Evidence for coexistence between calcitonin gene-related peptide and serotonin in the bulbospinal pathway in the monkey

By the use of the indirect immunofluorescence and in situ hybridization techniques, the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and CGRP mRNA was studied in the spinal cord as well as in the midline raphe nuclei and the hypoglossal nucleus in the medulla oblongata of the monkey (Macaca fascicularis). In the spinal cord only a few large neurons in the motor nucleus contained CGRP-LI, while a majority of the neurons in the hypoglossal nucleus contained CGRP-LI. A relatively dense innervation by CGRP-immunoreactive (IR) fibers was also seen close to cell bodies and proximal dendrites of large neurons in the motor nucleus, especially in its ventral part. 5-Hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-IR varicosities were also observed in a similar position around large neurons in the motor nucleus. Double labeling disclosed that the majority of CGRP-IR axon terminals also contained 5-HT-LI. Expression of CGRP mRNA was found in neurons in the medullary midline raphe nuclei and in large neurons in the motor nucleus at the cervical spinal cord level. In adjacent sections of the medulla oblongata, CGRP-labeled neurons in the midline raphe nuclei also expressed preprotachykinin mRNA. The present results show that CGRP- and 5-HT-LI coexist in fibers within the motor nucleus of the monkey spinal cord and that this coexistence is probably due to the presence of CGRP in the descending bulbospinal, serotonergic pathway.     

单侧隐睾大鼠生殖股神经神经核改变

目的 研究在单侧隐睾大鼠背根神经节中,生殖股神经神经核的改变.方法 应用辣根过氧化酶(HRP)逆行示踪与免疫荧光相结合的双重技术,观察注射17-β雌二醇获得隐睾大鼠动物模型GFN传导及其递质免疫反应细胞的变化.取单侧隐睾大鼠12只设为A组;取12只单侧隐睾大鼠在出生后第30天切断隐睾侧生殖股神经(GFN)设为B组;另取正常同日龄大鼠10只设为C组.分别在日龄第58天隐睾侧GFN末梢用HRP逆行示踪.48 h后对SD大鼠灌注固定后,取大鼠的T12～L3脊髓及背根神经结,脱水后冰冻切片显色,并用免疫荧光标记CGRP.结果 HRP标记的生殖股神经在L1,L2水平的背根神经节感觉核中发现,A组HRP阳性神经元表达平均灰度和阳性单位较B组和C组要高有统计学意义(P<0.05),B组和C组比较(P＞0.05);CGRP免疫荧光结果中A组较B组、C组表达要少(P<0.05).结论 单侧隐睾大鼠生殖股神经感觉核神经元增多,运动核相对减少,分泌的神经递质CGPR减少.     

Sexually dimorphic expression of calcitonin gene-related peptide (CGRP) immunoreactivity by rat mediobasal hypothalamic neurons

Although the hypothalamic arcuate nucleus is a sexually dimorphic region of the rat brain, there are no reports of sex differences in the number of neurons containing specific neuropeptides within this structure. As cells synthesizing calcitonin gene-related peptide (CGRP) have been shown to exhibit sex differences in other steroid-receptive regions of the rat brain, we examined whether the CGRP-immunoreactive cells located in the mediobasal hypothalamus may also be sexually dimorphic. Immunostaining of sections from male and female colchicine-treated rats revealed a small population of CGRP-immunoreactive cells distributed throughout the arcuate nucleus. Immunoreactive cells were also detected in the lateral hypothalamic perifornical region, dorsomedial, posterior periventricular and ventral tuberomammillary nuclei, and zona incerta. Cell count analysis revealed approximately twice as many CGRP-immunoreactive cell profiles in the rostral (P < 0.01), middle (P < 0.001), and caudal (P < 0.01) thirds of the arcuate nucleus of male rats compared with females. A significant sex difference in immunoreactive cell numbers (male > female) was also detected within the caudal dorsomedial nucleus (P < 0.05) but not in the posterior periventricular nucleus, perifornical region and zona incerta. Although fibers immunoreactive for CGRP were identified in low density throughout the mediobasal hypothalamus, only female rats displayed prominent fiber staining in the periventricular region. Double-labelling immunofluorescence experiments revealed that the CGRP-immunoreactive cells within the zona incerta, but not the hypothalamus, were also immunoreactive for tyrosine hydroxylase; at least 60% of the A13 dopaminergic neurons co-express CGRP. These results provide evidence that sex differences exist in the number of specific neuropeptide-synthesizing cells within the hypothalamic arcuate nucleus and provide further examples of cell populations expressing CGRP immunoreactivity in a sexually dimorphic manner. © 1996 Wiley-Liss, Inc.     

In situ hybridization localization of choline acetyltransferase mRNA in adult rat brain and spinal cord

The cellular distribution of choline acetyltransferase (ChAT) mRNA within the adult rat central nervous system was evaluated using in situ hybridization. In forebrain, hybridization of a ³⁵S-labeled rat ChAT cRNA densely labeled neurons in the well-characterized basal forebrain cholinergic system including the medial septal nucleus, diagonal bands of Broca, nucleus basalis of Meynert and substantia innominata, as well as in the striatum, ventral pallidum, and olfactory tubercle. A small number of lightly labeled neurons were distributed throughout neocortex, primarily in superficial layers. No cellular labeling was detected in hippocampus. In the diencephalon, dense hybridization labeled neurons in the ventral aspect of the medial habenular nucleus whereas cells in the lateral hypothalamic area and supramammillary region were more lightly labeled. Hybridization was most dense in neurons of the motor and autonomic cranial nerve nuclei including the oculomotor, Edinger-Westphal, and trochlear nuclei of the midbrain, the abducens, superior salivatory, trigeminal, facial and accessory facial nuclei of the pons, and the hypoglossal, vagus, and solitary nuclei and nucleus ambiguus of the medulla. In addition, numerous cells in the pedunculopontine and laterodorsal tegmental nuclei, the ventral nucleus of the lateral lemniscus, the medial and lateral divisions of the parabrachial nucleus, and the medial and lateral superior olive were labeled. Occasional labeled neurons were distributed in the giantocellular, intermediate, and parvocellular reticular nuclei, and the raphe magnus nucleus. In the medulla, light to moderately densely labeled cells were scattered in the nucleus of Probst's bundle, the medial vestibular nucleus, the lateral reticular nucleus, and the raphe obscurus nucleus. In spinal cord, the cRNA densely labeled motor neurons of the ventral horn, and cells in the intermediolateral column, surrounding the central canal, and in the spinal accessory nucleus. These results are in good agreement with reports of the immunohistochemical localization of ChAT and provide further evidence that cholinergic neurons are present within neocortex but not hippocampus.     

Calcitonin gene-related peptide in afferents to the cat's cerebellar cortex: distribution and origin.

In the present study, the distribution and origin of calcitonin gene-related peptide (CGRP) were analyzed in the cat's cerebellum. Following incubation in an antibody generated against rat CGRP and processing with the peroxidase anti-peroxidase (PAP) technique, CGRP immunoreactivity (IR) is found in profiles that have morphological characteristics of both simple and complex mossy fibers. However, all mossy fibers are not CGRP-positive. Further, CGRP-IR mossy fibers have a heterogeneous distribution in the cerebellum. In the vermis, the majority of immunoreactive profiles are in lobules VII, VIII, and the dorsal folia of IX. In anterior vermal lobules, only scattered terminals, located primarily at the apex and along the shoulder of the folia, are present. Laterally, CGRP-IR mossy fibers are located in the paramedian lobule, paraflocculus, and crus II. No CGRP fibers or varicosities are observed in any of the cerebellar nuclei. However, CGRP-positive cell bodies are scattered throughout the nuclear neuropil. A double label technique revealed that CGRP-IR mossy fibers arise from neurons located in the lateral reticular nucleus, external cuneate nucleus, inferior vestibular nucleus, and basilar pons. The present findings, taken together with previous data, indicate that cerebellar afferents are chemically heterogeneous. The findings of the present study suggest that precerebellar nuclei that give rise to the mossy fibers that contain CGRP have the potential for playing a complex role in modulating circuitry in the cerebellar cortex of the cat.     

Localization of GABAA-receptor alpha 1 subunit mRNA-containing neurons in the lower brainstem of the rat

The localization of gamma-aminobutyric acid-A (GABAA) receptors (GABAA-R) in the lower brainstem of the rat was examined by means of in situ hybridization histochemistry using an oligonucleotide probe to the sequence of the alpha 1 subunit (GABAA-R alpha 1). Strongly labeled neurons were found in the cranial motor nuclei, the dorsal motor nucleus of the vagus, reticular formation (large neurons), lateral vestibular nucleus, dorsal nucleus of the lateral lemniscus, central nucleus of the inferior colliculus, intermediate and white layers of the superior colliculus, red nucleus and substantia nigra. In addition, moderately labeled cells were abundant in the nucleus of the solitary tract, medial and inferior vestibular nuclei, parabrachial area, dorsal and ventral tegmental nuclei of Gudden, central gray matter, ventral nucleus of the lateral lemniscus, and reticular formation (small neurons). This study has therefore revealed some of the target neurons of GABA-containing fibers in the lower brainstem.     

Rat facial motoneurons express increased levels of calcitonin gene-related peptide mRNA in response to axotomy

The expression and localization of the mRNA encoding the calcitonin gene-related peptide (CGRP) were analyzed in the rat facial nucleus after axotomy by Northern blot analysis and by in situ hybridization histochemistry (ISH) using a synthetic 32P-labeled oligonucleotide probe. Northern blot analysis revealed the presence of the 1.2 kb CGRP mRNA in RNA extracted from the facial nucleus. This mRNA species was strongly increased after axotomy of the facial nerve. By ISH increased levels of CGRP mRNA were observed as soon as 16 hr after axotomy compared with the unoperated nucleus. CGRP mRNA could be localized in more than 50% of the motoneurons. Three populations of motoneurons with no, moderate, or strong labeling for CGRP mRNA could be distinguished. Peak expression of CGRP mRNA during the first 48 hr was followed by a decline to moderate levels at day 4 after lesion, and to almost basal levels at days 7 and 9. These data demonstrate that axotomy of the facial nerve leads to an early and strong induction of CGRP gene expression in motoneurons of the facial nucleus.     

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.     

Cranial motor neurons contain either galanin- or calcitonin gene-related peptidelike immunoreactivity

The demonstration of coexistence of a peptide or peptides in neurons that produce a small molecule neurotransmitter has become increasingly frequent. The calcitonin gene-related peptide (CGRP) is known to be colocalized in the cholinergic neurons of both cranial and spinal motor nuclei. The present study demonstrates that all somatic motor cranial nerve nuclei contain CGRP- and galaninlike immunoreactivity. The perikaryal content of both peptides is increased by colchicine pretreatment and by transecting axons arising from the perikarya, and both peptides are found in nerve fibers innervating striated musculature. CGRP- and galaninlike immunoreactivity appear to be present in different populations of neurons. In contrast to CGRP, galaninlike immunoreactivity was not detected in spinal motor neurons. These observations suggest that galanin and CGRP participate in the process of synaptic transmission at the neuromuscular junction of cranial motor neurons.     

Feeding-related immune responsive brain stem neurons: association with CGRP

Using dual-labeling in situ hybridization histochemistry, the neurotransmitter expression of immune-responsive neurons in the pontine parabrachial nucleus, a major relay for interoceptive information, was investigated. Intravenous injection of bacterial wall lipopolysaccharide resulted in dense c-fos mRNA expression in the external lateral parabrachial nucleus, and a majority of the c-fos expressing cells also expressed calcitonin gene-related peptide (CGRP) mRNA. In contrast CGRP-positive cells in the adjoining external medial subnucleus were c-fos negative. Taken together with previous hodological and behavioral studies, these data suggest that CGRPergic parabrachial neurons may mediate lipopolysaccharide-induced anorexia by means of their projection to central nucleus of the amygdala.     

Calcitonin gene-related peptide in monkey spinal cord and medulla oblongata.

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers and cell bodies was studied in the spinal cord and the medulla oblongata of the grey monkey (Macaca fascicularis) using peroxidase-antiperoxidase (PAP) immunohistochemistry. At all levels of the spinal cord many CGRP-IR motoneurons and fibers were seen in the motor nuclei. In the medulla, CGRP-IR cell bodies were encountered in nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus, nucleus reticularis lateralis as well as in the area dorsal to the inferior olive. Bulbar motoneurons were much more intensely stained than spinal cord motoneurons, indicating higher levels of CGRP-like immunoreactivity (LI) at the medullary level. The concentration of CGRP-LI measured by radioimmunoassay showed higher levels in the dorsal quadrants as compared to the ventral quadrants, but the dorsal/ventral ratio was lower than has previously been reported from the rat. The present results demonstrate that using the PAP technique CGRP-LI can be visualized in a larger number of spinal cord motoneurons of the monkey than earlier revealed by immunofluorescence. Moreover, the finding supports the view that the CGRP-IR nerve endings in the spinal motor nuclei originate from cell bodies in the medullary raphe nuclei.     

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.     

Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization

To determine the localization in rat brain and spinal cord of individual neurons that contain the messenger RNA coding for the opioid peptide precursor preproenkephalin, we performed in situ hybridization with a tritiated cDNA probe complementary to a protion of preproenkephalin mRNA. We observed autoradiographic signal over the cytoplasm of neurons of many regions of the central nervous system. Several types of controls indicated specificity of the labeling. Neurons containing preproenkephalin mRNA were found in the piriform cortex, ventral tenia tecta, several regions of the neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (especially central nucleus, with fewer labeled neurons in all other nuclei), hippocampal formation, anterior hypothalamic nucleus, perifornical region, lateral hypothalamus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, dorsal and ventral premamillary nuclei, medial mamillary nucleus, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral parabrachial nuclei, pontine and medullary reticular formation, several portions of the raphe nuclei, nucleus of the solitary tract, nucleus of the spinal trigeminal tract (especially substantia gelatinosa), ventral and dorsal cochlear nuclei, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, gracile nucleus, superior olive, nucleus of the trapezoid body, some deep cerebellar nuclei, Golgi neurons in the cerebellum, and most laminae of the spinal cord. In most of these brain regions, the present results indicate that many more neurons contain preproenkephalin mRNA than have been appreciated previously on the basis of immunocytochemistry.     

Light microscopic localization of calcitonin gene-related peptide in the normal feline trigeminal system and following retrogasserian rhizotomy

Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been implicated in the transmission and modulation of primary afferent nociceptive stimuli. In this study, we describe the light microscopic distribution of CGRP immunoreactivity (IR) within the feline trigeminal ganglion and trigeminal nucleus of normal adult subjects and in subjects 10 and 30 days following complete retrogasserian rhizotomy. Within the trigeminal ganglion of normal subjects, cell bodies and fibers showed CGRP-IR, whereas immunoreactive fibers were rare in the central root region. Within the normal spinal trigeminal and main sensory nuclei, CGRP-IR was seen to form a reproducible pattern that varied between the different nuclei. Following rhizotomy, most, but not all, of the CGRP-IR was lost from the spinal trigeminal and main sensory nuclei, except in regions where the upper cervical roots and cranial nerves VII, IX, and X project into the trigeminal nucleus. The pattern seen at 10 days contained more CGRP-IR than that seen at 30 days and suggests that degenerating fibers still show CGRP-IR. In contrast to the decrease seen in the nuclei after rhizotomy, examination of the central root that was still attached to the trigeminal ganglion showed an increase in CGRP-IR within fibers, some of which ended in growth conelike enlargements. Rhizotomy induced a dramatic increase in CGRP-IR within trigeminal motoneurons and their fibers, which was strongest 10 days after rhizotomy and weaker at 30 days, which was still stronger than normal. These results indicate that the majority of CGRP-IR found in the trigeminal nucleus originates from trigeminal primary afferents and that an upregulation of CGRP-IR occurs in trigeminal motoneurons and in regenerating fibers in the part of the central root that was still attached to the ganglion. In addition, the persistence of CGRP-IR fibers in the trigeminal nucleus provides one possible explanation for the preservation of pain in humans following trigeminal rhizotomy. © 1996 Wiley-Liss, Inc.     

Axonal projections from the parasubthalamic nucleus

The parasubthalamic nucleus (PSTN) is a differentiation of the lateral hypothalamic area, characterized by a distinct population of neurons expressing beta-preprotachykinin (beta-PPT) mRNA. The axonal projections from the PSTN have been analyzed with the PHAL anterograde tract tracing method in rats. The results indicate that the cell group is distinguished by massive projections to parasympathetic preganglionic neurons in the brainstem (especially in the salivatory nuclei and dorsal motor nucleus of the vagus nerve) and to parts of the parabrachial nucleus and nucleus of the solitary tract that relay viscerosensory and gustatory information. In addition, the PSTN projects to cortical parts of the cerebral hemisphere (infralimbic, agranular insular, postpiriform transition and lateral entorhinal areas, and posterior basolateral amygdalar nucleus)-directly and also indirectly via thalamic feedback loops involving the paraventricular and mediodorsal nuclei-and to nuclear parts of the cerebral hemisphere (central amygdalar nucleus, striatal fundus, rhomboid nucleus of the bed nuclei of the stria terminalis, and substantia innominata). The PSTN is thus positioned to influence directly many cerebral hemisphere and hindbrain components of the central parasympathetic control network that is active, for example, during feeding behavior and cardiovascular regulation.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish,scyliorhinus canicula L

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat α-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution. © 1995 Wiley-Liss, Inc.     

Localization, origin and fine structure of calcitonin gene-related peptide-containing fibers in the vestibular end-organs of the rat

The localization, origin and fine structure of nerve terminals immunoreactive for calcitonin gene-related peptide (CGRP) were examined in the vestibular end-organs of rats using immunocytochemistry. Many CGRP-like immunoreactive (CGRP-IR) fibers were observed in the vestibular sensory epithelial layer. By electronmicroscopy, CGRP-IR terminals were found to make synaptic contacts with the chalyces of the vestibular nerves terminating on type I cells. The origin of these vestibular CGRP-IR fibers was examined by a combination of a retrograde fiber tracing technique (using Fast blue) and immunocytochemistry. Injections of the tracer into the vestibular cistern, resulted in fast blue-labeled neurons bilaterally in the area dorsolateral to the genu of the facial nerve; these labeled neurons also contained CGRP. These findings indicate that CGRP-IR fibers originate bilaterally from the area dorsolateral to the genu of the facial nerve and that CGRP plays a modulatory role in the transmission of vestibular information from type I cells.