Mating-activated nitric oxide-producing neurons in specific brain regions in the female rat

Nitric oxide (NO)-containing neurons have been localized in various parts of the central nervous system including the hypothalamus. NO plays an important role in the regulation of reproductive activities including sexual behavior and pituitary hormone secretion. To test the hypothesis that NO-containing neurons in specific brain areas may respond to the stimulus of mating and participate in integrating the tactile information in the hypothalamus, this study used Fos as a marker of neuronal activity. Proestrous rats receiving intromissions (mated group) from males or mounts-without-intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 90 min after the beginning of mating or mounting. NOergic neurons were labeled by histochemical reaction for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). The presence of activated NO-producing (double-stained NADPH-d/Fos) neurons was quantitatively assessed in several brain areas before and after mating. The results showed that mating-with-intromissions induced a significant increase in the percentage of NADPH-d/Fos colabeled neurons in the medial preoptic area (mPOA) and the magnocellular component of the paraventricular nucleus (PVNm) compared to mounts-without-intromission or control treatment. Both mating and mounting induced Fos expression in NADPH-d-positive cells in the ventromedial nucleus of hypothalamus (VMN). In contrast, the expression of Fos in the NADPH-d-positive neurons in the supraoptic nucleus (SON) and the parvocellular portion of the paraventricular nucleus (PVNp) was not influenced by either mating or mounting although abundant NO-containing neurons were found in the two brain areas. The second experiment of the study examined whether NOergic neurons in these brain areas are influenced directly by estrogen by determining the number of NADPH-d-positive neurons that contained the estrogen receptor (ER), the classical ER. Double labeled NADPH-d/ER neurons were observed in several brain areas including the mPOA and VMN while few, if any, NADPH-d-positive neurons in the SON, PVNm or PVNp contained ER. The results suggest that the activated NOergic neurons in these brain areas may be involved in processing and integrating the mating stimulus. Further investigation is required to determine the physiological role of the mating-activated NOergic activity in specific mating-induced changes in reproductive neuroendocrinology.     

Changing patterns of Fos expression in brainstem catecholaminergic neurons during the rat oestrous cycle

Brainstem catecholaminergic neurons are believed to play an important role in the activation of luteinising hormone-releasing hormone (LHRH) neurons on the afternoon of proestrus which results in the luteinising hormone (LH) surge. To examine the respective roles of brainstem A1 and A2 neurons and the adjoining C1 and C2 adrenergic cells at this time, we have examined the patterns of Fos-immunoreactivity within tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT) neurons during diestrus and proestrus. Initial studies demonstrated that the LH surge commenced at approximately 15:00 h in proestrous animals and that peak plasma levels of LH were observed between 16:00 and 17:00 h. Groups of cycling female rats (n = 6) were then perfused between 09:00 and 11:00 (diestrus early) and 18:00 to 19:30 h (diestrus late) on diestrus and at the same times on proestrus (proestrus early and proestrus late). Double-labelling immunocytochemistry revealed little Fos expressions by adrenergic neurons of the C1 or C2 cell groups and this did not change significantly between any of the experimental groups. Analysis of the A2 region was divided into rostral, middle and caudal divisions and all regions showed a significant (P < 0.01) increase in the number of Fos-expressing TH neurons (up to 35% of TH cells) in proestrus early animals compared with diestrus and proestrus late rats. In the A1 region, a significant increase in the number of TH neurons expressing Fos ( 33%) was detected in both proestrus early (P < 0.05) and diestrus early (P < 0.01) rats compared with animals perfused in the late afternoon ( 12%). These results indicate that TH-immunoreactive neurons in both A1 and A2 cell groups are activated on the morning of proestrus prior to the LH surge whilst the C1 and C2 adrenergic neurons express little Fos throughout. The morning increase in Fos-expression by TH neurons within the A1 region on both diestrus and proestrus indicates a circadian pattern of activation for A1 noradrenergic cells and suggests different roles for the A1 and A2 cell groups in regulating the activity of LHRH neurons on proestrus.     

Flurothyl-induced seizures in rats activate Fos in brainstem catecholaminergic neurons

Autonomic changes accompany seizures in both animals and humans. While ictal autonomic dysfunction can be life-threatening, the participating neural networks involved are poorly understood. In this study we examined the activation of Fos following generalized seizures in brainstem structures known to mediate autonomic function. Adult female rats were sacrificed 2 h after flurothyl-induced seizures. Double-immunostaining for c-Fos and dopamine-beta-hydroxylase (DBH), and c-Fos and phenylethanol-N-methyl-transferase (PNMT) were performed in brainstem slices. Numbers of DBH-labeled neurons expressing Fos-like immunoreactivity (FLI) (DBH/Fos) and PNMT labeled neurons expressing FLI (PNMT/Fos) were counted in the noradrenergic (A1, A2, A5, A7) and adrenergic (C1, C2) cell groups localized in pons and medulla oblongata. Among the experimental animals, the highest degree of co-localization of DBH/Fos neurons was observed in the locus coeruleus (A6; 87.7%), and in the A1(72.8%) cell group located in the caudal ventrolateral medulla (VLM). No co-localization of DBH/Fos neurons was observed in control animals. The highest degree of co-localization of PNMT/Fos neurons was observed in the C1 adrenergic cell group (84.2%) located in the rostral VLM. Control animals showed very few (5.5%) PNMT/Fos co-localized neurons in the C1 adrenergic cell group. Our results indicate that flurothyl-induced generalized seizures in rats activate catecholaminergic neurons in the pons and medulla oblongata. Further studies are necessary to determine whether activation of brainstem catecholaminergic neurons contribute to the autonomic manifestations that frequently accompany epileptic seizures.     

Activation of brainstem catecholaminergic neurons during voluntary diving in rats

Underwater submergence produces a complex autonomic response that includes apnea, a parasympathetically-mediated bradycardia, and a sympathetically-mediated increase in total peripheral resistance (TPR). The present study was designed to identify brainstem catecholaminergic neurons that may be involved in producing the increased TPR during underwater submergence. Twelve male Sprague-Dawley rats were trained to voluntarily dive 5 m through an underwater maze. On the day of the experiment the rats were randomly separated into a Diving group that repetitively dived underwater, a Swimming group that repetitively swam on the surface of the water, and a Control group that remained in their cages. After the experiment the brainstems of the rats were immunohistologically processed for Fos as an indicator of neuronal activation, and for tyrosine hydroxylase (TH) as an indentifier of catecholaminergic neurons. Neurons labeled with both Fos and TH identified activated catecholaminergic neurons. In Diving rats there was increased Fos+TH labeling in A1, C1, A2, A5, and sub-coeruleus, as well as globosa neurons in the lateral A7 region compared with Control rats, and in A1, C1 and A5 compared with Swimming rats. In Swimming rats Fos+TH labeling was significantly increased in caudal A1, A5, sub-coeruleus and globosa neurons compared with Control rats. These data suggest that selective groups of catecholaminergic neurons within the brainstem are activated by voluntary underwater submergence, and some probably contribute to the sympathetically-mediated increase in vascular tone during diving.     

Neurochemical development of the brainstem catecholaminergic cell groups in rat

The postnatal development of tyrosine hydroxylase activity has been studied in the brainstem catecholaminergic cell groups (A1C1, A2C2, A5, A6, A7), involved in cardiorespiratory control. In rat, at birth and at postnatal days P3, P7, P14, P21 ant P68, we used a microdissection technique followed by in vivo measurement of the tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in catecholamine synthesis. There is two successive marked increases in TH activity: at P3 in every catecholaminergic cell groups (A1C1, +225%; A2C2, +300%; A5, +190%; A6, +205% compared to birth) and during the third postnatal week with a peak of TH activity at P14 (A6, +90% above the P7 level) or at P21 (A1C1, +715%; caudal A2C2, +585%; rostral A2C2, +15%; A5, +445%; A7, +180% compared to P7). The data suggest the existence of two temporal windows during the neurochemical development of the catecholaminergic cell groups, which correspond to two metabolic transitions. The first one could be related to the intra-, extrauterine transition and the second one, to a deep energetic phase of maturation in the rat brain, closely related to the maturation of cardiorespiratory processes.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Nicotine activates NPY and catecholaminergic neurons in brainstem regions involved in ACTH secretion

Nicotine rapidly and potently stimulates ACTH secretion via a centrally mediated mechanism. The purpose of the current study was to identify the phenotype of nicotine-sensitive neurons in brainstem catecholaminergic regions previously shown to be responsive to nicotine. Immunocytochemical double-labeling was used to detect c-Fos expression in neurons positive for activin, galanin, or neuropeptide Y (NPY), in comparison to those containing tyrosine hydroxylase (TH, catecholaminergic biosynthetic enzyme). These neuropeptides were chosen because (1) each is located in nicotine-sensitive brainstem regions, (2) neurons containing each of these peptides project to the hypothalamic paraventricular nucleus, and (3) each has been shown to affect ACTH secretion. Freely moving, adult, male rats received an intravenous (i.v.) infusion of saline or nicotine (0.045 mg/kg over 30 s or 0.135 mg/kg over 90 s) and were cardiac perfused 60 min thereafter. Nicotine significantly increased c-Fos expression in a dose-dependent manner in the brainstem regions examined. In nucleus tractus solitarius (NTS)-A2 and NTS-C2, both NPY⁺ and TH⁺ neurons responded to the lower dose of nicotine, whereas the activin and galanin neurons in these regions were unresponsive to either dose of nicotine. In contrast, the higher dose of nicotine was required to activate NPY⁺ neurons in the A1 region and both NPY⁺ and galanin⁺ neurons in the locus coeruleus; the C1 region was unresponsive to nicotine. Since plasma ACTH is elevated by the low dose of nicotine and only NTS neurons are activated by this dose, NPY projections from the NTS are likely to contribute to nicotine-stimulated ACTH secretion, in addition to the previously described catecholaminergic neurons.     

Gastric electrical stimulation increases ghrelin production and inhibits catecholaminergic brainstem neurons in rats

Gastric electrical stimulation (GES) is a new therapeutic option for functional dyspepsia and gastroparesis. In addition to ameliorating nausea and vomiting, GES results in improved appetite which is not always associated with accelerated gastric emptying. To explore the central and peripheral factors underlying GES‐associated improvement of appetite we developed a GES model in anaesthetized Wistar rats. During laparotomy, two electrodes were implanted into the stomach and high‐frequency low‐energy GES (14 Hz, 5 mA) was applied. The effects of 1 h GES were compared with sham stimulation. After GES, c‐Fos expression was increased in the mucosal and submucosal layers of the stimulated area (174%). In the stomach, GES increased ghrelin mRNA (178%) and doubled the number of ghrelin‐positive cells, resulting in elevated plasma levels of ghrelin (2.3 ± 0.2 vs. 1.6 ± 0.2 ng/mL). In the arcuate nucleus of the hypothalamus, GES increased c‐Fos (277%) and agouti‐related protein (AgRP) mRNA expression (135%). GES reduced the number of c‐Fos‐positive cells throughout the nucleus of the solitary tract (between 93 and 75% from rostral to caudal levels) including catecholaminergic neurons (81% at caudal level). Gastric emptying, plasma glucose and heart rate variability were not affected by GES. This study shows that GES may improve appetite via stimulation of main orexigenic pathways, including ghrelin production in the stomach and AgRP in the hypothalamus, as well as by reducing the activity of catecholaminergic brainstem neurons.     

Overlap in the distribution of cholinergic and catecholaminergic neurons in the upper brainstem of the ferret

The distribution of catecholaminergic and cholinergic neurons in the upper brainstem of the ferret were mapped by staining immunohistochemically two adjacent series of sections of brainstem for tyrosine hydroxylase and choline acetyltransferase, respectively. As in other species, large numbers of tyrosine-hydroxylase-positive neurons are localized in the ventral tegmental area (A10), the substantia nigra (A9), and in A8. Tyrosine-hydroxylase-positive neurons in the dorsolateral pontine tegmentum (A4, A6, and A7--the locus coeruleus complex) of the ferret are rather diffusely distributed, as has been observed in other carnivore species such as the cat and the dog, but unlike the cat, these cells in the ferret display a relative uniformity in size and morphology. Choline-acetyltransferase-positive neurons which extend in the ferret's pedunculopontine tegmental nucleus and ventral parabrachial area (Ch5) are relatively large cells that stain intensely for choline acetyltransferase, and their dendrites form prominent bundles in regions where unstained fibre tracts are prevalent. Choline-acetyltransferase-positive neurons distributed in the laterodorsal tegmental nucleus (Ch6) are smaller than the cholinergic cells of Ch5, and they stain less intensely for choline acetyltransferase. Rostrally, there is little overlap between the catecholaminergic cell groups A8, A9, and A10 and the cholinergic cell groups of Ch5 and Ch6. Caudally, the Ch5 neurons extend some considerable extent into the locus coeruleus complex. In the region of overlap, no cells with staining for both tyrosine hydroxylase and choline acetyltransferase were observed, as was ascertained with a double staining method employing a combination of tyrosine hydroxylase immunofluorescence and choline acetyltransferase peroxidase-antiperoxidase immunohistochemistry. In conclusion, the ferret has a typically carnivore pattern for the distribution of catecholaminergic cells in the upper brainstem, and there is a significant overlap between the catecholaminergic and cholinergic cell groups in the dorsolateral pontine tegmentum.     

Expression of Fos immunoreactivity in some catecholaminergic brainstem neurons in rats following high-altitude exposure

This study examined the response of neurons of the cardiorespiratory centers, i.e., the nucleus tractus solitarius and the ventrolateral medulla as well as the area postrema in adult and postnatal rats subjected to high-altitude exposure at 4,000 m and 8,000 m. In adult control rats, sporadic Fos-positive neurons were detected in the above-mentioned areas. On exposure to 4,000 m altitude, the number of Fos-positive neurons was noticeably increased. At 8,000 m, the incidence of labeled cells was markedly increased, with many of them doubly labeled for tyrosine hydroxylase. In postnatal rats, Fos expression was not detected in these areas in either control rats or rats exposed to 4,000 m altitude. Fos-positive cells, however, were observed in the these areas in postnatal rats exposed to 8,000 m. In the latter, tyrosine hydroxylase labeling was observed in some Fos-positive cells in the nucleus tractus solitarius and ventrolateral medulla. In rats killed at 24 hr after exposure to high altitude, Fos expression in both the adult and the postnatal rats was comparable to that in their corresponding control rats. Present results suggest that Fos expression in various brainstem areas was induced by reduced oxygen tension in the ambient air at high altitude. Double labeling of some Fos-positive neurons with tyrosine hydroxylase indicates an increased sympathetic activation, which may be involved in the mediation of cardiorespiratory responses to hypoxia. This, however, was less evident in the postnatal animals. It is possible that the peripheral chemoreceptors or the regulation of autonomic functions is not fully developed in this age group.     

A brainstem atlas of catecholaminergic neurons in man,using melanin as a natural marker

The present paper provides an atlas showing the distribution of melanin-containing nerve cells in the human brainstem. It was found that neuromelanin, which can be viewed as a waste product of catecholamine metabolism, is suitable as a natural marker for catecholaminergic neurons in the medulla oblongata, pons, and the mesencephalon of the adult human brain. Within these areas of the brain, there is a striking similarity between the location of melanin and the catecholamine cell bodies described in various animals and in human fetuses, whereas no melanin was found in the diencephalic dopaminergic cell groups. Cell counts from the center of each area showed that the mean density of melanin-containing perikarya varied considerably between the different areas.     

Immunocytochemical characterization of catecholaminergic neurons in the rat striatum following dopamine-depleting lesions

It is possible either permanently or transiently to deplete the rat striatum of dopamine. Following such depletions, striatal neurons immunoreactive for tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) or dopamine appear. The presence of dopamine-producing neurons in the striatum has relevance for the treatment of Parkinson's disease, but whether these catecholaminergic phenotypes all produce dopamine is unclear. In the present study we establish that after unilateral 6-hydroxydopamine lesions or methamphetamine administration, striatal TH-immunoreactive neurons differ in size, morphology and location from those that are immunopositive for AADC or dopamine. The TH-positive cells which were localized either to ventral parts of the striatum or to the central and dorsal areas of the caudate-putamen generally have the morphological features of projection neurons, whereas those containing AADC or dopamine were confined to subcallosal positions in the dorsal medial quadrant of the caudate-putamen and resemble small, local-circuit neurons. The fact that AADC-immunoreactive neurons overlap in size, morphology and location with the cells that produce dopamine suggests strongly that this population is dopaminergic. However, the simultaneous appearance of neurons that contain the TH enzyme but clearly do not make dopamine raises questions about the functional role of these cells and the cellular mechanisms responsible for their induction following striatal dopamine loss.     

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry.

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus

Immunohistochemical methods were used in the rat to plot the distribution of neuropeptide Y (NPY) immunoreactivity in the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus, and a combined retrograde transport-double immunohistochemical labeling technique was used to determine the extent to which NPY immunoreactivity is coexpressed in brainstem cell groups that stain with antisera to phenylethanolamine-N-methyltransferase (PNMT; a marker for adrenergic neurons) or dopamine-beta-hydroxylase (DBH; a marker for adrenergic and noradrenergic neurons) and also project to the PVH. The results confirm the existence of a major NPY-immunoreactive pathway that is in a position to influence each major class of output neurons in the PVH. Thus, most parts of the parvicellular division receive a dense input that is similar to, though somewhat more extensive than, the one stained by DBH antisera. However, in the magnocellular division catecholaminergic inputs are preferentially associated with vasopressinergic neurons, while NPY-stained fibers tend to be more evenly distributed in regions containing both oxytocinergic and vasopressingergic neurons, and their density appear to be lower than that of DBH-stained fibers. In the SO, only a moderate NPY-stained input was apparent, while, as described previously, DBH-immunoreactive fibers are rather dense and are preferentially distributed in vasopressinergic regions of the nucleus. The results of combined retrograde transport-double immunohistochemical labeling experiments may be summarized as follows: the vast majority of cells in the medulla that were retrogradely labeled after discrete implants of the fluorescent tracer true blue into the PVH, and were PNMT-immunoreactive, also stained for NPY. However, less extensive co-localization was detected in noradrenergic cell groups of the caudal medulla. About 60% of the retrogradely labeled-DBH positive cells in the A1 cell group were also NPY-positive, while those in the caudal part of the nucleus of the solitary tract (the A2 cell group) usually failed to stain with anti-NPY. Similarly, in the locus coeruleus (the A6 cell group) where virtually all retrogradely labeled neurons were DBH-positive, only rarely were triply labeled cells detected. These results suggest that NPY immunoreactivity is extensively co-contained within adrenergic neurons of the C1, C2, and C3 groups that project to the PVH, while the correspondence in noradrenergic cell groups is less complete, and generally limited to a subset of neurons in the A1 cell group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transmitter sensitivities in oval neurons from rat brainstem cultures

Using cultures of dissociated neurons from the lower brainstem of 14-15-day-old rat embryos, we studied a specific relation between neuronal activities of small-sized oval neurons and their sensitivities to various transmitter substances. Oval neurons were characterized by non-effective responses to acetylcholine and a facilitatory response to both leucine- and methionine-enkephalin.     

颈动脉注射辣椒素激活脑干心血管相关核团的儿茶酚胺神经元

在外周压力感受器去神经支配的大鼠上,用Ｆos蛋白和酪氨酸羟化酶（ＴＨ）的双重免疫组化方法,研究辣椒素的效应是否通过激活脑干核团内儿茶酚胺能神经元而诱发。结果显示,颈动脉注射辣椒素诱发脑干中最后区（ＡＰ）、孤束核（ＮＴＳ）、巨细胞旁外侧核（ＰＧＬ）和蓝斑（ＬＣ）等多个部位出现大量ＦOS样免疫反应（ＦＬＩ）神经元和双标神经元,辣椒素受体阻断剂钌红（ＲＲ）或ＮＭＤＡ受体阻断剂ＭＫ－８０１可明显减弱此效应。以上结果表明,辣椒素的兴奋效应通过激活儿茶酚胺能神经元而诱发,辣椒素受体和／（或）谷氨酸介导这一效应。     

A brainstem atlas of catecholaminergic neurons and serotonergic perikarya in a pygmy primate (Cebuella pygmaea).

The present paper provides a brainstem atlas showing the distribution of catecholaminergic cells and processes, as well as serotonergic perikarya, in the pygmy marmoset. The findings revealed by the Falck and Hillarp histofluorescence method conform in essential details to what has been described in rodents. These and other comparative data indicate that a prototypical pattern of organization of aminergic systems has been retained in the evolution of primates.     

Generation patterns of immunocytochemically identified cholinergic neurons in rat brainstem

Combined [3H]thymidine autoradiographic and choline acetyltransferase (ChAT)-immunocytochemical techniques were used to answer questions concerning the generation of specific classes and subclasses of cholinergic neurons in rat brainstem. First, the generation of rostrally and caudally located neurons of the same class (i.e. somatic efferent oculomotor and hypoglossal nuclei, respectively) were compared. Results indicated that, although embryonic day 11 (E11) was the peak birthday for both nuclei, hypoglossal neurons were generated significantly earlier than oculomotor neurons, indicating a caudorostral generation gradient for brainstem somatic motor nuclei. Second, the generation patterns of 3 different subclasses of motor neurons at the same brainstem level were compared; namely those of the somatic efferent hypoglossal nucleus (XII), the general visceral efferent dorsal nucleus of the vagus (X), and the predominantly special visceral efferent nucleus ambiguus. All 3 subclasses of cholinergic cells had the same peak day (E11) and overall period of generation (E11-12). However, statistical analyses indicated a precocious generation of nucleus ambiguus, but no developmental differences between N, XII and N. X. It is suggested that nucleus ambiguus is formed earlier than N. XII and N. X, due to its more ventral location within a ventrodorsal neurogenetic gradient. Third, the generation patterns of different classes of large cholinergic neurons were examined. Specifically, the birthdays of cholinergic non-motor projection neurons of the pedunculopontine-laterodorsal tegmental nuclei (PPT-LDT) were contrasted to those of the cholinergic brainstem motor neurons. The peak birthdays of both rostrally and caudally located motor neurons were two days earlier than those of the PPT-LDT neurons. Thus, large cholinergic cells projecting to peripheral targets are born significantly earlier than those projecting within the CNS, even though the former are located more rostrally on the caudorostral neurogenetic gradient. This represents an apparent exception to the emerging rule that cholinergic neurons obey the general gradients of neurogenesis manifest in the regions of the central nervous system where they reside.     

The central distribution of vagal catecholaminergic neurons which project into the abdomen in the rat

A double labeling technique employing retrograde labeling of vagal neurons with horseradish peroxidase from injections into the stomach wall and immunocytochemistry for dopamine-beta-hydroxylase revealed catecholaminergic neurons in the medulla oblongata which project into the abdomen. The great majority of such neurons were located in the dorsal motor nucleus of the vagus, particularly in its rostral third.