Brain
Research
Bulletin.
0361-9230191 S3.00 + .OO
C Pergamon Press pk. 1991. Printed in the U.S.A
Vol. 26. pp. 269-277.
Effects of Parabrachial Stimulation on Angiotensin and Blood Pressure Sensitive Area Postrema Neurons SOPHIA PAPAS AND ALASTAIR Department
of Physiology,
Queen’s University, Received
V. FERGUSON’
Kingston,
Onturio, Canadu, K7L 3N6
26 July 1990
S. AND A. V. FERGUSON. Effects of parabrcrchial stimulation on cqiotensin and blood pressure sensitive UWN posBRAIN RES BULL 26(2) 269-277, 1991 .-Subpopulations of neurons in the area postrema (AP) and commissural nucleus tractus solitarius (NTS) have been identified according to their responses to systemic angiotensin-II (ANG-II) and increases in blood pressure (BP). In order to further define the functional connections of these subpopulations of cells, electrophysiological single unit recording studies have been done to determine the orthodromic effects of parabrachial nucleus (PBN) stimulation on these functionally defined cell groups. Orthodromic effects were seen in a similar proportion of ANG-II sensitive neurons in the AP (31.5’%1 and NTS (31%). PBN stimulation influenced a similar percentage of BP sensitive neurons in the AP (35%). although a larger proportion of this group of NTS cells was affected (55.5%). Twenty-five percent of ANG-II/BP sensitive neurons in the AP were otthodromically influenced, and 71.57~ of this group of NTS neurons were affected by PBN stimulation. Small proportions of the neurons in the unaffected subpopulation of AP (10%) and NTS (27%) were also orthodromically affected by PBN stimulation. The remaining neurons in each group were not affected. This study suggests that there is no apparent preferential distribution of excitatory or inhibitory PBN efferents to any of the identified subpopulations of AP and NTS neurons. PAPAS.
frcm~ neurons.
Nucleus tractun solitartus
Cardiovascular
Electrical
stimulation
peripheral input is through its neural connections. Both the AP and the NTS receive vagal projections from the heart (24) and are known to receive baroreceptor information from both the carotid sinus and aortic depressor nerves (11, 12, 25. 3 1, 37, 42). A large proportion of the efferent projections of the AP and NTS travel to the lateral division of the parabrachial nucleus (PBN) (22, 29, 35, 38, 40), a collection of small neurons surrounding the superior cerebellar peduncle in the pans. In return. both the AP (38) and NTS (36) receive a relatively light projection from this region, which has been extensively implicated in cardiovascular control mechanisms (9, 20, 27, 39, 43, 44). Functional analysis of these connections using electrophysiological techniques have both confirmed their existence (14, 19, 20, 32) and demonstrated both excitatory and inhibitory PBN input to AP and NTS neurons (14, 20, 32). Thus. the AP and NTS provide entry points for peripheral input to the brain. Furthermore, their close reciprocal ties with the PBN. a structure which appears to act as an autonomic relay centre (9, 20, 22), suggest a pathway for this ascending visceral information, as well as a route by which parabrachial efferents may modify incoming information. Electrophysiological techniques have allowed us to identify distinct functional cell populations in the AP and NTS which are influenced by peripheral information: neurons specifically affected by systemic ANG-II, another group
FOR several decades. the primary role attributed to the area postrema (AP) has been that of chemosensitive trigger zone in the emetic reflex (3). More recently, however, this region has been implicated in various other physiological functions, such as control of dietary intake, respiration and cardiovascular regulation [see (2) for review]. The AP is a unique structure in that it can relay peripheral information to the central nervous system (CNS) in several ways. It is the most caudal of the circumventricular organs. overlying the nucleus tractus solitarius (NTS) on the medulla oblongata. The AP is a highly vascularized region which lacks a normal blood-brain barrier (46), and consequently may allow blood-borne substances to exert their effects on the CNS. In fact, it has been suggested that the central cardiovascular effects of angiotensin-11 (ANG-II) are mediated via the AP (16,23). This view is supported anatomically by the high concentration of ANG-II receptors in both the AP and the NTS (21,26), and by studies which have related pressor (6, 7, 17) and drinking (30) responses to the interaction of ANG-II with the AP and NTS. In addition, electrophysiological studies have demonstrated the existence of neurons in these two structures whose activities are specifically altered in response to intravenously (33) or iontophoretically (5) administered ANG-II. An alternate route by which the AP may receive and modify ‘Requests for reprints should be addressed
Rat
to Dr. A. V. Ferguson.
269
FIG. I. The photomic#~pb in (A) shows an exampfe of a recording electrode tract in the AP and NTS, as determined histologically. An example of a stimulating electrode site in the rostra1 dorsaf PBN is shown in (B). In each case, the ~ITOWindicates the location of the electrode tip. DPBN-dorsal PBN, SCP-superior cerebellar peduncle, VPBN- ventral PBN.
PARABRACHIAL
INFLUENCES
ON AREA POSTREMA
271
NEURONS
TABLE I
TABLE 2
SUMMARY OF THE DIFFERING RESPONSES OF AP AND NTS NEURONS TO ANG-II AND ADRENERGIC AGONISTS, AND THE FUNCTIONAL CLASSIFICATIONS OF THESE NEURONS ACCORDING TO SUCH RESPONSES-EXCITATION (EXC). INHIBITION (INH). NO EFFECT (NE)
MEAN LATENCIES AND DURATIONS OF RESPONSES OF ANG-II SENSITIVE, BP SENSITIVE. ANG-II/BP SENSITIVE. AND UNAFFECTED NEURONS IN THE AP AND NTS TO PBN STIMULATION-EXCITATION (EXC). INHIBITION (INH)
Classification of AP or NTS Neuron
ANG-II Sensitive BP Sensitive ANG-II/BP
Sensitive
Unaffected
Effect of ANG-II
EXC INH EXC INH EXC INH NE
Effect of Adrenergic Agonist
NE NE EXC
Cell Classification
ANG-II Sensitive AP Neurons NTS Neurons
INH
INH EXC NE
BP Sensitive AP Neurons NTS Neurons
of cells which by a systemic
respond to increases adrenergic agonist,
in blood pressure (BP) elicited and a third group of neurons
which respond to both ANG-II and increase in BP (33). In order to further define the physiological nature of these subpopulations of neurons, it is important to determine their neural connections. as well as the specificity of such connections to these functional subpopulations of neurons. In this study, neurons in the rat AP and NTS were classified according to their responses to systemic ANG-II or to an increase in BP elicited by both ANG-II and an adrenergic agonist. The PBN was stimulated electrically to determine if these identified AP and NTS cells received input from this region, and to examine the specific types of otthodromic influences received by functional subpopulations of AP and NTS neurons. METHOD
Male Sprague-Dawley rats (150-300 g) were anaesthetized with sodium pentobarbital (65 mg/kg). Each animal was fitted with an indwelling femoral arterial catheter (PE 50 Intramedic) to monitor BP, femoral and jugular venous catheters (PE 50 Intramedic) to administer agents or supplements of sodium pentobarbital when required, and an endotrachial tube to facilitate breathing. During the experiment, a feedback-controlled heating blanket was used to maintain body temperature at 37 ? 1°C. Animals were placed in a Kopf stereotaxic frame and a stimulating electrode positioned in the PBN according to the stereotaxic coordinates of Paxinos and Watson (34). A small burr hole was made 8.0 mm posterior to bregma and 1.7 mm lateral to the sagittal midline, and a monopolar or concentric bipolar stimulating electrode (Rhodes Medical Instruments) was lowered 6.5 mm ventral to the dura and attached to the skull using a jeweller’s screw and dental cement. The stimulating electrode was connected to an isolated stimulation unit programmed to deliver 0. I-ms current pulses, controlled by a digitimer. In order to expose the AP, the head of the animal was positioned vertically and a midline incision made. Both the atlantooccipital membrane and dura covering the AP were removed and the dorsal surface of the AP was then clearly visible. A pressure foot was positioned over the AP to allow for more stable recordings. Extracellular single unit recordings were obtained using NaCl (I .O M) filled glass recording electrodes (resistance 15-35 Ma, tip diameter 0.05, Table 2) seen between any of the subpopulations of neurons in the AP and NTS. However, a large range of times was recorded. This range of latency and duration times may be due to the various conduction velocities of neurons travelling from the PBN to the AP and NTS, and are dependent on the functional groups of PBN efferents activated during stimulation.
Various proportions of all of the groups of AP and NTS ncurons classified according to their responses to systemic ANG-II or adrenergic agonist were influenced by PBN stimulation. Both BP sensitive and ANG-II/BP sensitive cells in the NTS received a relatively stronger afferent input than did the ANG-II sensitivae and unaffected neurons in the NTS. There was, however, no apparent preferential distribution of PBN excitatory or inhibitory efferent information to any of the functional subpopulations identified in the AP or NTS. Further studies will be necessary to determine whether such afferent inputs from the PBN may be preferentially distributed to separate populations of AP and NTS neurons with different efferent projections. ACKNOWLEDGEMENTS The authors wish to thank Pauline Smith for her excellent technical help. This work was supported by the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario.
REFERENCES 1. Berk, M. L.; Finkelstein, J. A. Afferent projections to the preoptic area and hypothalamic regions in the rat brain. Neuroscience 6: 160 I1624; 1981. 2. Borison, H. L. Area postrema: Chemoreceptor circumventricular organ of the medulla oblongata. Prog. Neurobiol. 32:351-390; 1989. 3. Borison, H. L.; Wang, S. C. Physiology and pharmacology of vomiting. Pharmacol. Rev. 5:193-230; 1953. 4. Brooks, M. J.; Hubbard, J. I.; Sirett, N. E. Extracellular recording in rat area postrema in vitro and the effects of cholinergic drugs, serotonin and angiotensin II. Brain Res. 261:85-90; 1983. 5. Carpenter, D. 0.; Btiggs, D. B.; Knox, A. P.; Strominger, N. Excitation of area postrema neurons by transmitters, peptides, and cyclic nucleotides. J. Neurophysiol. 59:358-369; 1988. 6. Casto, R.; Phillips, M. I. Mechanism of pressor effects by angiotensin in the nucleus tractus solitarius of rats. Am. J. Physiol. 247: R75-R81; 1984. 7. Casto, R.; Phillips, M. I. Cardiovascular actions of microinjections of angiotensin II in the brain stem of rats. Am. J. Physiol. 246: R811-R816; 1984. 8. Casto, R.; Phillips, M. I. Angiotensin II attenuates baroreflexes at nucleus tractus solitatius of rats. Am. J. Physiol. 250:R193-R198; 1986. 9. Cechetto, D. F.; Calaresu, F. R. Parabrachial units responding to stimulation of buffer nerves and forebrain in the cat. Am. J. Physiol. 245:R811-R819; 1983. 10. Ciriello, J. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci. Lea. 36:37-42; 1983. 11. Ciriello, J.; Htycyshyn, A. W.; Calaresu, F. R. Horseradish peroxidase study of brain stem projections of carotid sinus and aortic depressor nerves in the cat. J. Auton. Nerv. Syst. 4:43-61; 1981. 12. Davies, R. 0.; Kalia, M. Carotid sinus nerve projections to the brain stem in the cat. Brain Res. Bull. 6:531-541; 1981. 13. Diz, D. I.; Barnes, K. L. ; Ferrario, C. M. Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla. Brain Res. Bull. 17:497-505; 1986. 14. Felder, R. B.; Mifflin, S. W. Modulation of carotid sinus afferent input to nucleus tractus solitarius by parabrachial nucleus stimulation. Circ. Res. 63:35-49; 1988. 15. Ferrario, C. M.; Barnes, K. L.; Diz, D. I. Involvement of the area postrema in blood pressure regulation. FASEB. J. 2:A922; 1988. 16. Ferrario, C. M.; Gildenberg, P. L.; McCubbin, J. W. Cardiovascular effects of angiotensin mediated by the central nervous system. Circ. Res. 30:257-262; 1972. 17. Fink, G. D.; Bruner, C. A.; Mangiapane, M. L. Area postrema is critical for angiotensin-induced hypertension in rats. Hypertension 9: 355-361; 1987. 18. Fulwiler, C. E.; Saper, C. B. Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev. 7:229-259; 1984.
19. Granata, A. R.; Kitai, S. T. Intracellular study of nucleus parabrachialis and nucleus tractus solitarii interconnections. Brain Res. 492: 281-292; 1989. 20. Hamilton, R. B.; Ellenberger, H.; Liskowsky, D.; Schneiderman, N. Parabrachial area as mediator of bradycardia in rabbits. J. Auton. Nerv. Syst. 4:261-281; 1981. 21. Healy, D. P.; Rettig, R.; Nguyen, T.; Printz, M. P. Quantitative autoradiography of angiotensin II receptors in the rat solitary-vagal area: effects of nodose ganglionectomy or sinoaortic denervation. Brain Res. 484:1-12; 1989. 22. Herbert, H.; Moga, M. M.; Saper, C. B. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J. Comp. Neurol. 293:_54O-580; 1990. 23. Joy, M. D.; Lowe, R. D. Evidence that the area postrema mediates the central cardiovascular response to angiotensin II. Nature 228: 1303-1304;1970. 24. Kalia, M.; Mesulam, M-M. Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches. J. Comp. Neurol. 193:467-508; 1980. 25. Kalia, M.; Welles, R. V. Brain stem projections of the aortic nerve in the cat: a study using tetramethyl benzidine as the substrate for horseradish peroxidase. Brain Res. 188:23-32; 1980. 26. Mendelsohn, F. A. 0.; Quirion, R.; Saavedra, I. M.; Aguilera, G. Autoradiographic localization of angiotensin 11 receptors in rat brain. Proc. Natl. Acad. Sci. USA 81:1575-1579; 1984. 27. Mraovitch, S.; ladecola, C.; Ruggiero, D. A.; Reis, D. J. Widespread reductions in cerebral blood flow and metabolism elicited by electrical stimulation of the parabrachial nucleus in rat. Brain Res. 341:283-296; 1985. 28. Mraovitch, S.; Kumada, M.; Reis, D. J. Role of the nucleus parabrachialis in cardiovascular regulation in cat. Brain Res. 232:57-75; 1982. 29. Norgren, R. Projections from the nucleus of the solitary tract in the rat. Neuroscience 3:207-218; 1978. 30. Ohman, L. E.; Johnson, A. K. Brain stem mechanisms and the inhibition of angiotensin-induced drinking. Am. J. Physiol. 256:R264R269; 1989. 31. Panneton, W. M.; Loewy, A. D. Projections of the carotid sinus nerve to the nucleus of the solitary tract in the cat. Brain Res. 191: 239-244; 1980. 32. Papas, S.; Ferguson, A. V. Electrophysiological characterization of reciprocal connections between the parabrachial nucleus and the area postrema in the rat. Brain Res. Bull. 24:577-582; 1990. 33. Papas, S.; Smith, P.; Ferguson, A. V. Electtophysiological evidence that systemic angiotensin influences rat area postrema neurons. Am. J. Physiol. 258:R7&R76; 1990. 34. Paxinos, G.; Watson, C. In: The rat brain in stereotaxic coordinates.
PARABRACHIALlNFLUENCESONAREAPOSTREh4ANEURONS
277
New York: Academic Press; 1982. 35. Ricardo. 3. A.: Koh, E. T. Anatomical evidence of direct projections
from the nucleus of the solitary tract to the hypothalamus. amygdala, and other forebrain structures in the rat. Brain Res. 153:1-X; 1978. 36. Saper. C. 3.; Loewy, A. D. Efferent conuections of the parabrachia~ nucleus in the rat. Brain Res. 197:291-317: 1980. 37.Seiders, E. P.; Stuesse, S. L. A horseradish peroxidase investigation of carotid sinus nerve components in the rat, Neurosci. Lett. 46:1318; 1984. 3x.Shapiro. R. E.; Miselis, R. R. The central neural connecti(~ns of the area postrema of the rat. J. Comp. Neural. 234:344-364; 1985. 39. Sved. A. F. Pontine pressor sites which release vasopressin. Brain Res. X9:143-1.50; 1986. 40. van der Kooy, D.: Koda, L. Y. Organization of the projections of a ~ir~umven~icular organ: The area postrema in the rat. J. Camp. Neurol. 219:328-338: 1983. 41. Voshart, K.: van der Kooy, D. The organization of the efferent projections of the parabrachial nucleus to the forebrain in the rat: a ret-
42 43 44 45
46
rograde fluorescent double-labeling study. Brain Res. 212:271-286; 1981. Wallach. J. H.: Loewy, A. D. Projections of the aortic nerve fn the nucleus tractus solitarius in the rabbit. Brain Res. 188:247-25 I: 198@. Ward. D. G. Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure. Brain Res. 462:X%-390: 1988. Ward. D. G. Neurons in the parabrachial nuclei respond to hcmorrhage. Brain Res. 491 :X0-92; 1989. Ward, D. G.; Adair, J. R.: Schramm, L. P.: Gann, D. S. Parabrachial pons mediates hyPothalamically induced renal vaso~onst~~t~on. Am. J. Physiol. 234:R223-R228: 1978. Wislocki, G. B.; Leduc, E. H. Vital staining of the hematoencephalit barrier by silver nitrate and trypan blue, and cytological comparisons of the neurohypophys~s. pineal body, area postrema. ~nter~olumn~ tuber& and sup~doptic crest. J. Camp. Neural, 96: 371-417: 1952.
Research
Bulletin.
0361-9230191 S3.00 + .OO
C Pergamon Press pk. 1991. Printed in the U.S.A
Vol. 26. pp. 269-277.
Effects of Parabrachial Stimulation on Angiotensin and Blood Pressure Sensitive Area Postrema Neurons SOPHIA PAPAS AND ALASTAIR Department
of Physiology,
Queen’s University, Received
V. FERGUSON’
Kingston,
Onturio, Canadu, K7L 3N6
26 July 1990
S. AND A. V. FERGUSON. Effects of parabrcrchial stimulation on cqiotensin and blood pressure sensitive UWN posBRAIN RES BULL 26(2) 269-277, 1991 .-Subpopulations of neurons in the area postrema (AP) and commissural nucleus tractus solitarius (NTS) have been identified according to their responses to systemic angiotensin-II (ANG-II) and increases in blood pressure (BP). In order to further define the functional connections of these subpopulations of cells, electrophysiological single unit recording studies have been done to determine the orthodromic effects of parabrachial nucleus (PBN) stimulation on these functionally defined cell groups. Orthodromic effects were seen in a similar proportion of ANG-II sensitive neurons in the AP (31.5’%1 and NTS (31%). PBN stimulation influenced a similar percentage of BP sensitive neurons in the AP (35%). although a larger proportion of this group of NTS cells was affected (55.5%). Twenty-five percent of ANG-II/BP sensitive neurons in the AP were otthodromically influenced, and 71.57~ of this group of NTS neurons were affected by PBN stimulation. Small proportions of the neurons in the unaffected subpopulation of AP (10%) and NTS (27%) were also orthodromically affected by PBN stimulation. The remaining neurons in each group were not affected. This study suggests that there is no apparent preferential distribution of excitatory or inhibitory PBN efferents to any of the identified subpopulations of AP and NTS neurons. PAPAS.
frcm~ neurons.
Nucleus tractun solitartus
Cardiovascular
Electrical
stimulation
peripheral input is through its neural connections. Both the AP and the NTS receive vagal projections from the heart (24) and are known to receive baroreceptor information from both the carotid sinus and aortic depressor nerves (11, 12, 25. 3 1, 37, 42). A large proportion of the efferent projections of the AP and NTS travel to the lateral division of the parabrachial nucleus (PBN) (22, 29, 35, 38, 40), a collection of small neurons surrounding the superior cerebellar peduncle in the pans. In return. both the AP (38) and NTS (36) receive a relatively light projection from this region, which has been extensively implicated in cardiovascular control mechanisms (9, 20, 27, 39, 43, 44). Functional analysis of these connections using electrophysiological techniques have both confirmed their existence (14, 19, 20, 32) and demonstrated both excitatory and inhibitory PBN input to AP and NTS neurons (14, 20, 32). Thus. the AP and NTS provide entry points for peripheral input to the brain. Furthermore, their close reciprocal ties with the PBN. a structure which appears to act as an autonomic relay centre (9, 20, 22), suggest a pathway for this ascending visceral information, as well as a route by which parabrachial efferents may modify incoming information. Electrophysiological techniques have allowed us to identify distinct functional cell populations in the AP and NTS which are influenced by peripheral information: neurons specifically affected by systemic ANG-II, another group
FOR several decades. the primary role attributed to the area postrema (AP) has been that of chemosensitive trigger zone in the emetic reflex (3). More recently, however, this region has been implicated in various other physiological functions, such as control of dietary intake, respiration and cardiovascular regulation [see (2) for review]. The AP is a unique structure in that it can relay peripheral information to the central nervous system (CNS) in several ways. It is the most caudal of the circumventricular organs. overlying the nucleus tractus solitarius (NTS) on the medulla oblongata. The AP is a highly vascularized region which lacks a normal blood-brain barrier (46), and consequently may allow blood-borne substances to exert their effects on the CNS. In fact, it has been suggested that the central cardiovascular effects of angiotensin-11 (ANG-II) are mediated via the AP (16,23). This view is supported anatomically by the high concentration of ANG-II receptors in both the AP and the NTS (21,26), and by studies which have related pressor (6, 7, 17) and drinking (30) responses to the interaction of ANG-II with the AP and NTS. In addition, electrophysiological studies have demonstrated the existence of neurons in these two structures whose activities are specifically altered in response to intravenously (33) or iontophoretically (5) administered ANG-II. An alternate route by which the AP may receive and modify ‘Requests for reprints should be addressed
Rat
to Dr. A. V. Ferguson.
269
FIG. I. The photomic#~pb in (A) shows an exampfe of a recording electrode tract in the AP and NTS, as determined histologically. An example of a stimulating electrode site in the rostra1 dorsaf PBN is shown in (B). In each case, the ~ITOWindicates the location of the electrode tip. DPBN-dorsal PBN, SCP-superior cerebellar peduncle, VPBN- ventral PBN.
PARABRACHIAL
INFLUENCES
ON AREA POSTREMA
271
NEURONS
TABLE I
TABLE 2
SUMMARY OF THE DIFFERING RESPONSES OF AP AND NTS NEURONS TO ANG-II AND ADRENERGIC AGONISTS, AND THE FUNCTIONAL CLASSIFICATIONS OF THESE NEURONS ACCORDING TO SUCH RESPONSES-EXCITATION (EXC). INHIBITION (INH). NO EFFECT (NE)
MEAN LATENCIES AND DURATIONS OF RESPONSES OF ANG-II SENSITIVE, BP SENSITIVE. ANG-II/BP SENSITIVE. AND UNAFFECTED NEURONS IN THE AP AND NTS TO PBN STIMULATION-EXCITATION (EXC). INHIBITION (INH)
Classification of AP or NTS Neuron
ANG-II Sensitive BP Sensitive ANG-II/BP
Sensitive
Unaffected
Effect of ANG-II
EXC INH EXC INH EXC INH NE
Effect of Adrenergic Agonist
NE NE EXC
Cell Classification
ANG-II Sensitive AP Neurons NTS Neurons
INH
INH EXC NE
BP Sensitive AP Neurons NTS Neurons
of cells which by a systemic
respond to increases adrenergic agonist,
in blood pressure (BP) elicited and a third group of neurons
which respond to both ANG-II and increase in BP (33). In order to further define the physiological nature of these subpopulations of neurons, it is important to determine their neural connections. as well as the specificity of such connections to these functional subpopulations of neurons. In this study, neurons in the rat AP and NTS were classified according to their responses to systemic ANG-II or to an increase in BP elicited by both ANG-II and an adrenergic agonist. The PBN was stimulated electrically to determine if these identified AP and NTS cells received input from this region, and to examine the specific types of otthodromic influences received by functional subpopulations of AP and NTS neurons. METHOD
Male Sprague-Dawley rats (150-300 g) were anaesthetized with sodium pentobarbital (65 mg/kg). Each animal was fitted with an indwelling femoral arterial catheter (PE 50 Intramedic) to monitor BP, femoral and jugular venous catheters (PE 50 Intramedic) to administer agents or supplements of sodium pentobarbital when required, and an endotrachial tube to facilitate breathing. During the experiment, a feedback-controlled heating blanket was used to maintain body temperature at 37 ? 1°C. Animals were placed in a Kopf stereotaxic frame and a stimulating electrode positioned in the PBN according to the stereotaxic coordinates of Paxinos and Watson (34). A small burr hole was made 8.0 mm posterior to bregma and 1.7 mm lateral to the sagittal midline, and a monopolar or concentric bipolar stimulating electrode (Rhodes Medical Instruments) was lowered 6.5 mm ventral to the dura and attached to the skull using a jeweller’s screw and dental cement. The stimulating electrode was connected to an isolated stimulation unit programmed to deliver 0. I-ms current pulses, controlled by a digitimer. In order to expose the AP, the head of the animal was positioned vertically and a midline incision made. Both the atlantooccipital membrane and dura covering the AP were removed and the dorsal surface of the AP was then clearly visible. A pressure foot was positioned over the AP to allow for more stable recordings. Extracellular single unit recordings were obtained using NaCl (I .O M) filled glass recording electrodes (resistance 15-35 Ma, tip diameter 0.05, Table 2) seen between any of the subpopulations of neurons in the AP and NTS. However, a large range of times was recorded. This range of latency and duration times may be due to the various conduction velocities of neurons travelling from the PBN to the AP and NTS, and are dependent on the functional groups of PBN efferents activated during stimulation.
Various proportions of all of the groups of AP and NTS ncurons classified according to their responses to systemic ANG-II or adrenergic agonist were influenced by PBN stimulation. Both BP sensitive and ANG-II/BP sensitive cells in the NTS received a relatively stronger afferent input than did the ANG-II sensitivae and unaffected neurons in the NTS. There was, however, no apparent preferential distribution of PBN excitatory or inhibitory efferent information to any of the functional subpopulations identified in the AP or NTS. Further studies will be necessary to determine whether such afferent inputs from the PBN may be preferentially distributed to separate populations of AP and NTS neurons with different efferent projections. ACKNOWLEDGEMENTS The authors wish to thank Pauline Smith for her excellent technical help. This work was supported by the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario.
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