Ruth L. Stornetta

Respiratory control by ventral surface chemoreceptor neurons in rats.

A long-standing theory posits that central chemoreception, the CNS mechanism for CO2 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 CO2 in vivo, whereas serotonergic neurons are not. Their CO2 sensitivity is unaffected by pharmacological blockade of the respiratory pattern generator and persists without carotid body input. RTN CO2-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 CO2. Their pH sensitivity is intrinsic and involves a background K+ current. In short, the CO2-sensitive neurons of the RTN are good candidates for the long sought-after VMS chemoreceptors.

Distribution of α2C‐adrenergic receptor‐like immunoreactivity in the rat central nervous system

The distribution of α2C‐adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70‐amino‐acid polypeptide portion of the third intracellular loop of the α2C‐AR fused to glutathione‐S‐transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]‐photoaffinity‐labeled α2‐AR and by immunohistochemical labeling of COS cells expressing the individual rat α2‐AR subtypes. In both cases the antibody recognized only the α2C‐AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, α2C‐AR‐like immunoreactivity (α2C‐AR‐LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of α2C‐LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Central autonomic system

Abstract The central autonomic system is defined by the cell groups that receive direct or disynaptically relayed input from the nucleus of the solitary tract, or that contribute projections to autonomic preganglionic neurons or their presynaptic inputs. The cell groups identified by these properties also project extensively among themselves, forming a true network. In this chapter, we identify the cell groups from the medulla and spinal cord through the brainstem, hypothalamus, thalamus, amygdala, and cortex that contribute to the central autonomic system. We then review the connections within the central autonomic network; the pathways that provide visceral sensory input to, and visceral motor output from the network; and the connections of the central autonomic system with cognitive, endocrine, and behavioral systems that must be integrated with autonomic responses. We also consider the neurotransmitters used by this network, and how they contribute to its functions.

Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2‐sensitive neurons in rats

The rat retrotrapezoid nucleus (RTN) contains pH‐sensitive neurons that are putative central chemoreceptors. Here, we examined whether these neurons respond to peripheral chemoreceptor stimulation and whether the input is direct from the solitary tract nucleus (NTS) or indirect via the respiratory network. A dense neuronal projection from commissural NTS (commNTS) to RTN was revealed using the anterograde tracer biotinylated dextran amine (BDA). Within RTN, 51% of BDA‐labelled axonal varicosities contained detectable levels of vesicular glutamate transporter‐2 (VGLUT2) but only 5% contained glutamic acid decarboxylase‐67 (GAD67). Awake rats were exposed to hypoxia (n= 6) or normoxia (n= 5) 1 week after injection of the retrograde tracer cholera toxin B (CTB) into RTN. Hypoxia‐activated neurons were identified by the presence of Fos‐immunoreactive nuclei. CommNTS neurons immunoreactive for both Fos and CTB were found only in hypoxia‐treated rats. VGLUT2 mRNA was detected in 92 ± 13% of these neurons whereas only 12 ± 9% contained GAD67 mRNA. In urethane–chloralose‐anaesthetized rats, bilateral inhibition of the RTN with muscimol eliminated the phrenic nerve discharge (PND) at rest, during hyperoxic hypercapnia (10% CO2), and during peripheral chemoreceptor stimulation (hypoxia and/or i.v. sodium cyanide, NaCN). RTN CO2‐activated neurons were recorded extracellularly in anaesthetized intact or vagotomized rats. These neurons were strongly activated by hypoxia (10–15% O2; 30 s) or by NaCN. Hypoxia and NaCN were ineffective in rats with carotid chemoreceptor denervation. Bilateral injection of muscimol into the ventral respiratory column 1.5 mm caudal to RTN eliminated PND and the respiratory modulation of RTN neurons. Muscimol did not change the threshold and sensitivity of RTN neurons to hyperoxic hypercapnia nor their activation by peripheral chemoreceptor stimulation. In conclusion, RTN neurons respond to brain P  CO 2 presumably via their intrinsic chemosensitivity and to carotid chemoreceptor activation via a direct glutamatergic pathway from commNTS that bypasses the respiratory network. RTN neurons probably contribute a portion of the chemical drive to breathe.

Expression of Phox2b by Brainstem Neurons Involved in Chemosensory Integration in the Adult Rat

Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.

Vesicular glutamate transporter DNPI/VGLUT2 mRNA is present in C1 and several other groups of brainstem catecholaminergic neurons

The mouse glutamate vesicular transporter VGLUT2 has recently been characterized. The rat homolog of VGLUT2, differentiation‐associated Na+/Pi cotransporter (DNPI), was examined using a digoxigenin‐labeled DNPI/VGLUT2 cRNA probe in the present study to determine which, if any, of the various groups of pontine or medullary monoaminergic neurons express DNPI/VGLUT2 mRNA and, thus, are potentially glutamatergic. DNPI/VGLUT2 mRNA was widely distributed within the brainstem and seemed exclusively neuronal. By using a double in situ hybridization method, the presence of the mRNA for DNPI/VGLUT2 and glutamic acid decarboxylase (GAD)‐67 was mutually exclusive. By combining DNPI/VGLUT2 mRNA detection and conventional immunohistochemistry, DNPI/VGLUT2 mRNA was undetectable in lower brainstem cholinergic and serotonergic cells, but it was present in several tyrosine hydroxylase‐immunoreactive (TH‐ir) cell groups. DNPI/VGLUT2 mRNA was detected in most of the adrenergic neurons of the C1, C2, and C3 groups (75–80% of TH‐ir neurons), in the A2 noradrenergic group (80%), and in vast numbers of area postrema cells. Within the A1 region, many fewer TH‐ir cells contained DNPI/VGLUT2 (16%). Finally, DNPI/VGLUT2 mRNA was undetectable in the pontine noradrenergic cell groups (A5 and A6/locus coeruleus). In conclusion, the general pattern of DNPI/VGLUT2 expression and its exclusion from GABAergic, cholinergic, and serotonergic neurons supports the notion that DNPI/VGLUT2 mRNA identifies a subset of glutamatergic neurons in the lower brainstem. Within this region several catecholaminergic cell groups appear to be glutamatergic, including but not limited to the adrenergic cell groups C1–C3. Based on the present evidence, the noradrenergic cell groups of the pons (A5 and A6) do not contain either known vesicular glutamate transporter and are most likely not glutamatergic. J. Comp. Neurol. 444:191–206, 2002.

Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2

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 (≈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. J. Comp. Neurol. 465:593–603, 2003.

A group of glutamatergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Bötzinger complex.

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‐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 (<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. J. Comp. Neurol. 455:499–512, 2003.

Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla

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. J. Comp. Neurol. 444:207–220, 2002.

The organization of two new cortical interneuronal circuits

Deciphering the interneuronal circuitry is central to understanding brain functions, yet it remains a challenging task in neurobiology. Using simultaneous quadruple-octuple in vitro and dual in vivo whole-cell recordings, we found two previously unknown interneuronal circuits that link cortical layer 1–3 (L1–3) interneurons and L5 pyramidal neurons in the rat neocortex. L1 single-bouquet cells (SBCs) preferentially formed unidirectional inhibitory connections on L2/3 interneurons that inhibited the entire dendritic-somato-axonal axis of ∼1% of L5 pyramidal neurons located in the same column. In contrast, L1 elongated neurogliaform cells (ENGCs) frequently formed mutual inhibitory and electric connections with L2/3 interneurons, and these L1-3 interneurons inhibited the distal apical dendrite of >60% of L5 pyramidal neurons across multiple columns. Functionally, SBC→L2/3 interneuron→L5 pyramidal neuronal circuits disinhibited and ENGC↔L2/3 interneuron→L5 pyramidal neuronal circuits inhibited the initiation of dendritic complex spikes in L5 pyramidal neurons. As dendritic complex spikes can serve coincidence detection, these cortical interneuronal circuits may be essential for salience selection.