to determine the functional significance of individual ER domains in the brain. We previously established that full-length rodent ERβ (i.e. rERβ1) has constitutive transcriptional activity in neuronal cells in the absence of ligand. By contrast to the rodent splice variants, the human ERβ splice variants used in the present study contain varying length truncations of exon 8, which encodes for the E/F domains. Our results reveal that, in neuronal cells, each human-specific ERβ splice variant constitutively activated promoters mediated by a canonical oestrogen response element and repressed promoters mediated by activator protein-1 sites via p38 activity. From these data, we conclude that the C-terminus, encoding the AF-2 region and F domain, is not essential for the constitutive properties of human ERβ. Taken together, these studies show that human-specific ERβ variants are constitutively active and also provide novel insight into the contributions of the functional domains of ERβ towards mediating constitutive transcription at various promoters in neuronal cells.
oestrogen receptor β; splice variants; ERE; AP-1; vasopressin; neurones
Cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. Here, we review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process.
cAMP; cGMP; protein kinase A; protein kinase G; voltage-gated channels
Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3 (Kv1.3−/−), increases olfactory sensitivity and discriminatory ability, and causes resistance to diet-induced obesity (DIO) in mice. The objective of the present study was to determine if the enhanced olfactory ability of the Kv1.3−/− mouse contributes to the resistance to DIO. Kv1.3+/+ and Kv1.3−/− mice were subject to bilateral olfactory bulbectomy (OBX) or sham surgery at 9 weeks of age and placed on either a control chow diet (CF) or a 32% moderately high-fat diet (MHF). Caloric and water intake, locomotor activity, and oxygen consumption were monitored after 5 weeks of diet treatment. At the end of 26 weeks of diet treatment, fat pad weight and blood chemistry were evaluated. Kv1.3+/+ mice exhibited a significant increase in weight, adiposity, fasting glucose and fasting leptin in response to the MHF-diet, with or without OBX. When treated with a MHF-diet, Kv1.3−/− mice gained significantly less weight than Kv1.3 +/+ mice and exhibited a significant increase in light phase metabolism. OBX of Kv1.3−/− mice prevented the resistance to DIO and concomitant upregulation of light phase metabolism while decreasing dark phase metabolism and total energy expenditure. These findings suggest that pathways activated in Kv1.3 −/− that increased energy expenditure and led to resistance to DIO are olfactory bulb dependent. Thus, these findings add to a growing body of evidence suggesting that the olfactory system can modulate pathways involved in the regulation of energy balance.
Olfactory bulbectomy; diet-induced obesity; super-smeller; potassium channel; oxygen consumption
The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurons. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurons. The aim of this study was to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0kb promoter activity in PR-B expressing cells, but a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the −1403 to −1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation (ChIP) analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposite effects on PR-B mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene and that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.
catecholamine; tyrosine hydroxylase; progesterone receptor; gene expression; transcriptional regulation; steroid hormones
Exposure to stress during early development causes long-lasting alterations in behavior and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotropin releasing hormone (CRH). To determine whether early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8-week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post-birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH hnRNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percent of methylation in 2 CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP-responsive element (CRE) at −230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel-shift assays, using nuclear proteins from forskolin treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band which was supershifted by phospho-CREB antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra-CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.
maternal deprivation; corticotropin releasing hormone; DNA methylation; HPA axis; CRH
GnRH neurones fire spontaneous bursts of action potentials, but little is understood about the underlying mechanisms. Here we show evidence for two types of bursting/oscillation driven by different mechanisms. Properties of these different types are clarified using mathematical modeling and a recently developed active-phase/silent-phase correlation technique. The first type of GnRH neurone (1–2%) exhibits slow (~0.05Hz) spontaneous oscillations in membrane potential. Action potential bursts are often observed during oscillation depolarization, but some oscillations were entirely subthreshold. Oscillations persist after blockade of fast sodium channels with TTX and blocking receptors for ionotropic fast synaptic transmission, indicating they are intrinsically generated. In the second type of GnRH neurone, bursts were irregular and TTX caused a stable membrane potential. The two types of bursting cells exhibited distinct active-phase/silent-phase correlation patterns, which is suggestive of distinct mechanisms underlying the rhythms. Further studies of type 1 oscillating cells revealed that the oscillation period was not affected by current or voltage steps, although amplitude was sometimes damped. Oestradiol, an important feedback regulator of GnRH neuronal activity, acutely and markedly altered oscillations, specifically depolarizing the oscillation nadir and initiating or increasing firing. Blocking calcium-activated potassium channels, which are rapidly reduced by oestradiol, had a similar effect on oscillations. Kisspeptin, a potent activator of GnRH neurones, translated the oscillation to more depolarised potentials, without altering period or amplitude. These data show that there are at least two distinct types of GnRH neurone bursting patterns with different underlying mechanisms.
burst; oscillation; hypothalamus; neuroendocrine; parabolic
Thyroid transcription factor 1 (TTF1), a member of the NK family of transcription factors required for basal forebrain morphogenesis, functions in the postnatal hypothalamus as a transcriptional regulator of genes encoding neuromodulators and hypophysiotrophic peptides. One of these peptides is gonadotropin-releasing hormone (GnRH). Here we show that Ttf1 mRNA abundance vary in a diurnal and melatonin-dependent fashion in the preoptic area (POA) of the rat, with maximal Ttf1 expression attained during the dark phase of the light/dark cycle, preceding the nocturnal peak in GnRH mRNA content. GnRH promoter activity oscillates in a circadian manner in GT1-7 cells, and this pattern is enhanced by TTF1 and blunted by siRNA-mediated Ttf1 gene silencing. TTF1 trans-activates GnRH transcription by binding to two sites in the GnRH promoter. Rat GnRH neurons in situ contain key proteins components of the positive (BMAL1, CLOCK) and negative (PER1) limbs of the circadian oscillator, and these proteins repress Ttf1 promoter activity in vitro. In contrast, Ttf1 transcription is activated by CRY1, a clock component required for circadian rhythmicity. In turn, TTF1 represses transcription of Rev-erbα, a heme receptor that controls circadian transcription within the positive limb of the circadian oscillator. These findings suggest that TTF1 is a component of the molecular machinery controlling circadian oscillations in GnRH gene transcription.
circadian rhythm; GnRH gene expression; clock genes; TTF1; transcriptional control
We have previously has demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone (P) in the facilitation of female reproductive behavior in oestradiol benzoate (EB)-primed female rats and mice. We also reported that both P- and SKF-initiated signalling was mediated by cAMP-dependent PKA signal transduction cascade. As the rapid effects of P are also mediated by calcium-dependent kinases, calcium and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKCin the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, the two regions implicated in the regulation of female reproductive behavior in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro. We report that in contrast to P-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not due to changes in PKC protein levels or phosphorylation status. These data indicate that P and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.
Emerging evidence support a role of purinergic P2X3 receptors in modulating nociceptive signaling in sensory neurons. Previously we showed that DRG neurons (L1-S1) express both ERα and ERβ receptors. In this study we investigated the expression of P2X3 receptors and the effect of 17β-estradiol (E2) on ATP-induced [Ca2+]i increase in DRG neurons collected from C57Bl/6J, ERαKO and ERβKO mice. Our data showed a significant decrease for P2X3 in ERαKO (all levels) and ERβKO (mostly observed in L1, L2, L4, and L6). Furthermore, 17β-estradiol (100 nM) significantly attenuated the ATP (10 μM)-induced [Ca2+]i in C57Bl/6J mice. ERs antagonist ICI 182,780 (1μM) blocked this attenuation. Homomeric P2X3 receptors are plentifully expressed in DRG neurons and contribute to nociceptive signals. α,β-me ATP which is a specific agonist of P2X2/3 receptors showed similar responses to the ATP-induced calcium increase in knock-out mice. A membrane-impermeable E-6-BSA (1μM) had the same effect as E2 suggesting action on the membrane. In DRG neurons from ERβKO and WT miceE2 attenuated the ATP/α,β-me ATP-induced [Ca2+]i fluxes but in DRG neurons from ERαKO mice, this hormone had no effect suggesting that this attenuation depends on membrane-associated ERα receptors. Together our data indicate an interaction between P2X3 and membrane-associated ERα in primary sensory neurons that may represent a novel mechanism to explain sex differences observed in clinical presentation of visceral nociceptive syndromes.
17β-estradiol (E2); DRG; P2X3; ATP; Ca2+; ERα/ERβ
Incidence of ovulatory disorders is common in obese animal models. The mechanism behind this effect is not clear. We hypothesized that high fat (HF) diet induces alterations in neuroendocrine mechanisms resulting in anovulation in diet-induced obese (DIO) animals.
Adult female DIO and diet-resistant (DR) rats were fed either chow or HF diet (45% calories from fat) for 6 weeks. Oestrous cyclicity and body weight were monitored regularly. At the end of treatment, rats were implanted with a jugular catheter to monitor luteinising hormone (LH) levels on the day of prooestrous. Rats were sacrificed on the following prooestrous, their brains and ovaries were collected. Plasma from trunk blood was analyzed for oestradiol and leptin concentrations. Ovaries were fixed and sectioned for histological analysis. Brains were removed, frozen and sectioned and norepinephrine (NE) concentrations in discrete hypothalamic areas were measured using HPLC-EC.
HF diet affected oestrous cyclicity in both DIO and DR rats with the effect being more pronounced in DIO animals. HF diet increased leptin levels in both DIO and DR rats. Oestradiol levels were low in the DIO-HF group. NE levels in the hypothalamus were unaffected by HF diet or genotype. A normal LH surge was observed in DR-Chow rats and LH levels were low in the rest of the groups.
DIO rats have an inherently reduced reproductive capacity and exposure to a HF diet decreases it further. A reduction in oestradiol and LH surge levels could contribute to this effect, however the underlying mechanisms need to be studied further.
leptin; obesity; oestradiol; norepinephrine; oestrous cycles
We examined the role of the androgen receptor (AR) in the investigatory behaviour of conspecifics using mice carrying the testicular feminisation mutation (XTfmY). Responses to members of the same and opposite sex were evaluated in a habituation / dishabituation task. Adult mice were gonadectomised and treated with oestradiol (E2) or testosterone. After E2 treatment, regardless of the sex of the stimulus mouse, wild-type (WT) males engaged in significantly more investigation than WT females. XTfmY males treated with E2 showed ‘male-like’ behaviour in response to a male but behaved ‘female-like’ when the stimulus was a female. Because WT and XTfmY males behaved the same in response to another male, we used two additional mouse models to ask whether sex chromosomes were responsible for this phenomenon. Regardless of sex chromosome complement, gonadal males displayed high levels of investigation. When mice were treated with testosterone, investigation by WT females was enhanced, which eliminated the sex differences. Most strikingly, XTfmY males receiving testosterone-treatment increased the investigation of females to levels equal to those shown by WT mice. Given that testosterone, but not its metabolite E2, caused XTfmY males to investigate female conspecifics at high levels, it is plausible that nonclassical actions of AR, and / or activation of a novel AR, may be involved in this behaviour. Taken together, our data show that AR activation during adulthood is not required for males to investigate mice of either sex. However, ‘male-like’ levels of investigation of a female stimulus may depend on neonatal activation of the classic nuclear AR.
androgen receptor; testicular feminisation mutation; CAIS; sex differences; social investigation; complete androgen insensitivity
The supraoptic nucleus (SON) is a particularly good model for the study of cell-specific gene expression since it contains two distinct neuronal phenotypes, the oxytocin (OXT) and vasopressin (AVP) synthesizing magnocellular neurons (MCNs). The MCNs are found in approximately equal numbers and selectively express either the OXT or the AVP gene in about 97% of the MCN population in the SON. An unresolved issue has been to determine what mechanisms are responsible for the highly selective regulation of the cell-type specific expression of OXT and AVP genes in the MCNs. Previous attempts to address this question used various bioinformatic and molecular approaches, which included using heterologous cell lines to study the putative cis-elements in the OXT and AVP genes, and the use of OXT and/or AVP transgenes in transgenic rodents. The data from all of the above studies identified a region <0.6kbp upstream of OXT exon I and about 3kb upstream of AVP exon I as being sufficient to produce cell-specific expression of the OXT and AVP genes, respectively, but failed to identify the specific cis-domains responsible for the MCN-specific gene expression. An alternative experimental approach to perform promoter deletion analysis in vivo, that is to use stereotaxic viral vector gene transfer into the SON in order to further dissect the cis-elements in the OXT and AVP genes, will be described here. This in-vivo method uses Adeno-Associated Viral (AAV) vectors expressing OXT-promoter deletion constructs and utilizes the enhanced green fluorescent protein (EGFP) as the reporter. The AAV constructs are stereotaxically injected into the rat brain above the SON and 2 weeks post injection the rats are sacrificed and assayed for EGFP expression. Using this method it has been possible to identify specific regions upstream of the transcription start site (TSS) in the OXT and AVP gene promoters which are responsible for conferring the cell-type specificity of the OXT and AVP gene expression in the SON.
cell-type specific gene expression; transcription factors; transcription factor binding sites; magnocellular neurons; phenotype
Synaptic activity in magnocellular neurosecretory neurones is influenced by the retrograde (i.e., somatodendritic) release of vasopressin, oxytocin and cannabinoids (CBs). For oxytocin neurones, oxytocin exerts constitutive effects on presynaptic activity through its ability to release CBs postsynaptically. In the present study we examined evoked and spontaneous inhibitory postsynaptic currents (IPSCs) in identified vasopressin (VP) neurones in coronal slices from virgin rats to determine: 1) the extent to which CBs may also tonically modulate VP synaptic activity; and 2) to determine if depolarization induced suppression of inhibition was present in VP neurones, and if so, whether it was mediated by VP or CBs. The CB1 antagonists AM251 (1 μM) and SR14171 (1 μM) consistently increased the frequency of spontaneous IPSCs (sIPSCs) in VP neurones without affecting their amplitude, suggesting a tonic CB presence. This effect on frequency was independent of action potential activity, and blocked by chelating intracellular calcium with10 mM EGTA. AM251 also increased the amplitude of evoked IPSCs (eIPSCs) and decreased the paired-pulse ratio (PPR) in VP neurones- effects that were completely blocked with even low (1 mM EGTA) internal calcium chelation. Bouts of evoked firing of VP neurones consistently suppressed sIPSCs, but had no effect on eIPSCs or the PPR. This depolarization-induced suppression of IPSCs was reduced by AM251, and was totally blocked by 10 μM of the mixed vasopressin/oxytocin antagonist, Manning Compound. We then tested the effect of vasopressin on IPSCs while blocking CB1 receptors. Vasopressin (10-100 nM) inhibited sIPSC frequency, but had no effect on sIPSC or eIPSC amplitudes, or on the PPR, in the presence of AM251. Together these results suggest a tonic, presynaptic inhibitory modulation of IPSCs in VP neurones by CBs that is largely dependent on postsynaptic calcium, and an inhibitory effect of VP on IPSCs that is independent of CB release.
supraoptic; paraventricular; GABA; vasopressin; oxytocin
Classically, glia have been regarded as non-excitable cells that provide nourishment and physical scaffolding for neurons. However, it is now generally accepted that glia are active participants in brain function that can modulate neuronal communication via several mechanisms. Investigations of anatomical plasticity in the magnocellular neuroendocrine system of the hypothalamic paraventricular and supraoptic nuclei led the way in the development of much of our understanding of glial regulation of neuronal activity. In this review, we provide an overview of glial regulation of magnocellular neuron activity from a historical perspective of the development of our knowledge of the morphological changes in evident in the paraventricular and supraoptic nuclei and focus on recent data from the authors’ laboratories that were presented at the 9th World Congress on Neurohypophysial Hormones and that have contributed to our understanding of the multiple mechanisms by which glia modulate the activity of neurons, including: gliotransmitter modulation of synaptic transmission; trans-synaptic modulation by glial neurotransmitter transporter regulation of neurotransmitter spillover; and glial neurotransmitter transporter modulation of excitability by regulation of ambient neurotransmitter levels and their action on extrasynaptic receptors. The magnocellular neuroendocrine system secretes oxytocin and vasopressin from the posterior pituitary gland to control birth, lactation and body fluid balance and we finally speculate as to whether glial regulation of individual magnocellular neurons might co-ordinate population activity to respond appropriately to altered physiological circumstances.
Paraventricular nucleus; supraoptic nucleus; oxytocin; vasopressin; dehydration; lactation
The cycle of chronic cocaine (CC) use and withdrawal results in increased anxiety, depression and disrupted stress-responsiveness. Oxytocin and corticosterone (CORT) interact to mediate hormonal stress responses and can be altered by cocaine use. These neuroendocrine signals play important regulatory roles in a variety of social behaviours, specifically during the postpartum period, and are sensitive to disruption by CC exposure in both clinical settings and preclinical models. To determine whether CC exposure during pregnancy affected behavioural and hormonal stress response in the early postpartum period in a rodent model, Sprague-Dawley rats were administered cocaine daily (30 mg/kg) throughout gestation (days 1–20). Open field test (OFT) and forced swim test (FST) behaviours were measured on postpartum day 5. Plasma CORT concentrations were measured prior to and following testing throughout the test day, while plasma and brain oxytocin concentrations were measured post-testing only. Results indicated increased CORT response following the OFT in CC-treated dams (p≤ 0.05). CC-treated dams also exhibited altered FST behaviour (p≤ 0.05), suggesting abnormal stress responsiveness. Peripheral, but not central, oxytocin levels were increased by cocaine treatment (p≤ 0.05). Peripheral oxytocin and CORT increased following the FST regardless of treatment condition (p≤ 0.05). Changes in stress-responsiveness, both behaviourally and hormonally may underlie some deficits in maternal behaviour, thus a clearer understanding of CC’s effect on the stress response system may potentially lead to treatment interventions which could be relevant to clinical populations. Additionally, these results indicate that CC treatment can have long-lasting effects on peripheral oxytocin regulation in rats, similar to changes observed in persistent social behaviour and stress-response deficits in clinical populations.
cocaine; postpartum; stress; oxytocin; corticosterone
The magnocellular neurons in the supraoptic nucleus project to the neural lobe and release vasopressin and oxytocin into the peripheral circulation where they act on the kidney to promote fluid retention or stimulate smooth muscles in the vasculature, uterus and mammary glands to support blood pressure, promote parturition, or induce milk let-down respectively. Hormone release is regulated by complex afferent pathways carrying information about plasma osmolality, blood pressure and volume, cervical stretch, and suckling. These afferent pathways utilize a broad array of neurotransmitters and peptides that activate both ligand-gated ion channels and G-protein coupled receptors (GPCRs). The ligand-gated ion channels induce rapid changes in membrane potential resulting in generation of action potentials, initiation of exocytosis, and release of hormone into the periphery. In contrast, the GPCRs activate a host of diverse signaling cascades that modulate action potential firing and regulate other cellular functions required to support hormone release (e.g. hormone synthesis, processing, packaging and trafficking). The diversity of these actions is critical for integration of the distinct regulatory signals into a response appropriate for maintaining homeostasis. This review will describe several diverse roles of GPCRs in magnocellular neurons focusing primarily on adrenergic, purinergic, and peptidergic (neurokinin and angiotensin) receptors.
vasopressin; oxytocin; supraoptic nucleus; adrenergic; purinergic; neurokinin; angiotensin
A growing body of evidence supports carbon monoxide (CO) as a gas neurotransmitter within the central nervous system. While CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying CO actions within the magnocellular neurosecretory system remain largely unknown. In this study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as CO actions on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs) in euhydrated (EU) and 48h water-deprived rats (48WD). Our results show expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in the supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalization with OT and VP neurones within SON and PVN were significantly enhanced in 48WD rats. Inhibition of HO activity (CrMP 20μM) resulted in a slight membrane hyperpolarization in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarization, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found the expression and actions of CO to be enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.
supraoptic; paraventricular; hypothalamus; dehydration; neuroendocrine
ATP-induced ionic currents were investigated in isolated terminals and somata of the Hypothalamic Neurohypophysial System (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence; EC50 of 50 μM in somata vs. 9.6 μM in terminals. The ATP induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a P2X receptor. However, the suramin inhibition was significantly different in the two HNS compartments: IC50 of 3.6 μM in somata vs 11.6 μM in terminals. Also, both HNS compartments show significantly different responses to the purinergic receptor agonists ATP-γ-S and Benzoyl-benzoyl-ATP. Finally, there was an initial desensitization to ATP upon successive stimulations in the terminals which was not observed in the somata. These differences in EC50, inactivation, desensitization, and agonist sensitivity in terminals vs. somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurons.
Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4, and 7 but not 6 have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and OT terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals vs. somata of these CNS neurons.
neuropeptides; oxytocin; vasopressin; P2X receptors
Bursts of action potentials are crucial for neuropeptide release from the Hypothalamic Neurohypophysial System (HNS). The biophysical properties of the ion channels involved in release of these neuropeptides cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only AVP release from HNS terminals, whereas, its metabolite adenosine, via A1 receptors acting on transient Ca2+ currents, inhibits both AVP and OT secretion. Thus, purinergic feedback-mechanisms have been proposed to explain bursting efficacy at HNS terminals.
Therefore, in the present study we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically stimulated neuropeptide release. Intact neurohypophyses (NH) from wild type (WT), P2X3 rKO, P2X2&3 rKO and P2X7 rKO mice were electrically stimulated with four 25 second bursts (3V at 39Hz) separated by 21 second interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or PPADS. These frequencies, number of bursts, and voltages were determined to maximize both AVP and OT release by electrical stimulations.
Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically stimulated AVP release. A similar inhibition by suramin was observed in electrically stimulated NH from P2X3 and P2X7 rKO mice but not P2X2&3 rKO mice, indicating that endogenous ATP facilitation of electrically stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Surprisingly, electrically stimulated OT release from WT, P2X3, P2X2&3 and P2X7 rKO mice was potentiated by suramin, indicating non-purinergic effects by this “selective” antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive-feedback mechanism to differentially modulate neuropeptide release from CNS terminals.
neuropeptides; oxytocin; vasopressin; P2X receptors; Knock outs
The purpose of this study was to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurons of the paraventricular (PVN) and supraoptic nucleus (SON) in an animal model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. In these studies we used chronic bile duct ligation (BDL) in the rat, a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. This study tested the hypothesis that changes in TRPV channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilized laser capture microdissection of AVP neurons in the PVN and SON and Western blot analysis from brain punches. Laser capture microdissection and qRT-PCR demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL as compared to sham ligated controls. AVP transcription was also increased as determined using intron specific primers to measure heteronuclear RNA. Immunohistochemistry demonstrated increased AVP and TRPV2 positive cells in both the PVN and SON after BDL. Also, there was an increased co-expression of TRPV2 and AVP cells after BDL. However, there was no change in the colocalization counts of TRPV2 and OXY in both the magnocellular regions evaluated. In the SON but not the PVN, transcription levels of TRPV4 was also significantly increased in BDL rats Western Blot analysis of punches containing the PVN and SON revealed that TRPV2 protein content was significantly increased in these brain regions in BDL rats compared to sham. Our data suggests that regionally specific changes in TRPV expression in the MNC AVP neurons could alter their osmosensing ability.
vasopressin; laser capture microdissection; TRPV; bile duct ligation
The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22,333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed thanks to genomic studies. This complexity is evident at a number of levels: (1) cis-regulatory sequences; (2) non-coding and anti-sense mRNAs, most of which have no known function; (3) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; (4) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamo-neurohypophyseal system (HNS). Five unique stories explain: (1) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; 2) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (3) the use of transgenic lines that express fusion transgenes that enable (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, their neuroanatomy, electrophysiology, and activation upon exposure to any given stimulus; (4) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (5) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours.
genome; transcriptome; proteome; neuropeptidome; hypothalamo-neurohypophyseal system; oxytocin; vasopressin; transgenic rats; viral vectors
In the female rat, a complex interplay of both stimulatory and inhibitory hypothalamic factors controls the secretion of prolactin. Prolactin regulates a large number of physiological processes from immunity to stress. In the following review, we have chosen to focus on the control of prolactin secretion in the female rat in response to suckling, mating and ovarian steroids. In all three of these states, dopamine, released from neurones in the mediobasal hypothalamus, is a potent inhibitory signal regulating prolactin secretion. Early research has determined that the relief of dopaminergic tone is not enough to account for the full surge of prolactin secretion observed in response to the suckling stimulus, launching a search for possible prolactin-releasing factors. This research has since broadened to include searching for prolactin-releasing factors controlling prolactin secretion following mating or ovarian steroids. A great deal of literature has suggested that this prolactin-releasing factor may include oxytocin. Oxytocin receptors are present on lactotrophs. These oxytocin receptors respond to exogenous oxytocin and antagonism of endogenous oxytocin inhibits lactotroph activity. In addition, the pattern of oxytocin neuronal activity and oxytocin release correlate with the release of prolactin. Here we suggest that not only is oxytocin stimulating prolactin secretion, but we also hypothesize that prolactin secretion is controlled by a complex network of positive (oxytocin) and negative (dopamine) feedback loops. In the present review, we will discuss this literature and attempt to describe the circuitry we believe may be responsible for controlling prolactin secretion.
Pregnancy and lactation produce a plethora of hormonal changes in females that promote maternal care of offspring. Males in the biparental marmoset species, (Callithrix jacchus), demonstrate high levels of parenting behaviour and express enhanced circulating reproductive hormones. Furthermore, these hormonal changes are influenced by paternal experience. In order to determine if the paternally experienced male marmoset has altered neurocrine hypothalamic release, as the maternal females does, we examined the release of several reproductive neurocrines, dopamine (DA), oxytocin (OT) and vasopressin (AVP) and prolactin (PRL), in cultured explants of the hypothalamus of paternally experienced male marmosets compared with naïve, paternally inexperienced males. DA levels secreted from the isolated hypothalamus were significantly lower in the experienced males while OT and PRL levels were significantly higher than levels found in inexperienced males. PRL levels decreased rapidly in the hypothalamic media suggesting PRL production occurs elsewhere. AVP levels did not change. Stimulation of the cultured explants with oestradiol significantly decreased DA levels in the inexperienced males but did not alter the other neurocrines suggesting a direct effect of oestradiol on DA suppression in the hypothalamus. While other factors such as age and rearing experience with siblings may play a role in hypothalamic neurocrine levels, these results demonstrate that paternal experience may impact the secretion of neurocrines in a male biparental primate.
paternal experience; prolactin; OT; oestradiol; dopamine; vasopressin; hypothalamus