Water deprivation activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the rostral ventrolateral medulla.

Elevated sympathetic outflow contributes to the maintenance of blood pressure in water-deprived rats. The neural circuitry underlying this response may involve activation of a pathway from the hypothalamic paraventricular nucleus (PVH) to the rostral ventrolateral medulla (RVLM). We sought to determine whether the PVH-RVLM projection activated by water deprivation is glutamatergic and/or contains vasopressin- or oxytocin-neurophysins. Vesicular glutamate transporter 2 (VGLUT2) mRNA was detected by in situ hybridization in the majority of PVH neurons retrogradely labeled from the ipsilateral RVLM with cholera toxin subunit B (CTB; 85% on average, with regional differences). Very few RVLM-projecting PVH neurons were immunoreactive for oxytocin- or vasopressin-associated neurophysin. Injection of biotinylated dextran amine (BDA) into the PVH produced clusters of BDA-positive nerve terminals within the ipsilateral RVLM that were immunoreactive (ir) for the VGLUT2 protein. Some of these terminals made close appositions with tyrosine-hydroxylase-ir dendrites (presumptive C1 cells). In water-deprived rats (n=4), numerous VGLUT2 mRNA-positive PVH neurons retrogradely labeled from the ipsilateral RVLM with CTB were c-Fos-ir (16-40%, depending on PVH region). In marked contrast, few glutamatergic, RVLM-projecting PVH neurons were c-Fos-ir in control rats (n=3; 0-3%, depending on PVH region). Most (94% +/- 4%) RVLM-projecting PVH neurons activated by water deprivation contained VGLUT2 mRNA. In summary, most PVH neurons that innervate the RVLM are glutamatergic, and this population includes the neurons that are activated by water deprivation. One mechanism by which water deprivation may increase the sympathetic outflow is activation of a glutamatergic pathway from the PVH to the RVLM.

Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column.

Markers of serotonergic, gamma-aminobutyric acid (GABA)-ergic (glutamic acid decarboxylase, 67 kDa isoform; GAD-67), and glutamatergic transmission (vesicular glutamate transporter 3; VGLUT3) have been detected in presumed sympathetic premotor neurons of the medullary raphe, a region that controls sympathetic tone to brown fat, skin blood vessels, and heart. In this study, the degree of coexpression of these markers was examined in raphe neurons by simultaneous histological detection of tryptophan hydroxylase (TrpOH) immunoreactivity with GAD-67 mRNA and VGLUT3 mRNA. Over half (52%) of the VGLUT3 mRNA-positive neurons expressed one or both of the other markers. The proportion of VGLUT3 neurons containing at least one of the other two markers was even higher (89%) for VGLUT3 spinally projecting neurons. VGLUT3 neurons containing markers for both serotonin and GABA were especially numerous (50-72%, depending on rostrocaudal level) within the marginal layer of raphe pallidus and the parapyramidal region. The dual GABAergic and glutamatergic nature of some bulbospinal raphe neurons was suggested by the presence of nerve terminals immunoreactive (ir) for both VGLUT3 and GABA in the intermediolateral cell column (IML) as detected by electron microscopy. VGLUT3-ir terminals formed approximately equal numbers of symmetric and asymmetric synapses onto presumed preganglionic neurons (nitric oxide synthase-ir profiles) or GABA-ir dendrites in IML, and terminals immunoreactive for both VGLUT3 and GABA always formed symmetric synapses. These data suggest that medullary raphe VGLUT3 neurons could inhibit sympathetic outflow and that their spinal targets include both preganglionic neurons and GABAergic interneurons.

Respiratory control by ventral surface chemoreceptor neurons in rats.

A long-standing theory posits that central chemoreception, the CNS mechanism for CO(2) detection and regulation of breathing, involves neurons located at the ventral surface of the medulla oblongata (VMS). Using in vivo and in vitro electrophysiological recordings, we identify VMS neurons within the rat retrotrapezoid nucleus (RTN) that have characteristics befitting these elusive chemoreceptors. These glutamatergic neurons are vigorously activated by CO(2) in vivo, whereas serotonergic neurons are not. Their CO(2) sensitivity is unaffected by pharmacological blockade of the respiratory pattern generator and persists without carotid body input. RTN CO(2)-sensitive neurons have extensive dendrites along the VMS and they innervate key pontomedullary respiratory centers. In brainstem slices, a subset of RTN neurons with markedly similar morphology is robustly activated by acidification and CO(2). Their pH sensitivity is intrinsic and involves a background K(+) current. In short, the CO(2)-sensitive neurons of the RTN are good candidates for the long sought-after VMS chemoreceptors.

Detection of amino acid and peptide transmitters in physiologically identified brainstem cardiorespiratory neurons.

Most of the CNS neurons that regulate circulation and respiration reside in regions of the brain characterized by extreme cellular heterogeneity (nucleus of the solitary tract, reticular formation, parabrachial nuclei, periaqueductal gray matter, hypothalamus, etc.). The chemical neuroanatomy of these regions is correspondingly complex and teasing out specific circuits in their midst remains a problem that is usually very difficult if not impossible to solve by conventional tract-tracing methods, Fos methodology or electrophysiology in slices. In addition, identifying the type of amino acid or peptide transmitter used by electrophysiologically recorded neurons has been until recently an especially difficult task either for lack of a specific marker or because such markers (many peptides for example) are exported to synaptic terminals and thus undetectable in neuronal cell bodies. In this review, we describe a general purpose method that solves many of these problems. The approach combines juxtacellular labeling in vivo with the histological identification of mRNAs that provide definitive neurochemical phenotypic identification (e.g. vesicular glutamate transporter 1 or 2, glutamic acid decarboxylase). The results obtained with this method are discussed in the general context of amino acid transmission in brainstem cardiorespiratory pathways. The presence of markers of amino acid transmission in specific aminergic pre-sympathetic neurons is especially emphasized as is the extensive co-localization of markers of GABAergic and glycinergic transmission in the brainstem reticular formation.