Neurochemical characterization of neurons expressing melanin‐concentrating hormone receptor 1 in the mouse hypothalamus

Melanin‐concentrating hormone (MCH) is a hypothalamic neuropeptide that acts via MCH receptor 1 (MCHR1) in the mouse. It promotes positive energy balance; thus, mice lacking MCH or MCHR1 are lean, hyperactive, and resistant to diet‐induced obesity. Identifying the cellular targets of MCH is an important step to understanding the mechanisms underlying MCH actions. We generated the Mchr1‐cre mouse that expresses cre recombinase driven by the MCHR1 promoter and crossed it with a tdTomato reporter mouse. The resulting Mchr1‐cre/tdTomato progeny expressed easily detectable tdTomato fluorescence in MCHR1 neurons, which were found throughout the olfactory system, striatum, and hypothalamus. To chemically identify MCH‐targeted cell populations that play a role in energy balance, MCHR1 hypothalamic neurons were characterized by colabeling select hypothalamic neuropeptides with tdTomato fluorescence. TdTomato fluorescence colocalized with dynorphin, oxytocin, vasopressin, enkephalin, thyrothropin‐releasing hormone, and corticotropin‐releasing factor immunoreactive cells in the paraventricular nucleus. In the lateral hypothalamus, neurotensin, but neither orexin nor MCH neurons, expressed tdTomato. In the arcuate nucleus, both Neuropeptide Y and proopiomelanocortin cells expressed tdTomato. We further demonstrated that some of these arcuate neurons were also targets of leptin action. Interestingly, MCHR1 was expressed in the vast majority of leptin‐sensitive proopiomelanocortin neurons, highlighting their importance for the orexigenic actions of MCH. Taken together, this study supports the use of the Mchr1‐cre mouse for outlining the neuroanatomical distribution and neurochemical phenotype of MCHR1 neurons. J. Comp. Neurol. 521:2208–2234, 2013. © 2012 Wiley Periodicals, Inc.     

Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmission

Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as gamma-aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5-hydroxytryptamine, 5-HT). The dorsal raphé nucleus (DRN) contains approximately 50% of the 5-HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5-HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor-1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1-immunoreactivity (IR) was detected in a substantial population of 5-HT-immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1-IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5-HT-IR to that in the rat, but GalR1-IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1-IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5-HT neuron activity in the DRN is primarily regulated via 5-HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward-rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5-HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5-HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression.     

Kisspeptin neurons co-express met-enkephalin and galanin in the rostral periventricular region of the female mouse hypothalamus

It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neurotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experiments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropeptides to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28-38% of kisspeptin neurons and 58-68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nucleus were also found to express galanin but not mENK. These observations indicate that, like the kisspeptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuropeptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neurons can modulate the activity of their afferent neurons.     

Anatomical characterization of bombesin receptor subtype‐3 mRNA expression in the rodent central nervous system

Bombesin receptor subtype‐3 (BRS‐3) is an orphan G‐protein‐coupled receptor (GPCR) involved in the regulation of energy homeostasis. Mice deficient in BRS‐3 develop late‐onset mild obesity with metabolic defects, while synthetic agonists activating BRS‐3 show antiobesity profiles by inhibiting food intake and increasing metabolic rate in rodent models. The molecular mechanisms and the neural circuits responsible for these effects, however, remain elusive and demand better characterization. We report here a comprehensive mapping of BRS‐3 mRNA in the rat and mouse brain through in situ hybridization. Furthermore, to investigate the neurochemical characteristics of the BRS‐3‐expressing neurons, double in situ hybridization was performed to determine whether BRS‐3 colocalizes with other neurotransmitters or neuropeptides. Many, but not all, of the BRS‐3‐expressing neurons were found to be glutamatergic, while few were found to be cholinergic or GABAergic. BRS‐3‐containing neurons do not express some of the well‐characterized neuropeptides, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin/hypocretin, melanin‐concentrating hormone (MCH), thyrotropin‐releasing hormone (TRH), gonadotropin‐releasing hormone (GnRH), and kisspeptin. Interestingly, BRS‐3 mRNA was found to partially colocalize with corticotropin‐releasing factor (CRF) and growth hormone‐releasing hormone (GHRH), suggesting novel interactions of BRS‐3 with stress‐ and growth‐related endocrine systems. Our study provides important information for evaluating BRS‐3 as a potential therapeutic target for the treatment of obesity. J. Comp. Neurol. 521:1020–1039, 2013. © 2012 Wiley Periodicals, Inc.     

Lumbar dorsal root ganglia of the cat: a quantitative study of peptide immunoreactivity and cell size

The purpose of the present study was to quantify the extent to which several peptides and serotonin coexist with substance P or somatostatin in selected lumbar dorsal root ganglia of the cat. The technique for the simultaneous visualization of two antigens by immunofluorescence was used to investigate the coexistence of neuropeptides in the lumbar dorsal root ganglia of colchicine-treated cats. Perikarya immunoreactive for calcitonin gene-related peptide, galanin, leu-enkephalin, somatostatin, and substance P were visualized in both the lumbar 5 and 6 dorsal root ganglia. In contrast, no immunoreactivity was observed for adipokinetic hormone, bombesin, dynorphin A, met-enkephalin, oxytocin, tyrosine hydroxylase, thyrotropin-releasing hormone, vasopressin, vasoactive intestinal peptide, or serotonin in either ganglion examined. Substance P coexisted with calcitonin-gene-related peptide, somatostatin, and leu-enkephalin. Somatostatin was colocalized with calcitonin gene-related peptide, leu-enkephalin, and substance P but coexisted with galanin minimally. The cell area of immunoreactive perikarya was also examined. Data concerning the cross-sectional area of immunoreactive cells indicated that somatostatin-immunoreactive perikarya were generally the largest population observed (up to approximately 6,000 microns2). Somatostatin and calcitonin gene-related peptide, as well as substance P and calcitonin gene-related peptide, coexisted in populations of cell bodies that had a smaller size (less than 2,000 microns2). These results suggest that certain peptides which coexist in the dorsal root ganglia may provide histochemical markers for functional groups of primary afferent neurons.     

Exchange protein directly activated by cAMP 2 is required for corticotropin‐releasing hormone‐mediated spine loss

Corticotropin‐releasing hormone is produced in response to acute and chronic stress. Previous studies have shown that activation of the corticotropin‐releasing hormone receptor 1 (CRHR1) by corticotropin‐releasing hormone results in the rapid loss of dendritic spines which correlates with cognitive dysfunction associated with stress. Exchange protein directly activated by cAMP (EPAC2), a guanine nucleotide exchange factor for the small GTPase Rap, plays a critical role in regulating dendritic spine morphology and has been linked with CRHR1 signalling. In this study, we have tested whether EPAC2 links corticotropin‐releasing hormone with dendritic spine remodelling. In primary rat cortical neurons, we show that CRHR1 is highly enriched in the dendritic spines. Furthermore, we find that EPAC2 and CRHR1 co‐localize in cortical neurons and that acute exposure to corticotropin‐releasing hormone induces spine loss. To establish whether EPAC2 was required for corticotropin‐releasing hormone–mediated spine loss, we knocked‐down EPAC2 in cortical neurons using a short hairpin RNA‐mediated approach. In the presence of Epac2 knocked‐down, corticotropin‐releasing hormone was no longer able to induce spine loss. Taken together, our data indicate that EPAC2 is required for the rapid loss of dendritic spines induced by corticotropin‐releasing hormone and may ultimately contribute to responses to acute stress.     

Cerebrospinal Fluid Levels of Neuropeptides, Cortisol, and Amino Acids in Patients with Epilepsy

Summary: We measured lumbar cerebrospinal fluid (CSF) levels of somatostatin, cholecystokinin, neurotensin, atrial natriuretic factor, vasoactive inhibitory peptide, neuropeptide Y, adrenocorticotrophic hormone, corticotropin releasing hormone, β-endorphin, metenkephalin, cortisol, alanine, glycine, aspartate, glutamate, taurine, and γ-aminobutyric acid in 25 inpatients with epilepsy at known interictal and postictal times and in 11 neurologically normal volunteers. There were no significant differences between interictal or postictal complex partial seizures (CPS), postictal generalized tonic-clonic seizures (GTC), and control CSF neuropeptide, cortisol, and amino acid (AA) levels. However, there were nonsignificant trends for CSF levels of several neuropeptides to be increased after CPS and GTC as compared with interictal baseline levels. There were significant correlations between levels of certain CSF neuropeptides or (AAs) and serum antiepileptic drug (AED) levels. Several correlations were noted between CSF levels of AAs, including a correlation between the excitatory neurotransmitters aspartate and glutamate identified only after CPS.     

Colocalization of serotonin receptor subtypes 5-HT2A, 5-HT2C,and 5-HT6 with neuropeptides in rat striatum

There are two primary output pathways from the striatum: a projection to the globus pallidus, and a projection to the substantia nigra. Certain striatally expressed neuropeptides are differentially distributed between these two pathways. Specifically, enkephalin is expressed in striatopallidal neurons, whereas substance P and dynorphin are expressed in striatonigral neurons. Several serotonin receptors are also prominently expressed in the striatum, but little is known about how they fit into the molecular neuroanatomy described above. We used double-label in situ hybridization to determine the striatal distribution of the mRNAs of the serotonin2A (5-HT2A), serotonin2C (5-HT2C), and serotonin6 (5-HT6) receptors in relation to enkephalin, substance P, and dynorphin expressing output neurons. Rat brain sections were simultaneously hybridized with an ³⁵S riboprobe for one of the serotonin receptors and a digoxygenin labeled riboprobe for one of the neuropeptides. Sections were examined by using brightfield microscopy, and the degree of colocalization of the two mRNAs determined. All the serotonin receptors colocalized extensively with all three of the neuropeptides examined. None of the serotonin receptors showed preferential colocalization in striatopallidal (enkephalin containing), or striatonigral (substance P or dynorphin containing) cells. The 5-HT2A and 5-HT2C mRNAs displayed a differential distribution with regard to the scattered islands of strongly dynorphin mRNA positive cells, which are thought to reside in the striatal patch compartment. Within these islands, 5-HT2C mRNA expression was much higher than in surrounding areas. 5-HT2A mRNA showed the opposite pattern with decreased expression over dynorphin rich cell clusters. © 1996 Wiley-Liss, Inc.     

Multiple neurotransmitters in the tuberomammillary nucleus: comparison of rat, mouse, and guinea pig.

Tuberomammillary neurons in the posterior hypothalamus are the sole source of neuronal histamine in adult mammalian brain. In the rat, these cells are reported to contain immunoreactivity for gamma-aminobutyric acid (GABA) and several neuropeptides. We compared the presence of these substances in the tuberomammillary cells of the rat, mouse, and guinea pig. In all three species, all histamine-immunoreactive neuronal cell bodies were positive for GABA. This suggests that GABAergic transmission may be important in tuberomammillary function. No cell bodies immunoreactive for thyrotropin releasing hormone (TRH) were found in the guinea pig or mouse tuberomammillary area. In contrast, about 14% of the histamine-immunoreactive tuberomammillary cells in the rat were TRH-positive. These cells were small or medium-sized and were located only in the medial part of the tuberomammillary complex. An antibody against porcine galanin stained about 45% of the tuberomammillary cell bodies in the rat and about 28% in the mouse, but none in the guinea pig. A large proportion of the cells in the rat and mouse, but none in the guinea pig, were positive for met-enkephalin-arg-phe. In contrast, all histamine-containing tuberomammillary cells in the guinea pig, but none in the rat or mouse, were immunoreactive for met-enkephalin. This may indicate a different expression of proenkephalin-derived peptides in the tuberomammillary neurons in these species. Some substance P-immunoreactive cell bodies were located in the tuberomammillary area in all three species. However, only 3% of the histamine-immunoreactive cell bodies in the rat and mouse but none in the guinea pig were substance P-positive. The neurochemical properties of the tuberomammillary nucleus that exhibited species commonality deserve to be studied neurochemically and electrophysiologically in order to determine the functional relevance of coexisting transmitters in this nucleus.     

Neuropeptides in the human superior cervical ganglion

Superior cervical ganglia from 7 human cadavers (3–7 h post mortem) were immunostained for tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and 14 different neuropeptides. The results show that ganglionic cells contain TH, DBH, neuropeptide Y (NPY), somatostatin, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP). These substances were present predominantly within large ganglionic cells. Inside the ganglion, the number and topographical distribution of various types of immunoreactive cells differed from one another. NPY and CGRP immunoreactivities were found in some TH-positive cells, but that co-localization never exceeded the 30% of the TH cells. Leu-enkephalin showed a weak immunoreactivity, which was restricted to fibers or varicosities. Neuropeptides like substance P, dynorphin A and B, cholecystokinin, galanin, corticotropin-releasing factor, thyreotropin-releasing hormone, angiotensin II and neurotensin showed no immunoreactivity in the human superior cervical ganglion.     

Neurotransmitter modulation of calcium current in rat spinal cord neurons

The modulation of Ca2+ currents by neurotransmitters was studied in freshly dissociated rat spinal cord neurons, using the whole-cell patch-clamp technique. GABA, baclofen, adenosine, ATP, serotonin, norepinephrine, somatostatin, and dynorphin A inhibited the current through Ca2+ channels in a substantial fraction of cells, while substance P, vasoactive intestinal polypeptide, [D-ala2,d-leu5]-enkephalin, cholecystokinin-8 (sulfated), calcitonin gene-related peptide, angiotensin II, neurotensin, vasopressin, and thyrotropin-releasing hormone had no effect. In the case of baclofen, the inhibition is mediated, at least in part, by a GTP-binding protein. Suppression of Ca2+ current by neurotransmitters may represent a mechanism of presynaptic inhibition in the spinal cord.     

Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.     

The role of peptides in treatment of psychiatric disorders

About 25 years ago the observation that neuropeptides serve as signalling molecules in the nervous system generated great expectations for drug industry. In this article the progress made since then in exploiting neuropeptide systems pharmacologically in psychiatry is highlighted. In affective disorders a number of neuropeptides seem to be causally involved in development and course of illness, especially corticotropin releasing hormone (CRH), vasopressin (AVP) and substance P, whose receptors are now targeted with small molecules designed to reduce depressive and anxiety symptoms. Although not exactly neuropeptides, also neurotrophins, may have a distinct role in antidepressant action and possibly also in causation of depression. Schizophrenia-like symptoms are caused by neurotensin (NT), supporting the notion that drugs interfering with NT systems are potential antipsychotics. Finally, sleep disorders, currently treated with hypnotics, that have serious adverse effects can be targeted with neuropeptides. According to the work by Axel Steiger several neuropeptides even if peripherally administered produce improvements of quality of sleep. All these observations call for intensified application of novel research tools necessary to exploit the potential of neuropeptide systems as psychopharmaceutical targets.     

Chemical anatomy of the hypothalamus

The neuropeptide field has witnessed considerable research interest over the past decade, and a growing body of anatomic, biochemical, and electrophysiologic data have since emerged, supporting the existence and putative neuromodulatory function of a large variety of these peptide hormones in several extrahypothalamic brain regions. It is now evident that neuropeptides not only fulfill criteria required of putative neurotransmitters, but more generally act as modulators of neuronal activity. The author discusses vasopressin and oxytocin pathways, corticotropin releasing factor, atrial natriuretic factor, thyrotropin releasing hormone, somatostatin, motilin, growth hormone releasing factor, dopamine, gonadotropin releasing hormone, and substance P.     

Neuropeptides and sexual behaviour

Many neuropeptides are involved in the control of sexual behaviour at the central level. Among these, the most studied are adrenocorticotropin, -melanocyte stimulating hormone, oxytocin and opioid peptides. This attempt to review old and new neuropharmacological, biochemical and psychobiological studies in this field, shows that all these neuropeptides apparently facilitate sexual behaviour, except for opioid peptides, which inhibit sexual performance, in most of the species studied so far (rats, mice, monkeys and humans). However, gonadotropin-releasing hormone, corticotropin releasing factor, neuropeptide Y, galanin, cholecystokinin, substance P and vasoactive intestinal peptide may be also involved in the control of sexual behaviour. Apparently, corticotropin releasing factor, neuropeptide Y and cholecystokinin inhibit, while substance P and vasoactive intestinal peptide facilitate, sexual behaviour. In contrast, gonadotropin-releasing hormone has been reported to exert a facilitative, inhibitory or no effect at all on sexual behaviour. Galanin was also shown either to facilitate or inhibit sexual behaviour. The above-mentioned putative role of the neuropeptides in sexual behaviour derives mainly from studies done in rats. In these studies, neuropeptides, their antisera or drugs that act as agonists or antagonists of neuropeptide receptors, were tested for their effect on sexual behaviour after systemic, intracerebroventricular, or intracerebral administration. The latter were infused into brain areas relevant for sexual behaviour, such as the medial preoptic area, and the ventromedial and paraventricular nuclei of the hypothalamus. The above studies show that little information is available on the mechanisms by which neuropeptides influence sexual behaviour. Also unclear is whether the above neuropeptides influence the anticipatory phase (sexual arousal and/or motivation) or the consummatory phase (performance) of sexual behaviour, except for opioid peptides. New information about the role of neuropeptides may come from the application of molecular biology and genetic manipulation techniques to the study of sexual behaviour. Of these, FOS protein determination, antisense oligonucleotides aimed at the neutralisation of neuropeptide and/or neuropeptide receptor mRNAs in specific brain areas, and gene ablation seem the most promising. Although still in the early stages, it is likely that these methodologies will provide new insights into the role of neuropeptides in the control of sexual behaviour.     

Immunocytochemical evidence for the existence of substance P receptor (NK1) in serotonin neurons of rat and mouse dorsal raphe nucleus

In addition to its neurotransmitter/modulator role in pain perception, substance P (SP) is involved in a regulation of mood, as antagonists of its neurokinin-1 receptor (NK1r) have been found to have antidepressant-like effects in humans. In rodents, treatment with NK1r antagonists has been shown to increase the firing of dorsal raphe nucleus (DRN) serotonin (5-hydroxytryptamine, 5-HT) neurons and to induce a desensitization of their 5-HT1A autoreceptors, suggesting local interactions between the SP and 5-HT systems. To search for the presence of NK1r on 5-HT neurons of the DRN, we used light and electron microscopic immunocytochemistry, as well as confocal microscopy, after single- and double-labelling of NK1r and of the biosynthetic enzyme of 5-HT, tryptophan hydroxylase (TpOH). A significant number of 5-HT (TpOH-positive) cell bodies and dendrites endowed with NK1r were thus demonstrated in the caudal part of rat and mouse DRN. As visualized by electron microscopy after gold immunolabelling, NK1r was mostly cytoplasmic in 5-HT neurons, while predominating on the plasma membrane in the case of TpOH-negative dendrites. The proportion of NK1r observed on the plasma membrane of 5-HT neurons was, however, slightly higher in mouse than rat. Thus, in both rat and mouse DRN, a subpopulation of 5-HT neurons is endowed with NK1r receptors and may be directly involved in the antidepressant-like effects of NK1r antagonists. These 5-HT neurons represent a new element in the neuronal circuitry currently proposed to account for the role of SP in mood regulation.     

Activation of galanin and cholecystokinin receptors in the lumbosacral spinal cord is required for ejaculation in male rats

The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic and motor neurons in the lumbosacral spinal cord. LSt cells regulate ejaculatory reflexes by release of neuropeptides that are co‐expressed in their axons, as previously demonstrated for gastrin‐releasing peptide and enkephalin. Here, the role of two other neuropeptides co‐expressed in LSt cells for ejaculatory reflexes is demonstrated: galanin and cholecystokinin (CCK). Adult male rats were anesthetized, spinalized, and received intrathecal infusions of galanin receptor antagonist Galantide (1 or 10 nmol) or CCK receptor antagonist proglumide (71 or 714 nmol). The dorsal penile nerve (DPN) was electrically stimulated to trigger ejaculatory reflexes and seminal vesicle pressure (SVP) and rhythmic contractions of the bulbocavernosus muscle (BCM) were analyzed as parameters of emission and expulsion respectively. Treatment with galanin or CCK antagonists significantly reduced SVP increases and BCM bursting, demonstrating that galanin and CCK are required for ejaculation. Next, anesthetized, spinalized males received intrathecal infusions of galanin (0.15 or 0.3 nmol) or CCK_(26–33) (4.35 nmol) and effects on subthreshold DPN stimulations were determined. Intrathecal infusions of galanin or CCK facilitated ejaculatory reflexes induced by subthreshold DPN stimulation in all animals, but did not trigger ejaculatory reflexes in the absence of DPN stimulation. Together, these results demonstrate that galanin and CCK both act in the spinal ejaculation generator to regulate ejaculation. However, effects of galanin and CCK were dependent on DPN stimulation, suggesting that these neuropeptides may act in concert with other LSt co‐expressed neuropeptides.     

Neurotransmitter diversity in pre‐synaptic terminals located in the parvicellular neuroendocrine paraventricular nucleus of the rat and mouse hypothalamus

Virtually all rodent neuroendocrine corticotropin‐releasing‐hormone (CRH) neurons are in the dorsal medial parvicellular (mpd) part of the paraventricular nucleus of the hypothalamus (PVH). They form the final common pathway for adrenocortical stress responses. Their activity is controlled by sets of GABA‐, glutamate‐, and catecholamine‐containing inputs arranged in an interactive pre‐motor network. Defining the nature and arrangement of these inputs can help clarify how stressor type and intensity information is conveyed to neuroendocrine neurons. Here we use immunohistochemistry with high‐resolution 3‐dimensional image analyses to examine the arrangement of single‐ and co‐occurring GABA, glutamate, and catecholamine markers in synaptophysin‐defined pre‐synaptic terminals in the PVHmpd of unstressed rats and Crh‐IRES‐Cre;Ai14 transgenic mice: respectively, vesicular glutamate transporter 2 (VGluT2), vesicular GABA transporter (VGAT), dopamine β‐hydroxylase (DBH), and phenylethanolamine n ‐methyltransferase (PNMT). Just over half of all PVHmpd pre‐synaptic terminals contain VGAT, with slightly less containing VGluT2. The vast majority of terminal appositions with mouse CRH neurons occur non‐somatically. However, there are significantly more somatic VGAT than VGluT2 appositions. In the rat PVHmpd, about five times as many pre‐synaptic terminals contain PNMT than DBH only. However, because epinephrine release has never been detected in the PVH, PNMT terminals may functionally be noradrenergic not adrenergic. PNMT and VGluT2 co‐occur in some pre‐synaptic terminals indicating the potential for co‐transmission of glutamate and norepinephrine. Collectively, these results provide a structural basis for how GABA/glutamate/catecholamine interactions enable adrenocortical responses to fast‐onset interosensory stimuli, and more broadly, how combinations of PVH neurotransmitters and neuromodulators interact dynamically to control adrenocortical activity.