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Anatomical substrates for baroreflex sympathoinhibition in the rat
Brain Research Bulletin, Vol. 51, No. 2, pp. 107–110, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(99)00233-6
Proceedings of the Experimental Biology ’98 Symposium
Anatomical substrates for baroreflex sympathoinhibition in the rat Sue A. Aicher,* Teresa A. Milner, Virginia M. Pickel and Donald J. Reis Department of Neurology and Neuroscience, Division of Neurobiology, Weill Medical College of Cornell University, New York, NY, USA [Received 16 September 1999] ABSTRACT. The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS3 CVL3 RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks. © 2000 Elsevier Science Inc.
excitation of cardiovagal motor neurons. The sympathoinhibitory component is organized within the medulla oblongata and thoracic spinal cord, but can be modulated by supramedullary inputs to individual neurons within the reflex pathway. Three medullary nuclei subserve the baroreflex: (1) the nuclei of the solitary tract (NTS); (2) a functionally defined subregion of the caudal ventrolateral medulla (CVL); and (3) the rostral ventrolateral medullary reticular nuclei (RVL) containing tonically active sympathoexcitatory neurons. This paper will discuss the synaptic connectivity between these medullary regions and spinal sympathetic preganglionic neurons which mediate the reflex reduction in sympathetic outflow produced by stimulation of aortic and carotid baroreceptors. Nucleus of the Solitary Tract: First Synapse in the Baroreflex Pathway Baroreceptor afferents terminate primarily in the intermediate portion of the NTS subjacent to the area postrema in the dorsal medulla [11] (see Fig. 1). Here they form asymmetric (excitatory) synaptic contacts with second-order neurons in the NTS [6,45]. Physiological and anatomical findings indicate that baroreceptor afferents contain and release glutamate [44], as well as substance P and other neuropeptides [16,21,43]. Pharmacological analyses indicate that the excitation of NTS neurons produced by stimulation of baroreceptor afferents is mediated and/or modulated by both NMDA- and AMPA-type glutamate receptors [8,9,19,36,46]. In agreement are our observations that vagal afferents terminating within the intermediate NTS, contain NMDA-type glutamate receptors at both pre- and postsynaptic sites [6]. Since the vagal afferents were identified by injections of an anterograde tracer into the nodose ganglion, they would include cardiopulmonary and baroreceptor afferents that have their cell bodies in the nodose and project to the intermediate NTS. The postsynaptic NMDA receptors are often at extrasynaptic plasma membrane sites, and thereby may modulate rather than initiate postsynaptic activity. The pres-
KEY WORDS: Cardiovascular, Blood pressure, Nucleus tractus solitarius, Caudal ventrolateral medulla, Rostral ventrolateral medulla, Ultrastructure.
The arterial baroreceptor reflex is elicited by distortion, usually due to the arterial pulse wave, of stretch-sensitive receptors located in specialized regions of the carotid arteries and the aortic arch which are innervated by branches of the IX and Xth cranial nerves [15,41]. The principle cardiovascular responses to stimulation of these stretch receptors are a fall in arterial pressure and bradycardia. Hypotension results from inhibition of the tonic activity of spinal sympathetic preganglionic neurons and bradycardia from
* Address for correspondence: Sue A. Aicher, Department of Neurology and Neuroscience, Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, NY 10021, USA. Fax: ⫹1-212-988-3672; E-mail: [email protected]
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FIG. 1. Schematic summary of medullary regions known (left side) and suspected (right side) of being involved in baroreceptor reflex-induced sympathoinhibiton viewed from the dorsal aspect of the medulla oblongata (see text for details). Abbreviations: AP, area postrema; CVL, caudal ventrolateral medulla; GiDA, gigantocellular depressor area; IML, interomediolateral cell column; NTS, nuclei of the solitary tract; RVL, rostral ventrolateral medulla.
ence of presynaptic NMDA receptors suggests that these receptors may also regulate release of glutamate and possibly other neurotransmitters from vagal afferents. Electrophysiological studies indicate that distinct populations of NTS neurons receive monosynaptic or polysynaptic input from baroreceptor afferents [7,39,41], however, it is not certain whether neurons in NTS are segregated with regard to their efferent output. While NTS neurons project to many brainstem and forebrain regions which regulate sympathetic outflow, only two of these targets are essential for the expression of the baroreceptor reflex: the caudal ventrolateral medulla (CVL) [14] and the rostral ventrolateral medulla (RVL) [37]. Lesions of NTS, CVL, or RVL eliminate the baroreceptor reflex and lead to fulminating hypertension (NTS or CVL) or severe hypotension (RVL), thus all three regions are critical for the tonic and reflex regulation of sympathetic outflow. Other brainstem regions which regulate sympathetic outflow, such as the caudal raphe nuclei, are not necessary for the expression of the baroreceptor reflex [2,34]. The Caudal Ventrolateral Medulla The CVL has been functionally defined as a tonicaly active sympathoinhibitory vasodepressor region of the medullary reticular formation located adjacent to, but independent from, the A1 noradrenergic cell group [1,10]. CVL neurons are interneurons in the baroreceptor reflex arc [15,22,41] and are interposed between NTS neurons, which excite them, and vasomotor neurons of RVL, which they inhibit. Electrophysiological characteristics of CVL neurons are consistent with their purported role in the intramedullary reflex arc: they are tonically active, they discharge in relation to the cardiac cycle, they are excited by stimulation of baroreceptor afferents, and they are antidromically activated from RVL [22]. Anatomically, CVL neurons identified as projecting to the RVL are mono-
AICHER ET AL. synaptically innervated by NTS efferent terminals, which make primarily asymmetric (excitatory) synapses [1] and may contain glutamate [18,32]. There are however some complexities. First, CVL “baroreceptor interneurons,” i.e., neurons which are antidromically excited from RVL and orthodromically activated by stimulation of baroreceptors are not confined to the ventrolateral medulla, but extend dorsally from just below the NTS to near the medullary surface in a strip which may correspond to the lateral tegmental field [12]. There is a comparable distribution of neurons retrogradely labeled from the RVL [1]. Baroreceptor interneurons in this strip can be subdivided on the basis of their response latencies to electrical stimulation of baroreceptor afferents [22]. However, neurons with short and long latencies were randomly distributed throughout the field, indicating that there is not a simple incremental progression of a signal from NTS along a pathway projection ventrally to the base of the medulla. Rather, the data indicate that either populations of baroreceptor afferent fibers conduct at different latencies (i.e., myelinated or unmyelinated axons) [see Seagard, this issue] or that NTS neurons with monosynaptic and polysynaptic latencies to baroreceptor stimulation [39] project to distinct populations of CVL neurons. Pharmacological studies suggest that inhibition of RVL neurons by CVL input is mediated by the release of GABA. Recent morphological evidence indicates that CVL barosensitive neurons are GABAergic [10,31] and project directly to neurons in the RVL. CVL efferent terminals form symmetric (inhibitory) synaptic contacts with RVL reticulospinal neurons [23] and with RVL adrenergic neurons [5]. These synapses were often located on the somata and proximal dendrites of RVL neurons, suggesting that activation of this synapse can potently inhibit RVL neuronal activity [5]. Rostral Ventrolateral Medulla Many anatomical and physiological studies have demonstrated that barosensitive RVL neurons, including C1 neurons, project to sympathetic preganglionic neurons in the thoracic spinal cord and are a principal effector of the baroreceptor reflex [33,35,38,42]. Recent studies using juxtacellular labeling methods support the notion that many of these reticulospinal neurons are C1 adrenergic neurons [40]. However, lower estimates have been obtained using intracellular recording methods [25]. All of these studies support the notion that the RVL contains barosensitive neurons that project to the sympathetic motor neurons in thoracic spinal cord. While the GABAergic projection from the CVL to RVL neurons appears to mediate the baroreceptor reflex, adrenergic neurons in the C1 area are also monosynaptically innervated from the NTS [5]. This direct NTS-RVL projection is unlikely to be a component of the baroreflex pathway since the synaptic morphology of the projection is consistent with it being excitatory and NTS neurons projecting to the RVL do not express c-fos when baroreceptors are stimulated by elevating arterial pressure [10]. More likely, the pathway from NTS to RVL represents the anatomical substrate for the sympathetic excitation elicited by activation of arterial chemoreceptors [5,20] whose afferents, also carried in the IXth and Xth cranial nerves, terminate in similar regions of the NTS as baroreceptor afferents. Consistent with the hypothesis that the direct NTS-RVL pathway mediates the pressor response to chemoreceptor stimulation is the fact that chemoreceptor stimulation excites RVL neurons and bilateral lesions of the RVL abolish the chemoreceptor reflex.
ANATOMY OF BAROREFLEX SYMPATHOINHIBITION Sympathetic Preganglionic Neurons (SPNs): Final Output for Baroreflex Sympathoinhibition Stimulation of baroreceptor afferents reduces the activity of reticulospinal sympathoexcitatory neurons in the RVL, thereby withdrawing their tonic excitatory input to sympathetic preganglionic neurons. The morphology of the projection from the RVL to SPNs in the spinal cord is consistent with this being an excitatory relay. Both anterogradely labeled RVL efferents [33] and adrenergic terminals [29] form asymmetric or excitatory type contacts with dendrites in the intermediolateral cell column (IML). While the majority of the RVL reticulospinal neurons are phenotypically adrenergic, it appears that the principal excitatory neurotransmitter released from RVL neurons is glutamate. This is supported by findings that: (1) many RVL efferent terminals contain glutamate [33]; (2) glutamate is co-localized with catecholamine synthetic enzymes in RVL neurons [30]; and (3) excitation of SPNs by RVL stimulation can be blocked by local application of glutamate antagonists [17]. Many RVL efferent and adrenergic terminals in the spinal cord contact dendrites that contain NMDA-type glutamate receptors [Aicher, personal observation], supporting the idea that at least some of the sympathoexcitation from the RVL to preganglionic neurons is mediated through activation of NMDA receptors [18]. Stimulation of baroreceptor afferents reduces the activity of this tonic sympathoexcitatory pathway and lowers sympathetic output. Direct Reticulospinal Regulation of Baroreflex Activity: The Gigantocellular Depressor Area (GiDA) As discussed above, the NTS and CVL are both vasodepressor regions of the medulla oblongata which are essential relays within the baroreceptor reflex pathway and both mediate sympathoinhibition indirectly through inhibition of sympathoexcitatory neurons in the RVL. Some baroreceptor-mediated inhibition of SPNs however may be directly relayed over medullo-spinal pathways [13, 24,26]. Barosensitive neurons have been described in the lamina 10 region of the spinal cord but these are thought to be inhibitory interneurons [28] and may provide some of the GABAergic input to SPNs. GABAergic synapses are thought to represent as much as 50% of the synaptic input to SPNs [26] and some of this input may arise from reticulospinal projections [13,24,26]. We have recently described a region located within the medullary gigantocellular reticular formation, the GiDA, which may be a direct source of tonic inhibition to SPNs in the thoracic spinal cord. Chemical stimulation of the GiDA evokes depressor responses [4] and neurons in this area also generate tonic sympathoinhibition since local blockade of neuronal functions with exocitotoxins (e.g., kainic acid) elevates arterial pressure and sympathetic nerve activity and results in fulminating hypertension [2]. The tonic inhibition of sympathetic activity generated from GiDA differs from that generated from NTS and CVL in that it does not result from inhibiting RVL activity [2], but rather appears to be relayed over a direct monosynaptic projection to SPNs [3]. Bilateral lesions of the GiDA eliminate the baroreceptor reflex [2]. However, the absence of direct projections from the NTS to the GiDA indicates that this area is not simply a parallel pathway for baroreceptor-induced sympathoinhibition [2]. The blockade of the baroreflex produced by GiDA lesions appears to result from a withdrawal of tonic inhibition from the sympathetic preganglionic neurons, i.e., profound “disinhibition.” As a result of this disinhibition, SPNs can no longer be silenced by baroreceptor-induced withdrawal of excitatory drive from RVL. This phenomenon may be analogous to the blunting of baroreflexes seen during the hypothalamic defense response [15], which is thought to be mediated by GABAergic projections from the hypothalamus to NTS.
109 CONCLUSIONS The present data indicate that at least three brainstem regions, the NTS, the CVL, and the RVL, are necessary for normal expression of both tonic sympathetic tone and baroreflex modulation of sympathetic outflow. Current models indicate that these regions are connected in a serial fashion and that a direct projection from the RVL to sympathetic preganglionic neurons in the spinal cord is the final central substrate for baroreflex inhibition of sympathetic tone. However, there is much to be understood about the regulation of sympathetic activity. Neurons within the NTS, CVL, RVL, and IML all receive convergent excitatory and inhibitory signals initiated by physiological events and from other brain regions, including: the A5 cell group, the locus coeruleus, parabrachial nuclei, hypothalamus, amygdala, and cortex [15,27,41]. Therefore, the excitability of neurons in the network may be influenced not only by baroreceptor afferent activity but by other biological signals. This would suggest that projections which provide synaptic input to any cell in the reflex pathway may alter the expression of the reflex, as illustrated by the direct projection from the gigantocellular reticular formation to SPNs in the spinal cord. The connectivity and role of the GiDA in integration of autonomic function remain to be clarified, but reminds us that the anatomical substrates for this important autonomic reflex are likely to be multiple, parallel, and redundant. Also remaining to be resolved are the roles individual neurotransmitters which are prominent in the baroreflex pathway, including monoamines and neuropeptides which appear to be released by exocytosis from large dense core vesicles to affect the presynaptic release, as well as postsynaptic responses to, other neurotransmitters including glutamate. ACKNOWLEDGEMENTS
This work was supported by grants from NIH (HL18974 and HL56301) and by an Established Investigator Award from the American Heart Association (SAA). The authors would like to thank Dr. David Averill for arranging the symposium at Experimental Biology 1998 that was the impetus for this review.
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29. Milner, T. A.; Morrison, S. F.; Abate, C.; Reis, D. J. Phenylethanolamine N-methyltransferase containing terminals synapse directly on sympathetic preganglionic neurons in the rat. Brain Res. 448:205–222; 1988. 30. Minson, J.; Pilowsky, P.; Llewellyn-Smith, I. J.; Kaneko, T.; Kapoor, V.; Chalmers, J. Glutamate in spinally projecting neurons of the rostral ventral medulla. Brain Res. 555:326 –331; 1991. 31. Minson, J. B.; Llewellyn-Smith, I. J.; Chalmers, J. P.; Pilowsky, P. M.; Arnolda, L. F. c-fos identifies GABA-synthesizing barosensitive neurons in caudal ventrolateral medulla. NeuroReport 8:3015–3021; 1997. 32. Miyawaki, T.; Suzuki, S.; Minson, J.; Arnolda, L.; Chalmers, J.; Llewellyn-Smith, I. J.; Pilowsky, P. Role of AMPA/kainate receptors in transmission of the sympathetic baroreflex in rat CVLM. Am. J. Physiol. Regul. Integr. Comp. Physiol. 272:R800 –R812; 1997. 33. Morrison, S. F.; Callaway, J.; Milner, T. A.; Reis, D. J. Rostral ventrolateral medulla: A source of the glutamatergic innervation of the sympathetic intermediolateral nucleus. Brain Res. 562:126 –135; 1991. 34. Morrison, S. F.; Gebber, G. L. Raphe neurons with sympatheticrelated activity: Baroreceptor responses and spinal connections. Am. J. Physiol. Regul. Integr. Comp. Physiol. 246:R338 –R348; 1984. 35. Morrison, S. F.; Reis, D. J. Responses of sympathetic preganglionic neurons to rostral ventrolateral medullary stimulation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 261:R1247–R1256; 1991. 36. Ohta, H.; Talman, W. T. Both NMDA and non-NMDA receptors in the NTS participate in the baroreceptor reflex in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 267:R1065–R1070; 1994. 37. Reis, D. J.; Ross, C. A.; Ruggiero, D. A.; Granata, A. R.; Joh, T. H. Role of adrenaline neurons of ventrolateral medulla (the C1 group) in the tonic and phasic control of arterial pressure. Clin. Exp. Hypertens. A6:221–241; 1984. 38. Ross, C. A.; Ruggiero, D. A.; Joh, T. H.; Park, D. H.; Reis, D. J. Rostral ventrolateral medulla: Selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons. J. Comp. Neurol. 228:168 –185; 1984. 39. Scheuer, D. A.; Zhang, J.; Toney, G. M.; Mifflin, S. W. Temporal processing of aortic nerve evoked activity in the nucleus of the solitary tract. J. Neurophysiol. 76:3750 –3757; 1996. 40. Schreihofer, A. M.; Guyenet, P. G. Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J. Comp. Neurol. 387:524 –536; 1997. 41. Spyer, K. M. Central nervous mechanisms contributing to cardiovascular control. J. Physiol. (Lond.) 474:119; 1994. 42. Sved, A. F.; Mancini, D. L.; Graham, J. C.; Schreihofer, A. M.; Hoffman, G. E. PNMT-containing neurons of the C1 cell group express c-fos in response to changes in baroreceptor input. Am. J. Physiol. Regul. Integr. Comp. Physiol. 266:R361–R367; 1994. 43. Sykes, R. M.; Spyer, K. M.; Izzo, P. N. Central distribution of substance P, calcitonin gene-related peptide and 5-hydroxytryptamine in vagal sensory afferents in the rat dorsal medulla. Neuroscience 59:195–210; 1994. 44. Talman, W. T.; Perrone, M. H.; Reis, D. J. Evidence for L-glutamate as the neurotransmitter of primary baroreceptor afferent nerve fibers. Science 209:813– 815; 1980. 45. Velley, L.; Milner, T. A.; Chan, J.; Morrison, S. F.; Pickel, V. M. Relationship of Met-enkephalin-like immunoreactivity to vagal afferents and motor dendrites in the nucleus of the solitary tract: A light and electron microscopic dual labeling study. Brain Res. 550:298 –312; 1991. 46. Zhang, J.; Mifflin, S. W. Influences of excitatory amino acid receptor agonists on nucleus of the solitary tract neurons receiving aortic depressor nerve inputs. J. Pharmacol. Exp. Ther. 282:639 – 647; 1997.
PII S0361-9230(99)00233-6
Proceedings of the Experimental Biology ’98 Symposium
Anatomical substrates for baroreflex sympathoinhibition in the rat Sue A. Aicher,* Teresa A. Milner, Virginia M. Pickel and Donald J. Reis Department of Neurology and Neuroscience, Division of Neurobiology, Weill Medical College of Cornell University, New York, NY, USA [Received 16 September 1999] ABSTRACT. The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS3 CVL3 RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks. © 2000 Elsevier Science Inc.
excitation of cardiovagal motor neurons. The sympathoinhibitory component is organized within the medulla oblongata and thoracic spinal cord, but can be modulated by supramedullary inputs to individual neurons within the reflex pathway. Three medullary nuclei subserve the baroreflex: (1) the nuclei of the solitary tract (NTS); (2) a functionally defined subregion of the caudal ventrolateral medulla (CVL); and (3) the rostral ventrolateral medullary reticular nuclei (RVL) containing tonically active sympathoexcitatory neurons. This paper will discuss the synaptic connectivity between these medullary regions and spinal sympathetic preganglionic neurons which mediate the reflex reduction in sympathetic outflow produced by stimulation of aortic and carotid baroreceptors. Nucleus of the Solitary Tract: First Synapse in the Baroreflex Pathway Baroreceptor afferents terminate primarily in the intermediate portion of the NTS subjacent to the area postrema in the dorsal medulla [11] (see Fig. 1). Here they form asymmetric (excitatory) synaptic contacts with second-order neurons in the NTS [6,45]. Physiological and anatomical findings indicate that baroreceptor afferents contain and release glutamate [44], as well as substance P and other neuropeptides [16,21,43]. Pharmacological analyses indicate that the excitation of NTS neurons produced by stimulation of baroreceptor afferents is mediated and/or modulated by both NMDA- and AMPA-type glutamate receptors [8,9,19,36,46]. In agreement are our observations that vagal afferents terminating within the intermediate NTS, contain NMDA-type glutamate receptors at both pre- and postsynaptic sites [6]. Since the vagal afferents were identified by injections of an anterograde tracer into the nodose ganglion, they would include cardiopulmonary and baroreceptor afferents that have their cell bodies in the nodose and project to the intermediate NTS. The postsynaptic NMDA receptors are often at extrasynaptic plasma membrane sites, and thereby may modulate rather than initiate postsynaptic activity. The pres-
KEY WORDS: Cardiovascular, Blood pressure, Nucleus tractus solitarius, Caudal ventrolateral medulla, Rostral ventrolateral medulla, Ultrastructure.
The arterial baroreceptor reflex is elicited by distortion, usually due to the arterial pulse wave, of stretch-sensitive receptors located in specialized regions of the carotid arteries and the aortic arch which are innervated by branches of the IX and Xth cranial nerves [15,41]. The principle cardiovascular responses to stimulation of these stretch receptors are a fall in arterial pressure and bradycardia. Hypotension results from inhibition of the tonic activity of spinal sympathetic preganglionic neurons and bradycardia from
* Address for correspondence: Sue A. Aicher, Department of Neurology and Neuroscience, Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, NY 10021, USA. Fax: ⫹1-212-988-3672; E-mail: [email protected]
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FIG. 1. Schematic summary of medullary regions known (left side) and suspected (right side) of being involved in baroreceptor reflex-induced sympathoinhibiton viewed from the dorsal aspect of the medulla oblongata (see text for details). Abbreviations: AP, area postrema; CVL, caudal ventrolateral medulla; GiDA, gigantocellular depressor area; IML, interomediolateral cell column; NTS, nuclei of the solitary tract; RVL, rostral ventrolateral medulla.
ence of presynaptic NMDA receptors suggests that these receptors may also regulate release of glutamate and possibly other neurotransmitters from vagal afferents. Electrophysiological studies indicate that distinct populations of NTS neurons receive monosynaptic or polysynaptic input from baroreceptor afferents [7,39,41], however, it is not certain whether neurons in NTS are segregated with regard to their efferent output. While NTS neurons project to many brainstem and forebrain regions which regulate sympathetic outflow, only two of these targets are essential for the expression of the baroreceptor reflex: the caudal ventrolateral medulla (CVL) [14] and the rostral ventrolateral medulla (RVL) [37]. Lesions of NTS, CVL, or RVL eliminate the baroreceptor reflex and lead to fulminating hypertension (NTS or CVL) or severe hypotension (RVL), thus all three regions are critical for the tonic and reflex regulation of sympathetic outflow. Other brainstem regions which regulate sympathetic outflow, such as the caudal raphe nuclei, are not necessary for the expression of the baroreceptor reflex [2,34]. The Caudal Ventrolateral Medulla The CVL has been functionally defined as a tonicaly active sympathoinhibitory vasodepressor region of the medullary reticular formation located adjacent to, but independent from, the A1 noradrenergic cell group [1,10]. CVL neurons are interneurons in the baroreceptor reflex arc [15,22,41] and are interposed between NTS neurons, which excite them, and vasomotor neurons of RVL, which they inhibit. Electrophysiological characteristics of CVL neurons are consistent with their purported role in the intramedullary reflex arc: they are tonically active, they discharge in relation to the cardiac cycle, they are excited by stimulation of baroreceptor afferents, and they are antidromically activated from RVL [22]. Anatomically, CVL neurons identified as projecting to the RVL are mono-
AICHER ET AL. synaptically innervated by NTS efferent terminals, which make primarily asymmetric (excitatory) synapses [1] and may contain glutamate [18,32]. There are however some complexities. First, CVL “baroreceptor interneurons,” i.e., neurons which are antidromically excited from RVL and orthodromically activated by stimulation of baroreceptors are not confined to the ventrolateral medulla, but extend dorsally from just below the NTS to near the medullary surface in a strip which may correspond to the lateral tegmental field [12]. There is a comparable distribution of neurons retrogradely labeled from the RVL [1]. Baroreceptor interneurons in this strip can be subdivided on the basis of their response latencies to electrical stimulation of baroreceptor afferents [22]. However, neurons with short and long latencies were randomly distributed throughout the field, indicating that there is not a simple incremental progression of a signal from NTS along a pathway projection ventrally to the base of the medulla. Rather, the data indicate that either populations of baroreceptor afferent fibers conduct at different latencies (i.e., myelinated or unmyelinated axons) [see Seagard, this issue] or that NTS neurons with monosynaptic and polysynaptic latencies to baroreceptor stimulation [39] project to distinct populations of CVL neurons. Pharmacological studies suggest that inhibition of RVL neurons by CVL input is mediated by the release of GABA. Recent morphological evidence indicates that CVL barosensitive neurons are GABAergic [10,31] and project directly to neurons in the RVL. CVL efferent terminals form symmetric (inhibitory) synaptic contacts with RVL reticulospinal neurons [23] and with RVL adrenergic neurons [5]. These synapses were often located on the somata and proximal dendrites of RVL neurons, suggesting that activation of this synapse can potently inhibit RVL neuronal activity [5]. Rostral Ventrolateral Medulla Many anatomical and physiological studies have demonstrated that barosensitive RVL neurons, including C1 neurons, project to sympathetic preganglionic neurons in the thoracic spinal cord and are a principal effector of the baroreceptor reflex [33,35,38,42]. Recent studies using juxtacellular labeling methods support the notion that many of these reticulospinal neurons are C1 adrenergic neurons [40]. However, lower estimates have been obtained using intracellular recording methods [25]. All of these studies support the notion that the RVL contains barosensitive neurons that project to the sympathetic motor neurons in thoracic spinal cord. While the GABAergic projection from the CVL to RVL neurons appears to mediate the baroreceptor reflex, adrenergic neurons in the C1 area are also monosynaptically innervated from the NTS [5]. This direct NTS-RVL projection is unlikely to be a component of the baroreflex pathway since the synaptic morphology of the projection is consistent with it being excitatory and NTS neurons projecting to the RVL do not express c-fos when baroreceptors are stimulated by elevating arterial pressure [10]. More likely, the pathway from NTS to RVL represents the anatomical substrate for the sympathetic excitation elicited by activation of arterial chemoreceptors [5,20] whose afferents, also carried in the IXth and Xth cranial nerves, terminate in similar regions of the NTS as baroreceptor afferents. Consistent with the hypothesis that the direct NTS-RVL pathway mediates the pressor response to chemoreceptor stimulation is the fact that chemoreceptor stimulation excites RVL neurons and bilateral lesions of the RVL abolish the chemoreceptor reflex.
ANATOMY OF BAROREFLEX SYMPATHOINHIBITION Sympathetic Preganglionic Neurons (SPNs): Final Output for Baroreflex Sympathoinhibition Stimulation of baroreceptor afferents reduces the activity of reticulospinal sympathoexcitatory neurons in the RVL, thereby withdrawing their tonic excitatory input to sympathetic preganglionic neurons. The morphology of the projection from the RVL to SPNs in the spinal cord is consistent with this being an excitatory relay. Both anterogradely labeled RVL efferents [33] and adrenergic terminals [29] form asymmetric or excitatory type contacts with dendrites in the intermediolateral cell column (IML). While the majority of the RVL reticulospinal neurons are phenotypically adrenergic, it appears that the principal excitatory neurotransmitter released from RVL neurons is glutamate. This is supported by findings that: (1) many RVL efferent terminals contain glutamate [33]; (2) glutamate is co-localized with catecholamine synthetic enzymes in RVL neurons [30]; and (3) excitation of SPNs by RVL stimulation can be blocked by local application of glutamate antagonists [17]. Many RVL efferent and adrenergic terminals in the spinal cord contact dendrites that contain NMDA-type glutamate receptors [Aicher, personal observation], supporting the idea that at least some of the sympathoexcitation from the RVL to preganglionic neurons is mediated through activation of NMDA receptors [18]. Stimulation of baroreceptor afferents reduces the activity of this tonic sympathoexcitatory pathway and lowers sympathetic output. Direct Reticulospinal Regulation of Baroreflex Activity: The Gigantocellular Depressor Area (GiDA) As discussed above, the NTS and CVL are both vasodepressor regions of the medulla oblongata which are essential relays within the baroreceptor reflex pathway and both mediate sympathoinhibition indirectly through inhibition of sympathoexcitatory neurons in the RVL. Some baroreceptor-mediated inhibition of SPNs however may be directly relayed over medullo-spinal pathways [13, 24,26]. Barosensitive neurons have been described in the lamina 10 region of the spinal cord but these are thought to be inhibitory interneurons [28] and may provide some of the GABAergic input to SPNs. GABAergic synapses are thought to represent as much as 50% of the synaptic input to SPNs [26] and some of this input may arise from reticulospinal projections [13,24,26]. We have recently described a region located within the medullary gigantocellular reticular formation, the GiDA, which may be a direct source of tonic inhibition to SPNs in the thoracic spinal cord. Chemical stimulation of the GiDA evokes depressor responses [4] and neurons in this area also generate tonic sympathoinhibition since local blockade of neuronal functions with exocitotoxins (e.g., kainic acid) elevates arterial pressure and sympathetic nerve activity and results in fulminating hypertension [2]. The tonic inhibition of sympathetic activity generated from GiDA differs from that generated from NTS and CVL in that it does not result from inhibiting RVL activity [2], but rather appears to be relayed over a direct monosynaptic projection to SPNs [3]. Bilateral lesions of the GiDA eliminate the baroreceptor reflex [2]. However, the absence of direct projections from the NTS to the GiDA indicates that this area is not simply a parallel pathway for baroreceptor-induced sympathoinhibition [2]. The blockade of the baroreflex produced by GiDA lesions appears to result from a withdrawal of tonic inhibition from the sympathetic preganglionic neurons, i.e., profound “disinhibition.” As a result of this disinhibition, SPNs can no longer be silenced by baroreceptor-induced withdrawal of excitatory drive from RVL. This phenomenon may be analogous to the blunting of baroreflexes seen during the hypothalamic defense response [15], which is thought to be mediated by GABAergic projections from the hypothalamus to NTS.
109 CONCLUSIONS The present data indicate that at least three brainstem regions, the NTS, the CVL, and the RVL, are necessary for normal expression of both tonic sympathetic tone and baroreflex modulation of sympathetic outflow. Current models indicate that these regions are connected in a serial fashion and that a direct projection from the RVL to sympathetic preganglionic neurons in the spinal cord is the final central substrate for baroreflex inhibition of sympathetic tone. However, there is much to be understood about the regulation of sympathetic activity. Neurons within the NTS, CVL, RVL, and IML all receive convergent excitatory and inhibitory signals initiated by physiological events and from other brain regions, including: the A5 cell group, the locus coeruleus, parabrachial nuclei, hypothalamus, amygdala, and cortex [15,27,41]. Therefore, the excitability of neurons in the network may be influenced not only by baroreceptor afferent activity but by other biological signals. This would suggest that projections which provide synaptic input to any cell in the reflex pathway may alter the expression of the reflex, as illustrated by the direct projection from the gigantocellular reticular formation to SPNs in the spinal cord. The connectivity and role of the GiDA in integration of autonomic function remain to be clarified, but reminds us that the anatomical substrates for this important autonomic reflex are likely to be multiple, parallel, and redundant. Also remaining to be resolved are the roles individual neurotransmitters which are prominent in the baroreflex pathway, including monoamines and neuropeptides which appear to be released by exocytosis from large dense core vesicles to affect the presynaptic release, as well as postsynaptic responses to, other neurotransmitters including glutamate. ACKNOWLEDGEMENTS
This work was supported by grants from NIH (HL18974 and HL56301) and by an Established Investigator Award from the American Heart Association (SAA). The authors would like to thank Dr. David Averill for arranging the symposium at Experimental Biology 1998 that was the impetus for this review.
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