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Within the central nervous system there are three groups of neurons that express all of the enzymes required for generation of the catecholamine, adrenaline. The cell groups, called the C1, C2, and C3 cell groups, are located in the medulla oblongata. Their axons project widely throughout the CNS, from the sacral spinal cord to the olfactory nucleus. While they are predominantly studied within the context of a spinal projection to sympathetic preganglionic neurons, and a role in cardiovascular regulation, this review outlines data showing a wider range of projections and potential functions that suggest a much broader role for these neurons.

Brainstem catecholaminergic neurons play key roles in the autonomic, neuroendocrine, and behavioral responses to glucoprivation, yet the functions of the individual groups are not fully understood. Adrenergic C3 neurons project widely throughout the brain, including densely to sympathetic preganglionic neurons in the spinal cord, yet their function is completely unknown. Here we demonstrate in rats that optogenetic stimulation of C3 neurons induces sympathoexcitatory, cardiovasomotor functions. These neurons are activated by glucoprivation, but unlike the C1 cell group, not by hypotension. The cardiovascular activation induced by C3 neurons is less than that induced by optogenetic stimulation of C1 neurons; however, combined stimulation produces additive sympathoexcitatory and cardiovascular effects. The varicose axons of C3 neurons largely overlap with those of C1 neurons in the region of sympathetic preganglionic neurons in the spinal cord; however, regional differences point to effects on different sympathetic outflows. These studies definitively demonstrate the first known function of C3 neurons as unique cardiovasomotor stimulatory cells, embedded in the brainstem networks regulating cardiorespiratory activity and the response to glucoprivation.

Initially recognized for their importance in control of appetite, orexins (also called hypocretins) are neuropeptides that are also involved in regulating sleep, arousal, and cardiovascular function. Loss of orexin appears to be the primary cause of narcolepsy. Cells expressing the orexins are restricted to a discrete region of the hypothalamus, but their terminal projections are widely distributed throughout the brain. With the diversity of function and broad distribution of orexin terminals, it is not known whether the orexin cells constitute a homogeneous population. Because orexins produce neuroexcitatory effects, we hypothesized that orexin-containing neurons are glutamatergic. In the present study we used digoxigenin-labeled cRNA probes for the vesicular glutamate transporters, VGLUT1 and VGLUT2, for in situ hybridization studies in combination with immunohistochemical detection of orexin cell bodies in the hypothalamus. In general, cells in the hypothalamus expressed low levels of the vesicular glutamate transporters relative to other areas of the forebrain, such as the cortex and thalamus. Light labeling for VGLUT2 mRNA was detected in about 50% of the orexin-immunoreactive neurons, and a much smaller percentage (approximately 13%) of orexin-immunoreactive cells was found to express VGLUT1. Despite the fact that intense labeling for GAD67 mRNA was found in a large number of cells throughout the hypothalamus, none of the orexin-immunoreactive cells was found to be GABAergic. These findings, showing that many of the orexin neurons are glutamatergic, are consistent with the neuroexcitatory effects of orexin but suggest that another neurochemical phenotype may define the remaining subset of orexin neurons.

The baroreflex pathway might include a glutamatergic connection between the nucleus of the solitary tract (NTS) and a segment of the ventrolateral medulla (VLM) called the caudal ventrolateral medulla. The main goal of this study was to seek direct evidence for such a connection. Awake rats were subjected to phenylephrine- (PE-) induced hypertension (N=5) or received saline (N=5). Neuronal activation was gauged by the presence of Fos-immunoreactive (Fos-ir) nuclei. Fos-ir neurons that contained vesicular glutamate transporter 2 mRNA (glutamatergic neurons) or glutamic acid decarboxylase mRNA (GABAergic neurons) were mapped throughout the medulla oblongata. Saline-treated rats had very few Fos-ir neurons. In PE-treated rats, Fos-ir neurons were detected in both NTS and VLM. In NTS, 72% of Fos-ir neurons were glutamatergic and 26% were GABAergic. In the VLM, 41% of Fos-ir neurons were glutamatergic and 56% were GABAergic. In VLM, Fos-ir glutamatergic neurons were evenly distributed and were often catecholaminergic, whereas Fos-ir GABAergic cells were clustered around Bregma -13.0 mm. This region of the VLM was injected with Fluoro-Gold (FG) in eight rats, four of which received PE and the rest saline. Fos-ir NTS neurons retrogradely labeled with FG were detected only in PE-treated rats. These cells were exclusively glutamatergic and were concentrated within the NTS subnuclei that receive the densest inputs from arterial baroreceptors. In conclusion, PE, presumably via baroreceptor stimulation, induces Fos in glutamatergic and GABAergic neurons in both NTS and VLM. At least 29% of the Fos-ir glutamatergic neurons of NTS project to the vicinity of the VLM GABAergic interneurons that are presumed to mediate the sympathetic baroreflex.

The pre-Botzinger complex (pre-BotC) is a physiologically defined group of ventrolateral medullary neurons that plays a central role in respiratory rhythm generation. These cells are located in a portion of the rostral ventrolateral medulla (RVLM) that is difficult to identify precisely for lack of a specific marker. We sought to determine whether somatostatin (SST) might be a marker for this region. The rat pre-BotC area was defined as a 500-μm-long segment of ventrolateral medulla coextensive with the ventral respiratory group. This region was identified by juxtacellular labeling of neurons with respiratory-related activity and by its location rostral to the phrenic premotor neurons. It contained most of the SST-ir neuronal somata of the RVLM. These cells were small (107 μm2) and expressed high levels of preprosomatostatin mRNA. They were strongly neurokinin 1 receptor (NK1R)-ir and were selectively destroyed by saporin conjugated with an NK1R agonist (SSP-SAP). Most SST-ir neurons (>90%) contained vesicular glutamate transporter 2 (VGLUT2) mRNA, and terminals immunoreactive for SST and VGLUT2 protein were found in their midst. Few SST-ir neurons contained GAD-67 mRNA (

Many of the inspiratory augmenting (I-AUG) neurons of the rostral ventral respiratory group (rVRG) are premotor neurons that excite phrenic motor neurons during inspiration, probably by releasing glutamate. In the present study, we demonstrate that these neurons are indeed glutamatergic, in that their cell bodies contain vesicular glutamate transporter-2 (VGLUT2) mRNA and spinal terminals from neurons in the region of the rVRG contain VGLUT2 protein. We also demonstrate by using parallel in situ hybridization and immunocytochemical evidence that most rVRG inspiratory premotor neurons are enkephalinergic. After iontophoretic deposits of biotinylated dextran amine (BDA) in the area of the rVRG, many BDA-labeled terminals in the ventral horn of cervical spinal cord (C4-C5) were immunoreactive for enkephalin and VGLUT2. Injections of Fluoro-Gold amidst phrenic motor neurons in C4-C5 labeled neurons in the area of the rVRG that contained both VGLUT2 mRNA and preproenkephalin (PPE) mRNA as revealed by double in situ hybridization. Thirty-eight bulbospinal I-AUG neurons were recorded in the rVRG and filled with biotinamide by using the juxtacellular labeling technique. Every biotinamide-filled cell tested was positively labeled for VGLUT2 mRNA (n = 14), and most of the cells tested in a separate population exhibited PPE mRNA (16/18). We conclude that most of the phrenic inspiratory premotor neurons of the rVRG are glutamatergic neurons that may also release enkephalins.

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro-Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE(+) neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase-immunoreactive (TH-ir) and the latter constituted 19.4% of the bulbospinal TH-ir cells. We determined whether the bulbospinal PPE(+) RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG-labeled RVLM neurons after 2 hours of hydralazine-induced hypotension (to 73 +/- 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos-ir neurons by two- to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE(+) neurons were Fos-ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide-filled neurons were PPE(+) (10 of 17), and the PPE(+) BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE(+) BSBS RVLM neurons were TH-ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non-C1 BSBS RVLM neurons.

C3 neurons constitute one of three known adrenergic nuclei in the rat central nervous system (CNS). While the adrenergic C1 cell group has been extensively characterized both physiologically and anatomically, the C3 nucleus has remained relatively obscure. This study employed a lentiviral tracing technique that expresses green fluorescent protein behind a promoter selective to noradrenergic and adrenergic neurons. Microinjection of this virus into the C3 nucleus enabled the selective tracing of C3 efferents throughout the rat CNS, thus revealing the anatomical framework of C3 projections. C3 terminal fields were observed in over 40 different CNS nuclei, spanning all levels of the spinal cord, as well as various medullary, mesencephalic, hypothalamic, thalamic, and telencephalic nuclei. The highest densities of C3 axon varicosities were observed in Lamina X and the intermediolateral cell column of the thoracic spinal cord, as well as the dorsomedial medulla (both commissural and medial nuclei of the solitary tract, area postrema, and the dorsal motor nucleus of the vagus), ventrolateral periaqueductal gray, dorsal parabrachial nucleus, periventricular and rhomboid nuclei of the thalamus, and paraventricular and periventricular nuclei of the hypothalamus. In addition, moderate and sparse projections were observed in many catecholaminergic and serotonergic nuclei, as well as the area anterior and ventral to the third ventricle, Lamina X of the cervical, lumbar, and sacral spinal cord, and various hypothalamic and telencephalic nuclei. The anatomical map of C3 projections detailed in this survey hopes to lay the first steps toward developing a functional framework for this nucleus.

Vesicular monoamine transporter 2 (VMAT2) packages biogenic amines into large dense core and synaptic vesicles for either somatodendritic or synaptic release from neurons of the CNS. Whilst the distribution of VMAT2 has been well characterized in many catecholaminergic cell groups, its localization amongst C1 adrenergic neurons in the medulla has not been examined in detail. Within the rostral ventrolateral medulla (RVLM), C1 neurons are a group of barosensitive, adrenergic neurons. Rostral C1 cells project to the thoracic spinal cord and are considered sympathetic premotor neurons. The majority of caudal C1 cells project rostrally to regions such as the hypothalamus. The present study sought to quantitate the somatodendritic expression of VMAT2 in C1 neurons, and to assess the subcellular distribution of the transporter. Immunoreactivity for VMAT2 occurred in 31% of C1 soma, with a high proportion of these in the caudal part of the RVLM. Retrograde tracing studies revealed that only two of 43 bulbospinal C1 neurons contained faint VMAT2-immunoreactivity, whilst 88 +/- 5% of rostrally projecting neurons were VMAT2-positive. A lentivirus, designed to express green fluorescent protein exclusively in noradrenergic and adrenergic neurons, was injected into the RVLM to label C1 neurons. Eighty-three percent of C1 efferents that occurred in close proximity to sympathetic preganglionic neurons within the T(3) intermediolateral cell column contained VMAT2-immunoreactivity. These data demonstrate differential distribution of VMAT2 within different subpopulations of C1 neurons and suggest that this might reflect differences in somatodendritic vs. synaptic release of catecholamines.

According to a recent theory (Gray et al., 1999) the neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons of the ventral respiratory group (VRG) are confined to the pre-Botzinger complex (pre-BotC) and might be glutamatergic interneurons that drive respiratory rhythmogenesis. In this study we tested whether the NK1R-ir neurons of the VRG are glutamatergic. We also examined whether different groups of NK1R-ir neurons coexist in the VRG on the basis of whether these cells contain preproenkephalin (PPE) mRNA or project to the spinal cord. NK1R immunoreactivity was found in two populations of VRG neurons that are both predominantly glutamatergic because most of them contained vesicular glutamate transporter 2 mRNA (77 +/- 9%; n = 3). A group of small fusiform neurons (somatic cross section: 91 +/- 3.6 microm2) that has neither PPE mRNA nor spinal projections is primarily restricted to the pre-BotC. These cells may be the interneurons the destruction of which produces massive disruptions of the respiratory rhythm (Gray et al., 2001). The rest of the NK1R-ir neurons of the VRG are multipolar, are larger (somatic cross section: 252 +/- 15 microm2), and express high levels of PPE mRNA. Some of these cells located in the rostral half of the rostral VRG project to the spinal cord (C4 or T3). Using electrophysiological methods, we showed that these bulbospinal NK1R-ir neurons are slowly discharging inspiratory-augmenting neurons, suggesting that they may control phrenic or intercostal motor neurons. In summary, NK1R-expressing cells of the VRG are a heterogeneous group of predominantly glutamatergic neurons that include subpopulations of respiratory premotor neurons.

The main source of excitatory drive to the sympathetic preganglionic neurons that control blood pressure is from neurons located in the rostral ventrolateral medulla (RVLM). This monosynaptic input includes adrenergic (C1), peptidergic, and noncatecholaminergic neurons. Some of the cells in this pathway are suspected to be glutamatergic, but conclusive evidence is lacking. In the present study we sought to determine whether these presympathetic neurons express the vesicular glutamate transporter BNPI/VGLUT1 or the closely related gene DNPI, the rat homolog of the mouse vesicular glutamate transporter VGLUT2. Both BNPI/VGLUT1 and DNPI/VGLUT2 mRNAs were detected in the medulla oblongata by in situ hybridization, but only DNPI/VGLUT2 mRNA was present in the RVLM. Moreover, BNPI immunoreactivity was absent from the thoracic spinal cord lateral horn. DNPI/VGLUT2 mRNA was present in many medullary cells retrogradely labeled with Fluoro-Gold from the spinal cord (T2; four rats). Within the RVLM, 79% of the bulbospinal C1 cells contained DNPI/VGLUT2 mRNA. Bulbospinal noradrenergic A5 neurons did not contain DNPI/VGLUT2 mRNA. The RVLM of six unanesthetized rats subjected to 2 hours of hydralazine-induced hypotension contained tenfold more c-Fos-ir DNPI/VGLUT2 neurons than that of six saline-treated controls. c-Fos-ir DNPI/VGLUT2 neurons included C1 and non-C1 neurons (3:2 ratio). In seven barbiturate-anesthetized rats, 16 vasomotor presympathetic neurons were filled with biotinamide and analyzed for the presence of tyrosine hydroxylase immunoreactivity and/or DNPI/VGLUT2 mRNA. Biotinamide-labeled neurons included C1 and non-C1 cells. Most non-C1 (9/10) and C1 presympathetic cells (5/6) contained DNPI/VGLUT2 mRNA. In conclusion, DNPI/VGLUT2 is expressed by most blood pressure-regulating presympathetic cells of the RVLM. The data suggest that these neurons may be glutamatergic and that the C1 adrenergic phenotype is one of several secondary phenotypes that are differentially expressed by subgroups of these cells.