Clinical and Experimental Pharmacology and Physiology (1992) 19,335-338
SHORT COMMUNICATION
AXONAL PROJECTIONS FROM RESPIRATORY CENTRES TOWARDS THE ROSTRAL VENTROLATERAL MEDULLA IN THE RAT Paul Pilowsky, Bruce Wakefield, Jane Minson, Ida Llewellyn-Smith and John Chalmers
Department of Medicine and Centre f o r Neuroscience, Flinders University of South Australia, Bedford Park, South Australia, Australia (Received 13 December 1991)
SUMMARY 1. Efferent pathways from brainstem respiratory centres towards bulbospinal tyrosine hydroxylase immunoreactive neurons were identified in the rat using a combination of electrophysiology, retrograde and anterograde tract-tracing, and immunohistochemistry. 2. Varicose axons originating from respiratory centres were found in close apposition to bulbospinal tyrosine hydroxylase immunoreactive neurons in the ventrolateral medulla. 3. These findings support the idea that respiratory rhythms in sympathetic nerves may be due to a synaptic connection between brainstem respiratory neurons and bulbospinal tyrosine hydroxylase immunoreactive neurons of the C1 cell group.
Key words: bulbospinal pathways, C1 cell group, cholera toxin B, Phuseolus vulgaris-leucoagglutinin, tract-tracing.
INTRODUCTION Respiratory rhythmicity is commonly observed in sympathetic nerve activity and blood pressure (Adrian et al. 1932). This rhythmicity is not simply due to the activation of pulmonary stretch receptors or other peripheral inputs as it persists in paralysed and mechanically ventilated animals where the central inspiratory activity is unrelated to ventilation (Preiss & Polosa 1977; Bainton et al. 1985; Gilbey el al. 1986). The site at which this respiratory rhythmicity is imposed on the sympathetic outflow is unknown at
present. An important source of tonic activity in sympathetic preganglionic neurons is a compact group of bulbospinal neurons in the rostra1 ventrolateral medulla (RVLM; Brown & Guyenet 1984; Minson et al. 1987; Guyenet et al. 1989). A number of studies have demonstrated that many bulbospinal sympathoexcitatory neurons in the RVLM have a respiratory modulation of their ongoing discharge rate (McAllen 1987; Haselton & Guyenet 1989a, b). Correlative physiological and histological studies have suggested
Correspondence: Dr Paul Pilowsky, Dept of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
P. Pilowsky et al.
336
that a subpopulation of these neurons is likely to contain tyrosine hydroxylase immunoreactivity (Minson et al. 1987). One possible source of this respiratory modulation is the ventral respiratory group (VRG) of neurons. These neurons are responsible for the generation of normal respiratory rhythms (Ezure 1990; Pilowsky et al. 1990). Several recent reports have provided anatomical support for the notion that there is a projection from respiratory neurons in the VRG towards the C1 cell group (Ellenberger & Feldman 1990; Pilowsky et al. 1990), but there have been no reports of close appositions between the axons of neurons arising from the VRG and the somata or dendrites of bulbospinal neurons that project to the intermediolateral cell column.
METHODS Experiments were performed on Wistar-Kyoto rats anaesthetized with pentobarbitone sodium (60mgl kg, i.p). The diaphragmatic electromyogram was used to monitor the phases of the respiratory cycle. The dorsal surface of the medulla was exposed for injections of Phaseolus vulgaris leucoagglutinin (PHAL; Vector Laboratories, CA, USA). The thoracic spinal cord was exposed for injections of cholera toxin B-gold (CTB-G; Gilt Products, Adelaide, SA, Australia). After allowing 3-7 days for transport of the injected tracers, the rats were fixed by intravascular perfusion with Zamboni's fixative containing 0.05% glutaraldehyde, and the brain and spinal cord were removed for histological processing. Pathways from the VRG to the RVLM were revealed by injection of PHAL into sites in the ventral medulla where multi-unit inspiratory (n = 4) or expiratory (n = 4) activity could be recorded. PHAL was injected by iontophoresisusing a Midgard CS4 constant current unit (Transkinetics, Canton, MA, USA). The location of PHAL-labelled axon terminals was detected using immunohjsio-chemistry and a diahinobenzidinenickel reaction that resulted in a black reaction product. Spinally-projectingtyrosine hydroxylase immunoreactive (TH-IR) neurons were detected by a combination of retrograde tracing with CTB-G and immunohistochemistry using an antityrosine hydroxylase antiserum (Eugene Tech., Allendale, NJ, USA). CTB-G was injected into the intermediolateral cell column of the thoracic spinal cord. CTB-G within neurons in the medulla was detected by silver intensification of the colloidal gold particles. TH-IR was detected by
immunohisto-chemistry using a diaminobenzidineimidazole reaction that resulted in a brown reaction product.
RESULTS Projections from the VRG (PHAL immunoreactivity) were visualized as black varicose fibres (Fig. 1). Silver intensified gold particles appeared as black puncta within the cytoplasm of neurons, while TH-IR was observed as an homogeneous brown reaction product in the cytoplasm and dendrites of neurons, but not in the nucleus. Within the ventral medulla, most PH AL-labelled axons arising from sites of multi-unit inspiratory or expiratory activity were observed in a compact region that was ventral to the nucleus ambiguus pars cornpacta, and dorsal to the C1 cell group, This area corresponds to the location of the ventral respiratory group. PHAL-labelled varicosities were also found in the same site on the contralateral side of the medulla. Several motor nuclei received an input from PHALlabelled varicosities, including the hypoglossal nucleus, the facial nucleus, the nucleus ambiguus pars compacts, the phrenic motor nucleus and the ventral horn of the spinal cord. A much smaller projection from the VRG to the RVLM was also observed. This projection included several examples of close appositions between PHALlabelled varicosities and bulbospinal and non-bulbospinal tyrosine hydroxylase immunoreactive neurans (Fig. 1).
Fig. 1. Light micrographs of bulbospinal TH-immunoreactive neurons with PHAL-labelled axon varicosities in close apposition: (a) silver-intensifiedgold particles (CTBG) within the cytoplasm of a TH-immunoreactive neuron. Two PHAL-labelled varicosities are closely apposed to the soma; (b) two PHAL-labelled varicosities are closely apposed to the soma and a proximal dendrite. A thin intervaricose segment connects the varicosities. Scale bar (10 pm) applies to (a) and (b).
Cardiovascular respiratory interactions
DISCUSSION The precise neuronal circuitry responsible for the central control of blood pressure, breathing and the interactions between these vital functions, has not yet been fully described. Physiological studies have clearly demonstrated that there are populations of neurons in the ventral medulla that discharge in phase with the respiratory or cardiac cycle, and in some cases both. Since both rhythms can persist in the absence of peripheral inputs, it seems likely that the respiratory modulation of sympathetic discharge is supplied by respiratory neurons in the brainstem, rather than by peripheral afferent inputs. A likely source of the respiratory input has been suggested on the basis of the finding that neurons in the ventral respiratory group have an axonal projection towards the rostral ventrolateral medulla where bulbospinal sympathoexcitatory neurons are known to be located (Ellenberger & Feldman 1990; Pilowsky et aZ. 1990). In the present study we have extended these findings by demonstrating the existence of close appositions between the axon varicosities of neurons whose cell bodies are located in the ventral respiratory group and bulbospinal tyrosine hydroxylase immunoreactive neurons of the C1 cell group. These findings provide anatomical evidence for a direct connection between respiratory neurons in the ventral respiratory group of the rat and bulbospinal TH-IR neurons of the Cl cell group. Previous studies from this laboratory (Minson et al. 1987; Chalmers & Pilowsky 1991; Minson et al. 1991) and others (Morrison et al. 1988; Haselton & Guyenet 1989b) have suggested that C l neurons are likely to be a source of excitatory synaptic input to sympathetic preganglionic neurons in the intermediolateral cell column. The present results support the hypothesis that the respiratory modulation of blood pressure and sympathetic nerve activity is produced, at least in part, by an input from neurons in the VRG towards presympathetic neurons in the RVLM, some of which may be phenotypically adrenergic.
ACKNOWLEDGEMENTS We are grateful for the expert technical assistance of Ms R. Coffey, Ms C. Frisby, Ms M. McLaren and Mr A. Wright. This work was supported by grants from the National Health & Medical Research Council, the National Heart Foundation, and the National Sudden Infant Death Syndrome Council of Australia.
337
REFERENCES
Adrian, E. D., Bronk, D. W. & Phillips, G. (1932) Discharges in mammalian sympathetic nerves. Journal of Physiology (Lond.), 74, 115-133. Bainton, C. R., Richter, D. W., Seller, H., Ballantyne, D. & Klein, J. P. (1985) Respiratory modulation of sympathetic activity. Journal of the Autonomic Nervous System, 12, 77-90. Brown, D. L. & Guyenet, P. G. (1984) Cardiovascular neurons of brain stem with projections to spinal cord. American Journal of Physiology, 247, R 1009- 1016. Chalmers, J. P. & Pilowsky, P. M. (1991) Brainstem and
bulbospinal neurotransmitter systems in the control of blood pressure. Journal of Hypertension, 9,675-694. Ellenberger, H. H. &. Feldman, J. L. (1990) Brainstem connections of the rostral ventral respiratory group of the rat. Brain Research, 513,35-42. Ezure, K. (1990) Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Progress in Neurobiology, 35, 429 -450. Gilbey, M. P., Numao, Y. & Spyer, K. M. (1986) Discharge patterns of cervical sympathetic preganglionic neurones related to central respiratory drive in the rat. Journal of Physiology (Lond.), 378, 253-265. Guyenet, P. G., Haselton, J. R. & Sun, M-K. (1989) Sympathoexcitatory neurons of the rostroventrolateral medulla and the origin of the sympathetic vasomotor tone. Progress in Brain Research, 81, 105-116. Haselton, J. R. & Guyenet, P. G. (1989a) Central respiratory modulation of medullary sympathoexcitatory neurons in rat. American Journal of Physiology, 256, R739-750. Haselton, J. R. & Guyenet, P. G. (1989b) Electrophysiological characterization of putative Cl adrenergic neurons in the rat. Neuroscience, 30, 199-214. McAllen, R. M. (1987) Central respiratory modulation of subretrofacial bulbospinal neurones in the cat. Journal of Physiology (Lond.), 388,533-545. Minson, J. B., Chalmers, J. P., Caon, A. C. & Renaud, B. (1987) Separate areas of rat medulla oblongata with populations of serotonin- and adrenaline-containing neurons alter blood pressure after L-glutamate stimulation. Journal of the Autonomic Nervous System, 19, 39-50. Minson, J. B., Pilowsky, P. M., Llewellyn-Smith, I. J., Kaneko, T., Kapoor, V. & Chalmers, J. P. (1991) Glutamate in spinally projecting neurons of the rostral ventral medulla. Bruin Research, 555,326-331. Morrison, S . F., Milner, T. A. & Reis, D. J. (1988) Reticulospinal vasomotor neurons of the rat rostral ventrolateral medulla: Relationship to sympathetic nerve activity and the C1 adrenergic cell group. Journal of Neuroscience, 8, 1286-1301. Pilowsky, P. M., Jiang, C. & Lipski, J. (1990) An intracellular study of respiratory neurons in the rostral
338
P. Pilowsky et al.
ventrolateral medulla of the rat and their relationship to catecholamine-containingneurons. Journal of Comparative Neurology, 301,604-617.
Preiss, G. & Polosn, C. (1977) The relation between endtidal COZ and discharge patterns of sympathetic preganglionic neurons. Brain Research, 122,255-267.
SHORT COMMUNICATION
AXONAL PROJECTIONS FROM RESPIRATORY CENTRES TOWARDS THE ROSTRAL VENTROLATERAL MEDULLA IN THE RAT Paul Pilowsky, Bruce Wakefield, Jane Minson, Ida Llewellyn-Smith and John Chalmers
Department of Medicine and Centre f o r Neuroscience, Flinders University of South Australia, Bedford Park, South Australia, Australia (Received 13 December 1991)
SUMMARY 1. Efferent pathways from brainstem respiratory centres towards bulbospinal tyrosine hydroxylase immunoreactive neurons were identified in the rat using a combination of electrophysiology, retrograde and anterograde tract-tracing, and immunohistochemistry. 2. Varicose axons originating from respiratory centres were found in close apposition to bulbospinal tyrosine hydroxylase immunoreactive neurons in the ventrolateral medulla. 3. These findings support the idea that respiratory rhythms in sympathetic nerves may be due to a synaptic connection between brainstem respiratory neurons and bulbospinal tyrosine hydroxylase immunoreactive neurons of the C1 cell group.
Key words: bulbospinal pathways, C1 cell group, cholera toxin B, Phuseolus vulgaris-leucoagglutinin, tract-tracing.
INTRODUCTION Respiratory rhythmicity is commonly observed in sympathetic nerve activity and blood pressure (Adrian et al. 1932). This rhythmicity is not simply due to the activation of pulmonary stretch receptors or other peripheral inputs as it persists in paralysed and mechanically ventilated animals where the central inspiratory activity is unrelated to ventilation (Preiss & Polosa 1977; Bainton et al. 1985; Gilbey el al. 1986). The site at which this respiratory rhythmicity is imposed on the sympathetic outflow is unknown at
present. An important source of tonic activity in sympathetic preganglionic neurons is a compact group of bulbospinal neurons in the rostra1 ventrolateral medulla (RVLM; Brown & Guyenet 1984; Minson et al. 1987; Guyenet et al. 1989). A number of studies have demonstrated that many bulbospinal sympathoexcitatory neurons in the RVLM have a respiratory modulation of their ongoing discharge rate (McAllen 1987; Haselton & Guyenet 1989a, b). Correlative physiological and histological studies have suggested
Correspondence: Dr Paul Pilowsky, Dept of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
P. Pilowsky et al.
336
that a subpopulation of these neurons is likely to contain tyrosine hydroxylase immunoreactivity (Minson et al. 1987). One possible source of this respiratory modulation is the ventral respiratory group (VRG) of neurons. These neurons are responsible for the generation of normal respiratory rhythms (Ezure 1990; Pilowsky et al. 1990). Several recent reports have provided anatomical support for the notion that there is a projection from respiratory neurons in the VRG towards the C1 cell group (Ellenberger & Feldman 1990; Pilowsky et al. 1990), but there have been no reports of close appositions between the axons of neurons arising from the VRG and the somata or dendrites of bulbospinal neurons that project to the intermediolateral cell column.
METHODS Experiments were performed on Wistar-Kyoto rats anaesthetized with pentobarbitone sodium (60mgl kg, i.p). The diaphragmatic electromyogram was used to monitor the phases of the respiratory cycle. The dorsal surface of the medulla was exposed for injections of Phaseolus vulgaris leucoagglutinin (PHAL; Vector Laboratories, CA, USA). The thoracic spinal cord was exposed for injections of cholera toxin B-gold (CTB-G; Gilt Products, Adelaide, SA, Australia). After allowing 3-7 days for transport of the injected tracers, the rats were fixed by intravascular perfusion with Zamboni's fixative containing 0.05% glutaraldehyde, and the brain and spinal cord were removed for histological processing. Pathways from the VRG to the RVLM were revealed by injection of PHAL into sites in the ventral medulla where multi-unit inspiratory (n = 4) or expiratory (n = 4) activity could be recorded. PHAL was injected by iontophoresisusing a Midgard CS4 constant current unit (Transkinetics, Canton, MA, USA). The location of PHAL-labelled axon terminals was detected using immunohjsio-chemistry and a diahinobenzidinenickel reaction that resulted in a black reaction product. Spinally-projectingtyrosine hydroxylase immunoreactive (TH-IR) neurons were detected by a combination of retrograde tracing with CTB-G and immunohistochemistry using an antityrosine hydroxylase antiserum (Eugene Tech., Allendale, NJ, USA). CTB-G was injected into the intermediolateral cell column of the thoracic spinal cord. CTB-G within neurons in the medulla was detected by silver intensification of the colloidal gold particles. TH-IR was detected by
immunohisto-chemistry using a diaminobenzidineimidazole reaction that resulted in a brown reaction product.
RESULTS Projections from the VRG (PHAL immunoreactivity) were visualized as black varicose fibres (Fig. 1). Silver intensified gold particles appeared as black puncta within the cytoplasm of neurons, while TH-IR was observed as an homogeneous brown reaction product in the cytoplasm and dendrites of neurons, but not in the nucleus. Within the ventral medulla, most PH AL-labelled axons arising from sites of multi-unit inspiratory or expiratory activity were observed in a compact region that was ventral to the nucleus ambiguus pars cornpacta, and dorsal to the C1 cell group, This area corresponds to the location of the ventral respiratory group. PHAL-labelled varicosities were also found in the same site on the contralateral side of the medulla. Several motor nuclei received an input from PHALlabelled varicosities, including the hypoglossal nucleus, the facial nucleus, the nucleus ambiguus pars compacts, the phrenic motor nucleus and the ventral horn of the spinal cord. A much smaller projection from the VRG to the RVLM was also observed. This projection included several examples of close appositions between PHALlabelled varicosities and bulbospinal and non-bulbospinal tyrosine hydroxylase immunoreactive neurans (Fig. 1).
Fig. 1. Light micrographs of bulbospinal TH-immunoreactive neurons with PHAL-labelled axon varicosities in close apposition: (a) silver-intensifiedgold particles (CTBG) within the cytoplasm of a TH-immunoreactive neuron. Two PHAL-labelled varicosities are closely apposed to the soma; (b) two PHAL-labelled varicosities are closely apposed to the soma and a proximal dendrite. A thin intervaricose segment connects the varicosities. Scale bar (10 pm) applies to (a) and (b).
Cardiovascular respiratory interactions
DISCUSSION The precise neuronal circuitry responsible for the central control of blood pressure, breathing and the interactions between these vital functions, has not yet been fully described. Physiological studies have clearly demonstrated that there are populations of neurons in the ventral medulla that discharge in phase with the respiratory or cardiac cycle, and in some cases both. Since both rhythms can persist in the absence of peripheral inputs, it seems likely that the respiratory modulation of sympathetic discharge is supplied by respiratory neurons in the brainstem, rather than by peripheral afferent inputs. A likely source of the respiratory input has been suggested on the basis of the finding that neurons in the ventral respiratory group have an axonal projection towards the rostral ventrolateral medulla where bulbospinal sympathoexcitatory neurons are known to be located (Ellenberger & Feldman 1990; Pilowsky et aZ. 1990). In the present study we have extended these findings by demonstrating the existence of close appositions between the axon varicosities of neurons whose cell bodies are located in the ventral respiratory group and bulbospinal tyrosine hydroxylase immunoreactive neurons of the C1 cell group. These findings provide anatomical evidence for a direct connection between respiratory neurons in the ventral respiratory group of the rat and bulbospinal TH-IR neurons of the Cl cell group. Previous studies from this laboratory (Minson et al. 1987; Chalmers & Pilowsky 1991; Minson et al. 1991) and others (Morrison et al. 1988; Haselton & Guyenet 1989b) have suggested that C l neurons are likely to be a source of excitatory synaptic input to sympathetic preganglionic neurons in the intermediolateral cell column. The present results support the hypothesis that the respiratory modulation of blood pressure and sympathetic nerve activity is produced, at least in part, by an input from neurons in the VRG towards presympathetic neurons in the RVLM, some of which may be phenotypically adrenergic.
ACKNOWLEDGEMENTS We are grateful for the expert technical assistance of Ms R. Coffey, Ms C. Frisby, Ms M. McLaren and Mr A. Wright. This work was supported by grants from the National Health & Medical Research Council, the National Heart Foundation, and the National Sudden Infant Death Syndrome Council of Australia.
337
REFERENCES
Adrian, E. D., Bronk, D. W. & Phillips, G. (1932) Discharges in mammalian sympathetic nerves. Journal of Physiology (Lond.), 74, 115-133. Bainton, C. R., Richter, D. W., Seller, H., Ballantyne, D. & Klein, J. P. (1985) Respiratory modulation of sympathetic activity. Journal of the Autonomic Nervous System, 12, 77-90. Brown, D. L. & Guyenet, P. G. (1984) Cardiovascular neurons of brain stem with projections to spinal cord. American Journal of Physiology, 247, R 1009- 1016. Chalmers, J. P. & Pilowsky, P. M. (1991) Brainstem and
bulbospinal neurotransmitter systems in the control of blood pressure. Journal of Hypertension, 9,675-694. Ellenberger, H. H. &. Feldman, J. L. (1990) Brainstem connections of the rostral ventral respiratory group of the rat. Brain Research, 513,35-42. Ezure, K. (1990) Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Progress in Neurobiology, 35, 429 -450. Gilbey, M. P., Numao, Y. & Spyer, K. M. (1986) Discharge patterns of cervical sympathetic preganglionic neurones related to central respiratory drive in the rat. Journal of Physiology (Lond.), 378, 253-265. Guyenet, P. G., Haselton, J. R. & Sun, M-K. (1989) Sympathoexcitatory neurons of the rostroventrolateral medulla and the origin of the sympathetic vasomotor tone. Progress in Brain Research, 81, 105-116. Haselton, J. R. & Guyenet, P. G. (1989a) Central respiratory modulation of medullary sympathoexcitatory neurons in rat. American Journal of Physiology, 256, R739-750. Haselton, J. R. & Guyenet, P. G. (1989b) Electrophysiological characterization of putative Cl adrenergic neurons in the rat. Neuroscience, 30, 199-214. McAllen, R. M. (1987) Central respiratory modulation of subretrofacial bulbospinal neurones in the cat. Journal of Physiology (Lond.), 388,533-545. Minson, J. B., Chalmers, J. P., Caon, A. C. & Renaud, B. (1987) Separate areas of rat medulla oblongata with populations of serotonin- and adrenaline-containing neurons alter blood pressure after L-glutamate stimulation. Journal of the Autonomic Nervous System, 19, 39-50. Minson, J. B., Pilowsky, P. M., Llewellyn-Smith, I. J., Kaneko, T., Kapoor, V. & Chalmers, J. P. (1991) Glutamate in spinally projecting neurons of the rostral ventral medulla. Bruin Research, 555,326-331. Morrison, S . F., Milner, T. A. & Reis, D. J. (1988) Reticulospinal vasomotor neurons of the rat rostral ventrolateral medulla: Relationship to sympathetic nerve activity and the C1 adrenergic cell group. Journal of Neuroscience, 8, 1286-1301. Pilowsky, P. M., Jiang, C. & Lipski, J. (1990) An intracellular study of respiratory neurons in the rostral
338
P. Pilowsky et al.
ventrolateral medulla of the rat and their relationship to catecholamine-containingneurons. Journal of Comparative Neurology, 301,604-617.
Preiss, G. & Polosn, C. (1977) The relation between endtidal COZ and discharge patterns of sympathetic preganglionic neurons. Brain Research, 122,255-267.
