Differential effects of long-term hypoxia on norepinephrine turnover in brain stem cell groups V. SOULIER, J. M. COTTET-EMARD, J. PEQUIGNOT, F. HANCHIN, L. PEYRIN, AND J. M. PEQUIGNOT Unit6 de Recherches Associbe 1195, Centre National de la Recherche Scientifique, Fuculte’ de Me’decine Grange-Blanche, 69373 Lyon Cedex 08, Frunce SOULIER,V.,J.M. COTTET-EMARD,J. PEQUIGNOT,F. HANCHIN, L. PEYRIN,ANDJ. M. PEQUIGNOT. Differential effects of long-term hypoxia on norepinephrine turnover in brain stern cell groups. J. Appl. Physiol. 73(5): 1810-1814, 1992.-The influ-
enceof long-term hypoxia on noradrenergic cell groups in the brain stem was assessed by estimating the changesin norepinephrine (NE) turnover in Al, A2 (subdivided into anterior and posterior parts), AS, and A6 groupsin rats exposedto hypoxia (10% O,-90% N2) for 14 days. The NE turnover was decreasedin A5 and A6 groupsbut failed to change significantly in Al. The NE turnover was increasedin the posterior part of A2 and remained unaltered in the anterior part. In normoxic rats, the hypotensive drug dihydralazine induced a reverse effect, namely increased NE turnover in anterior A2 and no change in posterior A2. The neurochemicalresponsesto hypoxia were abolishedby transection of carotid sinusnerves. The results showthat long-term hypoxia exerts differential effects on the noradrenergic cell groups located in the brain stem. Peripheral chemosensoryinputs control the hypoxia-induced noradrenergic alterations. The A2 cell group displays a functional subdivision: the posterior part is influenced by peripheral chemosensoryinputs, whereasthe anterior part may be concerned with barosensitivity. Al cell group; A2 cell group; A5 cell group; locus coeruleus; nucleusof tractus solitarius; rat
STIMULATION of the sympathoadrenal system has long been recognized as a major component of adaptive responses to hypoxia, especially in regulation of cardiovascular events that accompany the hyperventilation (6,18, 25). Sympathetic neurons are under tonic control of preganglionic fibers, the cell bodies of which are located in the intermediolateral cell column. Furthermore the preganglionic sympathetic nerves receive both excitatory and inhibitory influences from noradrenergic cell groups located in the brain stem. However, it is still poorly understood how chemoreceptor inputs reach the sympathetic outflow. More generally, little is known about the central organization of pathways that lead to the neurophysiological adjustments required by hypoxia (5, 31). The decrease in arterial partial pressure of oxygen is primarily monitored by peripheral arterial chemoreceptors that evoke excitation of chemosensory fibers projecting in the brain stem, within the nucleus of tractus solitarius (NTS). The issue is further complicated by the presence of barosensory fibers running in the same nerves (carotid sinus and aortic nerves) as the chemosensory fibers and 1810
also ending in the NTS. Recent studies based on electrophysiological stimulations or discrete lesions of the NTS revealed separate sites of projection for the chemo- and barosensory fibers (8,14,21). These studies suggest that the chemosensory fibers project to an area located just caudal to the obex, whereas the barosensory fibers terminate in an area just rostra1 to the obex. From an anatomic description of the projection sites of rat carotid sinus nerve (13, 14), it can be inferred that the part of NTS that receives the carotid sinus nerve terminals corresponds to the AZ noradrenergic cell group. The A2 cell group and other noradrenergic cell groups located in the brain stem, including Al, A5, A6 (locus coeruleus), and A7, integrate a variety of reflex inputs. These catecholamine cell groups are involved in central regulation of sympathetic outflow (A2, A5, and A7), arousal reactions (A6), or neuroendocrine activity (Al) and thus may participate in central control of adjustments required by hypoxemia. The present study was designed 1) to define the influence of long-term hypoxia on different noradrenergic cell groups (Al, A2, A5, and A6), 2) to provide evidence for a functional subdivision of the A2 cell group, and 3) to examine whether the neurochemical responses to hypoxia are subsequent to a direct effect of hypoxia on the central nervous system or are mediated by peripheral chemoreceptors. METHODS
Animals. Male Sprague-Dawley rats (200-220 g) were housed in a temperature-controlled room at 22 t 1°C with a 1X2-h light-dark cycle and were allowed free access to food and water. The ambient altitude (150 m) corresponded to a barometric pressure of ~740 Torr. Chemodeneruation. Anesthesia was induced by halothane inhalation. Thirty-two rats were subjected to bilateral chemodenervation. The two carotid sinus nerves were transected at the point of branching from the glossopharyngeal nerve and at the cranial pole of the carotid body. The denervation was performed under a dissection microscope 1 wk before the normoxic (n = 12) or hypoxic (n = 20) exposure. Long-term exposure to hypoxia. Forty-four rats (24 intact and 20 chemodenervated) were kept in a Plexiglas chamber supplied with 10% O,-90% N,. The gas composition inside the chamber was maintained at 10 t 0.5%
0161-75671’92 $2.00 Copyright 0 1992 the American Physiological Society Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 12, 2019.
BRAIN
STEM
NOREPINEPHRINE
0,. The CO, expired by rats was eliminated by circulating the gas mixture from the chamber through soda lime. Metabolic water contained in expiratory gases was continuously trapped into a chilled tank. The temperature inside the chamber was 23 t 1°C. Animals were kept in the hypoxic chamber for 14 days. The duration of hypoxia was chosen according to previous results from our laboratory showing that hypoxia-induced stimulation of the sympathoadrenal system was maximal after 2 wk of exposure (6). Normoxic control rats (24 intact and 12 chemodenervated) were maintained in normoxic air in the same room and killed 14 days after beginning the experiment. Dihydralazine treatment. Twenty-four rats were given a daily subcutaneous injection (20 mg/kg) of the hypotensive drug dihydralazine (Nepressol, Ciba-Geigy Laboratories, Basel, Switzerland) for 14 days. This dose was shown previously to induce a marked drop of arterial blood pressure in normotensive rats (4). Control rats (n = 24) were given the equivalent volume of 0.9% saline. Estimation of the turnover of norepinephrine (NE). The NE turnover was estimated in catecholamine cell groups by blocking the catecholamine biosynthesis with amethyl-p-tyrosine methyl ester (AMPT, 250 mg/kg ip) injected 2.5 h before the hypoxic rats were killed (intact rats, n = 12, or chemodenervated rats, n = 10). Additional hypoxic rats (intact rats, n = 12, or chemodenervated rats, n = 10) received the equivalent volume of 0.9% saline. First, basal levels of NE in the different cell groups were measured in saline-treated rats. Then the amount of NE remaining 2.5 h after AMPT was subtracted, and the difference estimated the’ utilization (turnover) rate of NE in long-term hypoxia. NE turnover was expressed as picomoles per milligram protein per hour. The normoxic controls were treated in the same manner as the hypoxic rats: one group of intact (n = 12) or chemodenervated rats (n = 6) was given AMPT while another group of intact (n = 12) or chemodenervated rats (n = 6) received saline. To determine the influence of dihydralazine on NE turnover in the A2 cell group, the same procedure was applied to the dihydralazine-treated rats. Tissue preparation. The rats were killed by cervical dislocation and then decapitated. The brain was rapidly dissected out, frozen on dry ice, and stored at -8OOC. The brain stem was cut into serial frontal slices of 480-pm thickness. The noradrenergic cell groups Al, AZ, A5, and A6 were punched out according to the dissection procedure described by Palkovits and Brownstein (22). The A2 cell group was subdivided rostrocaudally into two half parts (3 punches each). These areas were referred to as “posterior part” and ‘
enceof long-term hypoxia on noradrenergic cell groups in the brain stem was assessed by estimating the changesin norepinephrine (NE) turnover in Al, A2 (subdivided into anterior and posterior parts), AS, and A6 groupsin rats exposedto hypoxia (10% O,-90% N2) for 14 days. The NE turnover was decreasedin A5 and A6 groupsbut failed to change significantly in Al. The NE turnover was increasedin the posterior part of A2 and remained unaltered in the anterior part. In normoxic rats, the hypotensive drug dihydralazine induced a reverse effect, namely increased NE turnover in anterior A2 and no change in posterior A2. The neurochemicalresponsesto hypoxia were abolishedby transection of carotid sinusnerves. The results showthat long-term hypoxia exerts differential effects on the noradrenergic cell groups located in the brain stem. Peripheral chemosensoryinputs control the hypoxia-induced noradrenergic alterations. The A2 cell group displays a functional subdivision: the posterior part is influenced by peripheral chemosensoryinputs, whereasthe anterior part may be concerned with barosensitivity. Al cell group; A2 cell group; A5 cell group; locus coeruleus; nucleusof tractus solitarius; rat
STIMULATION of the sympathoadrenal system has long been recognized as a major component of adaptive responses to hypoxia, especially in regulation of cardiovascular events that accompany the hyperventilation (6,18, 25). Sympathetic neurons are under tonic control of preganglionic fibers, the cell bodies of which are located in the intermediolateral cell column. Furthermore the preganglionic sympathetic nerves receive both excitatory and inhibitory influences from noradrenergic cell groups located in the brain stem. However, it is still poorly understood how chemoreceptor inputs reach the sympathetic outflow. More generally, little is known about the central organization of pathways that lead to the neurophysiological adjustments required by hypoxia (5, 31). The decrease in arterial partial pressure of oxygen is primarily monitored by peripheral arterial chemoreceptors that evoke excitation of chemosensory fibers projecting in the brain stem, within the nucleus of tractus solitarius (NTS). The issue is further complicated by the presence of barosensory fibers running in the same nerves (carotid sinus and aortic nerves) as the chemosensory fibers and 1810
also ending in the NTS. Recent studies based on electrophysiological stimulations or discrete lesions of the NTS revealed separate sites of projection for the chemo- and barosensory fibers (8,14,21). These studies suggest that the chemosensory fibers project to an area located just caudal to the obex, whereas the barosensory fibers terminate in an area just rostra1 to the obex. From an anatomic description of the projection sites of rat carotid sinus nerve (13, 14), it can be inferred that the part of NTS that receives the carotid sinus nerve terminals corresponds to the AZ noradrenergic cell group. The A2 cell group and other noradrenergic cell groups located in the brain stem, including Al, A5, A6 (locus coeruleus), and A7, integrate a variety of reflex inputs. These catecholamine cell groups are involved in central regulation of sympathetic outflow (A2, A5, and A7), arousal reactions (A6), or neuroendocrine activity (Al) and thus may participate in central control of adjustments required by hypoxemia. The present study was designed 1) to define the influence of long-term hypoxia on different noradrenergic cell groups (Al, A2, A5, and A6), 2) to provide evidence for a functional subdivision of the A2 cell group, and 3) to examine whether the neurochemical responses to hypoxia are subsequent to a direct effect of hypoxia on the central nervous system or are mediated by peripheral chemoreceptors. METHODS
Animals. Male Sprague-Dawley rats (200-220 g) were housed in a temperature-controlled room at 22 t 1°C with a 1X2-h light-dark cycle and were allowed free access to food and water. The ambient altitude (150 m) corresponded to a barometric pressure of ~740 Torr. Chemodeneruation. Anesthesia was induced by halothane inhalation. Thirty-two rats were subjected to bilateral chemodenervation. The two carotid sinus nerves were transected at the point of branching from the glossopharyngeal nerve and at the cranial pole of the carotid body. The denervation was performed under a dissection microscope 1 wk before the normoxic (n = 12) or hypoxic (n = 20) exposure. Long-term exposure to hypoxia. Forty-four rats (24 intact and 20 chemodenervated) were kept in a Plexiglas chamber supplied with 10% O,-90% N,. The gas composition inside the chamber was maintained at 10 t 0.5%
0161-75671’92 $2.00 Copyright 0 1992 the American Physiological Society Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 12, 2019.
BRAIN
STEM
NOREPINEPHRINE
0,. The CO, expired by rats was eliminated by circulating the gas mixture from the chamber through soda lime. Metabolic water contained in expiratory gases was continuously trapped into a chilled tank. The temperature inside the chamber was 23 t 1°C. Animals were kept in the hypoxic chamber for 14 days. The duration of hypoxia was chosen according to previous results from our laboratory showing that hypoxia-induced stimulation of the sympathoadrenal system was maximal after 2 wk of exposure (6). Normoxic control rats (24 intact and 12 chemodenervated) were maintained in normoxic air in the same room and killed 14 days after beginning the experiment. Dihydralazine treatment. Twenty-four rats were given a daily subcutaneous injection (20 mg/kg) of the hypotensive drug dihydralazine (Nepressol, Ciba-Geigy Laboratories, Basel, Switzerland) for 14 days. This dose was shown previously to induce a marked drop of arterial blood pressure in normotensive rats (4). Control rats (n = 24) were given the equivalent volume of 0.9% saline. Estimation of the turnover of norepinephrine (NE). The NE turnover was estimated in catecholamine cell groups by blocking the catecholamine biosynthesis with amethyl-p-tyrosine methyl ester (AMPT, 250 mg/kg ip) injected 2.5 h before the hypoxic rats were killed (intact rats, n = 12, or chemodenervated rats, n = 10). Additional hypoxic rats (intact rats, n = 12, or chemodenervated rats, n = 10) received the equivalent volume of 0.9% saline. First, basal levels of NE in the different cell groups were measured in saline-treated rats. Then the amount of NE remaining 2.5 h after AMPT was subtracted, and the difference estimated the’ utilization (turnover) rate of NE in long-term hypoxia. NE turnover was expressed as picomoles per milligram protein per hour. The normoxic controls were treated in the same manner as the hypoxic rats: one group of intact (n = 12) or chemodenervated rats (n = 6) was given AMPT while another group of intact (n = 12) or chemodenervated rats (n = 6) received saline. To determine the influence of dihydralazine on NE turnover in the A2 cell group, the same procedure was applied to the dihydralazine-treated rats. Tissue preparation. The rats were killed by cervical dislocation and then decapitated. The brain was rapidly dissected out, frozen on dry ice, and stored at -8OOC. The brain stem was cut into serial frontal slices of 480-pm thickness. The noradrenergic cell groups Al, AZ, A5, and A6 were punched out according to the dissection procedure described by Palkovits and Brownstein (22). The A2 cell group was subdivided rostrocaudally into two half parts (3 punches each). These areas were referred to as “posterior part” and ‘
