Cardiovascular sympathoinhibitory neurons form an extended longitudinal column in cat lateral medulla

328

Brain Research, 603 (1993) 328-332 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 25544

Cardiovascular sympathoinhibitory neurons form an extended longitudinal column in cat lateral medulla Colby W. Dempesy, Donald E. Richardson

a n d C h a r l e s J. F o n t a n a

Department of Neurosurgery, Tulane Unit,ersity School of Medicine, New Orleans, LA 70112 (USA) (Accepted 17 November 1992)

Key words: Medulla; Cat; Sympathoinhibition; AMPA; Kynurenic acid; Cardiovascular tone; Baroreflex

A search for vasomotor depressor sites in the lateral medullary brainstem of anesthetized cat using microinjections of AMPA has delineated a longitudinal column of sympathoinhibitory neurons consisting of the rostral depressor area, the caudal ventrolateral medulla, and a new intermediate depressor area. Systematic blockade of these three subregions with bilateral microinjections of kynurenic acid indicates a uniform involvement of the entire column with sympathetic baroreflex, but a slightly greater involvement of the anterior portion, over the posterior, with regulation of cardiovascular tone.

Medullary sympathoinhibitory neurons, which are involved in tonic and reflex regulation of sympathetic nerve activity and arterial pressure, are generally considered to be located in the caudal ventrolateral medulla ( C V L M ) . 17'18'19'23 Evidence from several species suggests that these neurons send inhibitory projections to the sympathoexcitatory neurons of the rostral ventral lateral medulla (RVLM), and by this means modulate the medullary drive to the spinal sympathetic preganglionic neurons. 1'2~3"2a The origin of the tonic activity of the CVLM neurons is unknownJ 8 But these neurons are thought to respond to baroreceptor activation via excitatory projections to the CVLM from the intermediate division of the nucleus solitarius (NTS), which receives afferent fibers from the baroreceptor a r e a s . 3A1'17'2°'22 There is controversy as to whether the N T S - C V L M - R V L M pathway constitutes the primary efferent arm of the baroreflex arc, or only serves to alter the reflex sensitivity. 8'9'13,16 The CVLM is described as confined in extent, lying caudal to the obex in the region between the nucleus ambiguus and the lateral reticular nucleus, and medial to the A1 area in rat and rabbitJ a°,19 It does not correspond to any known nucleus, and it may be anatomically and functionally heterogeneous. 7'9'16 In-

deed, recent evidence suggests that only the rostral CVLM is involved in the baroreflex. 13 Current reports indicate that CVLM function can sometimes be found anterior to the obex. 13'16 This extension of the CVLM is sufficiently rostral in rabbit that its location has even been referred to as intermediate between the RVLM and the C V L M J 6 Now our recent report of finding a vasodepressor area in cat located dorsal to the RVLM requires that the claimed domain of medullary sympathoinhibitory neurons be further reconsidered. 14 This new area, which we have named the rostral depressor area (RDA), is similar to the CVLM both in being limited in extent and in lying lateral to the sagittal region containing the compact division of the nucleus ambiguus. 5'14 Furthermore, the R D A and CVLM display similar sympathoinhibitory behaviors. 14'18 Thus it became reasonable to ask if these areas are actually two distinct regions, or whether they might be the rostral and caudal ends of a continuous array of sympathoinhibitory sites. The present report describes a systematic survey in cat medulla of the entire longitudinal region bounded by the R D A rostrally and the CVLM caudally, in order to identify subregions of sympathoinhibitory activity

Correspondence: C.W. Dempesy, Dept. of Neurosurgery, Tulane Medical School, 1430 Tulane Ave., New Orleans, LA 70112, USA. Fax: (1) (504) 5885793.

329 and to compare their roles in tonic and reflex regulation of cardiovascular function. We conclude that the vasomotor depressor neurons in cat occupy a fairly homogeneous longitudinal column more than 4 mm in length in the lateral medulla, and that subregions of this column differ only slightly in their regulatory influences. Data were obtained from 56 random-access cats (2.5-3.5 kg) of either sex. Surgical anesthesia was induced with pentobarbital sodium (25 m g / k g , i.m.) following tranquilization with ketamine (15 m g / k g , i.m.). The trachea was cannulated and connected to a respirator to provide artificial ventilation with oxygen-enriched room air. A femoral artery was cannulated and attached to a pressure transducer connected to a polygraph to provide continuous recording of mean blood pressure (BP) and heart rate (HR). A femoral vein was cannulated to permit drug injection into the vena cava. Supplemental doses of pentobarbital (20 mg, i.v.) were administered as needed, and pancuronium bromide (0.3 m g / k g , i.v.) was provided for neuromuscular blockade approximately every 2 h. Temperature was maintained with a heating pad. It is well known that depressor responses can be elicited by microinjection into the CVLM of a variety of excitatory amino acids. Although we had originally delineated the R D A by microinjection of L-glutamate

RDA

(GLU, 3.5-7 nmol), the same injection sites yielded similar acute hypotensive responses with N M D A (1530 pmol), A M P A (2-3 pmol) or kainic acid (1 pmol). For the present study, A M P A (a-amino-3-hydroxy-5methylisoxazole-4-propionic acid) was chosen for the microinjection survey for two reasons: (1) like GLU, it provides a rapid depressor response with recovery within minutes, whereas N M D A or kainic acid requires two to three times longer to complete the same sequence; and (2) The A M P A depressor response, but not the GLU, is blocked by kynurenic acid (KYN, 5 nmol). Microinjected KYN was employed in this report to provide a functional blockade of sympathoinhibitory areas, and its ability to block the A M P A depressor response provided a reliable means for adjusting dose level. Drugs were dissolved in artificial cerebral spinal fluid vehicle 17, and microinjections were performed with a 1-/xl syringe (0.47 mm needle diameter) equipped for side-vent loading. Injection volumes were 40 nl for A M P A and 100 nl for KYN. Baroreflex was generated with an i.v. bolus of phenylephrine sufficient (15-30 lzg) to induce an acute blood pressure rise of 35 mmHg. Baroreflex sensitivity (BRS) was calculated as the ratio of maximum acute H R decrease to maximum acute BP increaseJ 2'14'18 To ensure that only sympathetic responses were observed, either of two vagolytic procedures, bilateral vagotomy

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Fig. 1. Distribution of sympathoinhibitory sites detected by AMPA microinjection in three defined subregions of cat lateral medulla, and the corresponding group values of acute hypotension and bradycardia observed at each site. The subregions are based on the previously described rostral depressor area (RDA, P10.5-P12.0 mm) and caudal depressor area of the caudal ventrolateral medulla (CDA, P13.5-P15.0 mm), and the newly identified intermediate depressor area (IDA, P12.0-P13.5 mm). Baseline values of BP and HR (obtained after the vagal influence to the heart was eliminated) for the group AMPA-stimulated responses are RDA: 121_+4 mmHg and 194+ 6 bpm; IDA: 123+ 3 mmHg and 192_+5 bpm, and CDA: 127+ 4 mmHg and 194_+6 bpm. All cats were vagotomized or atropinized to ensure only sympathetic responses. 5Sp, spinal trigeminal nucleus; A, ambiguus nucleus; IO, nucleus of the inferior olive; LR, lateral reticular nucleus; NTS, nucleus solitarius; Py, pyramids; Rgc, nucleus reticularis gigantocellularis; Rpc, nucleus reticularis parvocellularis.

330 ever, as sites became somewhat less dense and less responsive caudally. In order to quantify these changes, an artificial division of the column was made into three subregions: the rostral depressor area (RDA, P10.5P12.0 mm), the intermediate depressor area (IDA, P12.0-P13.5 mm) and the caudal depressor area (CDA, P13.5-P15.0 mm). This division resulted in a R D A somewhat larger than in our previous report, a C D A very similar to the CVLM, and an I D A that has not been previously described. Each frontal section of Fig. 1 is typical of the subregion it represents and each displays on a single plane all the site locations found in that subregion. Although each subregion was equally searched, the sections reveal that site density and number tended to be greatest in the R D A and IDA, and least in the CDA. This variation is also revealed in the saggital display of Fig. 2, where a better sense of the columnar array can be gained. Of particular interest in Fig. 2 is how closely the column parallels the more medial longitudinal configuration of the nucleus ambiguus. Fig. 1 also displays the acute sympathetic hypotensive and bradycardic responses to A M P A stimulation by subregion. These group responses were: in the R D A (n = 37), a depression of 40 + 3 mmHg and a cardiac slowing of 22 + 2 bpm; in the I D A (n = 40), a depression of 28 + 3 mmHg and a cardiac slowing of 17 + 2 bpm; and in the C D A (n = 30), a depression of 22 + 3 mmHg and a cardiac slowing of 9 + 2 bpm. These data clearly reveal the greater intensities of these responses at the rostral end of the column, and their gradual diminution toward the caudal end. The three subregions were tested by local blockade to ascertain their involvement in sympathetic tonic and reflex regulation of cardiovascular function. The blockade was induced with bilateral microinjections of KYN (at an A M P A depressor site on the left side of the

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Fig. 2. Sagittal view of the cat sympathoinhibitory sites displayed in Fig. 1. The laterality of all sites lies between 3.25 and 4.25 mm, locating them on the lateral border of the nucleus ambiguus (A), whose longitudinal sweep is designated by dashed lines. RDA, IDA and CDA are the sympathoinbibitory subregions defined in Fig. 1. The plane of the obex is indicated at P13.8 mm. All cats were vagotomized or atropinized. LR, lateral reticular nucleus; LTF, lateral tegmental field. or atropinization (0.5-1 m g / k g , in saline, i.v.), were performed. Cats were euthanized with an overdose of pentobarbital, and brains were recovered for histological examination of injection sites (see Ref. 14 for details). Each group result is expressed as the mean + S.E.M. Statistical comparisons are based on paired data, with significance reported at P < 0.01. The systematic search for A M P A depressor responses from P10.5 to P15.0 mm in cat medulla (Atlas of Berman 5) revealed a columnar array of sympathoinhibitory sites, lying between depths - 7 and - 9 mm and lateral to the nucleus ambiguus. Although there had been expectation that the sites would tend to fall into isolated clusters as the search proceeded from rostral to caudal extremes, such variations did not emerge. The array was not completely regular, how-

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Fig. 3. Tonic levels of blood pressure and heart rate were increased by bilateral microinjection of kynurenic acid (KYN, 1 /xg) into the three subregions (RDA, IDA and CDA; see Figs. 1 and 2) of the sympathoinhibitorycolumn of vagotomizedor atropinized cat. However, the increases were significant only for the anterior half of the column. Bars labeled "PRE-" represent group values obtained before KYN intervention, while those labeled "POST-" are for values obtained after intervention.

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Fig. 4. Sympathetically mediated baroreflex induced by i.v. bolus of phenylephrine in vagotomized or atropinized cat was significantly reduced by bilateral microinjection of kynurenic acid (KYN, 1 /xg) into all three subregions (RDA, IDA and CDA; see Figs. 1 and 2) of the sympathoinhibitory column. Bars labeled "PRE-" represent group values for BRS ("baroreflex sensitivity", see text) obtained before KYN intervention, while those labeled "POST-" are for values obtained after intervention. Baseline values of blood pressure and heart rate for the group calculated sensitivities are RDA: 111 + 5 mmHg and 195 + 8 bpm; IDA: 131 +-4 mmHg and 219 + 11 bpm; and CDA: 134 + 6 mmHg and 210+ 13 bpm.

midline, and the homologous site on the right side of the midline). As displayed in Fig. 3, temporary increases in BP and H R occurred in all three subregions, but the rises were significant only in the R D A ( + 23% in BP and + 8 % in HR, for n = 14) and in the IDA ( + 2 6 % in BP, for n = 9). These rises peaked in 5-15 min following the blockade and then vanished in 20-40 min with the reappearance of the A M P A depressor responses. Fig. 4 shows that the blockade diminished baroreflex sensitivity (BRS) in all three subregions, and these losses were" significant in the R D A ( - 5 0 % , for n = 19), the IDA ( - 6 2 % , for n = 10) and the CDA ( - 5 1 % , for n = 8). Recovery of the baroreflex from the KYN blockade required 30-50 min. These data indicate that all three regions play a partial role in mediating the sympathetic baroreflex, but only the anterior half appears to have a significant role in the regulation of sympathetic cardiovascular tone. This study resolves the difficulty of two apparently separated sympathoinhibitory areas in cat medulla, viz., the R D A and the CVLM (or CDA), by finding an intermediate link, the IDA, that joins the array into a single longitudinal column of vasomotor depressor neurons extending from P10.5 to P15.0 mm. The coordinates of this column suggest that it lies on the lateral border of the ambiguus nucleus (AMB), an array of neuronal clusters covering a similar longitudinal sweep in the medulla. 5 Moreover, the two columns are so intimate that sympathetic depressor neurons may be intermixed with parasympathetic neurons in the external formation of the AMB. Although the AMB is quite heterogeneous both in function and anatomy, at least in rat 6, the present findings for the sympathoinhibitory column in cat suggest only slight variations down its length. Indeed, the stronger sympathetic regulation

exhibited by the anterior half of the column may simply follow from its greater density of neurons, permitting the A M P A and KYN injections to encounter more synapses. Perhaps a more significant heterogeneity of the depressor column will be found in its afferent and efferent connections. The question of the existence of an R D A and an I D A in species other than cat remains. There have only been occasional reports of sympathoinhibitory areas outside of the normally recognized CVLM in rat and rabbit. 4"13'16 However, it has been reported that vagolytic procedures uncover sympathetic mechanismsJ 6 Perhaps the sympathoinhibitory column has first emerged in this study because all cats were vagotomized or atropinized. Finally, the problem of including the sympathoinhibitory neurons that Gebber and Barman 15 have been studying for some time in the lateral tegmental field (LTF), medial to the ambiguus nucleus, should be resolved. It is possible that the R D A and the I D A can be shown to have medial extensions into the LTF, a region this study has ignored. The authors wish to thank Monique Cola for her assistance with histological preparations.

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