Brain Research, 517 (1990) 57-63 Elsevier
57
BRES 15469
Localization of vasodepressor neurons in the caudal ventrolateral medulla in the rabbit Yu-Wen Li and W.W. Blessing Departments of Medicine and Physiology, Centrefor Neuroscience, Flinders University of South Australia, Bedford Park, S.A. (Australia) (Accepted 17 October 1989) Key words: Caudal ventrolateral medulla; A1 cell group; Nucleus ambiguus; Sympathetic vasomotor tone; L-Glutamate; Rabbit
The precise distribution of vasodepressor neurons in the caudal ventrolateral medulla has been mapped in the rabbit using very small injections of L-glutamate (0.5 nmol in 25 nl or 0.1 nmol of 10 nl of Ringer solution). The vasodepressor neurons are discretely located in a circumscribed region of the lateral reticular formation, ventral and ventrolateral to the nucleus ambiguus, from 0.5 mm to 2.5 mm caudal to the obex. The results indicate that the vasodepressor neurons in the caudal ventrolateral medulla are confined to a relatively limited region, not corresponding to any defined nucleus.
INTRODUCTION The caudal ventrolateral medulla contains neurons whose activity reduces arterial pressure. The general position of these cells has been defined but the precise anatomical localization is not yet clear. Initial studies in rabbit and rat showed that the vasodepressor neurons are in the medullary area which contains the A1 noradrenaline-synthesizing neurons and these neurons were hypothesized to constitute the vasodepressor cells 3'23. The first study to question the specific localization of the vasodepressor neurons was conducted by D a y et al. 10 in the rat. These authors used focal electrical stimulation to excite the depressor neurons and found the vasodepressor sites to be concentrated in a segment of the nucleus ambiguus dorsomedial to the rostral one-third of the A1 cell group, extending caudally from the obex for approximately 0.5 mm 1°. They therefore suggested that the vasodepressor response could not be mediated by A1 neurons. Other studies in the rat used microinjections of excitatory pharmacological agents, stimulating cell bodies without affecting fibers of passage. These studies, using injection volumes of 100 nl, located the depressor region in the area containing the A1 cells 13A7'23. Studies using excitatory amino acid antagonists have located the depressor region in the same area 13'14'17. Our original study in the rabbit used both electrical stimulation and injections of L-glutamate in 250 nl of vehicle. More recently B o n h a m and Jeske 7 addressed the
question of the precise localization of the vasodepressor cells by microinjecting 1-12 nl of DL-homocysteic acid into the rat caudal ventrolateral medulla. They localized the vasodepressor neurons in a region extending caudally from the obex for approximately 1 mm. The region extended dorsoventrally for approximately 1.6 mm, from just ventral to the nucleus ambiguus, through the lateral reticular nucleus, to within 0.2 m m of the ventral surface of the brain. A t different rostrocaudal levels the vasodepressor neurons were either ventrolateral or medial to the A1 cells. In general B o n h a m and Jeske 7 locate the vasodepressor neurons more laterally and more ventrally than do D a y et al. 1°. Although their localization is different, Bonham and Jeske agree that the vasodepressor neurons are distinct from the A1 cells. Other evidence obtained in the rat and the rabbit also argued against identifying the vasodepressor neurons with the A1 cells 5'6'22'24. The present study, using small injections of L-glutamate and careful localization of injection sites, was conducted in the rabbit to obtain a more detailed picture of the localization of the vasodepressor neurons in this species. MATERIALS AND METHODS Thirty-eight male New Zealand White rabbits (2.5-3.0 kg) were anesthetized with urethan (1.4 g/kg, i.v.), paralyzed with pancuronium bromide (0.25 mg/kg i.v. with supplemental doses as necessary) and artificially ventilated with oxygen-enriched air. End tidal pCO 2 was kept in the range of 30-35 mm Hg. Rectal temperature
Correspondence: Y.-W. Li, Department of Medicine, Centre for Neuroscience, Hinders Medical Centre, Bedford Park, S.A. 5042, Australia. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
58
1.5ram
2oo1
II
ARTERIAL PRESSURE I00 (mm Hg)
lmln
lmln 0
E
L-glutamate (0.5 nmol in 25 nl)
200 "7
IV
III
ARTERIAL PRESSURE 100 I I (mm Hg)
1
OIi,
F
L-glutamate ( 0 5 nmol in 25 nl)
v
lmln *L-glutamate (0.1 nmol in lOnl)
V tltlltt
Imln * L-glutamate (01 nmol ~n l O n l )
]FIUI
* L-glutamate (01 nmol In lOnl)
Fig. 1. A and B are diagrams of hemisections of medulla 1 mm caudal to the obex showing approximate locations of injection sites (I-V) marked by black dots. C and D are darkfield and brightfield, respectively, photomicrographs showing the locations of these injection sites (I, II from different rabbits and III, IV, V from a single rabbit) demonstrated by silver particles. E and F are polygraph records showing arterial pressure (AP) responses after injections of L-glutamate (0.5 nmol in 25 nl) into sites I and II or L-glutamate (0.1 nmol in 10 nl) into sites III, IV, V. io, inferior olivary nucleus; nTS, nucleus tractus solitarius; nVsp, spinal nucleus of trigeminal nerve; nA, nucleus ambiguus; LRn, lateral reticular nucleus; XII, hypoglossal nucleus.
59 was maintained at 39 + 0.5 °C. Pulsatile arterial pressure (AP) was recorded with a Statham P23 ID transducer and a Grass model 7 polygraph. Heart rate (HR) was obtained from the phasic arterial signal using a Grass model 7P4F taehograph. The animals were placed in a modified Kopf stereotaxic holder and the medulla was exposed, with neck flexion adjusted such that the dorsal surface of the medulla was horizontal. Rostrocaudal, mediolateral and dorsoventral stereotaxic coordinates were defined with reference to the obex (defined as the rostral edge of area postrema in the midline), the midline and the dorsal medullary surface. L-Glutamate monosodium (Sigma Chemical Co.) was dissolved in Ringer solution. Unilateral injections were made by transmitting pressure from a hand-held microsyringe to a glass micropipette (tip o.d., 10-20 ~m) through connecting polyethylene tubing. The volume injected was monitored by measuring the movement of the fluid meniscus within the pipette with a binocular operating microscope equipped with a graticule in the eyepiece. The injectate also contained concanavalin A-gold (Sigma Chemical Co., 1:10 dilution) to enable subsequent localization of the injection site using silver intensification of gold9,15. In a first group of experiments 0.5 nmol L-glutamate in 25 nl Ringer solution was used. The medullary area explored extended from 0.0 mm to 3.0 mm caudal to the obex, from 2.0 mm to 3.5 mm lateral to the midline and from 2.0 mm to 3.5 mm ventral to the dorsal surface of the medulla. Effect on AP and HR was recorded. In a second group of experiments a subregion of the caudal ventrolateral medulla, from which large falls in AP were recorded in the first experiments, was selected and 0.1 nmol L-glutamate in 10 nl Ringer solution was used for injection. This subregion extended from 0.5 mm to 1.5 mm caudal to the obex, from 2.5 mm to 3.5 mm lateral to the midline and from 2.5 mm to 3.5 mm ventral to the dorsal surface of the medulla. In both groups of experiments the interval between injection sites was 0.5 mm. At least 6 injections were made at each one of the injection sites. In an individual rabbit only one injection was made at any site. In addition, because of the problem of diffusion of injectate along the tract made by the micropipette, only one injection was made in any one dorsoventral penetration. At the end of each experiment rabbits were perfused transcardially with buffered aldehyde fixative and the medulla was sectioned transversally on a Vibratome. Sections were processed using silver intensification of the gold particles9"15 and counterstained with Neutral red. Sections were examined with an Olympus BH2 microscope fitted with a darkfield condenser. Statistical analysis was performed using Student's two-tailed paired t-test to compare preand postinjection AP and HR values. All values are expressed as the means + S.E.M. The significance level was set at P < 0.01.
RESULTS L - G l u t a m a t e injections into the v a s o d e p r e s s o r region caused falls in A P and H R as previously d o c u m e n t e d 3. Fig. 1 shows e x a m p l e s of v a s o d e p r e s s o r responses and the a c c o m p a n y i n g histological localization of the injection sites. By including concanavalin A - g o l d with the injectate we were able to clearly define these sites, especially using darkfield illumination (Fig. 1C). W i t h brightfield illumination it was possible to identify the n e u r o n a l architecture in the injection site (Fig. 1D). In the first group of experiments with microinjections of 25 nl containing 0.5 nmol L-glutamate, occasional small d e p r e s s o r responses were o b s e r v e d at the level of the obex, but the p o o l e d results showed that no significant falls were o b s e r v e d at this level (Table I and Fig. 2A). A s
the injection sites b e c a m e m o r e caudal the d e p r e s s o r responses b e c a m e larger, with the v a s o d e p r e s s o r area increasing in d o r s o v e n t r a l and m e d i o l a t e r a l extent until at 1.0 m m caudal to the o b e x the d e p r e s s o r response reached the m a x i m u m level and the d e p r e s s o r region occupied the largest extent (Fig. 2C, Fig. 3A). Then, m o r e caudally in the m e d u l l a o b l o n g a t a , the depressor region gradually r e d u c e d in extent, associated with reduction in the magnitude of the v a s o d e p r e s s o r response (Table I and Fig. 3B). A t the level 3 m m caudal to the obex no significant d e p r e s s o r responses were observed. Thus significant d e p r e s s o r responses were o b s e r v e d only within a relatively limited region, extending from 0.5 m m caudal to 2.5 m m caudal to the obex, forming an irregularly cylindrical column. This column is located in the medullary reticular f o r m a t i o n , ventral and ventrolateral to the nucleus ambiguus and dorsal to the lateral reticular nucleus, situated in the v e n t r o l a t e r a l p o r t i o n of the area designated as subnucleus reticularis ventralis by Meessen and Olszewski 18. O u r histological analysis showed that the v a s o d e p r e s s o r region also included the dorsal margin of the lateral reticular nucleus and the caudal part of the nucleus ambiguus (Fig. 2 C - E ) . The largest depressor response was o b s e r v e d ventrolateral to the nucleus ambiguus, 1.0 m m caudal to the obex, 3.0 m m lateral to the midline and 3.0 m m b e l o w the dorsal surface of the m e d u l l a (Table I, Fig. 3 A , B ) . In the second group of e x p e r i m e n t s , using 0.1 nmol of L-glutamate in 10 nl, the d e p r e s s o r responses were r e d u c e d in m a g n i t u d e in c o m p a r i s o n to the 0.5-nmol dose (Table II). A t the site which y i e l d e d the largest d e p r e s s o r response with 0.5 nmol ( - 3 4 + 2 m m Hg) the smaller dose yielded a fall of 8 + 1 m m Hg, significantly less than the fall o b t a i n e d with the larger dose ( P < 0.01). A l t h o u g h the responses were smaller, significant reductions in A P were still e v o k e d from virtually all of the sites from which larger d e p r e s s o r responses were r e c o r d e d in the first group of experiments. C o n s e q u e n t l y the depressor region defined by the 0.1-nmol dose occupied a p p r o x i m a t e l y the same volume as the region defined by the 0.5-nmol dose. N o significant rises in either A P o r H R were o b s e r v e d at any of the e x a m i n e d r o s t r o c a u d a l levels in either the first or the second series of experiments. A t m a n y sites we o b s e r v e d decreases in H R up to a m a x i m u m fall of 71 + 17 beats/min from a resting rate of 319 __. 17 beats/min at caudal 1.5 mm, lateral 2.5 ram, ventral 3.5 mm. We e x a m i n e d the relationship b e t w e e n fall in A P and fall in H R for c o m b i n e d d a t a o b t a i n e d from all sites yielding significant v a s o d e p r e s s o r responses to injection of 0.5 nmol of L-glutamate in 25 nl. T h e correlation coefficient was 0.63 ( P < 0.01, n = 318 injections).
60
,5
¢ -1. .0 Fig. 2. Photomicrographs (left half of each section) and diagrams (right half of each section) of Nissl-stained transverse sections from 0.0 mm (A) to 3.0 mm (G) caudal to the obex showing location of the vasodepressor region mapped by injections of L-glutamate (0.5 nmol in 25 nl). Black circles represent significant reductions in AP (P < 0.01), open circles represent non-significant reductions in AP. Size of each circle is proportional to magnitude of reduction in A, from less than 10 mm Hg (smallest circle) to more than 30 mm Hg (largest circle), ap, area postrema; io, inferior olivary nucleus; nTs, nucleus tractus solitarius; nVsp, spinal nucleus of trigeminal nerve; nA, nucleus ambiguus; lrn, lateral reticular nucleus; XII, hypoglossal nucleus; pyr, pyramid.
DISCUSSION In the present study we have confirmed that vasodepressor neurons in the rabbit ventrolateral medulla are located in a circumscribed region of the medullary reticular formation. In contrast to studies in the rat 7A3, we did not observe any pressor responses in the region we examined. Pressor responses to L-glutamate injections in the rabbit have been o b t a i n e d rostral to the obex where s y m p a t h o e x c i t a t o r y v a s o m o t o r neurons have been characterized 8. In agreement with this finding we also
observed large increases in A P when we injected 0.5 nmol of L-glutamate into the rostral a r e a defined by D a m p n e y et al. 8. We have previously shown that neither vagal b l o c k a d e with peripherally acting muscarinic cholinergic antagonists nor c o m b i n e d vagal and cardiac sympathetic b l o c k a d e with muscarinic cholinergic antagonists and fl-adrenergic blockers alters the fall in A P o b t a i n e d with injection of L-glutamate into the caudal ventrolateral m e d u l l a 3 and direct recordings from peripheral sympathetic nerves d e m o n s t r a t e that L-glutamate injections into the caudal ventrolateral m e d u l l a cause a
61 profound inhibition of peripheral sympathetic tone 6. It is therefore reasonable to assume that the depressor responses observed in the present study reflect inhibition of sympathetic vasomotor tone. By using very small injections of L-glutamate and precise localization of injection sites we have more fully characterized the position of the vasodepessor cells. They are located just ventral to the nucleus ambiguus, extending rostrocaudally from 0.5 mm to 2.5 mm caudal to the obex. Throughout this longitudinal extent the vasodepressor cells are situated between the nucleus ambiguus and the lateral reticular nucleus. The region is not specifically designated in the available atlas in the rabbit TM and it is variously designated in the rat 1'16'2°. Previous detailed studies have shown that the A1 catecholamine-synthesizing neurons are situated in this region in the rabbit 2'4. Our present study shows that most
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Fig. 3. A : d e p r e s s o r r e s p o n s e s e l i c i t e d b y i n j e c t i o n s o f 0.5 n m o l L - g l u t a m a t e in 25 nl at v a r y i n g d i s t a n c e s f r o m the m i d l i n e a n d f r o m the d o r s a l surface of the m e d u l l a , at the c o r o n a l level 1.00 m m c a u d a l to the o b e x . B: d e p r e s s o r r e s p o n s e s elicited at v a r y i n g d i s t a n c e s f r o m the o b e x a n d f r o m the d o r s a l surface of the m e d u l l a , at the s a g i t t a l level 3.00 m m l a t e r a l to the m i d l i n e .
of the vasodepressor neurons are located just medial to the region containing the A1 cells. However the position of the vasodepressor neurons does overlap with the medial portion of the region containing the A1 neurons. Thus on localization criteria alone a subgroup of the A1 neurons could be involved in the vasodepressor response. This conclusion contrasts with that of Day et al. 1° and Bonham and Jeske 7. These authors found that the region from which the vasodepressor response was elicited did not overlap with the position of any A1 cells. However, the two groups of investigators differ in their description of the anatomical relationship between the position of the vasodepressor neurons and the position of the A1 cells. Day et al. 1° locate the vasodepressor neurons in the nucleus ambiguus, dorsomedial to the A1 cells. Bonham and Jeske 7 locate the vasodepressor neurons either ventrolateral to the A1 cells or medial to the A1 cells, depending on the particular rostrocaudal level. In our present study the vasodepressor region demonstrated with 0.1 nmol of L-glutamate in 10 nl was similar in extent to the region demonstrated with 0.5 nmol of L-glutamate in 25 nl. This suggests that the neurons responsible for the vasodepressor effect are spread throughout the vasodepressor region and are certainly not concentrated in the region containing the A1 cells. With 0.5 nmol of L-glutamate in 25 nl (a concentration of 20 mM) the fall in AP after injections in the centre of the depressor area was 34 + 1 mm Hg. In our original study L-glutamate was dissolved in 250 nl of vehicle. Such a large volume would presumably result in excitation of neurons situated at some distance from the center of the injection site. However, the fall in AP recorded using the same concentration of L-glutamate (ref. 3, Fig. 6 dose-response relationship) was of similar magnitude to the fall in the present study. This suggests that the vasodepressor neurons are present in a limited region of the caudal ventrolateral medulla, in agreement with the findings summarized in Fig. 2 of the present study. More detailed studies using the focal microinjection approach are unlikely to resolve the question as to the neurochemical identity of the vasodepressor neurons, particularly because neurons in the area often have extensive dendritic processes, sometimes extending as far as 1 mm from the perikaryon 4. This means that both focal electrical stimulation and local injections of neuroactive pharmacological agents could directly affect neuronal firing by an action some distance from the perikaryon. Fortunately, evidence obtained from alternative experimental models bears on the question of the identity of the vasodepressor cells. In both rabbits and rats it appears that these neurons act by inhibiting sympathoexcitatory neurons in the rostral ventrolateral medulla 6"24. This
62 TABLE I Changes (mean ± S.E.M.) in arterial pressure (in mm Hg) after injection of L-glutamate (0.5 nmol in 25 nl) in the caudal ventrolateral medulla oblongata of the rabbit. Each value is based on data obtained from 6 rabbits. *P < 0.01 compared with preinjection values.
Distance caudal Distancefrom to obex (mm) dorsalsurface of medulla (ram)
Distance from midline (ram) 2.0
2.5
3.0
3.5
-0.0
2.0 2.5 3.0 3.5
1~±2 1~±4 94±4 92±2
0±1 0±1 -8±3 -3±1
92±4 88±3 86±4 86±3
-2±2 0±1 -4±2 -7±2
87±4 93±3 90±2 88±5
0±1 -1±1 -4±1 -4±1
-0.5
2.0 2.5 3.0 3.5
90±3 92_+5 101±4 97±2
-3±1 -2±1 -1±1 -6±2
91_+3 85±4 91±4 88±5
-2±1 -5±2 -19±4" -15±5"
93±3 91±3 93±4 87±5
-0±1 -3±1 -19±4" -20±4*
98±4 97±4 97±5 95_+4
-4±4 -0±1 -6±1 -15±6"
-1.0
2.0 2.5 3.0 3.5
98±1 101_+4 95±2 95±4
-3±2 -6±2 -9±1" -4±2
91_+2 91±6 87±1 87±3
-5_+1 -19±4" -15_+1" -16±2"
93±2 93±6 101_+1 86_+7
-1±1 -25±4* -34_+1" -23±2*
96±1 90±4 96±1 95±2
0±1 -5±2 -7±2 -12±3"
-1.5
2.0 2.5 3.0 3.5
94±3 93_+3 98±2 100±3
-6±2 -8±1 -4±1 -7±4
85±3 93±2 97_+4 99±4
-4±2 -12±4" -21±5" -15_+3"
87±3 89±4 95±3 93±4
-6±3 18±4" -25±2* -16±2"
94±6 99±3 94_+3 92±4
-1±1 -4±3 -4±3 -2±2
-2.0
2.0 2.5 3.0 3.5
90±4 94±4 92±3 88±2
-3±1 -9±3* -6±2 -6±1
94±4 96±5 92±4 91_+4
-5_+2 -15±4" -12±2" -8±3
93±4 92±3 94±5 93_+4
-9_+1" -15±5" -10±4" -4±2
94±3 91±3 91±3 93±2
0±1 -3±1 -2±1 -2±1
-2.5
2.0 2.5 3.0 3.5
96±4 95±4 98±5 97±6
-5±1 -6±3 -3±2 -5±3
94_+2 1~±3 94±7 101±3
-5±1 -7±2 -10±2" -4±1
92_+5 99±3 93±3 96±4
-8±2* -8±1" -6_+2 -5±2
97_+3 98±3 103±2 99±2
-1±1 -6±3 -3_+2 -4±2
-3.0
2.0 2.5 3.0
98±5 93±4 94±4
-6±2 -4±1 -8±3
92±5 94±4 93±4
-3±1 -6±2 -2±1
98±5 93±4 92±4
-3_+1 -2±1 -4_+2
strongly suggests that the vasodepressor cells have a short projection to the rostral region. Double-label retrograde
99±2 96±2 1~±3 97±4
0±1 0±2 -2±2 -2±1
tracing studies have failed to show that any A1 cells project to the rostral sympathoexcitatory area in either
TABLE II Changes (mean + S.E.M.) in arterial pressure (in mm Hg) after injection of L-glutamate (0.1 nmol in 10 nl). Each value is based on data obtained from 6 rabbits. *P < 0.01 compared with preinjection values.
Distance from midline (mm)
Distance caudal to obex (mm)
Distance from dorsal surface of medulla (ram)
-0.5
2.5 3.0 3.5
95±3 93_+2 93±2
-4_+3 -7_+1" -11_+2"
100±3 100±3 98_+3
-3_+2 -8_+2* -12_+2"
-1.0
2.5 3.0 3.5
94+6 93+ 6 98+5
-6+1" - 8 + 1" -6__+2*
94+5 94+ 5 94_+5
-1.5
2.5 3.0 3.5
103 + 7 104+6 103+6
-7-+ 2* -11+4" -8+1"
100 + 5 99+6 97+5
2.5
3.0
3.5 98_+5 96±4 98±5
-4±3 -3±1 -3_+1
-7+1" - 9 + 1" -8_+1"
95+6 90+6 95+5*
-3__ +1 -2+ 1 -4_+1
- 7 + i* - 1 0 + 1" -5+1"
99 + 4 105+6 102+5
-3 + 1 -2+1 -1+1
63 the rabbit or the rat 5'22. F u r t h e r m o r e , pharmacological
some of which project to the rostral ventrolateral medulla
evidence suggests that the vasodepressor n e u r o n s inhibit
(manuscript in preparation) and it is therefore possible
the rostral n e u r o n s by activating G A B A e r g i c receptors in the rostral region 6. N e u r o a n a t o m i c a l studies using antibodies to glutamic acid decarboxylase ( G A D ) demonstrate that n e u r o n s which synthesize G A B A are spread widely throughout the rat medulla oblongata, including the ventrolateral medulla 19'21. The rabbit caudal ventro-
that the vasodepressor n e u r o n s are G A B A - s y n t h e s i z i n g cells. The location of the vasodepressor sites as defined in the present study is consistent with this hypothesis.
lateral medulla also contains G A D - p o s i t i v e neurons,
Acknowledgements. This work was supported by the National Health and Medical Research Council and The National Heart Foundation of Australia. We are grateful to Dr Guy Drolet for reading our manuscript.
REFERENCES 1 Andrezik, J.A., Chan-Palay, V. and Palay, S.L., The nucleus paragigantocellularis lateralis. Conformation and cytology, Anat. Embryol., 161 (1981) 356-371. 2 Blessing, W.W., Chalmers, J.P. and Howe, P.R.C., Distribution of catecholamine-containing cell bodies in the rabbit central nervous system, J. Comp. Neurol., 179 (1978) 407-423. 3 Blessing, W.W. and Reis, D.J., Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing A 1 noradrenergic cells, Brain Research, 253 (1982) 161-171. 4 Blessing, W.W., Howe, P.R.C., Joh, T.H., Oliver, J.R. and Willoughby, J.O., Distribution of tyrosine hydroxylase and neuropeptide Y-like immunoreactive neurons in rabbit medulla oblongata, with attention to colocalization studies, presumptive adrenaline-synthesizing perikarya, and vagal pre-ganglionic neurons, J. Comp. Neurol., 248 (1986) 285-300. 5 Blessing, W.W., Hedger, S.C., Joh, T.H. and Willoughby, J.O., Neurons in the area postrema are the only catecholaminesynthesizing cells in the medulla or ports with projections to the rostral ventrolateral medulla (Cl-area) in the rabbit, Brain Research, 419 (1987) 336-340. 6 Blessing, W.W., Depressor neurons in rabbit caudal medulla act via GABA receptors in rostral medulla, Am. J. Physiol., 254 (1988) H686-H692. 7 Bonham, A.C. and Jeske, I., Cardiorespiratory effects of DL-hOmocysteic acid in caudal ventrolateral medulla, Am. J. Physiol., 256 (1989) H688-H696. 8 Dampney, R.A.L., Goodchild, A.K., Robertson, L.G. and Montgomery, W., Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study, Brain Research, 249 (1982) 223-235. 9 Danscher, G., Localization of gold in biological tissue. A photochemical method for light and electron microscopy, H/stochemistry, 71 (1981) 81-88. 10 Day, T.A., Ro, A. and Renaud, L.P., Depressor area within caudal ventrolateral medulla of the rat does not correspond to the Al-catecholamine cell group, Brain Research, 279 (1983) 299-302. 11 Goodchild, A.K., Dampney, R.A.L. and Bandler, R., A method of evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system, J. Neurosci. Meth., 6 (1982) 351-363. 12 Gordon, F.J., Aortic reflexes are mediated by NMDA receptors
13 14 15
16 17 18 19
20 21
22
23
24
in caudal ventrolateral medulla, Am. J. Physiol., 252 (1987) R628-R633. Gordon, EJ. and McCann, L., Pressor responses evoked by microinjection of L-glutamate into the caudal ventrolateral medulla of the rat, Brain Research, 457 (1988) 251-258. Guyenet, EG., Filtz, T.M. and Donaldson, S.R., Role of excitatory amino acids in rat vagal and sympathetic baroreflexes, Brain Research, 407 (1987) 272-284. Hodgson, A.J., Minson, J., Chalmers, J. and Pilowsky, P., The use of microinjected colloidal gold and immunocytochemistry to localise pressor sites in the rostral medulla oblongata of the rat, Neurosci. Lett., 77 (1987) 125-130. Kalia, M. and Fuxe, K., Rat medulla oblongata. I. Cytoarchitectonic considerations, J. Comp. Neurol., 233 (1983) 285-307. Kubo, T. and Kihara, M., N-Methyl-D-aspartate receptors mediate tonic vasodepressor control in the caudal ventrolateral medulla of the rat, Brain Research, 451 (1988) 366-370. Meessen, H. and Olszewski, J., A Cytoarchitectonic Atlas of the Rhomencephalon of the Rabbit, S. Karger, Basel, New York, 1949. Mugnaini, E. and Oertel, W.H., An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4, GABA and Neuropeptides in the CNS, Part 1, Elsevier, Amsterdam, 1985, pp. 436-608. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. Ruggiero, D.A., Meeley, M.P., Anwar, M. and Reis, D.J., Newly identified GABAergic neurons in regions of the ventrolateral medulla which regulate blood pressure, Brain Research, 339 (1985) 171-177. Sun, M.-K. and Guyenet, P.G., Effect of clonidine and gamma-aminobutyric acid on the discharges of medulla-spinal sympathoexcitatory neurons in the rat, Brain Research, 368 (1986) 1-17. Wiilette, R.N., Barcas, P.P., Krieger, A.J. and Sapru, H.N., Vasopressor and depressor areas in the rat medulla: identification by microinjection of L-glutamate, Neuropharmacology, 22 (1983) 1071-1079. Willette, R.N., Punnen, S., Krieger, A.J. and Sapru, H.N., Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure, Brain Research, 321 (1984) 169-174.
57
BRES 15469
Localization of vasodepressor neurons in the caudal ventrolateral medulla in the rabbit Yu-Wen Li and W.W. Blessing Departments of Medicine and Physiology, Centrefor Neuroscience, Flinders University of South Australia, Bedford Park, S.A. (Australia) (Accepted 17 October 1989) Key words: Caudal ventrolateral medulla; A1 cell group; Nucleus ambiguus; Sympathetic vasomotor tone; L-Glutamate; Rabbit
The precise distribution of vasodepressor neurons in the caudal ventrolateral medulla has been mapped in the rabbit using very small injections of L-glutamate (0.5 nmol in 25 nl or 0.1 nmol of 10 nl of Ringer solution). The vasodepressor neurons are discretely located in a circumscribed region of the lateral reticular formation, ventral and ventrolateral to the nucleus ambiguus, from 0.5 mm to 2.5 mm caudal to the obex. The results indicate that the vasodepressor neurons in the caudal ventrolateral medulla are confined to a relatively limited region, not corresponding to any defined nucleus.
INTRODUCTION The caudal ventrolateral medulla contains neurons whose activity reduces arterial pressure. The general position of these cells has been defined but the precise anatomical localization is not yet clear. Initial studies in rabbit and rat showed that the vasodepressor neurons are in the medullary area which contains the A1 noradrenaline-synthesizing neurons and these neurons were hypothesized to constitute the vasodepressor cells 3'23. The first study to question the specific localization of the vasodepressor neurons was conducted by D a y et al. 10 in the rat. These authors used focal electrical stimulation to excite the depressor neurons and found the vasodepressor sites to be concentrated in a segment of the nucleus ambiguus dorsomedial to the rostral one-third of the A1 cell group, extending caudally from the obex for approximately 0.5 mm 1°. They therefore suggested that the vasodepressor response could not be mediated by A1 neurons. Other studies in the rat used microinjections of excitatory pharmacological agents, stimulating cell bodies without affecting fibers of passage. These studies, using injection volumes of 100 nl, located the depressor region in the area containing the A1 cells 13A7'23. Studies using excitatory amino acid antagonists have located the depressor region in the same area 13'14'17. Our original study in the rabbit used both electrical stimulation and injections of L-glutamate in 250 nl of vehicle. More recently B o n h a m and Jeske 7 addressed the
question of the precise localization of the vasodepressor cells by microinjecting 1-12 nl of DL-homocysteic acid into the rat caudal ventrolateral medulla. They localized the vasodepressor neurons in a region extending caudally from the obex for approximately 1 mm. The region extended dorsoventrally for approximately 1.6 mm, from just ventral to the nucleus ambiguus, through the lateral reticular nucleus, to within 0.2 m m of the ventral surface of the brain. A t different rostrocaudal levels the vasodepressor neurons were either ventrolateral or medial to the A1 cells. In general B o n h a m and Jeske 7 locate the vasodepressor neurons more laterally and more ventrally than do D a y et al. 1°. Although their localization is different, Bonham and Jeske agree that the vasodepressor neurons are distinct from the A1 cells. Other evidence obtained in the rat and the rabbit also argued against identifying the vasodepressor neurons with the A1 cells 5'6'22'24. The present study, using small injections of L-glutamate and careful localization of injection sites, was conducted in the rabbit to obtain a more detailed picture of the localization of the vasodepressor neurons in this species. MATERIALS AND METHODS Thirty-eight male New Zealand White rabbits (2.5-3.0 kg) were anesthetized with urethan (1.4 g/kg, i.v.), paralyzed with pancuronium bromide (0.25 mg/kg i.v. with supplemental doses as necessary) and artificially ventilated with oxygen-enriched air. End tidal pCO 2 was kept in the range of 30-35 mm Hg. Rectal temperature
Correspondence: Y.-W. Li, Department of Medicine, Centre for Neuroscience, Hinders Medical Centre, Bedford Park, S.A. 5042, Australia. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
58
1.5ram
2oo1
II
ARTERIAL PRESSURE I00 (mm Hg)
lmln
lmln 0
E
L-glutamate (0.5 nmol in 25 nl)
200 "7
IV
III
ARTERIAL PRESSURE 100 I I (mm Hg)
1
OIi,
F
L-glutamate ( 0 5 nmol in 25 nl)
v
lmln *L-glutamate (0.1 nmol in lOnl)
V tltlltt
Imln * L-glutamate (01 nmol ~n l O n l )
]FIUI
* L-glutamate (01 nmol In lOnl)
Fig. 1. A and B are diagrams of hemisections of medulla 1 mm caudal to the obex showing approximate locations of injection sites (I-V) marked by black dots. C and D are darkfield and brightfield, respectively, photomicrographs showing the locations of these injection sites (I, II from different rabbits and III, IV, V from a single rabbit) demonstrated by silver particles. E and F are polygraph records showing arterial pressure (AP) responses after injections of L-glutamate (0.5 nmol in 25 nl) into sites I and II or L-glutamate (0.1 nmol in 10 nl) into sites III, IV, V. io, inferior olivary nucleus; nTS, nucleus tractus solitarius; nVsp, spinal nucleus of trigeminal nerve; nA, nucleus ambiguus; LRn, lateral reticular nucleus; XII, hypoglossal nucleus.
59 was maintained at 39 + 0.5 °C. Pulsatile arterial pressure (AP) was recorded with a Statham P23 ID transducer and a Grass model 7 polygraph. Heart rate (HR) was obtained from the phasic arterial signal using a Grass model 7P4F taehograph. The animals were placed in a modified Kopf stereotaxic holder and the medulla was exposed, with neck flexion adjusted such that the dorsal surface of the medulla was horizontal. Rostrocaudal, mediolateral and dorsoventral stereotaxic coordinates were defined with reference to the obex (defined as the rostral edge of area postrema in the midline), the midline and the dorsal medullary surface. L-Glutamate monosodium (Sigma Chemical Co.) was dissolved in Ringer solution. Unilateral injections were made by transmitting pressure from a hand-held microsyringe to a glass micropipette (tip o.d., 10-20 ~m) through connecting polyethylene tubing. The volume injected was monitored by measuring the movement of the fluid meniscus within the pipette with a binocular operating microscope equipped with a graticule in the eyepiece. The injectate also contained concanavalin A-gold (Sigma Chemical Co., 1:10 dilution) to enable subsequent localization of the injection site using silver intensification of gold9,15. In a first group of experiments 0.5 nmol L-glutamate in 25 nl Ringer solution was used. The medullary area explored extended from 0.0 mm to 3.0 mm caudal to the obex, from 2.0 mm to 3.5 mm lateral to the midline and from 2.0 mm to 3.5 mm ventral to the dorsal surface of the medulla. Effect on AP and HR was recorded. In a second group of experiments a subregion of the caudal ventrolateral medulla, from which large falls in AP were recorded in the first experiments, was selected and 0.1 nmol L-glutamate in 10 nl Ringer solution was used for injection. This subregion extended from 0.5 mm to 1.5 mm caudal to the obex, from 2.5 mm to 3.5 mm lateral to the midline and from 2.5 mm to 3.5 mm ventral to the dorsal surface of the medulla. In both groups of experiments the interval between injection sites was 0.5 mm. At least 6 injections were made at each one of the injection sites. In an individual rabbit only one injection was made at any site. In addition, because of the problem of diffusion of injectate along the tract made by the micropipette, only one injection was made in any one dorsoventral penetration. At the end of each experiment rabbits were perfused transcardially with buffered aldehyde fixative and the medulla was sectioned transversally on a Vibratome. Sections were processed using silver intensification of the gold particles9"15 and counterstained with Neutral red. Sections were examined with an Olympus BH2 microscope fitted with a darkfield condenser. Statistical analysis was performed using Student's two-tailed paired t-test to compare preand postinjection AP and HR values. All values are expressed as the means + S.E.M. The significance level was set at P < 0.01.
RESULTS L - G l u t a m a t e injections into the v a s o d e p r e s s o r region caused falls in A P and H R as previously d o c u m e n t e d 3. Fig. 1 shows e x a m p l e s of v a s o d e p r e s s o r responses and the a c c o m p a n y i n g histological localization of the injection sites. By including concanavalin A - g o l d with the injectate we were able to clearly define these sites, especially using darkfield illumination (Fig. 1C). W i t h brightfield illumination it was possible to identify the n e u r o n a l architecture in the injection site (Fig. 1D). In the first group of experiments with microinjections of 25 nl containing 0.5 nmol L-glutamate, occasional small d e p r e s s o r responses were o b s e r v e d at the level of the obex, but the p o o l e d results showed that no significant falls were o b s e r v e d at this level (Table I and Fig. 2A). A s
the injection sites b e c a m e m o r e caudal the d e p r e s s o r responses b e c a m e larger, with the v a s o d e p r e s s o r area increasing in d o r s o v e n t r a l and m e d i o l a t e r a l extent until at 1.0 m m caudal to the o b e x the d e p r e s s o r response reached the m a x i m u m level and the d e p r e s s o r region occupied the largest extent (Fig. 2C, Fig. 3A). Then, m o r e caudally in the m e d u l l a o b l o n g a t a , the depressor region gradually r e d u c e d in extent, associated with reduction in the magnitude of the v a s o d e p r e s s o r response (Table I and Fig. 3B). A t the level 3 m m caudal to the obex no significant d e p r e s s o r responses were observed. Thus significant d e p r e s s o r responses were o b s e r v e d only within a relatively limited region, extending from 0.5 m m caudal to 2.5 m m caudal to the obex, forming an irregularly cylindrical column. This column is located in the medullary reticular f o r m a t i o n , ventral and ventrolateral to the nucleus ambiguus and dorsal to the lateral reticular nucleus, situated in the v e n t r o l a t e r a l p o r t i o n of the area designated as subnucleus reticularis ventralis by Meessen and Olszewski 18. O u r histological analysis showed that the v a s o d e p r e s s o r region also included the dorsal margin of the lateral reticular nucleus and the caudal part of the nucleus ambiguus (Fig. 2 C - E ) . The largest depressor response was o b s e r v e d ventrolateral to the nucleus ambiguus, 1.0 m m caudal to the obex, 3.0 m m lateral to the midline and 3.0 m m b e l o w the dorsal surface of the m e d u l l a (Table I, Fig. 3 A , B ) . In the second group of e x p e r i m e n t s , using 0.1 nmol of L-glutamate in 10 nl, the d e p r e s s o r responses were r e d u c e d in m a g n i t u d e in c o m p a r i s o n to the 0.5-nmol dose (Table II). A t the site which y i e l d e d the largest d e p r e s s o r response with 0.5 nmol ( - 3 4 + 2 m m Hg) the smaller dose yielded a fall of 8 + 1 m m Hg, significantly less than the fall o b t a i n e d with the larger dose ( P < 0.01). A l t h o u g h the responses were smaller, significant reductions in A P were still e v o k e d from virtually all of the sites from which larger d e p r e s s o r responses were r e c o r d e d in the first group of experiments. C o n s e q u e n t l y the depressor region defined by the 0.1-nmol dose occupied a p p r o x i m a t e l y the same volume as the region defined by the 0.5-nmol dose. N o significant rises in either A P o r H R were o b s e r v e d at any of the e x a m i n e d r o s t r o c a u d a l levels in either the first or the second series of experiments. A t m a n y sites we o b s e r v e d decreases in H R up to a m a x i m u m fall of 71 + 17 beats/min from a resting rate of 319 __. 17 beats/min at caudal 1.5 mm, lateral 2.5 ram, ventral 3.5 mm. We e x a m i n e d the relationship b e t w e e n fall in A P and fall in H R for c o m b i n e d d a t a o b t a i n e d from all sites yielding significant v a s o d e p r e s s o r responses to injection of 0.5 nmol of L-glutamate in 25 nl. T h e correlation coefficient was 0.63 ( P < 0.01, n = 318 injections).
60
,5
¢ -1. .0 Fig. 2. Photomicrographs (left half of each section) and diagrams (right half of each section) of Nissl-stained transverse sections from 0.0 mm (A) to 3.0 mm (G) caudal to the obex showing location of the vasodepressor region mapped by injections of L-glutamate (0.5 nmol in 25 nl). Black circles represent significant reductions in AP (P < 0.01), open circles represent non-significant reductions in AP. Size of each circle is proportional to magnitude of reduction in A, from less than 10 mm Hg (smallest circle) to more than 30 mm Hg (largest circle), ap, area postrema; io, inferior olivary nucleus; nTs, nucleus tractus solitarius; nVsp, spinal nucleus of trigeminal nerve; nA, nucleus ambiguus; lrn, lateral reticular nucleus; XII, hypoglossal nucleus; pyr, pyramid.
DISCUSSION In the present study we have confirmed that vasodepressor neurons in the rabbit ventrolateral medulla are located in a circumscribed region of the medullary reticular formation. In contrast to studies in the rat 7A3, we did not observe any pressor responses in the region we examined. Pressor responses to L-glutamate injections in the rabbit have been o b t a i n e d rostral to the obex where s y m p a t h o e x c i t a t o r y v a s o m o t o r neurons have been characterized 8. In agreement with this finding we also
observed large increases in A P when we injected 0.5 nmol of L-glutamate into the rostral a r e a defined by D a m p n e y et al. 8. We have previously shown that neither vagal b l o c k a d e with peripherally acting muscarinic cholinergic antagonists nor c o m b i n e d vagal and cardiac sympathetic b l o c k a d e with muscarinic cholinergic antagonists and fl-adrenergic blockers alters the fall in A P o b t a i n e d with injection of L-glutamate into the caudal ventrolateral m e d u l l a 3 and direct recordings from peripheral sympathetic nerves d e m o n s t r a t e that L-glutamate injections into the caudal ventrolateral m e d u l l a cause a
61 profound inhibition of peripheral sympathetic tone 6. It is therefore reasonable to assume that the depressor responses observed in the present study reflect inhibition of sympathetic vasomotor tone. By using very small injections of L-glutamate and precise localization of injection sites we have more fully characterized the position of the vasodepessor cells. They are located just ventral to the nucleus ambiguus, extending rostrocaudally from 0.5 mm to 2.5 mm caudal to the obex. Throughout this longitudinal extent the vasodepressor cells are situated between the nucleus ambiguus and the lateral reticular nucleus. The region is not specifically designated in the available atlas in the rabbit TM and it is variously designated in the rat 1'16'2°. Previous detailed studies have shown that the A1 catecholamine-synthesizing neurons are situated in this region in the rabbit 2'4. Our present study shows that most
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Fig. 3. A : d e p r e s s o r r e s p o n s e s e l i c i t e d b y i n j e c t i o n s o f 0.5 n m o l L - g l u t a m a t e in 25 nl at v a r y i n g d i s t a n c e s f r o m the m i d l i n e a n d f r o m the d o r s a l surface of the m e d u l l a , at the c o r o n a l level 1.00 m m c a u d a l to the o b e x . B: d e p r e s s o r r e s p o n s e s elicited at v a r y i n g d i s t a n c e s f r o m the o b e x a n d f r o m the d o r s a l surface of the m e d u l l a , at the s a g i t t a l level 3.00 m m l a t e r a l to the m i d l i n e .
of the vasodepressor neurons are located just medial to the region containing the A1 cells. However the position of the vasodepressor neurons does overlap with the medial portion of the region containing the A1 neurons. Thus on localization criteria alone a subgroup of the A1 neurons could be involved in the vasodepressor response. This conclusion contrasts with that of Day et al. 1° and Bonham and Jeske 7. These authors found that the region from which the vasodepressor response was elicited did not overlap with the position of any A1 cells. However, the two groups of investigators differ in their description of the anatomical relationship between the position of the vasodepressor neurons and the position of the A1 cells. Day et al. 1° locate the vasodepressor neurons in the nucleus ambiguus, dorsomedial to the A1 cells. Bonham and Jeske 7 locate the vasodepressor neurons either ventrolateral to the A1 cells or medial to the A1 cells, depending on the particular rostrocaudal level. In our present study the vasodepressor region demonstrated with 0.1 nmol of L-glutamate in 10 nl was similar in extent to the region demonstrated with 0.5 nmol of L-glutamate in 25 nl. This suggests that the neurons responsible for the vasodepressor effect are spread throughout the vasodepressor region and are certainly not concentrated in the region containing the A1 cells. With 0.5 nmol of L-glutamate in 25 nl (a concentration of 20 mM) the fall in AP after injections in the centre of the depressor area was 34 + 1 mm Hg. In our original study L-glutamate was dissolved in 250 nl of vehicle. Such a large volume would presumably result in excitation of neurons situated at some distance from the center of the injection site. However, the fall in AP recorded using the same concentration of L-glutamate (ref. 3, Fig. 6 dose-response relationship) was of similar magnitude to the fall in the present study. This suggests that the vasodepressor neurons are present in a limited region of the caudal ventrolateral medulla, in agreement with the findings summarized in Fig. 2 of the present study. More detailed studies using the focal microinjection approach are unlikely to resolve the question as to the neurochemical identity of the vasodepressor neurons, particularly because neurons in the area often have extensive dendritic processes, sometimes extending as far as 1 mm from the perikaryon 4. This means that both focal electrical stimulation and local injections of neuroactive pharmacological agents could directly affect neuronal firing by an action some distance from the perikaryon. Fortunately, evidence obtained from alternative experimental models bears on the question of the identity of the vasodepressor cells. In both rabbits and rats it appears that these neurons act by inhibiting sympathoexcitatory neurons in the rostral ventrolateral medulla 6"24. This
62 TABLE I Changes (mean ± S.E.M.) in arterial pressure (in mm Hg) after injection of L-glutamate (0.5 nmol in 25 nl) in the caudal ventrolateral medulla oblongata of the rabbit. Each value is based on data obtained from 6 rabbits. *P < 0.01 compared with preinjection values.
Distance caudal Distancefrom to obex (mm) dorsalsurface of medulla (ram)
Distance from midline (ram) 2.0
2.5
3.0
3.5
-0.0
2.0 2.5 3.0 3.5
1~±2 1~±4 94±4 92±2
0±1 0±1 -8±3 -3±1
92±4 88±3 86±4 86±3
-2±2 0±1 -4±2 -7±2
87±4 93±3 90±2 88±5
0±1 -1±1 -4±1 -4±1
-0.5
2.0 2.5 3.0 3.5
90±3 92_+5 101±4 97±2
-3±1 -2±1 -1±1 -6±2
91_+3 85±4 91±4 88±5
-2±1 -5±2 -19±4" -15±5"
93±3 91±3 93±4 87±5
-0±1 -3±1 -19±4" -20±4*
98±4 97±4 97±5 95_+4
-4±4 -0±1 -6±1 -15±6"
-1.0
2.0 2.5 3.0 3.5
98±1 101_+4 95±2 95±4
-3±2 -6±2 -9±1" -4±2
91_+2 91±6 87±1 87±3
-5_+1 -19±4" -15_+1" -16±2"
93±2 93±6 101_+1 86_+7
-1±1 -25±4* -34_+1" -23±2*
96±1 90±4 96±1 95±2
0±1 -5±2 -7±2 -12±3"
-1.5
2.0 2.5 3.0 3.5
94±3 93_+3 98±2 100±3
-6±2 -8±1 -4±1 -7±4
85±3 93±2 97_+4 99±4
-4±2 -12±4" -21±5" -15_+3"
87±3 89±4 95±3 93±4
-6±3 18±4" -25±2* -16±2"
94±6 99±3 94_+3 92±4
-1±1 -4±3 -4±3 -2±2
-2.0
2.0 2.5 3.0 3.5
90±4 94±4 92±3 88±2
-3±1 -9±3* -6±2 -6±1
94±4 96±5 92±4 91_+4
-5_+2 -15±4" -12±2" -8±3
93±4 92±3 94±5 93_+4
-9_+1" -15±5" -10±4" -4±2
94±3 91±3 91±3 93±2
0±1 -3±1 -2±1 -2±1
-2.5
2.0 2.5 3.0 3.5
96±4 95±4 98±5 97±6
-5±1 -6±3 -3±2 -5±3
94_+2 1~±3 94±7 101±3
-5±1 -7±2 -10±2" -4±1
92_+5 99±3 93±3 96±4
-8±2* -8±1" -6_+2 -5±2
97_+3 98±3 103±2 99±2
-1±1 -6±3 -3_+2 -4±2
-3.0
2.0 2.5 3.0
98±5 93±4 94±4
-6±2 -4±1 -8±3
92±5 94±4 93±4
-3±1 -6±2 -2±1
98±5 93±4 92±4
-3_+1 -2±1 -4_+2
strongly suggests that the vasodepressor cells have a short projection to the rostral region. Double-label retrograde
99±2 96±2 1~±3 97±4
0±1 0±2 -2±2 -2±1
tracing studies have failed to show that any A1 cells project to the rostral sympathoexcitatory area in either
TABLE II Changes (mean + S.E.M.) in arterial pressure (in mm Hg) after injection of L-glutamate (0.1 nmol in 10 nl). Each value is based on data obtained from 6 rabbits. *P < 0.01 compared with preinjection values.
Distance from midline (mm)
Distance caudal to obex (mm)
Distance from dorsal surface of medulla (ram)
-0.5
2.5 3.0 3.5
95±3 93_+2 93±2
-4_+3 -7_+1" -11_+2"
100±3 100±3 98_+3
-3_+2 -8_+2* -12_+2"
-1.0
2.5 3.0 3.5
94+6 93+ 6 98+5
-6+1" - 8 + 1" -6__+2*
94+5 94+ 5 94_+5
-1.5
2.5 3.0 3.5
103 + 7 104+6 103+6
-7-+ 2* -11+4" -8+1"
100 + 5 99+6 97+5
2.5
3.0
3.5 98_+5 96±4 98±5
-4±3 -3±1 -3_+1
-7+1" - 9 + 1" -8_+1"
95+6 90+6 95+5*
-3__ +1 -2+ 1 -4_+1
- 7 + i* - 1 0 + 1" -5+1"
99 + 4 105+6 102+5
-3 + 1 -2+1 -1+1
63 the rabbit or the rat 5'22. F u r t h e r m o r e , pharmacological
some of which project to the rostral ventrolateral medulla
evidence suggests that the vasodepressor n e u r o n s inhibit
(manuscript in preparation) and it is therefore possible
the rostral n e u r o n s by activating G A B A e r g i c receptors in the rostral region 6. N e u r o a n a t o m i c a l studies using antibodies to glutamic acid decarboxylase ( G A D ) demonstrate that n e u r o n s which synthesize G A B A are spread widely throughout the rat medulla oblongata, including the ventrolateral medulla 19'21. The rabbit caudal ventro-
that the vasodepressor n e u r o n s are G A B A - s y n t h e s i z i n g cells. The location of the vasodepressor sites as defined in the present study is consistent with this hypothesis.
lateral medulla also contains G A D - p o s i t i v e neurons,
Acknowledgements. This work was supported by the National Health and Medical Research Council and The National Heart Foundation of Australia. We are grateful to Dr Guy Drolet for reading our manuscript.
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