Orexin (hypocretin) gene expression in rat ependymal cells.

The expression of prepro-orexin (PPO) mRNA in the rat brain was investigated by in situ hybridization histochemistry. In the lateral and posterior hypothalamic areas, which are considered to produce exclusively PPO mRNA, we found high levels of PPO mRNA expressions. We also localized PPO mRNA hybridization signals at lower levels around the lateral ventricles, the third and fourth ventricle. Cellular analysis by emulsion autoradiography revealed the expression of PPO mRNA in the ependymal cell layer. Our results demonstrate that beside the lateral and posterior hypothalamus PPO mRNA is expressed in ependymal cells.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.     

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.     

Distribution of pituitary adenylate cyclase-activating polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain

To map in detail the brain areas in which pituitary adenylate cyclase-activating polypeptide (PACAP) may play a significant role in birds, the distribution of PACAP and PACAP type I receptor (PAC(1)-R) mRNA was examined throughout the entire chicken brain by using in situ hybridization histochemistry. Widespread distribution of both PACAP and its receptor mRNA was found. The telencephalic areas where the most intense signals for PACAP mRNA were found included the hyperstriatum accessorium, the hippocampus, and the archistriatum. In the diencephalon, a group of neurons that highly expressed PACAP mRNA was observed from the anterior medial hypothalamic nucleus to the inferior hypothalamic nucleus. Moderate expression was found in the paraventricular nucleus and the preoptic region. A second large group of neurons containing PACAP message was found within the nucleus dorsolateralis anterior thalami and extended caudally to the area around the nucleus ovoidalis and the nucleus paramedianus internus thalami. Furthermore, expression of PACAP message was observed within the bed nucleus of the pallial commissure, nucleus spiriformis medialis, optic tectum, cerebellar cortex, olfactory bulbs, and several nuclei within the brainstem (dorsal vagal and parabrachial complex, reticular formation). The highest expression of PAC(1)-R mRNA was found in the dorsal telencephalon, olfactory bulbs, lateral septum, optic tectum, cerebellum, and throughout the hypothalamus and thalamus. The presence of PACAP and PAC(1)-R mRNA in a variety of brain areas in birds suggests that PACAP mediates several physiologically important processes in addition to regulating the activity of the pituitary gland.     

Localization of calcitonin receptor mRNA in the mouse brain: coexistence with serotonin transporter mRNA

To elucidate the sites of and mechanisms of analgesic effect of centrally injected calcitonin, we examined expression of calcitonin receptor mRNA in the mouse brain by in situ hybridization techniques. Calcitonin receptor mRNA was expressed in various brain regions, including the preoptic area, dorsomedial hypothalamic nucleus, lateral hypothalamic area, periaqueductal gray, dorsal raphe nucleus, locus coeruleus, lateral parabrachial nucleus, gigantocellular reticular nucleus alpha part, lateral paragigantocellular nucleus, raphe magnus nucleus and solitary tract nucleus, which are known to play important roles in pain modulation. In addition, a double in situ hybridization technique demonstrated the intense expression of calcitonin receptor mRNA on serotonergic neurons in some raphe nuclei and the lateral paragigantocellular nucleus, suggesting the involvement of central serotonergic pathways in analgesic effect of calcitonin.     

Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain

We have used in situ hybridization and immunocytochemistry to map the cellular localization of NGF receptor (NGF-R) mRNA and protein in the adult rat brain. In addition to basal forebrain magnocellular neurons, NGF-R is widely expressed within the CNS, including neurons of the caudate/putamen, ventral premamillary nucleus, mesencephalic trigeminal nucleus, prepositus hypoglossal nucleus, raphe nucleus, nucleus ambiguous, and Purkinje cells of the cerebellum. Cells of the vestibulocochlear ganglion also contain NGF-R mRNA and protein. Ventricular subependymal cells and tanycytes are clearly stained by immunocytochemistry, yet only very weak hybridization is detectable in these cells. Also, greater amounts of NGF-R protein than of mRNA appear to be present in the glomeruli of the olfactory bulb, area postrema, and nucleus tractus solitarius. Areas that contain only NGF-R immunoreactive fibers and terminals can be distinguished from the cellular sites of NGF-R biosynthesis and include the suprachiasmatic nucleus, the principal olivary pretectal nucleus, the superior colliculus, the inferior olive, and the principal and spinal trigeminal nuclei. This study shows that NGF-R is widely expressed within individual neurons in different areas of the rat brain and identifies new potential CNS target sites of endogenous NGF.     

Anatomic relationships between aromatase and androgen receptor mRNA expression in the hypothalamus and amygdala of adult male cynomolgus monkeys

This study mapped the regional locations of cells expressing cytochrome P450 aromatase (P450AROM) and androgen receptor (AR) mRNAs in the adult male macaque hypothalamus and amygdala by in situ hybridization histochemistry using monkey-specific cRNA probes. High densities of P450AROM and AR mRNA-containing neurons were observed in discrete hypothalamic areas involved in the regulation of gonadotropin secretion and reproductive behavior. P450AROM mRNA-containing neurons were most abundant in the medial preoptic nucleus, bed nucleus of the stria terminalis, and anterior hypothalamic area, whereas AR mRNA-containing neurons were most numerous in the ventromedial nucleus, arcuate nucleus, and tuberomamillary nucleus. Moderate to heavily labeled P450AROM mRNA-containing cells were present in the cortical and medial amygdaloid nuclei, which are known to have strong reciprocal inputs with the hypothalamus. Heavily labeled P450AROM mRNA-containing cells were found in the accessory basal amygdala nucleus, which projects to the cingulate cortex and hippocampus, areas that are important in the expression of emotional behaviors and memory processing. In contrast to P450AROM, the highest density of AR mRNA labeling in the temporal lobe was associated with the cortical amygdaloid nucleus and the pyramidal cells of the hippocampus. All areas that contained P450AROM mRNA-expressing cells also contained AR mRNA-expressing cells, but there were areas in which AR mRNA was expressed but not P450AROM mRNA. The apparent relative differences in the expression of P450AROM and AR mRNA-containing neurons within the monkey brain suggests that T acts through different signaling pathways in specific brain areas or within different cells from the same region.     

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF. © 1995 Wiley-Liss, Inc.     

Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.     

Estrogen receptor beta and progesterone receptor mRNA in the intergeniculate leaflet of the female rat.

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor beta and progesterone receptor was carried out on sections containing the lateral geniculate body using [35S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor beta and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.     

Distribution of Galanin mRNA Containing Cells and Galanin Receptor Binding Sites in Human and Rat Hypothalamus

The distribution of cells containing galanin mRNA and that of galanin receptor binding sites were investigated using in situ hybridization histochemistry and receptor autoradiography in male rat hypothalamus and in postmortem hypothalamic tissues from control human brains. Oligonucleotide probes labelled with 32P were used for hybridization experiments. The specificity of the hybridization signal was ascertained using several probes, competition assays and Northern blot analysis. High levels of hybridization were found in the paraventricular, supraoptic and arcuate nuclei of rat and human hypothalamus. Human intermediate nuclei and scattered cells of the posterior perifornical nucleus also contained galanin mRNA. Galanin mRNA was also found in the dorsomedial nucleus of the rat. The distribution of galanin receptor sites was investigated by receptor autoradiography using 125I-labelled porcine galanin. The specificity of the binding was assessed by competition with different neuropeptides. While galanin blocked the binding at nanomolar concentrations, the other neuropeptides examined were ineffective at 10-7 M concentrations. The highest densities of galanin binding sites were seen in the preoptic area, ventromedial and lateral nuclei, of rat and human hypothalamus. In contrast, very low densities of binding sites were observed in the paraventricular, supraoptic and arcuate nuclei. Our results show that the distribution of neurons expressing galanin is complementary to that of galanin receptors in the rat and human hypothalamus. This suggests that receptors for galanin are not located on the cell bodies of galaninergic neurons, but are probably presynaptic on or postsynaptic to the processes of these cells.     

In situ hybridization detection of trka mRNA in brain: Distribution,colocalization with p75NGFR and up-regulation by nerve growth factor

In situ hybridization techniques were used to examine the distribution and the nerve growth factor (NGF) regulation of trkA mRNA in the adult rat brain in order to identify neurons in discrete regions of the brain that may be NGF responsive. In agreement with previous studies, trkA mRNA was detected within cells located in the medial septum (MS), diagonal band of Broca (DBB), and caudate. trkA mRNA was also detected in many other regions of the brain, including the nucleus basalis of Meynert, substantia innominata, paraventricular nucleus of the thalamus, interpeduncular nucleus, prepositus hypoglossal nucleus, vestibular nudei raphe obscuris, cochlear nucleus, sensory trigeminal nuclei, and gigantocellular as well as perigigantocellular neurons in the medullary reticular formation. By combining in situ hybridization detection of trkA mRNA with immunocytochemical detection ofp75^NGFR, it was determined that the vast majority (> 90%) of the trkA mRNA-containing cells detected in the MS and DBB also express p75^NGFR. Likewise, the vast majority of p75^NGFR-IR cells detected in the MS and DBB expressed trkA mRNA. Intracerebroventricular infusions of NGF into the third ventricle adjacent to the preoptic area resulted in a 58% increase in relative cellular levels of trkA mRNA in the horizontal limb of the DBB. These data provide evidence that both p75^NGFR and trkA are expressed by NGF-responsive neurons in the MS and DBB. In addition, we note that areas that contained trkA mRNA and that also have been reported to contain p75^NGFR are areas where high-affinity NGF binding sites have been observed autoradio-graphically, whereas areas that contain either trkA or p75^NGFR alone are areas where no high-affinity NGF binding has been reported. Together, these findings suggest that both trkA and p75^NGFR play an important role in the formation of high-affinity NGF receptors in brain and, furthermore, suggest that NGF may have physiological effects within many regions of the brain outside of the basal forebrain.     

Estrogen receptor β and progesterone receptor mRNA in the intergeniculate leaflet of the female rat

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor β and progesterone receptor was carried out on sections containing the lateral geniculate body using [³⁵S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor β and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.     

Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1.

Recent molecular cloning studies by our laboratory and others have identified the existence of a novel rat galanin receptor subtype, GALR2. In the present study, we examined the regional and cellular distribution of GALR2 mRNA in the rat central nervous system (CNS) by in situ hybridization. For comparative purposes, adjacent sections were probed for GALR1 mRNA expression. Our findings indicate that dorsal root ganglia express by far the highest levels of GALR2 mRNA in the rat CNS. Hybridization signal is mainly concentrated over small and intermediate primary sensory neurons. In spinal cord, the large alpha motoneurons of the ventral horn are moderately labeled and several small, but less intensely labeled, cells are scattered throughout the gray matter. In brain sections, the highest levels of GALR2 mRNA are detected in granule cells of the dentate gyrus, in the mammillary nuclei, and in the cerebellar cortex. Moderate levels of GALR2 mRNA are observed in the olfactory bulb, olfactory tubercle, piriform and retrospinal cortices, hypothalamus (namely the preoptic area, arcuate nucleus, and dorsal hypothalamic area), substantia nigra pars compacta, and sensory trigeminal nucleus. Moderate to weak hybridization signal is also present in several other hypothalamic nuclei, specific layers of the neocortex, periaqueductal gray, and several nuclei within the pons and medulla, including locus coeruleus, lateral parabrachial, motor trigeminal, pontine reticular, hypoglossal, vestibular complex, ambiguus, and facial and lateral reticular nuclei. This novel pattern of GALR2 distribution within the rat CNS differs considerably from that of GALR1, suggesting that specific physiologic effects of galanin may be ascribed to the GALR2 galanin receptor subtype.     

Galanin-R1 receptor in anterior and mid-hypothalamus: Distribution and regulation

The distribution and regulation of galanin-R1 receptor (GAL-R1-R) mRNA has been studied in the anterior and mid-diencephalon by using in situ hybridization. Moreover, possible colocalization of GAL-R1-R mRNA and prepro-galanin or vasopressin mRNAs has been analyzed at the cellular level using double in situ hybridization methodology. Many nuclei in the hypothalamus expressed GAL-R1-R mRNA, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Strong expression was also seen in the same sections in various areas outside of the diencephalon. The distribution patterns are similar to those described in earlier studies. Double labeling experiments showed GAL-R1-R mRNA in vasopressin neurons in the PVN and SON. Moreover, GAL-R1-R mRNA and prepro-galanin mRNA were colocalized in several hypothalamic nuclei. GAL-R1-R mRNA levels showed a high degree of plasticity. Thus, salt loading resulted in a marked increase in GAL-R1-R mRNA levels in the PVN and SON and a moderate decrease was seen during lactation. In contrast, hypophysectomy caused a decrease in GAL-R1-R mRNA levels. Differential effects of colchicine were recorded with a decrease of GAL-R1-R mRNA in the magnocellular hypothalamic neurons. After salt loading or during lactation, GAL-R1-R mRNA and prepro-galanin mRNA were regulated in parallel, whereas their levels changed in opposite directions after hypophysectomy and colchicine injection. In conclusion, GAL-R1-Rs are present in several hypothalamic nuclei, partly in neurons synthesizing galanin. The receptors are regulated in a specific fashion in the various nuclei, depending on the stimulus applied. The results suggest that the effect of galanin in the hypothalamus partly depends on the state of receptor expression. J. Comp. Neurol. 399:321–340, 1998. © 1998 Wiley-Liss, Inc.     

Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat

The melanocortin 4 receptor (MC4-R) plays a pivotal role in maintaining energy homeostasis in rodents and humans. For example, MC4-R deletion or mutation results in obesity, hyperphagia, and insulin resistance. Additionally, subsets of leptin-induced autonomic responses can be blocked by melanocortin receptor antagonism, suggesting that MC4-R-expressing neurons are downstream targets of leptin. However, the critical autonomic control sites expressing MC4-Rs are still unclear. In the present study, we systematically examined the distribution of MC4-R mRNA in the adult rat central nervous system, including the spinal cord, by using in situ hybridization histochemistry (ISHH) with a novel cRNA probe. Autonomic control sites expressing MC4-R mRNA in the hypothalamus included the anteroventral periventricular, ventromedial preoptic, median preoptic, paraventricular, dorsomedial, and arcuate nuclei. The subfornical organ, dorsal hypothalamic, perifornical, and posterior hypothalamic areas were also observed to express MC4-R mRNA. Within extrahypothalamic autonomic control sites, MC4-R-specific hybridization was evident in the infralimbic and insular cortices, bed nucleus of the stria terminalis, central nucleus of the amygdala, periaqueductal gray, lateral parabrachial nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus (DMV), and intermediolateral nucleus of the spinal cord (IML). By using dual-label ISHH, we confirmed that the cells expressing MC4-R mRNA in the IML and DMV were autonomic preganglionic neurons as cells in both sites coexpressed choline acetyltransferase mRNA. The distribution of MC4-R mRNA is consistent with the proposed roles of central melanocortin systems in feeding and autonomic regulation.