Serotonergic and glutamatergic neurons at the ventral medullary surface of the human infant: Observations relevant to central chemosensitivity in early human life

Abstract

Central chemoreception is the mechanism by which the brain detects the level of carbon dioxide (CO₂) in the arterial blood and alters breathing accordingly in order to maintain it within physiological levels. The ventral surface of the medulla oblongata (VMS) of animals has long been recognized as a site of chemosensitivity, culminating in the recent identification of chemosensitive serotonergic (5-HT) and glutamatergic (Glut) neurons in this region. In this study, we analyzed the distribution of 5-HT and Glut neurons and their receptors in the arcuate nucleus (Arc) at the VMS of the human infant, using single-and double-label immunohistochemistry with specific antibodies. We also examined the expression of astrocytes, as experimental evidence suggests that astrocytes mediate, at least in part, central chemosensitivity via 5-HT and/or Glut receptors. We identified a small number of 5-HT neurons (approximately 5% of Arc neurons), distributed over the entire extent of the VMS, a large number of Glut neurons (approximately 95% of Arc neurons) that localized almost exclusively to the medial Arc, and a large number of astrocytes distributed across the entire extent of the VMS. The Arc also contained 5-HT_1A, kainate (GluR5), and 5-HT_2A receptors, which localized predominantly to 5-HT neurons, glutamate neurons and astrocytes, respectively. Astrocytes also expressed the vesicular glutamate transporter 2 and low levels of 5-HT_1A and kainate (GluR5) receptors, indicating that astrocytes may store and release glutamate, possibly in response to stimulation by 5-HT and/or Glut. These observations suggest that important functional interactions exist between 5-HT, glutamate, and astrocytes in the Arc. They also support the idea that the Arc is homologous to chemosensitive zones at the VMS in experimental animals. These data are important towards delineating the role of the human Arc in modulation of homeostasis, and its dysfunction in brainstem-associated pathologies such as the sudden infant death syndrome.

Introduction

Central chemoreception is the mechanism by which the brain detects the level of carbon dioxide (CO₂) in the arterial blood and alters breathing accordingly in order to maintain CO₂ within physiological levels (Nattie, 2001). Central chemoreceptors are located primarily in the brainstem, and detect changes in CO₂ via attending changes in pH (Nattie, 2001, Rodman et al., 2001, Feldman et al., 2003, Richerson, 2004). Recently, both serotonergic (5-HT) (Wang et al., 2001, Richerson, 2004, Richerson et al., 2001, Messier et al., 2004, Nattie et al., 2004, Taylor et al., 2004) and glutamatergic (Glut) (Mulkey et al., 2004, Weston et al., 2004) neurons at the ventral medullary surface (VMS) of rats have been shown to exhibit properties of CO₂ chemoreceptors. Both 5-HT and Glut neurons increase their firing rate in response to elevated CO₂ and subsequently stimulate respiration through their projections to major respiratory regions in the brainstem and spinal cord. In addition, astrocytes may be central chemoreceptors (Higashi et al., 2001, Wellner-Kienitz et al., 1998), as specific lesions of astrocytes at the VMS in rats attenuate respiratory responses to elevated pCO₂ (Erlichman et al., 1998, Holleran et al., 2001). It is possible that “chemosensitive” 5-HT and Glut neurons are stimulated to fire by neurotransmitter released from astrocytes in response to elevated pCO₂, rather than any intrinsic chemosensitive nature. The roles of 5-HT neurons, Glut neurons, and astrocytes, and their interactions with each other in the mediation of chemosensitivity at the VMS remain unclear.

The positionally homologous region to the VMS in experimental animals in the human medulla oblongata includes neuron clusters labeled collectively as the arcuate nucleus. The Arc has been historically considered as a precerebellar nucleus that is composed, at least in part, of downwardly displaced neurons from the basis pontis, and is putatively involved in motor coordination (Rasmussen and Peyton, 1946). Evidence exists, however, to support a role for the Arc in respiratory chemosensitivity, including morphological and topographical homology between Arc neurons and neurons in the chemosensitive fields at the VMS in cats (Filiano et al., 1990), increased of firing of neurons at the VMS, demonstrated by functional neuroimaging upon exposure of human adult experimental subjects to carbon dioxide (CO₂) (Gozal et al., 1994), absence of the Arc in an autopsied infant with clinically documented central insensitivity to CO₂ (Folgering et al., 1979), and abnormalities in the Arc in sudden infant death syndrome (SIDS) (Filiano and Kinney, 1992, Matturri et al., 2000, Matturri et al., 2004, Gilson et al., 1994, Biondo et al., 2003, Panigrahy et al., 1997, Panigrahy et al., 2000, Kinney et al., 1995), a putative disorder of compromised responses to elevated CO₂ (Kinney et al., 2001).

Based upon these combined observations, and the recent observations that 5-HT and Glut neurons are CO₂ detectors at the VMS of the rat, we hypothesize that the Arc is homologous to the respiratory chemosensitive zones on the VMS in animals, and thereby contains homologous chemosensitive 5-HT and Glut neurons. Very little is known about the chemoarchitecture of the Arc, particularly in relationship to the idea that it contains at least two, and possibly more, neurotransmitter systems involved in the mediation of chemosensitivity, as well as possible neurotransmitter–astrocyte interactions in this and/or other functions of the nucleus. As a step towards testing our hypotheses, we used immunohistochemistry with the PH8 antibody to detect tryptophan hydroxylase (TPOH), and specific antibodies for vesicular glutamate transporter 2 (VGLUT2), and glial fibrillary acidic protein (GFAP), to map the relative distribution of 5-HT neurons, Glut neurons, astrocytes respectively in the Arc. In addition, using specific antibodies, we mapped the distribution of 5-HT_1A receptors, 5-HT_2A receptors, and GluR5 kainate receptors in the Arc to examine the potential circuitry among different neuronal and astrocyte subtypes in the mediation of chemosensitivity. We selected these receptors in particular because agonists selective for each of the 5-HT_1A, 5-HT_2A, and GluR5 receptors alter respiratory function when applied to the VMS or raphé of animals (Lalley et al., 1994, Gillis et al., 1989, Rose et al., 1995, Teng et al., 2003, Nattie et al., 1988). In addition, we previously identified reductions in 5-HT receptor binding using the broad radioligand [³H] lysergic acid diethyl amide (LSD), and kainate receptor binding using [³H] kainate, in the Arc in SIDS cases with tissue section autoradiography (Panigrahy et al., 1997, Panigrahy et al., 2000). Due to the focus of our laboratory on putative disorders of central chemosensitivity in early life, we analyzed in this study the chemoarchitecture of the VMS in human infants.