Identification of central nervous system sites involved in the water diuresis response elicited by central microinjection of nociceptin/ Orphanin FQ in conscious rats via c-Fos and inducible cAMP early repressor immunocytochemistry

Intracerebroventricular (i.c.v.) administration of the opioid-like peptide, nociceptin/Orphanin (nociceptin), in conscious rats produces diuretic and antinatriuretic effects. The present study utilised changes in Fos and inducible cAMP early repressor (ICER) immunocytochemistry expression to examine the central nervous (CNS) sites activated or inhibited, respectively, by central administration of nociceptin. Urine samples were collected during control (15 min) and after i.c.v. vehicle (5 microl, n = 12) or nociceptin (10 microg/5 microl; n = 12). Four additional urine samples (15-min) were collected after the i.c.v. injection. The brain was processed for Fos using a commercially available antibody (Oncogene AB-5) and for ICER using a polyclonal anti-ICER antibody raised in rabbits. In vehicle-injected conscious rats, renal excretion of water or sodium was not altered. However, nociceptin produced a rapid and marked increase in urine flow (V) and a decrease in urinary sodium excretion rate. In addition, i.c.v. nociceptin produced a significant increase in Fos staining in the dorsomedial nucleus of the hypothalamus, the perinuclear zone of the supraoptic nucleus, the organum vasculosum of the lamina terminalis (OVLT), the lateral preoptic area and the lateral hypothalamic area compared to control. By contrast, Fos expression decreased in the area postrema and locus coeruleus compared to controls. Furthermore, ICER staining was significantly increased in the perinuclear zone of the supraoptic nucleus, supraoptic nucleus, median preoptic nucleus, OVLT, medial preoptic area, central nucleus of the amygdala, and medial nucleus of the solitary tract. Together, central opioid receptor-like type 1 activation in these CNS regions may participate in the neural pathways involved in the diuretic and antinatriuretic effects of nociceptin.     

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.     

CNS cell groups projecting to pancreatic parasympathetic preganglionic neurons

The CNS cell groups that project to the pancreatic parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the pancreas of C8 spinal rats and after 6 days survival, the animals were perfused and their brains processed for immunohistochemical detection of PRV. Parasympathetic preganglionic neurons of the dorsal vagal nucleus were retrogradely labeled with PRV. Several CNS cell groups consistently contained transneuronally labeled neurons. In the medulla oblongata, labeled neurons were found in the nucleus tractus solitarius, area postrema, paratrigeminal nucleus, lateral paragigantocellular reticular nucleus, raphe pallidus and obscurus nuclei, C3 region and scattered cells in the ventral medullary reticular formation. In the pons, the A5 cell group, Barrington's nucleus and the subcoeruleus region contained labeled neurons. Only an occasional labeled cell was identified in the parabrachial nucleus. In the midbrain, almost no labeling was found except for an occasional neuron in the central gray matter. In the diencephalon, labeling was found in the paraventricular hypothalamic nucleus (PVN) as well as in the lateral hypothalamic nucleus at two levels (one at the level of the PVN and the other at the level of the subthalamic nucleus). in addition, the perifornical and dorsal hypothalamic nuclei contained labeled neurons. A few cells were found in the peripheral part of the dorsomedial hypothalamic nucleus. No labeling was seen in the ventromedial hypothalamic nucleus. In the telencephalon, the central amygdaloid nucleus and the bed nucleus of the stria terminalis were labeled.     

Nitrous oxide-induced c-Fos expression in the rat brain

Induction of c-Fos has previously been used to map locations of cells in the central nervous system (CNS) that are activated by ethanol administration. Only a few studies examining a restricted range of CNS areas have identified brain areas activated by nitrous oxide (N(2)O). Because ethanol and N(2)O have overlapping physiological, psychological and behavioral effects, we hypothesized that these drugs act on similar sites in the CNS. To test this hypothesis, we assessed c-Fos-like immunoreactivity in brain slices from male Long-Evans rats that received a 2-h exposure of 0, 20, 40 or 60% N(2)O (n=5 each) immediately prior to sacrifice. N(2)O administration produced significant (P<0.05) dose-related increases of c-Fos expression in several forebrain regions, including the hypothalamic supraoptic and paraventricular nuclei, the thalamic paraventricular nucleus, the amygdala, and in retrosplenial cortex. In the midbrain, N(2)O caused significant dose-related c-Fos expression in the Edinger-Westphal nucleus. Finally, the pontine locus coeruleus, and two medullary regions, the nucleus of the solitary tract and ventrolateral medulla, also showed significant dose-related N(2)O-induced c-Fos expression. Most of the brain areas identified as targets of N(2)O are also activated by ethanol administration. The overlapping pattern of c-Fos induced by ethanol and N(2)O suggests that these drugs may cause comparable central activity by acting on similar neuronal pathways.     

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.     

Distribution of preproadrenomedullin mRNA in the rat central nervous system and its modulation by physiological stressors

Adrenomedullin (ADM), encoded by the preproadrenomedullin (ppADM) gene, exerts multiple effects in a wide variety of peripheral and central tissues. Although ADM-like immunoreactivity has been shown to be widely distributed throughout the rat central nervous system (CNS), the detailed distribution of ppADM gene expression in the CNS and its modulation by physiological stimuli remain unknown. In our study, in situ hybridization was used to localize ppADM mRNA in the rat brain and to quantify its levels after exposure to different stressors including lipopolysaccharide (LPS; 100 microg/kg, iv), restraint stress (2 cycles of 1 hour restraint/1 hour rest), and 24 hours of dehydration. In addition, Fos immunoreactivity was used to identify the activation of neurons in response to LPS. Our results show that ppADM mRNA is widely distributed throughout the rat CNS, with especially high levels in autonomic centers including the hypothalamic paraventricular nucleus (PVN), hypothalamic supraoptic nucleus (SON), locus coeruleus, ventrolateral medulla, and intermediolateral cell column of the spinal cord. Furthermore, LPS inhibits ppADM gene expression in the parvocellular PVN (pPVN), magnocellular PVN (mPVN), SON, dorsal motor nucleus of the vagus, and area postrema among examined regions; restraint stress reduces ppADM mRNA levels in the pPVN, mPVN, SON, nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, and subfornical organ; 24 hours of water deprivation decreases ppADM gene expression only in the mPVN and SON. Taken together, our results suggest that ADM is involved in the regulation of the hypothalamo-neurohypophysial system, the hypothalamo-pituitary-adrenal axis, and central autonomic functions.     

Corticotropin releasing factor-like immunoreactivity in the rat brain as revealed by a modified cobalt-glucose oxidase-diaminobenzidine method

A cobalt-glucose-oxidase diaminobenzidine (Co-GOD) method, employing a specific antiserum against rat corticotropin releasing factor (CRF), was applied to determine immunohistochemically a widespread and detailed localization of corticotropin releasing factor-like immunoreactivity (CRFI) in the rat brain. Besides the CRFI cells in the paraventricular hypothalamic nucleus that project to the median eminence, CRFI cells were demonstrated in many brain regions, including the olfactory bulb, cerebral cortex, septal nuclei, hippocampus, amygdala, thalamic nuclei, medial hypothalamic nuclei, lateral hypothalamic area, perifornical area, central gray, cuneiform nucleus, inferior colliculus, raphe nuclei, mesencephalic reticular formation, laterodorsal tegmental nucleus, locus coeruleus, parabrachial nuclei, mesencephalic tract of the trigeminal nerve, pontine reticular formation, lateral superior olive, vestibular nuclei, prepositus hypoglossal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, lateral reticular nucleus, nucleus of the spinal tract of the trigeminal nerve, external cuneate nucleus, inferior olive, and medullary reticular formation. CRFI-reacting neural processes were also detected in these same areas. In particular, the median eminence, lateral septum, bed nucleus of the stria terminalis, mesencephalic reticular formation, parabrachial nuclei, and nucleus of the solitary tract contained large numbers of CRFI fibres. The widespread localization of CRFI demonstrated in the present study strongly suggests that CRF, like many other neurohormones and peptides, may act as a neurotransmitter and/or neuromodulator in numerous extrahypothalamic circuits, as well as participate in neuroendocrine regulation.     

Effect of oxytocin on acupuncture analgesia in the rat

Oxytocin has been demonstrated to be involved in pain modulation. Acupuncture analgesia is a very useful clinical tool for pain relief, which has over 2500-year history in China. The present study investigated the role of oxytocin in acupuncture analgesia in the rat through oxytocin administration and measurement. Central administration of oxytocin (intraventricular injection or intrathecal injection) enhanced acupuncture analgesia, while central administration of anti-oxytocin serum weakened acupuncture analgesia in a dose-dependent manner. However, intravenous injection of oxytocin or anti-oxytocin serum did not influence acupuncture analgesia. Electrical acupuncture of "Zusanli" (St. 36) reduced oxytocin concentration in the hypothalamic supraoptic nucleus, and elevated oxytocin concentration in the hypothalamic suprachiasmatic nucleus, hypothalamic ventromedial nucleus, thalamic ventral nucleus, periaqueductal gray, raphe magnus nucleus, caudate nucleus, thoracic spinal cord and lumbar spinal cord, but did not alter oxytocin concentration in the hypothalamic paraventricular nucleus, anterior pituitary, posterior pituitary and plasma. The data suggested that oxytocin in central nervous system rather than in peripheral organs is involved in acupuncture analgesia.     

Comparative topography of projections from the mesodiencephalic junction to the inferior olive, vestibular nuclei, and upper cervical cord in the cat

Distributions of neurons located in the central rostral mesencephalon and caudal diencephalon that project to the upper cervical spinal cord, vestibular nuclei, or inferior olive were studied in the cat by using retrograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Afferent sources to all of these targets were observed in the interstitial nucleus of Cajal (INC), the region surrounding the fasciculus retroflexus (PF), and the nucleus of the fields of Forel (NFF). Three-dimensional reconstruction revealed differences in densities of cells projecting from these common areas. Spinal projecting cells were present in slightly greater numbers in the caudal two-thirds of the INC, whereas those projecting to the vestibular complex were more numerous in the rostral two-thirds of this nucleus. A relatively smaller number of olivary projecting cells were dispersed throughout the INC. Olivary afferent sources outnumber those with spinally directed or vestibularly directed axons in the PF region. In the fields of Forel, cells projecting to the vestibular nuclei or inferior olive were concentrated medially, whereas cells projecting to the spinal cord appeared both medially and laterally. Each type of afferent source was also seen in the nucleus of the posterior commissure and the posterior ventral lateral hypothalamic area. Unique sources of afferents to the inferior olive were observed in the parvicellular red nucleus (ipsilateral to the injections) and the anterior and posterior pretectal nuclei. A large number of labeled neurons was seen in the nucleus of Darkschewitsch after injections of tracer into the inferior olive, but this projection did not appear to be unique, as small numbers of labeled cells were also seen after injections into the cervical spinal cord. The Edinger-Westphal nucleus and the adjacent somatic oculomotor nucleus contained cells which projected separately to the spinal cord or the vestibular complex, and the superior colliculus contained cells which projected separately to the contralateral spinal cord or the contralateral inferior olive. In this study, it was also noted that neurons in the medial terminal nucleus of the accessory optic tract projected to the ipsilateral inferior olive or to the contralateral vestibular complex. These differences in locations and densities of cells projecting to the cervical spinal cord, vestibular complex, and inferior olive may underlie functional specializations in these areas in relation to vertical eye and head movement control and to neural systems controlling postural adjustments accompanying limb movements.     

CNS cell groups projecting to the submandibular parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study.

The retrograde transneuronal viral tracing method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons controlling the submandibular gland in the rat. A genetically engineered beta-galactosidase expressing Bartha strain of pseudorabies virus (PRV) was injected into the submandibular gland of rats. After 4 days, PRV infected tissues were reacted with the Bluo-Gal substrate (halogenated indolyl-beta-D-galactoside) and labeled cell bodies were identified throughout the brain. In the medulla oblongata, cell body labeling was seen in the superior salivatory nucleus, and throughout the medullary reticular formation as well as in the nucleus of the solitary tract, spinal trigeminal nucleus, and deep cerebellar nuclei. In the pons, PRV labeled neurons were found bilaterally in the locus ceruleus, subceruleus region, and parabrachial complex. In the mesencephalon, labeled cells were found in the Edinger-Westphal nucleus, deep mesencephalic nucleus, and central grey matter. Several hypothalamic regions were labeled including the lateral, perifornical and paraventricular hypothalamic nuclei. In the telencephalon, PRV-positive cell bodies were observed in the substantia innominata, bed nucleus of the stria terminalis and central nucleus of the amygdala. The results suggest that widespread areas of the CNS are involved in control of salivation.     

Distribution of nerve growth factor-like immunoreactive neurons in the adult rat brain following colchicine treatment.

Using immunohistochemical techniques, we have previously localized nerve growth factor (NGF)-like immunoreactivity in the normal adult rat central nervous system (CNS) exclusively in the hippocampal mossy fiber region and within basal forebrain cholinergic neurons--a cell population believed to be primary NGF consumers within the CNS. In the present investigation, we have attempted to identify potential producers of NGF by pretreating animals with colchicine. Such a treatment would be expected to block microtubule-assisted neuritic transport mechanisms, thus preventing the accumulation of antigens normally obtained by retrograde transport and forcing the accumulation of cell products normally exported anterogradely. Forty-eight hours after colchicine administration within their innervation territories, basal forebrain cholinergic neurons showed a marked loss of NGF-like immunoreactivity. Conversely, following colchicine treatment, many new populations of NGF-like immunoreactive cells were detected, several of which have been previously observed with in situ hybridization techniques for NGF mRNA. Many NGF-like immunoreactive populations, however, were not previously recognized by in situ hybridization methods, including cells of the striatum, reticular thalamic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, lateral and medial septum, substantia innominata, and nucleus basalis. Furthermore, evidence is provided that colchicine-blocked, NGF-like immunoreactive neurons within the basal forebrain are not cholinergic, thus reinforcing the hypothesis that trophic support for these NGF-dependent neurons may be derived from distant and local sources. The distinctive distribution of NGF-like immunoreactive cells observed in this study strongly correlates with the reported distribution of NGF mRNA in CNS neurons, thus suggesting that our antibodies are uniquely recognizing NGF and not other related neurotrophins.     

Widespread expression of Fos protein induced by acute haloperidol administration in the rat brain

The effect of acute haloperidol administration on Fos protein expression was examined immunohistochemically in discrete regions of the rat brain. Male Wistar rats were injected subcutaneously (s.c.) with 0.1, 0.25, or 1.0 mg/kg of haloperidol. Two h after the injection, the rats were perfused, and the numbers of Fos immunoreactive neurons were counted in 24 brain regions. In contrast to the limited changes in Fos immunoreactivity at the low dose of haloperidol (0.1 mg/kg), the moderate dose (0.25 mg/kg) induced widespread increases in Fos-positive neurons in the rat brain. Large increases were produced in the caudate-putamen, nucleus accumbens, central amygdaloid nucleus, dorsomedial hypothalamic nucleus, hippocampus CA1 and substantia nigra pars compacta. Moderate increases were observed in the entorhinal cortex, lateral septum, lateral habenula, lateral amygdaloid nucleus, dentate gyrus, and mesencephalic central grey. Mild increases were induced in the anterior cingulate, temporal, occipital and perirhinal cortex, and central medial thalamic nucleus. The distribution of changes in Fos immunoreactivity at the high dose of haloperidol (1.0 mg/kg) were comparable to their distribution at the moderate dose. These findings indicate that the effect of acute haloperidol on Fos expression is widely distributed in the rat brain beyond the previously known dopamine-rich areas at the dose which produces plasma levels equivalent to those within the therapeutic range used clinically in humans. Further studies on the effects of chronic antipsychotic treatment are needed in order to identify the sites of the therapeutic action of antipsychotic drugs.     

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the development and function of the blood–brain barrier

Prenatal exposure to environmental chemicals such as dioxins is known to have adverse effects on the developing central nervous system (CNS) in mammals. Because the fetal blood–brain barrier (BBB) is immature, dioxins are thought to exert their toxic effects on the CNS by crossing the BBB and acting on neural cells directly. However, little is known whether dioxins alter the BBB. In this study, to investigate the effects of dioxins on BBB function, we exposed an in vitro BBB system comprising rat endothelial cells, astrocytes, and pericytes to the toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either before or after BBB formation. We assessed BBB permeability and the function of tight junctions by measuring transendothelial electric resistance (TEER) values following exposure. Subsequently, total RNA and proteins were obtained from the cells for analysis. TEER values following TCDD exposure before but not after BBB formation were lower than those of the control group. We also observed that the expression of the tight junction proteins ZO-1 and claudin-5 was suppressed following TCDD exposure. To examine the cause of this reduction in protein levels, we performed a real-time quantitative polymerase chain reaction assay and observed low expression of the glial cell line-derived neurotrophic factor (GDNF) mRNA in the exposed groups. Moreover, to rescue the effects of TCDD, we applied extrinsic GDNF with TCDD. The several disruptions caused by TCDD were rescued by the GDNF addition. Our findings suggest that exposure to TCDD during BBB formation disrupts and impairs BBB function in part by the suppression of GDNF action, which may contribute to the adverse effects of TCDD on the fetal CNS.     

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors.     

Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure

The thermogenic activity of interscapular brown adipose tissue (IBAT) in response to physiologic stimuli, such as cold exposure, is controlled by its sympathetic innervation. To determine which brain regions might be involved in the regulation of cold-evoked increases in sympathetic outflow to IBAT, the present study compared central nervous system (CNS) areas activated by cold exposure with brain regions anatomically linked to the sympathetic innervation of IBAT. Immunocytochemical localization of Fos was examined in the brains of rats exposed to 4 degrees C for 4 hours. In a separate group of rats, the neural circuit involved in IBAT control, including the location of sympathetic preganglionic neurons in the spinal cord, was characterized with pseudorabies virus, a retrograde transynaptic tracer. Central noradrenergic and serotonergic groups related to the sympathetic outflow to IBAT also were identified. Localization of viral antigens at different survival times (66-96 hours) revealed infection in circumscribed CNS populations, but only a subset of the regions comprising this circuitry showed cold-evoked Fos expression. The raphe pallidus and the ventromedial parvicellular subdivision of the paraventricular hypothalamic nucleus (PVH), both infected at early survival times, were the main areas containing sympathetic premotor neurons activated by cold exposure. Major cold-sensitive areas projecting to spinal interneurons or to regions containing sympathetic premotor neurons, which became infected at intermediate intervals, included lateral hypothalamic, perifornical, and retrochiasmatic areas, anterior and posterior PVH, ventrolateral periaqueductal gray, and Barrington's nucleus. Areas infected later, most likely related to reception of cold-related signals, comprised the lateral preoptic area, parastrial nucleus, dorsomedial hypothalamic nucleus, lateral parabrachial nucleus, and nucleus of the solitary tract. These interconnected areas, identified by combining functional and retrograde anatomic approaches, likely constitute the central circuitry responsible for the increase in sympathetic outflow to IBAT during cold-evoked thermogenesis.     

Elucidation of the anatomy of a satiety network: Focus on connectivity of the parabrachial nucleus in the adult rat

We hypothesized that brain regions showing neuronal activation after refeeding comprise major nodes in a satiety network, and tested this hypothesis with two sets of experiments. Detailed c‐Fos mapping comparing fasted and refed rats was performed to identify candidate nodes of the satiety network. In addition to well‐known feeding‐related brain regions such as the arcuate, dorsomedial, and paraventricular hypothalamic nuclei, lateral hypothalamic area, parabrachial nucleus (PB), nucleus of the solitary tract and central amygdalar nucleus, other refeeding activated regions were also identified, such as the parastrial and parasubthalamic nuclei. To begin to understand the connectivity of the satiety network, the interconnectivity of PB with other refeeding‐activated neuronal groups was studied following administration of anterograde or retrograde tracers into the PB. After allowing for tracer transport time, the animals were fasted and then refed before sacrifice. Refeeding‐activated neurons that project to the PB were found in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamic area; arcuate, paraventricular, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; parasubthalamic nucleus; central amygdalar nucleus; area postrema; and nucleus of the solitary tract. Axons originating from the PB were observed to closely associate with refeeding‐activated neurons in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamus; paraventricular, arcuate, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; central amygdalar nucleus; parasubthalamic nucleus; ventral posterior thalamic nucleus; area postrema; and nucleus of the solitary tract. These data indicate that the PB has bidirectional connections with most refeeding‐activated neuronal groups, suggesting that short‐loop feedback circuits exist in this satiety network. J. Comp. Neurol. 524:2803–2827, 2016. © 2016 Wiley Periodicals, Inc.     

Identification of central sites involved in butorphanol-induced feeding in rats

Butorphanol (BT), a mixed kappa- and mu-opioid receptor agonist, induces vigorous food intake in rats. Peripheral injection of BT seems to increase food intake more effectively than intracerebroventricular administration. To further elucidate the nature of BT's influence on consummatory behavior, we examined which feeding-related brain areas exhibit increased c-Fos immunoreactivity (IR) following subcutaneous injection of 4 mg/kg body weight BT, a dose known to induce a maximal orexigenic response. We also evaluated whether direct administration of BT into the forebrain regions activated by peripheral BT injection affects food intake. Peripheral BT administration induced c-Fos-IR in the hypothalamic paraventricular nucleus (PVN), central nucleus of the amygdala (CeA), and nucleus of the solitary tract (NTS). However, 0.1-30 microg BT infused into the CeA, failed to increase food intake 1, 2, and 4 h after injection. Only the highest dose of BT (30 microg) injected into the PVN increased feeding. These results suggest that the PVN, CeA, and NTS mediate the effects of peripherally-injected BT. The PVN or CeA are probably not the main target sites of immediate BT action.     

Localization of the urotensin II receptor in the rat central nervous system

The vasoactive peptide urotensin II (UII) is primarily expressed in motoneurons of the brainstem and spinal cord. Intracerebroventricular injection of UII provokes various behavioral, cardiovascular, motor, and endocrine responses in the rat, but the distribution of the UII receptor in the central nervous system (CNS) has not yet been determined. In the present study, we have investigated the localization of UII receptor (GPR14) mRNA and UII binding sites in the rat CNS. RT-PCR analysis revealed that the highest density of GPR14 mRNA occurred in the pontine nuclei. In situ hybridization histochemistry showed that the GPR14 gene is widely expressed in the brain and spinal cord. In particular, a strong hybridization signal was observed in the olfactory system, hippocampus, olfactory and medial amygdala, hypothalamus, epithalamus, several tegmental nuclei, locus coeruleus, pontine nuclei, motor nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, inferior olive, cerebellum, and spinal cord. Autoradiographic labeling of brain slices with radioiodinated UII showed the presence of UII-binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex. Intense expression of the GPR14 gene in some hypothalamic nuclei (supraoptic, paraventricular, ventromedian, and arcuate nuclei), in limbic structures (amygdala and hippocampus), in medullary nuclei (solitary tract, dorsal motor nucleus of the vagus), and in motor control regions (cerebral and cerebellar cortex, substantia nigra, pontine nuclei) provides the anatomical substrate for the central effects of UII on behavioral, cardiovascular, neuroendocrine, and motor functions. The occurrence of GPR14 mRNA in cranial and spinal motoneurons is consistent with the reported autocrine/paracrine action of UII on motoneurons.     

Capsaicin-induced neuronal degeneration: silver impregnation of cell bodies, axons, and terminals in the central nervous system of the adult rat

Capsaicin is a neurotoxic substance valued in neurobiological research because of its ability to selectively damage small unmyelinated primary sensory neurons. Previous work has indicated that systemic capsaicin administration causes permanent neuronal degeneration in neonatal rats, but evidence that capsaicin has a similar effect in adults is equivocal and incomplete. Therefore, we used silver impregnation, a method that labels degenerating neurons, to examine the central nervous system of adult rats after systemic capsaicin treatment. Adult rats were injected with a single intraperitoneal dose of capsaicin (50 or 90 mg/kg) or vehicle solution and killed 6, 12, 18, 24, 48, 96, or 240 hours later. Sections of brain and spinal cord were stained with the Carlsen-de Olmos cupric silver method. As reported previously, stained sections revealed degeneration in areas known to be innervated by small-diameter primary sensory fibers: the substantia gelatinosa of the spinal cord dorsal horn and spinal trigeminal nucleus, the solitary nucleus and tract, and the lateral borders of the area postrema. In addition, axon and terminal degeneration was observed in several discrete forebrain and hindbrain areas not previously associated with capsaicin-induced degeneration in either adult or neonatal rats: the inferior olive, the olivary pretectal nucleus, the interpeduncular nucleus, the suprachiasmatic nucleus, and the lateral septum/medial accumbens region. Furthermore, degenerating cell bodies were observed in the intrafascicular nucleus of the ventromedial midbrain tegmentum, in the supramammillary nucleus, and in the posterior hypothalamic area. Unilateral nodose ganglionectomy produced terminal staining on the denervated side very similar to that induced bilaterally by capsaicin. In addition, unilateral nodose ganglionectomy 1 month prior to capsaicin injection greatly attenuated staining in the ipsilateral nucleus of the solitary tract, confirming the hypothesis that capsaicin damages vagal sensory neurons innervating this nucleus. Capsaicin-induced damage in adult rats was long-lasting, since the second of two capsaicin treatments spaced 4.5 months apart produced no additional degeneration.