Ir Neurons Research Articles

Central blockade of nitric oxide transmission impairs exercise-induced neuronal activation in the PVN and reduces physical performance.

The blockade of central nitric oxide (NO) signaling modifies the thermoregulatory and metabolic adjustments that occur during exercise, thereby impairing physical performance. However, the brain areas involved in this response remain unknown. Nitrergic neurons are present in the hypothalamic areas that are activated during exercise and participate in autonomic and neuroendocrine responses, such as, the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). To investigate whether brain NO signaling affects thermoregulation during exercise through the activation of hypothalamic neurons, rats underwent acute submaximal treadmill exercise (18 mmin(-1), 5% inclination) until fatigue received an intracerebroventricular injection of 1.43 μmol Nω-nitro-l-arginine metil ester (L-NAME), a nitric oxide synthase inhibitor, or saline (SAL). Skin tail temperature (Tsk) and internal body temperature (Ti) were continuously recorded and c-Fos expression was determined in the PVN and the SON. L-NAME treatment reduced physical performance by 48%, which was positively correlated with tail vasodilation capacity, which was reduced by 28%, and negatively correlated with heat storage rate (HSR), which was increased by 38%. Physical exercise until fatigue increased the number of c-Fos-immunoreactive (ir) neurons in the PVN and the SON. L-NAME-treatment significantly reduced the exercise-induced c-Fos expression in the PVN, whereas it had no effect in the SON. Interestingly, the number of c-Fos-ir neurons in the PVN was closely correlated with physical performance and inversely associated with HSR. Thus, the inhibition of central NO attenuates neuronal activation induced by exercise in the PVN, impairs the autonomic regulation of heat dissipation, and anticipates the fatigue. Brain NO seems to play a role in exercise performance through the regulation of neuronal activation in the PVN, but not in the SON, although the SON neurons are also activated by running exercise. Moreover, this role in performance mediated by neuronal activation in the PVN can be related with the improvement of thermoregulatory adjustments that occur during exercise.

Changes in RFamide-related peptide-1 (RFRP-1)-immunoreactivity during postnatal development and the estrous cycle.

GnRH is a key player in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland. It has been shown that the mammalian counterpart of the avian gonadotropin inhibitory hormone named RFamide-related peptide (RFRP) is expressed in hypothalamic neurons that innervate and inhibit GnRH neurons. The RFRP precursor is processed into 2 mature peptides, RFRP-1 and RFRP-3. These are characterized by a conserved C-terminal motif RF-NH2 but display highly different N termini. Even though the 2 peptides are equally potent in vitro, little is known about their relative distribution and their distinct roles in vivo. In this study, we raised an antiserum selective for RFRP-1 and defined the distribution of RFRP-1-immunoreactive (ir) neurons in the rat brain. Next, we analyzed the level of RFRP-1-ir during postnatal development in males and females and investigated changes in RFRP-1-ir during the estrous cycle. RFRP-1-ir neurons were distributed along the third ventricle from the caudal part of the medial anterior hypothalamus throughout the medial tuberal hypothalamus and were localized in, but mostly in between, the dorsomedial hypothalamic, ventromedial hypothalamic, and arcuate nuclei. The number of RFRP-1-ir neurons and the density of cellular immunoreactivity were unchanged from juvenile to adulthood in male rats during the postnatal development. However, both parameters were significantly increased in female rats from peripuberty to adulthood, demonstrating prominent gender difference in the developmental control of RFRP-1 expression. The percentage of c-Fos-positive RFRP-1-ir neurons was significantly higher in diestrus as compared with proestrus and estrus. In conclusion, we found that adult females, as compared with males, have significantly more RFRP-1-ir per cell, and these cells are regulated during the estrous cycle.

Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum.

The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyze the localization and morphology of neurons expressing parvalbumin (PV), calretinin (CR), NPY, and somatostatin (SOM) in the claustrum of man (# 5), chimpanzee (# 1) and crab-eating monkey (# 3). Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir) neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY), was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex.

Dopamine and prolactin involvement in the maternal care of chicks in the native Thai hen (Gallus domesticus)

The dopaminergic (DAergic) system plays a pivotal role in incubation behavior via the regulation of prolactin (PRL) secretion in birds, however the role of the DA/PRL system in rearing behavior is poorly understood. The objective of this study was to investigate the relationship between the DA/PRL system and rearing behavior in a gallinaceous bird, the native Thai chicken. Incubating native Thai hens were divided into two groups. In the first group, hens were allowed to care for their chicks (rearing hens; R). In the second group, hens were deprived of their chicks immediately after hatching (non-rearing hens; NR). In both groups, blood samples and brain sections were collected at different time points after the chicks hatched (days 4, 7, 10, 14, 17, 21, 24, and 28; 6 hens/time point/group). In this study, tyrosine hydroxylase (TH) was used as a marker for DAergic neurons. The numbers of TH-immunoreactive (-ir) neurons in the nucleus intramedialis (nI) and in the nucleus mamillaris lateralis (ML), which regulate the vasoactive intestinal peptide (VIP)/PRL system, were determined in R and NR hens utilizing immunohistochemical techniques. Plasma PRL levels were determined by enzyme-linked immunosorbent assays. The results revealed that both the number of TH-ir neurons in the nI and the plasma PRL levels were significantly higher in the R hens compared with the NR hens during the first 14days of chick rearing (P<0.05). However, there was no significant change in the DAergic activity in the ML in either the R or NR groups throughout the 28-day rearing periods. These results suggest that the DA/PRL system is involved in early rearing behavior. The additional decline in DAergic activity and plasma PRL levels during the disruption of rearing behavior further supports their involvement in rearing behavior in this equatorial precocial species.

Distribution of gastrin‐releasing peptide in the rat trigeminal and spinal somatosensory systems

Gastrin-releasing peptide (GRP) has recently been identified as an itch-specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP-immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small- and medium-sized type cells. In both the TG and DRG, many GRP-ir neurons also expressed substance P and calcitonin gene-related peptide, but not isolectin B4 . The different proportions of GRP and transient receptor potential vanilloid 1 double-positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP-ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP-ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP-ir terminals contained clear microvesicles and large dense-cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP-dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn.

Numbers of pH‐sensitive K+ channel‐immunoreactive neurons are reduced in CO2‐insensitive Brown Norway rats in select brainstem nuclei associated with central respiratory chemoreception (1092.8)

Brown Norway (BN) rats display an extremely low hypercapnic ventilatory response (HCVR) compared to other strains such as the Dahl S (SS) rat. Multiple pH‐sensitive potassium (K+) channels are expressed within multiple brainstem chemosensory nuclei and may contribute to CO2‐H+ detection/signaling in chemosensitive neurons. Herein we tested the hypothesis that there are selective reductions in the numbers of neurons expressing pH‐sensitive Kv1.4, Kir 2.3, and TASK‐1 K+ channels in the chemosensory nucleus of the solitary tract (NTS) but not in the pre‐Botzinger Complex (pre‐BötC), nucleus ambiguus (NA) or hypoglossal nucleus (XII) of BN but not SS rats. Medullary sections (25 µm) from male and female BN (n=14) and SS (n=15) rats were stained with cresyl violet or primary antibodies targeting KV1.4, Kir2.3, or TASK‐1 and the numbers of K+ channel‐immunoreactive (‐ir) neurons were quantified. There was little difference among female and male rats within each strain in all nuclei studied. However, within the NTS there were significantly fewer Kir2.3, (47.46% ± 1.83%; TASK‐1 57.46% ± 2.17%; and Kv1.4 64.11% ± 1.6% observed in BN rats as compared to SS rats. In contrast, the numbers of K+ channel‐ir neurons did not significantly (P&lt; 0.05) differ within the NA and the hypoglossal nucleus, with some significant (P &lt; 0.05) differences observed in the Medullary Raphe and the pre‐Botzinger complex. Fewer pH sensitive K+ channel‐immunoreactive neurons is evidence that at least partly explains the lower hypercapnic response in BN rats compared to that in SS rats.Grant Funding Source: Supported by the Department of Veterans Affairs and NIH HL25739

The claustrum of the bottlenose dolphin Tursiops truncatus (Montagu 1821).

The mammalian claustrum is involved in processing sensory information from the environment. The claustrum is reciprocally connected to the visual cortex and these projections, at least in carnivores, display a clear retinotopic distribution. The visual cortex of dolphins occupies a position strikingly different from that of land mammals. Whether the reshaping of the functional areas of the cortex of cetaceans involves also modifications of the claustral projections remains hitherto unanswered. The present topographic and immunohistochemical study is based on the brains of eight bottlenose dolphins and a wide array of antisera against: calcium-binding proteins (CBPs) parvalbumin (PV), calretinin (CR), and calbindin (CB); somatostatin (SOM); neuropeptide Y (NPY); and the potential claustral marker Gng2. Our observations confirmed the general topography of the mammalian claustrum also in the bottlenose dolphin, although (a) the reduction of the piriform lobe modifies the ventral relationships of the claustrum with the cortex, and (b) the rotation of the telencephalon along the transverse axis, accompanied by the reduction of the antero-posterior length of the brain, apparently moves the claustrum more rostrally. We observed a strong presence of CR-immunoreactive (-ir) neurons and fibers, a diffuse but weak expression of CB-ir elements and virtually no PV immunostaining. This latter finding agrees with studies that report that PV-ir elements are rare in the visual cortex of the same species. NPY- and somatostatin-containing neurons were evident, while the potential claustral markers Gng2 was not identified in the sections, but no explanation for its absence is currently available. Although no data are available on the projections to and from the claustrum in cetaceans, our results suggest that its neurochemical organization is compatible with the presence of noteworthy cortical inputs and outputs and a persistent role in the general processing of the relative information.

Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI

Traumatic brain injury (TBI) causes 10–20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague–Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM–VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p<0.001 as compared to controls) and 84% contralaterally (p<0.05). Accordingly, the volume of the ipsilateral RT was 58% of that in controls ipsilaterally (p<0.001) and 90% contralaterally (p>0.05). Also, the volume of the VPM–VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate to the inclusion of the hypothalamus into the tissue analyzed with RT-PCR arrays. In controls, the lower the number of PARV-ir neurons in the RT, the higher the seizure susceptibility in the PTZ test. Rats with TBI showed seizure susceptibility comparable to that in controls with the lowest number of PARV-ir neurons in the RT. Our data show that the RT and VPM–VPL undergo remarkable degeneration after lateral-FPI which results in reorganization of PARV-ir terminals in the VPM–VPL. The contribution of RT damage to seizure susceptibility and post-traumatic epileptogenesis deserves further studies.

Distribution of vasopressin, oxytocin and vasoactive intestinal polypeptide in the hypothalamus and extrahypothalamic regions of tree shrews

Vasopressin (VP), oxytocin (OXT) and vasoactive intestinal polypeptide (VIP) in the brain modulate physiological and behavioral processes in many vertebrates. Day-active tree shrews, the closest relatives of primates, live singly or in pairs in territories that they defend vigorously against intruding conspecifics. However, anatomy concerning peptidergic neuron distribution in the tree shrew brain is less clear. Here, we examined the distribution of VP, OXT and VIP immunoreactivity in the hypothalamus and extrahypothalamic regions of tree shrews (Tupaia belangeri chinensis) using the immunohistochemical techniques. Most of VP and OXT immunoreactive (-ir) neurons were found in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. In addition, VP-ir or OXT-ir neurons were scattered in the preoptic area, anterior hypothalamic areas, dorsomedial hypothalamic nucleus, stria terminalis, bed nucleus of the stria terminalis and medial amygdala. Interestingly, a high density of VP-ir fibers within the ventral lateral septum was observed in males but not in females. Both VP-ir and VIP-ir neurons were found in different subdivisions of the suprachiasmatic nucleus (SCN) with partial overlap. VIP-ir cells and fibers were also scattered in the cerebral cortex, anterior olfactory nucleus, amygdala and dentate gyrus of the hippocampus. These findings provide a comprehensive description of VIP and a detailed mapping of VP and OXT in the hypothalamus and extrahypothalamic regions of tree shrews, which is an anatomical basis for the participation of these neuropeptides in the regulation of circadian behavior and social behavior.

Neuroendocrine responses to social isolation and paternal deprivation at different postnatal ages in mandarin voles

Neonatal isolation and paternal deprivation have long lasting effects on the behavior and neuroendocrine system at adulthood. Whether these effects at adulthood are induced by neonatal changes in relevant neuroendocrine parameters lead by these early-life social experiences is not well understood. Whether monogamous rodents exhibit a stress hypo-responsive period (SHRP) also remains unclear. Using the monogamous mandarin vole, we found that 30 min of isolation did not affect levels of corticosterone (CORT) and adrenocorticotropin (ACTH) at postnatal days 8, 10, and 12 displaying a SHRP, but increased these at postnatal days 4, 14, 16, and 18. Isolation increased vasopressin (AVP)-ir neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) from postnatal days 4 to 12, and up-regulated oxytocin (OT)-ir neurons in the PVN at postnatal days 4 and 8 and SON at postnatal day 4. Paternally deprived pups showed increase in ACTH and CORT after 30 min of social isolation from postnatal days 8 to 14, increase in AVP-ir neurons in the PVN from postnatal days 10 to 14, reduction in OT-ir neurons in the PVN from postnatal days 10 to 14 and in the SON at postnatal days 12 and 14. These results indicate that monogamous mandarin voles display a short SHRP which can be disrupted by paternal deprivation. Central AVP and OT levels may also be altered by paternal deprivation and social isolation. We propose that changes in these neuroendocrine parameters induced by early-life social experiences such as those tested here persist and result.

The Novel Endocannabinoid Receptor GPR18 Is Expressed in the Rostral Ventrolateral Medulla and Exerts Tonic Restraining Influence on Blood Pressure

Systemic administration of the G-protein-coupled receptor 18 (GPR18) agonist abnormal cannabidiol (Abn CBD) lowers blood pressure (BP). Whether GPR18 is expressed in the central nervous system (CNS) and plays a role in BP control is not known despite the abundance of the GPR18 ligand N-arachidonoyl glycine (NAGly) in the CNS. Therefore, we first determined whether GPR18 is expressed in the presympathetic tyrosine hydroxylase (TH) immunoreactive (ir) neurons of the brainstem cardiovascular regulatory nuclei. Second, we investigated the impact of GPR18 activation and blockade on BP and heart rate (HR) and neurochemical modulators of sympathetic activity and BP. Immunofluorescence findings revealed GPR18 expression in TH-ir neurons in the rostral ventrolateral medulla (RVLM). Intra-RVLM GPR18 activation (Abn CBD) and blockade (O-1918, 1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-,cyclohexen-1-yl]benzene) elicited dose-dependent reductions and elevations in BP, respectively, along with respective increases and decreases in HR in conscious male Sprague-Dawley rats. RVLM GPR18 activation increased neuronal adiponectin (ADN) and NO and reduced reactive oxygen species (ROS) levels, and GPR18 blockade reduced neuronal ADN and increased oxidative stress (i.e., ROS) in the RVLM. Finally, we hypothesized that the negligible hypotensive effect caused by the endogenous GPR18 ligand NAGly could be due to concurrent activation of CB(1)R in the RVLM. Our findings support this hypothesis because NAGly-evoked hypotension was doubled after RVLM CB(1)R blockade (SR141716, rimonabant). These findings are the first to demonstrate GPR18 expression in the RVLM and to suggest a sympathoinhibitory role for this receptor. The findings yield new insight into the role of a novel cannabinoid receptor (GPR18) in central BP control.

Morphological properties of tectal neurons that project to the nucleus geniculatus lateralis, pars ventralis (GLv) and the surrounding ventral thalamus in chicks

Layer 10 neurons of the chick tectum were morphologically investigated. The layer 10 neurons displayed heterogeneous immunoreactivities to calcium-binding proteins (CaBPs). Calbindin (CB)-immunoreactive (ir) neurons had pyramidal or round somata, primarily found in layers 5, 9, and 13. Parvalbumin (PV)-ir neurons were of various shapes with small to large somata (109.7±48.6μm(2)) that were located mainly in layers 4 and 10. Calretinin (CR)-ir neurons had small to middle-sized somata (79.3±9.7μm(2)) located primarily in layers 10 and 13, and most of them were similar to typical radial cells in size and shape. Two distinct types of neurons that projected to the nucleus geniculatus lateralis, pars ventralis (GLv) and ventral thalamus were demonstrated in layer 10. Type 1 cells had small to middle-sized somata (74.3±33μm(2)), and each cell had a single apical dendrite that ramified into bush-like branches in layer 7. These cells corresponded to CR-ir neurons and radial cells in size and shape. Type 2 cells had larger somata (124.7±52.6μm(2)), and their shapes were pyramidal, polygonal, or oval. They had multiple obliquely ascending dendrites that ramified into bush-like branches in layer 7. These cells often appeared similar to PV-ir neurons.

Socially regulated reproductive development: Analysis of GnRH‐1 and kisspeptin neuronal systems in cooperatively breeding naked mole‐rats (Heterocephalus glaber)

In naked mole-rat (NMR) colonies, breeding is monopolized by the queen and her consorts. Subordinates experience gonadal development if separated from the queen. To elucidate the neuroendocrine factors underlying reproductive suppression/development in NMRs, we quantified plasma gonadal steroids and GnRH-1- and kisspeptin-immunoreactive (ir) neurons in subordinate adults and in those allowed to develop into breeders, with or without subsequent gonadectomy. In males and females, respectively, plasma testosterone and progesterone are higher in breeders than in subordinates. No such distinction occurs for plasma estradiol; its presence after gonadectomy and its positive correlation with adrenal estradiol suggest an adrenal source. Numbers of GnRH-1-ir cell bodies do not differ between gonad-intact breeders and subordinates within or between the sexes. As in phylogenetically related guinea pigs, kisspeptin-ir processes pervade the internal and external zones of the median eminence. Their distribution is consistent with actions on GnRH-1 neurons at perikaryal and/or terminal levels. In previously investigated species, numbers of kisspeptin-ir cell bodies vary from substantial to negligible according to sex and/or reproductive state. NMRs are exceptional: irrespective of sex, reproductive state, or presence of gonads, substantial numbers of kisspeptin-ir cell bodies are detected in the rostral periventricular region of the third ventricle (RP3V) and in the anterior periventricular (PVa), arcuate, and dorsomedial hypothalamic nuclei. Nevertheless, the greater number in the RP3V/PVa of female breeders compared with female subordinates or male breeders suggests that emergence from a hypogonadotrophic state in females may involve kisspeptin-related mechanisms similar to those underlying puberty or seasonal breeding in other species.

Immunohistochemical distribution of calretinin and calbindin (D‐28k) in the brain of the cladistian Polypterus senegalus

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes.

Neurokinin-1 Receptor-Expressing Neurons That Contain Serotonin and Gamma-Aminobutyric Acid in the Rat Rostroventromedial Medulla Are Involved in Pain Processing

The rostroventromedial medulla (RVM) is an important area of the endogenous pain-regulating system, in which 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) are 2 main transmitters involved in pain modulation. However, whether 5-HT and GABA are colocalized is poorly understood. By using glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse, we confirmed the colocalization of 5-HT and GABA in the RVM, with a main distribution in the raphe magnus nucleus and paragigantocellular reticular nucleus. Interestingly, more than half (51.6%) of the 5-HT/GABA-immunoreactive (ir) neurons expressed neurokinin-1 receptors (NK-1R) and one-third (30.1%) of the 5-HT/GABA/NK-1R-ir neurons projected to the spinal cord, suggesting that substance P (SP) should regulate the activity of 5-HT/GABA-ir spinal cord-projecting neurons. By combining retrograde and anterograde tracing methods, we observed that the cuneiform nucleus, dorsal raphe nucleus, and lateral periaqueductal gray are the main origin nuclei for the SP-ir fibers and terminals in the RVM. Finally, after formalin injection into the mice hind paw, 29.2% SP-ir RVM-projecting neurons from supra-RVM nuclei and 33.1% NK-1R-ir spinal cord-projecting neurons in the RVM were activated. The present study provides potent morphological evidence that 5-HT and GABA are coexistent in RVM-spinal cord-projecting neurons that are also regulated by SPergic projections. PerspectiveThe results will greatly enhance our understanding for the modulation of nociceptive information in the descending pain-regulating system.

Mesotocin and maternal care of chicks in native Thai hens (Gallus domesticus)

Oxytocin (OT) is known to induce and regulate maternal behaviors in mammals via the supraoptic nucleus and paraventricular nucleus (PVN), whereas the function of mesotocin (MT; the avian homolog of OT) is poorly understood in birds. To elucidate the association of MT and the regulation of maternal behaviors in birds, we studied changes in the number of MT-immunoreactive (ir) neurons in native Thai chickens using immunohistochemistry. We observed that MT-ir neurons and fibers appeared in discrete regions located close to the third ventricle from the level of the preoptic area through the anterior hypothalamus with an abundance observed in the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and PVN. The number of MT-ir neurons was low in the SOv, POM, and PVN of non-laying hens, but it increased gradually when the hens entered the laying stage, and peaked in incubating and rearing hens. We compared the number of MT-ir neurons in the SOv, POM, and PVN of native Thai hens rearing chicks (R) with that of non-rearing chicks (NR). The number of MT-ir neurons was high in the R hens, but low in the NR hens in these nuclei. For the first time, these results indicate that the association between the MT neurons and the presence of chicks might, in part, play a role in the neuroendocrine reorganization to establish and maintain maternal behaviors in native Thai chickens. MTergic activity is likely related to the contribution of rearing behavior in this equatorial precocial species.

Forebrain GABAergic projections to locus coeruleus in mouse

The noradrenergic locus coeruleus (LC) regulates arousal, memory, sympathetic nervous system activity, and pain. Forebrain projections to LC have been characterized in rat, cat, and primates, but not systematically in mouse. We surveyed mouse forebrain LC-projecting neurons by examining retrogradely labeled cells following LC iontophoresis of Fluoro-Gold and anterograde LC labeling after forebrain injection of biotinylated dextran amine or viral tracer. Similar to other species, the central amygdalar nucleus (CAmy), anterior hypothalamus, paraventricular nucleus, and posterior lateral hypothalamic area (PLH) provide major LC inputs. By using mice expressing green fluorescent protein in γ-aminobutyric acid (GABA)ergic neurons, we found that more than one-third of LC-projecting CAmy and PLH neurons are GABAergic. LC colocalization of biotinylated dextran amine, following CAmy or PLH injection, with either green fluorescent protein or glutamic acid decarboxylase (GAD)65/67 immunoreactivity confirmed these GABAergic projections. CAmy injection of adeno-associated virus encoding channelrhodopsin-2-Venus showed similar fiber labeling and association with GAD65/67-immunoreactive (ir) and tyrosine hydroxylase (TH)-ir neurons. CAmy and PLH projections were densest in a pericoerulear zone, but many fibers entered the LC proper. Close apposition between CAmy GABAergic projections and TH-ir processes suggests that CAmy GABAergic neurons may directly inhibit noradrenergic principal neurons. Direct LC neuron targeting was confirmed by anterograde transneuronal labeling of LC TH-ir neurons following CAmy or PLH injection of a herpes virus that expresses red fluorescent protein following activation by Cre recombinase in mice that express Cre recombinase in GABAergic neurons. This description of GABAergic projections from the CAmy and PLH to the LC clarifies important forebrain sources of inhibitory control of central nervous system noradrenergic activity.

Neuropeptide S Facilitates Mice Olfactory Function through Activation of Cognate Receptor-Expressing Neurons in the Olfactory Cortex

Neuropeptide S (NPS) is a newly identified neuromodulator located in the brainstem and regulates various biological functions by selectively activating the NPS receptors (NPSR). High level expression of NPSR mRNA in the olfactory cortex suggests that NPS-NPSR system might be involved in the regulation of olfactory function. The present study was undertaken to investigate the effects of intracerebroventricular (i.c.v.) injection of NPS or co-injection of NPSR antagonist on the olfactory behaviors, food intake, and c-Fos expression in olfactory cortex in mice. In addition, dual-immunofluorescence was employed to identify NPS-induced Fos immunereactive (-ir) neurons that also bear NPSR. NPS (0.1–1 nmol) i.c.v. injection significantly reduced the latency to find the buried food, and increased olfactory differentiation of different odors and the total sniffing time spent in olfactory habituation/dishabituation tasks. NPS facilitated olfactory ability most at the dose of 0.5 nmol, which could be blocked by co-injection of 40 nmol NPSR antagonist [D-Val5]NPS. NPS administration dose-dependently inhibited food intake in fasted mice. Ex-vivo c-Fos and NPSR immunohistochemistry in the olfactory cortex revealed that, as compared with vehicle-treated mice, NPS markedly enhanced c-Fos expression in the anterior olfactory nucleus (AON), piriform cortex (Pir), ventral tenia tecta (VTT), the anterior cortical amygdaloid nucleus (ACo) and lateral entorhinal cortex (LEnt). The percentage of Fos-ir neurons that also express NPSR were 88.5% and 98.1% in the AON and Pir, respectively. The present findings demonstrated that NPS, via selective activation of the neurons bearing NPSR in the olfactory cortex, facilitates olfactory function in mice.

Vesicular glutamate transporter 1‐immunoreactive sensory neurons in the rat intrinsic cardiac ganglia

Sensory neurons that reside in intrinsic cardiac ganglia (ICG) have an important role in monitoring the cardiac milieu for regional cardiac dynamic control. We recently described sensory neurons in ICG that project to the cardiac ventricular wall and currently are evaluating their neurochemical phenotype. Since many primary sensory neurons are glutamatergic, we hypothesized that ICG sensory neurons are glutamatergic, utilizing vesicular glutamate transporter 1 (VGLUT1) to package glutamate into synaptic vesicles. In the present study, the rat ventricle wall was injected (retro‐diaphragmatic, 8μl) with the retrograde tracer wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP). After 72h, rats were euthanized and transcardially perfused with fixative. ICG were processed for immunohistochemistry studies. Our results demonstrated that: (1) VGLUT1‐immunoreactive (ir) neurons were present in ICG; (2) Some VGLUT1‐ir neurons were labeled from the ventricles with WGA‐HRP. Our previous studies indicated that sensory ICG neurons contain glutaminase, the synthetic enzyme for glutamate. These and the current results indicate that ICG sensory neurons are glutamatergic in neurochemical phenotype. Further investigation of VGLUTs during cardiac physiological and pathological conditions will help in deciphering the function of glutamate as a neurotransmitter in the ICG. Supported by NIH AR47410 (KEM).Grant Funding Source: NIH AR47410

Distribution of dopamine receptors D1- and D2-immunoreactive neurons in the dorsal motor nucleus of vagus in rats

The dorsal motor nucleus of vagus (DMV) plays an important role in the regulation of gastrointestinal function. Dopamine (DA) exerts potent neuromodulatory effects on the motoneurons in the DMV via dopamine receptors (DRs). However, the distribution of DRs and their neurochemical phenotypes in the DMV are unclear. In the present study, the distribution of DRs D1- and D2-immunoreactive (IR) neurons and their neurochemical phenotypes in the DMV of rats were investigated using a double-labeling immunofluorescence technique combined with confocal microscopy. The results indicated that a considerable quantity of D1 and D2 was expressed throughout the DMV. A large amount of choline acetyltransferase (ChAT)-IR and a few tyrosine hydroxylase (TH)-IR neurons were observed in the DMV. Nearly all of the neurons were also D1-IR and D2-IR. In conclusion, the present study demonstrates the wide distribution of D1 and D2 in the cholinergic and catecholaminergic neurons in the DMV of rats. The DRs might play an important role in the regulation of DA on the activity of cholinergic and catecholaminergic neurons in the DMV.