Monoaminergic neurons, chemosensation and arousal

Respiration Physiology 129 (2001) 191– 209 www.elsevier.com/locate/resphysiol

Monoaminergic neurons, chemosensation and arousal M.A. Haxhiu a,b,c,*, F. Tolentino-Silva b, G. Pete a, P. Kc a, S.O. Mack a a

Department of Physiology and Biophysics, Howard Uni6ersity College of Medicine, 520 W Street, N.W. Washington, DC 20059, USA b Department of Pediatrics, Case Western Reser6e Uni6ersity, Cle6eland, OH 44106, USA c Department of Medicine, Case Western Reser6e Uni6ersity, Cle6eland, OH 44106, USA Accepted 16 May 2001

Abstract In recent years, immense progress has been made in understanding central chemosensitivity at the cellular and functional levels. Combining molecular biological techniques (early gene expression as an index of cell activation) with neurotransmitter immunohistochemistry, new information has been generated related to neurochemical coding in chemosensory cells. We found that CO2 exposure leads to activation of discrete cell groups along the neuraxis, including subsets of cells belonging to monoaminergic cells, noradrenaline-, serotonin-, and histamine-containing neurons. In part, they may play a modulatory role in the respiratory response to hypercapnia that could be related to their behavioral state control function. Activation of monoaminergic neurons by an increase in CO2/H+ could facilitate respiratory related motor discharge, particularly activity of upper airway dilating muscles. In addition, these neurons coordinate sympathetic and parasympathetic tone to visceral organs, and participate in adjustments of blood flow with the level of motor activity. Any deficit in CO2 chemosensitivity of a network composed of inter-related monoaminergic nuclei might lead to disfacilitation of motor outputs and to failure of neuroendocrine and homeostatic responses to life-threatening challenges (e.g. asphyxia) during sleep. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chemosensitivity, central; Control of breathing, central chemosensitivity; Deficiency, a2A-adrenergic; Mammals, mouse; Neurons, catecholaminergic, serotonergic, histaminergic; Protein, c-Fos; Receptor, a2A-adrenergic

1. Introduction In this chapter we will discuss chemosensitive properties of monoaminergic neurons and their possible function as chemosensors. Functionally, * Corresponding author. Tel.: + 1-202-806-6330; fax: +1202-806-4479. E-mail address: [email protected] (M.A. Haxhiu).

it is a difficult task to ascribe specific CO2-induced responses to particular monoaminergic neurotransmitter(s). Hence, we will attempt to highlight their integrative role, including their involvement in behavioral state control. Over the last 40 years, intensive research has been performed on localization of chemosensory cells. These studies demonstrated that chemosensitive neural elements of the ventrolateral surface

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of the medulla play a pivotal role in regulation of respiratory activity. Although there are recent data demonstrating the presence of chemosensitive neurons in regions outside of the ventrolateral aspect of the medulla oblongata (Coates et al., 1993), physiological studies have established the primary importance of the ventral medullary chemosensitive areas, located just beneath the surface of the ventrolateral aspect of the medulla oblongata (Mitchell et al., 1963; Schla¨ fke and Loeschcke, 1967; Loeschcke et al., 1970; Trouth et al., 1973). Based on CO2-induced expression of early genes, we assumed that central chemosensory neurons are components of neuronal networks involved in the regulation of diverse functions, including the sleep/wakefulness cycle. Conceivably, a single neurotransmitter (i.e. acetylcholine) cannot mediate all physiological responses to an increase or decrease in CO2/H+ content. Since physiologic responses are highly coordinated and coherently assembled, it is expected that a number of different chemicals may participate in transmission of chemical information, and in dynamic control of final motor and behavioral responses. In this line of thinking, it could be considered that central chemosensory cells in the brainstem reticular formation and suprapontine regions are neurotransmitter-specific containing neurons that mediate ventilatory and autonomic nervous system responses to an increased concentration of CO2 or H+ via local projections, and/or axons that ascend to the forebrain or descend to the spinal cord. Thus, the response to hypercapnia may be modulated at different levels of organization. In the mammalian central nervous system, monoaminergic neurons are well developed and provide widespread projections throughout the entire brain (Jacobs and Fornal, 1999; Smeets and Gonzalez, 2000). Hence, it is hypothesized that a subpopulation of these neurons that belong to dissociable neurotransmitter specific monoaminecontaining cell groups could sense changes in concentrations of CO2 or H+ and use their transmitter(s) content to relay information that modulates responses to CO2 or H+ in a concentration-dependent manner.

2. Expression of c-fos gene in monoaminergic neurons as a cellular marker of neuronal activity Until recently, chemosensitive traits of noradrenaline-, serotonin-, and histamine-containing neurons have not been studied. This is partly because large numbers of functionally active cells under awake, unsedated experimental conditions cannot be sampled using established methods for identifying chemosensory neurons such as the single-cell recording technique (Dean et al., 1990; Richerson, 1995; Kawai et al., 1996; Oyamada et al., 1998). One way to circumvent this difficulty is to determine hypercapnia-induced expression of immediate-early genes encoding transcription factors such as members of the fos (c-fos, fos B, fra-1, fra-2), jun (c-jun, jun B, jun D) and Krox [Krox-20 (Erg-2), Krox-24 (Erg-1), zif/268] families. The c-fos gene, and its product Fos protein (c-Fos), have been used to identify activated neurons within the central nervous system (CNS). This gene is rapidly and transiently expressed within the cell nucleus following cell activation by different stimuli; c-fos gene expression is inducible and it serves as a high resolution marker for activated neurons, but it is not equivalent to electrophysiological recordings. Following cell stimulation, c-fos gene expression at the message level (mRNA) can be detected within minutes. Production of c-Fos protein, however, in an amount observable by immunohistochemistry, requires more time. The c-Fos protein appears 30 min after the end of a stimulus and reaches its maximum one to 2 h following stimulation (Morgan and Curran, 1986; Bullitt, 1990; Sheng and Greenberg, 1990). Furthermore, there is growing evidence suggesting that neurotrophins (i.e. brain-derived neurotrophic factor and neurotrophin 3) produce a rapid increase in c-Fos mRNA expression (Kim et al., 2000). At central synapses, neutrophins enhance synaptic transmission (Kang and Schuman, 1995), but suppress inhibitory transmission (Tanaka et al., 1997), and the net effect is cell activation. However, not all activated neurons express c-Fos. For example, elevated CO2/H+ concentration, via chemosensory neurons, activate phrenic and hypoglossal motoneurons, but these cells do not express c-Fos

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following exposure to hypercapnic loading (Haxhiu et al., 1996). Apart from these limitations, such as sensitivity and specificity, the advantage of using c-fos expression in a functionally-oriented anatomical approach is that activity of a large number of cells can readily be identified under conditions, which allow recording of functional changes. In the last 10 years, we used this method to identify neurons within the brainstem that may be involved in sensing changes in CO2/H+ . With this method, we were able to characterize neurotransmitter content, and signal transduction pathways involved in CO2/H+-induced cell activation (Haxhiu et al., 1992a, 1996; Kuo et al., 1998). Previously, we and others demonstrated that unsedated animals, including rats of different postnatal ages, exposed to CO2 expressed c-Fos at putative chemoreceptor sites (Haxhiu et al., 1992a, 1996; Sato et al., 1992; Teppema et al., 1997; Belegu et al., 1999). It could be presumed that neurons responding to hypercapnic loading by c-fos expression are chemosensory cells. This assumption is supported by findings related to the distribution of neurons activated by elevated CO2 and/or H+ ion content of extracellular fluid along the neuraxis (Haxhiu et al., 1996), based on metabolic mapping (Ciriello et al., 1985), mapping of the pH decrease during systemic hypercapnic loading (Arita et al., 1989), and on functional magnetic resonance imaging (Gozal et al., 1994). Furthermore, the distribution pattern of hypercapnia-induced c-fos expression in the brainstem and diencephalon corresponds well with recently identified chemosensitive sites based on the response to local changes in CO2/H+ concentrations, or topical application of acetazolamide (Dean et al., 1990; Dillon and Waldrop, 1992; Coates et al., 1993; Richerson, 1995; Bernard et al., 1996; Kawai et al., 1996; Pineda and Aghajanian, 1997 Oyamada et al., 1998). It has been shown that synaptic inputs are not required for excitatory effects of CO2/H+ signals on a subset of nucleus tractus solitarius (NTS) or caudal raphe neurons (Fukuda et al., 1978; Dean et al., 1990; Neubauer et al., 1991; Richerson, 1995), and for c-Fos expression in these regions (Haxhiu et al., 1996). However, c-fos expression

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induced by systemic hypercapnia can be due to synaptically activated cells. The subset of ventral medullary neurons, which are located within chemosensitive fields, possesses extensive axonal arborization and exerts wide-ranging influences over neurons in virtually all CNS nuclei, including intrinsic chemosensitive locus coeruleus neurons, the majority of which express c-fos when stimulated by CO2 or H+ (Haxhiu et al., 1996). Hence, intrinsic chemosensitivity of monoaminergic neurons and synaptic input to these cells can contribute to c-Fos expression when they are induced by hypercapnic stress. Hypercapnia may hyperpolarize the neurons that provide inhibitory inputs to respiratory-related excitatory interneurons, thereby reducing the source of inhibitory influence and increasing the strength of excitatory inputs to respiratory-related networks. Therefore, the CNS neurons inhibited by CO2 may play a role as inhibitory chemoreceptors (Richerson, 1995). Inhibited neurons do not express c-fos (Chan and Sawchenko, 1994), hence, their neurotransmitter(s) content cannot be visualized by this technique. Knowing the usefulness and limitations of c-fos gene and c-Fos protein expression as cellular markers of activated neurons within the CNS, we determined whether monoamine-containing neurons respond to an increase in CO2 or H+ concentration and how these cells may affect the ventilatory response to hypercapnia. The method used to identify expression of c-Fos protein following exposure to hypercapnia in monoaminergic neurons has been described previously (Haxhiu et al., 1996).

3. Anatomical, chemical, and physiological bases for chemosensitivity in monoaminergic neurons In the mammalian central nervous system, monoaminergic pathways represent key components of the reticular activating system and are implicated in diverse physiological functions, including behavioral state control (McGinty and Harper, 1976; Aston-Jones and Bloom, 1981). While subgroups of catecholaminergic and serotonergic neurons are located in the brainstem, histamine-containing neurons are found in the

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hypothalamus (Watanabe et al., 1983; Panula et al., 2000). A subpopulation of monoaminergic neurons and their nerve processes are located just beneath the surface of the brainstem and ventricles, suggesting that monoaminergic neurons could be involved in sensing changes in pH, concentrations of diverse hormones or other chemical compounds in the cerebrospinal fluid. Monoaminergic systems in the brain exert different physiological and behavioral modulatory influences on their widespread targets. These effects are related primarily to central regulation of autonomic functions, motor activity, and the sleep-wake-arousal cycle (Jouvet, 1972; Aston-Jones and Bloom, 1981; Jacobs et al., 1990).

3.1.1. Catecholaminergic neurons Since the pioneering work of Von Euler (1946) demonstrating the presence of catecholamines in nerve terminals, numerous studies have shown the role of catecholamines in regulation of autonomic functions. The major catecholamines used by the nervous system are, dopamine; noradrenaline; and adrenaline. These substances are synthesized in the central nervous system from tyrosine, a dietary amino acid. Tyrosine is converted to dihydroxyphenylalanine (DOPA) through addition of a hydroxyl group to the catechol ring by the enzyme tyrosine hydroxylase (TH). The enzyme TH is the rate limiting enzyme in catecholamine synthesis. DOPA is then converted to dopamine by decarboxylation of the amine group by the enzyme DOPA decarboxylase. Dopamine is converted to noradrenaline by the enzyme dopaminebeta-hydroxylase through the addition of a hydroxyl group to the carbon atom nearest to the catechol ring. Noradrenaline in the periphery, and to some extent in the CNS, is converted to adrenaline by methylation of the terminal amide group by the enzyme phenylethanolamine Nmethyltransferase (for review see Moore and Bloom, 1979; Smeets and Gonzalez, 2000). The availability of antisera against specific enzymes involved in catecholamine synthesis and against specific catecholamines themselves, allowed for a more precise determination of cate-

cholamine expression. In general, six main groups of catecholamine cells are recognized in the brains of vertebrates, (1) a caudal rhombencephalic group (A1 –A3/C1 –C3); (2) a rostral rhombencephalic group (A4–A7); (3) a mesencephalic group (A8 –A10); (4) a diencephalic group (A11– A15); (5) an olfactory bulb group (A16); and (6) a retinal group (A17).

3.2. Chemosensory traits in noradrenergic and dopaminergic neurons 3.2.1. Medullary noradrenergic neurons The distribution of the norepinephrine-containing neurons with the brainstem is presented in Fig. 1. Our studies demonstrated that noradrenaline-containing neurons are part of the networks that sense changes in arterial CO2. In these

Fig. 1. (A) and (B), coronal sections show the locations of noradrenaline-containing neurons that expressed c-Fos induced by hypercapnic stress. (C), schematic drawing representing the distribution of catecholaminergic neurons on the sagittal plane of the rat brain. A1/C1, noradrenaline cell group; A2/C2, noradrenaline cell group; A5, noradrenaline cell group; A6, noradrenaline cell group; A7, noradrenaline cell group; LC, locus coeruleus; NTS, nucleus tractus solitarius; PVN, paraventricular hypothalamic nucleus; SHN, supraoptic hypothalamic nucleus.

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experiments, as previously described (Haxhiu et al., 1996), we examined c-fos expression in catecholaminergic neurons following exposure of unanesthetized rats to hypercapnic stress. Breathing a gas mixture with elevated CO2 (15% CO2, 21% O2 and 64% N2, or 15% CO2 in O2) for 60 min, induced activation of the c-fos gene in widespread regions of the CNS, as indicated by the expression of Fos-like immunoreactive protein. Similar results were obtained in carotid body denervated animals. Colocalization studies of tyrosine hydroxylase (TH) and c-Fos revealed that in the brainstem, 73–85% of noradrenaline-containing cells expressed Fos immunoreactivity. Double-labeled neurons were found in the ventrolateral medullary reticular formation (A1 noradrenaline cells) and in the dorsal aspect of the medulla oblongata (A2 noradrenaline cells). An example of c-Fos expression in the A1 cell group is shown in Fig. 2. A drawing showing its location is presented in panel A of Fig. 1. Noradrenaline-containing neurons (A1 and A2 cell groups), project to the rostral ventrolateral medulla and to the hypothalamus, and they are involved in cardiovascular and neuroendocrine functions (for review see Moore and Bloom, 1979; Smeets and Gonzalez, 2000).

3.2.2. The pontine noradrenergic neurons In the pons, following exposure to hypercapnia, 79 – 85% of noradrenergic neurons expressed cFos (Haxhiu et al., 1996). These neurons were localized in three distinct groups (Fig. 1 panels B and C) along the ventrolateral margin of the pontine tegmentum (A5 group), dorsal and lateral to the midline and beneath the fourth ventricle, (A6 group, Figs. 1 and 2), and ventrolaterally to the locus coeruleus (A7 group). While the A5 and A7 cell groups project to the medulla oblongata and spinal cord, the locus coeruleus (A6) has extensive projections to the cerebral cortex, cerebellum, as well as descending projections to the medulla oblongata and spinal cord (Moore and Bloom, 1979; Smeets and Gonzalez, 2000). 3.2.3. The suprapontine noradrenergic and dopaminergic neurons Exposure to CO2 did not induce c-Fos expres-

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sion in the majority of dopaminergic neurons located in the retrorubral field (A8 group) or in the substantia nigra (A9 group). However, in response to CO2 loading, a subpopulation of THcontaining neurons seen in the periaqueductal central gray (A10 group) and in hypothalamic nuclei (A11–A15 cell groups) expressed c-Fos like immunoreactivity. Hypothalamic dopaminergic neurons in the A11 and A13 cell groups, a small number of which respond to hypercapnia, project to caudal brainstem and spinal cord regions involved in central regulation of autonomic functions. Neurons in the A12 and A14 groups participate in endocrine control. Catecholaminergic neurons in the telencephalon (A 16 group) and retina (A17 group) were not examined. The lack of hypercapnia-induced expression of c-Fos protein in two major suprapontine catecholaminergic cell groups (A8 and A9 dopaminecontaining cells) may suggest that these dopamine-containing cell groups do not play a significant role in mediation of hypercapnia-induced changes in respiration and arousal reactions.

3.2.4. Noradrenergic chemosensiti6e neurons and central regulation of respiration In general, neurotransmission occurs within specialized synaptic junctions (synaptic transmission) or outside conventional synapses (volume transmission; Callado and Stamford, 2000). Cellular targets of noradrenaline-containing chemosensory cells are interneurons, motoneurons of cranial nerves, and/or respiratory related bulbospinal and spinal motoneurons (Haxhiu et al., 1993; Dobbins and Feldman, 1994). The response by these targeted neurons to released noradrenaline may depend on expression of adrenergic receptors of the alpha or beta type and their specific subtypes. Many modulatory effects of noradrenaline are attributable to alpha 1-adrenergic receptors. At least three alpha 1 receptor subtypes identified by molecular criteria are designated as alpha 1A, alpha 1B, and alpha 1D. Studies related to the distribution of the alpha 1A adrenergic receptor mRNA showed that this receptor subtype is

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Fig. 2. Upper panels, brightfield (left c-Fos) and fluorescent (right) photomicrographs in the caudal region of the medulla oblongata containing A1 noradrenaline neurons of a rat exposed to hypercapnia. Lower panels: brightfield (left) and fluorescent (right) photomicrographs in the pons containing A6 noradrenaline neurons of a rat exposed to hypercapnia. A1 and A6 neurons containing TH (tyrosine hydroxylase) and expressing c-Fos are marked with thick, closed arrows. Neurons containing TH but not expressing Fos, and TH-negative cells are indicated by open, light and dark arrows, respectively. Scale bar = 20 mm.

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highly expressed in suprapontine regions, brainstem reticular nuclei, and several cranial nerve motor nuclei, indicating the neuromodulatory effects of noradrenaline in processes such as arousal, neuroendocrine control, sensorimotor and autonomic regulation, and the stress response (Domyancic and Morilak, 1997). In addition, previous studies have reported that the alpha 1adrenergic system activates both the central respiratory command and spinal inspiratory output neurons, and increases motoneuronal excitability, probably through a decrease in postsynaptic leak K+ conductance (Morin et al., 2000). The alpha 2-adrenoreceptors have been found in many locations and they mediate a variety of functional responses. Studies on prejunctional alpha 2-adrenergic receptors suggested that there is more than a single alpha 2-adrenergic subtype (Hudson et al., 1999). The subdivision of the alpha 2-adrenoreceptors into four subtypes is based primarily on radioligand binding characteristics in native tissue homogenates. In the rat, the primary alpha 2-adrenergic receptor is alpha 2D (relatively low affinity for yohimbine and rauwolscine); in the rabbit, the orthologous alpha 2A-adrenoreceptor subtype (relatively high affinity for yohimbine and rauwolscine) is predominate. Prejunctional alpha 2-adrenoreceptors are located on many peripheral and central nerve terminals where their activation inhibits neurotransmitter release. These receptors are also present on neuronal cell bodies where they mediate hyperpolarization and inhibition of firing rate. Recently, it was shown that both the alpha 2Aand alpha 2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha 2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha 2C-subtype modulates neurotransmission at lower levels of nerve activity (Hein et al., 1999). Alpha 2-adrenergic receptor antagonists may produce an opposite action from an agonist, suggesting that alpha 2-adrenoreceptors may be tonically active. However, there are considerable differences in the receptor reserves among the alpha 2-adreno-

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receptors subtypes, which may explain variations in physiological responses. The role of noradrenaline-containing chemosensory neurons in control of the respiratory drive response to an increased concentration of CO2 or H+ is not well understood. Inputs from activated brainstem catecholaminergic neurons may affect the response to hypercapnia via projections to the respiratory related network and activation of different types of adrenergic receptors expressed by these cells. The action of a CO2-induced release of noradrenaline at targeted sites may represent a summation of excitatory and inhibitory influences on respiratory related neurons. Recently, we studied the respiratory effects elicited by chemical interventions on locus coeruleus neurons. In rats, microinjection of glutamate into the locus coeruleus significantly elevated diaphragm minute electromygraphic activity, by increasing amplitude and discharge frequency. On the other hand, microinjection of GABA into the same region decreased respiratory drive by decreasing diaphragm activity and slowing frequency discharge (Chavez et al., 1998). Conceivably, a hypercapnia-induced increase in activity of locus coeruleus neurons will lead to the release of noradrenaline at targeted sites. Noradrenaline, in turn, may activate alpha 1-adrenergic receptors on excitatory interneurons that project to inspiratory related bulbospinal neurons, and/or rhythm generating cells, causing an increase in cell excitability, cell depolarization, and augmented respiratory drive. This assumption is in agreement with findings that activation of the alpha 1-adrenergic system increases respiratory drive (Morin et al., 2000). Noradrenaline may have similar effects if it activates alpha 2-adrenergic receptors located on inhibitory neurons that project to the respiratory rhythm generating network and/or phrenic pre-motor cells. This response will remove inhibitory inputs that lead to facilitation of excitatory signals with a subsequent increase in breathing activity. Other noradrenaline-containing neurons that are activated by an increase in CO2 or H+ concentration may affect respiratory drive. Neurons of the A5 group project to motoneurons of cranial nerves and bulbospinal inspiratory cells (Hax-

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hiu et al., 1993; Dobbins and Feldman, 1994). The A5 cell group is the potential source of the noradrenergic inhibitory drive to the medullary respiratory network (Hilaire et al., 1989; Jodkowski et al., 1997). Similarly, the A1 cell group provides inhibitory inputs to the medullary network involved in regulation of respiratory timing. In mice, we found that activation of these sites invariably induces a decrease in breathing frequency and arterial pressure, as was also described in rats (Bonham and Jeske, 1989; Tolentino-Silva et al., 1997). Our current findings show that the decrease in minute diaphragm activity (analog to minute ventilation) was entirely due to changes in respiratory timing such as prolongation of expiratory duration (Fig. 3). Microinjection of SK and F-86-466 (an alpha 2-adrenergic receptor blocker) into the rostral ventrolateral medulla prior to stimulation of the caudal ventrolateral medulla significantly reduced changes in expiratory duration but had little effect on arterial pressure responses, indicating that activation of the A1 noradrenergic cell group induces prolongation of expiratory duration via activation of alpha 2-adrenergic receptors (Tolentino-Silva et al., 2000). Studies on the role of noradrenaline in transmission of signals from activated chemosensory cells to the respiratory network have been hampered by a lack of specific pharmacological probes for alpha 2-adrenergic receptor subtypes. These difficulties can be circumvented using genetically engineered mice with a functional deficit of a specific receptor subtype, such as mice with an inactive mutant form of the alpha 2A-adrenergic receptor subtype (MacMillan et al., 1996). Using functionally alpha 2A-adrenergic receptor deficient mice, we studied the role of the noradrenaline-alpha 2A-adrenergic signal transduction pathway in CO2-induced changes in respiratory drive by examining the ventilatory response to hypercapnic loading. Exposure to 7% CO2 in O2 caused a significantly larger increase in minute ventilation in functionally alpha 2A-adrenergic receptor deficient mice than in control, C57Bl6 wild type mice (Fig. 4). The increases in tidal volume were identical, whereas the increase in breathing frequency was higher in mice with the

mutant form of the alpha 2A-adrenergic receptor than in control animals. These findings indicate that the release of noradrenaline and activation of functional alpha 2A-adrenergic receptor subtypes play a modulatory role in the frequency response to hypercapnia. This is in agreement with data showing that hypercapnia-induced long-term depression of respiratory activity requires alpha 2adrenergic receptors (Bach and Mitchell, 1998). Similarly, it was found that a decline in breathing frequency in rats after exposure to hypoxic stress is mediated mainly via activation of alpha 2adrenoreceptors (Bach et al., 1999).

3.2.5. Noradrenergic chemosensiti6e neurons and beha6ioral state Breathing is impaired by the loss of wakefulness that accompanies sleep. The neural mechanisms that cause state-dependent changes in respiratory control remain poorly understood. It has been shown that cholinergic mechanisms in the medial pontine reticular formation cause state-dependent reductions in normocapnic minute ventilation and in the ventilatory response to hypercapnia (Lydic et al., 1991). Activation of sleep promoting sites also reduces the discharge of monoamine-containing neurons, including noradrenaline-containing chemosensory neurons in the locus coeruleus, which play an integral role in generating a critical level of thalamocortical activation necessary for arousal. Chemical respiratory stimuli can induce waking from sleep, but until now the specific mechanisms involved have not been established. Recent evidence suggests that both rapid and slow increases in end tidal CO2 lead to arousal in humans in the absence of changes in respiratory mechanoreceptor activity (Ayas et al., 2000). This may occur via activation of locus coeruleus noradrenaline-containing neurons that overcome sleep related inhibitory inputs, and send parallel signals to the medullary respiratory network for adjustment of ventilatory drive, and to CNS structures responsible for arousal. The coeruleo-cortical and cholinergic systems are implicated in different forms of behavioral arousal. Recently, it was shown that the number of brainstem adrenaline and noradrenaline neu-

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Fig. 3. (A), coronal section showing the site of microinjection of 10 nl of 2% Evans Blue. (B), Tyrosine hydroxylase-containing neurons in the mouse caudal ventrolateral medulla (CVLM, A1 cell group). (C), recording of the integrated electromyographic activity of the diaphragm [ DEMG] and of pulsatile blood pressure (BP) following administration of 10 nl of a 50 mmol solution of glutamate into the CVLM.

rons is decreased in Sudden Infant Death Syndrome (SIDS); this decrease is closely correlated with brainstem gliosis (Obonai et al., 1998). Furthermore, in SIDS victims, there is a deficit in catecholaminergic innervation of the diencephalon and basal ganglia, suggesting impair-

ment of the development of the neuronal connection from the brainstem (Ozawa et al., 1999). Hence, catecholaminergic changes may underlie sleep related alterations in respiratory and cardiovascular control, and may cause failure to arouse during prolonged sleep apnea, with a con-

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sequent sudden, unexpected death. This concept is supported by findings in infants of substanceabusing mothers. Substances such as cocaine affect the development of monoaminergic pathways (Mayes, 1999). These infants have an increased risk of SIDS, manifest abnormal sleeping ventilatory patterns, and require a significantly longer exposure to hypercapnia before arousal (Ward et al., 1992). The inability to arouse from sleep in response to asphyxia may also be the underlying abnormality explaining numerous other behavioral deficits reported in infant groups with symptomatic apneas. Furthermore, in infants with chronic oxygenation abnormalities and victims of sudden, unexpected infant death, decreased muscarinic binding in the arcuate nucleus has been shown, which might contribute to a failure of responses to cardiopulmonary changes during sleep (Kinney et al., 1995). An arousal response deficit to asphyxia may be a critically important and fundamental pathophysiological component of changes that lead to the occurrence of sudden death during sleep (Hunt, 1989). We assume that impaired cholinergic brainstem mechanisms in these infants lead to a decline in activation of locus coeruleus noradrenaline-containing neurons and diminished arousal response to life threatening events, such as prolonged apneas. Hypercapnic arousal responses are altered in a variety of respiratory disorders, including Prader –Willi Syndrome (PWS), a disease characterized by a number of abnormalities of hypothalamic function, such as hyperphagia, short

stature, temperature instability, hypogonadotropic hypogonadism, and neurosecretory growth hormone deficiency. Patients with PWS are reported to have sleep-disordered breathing, a blunted hypercapnic ventilatory response, and a significantly higher arousal threshold to hypercapnia compared with the controls, which may further contribute to sleep-disordered breathing in PWS patients (Livingston et al., 1995). Structural alterations in the hypothalamic paraventricular nucleus are observed in these patients (Swaab et al., 1995), suggesting that a deficit in noradrenaline-containing neurons-PVN pathways may contribute to an arousal deficit and CO2 retention. Congenital central hypoventilation syndrome (CCHS, Ondine’s curse) is generally thought to be due to insensitivity of the central chemoreceptors to carbon dioxide. Children with CCHS have absent ventilatory responses to both hypercapnia and hypoxia, suggesting either abnormal central and peripheral chemoreceptor function or abnormal central integration of chemoreceptor input. Since ventilatory and arousal responses to respiratory stimuli are distinct from each other, if children with CCHS have complete chemoreceptor dysfunction, one would predict that both ventilatory and arousal responses to respiratory stimuli would be abnormal. However, if they have abnormal central integration of chemoreceptor input for ventilation, they may still be aroused by respiratory stimuli despite the absence of a ventilatory response. Children or adults with CCHS who

Fig. 4. Percent changes in tidal volume (DVT) and in frequency (Df) in C7Bl6 wild type (open circle) and 2-adrenergic receptor functionally deficit mice (closed circle). *PB 0.05 between mouse strains.

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have little or no arousal sensitivity to hypercapnic stress may have an altered catecholaminergic system, but subjects with CCHS that are aroused by hypercapnia may possess some central nonrespiratory-related chemoreceptor functions (Marcus et al., 1991). In addition, children with obstructive sleep apnea syndrome have a blunted arousal response to hypercapnia (Marcus et al., 1998). We assume that in these individuals, anatomical or functional alterations in noradrenaline contaning cells may exist.

4. Serotonergic neurons There is increasing evidence suggesting that midline neurons play a role in the ventilatory response to hypercapnia (see Nattie, and Richerson, in this volume). A subpopulation of these cells (25%) expresses serotonergic traits (Mason, 1997) and is involved in diverse physiological functions. A regulatory role is witnessed by findings that neurons of this system are localized in the medial aspect of the brainstem, the most primitive portion of the CNS; they develop in early ontogeny, and are largely conserved. Furthermore, their axonal projections and terminal arborizations invade almost the entire neuraxis, from the most caudal segments of the spinal cord to the frontal cortex (Jacobs and Fornal, 1999), as schematically presented in Fig. 5.

4.1. Chemosensory traits in serotonergic neurons Using c-Fos protein as a marker of cell activation for increased concentrations of CO2 or H+, we found that hypercapnic loading activates a subpopulation of serotonin-containing cells within caudal midline nuclei. Activated serotonergic cells were observed in the following serotonergic groups: raphe pallidus (B1 group) and its lateral extension (parapyramidal serotonergic cells), raphe obscurus (B2 group), and raphe magnus (B3 group). Fos-labeled cells were also seen in the dorsal raphe nucleus (B 7). An example of CO2induced c-Fos expression in serotonergic neurons of the caudal raphe is shown in Fig. 5.

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4.2. Serotonergic chemosensiti6e neurons and central regulation of respiration Observations that a subpopulation of serotonergic neurons is activated by hypercapnic loading indicate that these neurons could be chemosensitive cells, which is in agreement with data showing that raphe neurons in vitro exhibit CO2 dependent changes in membrane potential and in firing rate (Richerson, 1995; Wang et al., 1998). The functional significance of these findings could be derived from neuroanatomical and physiological experiments. Namely, projections emanating from serotonin-containing neurons of the caudal raphe nuclei to the NTS, ventrolateral medulla, and the spinal cord have been demonstrated (Loewy and McKellar, 1981; Holtman et al., 1990; Sasek et al., 1990). Furthermore, serotonin-immunoreactive boutons synapse with phrenic motoneurons, respiratory related neurons of the ventral and dorsal respiratory groups (Holtman, 1988), and motoneurons of cranial nerves, including airway-related vagal preganglionic neurons (Haxhiu et al., 1993). Focal acidification of raphe nuclei by microinjection of acetalozamide in anesthetized, vagotomized animals increases the amplitude of the integrated phrenic moving average (Bernard et al., 1996). Extensive chemical lesioning of midline neurons in anesthetized and decerebrate piglets reduces the response of the phrenic and hypoglossal nerves to progressive hypercapnia with hypoglossal nerve activity being substantially more affected than phrenic nerve discharge (Dreshaj et al., 1998). Chemical stimulation of the raphe pallidus produces an increase in respiratory output, preferentially to the genioglossus muscle but inhibits cholinergic outflow to the airways (Haxhiu et al., 1998). It is conceivable that a decrease in serotonergic activity may lead to an imbalance between the activity of upper airway dilating and chest wall pumping muscles that may lead to upper airway obstruction, and withdrawal of inhibitory inputs to cholinergic neurons, causing bronchoconstriction. Conceivably, the effects of midline neurons on the ventilatory response to CO2 could be partly mediated via release of other transmitters such as substance P. Recently, we

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Fig. 5. (A), coronal section indicates the site of 5-HT-containing midline neurons that expressed c-Fos when exposed to CO2. (B), Schematic drawing shows distribution of serotonergic neurons on the sagittal plane of the rat brain. Rpa, raphe pallidus; TH, thalamus; H, hypothalamus; B1-9 serotonin cell groups. (C), 5-Hydroxytryptamine (5-HT) and Fos expression in midline neurons of the Rpa following exposure to hypercapnia. Closed arrows indicate a serotonergic neuron expressing both 5HT (left panel, fluorescent) and Fos (right panel, brightfield). Open arrow shows a 5-HT-containing neuron that does not express Fos. Asterisk indicates a c-Fos positive and 5-HT negative neuron. Scale bar =20 mm.

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showed that substance P-containing neurons within raphe nuclei are activated by hypercapnic loading (Pete et al., 2000).

4.3. Serotonergic chemosensiti6e neurons and beha6ioral state It has been shown that the activity of serotonincontaining neurons is decreased during sleep. Entering slow wave sleep (non-REM), neuronal activity slows to approximately 50% of the quiet waking level and loses its regularity. Finally, during rapid eye movement (REM) sleep, most serotonin neurons become nearly quiescent, via activation of GABAergic input (Jacobs and Fornal, 1999). Furthermore, serotonin levels in the hypoglossal nucleus region and hypoglossal nerve activity are reduced during carbachol-induced REM sleep (Kubin et al., 1994). Stimulation of serotonin-containing neurons is associated with inhibition of airway-related vagal preganglionic cells, but excitation of hypoglossal motoneurons. With in situ voltametry, we observed a release of serotonin and a decrease in airway smooth muscle tone following stimulation of midline neurons. These actions were diminished by blockade of serotonergic receptors (Haxhiu et al., 1998). Furthermore, the data suggest that behavioral state related changes in serotonergic inputs may explain oscillations in the patency of upper airways and in airway smooth muscle tone across the wake/sleep/ arousal cycle. During sleep, diminished activity of serotonin neurons may lead to a decrease in airway patency. Entering sleep and in non-REM or REM sleep, respiratory drive to upper airway dilating muscles preferentially is decreased (for review see Dempsey et al., 1996). In humans, this may lead to sleep obstructive apnea, hypopnea, and autonomic stress (Cherniack, 1981; Dempsey et al., 1996). In addition, during sleep, an increase in cholinergic outflow to the tracheobronchial system is observed, which is greatly amplified in disease states such as bronchial asthma (Ballard, 1999; Lewis, 1999). Taken together, these findings indicate that sleep-producing neurons may utilize specific neuronal networks that inhibit the activity of serotonergic neurons, influencing upper airway patency and cholinergic outflow to the airways.

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Serotonin exerts modulatory effects in many brain nuclei involved in regulation of motor, sensory, and behavioral state responses. Diverse actions of serotonin are due to the existence of at least 15 different 5-hydroxytryptamine (5-HT) receptor subtypes. For example, activation of 5HT2A receptors induces cell depolarization, while activation of 5-HT1A has an opposite effect (Andrade, 1998). Hypoglossal motoneurons and motor cells innervating pharyngeal and laryngeal muscles express 5-HT2A receptors that provide tonic excitatory inputs in the awake state (Fay and Kubin, 2000). During sleep, particularly entering REM sleep, the activity of serotonergic receptors reaches its nadir. Microinjection of 5HT into the hypoglossal nucleus can significantly attenuate the REM sleep-like suppression of XII nerve activity, in part, by substituting for decreased endogenous 5%-HT in the XII nucleus (Kubin et al., 1994) The 5-HT1A receptors are expressed throughout the CNS (Andrade, 1998), including airway-related vagal preganglionic neurons (Haxhiu et al., 1998). In most of these nuclei, the receptor functions postsynaptically, responding to serotonin release from raphe nuclei projections. The most obvious effect of serotonin, which is mediated by 5-HT1A receptors, is membrane hyperpolarization. This membrane-delimited signaling mechanism involves a G protein of the Gi/Go family, opening of inwardly rectifying potassium channels, and inhibition of calcium currents (Andrade, 1998). Alterations in serotonergic pathways may cause failure of homeostatic responses to life-threatening challenges (e.g. asphyxia, hypercapnia) during sleep (Panigrahy et al., 2000). In children or adults, altered serotonergic pathways may contribute to the severity of obstructive sleep apnea syndrome and nocturnal asthma.

5. Histaminergic neurons Histaminergic neurons in the adult vertebrate brain are confined to the posterior hypothalamic area. Scattered groups of these neurons are referred to as the tuberomammillary nucleus. These cells give rise to widespread projections extending

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via the basal forebrain to the cerebral cortex, as well as to the thalamus and pontomesencephalic tegmentum. The morphological features of these neurons suggest that the histaminergic system acts as a regulatory network for whole-brain activity. Indeed, this amine regulates hormonal functions, sleep, food intake, thermoregulation and locomotor changes (Panula et al., 2000). The histaminergic system is also involved in the control of arousal. In addition to their ascending axons, these neurons send heavy descending inputs to the mesopontine tegmentum, which plays a key role in cortical activation (Lin et al., 1996), and to NTS subnuclei (Airaksinen and Panula, 1988; Panula et al., 1989), which are known to play an important role in cardiovascular control.

5.1. Chemosensory traits in histaminergic neurons We assumed that histaminergic neurons may partly mediate changes in behavioral state in response to changes in CO2 and H+ within the brain, and the physiological effects on the respiratory and cardiovascular systems could be related to behavioral state and arousal. We tested our hypothesis by examining c-Fos expression in histamine-containing neurons. The results showed that a subset of histamine-containing cells is activated by hypercapnic loading. An example is shown in Fig. 6.

5.2. Histaminergic chemosensiti6e neurons and central regulation of respiration The functional significance of chemosensory traits in histaminergic neurons is not known. However, it is expected that activation of histamine-containing cells by an increase in CO2 and/or H+ may affect central respiratory drive, via activation of NTS neurons which are heavily innervated by histaminergic fibers (Airaksinen and Panula, 1988; Panula et al., 1989). Furthermore, a pharmacological study of respiratory rhythm in isolated brainstem-spinal cord preparations of newborn rats showed that histamine

increases the frequency of spontaneous periodic depolarization (Murakoshi et al., 1985), acting via H-1 receptors. There is pharmacological evidence suggesting that most of the centrally acting drugs, i.e. benzodiazapines, barbiturates, and ethanol, share an antihistaminergic effect (Pollard et al., 1973; Sawynok et al., 2001). These substances decrease central histaminergic transmission acting pre-(decrease of the turnover rate of histamine) or postsynaptically (H1-receptor blockade) that could contribute to sedation and sleep related disturbances, such as the occurrence of complete obstructive sleep apnea in heavy snorers (Guilleminault, 1990). Sleep related disturbances following administration of sedatives could be a consequence of preferential inhibition of hypoglossal nerve activity and upper airway dilating muscles (Haxhiu et al., 1986, 1992b).

5.3. Histaminegic chemosensiti6e neurons and beha6ioral state It is well established that histaminergic neurons of the tuberomamillary nucleus are involved in behavioral state regulation. These cells express the highest discharge rate during waking and are virtually silent during nonREM and REM sleep (Vanni-Mercier et al., 1984; Lin et al., 1996). Activation of histamine-containing neurons inhibits basal forebrain-preoptic cells involved in the generation of nonREM sleep. Conversely, blockade of histamine synthesis in this region promotes sleep, and decreases wakefulness. Hence, it is expected that alterations in central histaminergic control may contribute to an arousal deficit upon exposure to hypercapnia. The link between sleep-related respiratory disorders in infants and maternal smoking during pregnancy (Rintahaka and Hirvonen, 1986; Kraus et al., 1989) could be partly due to interference of nicotine with the development of histaminergic system. Nicotine inhibits histamine-N-methyltransferase (Gairola et al., 1988), leading to altered histaminergic transmission, arousal deficit, and possibly SIDS.

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Fig. 6. (A), the coronal section shows the site of histidine decarboxylase-containing neurons that expressed c-Fos by hypercapnic stress. (B), the drawing represents the distribution of histaminergic neurons on the sagittal plane of the rat brain. VTM, ventral tuberomamillary histamine cell group; TH, thalamus; H, hypothalamus; TM, tuberomammilary nucleus. C: Histidine decarboxylase (HD) and Fos immunoreactivity of VTM neurons in a rat exposed to hypercapnia. Closed arrows indicate a histaminergic neuron expressing both histidine decarboxylase (left panel, fluorescent) and Fos (right panel, brightfield). Open arrow shows a histidine decarboxylase-containing neuron that does not express Fos. Asterisk indicates a c-Fos positive and histidine decarboxylase negative neuron. Scale bar =20 mm.

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6. Summary In conclusion, CO2 exposure leads to activation of discrete cell groups along the neuraxis, including subsets of cells belonging to monoaminergic systems, noradrenaline-; serotonin-; and histamine-containing neurons. In part, their modulatory role in the respiratory response to hypercapnia could be related to their behavioral state control function. Activation of monoaminergic neurons by an increase in the concentration of CO2 and/or H+ will facilitate respiratory related motor activity, particularly of upper airway dilating muscles. In addition, these neurons coordinate sympathetic and parasympathetic tone to visceral organs, and participate in adjustments of blood flow with the level of motor activity. Any deficit in CO2 chemosensitivity of a network composed of inter-related monoaminergic nuclei might lead to disfacilitation of motor outputs and to failure of neuroendocrine and homeostatic responses to life-threatening challenges (e.g. asphyxia, hypercapnia) during sleep. In children and adults, chemosensory dysfunction of monoaminergic neurons may contribute to the pathophysiology of the obstructive sleep apnea syndrome and to worsening of nocturnal asthma.

Acknowledgements This work was supported by NIH grants HL50527 and I U 54 NS 39407.

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