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.

Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting–based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons. We identified correlative populations in mouse and human glioma and found that the emergence of specific subpopulations during tumor progression corresponded with the onset of seizures and tumor invasion. In sum, we have identified subpopulations of astrocytes in the adult brain and their correlates in glioma that are endowed with diverse cellular, molecular and functional properties. These populations selectively contribute to synaptogenesis and tumor pathophysiology, providing a blueprint for understanding diverse astrocyte contributions to neurological disease.

The pre-Bötzinger complex (pre-BötC) 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-BötC area was defined as a 500-microm-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 microm(2)) 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 (<1%) or preproenkephalin mRNA (6%). Retrograde labeling experiments demonstrated that over 75% of the SST-ir neurons project to the contralateral pre-BötC area, but none projects to the spinal cord. In conclusion, the RVLM contains many neurons that express preprosomatostatin mRNA. A subgroup of these cells contains high levels of SST and NK1R immunoreactivity in their somata. These glutamatergic interneurons identify a narrow region of the RVLM that appears to be coextensive with the pre-BötC of adult rats.

Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

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.