Temporal modulation of antinociception by reciprocal connections between the dorsomedial medulla and parabrachial region

BrainResearchBulletin,Vol. 37, No. 5, pp. 467-474, 1995 Copyright© 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0361-9230/95 $9.50 + .00

Pergamon 0361-9230(95)00026-7

Temporal Modulation of Antinociception by Reciprocal Connections Between the Dorsomedial Medulla and Parabrachial Region LEDA MENESCAL-DE-OLIVEIRA1 AND ANE-VFE HOFFMANN

Department of Physiology, Faculty of Medicine of Ribeir~o Preto, University of S~o Paulo, 14049-900 Ribeir~o Preto, SP, Brazil [Received 2 July 1993; Accepted 20 December 1994] ABSTRACT: Microinjection of carbachol into the dorsal parabrachial region (PBRd) of guinea pigs induces analgesia from the 5th to the 15th min postinjecUon, as evaluated by the reduction of the vocalization in response to an electric shock applied to one paw. When reversible blockade of the dorsomedial medulla or specifically of the nucleus tractus solitarius (NTS) is parformed with xylocaine 5 min after microinjection of carbachol into the PBRd, the analgesic effect continues up to the 45th and to the 60th min, respectively. Blockade of the dorsomedial medulia is achieved by topical application of xylocaine to the area postbrema (AP) or microinjection of the drug into the NTS. A prolongation of the duration of the analgesic effect also occurs after the inverse procedure, i.e., after reversible blockade of the PBRd 5 rain after topical application of carbachol (1 pg/pl) to the AP or microinjection of cerbachol into the NTS. In this case, the analgesic action, which lasted up to 30 min when carbachol was applied to the AlP and 60 rain when microinjected into the NTS, was prolonged up to 60 min and to 80 min, respectively, after reversible blockade of PBR. The present data suggest that the reciprocal connections between the different regions of the dorsomedial medulla and the PBR play an important role in the modulation of the duration of the analgesic effect, and that this fact may be of adaptive importance in the defensive analgesia that occurs in the con~ontation between prey and predator.

and PBR [3,14,24,38,55,59] were confirmed by other authors. Anterograde and retrograde axon labeling techniques demonstrated that the two main projections of the AP seem to be to the subjacent NTS and to the parabrachial region [35]. Projections toward the AP originating both from the NTS and the parabrachial region were described [58,60]. More recently, the reciprocal connections between AP and PBR have been characterized electrophysiologically [51 ]. In terms of function, the AP has been considered to be a chemoreceptor zone, a vomit-triggering zone [5,6], a structure inducing electrocortical synchronization by 5-HT [7,33] and participating in cardiovascular regulation [1,17]. Together with the NTS, the AP receives afferent visceral information via the vagus, thus being involved in the integration of neurally and humorally mediated visceral sensations [9,30,34]. It has been shown recently that the nucleus tractus solitarius (NTS), a destination of most vagal afferent fibers [30], is an important relay for the modulation of nociception produced by vagal afferent stimulation (VAS) [53,54]. Microinjection of glutamate into, or electrical stimulation of, the NTS inhibits spinal dorsal horn neurons and nociceptive reflexes [11,36,48,53,54], and local anesthesia of the NTS abolishes or significantly attenuates these VAS-produced effects [53,54]. Our group reported for the first time the involvement of AP and NTS in the modulation of nociceptive responses in guinea pigs [42]. Our findings were confirmed by others [50] who observed an increase in tail flick latency in rats as the result of morphine microinjection into the NTS. The parabrachial region also participates in nociceptive regulation [21]. These authors, using localised electrolytic lesions, showed that the PBR is involved in the analgesic action of morphine in rats submitted to the hot-plate and tail-flick tests. Carbachol microinjection into the PBR of cats [23,31,32] produced analgesia, possibly by the activation of direct and/or indirect inhibitory descending pathways, which may act in the suppression of nociceptive information at the spinal level. The involvement of the PBR in the processing of painful information is partially facilitated by the projection of nociceptive information originating from dense inputs from lamina I neurons of the dorsal horn of the spinal cord [25]. Projections of specific nociceptive neurons toward the midbrain (including the PBR) and the interconnections of these midbrain regions with the hypothalamus and

KEY WORDS: Parabrachial region, Dorsomedial medulla, Area postroma, Nucleus tractus solitarius, Reciprocal connections, Carbachol, Analgesia, Temporal potentiation, Guinea pigs.

INTRODUCTION There is evidence of reciprocal connections between the dorsal parabrachial region (PBRd) and a distinct region in the dorsomedial medulla that incorporates the nucleus of the tractus solitarius (NTS) as well as the area postrema (AP). The neuronal projections of the area postrema (AP), first studied by Morest [49], mainly point toward neighboring portions of the nucleus tractus solitarius (NTS). Later studies [38,55] have shown that the AP and NTS massively project toward the nuclei of the parabrachial region (PBR), although less abundant connections with other structures of the brain stem are also present. The existence of connections between AP and PBR [8,35,55,60,61[ and NTS To whom requests for reprints should be addressed. 467

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limbic regions suggest that the spinal-mesencephalic route of the lamina I plays an important role in the organization of the autonomic and affective responses to pain [26,39]. Recent electrophysiological data have indicated that spinal-parabrachial neurons predominantly transmit specific nociceptive information [4,37]. Considering that structures of the dorsomedial medulla (AP and NTS) and the PBR are involved in pain modulation and that they have reciprocal connections, the present study was undertaken to investigate the functional significance of the interconnections of regions of the dorsomedial medulla with PBR in the presence of a noxious stimulus in guinea pigs. Because these regions have cholinergic receptors, we observed the action of cholinergic stimulation of each region on pain modulation separately and in the presence of functional exclusion of the other with xylocaine. METHOD

Animals The study was conducted in concordance with the guidelines of the Ethics Committee of the International Association for the Study of Pain [63]. Sixty-five male guinea pigs (Cavia porcellus), weighing 450 to 500 g, were used.

Experiment I The animals were divided into five experimental groups: group 1 (n = 8) unilaterally microinjected with carbachol (1 #g/ 0.2 #1) into the dorsal parabrachial region (PBRd); group 2 (n = 8) unilaterally microinjected with carbachol ( 1 tzg/0.2 #l) into the PBRd and submitted 5 min later to topical application of 2% xylocaine to the AP for 10 min; group 3 (n = 8) unilaterally microinjected with carbachol (1 ,ug/0.2 ~tl) into the PBRd and submitted 5 min later to microinjection of 0.2 #1 of 2% xylocaine into the NTS ipsilateraly; group 4 (n = 9) microinjected into the PBRd with 0.2 #1 saline; group 5 (n = 8) injected with 0.2 #1 xylocaine only into the PBRd.

Experiment H This experiments was also carried out on eight experimental groups: group 1 (n = 8) submitted to topical application of carbachol (1 izg//zl) to the AP for 2 min; group 2 (n = 8) submitted to topical application of carbachol (1 #g/#l) to the AP, followed 5 min later by microinjection of 0.2 #1 2% xylocaine into the PBRd; group 3 (n = 8) submitted to topical application of saline to the AP; group 4 (n = 8) submitted to topical application of 0.2/A of 2% xyiocaine to the AP; group 5 (n = 9) unilaterally microinjected with carbachol ( 1 #g/0.2 #1) into the nucleus tractus solitarius (NTS); group 6 (n = 8) unilaterally microinjected with carbachol (1 #g/0.2 #1) into the NTS and submitted 5 min later to microinjection of 0.2 ~1 of 2% xylocaine into the PBRd ipsilaterally; group 7 (n = 7) unilaterally microinjected into the NTS with 0.2 ~1 saline; group 8 (n = 7) injected unilaterally with 0.2 #1 xylocaine only into the NTS.

Animal Preparation After anesthesia (40 mg/kg pentobarbital sodium, IP), the animal was fixed to the Kopf stereotaxic apparatus, with the buccal piece 21.4 mm below the interauricular line. A guide cannula measuring 0.5 mm in outer diameter and 14 mm long, was introduced into the brain, fixed to the skull with dental acrylic, and kept patent with a mandril to avoid obstructions. The following coordinates of the guinea pig brain atlas [57] were used for the PBRd: 12.4 to 13.4 mm caudal to the bregma; 2.4 to 2.5 mm lateral to the midline, and 6.5 to 6.7 mm below

the cortical surface. In this situation, the guide cannula was located 1 mm above the PBRd. For the NTS, the coordinates were 19.2 to 20.5 mm caudal to the bregma (guide cannula at a 24 ° angle), 0.6 to 0.8 mm lateral to the mediline, and 12.3 a 12.5 mm below the cortical surface. After 1 week, the animals of group 2 in Experiment I and the animals of groups 1, 2, 3, and 4 in Experiment II were anesthetized with sodium pentobarbital (40 mg/ kg IP) and submitted to surgical preparation for AP exposure according to a previously described method [42]. A pair of uninsulated stainless steel electrodes (0.3 mm in diameter) was introduced subcutaneously into the thigh region 1 h before the experiment. The animal was then placed in an acrylic box lined with nylon foam where some movement was possible. During the experiment and after 20 min of habituation of the animal to the experimental situation, the electrode was connected to an electronic stimulator that liberated pulses (square waves, 100 Hz frequency, 0.5 ms duration) of varying intensity (0.5 to 4.0 V) sufficient to induce vocalization (VOC), which is interpreted as a manifestation of pain. Guinea pigs are not commonly utilized in experiments destined to the study of the neural mechanisms of analgesia. Analgesic tests currently used in rats and mice are not applicable to guinea pigs because of anatomical reasons (tail flick) or because they have not been standardized (hot plate and writhing). In our laboratory, we have been using for some years tests that have proved to be appropriate for guinea pigs [42,43]. In the presence of acute painful stimuli (electric shock), semirestrained guinea pigs emit a vocalization response and a brief but intense motor response consisting of a body shake. Once the threshold value was established, voltage was maintained at a constant level throughout the experiment. Vocalization is considered to be an objective and specific pain indicator in animals submitted to a noxious stimulus [20]. Undisturbed guinea pigs adapted to the experimental situation are normally quiet and silent. Electrical shocks (3 s duration) induced brief motor and vocalization responses that did not persist in the intervals between stimuli. Vocalization was converted into electrical signals by an Aiwa DM-64 microphone, and the envelope of the sound obtained by a peak detector circuit was also recorded with a Nihon Kolden polygraph. In the polygraphic recording of VOC, peak amplitude is proportional to the intensity of animal vocalization. The mean of the peaks of each response is a reliable index of the magnitude of vocalization. The peak amplitude of the graphic recordings of vocalization was measured in milimeters, and the mean of each response was used for quantitative evaluation. Chemical stimulation of the AP was performed by topical application of a small cotton wick 0.5 mm in diameter soaked in the appropriate solution and placed in contact with the AP, under visual control with a stereoscopic microscope. Despite these precautions, we cannot exclude the possibility that the solution diffused to nearby regions of the dorsomedial medulla, including the NTS itself. For this reason, when we talk about results caused by drug application to the AP, these results will be interpreted as being due to the involvement of the AP and of structures of the dorsomedial medulla close to it. The wick soaked in the carbachol solution was left in touch with the AP for 2 min, and the same procedure was adopted for the groups in which saline and xylocaine were applied to the AP. The microinjections into the PBR and into the NTS were performed with a 30-gauge Mizzy needle segment coupled to a l0 #1 Hamilton syringe through PE-10 polyethylene tubing. The microinjection cannula was introduced into the guide cannula at a depth exceeding the latter by 1 mm.

Drugs The tbllowing drugs and solutions were used: carbachol (Merck Sharp & Dohme) dissolved in sterile saline at a concentration of 1/~g/#l, 2% xylocaine (Astra), and physiological saline.

P A R A B R A C H I A L REGION, D O R S O M E D I A L MEDULLA, AND A N A L G E S I A

Experimental Procedure

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To obtain basal control responses, the nociceptive thresholds were established and five noxious stimulations were performed at random intervals ranging from 1 to 5 min. After a control "recording of the vocalization, group 1 animals (n = 8) in Experiment I and group 5 of Experiment II (n = 9) were submitted to ipsilateral microinjection of 1 #g/0.2/zl carbachol into the dorsal PBR and into the NTS, respectively. Starting at that time, peripheral noxious stimuli were applied at 2, 5, 10, 15, 30, 45, 60, 80, and 90 min. The experiment was considered to have ended when the response emitted by the animal returned to levels close to basal ones. In groups 2 (n = 8) and 3 (n = 8) of Experiment I, the procedure adopted was similar to that for group 1 of Experiment I, except that, 5 min after carbachol microinjection (1 #g/0.2 #1) into the PBR, these animals received topical application of xylocaine to the AP for 10 min. (group 2) and ipsilateral microinjection of xylocaine into the NTS (group 3). Groups 2 (n = 8) and 6 (n = 8) of Experiment II were submitted to the inverse procedure, i.e., carbachol was first applied to the AP (group 2) or microinjected into the NTS (group 6), and after 5 min both groups received a microinjection of 2% xylocaine into the PBRd. Groups 1, 3, and 4 of Experiment II were submitted to topical stimulation of the AP with carbachol (1 #g/#l), saline, and xylocaine, respectively. Groups 5, 7, and 8 of Experiment II received an ipsilateral injection of carbachol, saline, and xylocaine into the NTS, respectively. The vocalization was recorded continuously on a polygraph throughout the experiment.

Histological Verification At the end of the experiment, 0.2 #1 2% Methylene blue was injected into the same PBR and into the same NTS sites in which the microinjections had been performed to facilitate the determination of the exact sites of microinjection after histological processing of the brains. The animals were then anesthetized and perfused intracardially with 20 ml saline followed by 20 ml 10% formalin. After histological processing, the path of the guide cannula and the microinjection site were determined with the aid of a microscope.

Statistical Analysis Analysis of the results was performed quantitatively for the vocalization response. The amplitude of the vocalization responses was measured in milimeters directly from the polygraphic tracings. All data are expressed as mean --- SEM. Mean values were compared by analysis of variance and by the Scheff6's test to determine statistical differences between any two groups, p < 0.05 was considered to be statistically significant. RESULTS

Experiment I Carbachol microinjected unilaterally into the PBRd of guinea pigs submitted to a ipsilateral peripheral noxious stimulus (group 1) produced antinociception, as shown by a decrease in mean vocalization amplitude (Fig. 1, CPB). One-way A N O V A showed a significant difference in mean vocalization response at 5 min, F(3, 31) = 3.07, p < 0.03, at 10 min, F(3, 31) = 11.05, p < 0.0008, and 15 rain, F(3, 31) = 7.77,p < 0.005, when compared with the saline group at the same time intervals (Fig. 1). The antinociceptive effect of carbachol on the PBRd lasted up to 45 rain in group 2, which had been submitted to concomitant AP blockade with xylocaine (Fig. 1, CPBXAP). One-way A N O V A showed a significant difference in mean vocalization response in group 2 at 5 min, F(3, 31) = ll.05, p < 0.0001, at 15 min, F(3,

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FIG. I. Effect of carbachol (1 #g/0.2 #1) microinjected unilaterally into the PBRd. The ordinate indicates (in mm) the mean vocalization amplitude (M. VOC. AMPL.) of the animal groups that received saline in the PBR (SALPB, n = 9), carbachol in the PBR (CPB, n = 8), carbachol in the PBR plus xylocaine in the AP (CPBXAP, n = 8) and carbachoi in the PBR plus xylocaine in the NTS (CPBXNT, n = 8). The abscissa shows the intervals in minutes (TIME min) at which vocalization amplitude was measured in the presence of a peripheral noxious stimulus; 0 = mean control values for each experimental situation. *p < 0.05 compared to SALPB. The vertical bars indicate the SEM.

31) = 7.55,p < 0.0001, at 30 rain, F(3, 31) = 6.62,p < 0.0006, and at 45 min, F(3, 31) = 5.08, p < 0.002. In group 3 (Fig. 1, CPBXNT), which was submitted to NTS blocakde with 2% xylocaine, the antinociceptive effect lasted as long as 60 min. In this group, mean vocalization amplitude was significantly reduced in response to application of a noxious stimulus at 15 min (t = 2.93, p < 0.05), at 10 min (t = 3.14,p < 0.001), at 15 min (t = 3.89, p < 0.004), at 30 min (t = 3.68, p < 0.008), at 45 min (t = 3.42, p < 0.007), and at 60 min (t = 2.85, p < 0.05) when compared with the saline group at the same time intervals (Fig. 1, CPBXNT). Microinjection of xylocaine alone into the PBR (Fig. 2, XILPB) did not change the magnitude of the vocalization response, which did not differ statistically from those observed after microinjection of saline into the PBR. Animals in which carbachol was injected into the inferior colliculus, lateral lemniscus, and cuneiform nucleus did not present changes in vocalization induced by the noxious stimulus.

Experiment H Topical application of carbachol to the AP of guinea pigs submitted to a peripheral noxious stimulus for 2 min induced a statistically significant reduction of the vocalization response at the times tested from 2 to 30 min (Fig. 3A, CAP). One-way A N O V A showed a significant difference in mean vocalization response at 2 min, F(2, 21) = 14.76, p < 0.001, at 5 min, F(2, 21) = 10.57, p < 0.0006, at 10 min, F(2, 21) = 8.47, p < 001, at 15 min, F(2, 21) = 13.86, p < 0.0001, and 30 rain, F(2, 21) = 10.78, p < 0.0005. Mean vocalization amplitude was 14.4 _ 0.5 mm before treatment and decreased to 6.0 +_ 1.5 mm 2 min after carbachol (Fig. 3A, CAP).

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FIG. 2. Comparison of the effect of unilateral microinjection of (1.2/.tl xylocaine (XILPB, n = 8) and of saline (SALPB, n = 8) into the PBRd on mean vocalization amplitude (M. VOC. AMPL.) in response to the application of a peripheral noxious stimulus. The abscissa shows the intervals in minutes (TIME min) at which vocalization amplitude was measured in the presence of a peripheral noxious stimulus; C represents mean control values. The vertical bars indicate the SEM.

Topical stimulation of the AP with carbachol followed by 2% xylocaine microinjection into the dorsal PBR 5 min later induced a reduction in vocalization response at all times tested from 5 to 60 min (Fig. 3A, CAPXPB). This reduction was statistically significant at 5 min (t = 3.85, p < 0.003), 10 min (t = 3.29, p < 0.01), 15 min (t = 4.98, p < 0.0003), 30 min (t = 4.74, p < 0.0006), 45 rain (t = 3.68, p < 0.006), and 60 min (t = 3.41, p < 0.01) when compared to the animals topically treated with saline alone in the AP. It can be seen (Fig. 3A, C A P X P B ) that the basal vocalization amplitude was 16.0 _+ 1.8 mm and that its magnitude decreased to 3.8 _+ 2. I, 5.9 _+ 1.4, 4.2 _+ 1.0, 4.6 _4- 2.1 and 6,8 _+ 1.9 mm at 5, 10, 30, 45, and 60 min after treatment, respectively. In contrast, the vocalization responses caused by xylocaine topically applied to the AP (Fig. 4A, XILAP) did not differ statistically from those observed after saline (Fig. 4A, SALAP). Unilateral microinjection of carbachol (1 /.zg/0.2/.zl) into the NTS produced antinociception shown by a decrease in vocalization amplitude in guinea pigs submitted to a peripheral noxious stimulus. The antinociceptive effect of carbachol lasted 60 min, One-way A N O V A showed a significant difference in mean vocalization response at 2 min, F(2, 21) = 4.32, p < 0.006, at 5 min, F(2, 21) = 6.75, p < 0.005, at 10 min, F(2, 21) = 7.01, p < 0.005, at 15 min, F(2, 21) = 6.63, p < 0.005, at 30 min, F(2, 21) = 3.97, p < 0.007, at 45 min F(2, 21) = 3.82, p < 0.01, and at 60 min F(2, 21) = 3.71, p < 0.05 (Fig. 3B, CNT). The duration of the antinociceptive effect of carbachol microinjection into the NTS followed by simultaneous blockade of the PBRd with xylocaine lasted as long as 80 min. The antinociceptive effect represented by the reduction in vocalization amplitude was statistically significant at 5 rain (t = 3.12, p < 0.02), at 10 min (t = 3.96, p < 0.01), at 15 min (t = 2.96, p < 0.04), at 30 min (t = 2.87, p < 0.05), at 45 min (t = 2.88, p < 0.05), at 60 min (t = 2.87, p < 0.05), and at 80 min (t = 2.89, p < 0.05) when compared with the group microinjected with saline (Fig. 3B,

The present results show the occurrence of temporal potentiation of the antinociceptive effect of carbachol microinjected into the dorsal PBR brought about by subsequent xylocaine application to the AP or microinjection into the NTS of guinea pigs. An increased duration of the effect of carbachol was also clear when the inverse maneuver was used, i.e., when carbachol was applied to the AP or microinjected into the NTS and xylocaine was microinjected into the dorsal PBR 5 min after carbachol. The procedure used by us to apply drugs to the AP does not exclude the possibility that the drugs diffused to nearby regions, including the NTS. For this reason, it is implied that the effects caused by drug application may be due to activation or blockade of the dorsomedial medulla as a whole. The antinociceptive effect of carbachol injected into the dorsal PBR, quantitatively demonstrated in our experiments by the reduction in vocalization amplitude in animals submitted to a peripheral noxious stimulus, lasted 15 min, but was prolonged to 45 min after reversible dorsomedial medulla blockade with xylocaine and to 60 min after blockade of the NTS. Several studies have shown the participation of the AP [42,44], the NTS [11,36,48,50], and the PBR [22,31,32] in the modulation of nociceptive responses. In our laboratory, we have observed that chemical stimulation of the AP with noradrenaline and 5-HT reduces the motor defense and vocalization responses of guinea pigs to a peripheral noxious stimulus [42,44]. Although the duration of the antinociceptive effect of carbachol application to the AP (30 min) differed from that observed after carbachol microinjection into the NTS (60 min), in the present experiment we cannot rule out the possibility that the antinociception observed after carbachoi application was partially due to diffusion of the drug toward the NTS and or to connections between these two structures. Electrical stimulation of the PBR of cats [t0] produces a profound nociceptive suppression that is not attenuated by systemic naloxone administration, indicating that the opioid mechanisms may not be involved in this type of analgesia. An identical observation was also reported by Katayama et al. [31] when they bilaterally microinjected carbachol into the parabrachial region of cats. Similarly to the findings reported by Katayama et al. [31 ], the analgesia induced by carbachol in our experiments was not observed when microinjection was performed at sites close to the dorsal PBR, such as the inferior colliculus, lateral lemniscus, and cuneiform nucleus. However, the difference between our data and those reported by the above authors is mainly related to the duration of the analgesic effect of carbachol microinjected into the dorsal PBR. This may have been due to the use of animals of different species (guinea pigs in our case), to the doses of carbachol employed, to the different analgesia tests used, and to the fact that our microinjections were performed unilaterally while the above authors used bilateral microinjections. As regards the analgesic tests, the vocalization response used by us is integrated at supraspinal levels and is considered by Guzman et al. [20] to be the most specific central indicator of a painful sensation, thus

PARABRACHIAL

REGION, DORSOMEDIAL

MEDULLA, AND ANALGESIA

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FIG. 3. (A) Effects of application of saline to the area postrema (SALAP, n = 8), of carbachol to the area postrema (CAP, n = 8), and of carbachol to the area postrema plus xylocaine to the dorsal parabrachial region (CAPXPB, n = 8). (B) effects of microinjection of saline unilaterally into the nucleus tractus solitarius (SALNT, n = 7), of carbachol into the NTS (CNT, n = 9), and of carbachol into the nucleus tractus solitarius plus xylocaine ipsilaterally into the dorsal parabrachial region (CNTXPB, n = 8) on mean vocalization amplitude (M. VOC. AMPL.) in response to the application of a peripheral noxious stimulus to the paw of awake guinea pigs. The abscissa shows the intervals in minutes (TIME min) at which vocalization amplitude (in mm) was measured in the presence of a peripheral noxious stimulus; 0 represents the mean control values. *p < 0.05 compared to SALAP (A) and to SALNT (B). The vertical bars indicate the SEM.

r e p r e s e n t i n g a m o r e c o m p l e x r e s p o n s e than a reflex integrated at the spinal level. T h e parabrachial nucleus receives abundant afferences from the limbic s y s t e m and spinal cord and sends m a n y fibers (possibly cholinergic ones) to the N R M and to the adjacent reticular formation, a fact that explains its i n v o l v e m e n t in the control o f

nociception b e c a u s e inhibitory d e s c e n d i n g p a t h w a y s are k n o w n to originate in the latter structures. H a w s et al. [22] also d e m onstrated the i n v o l v e m e n t o f n e u r o n s o f the parabrachial region in the modulation o f nociceptive transmission at the level o f the dorsal horn o f the spinal cord. This m a y o c c u r both via a direct p a t h w a y or indirectly t h r o u g h n e u r o n s o f the rostroventral me-

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FIG. 4. (A) Comparison of the effect of topical application of xylocaine (XILAP, n = 8) and of saline (SALAP, n = 8) to the AP, and (B) comparison of the effect of unilateral microinjection of xylocaine (XILNT, n = 7) and of saline (SALNT, n = 7) into the NTS on mean vocalization amplitude (M. VOC. AMPL.) in response to the application of a peripheral noxious stimulus to awake guinea pigs. The abscissa shows the intervals in minutes (TIME min) at which vocalization amplitude (in mm) was measured. 0 represents the mean control values, and the vertical bars indicate the SEM.

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MIDBRAIN 12,4

PONS 13,4

FIG. 5. Schematic drawing of frontal sections obtained on two planes representative of the midbrain and of the pons reproduced from the Rossner guinea pig atlas, with the projection of the sites where microinjections were made. Individual microinjection sites for group 2 (asterisks) and group 3 (open circles) of Experiment ! are indicated on the left side. Individual microinjection sites for group 2 (asterisks) of Experiment 11 are indicated on the right side. BC = brachium conjuntivum; Aq. S. aqueductus Sylvii.

and Ritter [ 14] demonstrated that lesion of the lateral PBR of rats on both sides prevents overingestion caused by destruction of the AP. These investigators raised the possibility that the AP neurons that were destroyed, under normal conditions should modulate the activity of neurons of the lateral PBR, which integrate and transmit chemosensory information to the forebrain. Also, Felder and Mifflin [16] first demonstrated that the parabrachial nuclei may have a powerful modulatory action on the activity of NTS neurons that process cardiovascular inputs. Our results also indicate the possibility of a functional connection between regions of the dorsomedial medulla and PBR in terms of pain modulation. The duration of the antinociceptive effect of carbachol applied to the AP was duplicated by xylocaine injection into the PBR, indicating the removal of a tonic inhibition. On the other hand, reversible blockade of the PBR with xylocaine alone did not change vocalization amplitude when compared to the saline group. This indicates that the temporal potentiation observed when the dorsomedial medulla is stimulated by topical application of carbachol to the AP during reversible blockade of PBR is due to an interaction of the two regions and not to blockade of PBR alone. Our data indicate that modulation between the dorsomedial medulla and PBR is reciprocal because the same temporal potentiation was obtained in animals in which xylocaine was applied to the AP and carbachol to the dorsal PBR. The same reciprocity with respect to the temporal effect was observed between the NTS and PBRd, although with different durations. The duration of the analgesic effect of carbachol microinjected unilaterally into the NTS (60 min) was extended to 80 min after ipsilateral blockade of the PBRd with

19,0 dulla (RVM) towards which the parabrachial region sends projections. Wamsley et al. [62] detected a high density of muscarinic receptors in the dorsal parabrachial region, which suggests that the antinociceptive effects of carbachol in this region may be mediated by such receptors. Previous experiments in our laboratory demonstrated that analgesia observed after carbachol microinjection into the PBRd was abolished by pretreatment with atropine [46]. Direct projections of most lamina I neurons to the PBR via the dorsolateral funiculus were demonstrated in cats 127] and later in rats [28]. In the latter study, these investigators suggested that this projection system may be important for some basic components of the nociceptive response and, therefore, may be involved in mechanisms of pain modulation and in autonomic reflex activation. Based on the findings of Hylden et al. [28], we may assume that the modulation of ascending nociceptive information may be controlled by carbachol microinjection into midbrain sites, as done in the present study. Thus, the antinociceptive effect of carbachol may be due to the prevention of the ascension of nociceptive information to the thalamus or to the activation of a cholinergic descending inhibitory pathway. Basbaum and Fields [2] demonstrated the activation of descending modulatory systems through connections of the PBR with the nucleus raphe magnus. It, thus, appears that the modulation exerted by the PBR may occur not only at the spinal but also at the supraspinal level. The interconnections of the AP with the PBR and of the NTS with the PBR have been reported in several neuroanatomical studies [19,38,47,55,60]. The functional importance of these interconnections has been reported by some investigators. Edwards

19,6

FLM

/

20,5

FIG. 6. Schematic drawing of frontal sections obtained on three planes representative of the medulla reproduced from the Rossner guinea pig atlas, with the projection of the sites where microinjections were made. Individual microinjection sites for group 6 (asterisks) and group 7 (open circles) of Experiment II are indicated on the left side. Individual microinjection sites for group 5 (open circles) of Experiment II are indicated on the right side. NTS = nucleus tractus solitarius; MRF = nucleus magnocellularis formationis reticularis; FLM = Fasciculus longitudinalis medialis.

P A R A B R A C H I A L REGION, D O R S O M E D I A L M E D U L L A , A N D A N A L G E S I A

xylocaine. Conversely, the analgesic effect of carbachol applied to the P B R d (15 min) was extended to 60 min after ipsilateral microinjection of xylocaine into the NTS. Therefore, the reciprocal inhibitory action of the dorsomedial medulla in general and of the NTS in particular and P B R permits to control the duration of the analgesic effect. One of the situations in which analgesic mechanisms are activated is the prey/ predator confrontation [56]. There is evidence that the PBR modulates the tonic immobility (TI) response, which is a component of the defensive behavior, in rabbits [18] and guinea pigs [45]. Katayama et al. [32] also observed attack/escape behavior in cats due to chemical stimulation of the P B R with carbachol. Several studies have shown the importance of the emotional component in the modulation of TI [13,52] and of analgesia [15,29]. The fact that the PBR is connected to limbic and hypothalamic regions [19,26,40,59] may possibly be important in triggering the reciprocal temporal control between P B R and A P and P B R and NTS as a function of emotionality. On the other hand, humoral factors that are altered or released during defensive behavior may modify neuron firing in the AP, a chemosensitive region devoid of a barrier. The NTS is a site of convergence o f information originating from interoceptors, including baroreceptors. Many experiments have demonstrated that the baroreceptors, which may be stimulated during a situation of confrontation, may modulate a response to a noxious stimulus [12,41]. Thus, due to this interconnections, the combination of emotionality, humoral factors, and visceral sensation may determine the duration of the analgesic effect. The temporal modulation of the analgesic response may have an important adaptive function in confrontation situations because the longer or shorter duration of the analgesic effect may reinforce the defensive response, depending on external conditions. ACKNOWLEDGEMENTS The authors acknowledge the financial support of CNPq and FAEPA and the technical assistance of Mrs. Mariulza Rocha Brentegani and Mrs. Aparecida de Souza Fire Pereira, Maria Aparecida Protti de Andrade, and Rubens Fernando de Melo.

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