81

Pain, 42 (1990) 81-91 Elsevier PAIN 01599

Comparison of the influence of rostra1 and caudal raphe neurons on the adrenal secretion of catecholamines and on the release of adrenocorticotropin in the cat David A. Bereiter and Donald S. Cam 1 Section of ~~objolo~

and ~epartrn~~t of Surgery, Brown University/

Rhode Island hospital,

Providence, RI 02903 (U. X.4.)

(Received 4 October 1989, revision received 3 January 1990, accepted 10 January 1990)

Neuroendocrine and autonomic responses were assessed in chloralose-anesthetized cats after chemical stimulation of medial brain-stem regions, including those that influence nociceptive input to the medullary or spinal dorsal horn. ~~roinj~tions of L-glutamate (0.5 M, 160 nl) were directed at the following rostra1 and caudal raphe nuclei: the periaqueductal gray (PAG), the dorsal raphe nucleus (DR), the raphe magnus (RM), and the raphe obscurus/raphe pallidus (Ro/Rpa). Activation of DR neurons evoked a significant incmase in the adrenal secretion of epinephrine ( +2.6 f 1.1 ng/min, P < 0.01) that returned towards prestimulus values by 6 mm, whereas ~cro~jections into other raphe nuclei had no consistent effect. Activation of Ro/Rpa neurons evoked an increase in the plasma concentration of adrenocorticotropin (ACTH, +47.9 f 12.3 pg/ml, P < O.Ol), whereas microinjections into other raphe nuclei did not affect ACTH. Arterial pressure increased significantly after activation of PAG ( + 7.5 It: 2.1 mm Hg, P < 0.01) or of DR (+4.8 f 2.0 mm Hg, P ( 0.05) neurons, whereas heart rate increased significantly (P < 0.05) after stimulation of cells within the Ro/Rpa. Glutamate microinjections within the RM, a raphe nucleus that exerts a significant descending influence on nociceptive input to the medullary and to the spinal dorsal horns, had no consistent effect on any measured variable. No evidence was seen to suggest that chemi& activation of neurons within raphe nuclei ~bit~ the adrenal secretion of cat~hol~nes or inhibited the retease of ACTH. The results indicated that glutamate activation of neurons within different raphe nuclei evoked non-uniform effects on neuroendocrine and autonomic function. Further, these data suggested that the neural substrate underlying the control of the adrenal secretion of cateeholamines and of the release of ACTH in response to activation of raphe neurons is likely distinct from that which con~bu~s to the descending influence on noeiceptive input to the medullary and spinal dorsal horn.

S-

Key wordsr Adrenal medulla; Adren~~i~tropin; Raphe nuclei

Cardiovascular function; ~pin~~ne;

Introduction

Medial brain-stem regions ~pe~aqueduct~ gray (PAG) and raphe nuclei} contribute

significantly

’ Present address: Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. Correspondence to: David A. Bereiter, Ph.D., Brown University/Rhode Island Hospital, Neuroendocrine Laboratory, Division of SurgicaJ Research, Providence, RJ 02903, U.S.A.

03~3959/~/$03.50

Norepinephrine;

Pe~aqu~u~t~

gray;

to the descending control of nociceptive input to the medullary and spinal dorsal horns [12,40]. Further, PAG and raphe neurons have also been implicated in the control of the release of anterior pituitary hormones [37], in the regulation of sympathetic preganglionic neuron activity [13,15,23], and in the control of cardiovascular function ~1,10,1~,24,38,42]. Although the relations~p between the underlying central neural substrate for raphe-induced effects on nociceptive input and that for effects on autonomic function is not well

@ 1990 Elsetier Science Publishers B.V. {Biomedical Division)

82

defined, a significant interaction between cardiovascular function and nociceptor activity has been suggested [22,25,29]. The effects of electrical stimulation of raphe nuclei on autonomic function have been described [1,13,15,25,33,38,39,42]; however, few studies have compared the relative influence of selective activation of neuronal cell bodies within different raphe nuclei. Thus, the present study sought to define the effects of local activation of raphe neurons on neuroendocrine activity and on autonomic function without activation of fibers of passage. To assess the relative importance of rostra1 and caudal raphe regions in the control of neuroendocrine and autonomic function, raphe cell bodies were activated selectively by microinjections of L-glutamate [17] in chloralose-anesthetized cats. Microinjections were directed at rostra1 raphe regions (PAG, DR) as well as more caudal raphe nuclei (RM, Ro/Rpa). Peripheral plasma concentrations of ACTH were determined to assess anterior pituitary function, whereas adrenal venous blood was sampled directly to assess the adrenal secretory rate of catecholamines and to estimate the effects of raphe stimulation on adrenal venous blood flow. The results indicated that glutamate microinjections into different raphe regions evoked significant effects on the adrenal secretion of epinephrine and on plasma concentrations of ACTH that were restricted to the DR and the Ro/Rpa regions, respectively. In contrast, raphe areas reported to have the greatest influence on nociceptive input to neurons in the brain-stem and the spinal cord (PAG and RM) had no significant effect on the adrenal secretion of catecholamines or on the release of ACTH.

Materials and methods General methods Adult cats of either sex (2.8-5.2 kg) were deprived of food overnight but were given free access to water. After induction with ketamine-HCl (30 mg/kg, i.m.), anesthesia was maintained by (Ychloralose (75 mg/kg, i.v.) given as a full dose prior to surgery. Supplemental doses of chloralose (7.5 mg/kg, i.v.) were given every 60 mm.

Catheters were placed in the descending aorta (via the femoral artery) to monitor arterial pressure, in the cephalic vein for counterbalanced infusions of normal saline during blood sampling and for administration of all drugs, and in the femoral vein for the collection of peripheral blood samples. The left lumboadrenal vein was catheterized distal to the lateral edge of the adrenal gland as reported previously [4]. Heart rate was monitored from a standard 3-lead electrocardiogram. After tracheostomy, animals were respired artificially with room air, expiratory CO, was monitored with an infrared detector (Anarad) and stroke volume was adjusted initially to maintain a normal range (3.04.5%). Animals were paralyzed with gallamine triethiodide (5 mg/kg, i.v.) after completion of all surgical procedures and supplemental doses (3 mg/kg, i.v.) were given hourly. Body temperature was kept at 38’C with a heating blanket. All surgical incisions were infiltrated with 2% lidocaine jelly. Brain-stem exposure, microinjection technique and histology After placement of all catheters, the animal was mounted in a stereotaxic frame (Kopf), the muscle overlying the occipital bone was reflected and a portion of the bone was removed to allow visualization of the obex as a reference point for the mediolateral plane. For those cats that received microinjections directed at more caudal raphe nuclei, the dura mater overlying the dorsal brainstem was cut and reflected. Microinjections were delivered via a 28-gauge stainless steel cannula. At least 1 h prior to injection, a 22-gauge guide cannula (with dummy cannula inserted) was positioned stereotaxically above the targeted brainstem site and directed at a 40” angle off vertical. The injection cannula protruded approximately 0.5 mm beyond the tip of the guide cannula and was substituted for the dummy cannula at least 30 min prior to injection. The injection cammla was attached to a thick-walled plastic connector and filled with artificial cerebrospinal fluid or with L-glutamate (0.5 M, pH 8.0 in cerebrospinal fluid) plus 3% Fast Green dye. This assembly was attached to a 1 ~1 syringe with a manual injector that delivered 20 nl per step. The patency of the

83

injection cannula was confirmed prior to placement in the brain-stem and again, upon removal from the brain at the end of the experiment. A bolus injection of anesthetic was given at the end of the experiment and the animal was perfused through the heart with normal saline followed by 10% buffered formalin. The sites of injection were identified in coronal sections (40 pm) from the deposit of Fast Green dye and mapped onto a series of brain-stem outlines adapted from Berman

l-39 and ACTH l-24, but not with either the N-terminal end (ACTH 1-13) or with the Cterminal end (ACTH 18-39) fragments of the ACTH molecule. Charcoal-stripped cat plasma was added to the ACTH l-39 standards (Bachem) to control for matrix effects [26]. Intra-assay and interassay coefficients of variation for a plasma pool with an ACTH concentration of 30 pg/ml were 6% and ll%, respectively.

PI.

Statistical analyses All responses were analyzed after first grouping the sites of microinjection within the medial brain-stem after the anatomical description of Berman [8]. Sites located within the periaqueductal gray (PAG) included coronal planes A3.3A1.6; sites located within the dorsal raphe nucleus (DR) included planes PO.2-P3.1; sites located within nucleus raphe magnus (RM) included planes P5.1-P7.7; sites located within raphe obscurus or raphe pallidus (Ro/Rpa) included planes P9.2-P11.6. Sites located outside of these raphe nuclear areas ( 2 200 pm, n = 14) were not included in the statistical analyses. However, the locations of all sites were encoded for the effect on adrenal secretion of epinephrine and for the release of ACTH as shown on the brain-stem outlines of Figs. 2 and 4, respectively. All data were collected from a total of 24 animals where the mean arterial pressure remained above 75 mm Hg and the patency of the injection cannula was verified at the end of the experiment. The adrenal secretion of catecholamines, plasma concentration of ACTH, adrenal blood flow, mean arterial pressure, heart rate, and the percent expiratory CO, responses to injections of L-glutamate within the 4 raphe areas were assessed by 2-way analysis of variance (raphe area and time) corrected for repeated measures [41]. Analyses were performed on the absolute change from the prestimulus value (mean of - 5 and 0 min values) and individual comparisons used the NewmanKeuls test after analysis of variance [41]. To code each microinjection site on the brain-stem outlines for the evoked change in adrenal secretion of epinephrine or for the change in plasma ACTH, the 95% confidence limits for the absolute difference between the prestimulus values ( - 5 and 0

Experimental design A total of 20 cats received l-3 microinjections of L-glutamate directed at the PAG or at 1 of 3 raphe nuclear areas. Microinjections were delivered in volumes of 160 nl at a rate of 20 nl/5 sec. Similarly, 4 additional cats received 7 microinjections of artificial CSF in 160 nl volumes. Peripheral venous (0.5-1.0 ml) and adrenal venous blood samples (0.4-1.0 ml) were collected over 20-60 set at - 5 and 0 min (prestimulus controls) and at 1, 3, 6 and 10 min after the onset of each microinjection. At least 60 min elapsed between each subsequent experimental period. Biochemical determinations Peripheral and adrenal venous blood samples were collected on ice in heparinized tubes. After centrifugation, the plasma was stored at -8O’C for subsequent analyses. The plasma concentrations of catecholamines were determined from 100 ~1 of plasma after alumina extraction by HPLC with electrochemical detection as described previously [ll]. The intra-assay and interassay coefficients of variation for a plasma pool containing 0.56 ng/ml of norepinephrine were 6.2 and 14.3% respectively; and for a plasma pool containing 2.52 ng/ml of epineplnine were 5.6 and 14.1%, respectively. Adrenal secretory rates of catecholamines were calculated as the product of plasma concentration (ng/ml) and adrenal plasma flow (ml/min) = ng/min for each catecholamine species. Plasma adrenocorticotropin (ACTH) was determined by direct radioimmunoassay [26] using an antibody generated against a conjugate of ACTH l-24 that reacts completely with ACTH

84

min) were determined for all sites of injection of L-glutamate (n = 55) both as an absolute difference and as a percentage difference. For the adrenal secretion of epinephrine the 95% confidence intervals for the prestimulus values were: A = 1.63 ng/min and A ‘%= 39.31%. Similarly, the 95% confidence intervals for plasma ACTH were: A = 17.5 pg/ml and A% = 14.6%. The symbols on the brain-stem outlines reflect the peak (maximum) evoked change in secretion with respect to both of these confidence intervals that was seen during the 10 min postinjection sampling period. All data in the text and in the figures represent the mean + S.E.M. The number of animals receiving control injections of artificial cerebrospinal fluid (7 sites from 4 cats) was limited and the means of these data were not assessed statistically. However, individual microinjections of CSF (160 nl) did not evoke significant changes in adrenal secretion of catecholamines or in the release of ACTH, when compared to the 95% confidence intervals, regardless of the site of injection. Spearman rank correlation (rs) analysis was used to assess the relationship between response variables. The peak change in adrenal secretion of catecholamines was examined in relation to the peak change in adrenal blood flow, in mean arterial pressure, and in plasma ACTH. Similarly, correlation analyses assessed the relationship between the peak change in plasma ACTH and in mean arterial pressure.

Results Adrenal secretion of catecholamines

The mean prestimulus adrenal secretory rates of epinephrine and of norepinephrine were 3.68 + 0.53 ng/min and 2.27 f 0.39 ng/min, respectively (n = 41). No significant differences were noted among the 4 groups of microinjections. The mean changes in adrenal secretion of epinephrine and in norepinephrine for each of the 4 anatomical groups are seen in Fig. 1. Microinjections of L-glutamate into the DR evoked a significant (P < 0.01) increase in the secretion of epinephrine by 3 min that returned towards prestimulus values by 6 min. Microinjections into Ro/Rpa caused a small increase in the secretion of epinephrine that was

u

-1

-2

ns I I 012345676910 Time

r

1

I

1

I

I

,

(min)

Fig. 1. Adrenal secretion of epinephrine and of norepinephrine in response to microinjections of L-glutamate (0.5 M, 160 nl) into the periaqueductal gray (open circles, n = 14). into the dorsal raphe (closed circles, n =13), into raphe magnus (open triangles, n = 6) or into raphe obscurus/raphe pallidus (closed value (epitriangles, n = 8). * * P < 0.01versus prestimulus nephrine = 3.61+ 0.53 ng/min, norepinephrine = 2.27 i 0.39 ng/min; n = 41). Shaded bar at time 0 indicates period of microinjection.

not statistically significant (P < 0.10). Glutamate activation of neurons within the PAG or within the RM had little or no effect on the adrenal secretion of epinephrine. The adrenal secretion of norepinephrine was not affected consistently by glutamate, although microinjections within the DR caused a small increase that was not significant (P < 0.10). The mean prestimulus epinephrine/ norepinephrine adrenal secretory ratio (2.65 + 0.41, n = 41) did not change after glutamate microinjections into any of the raphe nuclei (data not shown). The locations of all sites of microinjection of glutamate (n = 55), including those sites that were outside of the raphe nuclei (n = 14), are shown in Fig. 2. Each site was encoded for the maximum evoked change in the adrenal secretion of epinephrine seen during the 10 min postinjection sampling period. As seen in plane P2.1, 6 of the 13

85

the 8 sites within the Ro/Rpa (plane P11.6) increased the adrenal secretion of epinephrine.

P2.1

0

cs

*tb

P6.0

Fig. 2. Location of sites of ~croinj~tion of L-glutamate encoded for the maximum evoked change in the adrenal secretion of epinepluine. Sites determined to be outside of the raphe nuclei (n =14) are also included on the brain-stem outlines. Symbols: closed triangles = increase, closed circles = decrease, open circles = no change (see Methods for further description). Abbreviations: CS, n. centralis superior; DR, dorsal raphe n.; IO, inferior olive; mlf, medial lon~tudinal fasciculus; p, pyramidal tract; PAG, periaqueductal gray; RM, raphe magnus; Ro, raphe obscurus; Rpa, raphe pallidus; tb, trapezoid body; TD, dorsal tegmental n.; 3, oculomotor nucleus; 6, abducens nerve and nucleus; 7G, genu of facial nerve. Calibration bar = 500 pm.

microinjections of glutamate into the DR caused an increase in the adrenal secretion of epinephrine that exceeded the 95% confidence intervals. It was further noted that these 6 sites were located contiguously within the central portion of the DR. Sites of microinjection located in the PAG (plane A2.5), in the RM (plane P6.0) or in the Ro/Rpa (plane P11.6) had no consistent influence on the secretion of epinephrine, however, individual sites did cause significant increases. For example, 4 of

Release of adrenocorticotropin (ACTH) The mean prestimulus plasma concentration of ACTH was 97.5 fr 8.7 pg/ml (n = 41) and no significant differences between anatomical groups were noted. The changes in plasma ACTH in response to microinjections of L-glutamate are presented in Fig. 3. Microinjections within the Ro/Rpa evoked an increase (P < 0.01) in the release of ACTH, whereas glutamate activation of neurons in more rostra1 raphe areas had no significant effect. Although the increase in mean plasma concentration of ACTH in response to stimulation of the Ro/Rpa did not return towards prestimulus values during the 10 min sampling period, it had returned after 60 min. Five of the 8 experiments in which ~croinj~tions of glutamate were made within the Ro/Rpa were followed by at least one subsequent stimulus period. The mean prestimulus concentration of ACTH for these experiments was not elevated in comparison to that for the 3 cats that received the Ro/Rpa injection as the final stimulus of the experiment. As seen on the brain-stem outlines of Fig. 4, the ACTH response to activation of Ro/Rpa neurons was very consistent and 7 of 8 injections evoked an increase in plasma ACTH that exceeded the 95% confi-

l

601

I

I

1

I

I

I

I

I

*

T

a

012345678910 Time

(min)

Fig. 3. Responses of plasma concentrations of ACTH after microinjections of L-glutamate (0.5 M, 160 nl) into the periaqueductal gray (open circles, n =14), into the dorsal raphe (closed circles, n =13), into raphe magnus (open triangles, n = 6) or into raphe obscurus/raphe palhdus (closed triangles, n = 8). * P i 0.05, * * P < 0.01 versus prestimulus value (97.5 & 8.7 pg,/mL n = 41). Shaded bar at time 0 indicates period of

0

P2.1

‘*

! I cs

.--

ical groups were noted. Microinjections of glutamate caused a small overall increase in adrenal blood flow (P < 0.025) when assessed across all injections; however, individual comparisons revealed no si~ificant mean changes for any one anatomical group (data not shown}. The responses of mean arterial pressure to glutamate are seen in Fig, 5 (above). Mean arterial pressure increased by 1 min after microinjections within the PAG (P < 0.01) or within the DR (P < 0.05) and returned towards prestimulus values by 3 min, whereas glutamate activation of RM or of Ro/Rpa neurons had no significant effect. Small increases in heart rate (Fig. 5, below) were evoked after microinjections of glutamate within the Ro/Rpa (P < 0.05 ); however, individual comparisons revealed a significant increase only by 10 min after injection when compared to prestimulus values. The mean percent expiratory CO, was 3.51 + 0.04 (n = 41)

i

Fig 4. Location of sites of microinjection of L-glutamate encoded for the maximum evoked change in the plasma concentration of ACTH. Sites determined to be outside of the raphe nuclei (n =14) are also inchtded on the brain-stem outlines. Symbols: closed triangtes = increase, closed circles,= decrease, open circles = no change (see Methods and Fig. 2 for further description and abbreviations).

dence intervals for the variation in prestimulus concentrations (A 2 17.5 pg/ml; A% r 14.6%. n = 55). In contrast, only 2 of the 14 sites located within the PAG and only 3 of 13 sites located within the DR caused a significant increase in plasma ACTH when assessed against the 95% confidence intervals. None of the 6 sites located within the RM caused a significant change in the plasma concentration of ACTH. Cardiovascular responses The mean prestimulus values for adrenal blood flow, mean arterial pressure, and heart rate were 0.71 + 0.03 ml/mm, 103.1 i: 2.9 mm Hg, and 171.5 f 2.6 beats/mm (n = 41) respectively. No differences in prestimulus values between anatom-

-6 -8

012345678910 Time

(min)

Fig. 5. Mean arterial pressure and heart rate responses to microinjections of t_-glutamate (0.5 M, 160 nl) into the periaqueductal gray (open circles, n =14). into the dorsal raphe (closed circles, n =13), into raphe magnus (open triangles, n = 6) or into raphe obscurus/raphe pallidus (closed triangles, n =8). * Pi 0.05, * *P c:0.01 versus prestimulus value (mean arterial pressure = 103.15 2.9 mm Hg, heart rate = 171.5 + 2.6 beats/m@ n = 41). Shaded bar at time 0 indicates period of microinjection.

87

prior to stimulation and was not affected croinjections of glutamate.

by mi-

Correlation analyses To assess possible relationships between response variables after microinjections of glutamate, Spearman rank-order correlation coefficients (rs) were determined from the peak changes seen during the 10 min sampling period for each microinjection site. The peak change in adrenal secretion of epinephrine was not well correlated (r, = 0.289, P > 0.05) with changes in adrenal blood flow when assessed across all injections (n = 41); however, among those injections restricted to the DR (rs = 0.720, n = 13) or to the Ro/Rpa (rs = 0.786, n = 8) significant correlations were seen (P < 0.05). In contrast, the peak change in the adrenal secretion of norepinephrine was well correlated with the changes in adrenal blood flow (rs = 0.501, P < 0.01) when assessed across all injections (n = 41). Changes in the adrenal secretion of catecholamines were not correlated with evoked changes in mean arterial pressure. Also, the peak changes in the adrenal secretion of catecholamines were not correlated (r, I 0.300, n = 41) with the peak changes in plasma ACTH indicating that the contribution of raphe neurons to the control of the adrenal medulla and to the control of ACTH do not likely involve the same neural substrate. The peak change in plasma ACTH was not correlated with the peak change in mean arterial pressure (rs = -0.300, n = 41).

Discussion Microinjections of L-glutamate were directed at medial brain-stem areas to compare the relative contribution of neurons from different raphe nuclei on the adrenal secretion of catecholamines, on the release of ACTH, and on cardiovascular function. The results indicated that chemical activation of neurons within the PAG or within brain-stem raphe nuclei did not have widespread effects on the neuroendocrine and autonomic variables assessed. The adrenal secretion of epinephrine was increased significantly only after microinjections restricted to the DR, whereas the release

of ACTH was increased uniquely after stimulation of cells within the Ro/Rpa. Arterial pressure increased after microinjections into the PAG or into the DR, whereas small increases in heart rate were seen after activation of cells within the Ro/Rpa region. In contrast, microinjections within the RM, a raphe nucleus most often associated with descending control of nociceptive input to the spinal cord, had no significant effect on any of the measured variables. The PAG and raphe nuclei have long been considered as important in the central neural control of autonomic function. Anatomical studies have indicated direct projections from raphe nuclei to the intermediolateral cell column [2,3,20,34], as well as ascending projections to the hypothalamus [9,27,31,35]. Neurophysiological studies have revealed that caudal raphe neurons often display spontaneous activity related to the cardiac cycle and project to the thoracic spinal cord [24]. Electrical stimulation of brain-stem raphe nuclei evoke significant effects on cardiovascular function [1,23,38,42], on respiration function [21,33], on sympathetic efferent nerve activity [13,15,23] and on the release of ACTH [39]. However, a direct contribution from raphe neurons distinct from that due to fibers of passage cannot be determined by electrical stimuli. More recently, microinjections have been used to reveal that chemical activation of cell bodies within the dorsolateral PAG [lo], the DR [16,32] or the Ro/Rpa [21] can evoke significant changes in autonomic function. Possible effects of activation of raphe cell bodies on adrenomedullary function or on the release of ACTH were not assessed in these studies. Although the microinjection of glutamate provided a suitable technique to distinguish those responses evoked by neuronal cell bodies from those evoked by fibers of passage [16], it was important to confirm that the observed responses were not the result of non-specific damage to neural tissue. Microinjections of equal volumes of artificial CSF (160 nl) into various raphe nuclei had no significant effects on any measured variable. A second concern is that the diffusion of glutamate to sites distant from the raphe nuclei may have contributed to these responses. Although this cannot be excluded completely, sites

xx located beyond (2 200 pm) the boundaries of the raphe nuclei had no consistent effects on the adrenal secretion of catecholamines, on the release of ACTH, or on arterial pressure. Previously, we have reported that this microinjection technique adequately delineated the laminar organization of trigeminal subnucleus caudalis with respect to the control of adrenal secretion of catecholamines and the release of ACTH [5-71. Others have used volume injections somewhat larger (200 nl) than those used here to identify distinct groups of cells within the PAG ihat mediate characteristic autonomic and somatic response patterns [lo]. The effect of chemical activation of raphe neurons on the adrenal secretion of catecholamines has not been assessed previously by direct sampling of adrenal venous blood. The activation of DR neurons was unique among raphe nuclei in affecting the adrenal secretion of catecholamines. It was unexpected to find that activation of more caudal raphe neurons (Ro/Rpa) had no significant influence on the adrenal secretion of catecholamines, since serotonin fibers synapse with identified adrenal preganglionic neurons in the thoracic spinal cord [19] and the origin of these fibers is likely from the caudal raphe nuclei [34]. However, it should be noted, that 4 of the 8 sites located within the Ro/Rpa (see Fig. 2, plane P11.6) did cause a significant increase in the adrenal secretion of epinephrine when compared to the 95% confidence intervals for prestimulus values. The functional significance of an increase in the adrenal secretion of epinephrine evoked by activation of DR neurons is not known. Electrical or chemical stimulation of the DR caused prompt decreases in carotid arterial resistance in intact cats as well as in cats that were functionally adrenalectomized [16]. DR neurons may contribute to the regulation of pain or of autonomic responses associated with cerebral arterial spasm, since direct projection from the DR to the middle cerebral artery has been reported in the cat [36]. Although lesions of the DR alone had no effect on adrenal tyrosine hydroxylase activity, when combined with median raphe lesions, enzyme activity was increased significantly [28]. It is likely that the contribution of DR neurons to the control of the adrenal secretion of catecholamines involves a

multisynaptic pathway since DR neurons have relatively sparse direct projections to the spinal cord [9,27]. The peak increase in the adrenal secretion of epinephrine evoked by glutamate activation of DR neurons ( + 4.0 f 1.4 ng/min) was considerably less than that seen previously [5] after stimulation of lamina I-II neurons of trigeminal subnucleus caudalis ( + 21.8 f 8.3 ng/ min). Although these results suggest that the principal pathway from trigeminal subnucleus caudalis to the thoracic spinal cord does not involve a significant contribution from the DR, it cannot be excluded that DR neurons are less sensitive to glutamate than are lamina I-II neurons of subnucleus caudalis. However, those microinjections within the DR that did cause a significant increase in the secretion of epinephrine were located contiguously within the middle portion of the nucleus. Thus, the influence of a select group of DR neurons on the control of the adrenal medulla may have been underestimated since the statistical analyses included all sites within the DR. Considerable pharmacological evidence has suggested that serotonin and, by implication, raphe neurons contribute to the control of the release of ACTH [see ref. 371; however, the effect of activation of raphe neurons on the release of ACTH has not been reported. Electrical stimulation of the DR caused a decrease in the plasma concentrations of ACTH in the cat [39]. Whereas glutamate activation of DR neurons did not significantly change plasma concentrations of ACTH, it remains that electrical stimulation may have altered plasma ACTH by activation of fibers of passage. Glutamate activation of Ro/Rpa neurons was unique among the raphe areas examined and caused a significant and consistent increase in plasma ACTH. These results do not provide direct evidence for serotonin involvement in this response, since caudal raphe neurons likely contain multiple neurotransmitters [34]. Direct projections from raphe nuclei to the hypothalamus originate mainly from rostra1 raphe nuclei; however. sparse projections from caudal raphe neurons have also been noted [9,27,31,35]. Significant numbers of glucocorticoid receptors are located within the caudal raphe nuclei (Ro/Rpa) in the rat [14].

89

further

suggesting that raphe neurons contribute of the pituitary-adrenal axis. It is also possible that the changes in ACTH evoked by microinjections of glutamate into raphe nuclei may have been influenced by differences in the level of feedback inhibition via glucocorticoids. An important finding was the apparent dissociation between those raphe areas that had the greatest influence on the adrenal secretion of catecholamines (DR) or on plasma concentrations of ACTH (Ro/Rpa) versus those raphe areas reported to exert the greatest influence on nociceptive input to the spinal cord (PAG and RM). The descending inhibition of nociceptive input to the medullary and spinal dorsal horns ascribed to the PAG and RM has been well described [12,40]. Although the central neural substrate that underlies descending inhibition of noxious sensory input may share some common organization with those central pathways that contribute to the control of autonomic function [22,25,29], this relationship has not been well defined. Direct comparison of the thresholds for inhibition of evoked responses in spinal neurons were similar after PAG or RM stimulation, yet were significantly lower than the thresholds for inhibition evoked by stimulation of other raphe nuclei [18]. Electrical stimulation of the PAG and adjacent areas that inhibited the input of noxious sensory information to the lumbar spinal cord also affected autonomic function [25]. Similarly, electrical stimulation of sites within the PAG or within the RM that inhibited the jawopening reflex also inhibited respiration, pharyngeal reflexes, and neural activity within the nucleus tractus solitarius [33]. Although the present study did not assess the influence of glutamate microinjections on nociceptive input to the spinal cord, the absence of autonomic responses to injections within the RM and the diversity of neuroendocrine and autonomic response patterns seen after glutamate microinjections into other raphe nuclei suggested a selective functional output from distinct groups of raphe neurons. It is possible that neurons exist within the RM that can influence autonomic function but were not sensitive to glutamate as suggested by Goadsby et al. [16] for DR neurons. However, Sandkuhler et al. [30] have shown that microinjections of glutamate within to control

the PAG or the RM effectively inhibited nociceptive input to the lumbar spinal cord in the cat. Autonomic responses to these microinjections were not reported. No evidence was seen for glutamate-evoked inhibition of the adrenal secretion of catecholamines, of the release of ACTH or of autonomic function regardless of the raphe region stimulated. Although this is in contrast to many of the results obtained after electrical stimulation of raphe nuclei [1,15,38,39,42], sympathoexcitatory effects after caudal raphe stimulation have been reported [23]. The basis for this finding is not apparent; however, it cannot be excluded that differences in method of stimulation, location of microinjections, or choice and/or level of anesthesia may have contributed. In summary, glutamate activation of neurons within the PAG or within various raphe nuclei caused significant and selective changes in the adrenal secretion of catecholamines, in the release of ACTH, and in autonomic function. Increases in the adrenal secretion of catecholamines and in plasma concentrations of ACTH were evoked from distinctly different raphe regions (DR and Ro/Rpa, respectively). Glutamate injections into raphe regions most often associated with descending control of nociceptive input to the spinal cord (RM) did not evoke significant neuroendocrine or autonomic responses. These data indicate that neurons within specific raphe nuclei contribute selectively to the control of neuroendocrine and of autonomic function rather than having a generalized influence. Further, the central neural pathways that contribute to the control of nociceptive input to the spinal cord likely originate from raphe neurons distinct from those that contribute significantly to the control of the adrenal secretion of catecholamines, of the plasma concentration of ACTH, or of autonomic function.

Acknowledgements The authors thank Albert Benetti for his excellent technical assistance and Carol Cornell and Pat McDermott for their assistance with the biochemical determinations.

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This study was supported in part Grants NS-26137 and DK-26831.

by

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References I5 Adair, J.S., Hamilton, B.L., Scappaticci, K.A., Helke, C.J. and Gillis, R.A.. Cardiovascular responses to electrical stimulation of the medullary raphe area of the cat, Brain Res., 128 (1977) 141-145. Amendt, K., Czachurski, J., Dembowsky, K. and Seller, H.. Bulbospinal projections to the intermediolateral cell column: a neuroanatomical study. J. Auton. Nerv. Syst.. 1 (1979) 103-117. Basbaum, A.I., Clanton, C.H. and Fields. H.L.. Three bulbospinal pathways from the rostra1 medulla of the cat: an autoradiographic study of pain modulating systems. J. Comp. Neural., 178 (1978) 209-224. Bereiter, D.A., Engeland. W.C. and Gann, D.S., Peripheral venous catecholamines versus adrenal secretory rates after brain stem stimulation in cats. Am. J. Physiol., 251 (1986) E14-E20. Bereiter, D.A. and Gann, D.S.. Adrenal secretion of catecholamines evoked by chemical stimulation of trigeminal nucleus caudalis in the cat. Neuroscience. 25 (1988) 6977104. Bereiter, D.A. and Gann. D.S.. Glutamate activation of neurons within trigeminal nucleus caudalis increases adrenocorticotropin in the cat, Pain. 33 (1988) 341-348. Bereiter. D.A. and Gann, D.S., Substance P and GABAergic effects on adrenal and autonomic function evoked by microinjections into trigeminal subnucleus caudalis in the cat, Brain Res., 490 (1989) 307-319. Berman, A.L.. The Brain Stem of the Cat, University of Wisconsin Press, Madison. 1968. Bobillier, P.S., Seguin, S., PetitJean, F., Salve& D., Touret. M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography. Brain Res., 113 (1976) 449-486. Carrive, P., Bandler, R. and Dampney, R.A.L.. Somatic and autonomic integration in the midbram of the unanesthetized decerebrate cat: a distinctive pattern evoked by excitation of neurones in the subtentorial portion of the midbrain periaqueductal grey. Brain Res.. 483 (1989) 251~~ 258. Engeland, W.C.. Dempsher, D.P.. Byrne& G.J., Presnell. K. and Gann, D.S., The adrenal medullary response to graded hemorrhage in awake dogs. Endocrinology, 109 (1981) 153991544. Fields. H.L. and Basbaum. A.I.. Brainstem control of spinal pain-transmission neurons. Annu. Rev. Physiol.. 40 (I 978) 217-248. Futuro-Neto, H.A. and Coote, J.H., Desynchronized sleeplike pattern of sympathetic activity elicited by electrical stimulation of sites in the brainstem, Brain Res., 252 (1982) 269.-276.

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K., Harfstrand, A., Agnati, L.F., Yu, Z.Y., Cmtra. A., Wikstrom, A.C., Gkret. S.. Cantoni, E. and Gustafsson, J.A.. Immunocytochemical studies on the localization of glucocorticoid receptor immunoreactive nerve cells in the lower brain stem and spinal cord of the male rat using a monoclonal antibody against rat liver glucocorticoid receptor. Neurosci. Lett., 60 (1985) I 6. Gilhey, M.P.. Coote, J.H.. MacLeod, V.H. and Peterson, D.F.. lnhibition of sympathetic activity by stimulating m the raphe nuclei and the role of 5-hydroxytryptamine in this effect, Brain Res., 226 (1981) 131-142. Goadsby, P.J., Piper, R.D.. Lambert, G.A. and Lance, J.W.. Effect of stimulation of nucleus raphe dorsalis on carotid blood flow. II. The cat. Am. J. Physiol.. 248 (1985) R263-~ R269. Goodchild, A.K.. Dampney. R.A.L. and Bandler. R.. A method for 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. Griffith, J.L. and Gatipon, G.B., A comparative study of selective stimulation of raphe nuclei in the cat in inhibiting dorsal horn neuron responses to noxious stimulation, Brain Res., 229 (1981) 520-524. Holets, V. and Elde. R.. The differential distribution and relationship of serotoninergic and peptidergic fibers to \ympathoadrenal neurons in the intermediolateral cell column of the rat: a combined retrograde axonal transport and immunofluorescence study. Neuroscience, 7 (1982) 1155-1174. Holstege, G. and Kuypers. H.G.J.M., The anatomy of brain stern pathways to the spinal cord in the cat. A labeled ,mlino acid tracing study. Prog. Brain Rcs.. 57 (1982) 145.--175. Holtman, Jr.. J.R., Anastasi, N.C.. Norman. W.P. and Dretchen, K.L., Effect of electrical and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat, Brain Res.. 362 (1986) 214-220. Lewis, J.W., Baldrighi, G. and Akil, H.. A poxsible interface between autonomic function and pain control: opioid analgesia and the nucleus tractus solitarius. Bram Res.. 424 (1987) 65-70. McCall, R.B., Evidence for a serotonergically mediated \ympathoexcitatory response to stimulation of medullarv raphe nuclei. Brain Res.. 311 (1984) 131-139. Morrison, SF. and Gebber, G.L.. Raphe neurons with sympathetic-related activity: baroreceptor responses and spinal connections, Am. J. Physiol., 246 (1984) R338-R348. Morton. C.R. and Duggan, A.W.. Inhibition of spinal cardiovascular nociceptive transmission accompanies changes from stimulation in diencephalic ‘defence’ regions of cats. Behav. Brain Res.. 21 (1986) 183-18X. Nicholson, W.E., Davis, D.R.. Sherrell. B.J. and Grth. D.N., Rapid radioimmunoassay for corticotropm in unextracted human plasma. Clin. Chem., 30 (1984) 259-265. Pierce. ET.. Foote, W.E. and Hobson, J.A.. The efferent connection of the nucleus raphe dorsalis. Brain Res.. 107 11976) 137-144

91 28 Quik, M., Sourkes, T.L., Dubrovsky, B.O. and Gauthier, S., Role of the raphe nuclei in the regulation of adrenal tyrosine hydroxylase, Brain Res., 122 (1977) 183-190. 29 Randich, A. and Maixner, W., Interactions between cardiovascular and pain regulatory systems, Neurosci. Biobehav. Rev., 8 (1984) 343-367. 30 Sandkuhler, J., Helmchen, C., Fu, Q.-G. and Zimmermann, M., Inhibition of spinal nociceptive neurons by excitation of cell bodies or fibers of passage at various brainstem sites in the cat, Neurosci.. Lett., 93 (1988) 67-72. 31 Sawchenko, P.E., Swanson, L.W., Steinbusch, H.W.M. and Verhofstad, A.A.J., The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat, Brain Res., 277 (1983) 355-360. 32 Saxena, A.K., Saksena, A.K., Vrat, S. and Tangri, K.K., Presence of opioid receptors in mesencephalic nucleus dorsalis raphe concerned in cardiovascular regulation in cats, Naunyn-Schmiedeberg Arch. Pharmacol., 336 (1987) 81-86. 33 Sessle, B.J., Ball, G.J. and Lucier, G.E., Suppressive influences from periaqueductal gray and nucleus raphe magnus on respiration and related reflex activities and on solitary tract neurons, and effect of naloxone, Brain Res., 216 (1981) 145-161. 34 Strack, A.M., Sawyer, W.B., Platt, K.B. and Loewy, A.D., CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus, Brain Res., 491(1989) 274-296.

35 Takagi, H., Shiosaka, S., Tohyama, M., Senba, E. and Sakanaka, M., Ascending components of the medial forebrain bundle from the lower brain stem in the rat, with special reference to raphe and catecholamine cell groups. A study by the HRP method, Brain Res., 193 (1980) 315-337. 36 Tsai, S.-H., Lin, S.-Z., Wang, S.-D., Liu, J.-C. and Shih, C.-J., Retrograde localization of the innervation of the middle cerebral artery with horseradish peroxidase in cats, Neurosurgery, 16 (1985) 463-467. 37 Tuomisto, J. and Man&to, P., Neurotransmitter regulation of anterior pituitary hormones, Pharmacol. Rev., 37 (1985) 249-332. 38 Wang, SC. and Ranson, SW., Autonomic responses to electrical stimulation of the lower brain stem, J. Comp. Neurol., 71 (1939) 437-455. 39 Ward, D.G., Grizzle, W.E. and Gann, D.S., Inhibitory and facilitatory areas of the rostra1 pons mediating ACTH release in the cat, Endocrinology, 99 (1976) 1220-1228. 40 Willis, Jr., W.D., Anatomy and physiology of descending control of nociceptive responses of dorsal horn neurons: comprehensive review, Prog. Brain Res., 77 (1988) l-29. 41 Winer, B.J., Statistical Principles in Experimental Design, 2nd Edition, McGraw-Hill, New York, 1971, pp. 514-603. 42 Yen, C.-T., Blum, P.S. and Spath, Jr., J.A., Control of cardiovascular function by electrical stimulation within the medullary raphe region of the cat, Exp. Neurol., 79 (1983) 666-679.

Comparison of the influence of rostral and caudal raphe neurons on the adrenal secretion of catecholamines and on the release of adrenocorticotropin in the cat.

Neuroendocrine and autonomic responses were assessed in chloralose-anesthetized cats after chemical stimulation of medial brain-stem regions, includin...
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