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Pain, 42 (1990) 103-119 Elsevier

PAIN 01611

Antinociception and cardiovascular responses produced by electrical stimulation in the nucleus tractus solitarius, nucleus reticularis ventralis, and the caudal medulla Sue A. Aicher and Alan Randich ~epur~rne~~of Psychology, Unioersify ofiowu,Zawu City, IA (Received

18 October

1989, revision received

16 January

52242 (U.S.A.)

1990, accepted

30 January

1990)

In experiment 1, quantitative regional comparisons of the antinociceptive and cardiovascular responses produced by Summary electrical stimulation in the caudal medulla, including regions such as the nucleus tractus solitarius (NTS), nucleus reticularis ventralis (NRV), nucleus reticularis ~g~t~Ilul~s (NRGC), nucleus reticularis p~a~8ant~ellula~s (NRPGC), nucleus raphe obscurus (NRO), and medial portions of the lateral reticular nucleus (LRN), were made in the rat. Electrical stimulation in all of these regions resulted in inhibition of the nociceptive tail-flick reflex, although the threshold intensity for inhibition was greater for sites in NTS compared to many sites ventral to the NTS. Antinociception was generally accompanied by an increase in mean arterial blood pressure, with the exception of sites in the NRO, where depressor responses were evoked by stimulation. Detailed comparisons between the NTS and NRV revealed that greater intensities of electrical stimulation were required to produce antinociception for sites in the NTS as compared to the NRV. There were no significant differences in threshold intensities for antin~iception as a function of rostrocaudal subdivisions of the NT’S, but the lateral subdivision of the NTS was silently more efficacious than the medial subdivision. This mediolateral difference within NTS was primarily due to stimulation in medial sites producing overt movements in some animals, probably due to stimulation of adjacent midline nuclei or pathways. Within the NRV, thresholds for inhibition of the tail-flick reflex were greater for sites in the dorsal subdivision as compared to the ventral subdivision, which contains spinopetal projections from the NRM. The slopes of the lines of recruitment for inhibition of the tail-flick reflex at stimulation sites in either the NTS or NRV were both very steep, similar to other forms of antinociception. In experiment 2, the pulse duration of electrical stimulation was varied for sites of stimulation in the lateral NTS and NRV to generate strength-duration curves. This experiment confirmed that stimulation sites in the lateral NTS required greater current intensities to inhibit the tail-flick reflex than sites in the NRV. However, the chronaxies derived from the strength-duration functions for the NTS or NRV were both approximately 170 psec, indicating that the antinociceptive effects in these regions may not be exclusively due to the stimulation of fibers of passage. These results are discussed in terms of the role of the NTS, NRV, and caudal medulla in the modulation of nociceptive responses and cardiovascular function. Key words:

Antin~~ception:

Blood pressure;

Tail-flick;

(Rat)

Introduction Recently, it has been demonstrated that either electrical stimulation or glutamate microinjection

Correspondence to: Sue Aicher, Division of Neurobiology, Cornell University Medical College, 411 E. 69th St., New York, NY 10021, U.S.A. 0304-3959/90/%03.50

0 1990 Elsevier Science Publishers

in the caudal nucleus tractus solitarius (NTS) produces inhibition of both the tail-flick reflex evoked by noxious heat and responses of spinal dorsal horn neurons to noxious stimuli [11,26,31,42,46]. Pharmacological or electrical stimulation of either cervical or subdiaphragmatic vagal afferents, which terminate in the caudal NTS [22], produces similar inhibitory effects when assessed with the same response measures [1,4,28,37-41,43-45]. However,

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Division)

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these antinociceptive effects are not limited to the NTS region of the caudal medulla, since it has also been reported that electrical stimulation and/or glutamate microinjection in the nucleus reticularis ventralis (NRV), the caudal lateral reticular nucleus (LRN), the nucleus reticularis gigantocellularis (NRGC) and the ventral medulla adjacent to the inferior olive, also produce antinociception and/or inhibition of spinal dorsal horn neurons [13,27,42,49]. Thus. many regions of the caudal medulla are clearly involved in descending control of nociceptive reflexes. It is also well established that many of the regions of the caudal medulla noted above regulate cardiovascular function. Several theories have emphasized the importance of cardiovascular/ pain-regulatory interactions during normal physiological functioning, as well as in some disease states such as hypertension [see 39,541. Experimental evidence suggests that regions of the caudal medulla may be involved in mediating both antinociception and cardiovascular reflexes. For example, microinjections of glutamate in the caudal NTS that produce either antinociception or inhibition of spinal dorsal horn neuronal responses to noxious stimuli also decrease arterial blood pressure and heart rate in the rat, i.e.. stimulate the baroreceptor reflex [42,46]. On the other hand, antinociceptive effects produced by glutamate microinjection in the NRV are accompanied by an increase in arterial blood pressure [42]. Therefore, there are divergences in the cardiovascular responses that are produced by stimulation in these regions of the caudal medulla that support antinociception. The aim of the present experiments was to make quantitative regional comparisons of both antinociceptive and cardiovascular responses produced by electrical stimulation in the caudal medulla of the rat. The first experiment involved a systematic grid-analysis of the caudal medulla to determine the intensity of electrical stimulation necessary to produce inhibition of the tail-flick reflex. The changes in arterial blood pressure and heart rate produced by electrical stimulation in these regions were also quantitatively compared, since these have not been reported in previous studies of antinociception and have not been sys-

tematically studied on the basis of regional subdivisions. The second experiment generated strengthduration functions for both inhibition of the tailflick reflex and concomitant arterial blood pressure responses produced by electrical stimulation of sites in the NTS and NRV to assess potential neural substrates for these responses [36].

Methods Subjecrs Ninety-two male Sprague-Dawley mone Assay Labs., Chicago) weighing served as subjects in these experiments. were housed individually in wire mesh ad lib food and water and maintained LD cycle.

rats (Hor300-450 g Animals cages with on a 12:12

Apparutus The tail-flick reflex was evoked by a radiant heat stimulus provided by a projector lamp. The heat stimulus was housed in a metal casing and focused on the rat’s tail through a 1 cm X 0.3 cm opening. Onset and termination of each trial were automatically recorded by a digital timer. The radiant heat stimulus was identical for all subjects and temperature characteristics have been reported previously [ 11. The arterial blood pressure signal was obtained with a Century pressure transducer interfaced to an Apple II Plus microprocessor. The microprocessor used the FIRST system [50] and has been described in detail previously [l]. Gene& surgical and testing techniques Each rat was anesthetized with pentobarbital sodium (50 mg/kg i.p.) and an arterial catheter (Microline) was placed in either the left or right common carotid artery. Each rat was also implanted with a tracheal catheter (PE-100). All wound margins were liberally coated with an anesthetic ointment. Following catheterization, each rat was placed in a stereotaxic apparatus. with the head tilted to approximately 45” below the horizontal plane. An incision was made medially along the occipital bone through all layers

105

of the overlying skin and musculature. The tissue was reflected laterally to expose the atlanto-occipital membrane, which was then carefully cut. A portion of the occipital bone was removed to allow visualization of the dorsal medulla~ surface and placement of the stimulation electrode (see below). Rats were maintained in a lightly anesthetized state during all testing, with supplements of pentobarbital sodium (2.5 mg) administered i.p. as necessary to maintain this state as previously described [49]. The nociceptive test used in these experiments was the spinally mediated tail-flick reflex [9]. Baseline tail-flick reflex latencies elicited by the application of the noxious radiant heat to the tail ranged between 2.5 and 3.5 set, which corresponds to a skin temperature of approximately 45°C [32]. The cut-off latency for the application of the heat stimulus was 10 sec. The test trial tail-flick latency was converted to a percent maximum possible effect (% MPE) to indicate the change from the baseline latency relative to the cut-off latency. The formula for the % MPE is: (test trial latency - baseline latency)/ (10 set - baseline latency)

x

100.

Therefore, a % MPE value of 100 indicates that the test trial tail-flick reflex was not evoked within 10 set, whereas a % MPE value of 0 indicates the test trial latency was the same as the baseline latency. Brain stimulation and response measurement A paralene-C insulated tungsten electrode (0.13 mm diameter, Micro Probe) was used for brain stimulation with a needle inserted into the musculature of the right leg serving as the reference electrode (anode). Constant current monopolar cathodal stimulation at 100 Hz was generated with a Grass S48 stimulator and constant current source (Iowa Psychology Department Electronics Shop). The tail-flick reflex was tested 10 set after the onset of brain stimulation in each experiment. The brain stimulation terminated after either the tailflick occurred or the tail-flick latency reached 10 set, whichever occurred first. A new baseline

latency (2.5-3.5 set) was established prior to each test of electrical stimulation. The minimum current intensity necessary to produce inhibition of the tail-flick reflex to the 10 set cut-off latency, i.e., % MPE of 100, will be referred to as the “threshold” intensity. The heat stimulus after 10 set was approximately 53°C well above the temperature at which the baseline tail-flick occurred (45’C), making these stringent criteria for an antinociceptive effect. The tail-flick data will be presented in terms of the % MPE at the ~~re~~u~d intensity, as defined above. All mean arterial blood pressure and heart rate data presented in the results were recorded during the 10 set of brain stimulation prior to the application of noxious heat to the tail, thereby eliminating possible confounding with the cardiovascular responses evoked by the subsequent application of the noxious heat stimulus. The mean arterial blood pressure and heart rate values recorded 2 set prior to the beginning of each test of brain stimulation were designated as baseline values and the reported percent change measures were from these values. All rats were ventilated with room air during testing (60 strokes/mm) to prevent the possible confounding effects of apnea produced by electrical stimulation in some brain areas. Experiment I Sixteen placements on the left side of the dorsomedial medulla were designated as initial reference sites for analysis in this study. The calamus scriptorius (the caudal tip of the area postrema) served as the zero reference site and this site corresponds to 14.30 mm caudal to the interaural line [35]. The 16 reference placements consisted of sites that were 0.5 mm apart and extending from 0.5 mm caudal to 1.0 mm rostra1 to the calamus scriptorius, and 0.0-1.5 mm lateral to the calamus scriptorius. At each of these 16 reference sites, the electrode was lowered in 0.5 mm increments from the medullary surface for a test of electrical stimulation and the final ventral placement was 2.5 mm below the surface. Therefore, 5 ventral placements were tested at each of the 16 initial placements, resulting in a 4 x 4 x 5 matrix and a total of 80 separate stimulation sites. Each site of stimulation was tested in at least 6

rats (N = &g/site) and no rat was tested with more than 2 series of ventral tracks, i.e., 10 sites of stimulation. Fifty-seven rats received only 1 ventral track and 23 rats received 2 ventral tracks: the second track was placed at least 1 mm lateral to the first track to minimize tissue damage. In determining threshold, stimulation frequency (100 Hz) and pulse duration (100 psec) were held constant, while intensity was incremented from 10 to 200 PA (10 PA steps from 10 to 80 PA; 25 PA steps from 100 to 200 PA). When either the threshold for tail-flick inhibition had been determined or a maximal intensity of 200 PA had been tested, the electrode was advanced ventrally to the next brain site. In some cases, however, electrical stimulation in a brain area produced overt movements that made it inappropriate to conduct tail-flick tests, i.e., dorsal flexion of the body; flexion or extension of the hind limbs; or tonic flexion of the tail laterally. When electrical stimulation resulted in overt movements, the trial was terminated and that site was not tested further. For the purpose of data analysis at sites where overt movements were produced by stimulation, the last current intensity tested prior to the intensity that produced movements was considered the threshold intensity for that site. Since some sites produced movements and not all sites supported antinociception, both the threshold intensity in PA and the % MPE measures were compared in the analyses, and together these measures allow one to differentiate antinociceptive effects from these other outcomes (see Results). Experiment 2 Rats were catheterized and tested as described above, except that the arterial catheter was placed in the right axillary artery for this experiment. In this experiment, the threshold current intensity for inhibition of the tail-flick reflex was initially determined in 2 regions chosen for comparison: the lateral NTS and the NRV (N = 6/site). Only a single site was tested in each rat. The threshold intensity was initially established using the stimulation parameters described above (100 Hz, 100 psec, and intensity incremented from 10 to 200 PA). The threshold intensity was tested

3 times to ensure that the inhibition of the tail-flick reflex was reliable. After the initial threshold intensity had been established, strength-duration functions were generated by varying pulse duration and re-establishing threshold intensity. The frequency of stimulation was held constant (100 Hz) and 9 pulse durations were tested ranging from 50 to 800 psec. At each pulse duration, the minimum current necessary to produce inhibition of the tail-flick reflex to the 10 set cut-off latency was determined. The order of testing for the 9 pulse durations was randomized across rats. After all pulse durations had been tested, the threshold intensity at the initial pulse duration of 100 psec was retested and if the threshold had changed by more than 10 PA the animal was excluded from the experiment.

Histology At the conclusion of each experiment, the rat was sacrificed and a lesion was made at the most ventral brain site tested. Lesions were made by stimulation with dc current of 50 PA for 4 sec. The brain was removed and placed in 10% formalin for at least 3 days. Brains were frozen, sectioned at 40 pm on a cryostat, and stained with cresyl violet. For experiment 1, stimulation sites were reconstructed with the use of computer-assisted image analysis in 3 dimensions (University of Iowa Image Analysis Facility). The total distance of the electrode trajectory between the point of entry and the lesion was calculated and the 5 stimulation sites were placed at equidistant points along this trajectory. Means and standard deviations for the sites of stimulation were then computed and plotted in 3 dimensions for each group. For experiment 2, the lesion corresponded directly to the stimulation site, since only one site was tested in each animal.

Dutu unalysis The % MPE and percent change from baseline blood pressure or heart rate values were subjected to an analysis of variance (ANOVA) for each experiment. Newman-Keuls post-hoc comparisons of means were performed when required. Alpha was 0.05 for all analyses.

107

Results Experiment

1

Trackings The normative data for the threshold intensities for inhibition of the tail-flick reflex and ‘% MPE values are shown in Figs. 1-4 and the corresponding histology is shown in Fig. 5. In Figs. 1-4, each figure shows the mean data for the 4 lateral tracks (0.0 mm lateral to 1.5 mm lateral) at each of the 4 rostrocaudal placements (0.5 mm caudal to 1.0 mm rostral). Fig. 5 shows 3-dimensional reconstructions of the mean and standard deviation for each of 5 ventral stimulation sites along 4 lateral tracks. Each histological section in Fig. 5 shows the stimulation sites for the data shown in Figs. l-4. Several general observations can be derived from the tracking data presented in these figures. First, as noted previously, some sites of stimulation either did not produce inhibition of the tailflick reflex in all animals or produced overt movements. Thus, if the % MPE value for a site is less than 100, then either movements were produced by stimulation or a failure to inhibit the tail-flick reflex was obtained. These outcomes can be distinguished since stimulation in a site that did not produce inhibition or movements would result in a % MPE much less than 100 and a corresponding current intensity of approximately 200 PA, whereas movements were often seen at intensities less than the maximum tested. Figs. l-5 clearly show that the sites where movements were produced lie predominantly in the dorsomedial medulla in the vicinity of the hypoglossal nucleus. Another conclusion derived from these figures is that sites at least 1.0 mm lateral to the midline reliably produced inhibition of the tail-flick reflex at all placements. In many of these tracks, the threshold intensity for inhibition of the tail-flick reflex was greater at dorsal sites and decreased for more ventral sites. The dorsal sites were located in the nucleus reticularis dorsalis (NRD), NTS, nucleus reticularis parvocellularis (NRPC), or cuneate nucleus. The ventral sites were in or near the NRV, NRGC, nucleus retroambiguus (NRA), or LRN. Within medial tracks, inhibition of the tail-flick reflex could be obtained at ventral sites

that were predominantly located within the nucleus raphe obscurus (NRO). Although not illustrated (see below for regional the predominant cardiovascular reanalyses), sponse to electrical stimulation in virtually all medullary sites was an increase in mean arterial blood pressure. The magnitude of the pressor response ranged from less than 5% to 308, but was often confounded with differences in stimulation intensity since all measurements were anchored to the current intensity required for inhibition of the tail-flick reflex. The only exception occurred with sites of stimulation in the NRO, which produced a decrease in mean arterial blood pressure of lo20%. There was little or no change in heart rate during electrical stimulation of any of these medullary regions. Regional analyses Tail-flick. Detailed analyses were performed on sites of stimulation within either the NTS or the NRV using the tracking data just described. Each of these regions was divided into subregions as shown in Fig. 6. The NTS was divided into a medial and a lateral subdivision at each of the 4 rostrocaudal placements, where the medial subdivision included the medial and commissural subnuclei [23]. NRV was divided into a dorsal and a ventral subdivision regardless of rostrocaudal placement, corresponding approximately to the beta and alpha subnuclei of the NRV [33]. Fig. 6 shows all of the stimulation sites that were located in either the NTS (N = 67) or the NRV (N = 72). Fig. 7 shows the mean threshold intensities for inhibition of the tail-flick reflex at sites located within the designated subdivisions of NTS or NRV. An ANOVA of threshold current intensities as a function of the subdivisions within NTS revealed no significant differences for either rostrocaudal or mediolateral placement. However, an ANOVA of the % MPE values of the same subdivisions revealed a significant difference in the mediolateral factor, F (1, 59) = 4.1, indicating that inhibition of the tail-flick reflex was not always produced by stimulation at the threshold current intensity in some sites. This effect is primarily due to smaller % MPE values derived from medial sites and reflects overt movements, possi-

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bly due to stimulation of other midline structures or pathways. Together, the analyses of threshold current intensity and % MPE values indicate that the lateral NTS is more efficacious than the medial NTS in producing inhibition of the tail-flick reflex, but that there are no significant differences in efficacy as a function of the rostra]-caudal divisions examined. Since the stimulation intensity was systematically increased until the tail-flick reflex was inhibited to the cut-off latency, subthreshold current intensities were available for many sites, particularly for sites with higher thresholds. Therefore. the change in % MPE as a function of subthreshold intensity was examined for the NTS and NRV. For each animal, the intensity that was closest to, but not exceeding, either 0.25, 0.50. or 0.75 of the threshold intensity was recorded when available (i.e., if the threshold intensity was 20 PA, only the 0.50 fraction corresponding to 10 PA was tested). The threshold intensity for each animal corresponds to 1.0. Fig. 8 shows the % MPE as a function of the proportion of threshold intensity of stimulation for the medial and lateral subdivisions of NTS at each of the four rostrocaudal placements. Fig. 8 shows that there is an abrupt increase in %I MPE as threshold current intensity is approached compared to all subthreshold stimulation intensities, in spite of approximately equal steps in current intensity. A l-way ANOVA of the % MPE values at subthreshold and threshold intensities from subjects that had thresholds of 40 PA or greater (complete data set) showed a significant effect across stimulation intensities in each of these 8 groups. For 6 of these 8 groups, the ‘% MPE values at threshold intensity were significantly greater than the % MPE values at any subthreshold intensity, and none of the subthreshold ‘36MPE values differed

Fig. 1. Threshold intensity for inhibition of the tail-flick reflex (filled circles) and % MPE values (open squares) for lateral tracks that began 0.5 mm caudal to the calamus scriptorius. Each panel shows one lateral track (0.0, 0.5, 1.0 or 1.5 mm) and each of 5 depths within each track ( - 0.5 to - 2.5 mm) on the y axis.

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from each other, i.e., ordering of mean % MPE values is 0.25 = 0.50 = 0.75 < 1.0. Fig. 7 also shows the mean threshold intensities for the dorsal and medial subdivisions of NRV. A l-way ANOVA of the intensity thresholds showed a significant effect of dorsoventral position, F (1, 70) = 20.5, indicating that the threshold current intensity required to produce inhibition of the tail-flick reflex was greater for sites within the dorsal NRV as compared to the ventral NRV. The more effective sites lie predominantly within the alpha subdivision of this nucleus [33]. Inhibition of the tail-flick reflex was reliably produced by threshold intensities of stimulation within these sites. The top panel of Fig. 9 shows the % MPE values for subthreshold and threshold intensities of electrical stimulation within the NRV. As shown for sites within the NTS, there is also an abrupt increase in the % MPE value as threshold intensity is approached for sites within the NRV, even though the increments in intensity were approximately equal. Since only approximately 5% of the ventral group had thresholds of 40 PA or greater, this group was not analyzed further. Post-hoc analyses of the % MPE values across fractions of threshold intensity in the dorsal NRV revealed the following ordering of % MPE values: 0.25 = 0.50 < 0.75 < 1.0. Recruitment indices were also calculated for the medial and lateral divisions of NTS and the dorsal and ventral divisions of NRV. For each subject that showed inhibition of the tail-flick reflex to the 10 set cut-off, the slope of the change in % MPE as a function of stimulation intensity was determined for the portion of the curve that departed greater than 15% from baseline levels. The recruitment index (change in % MPE/lO PA step in stimulation intensity) was 63 for medial NTS, 70 for lateral NTS, 86 for dorsal NRV and 89 for ventral NRV. An ANOVA af the recruit-

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Fig. 2. Threshold intensity for inhibition of the tail-flick reflex (filled circles) and % MPE values (open squares) for lateral tracks that began at the calamus scriptorius. Each panel shows one lateral track (0.0, 0.5, 1.0 or 1.5 mm) and each of 5 depths within each track ( - 0.5 to - 2.5 mm) on the y axis.

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ment index for groups in either lateral or medial NTS versus dorsal or ventral NRV showed no significant difference as a function of the subdivisions within each region, but there was a significant main effect of site, F (1. 125) = 18.8. Stimulation sites in NRV had higher recruitment indices than sites in NTS, indicating that a greater change in tail-flick latency was produced by equivalent steps in intensity when stimulating in NRV as compared to NTS. This may seem at odds with the subthreshold data presented above and in Figs. 8 and 9, but it should be noted that the actual intensity steps for the subthreshold data for NTS are larger than for NRV since their thresholds were larger. A final analysis was conducted on the threshold intensities for sites within either the NTS or the NRV, collapsed across all subdivisions. A l-way ANOVA revealed that the threshold intensity for inhibition of the tail-flick reflex was greater for sites in the NTS as compared to the NRV. F (1. 137) = 68.3. Cardiovascular responses. Fig. 10 shows the percent change in arterial blood pressure following 10 set of electrical stimulation in either the medial or lateral NTS as a function of the proportion of threshold intensity. Only the 1.0 mm rostra1 subgroup in the medial NTS showed an increase in mean arterial blood pressure of greater than 10%. It should be noted, however, that arterial blood pressure increases were occasionally observed following the onset of stimulation in some subregions, but blood pressure returned towards baseline values during the 10 set of stimulation. perhaps due to baroreflex modulation. For sites in the lateral NTS, where inhibition of the tail-flick reflex was obtained most reliably at threshold intensities of stimulation, there was a pressor response of approximately 20%. At the lowest subthreshold intensity, there was virtually no change

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in blood pressure. As the intensity of stimulation was increased, there was a progressive increase in the magnitude of the pressor response, as opposed to the non-linear changes in the corresponding ‘% MPE values for these groups at the same intensities (cf., Fig. 10 vs. Fig. 8). A similar relationship was seen for stimulation in the NRV (Fig. 9) although the magnitude of the pressor response was less for sites in the ventral NRV. However, the intensity of stimulation was also significantly lower for sites in the ventral NRV as compared to either dorsal NRV or the NTS. 2

Strength-duration manipulations were used to examine the differences in threshold intensity for inhibition of the tail-flick reflex as a function of pulse duration for sites within either the lateral NTS or the NRV. The right panel of Fig. 11 shows the stimulation sites in the NTS and NRV that were used for the strength-duration analyses. The top left panel of Fig. 11 shows the mean threshold intensities for sites in either the lateral NTS or NRV, as a function of pulse duration. A mixed ANOVA of the intensity of stimulation necessary to produce inhibition of the tail-flick reflex to the 10 set cut-off latency was performed on the 2 groups as a function of the 9 pulse durations tested. The between-subject factor in this analysis was site (NTS or NRV) while the within-subject factor was pulse duration (50-800 psec). This analysis revealed significant main effects of site, F (1, 10) = 19.8, and pulse duration, F (1, 10) = 71.7, as well as a significant site x pulse duration interaction, F (8, 80) = 13.1. Post-hoc analyses revealed that the threshold intensity to produce inhibition of the tail-flick reflex was greater for sites within the lateral NTS than for sites within the NRV at all pulse durations tested.

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Fig. 4. Threshold intensity for inhibition of the tail-flick reflex (filled circles) and % MPE values (open squares) for lateral tracks that began 1.0 mm rostra1 to the calamus scriptorius. Each panel shows one lateral track (0.0, 0.5, 1.0 or 1.5 mm) and each of 5 depths within each track (- 0.5 to - 2.5 mm) on the y axis.

The bottom left panel of Fig. 11 shows the threshold current intensities for the 9 pulse durations tested at sites within either the NTS or NRV expressed as a proportion of the rheobase current. The rheobase current is defined in the present context as the intensity of stimulation necessary to produce inhibition of the tail-flick reflex to the cut-off latency at a long pulse duration, where the intensity has reached a plateau [36]. As shown in Fig. 7 1, the threshold intensity plateaus at pulse durations of approximately 600-800 psec. Therefore, the threshold intensity at the 800 psec pulse duration (approximately 30 PA for NTS and 15 PA for NRV) was designated as the rheobase current for each rat, i.e., 1.0 r. The threshold intensities at all other pulse durations were then converted to a proportion of this value for each subject and then averaged for each pulse duration to produce the values shown in Fig.. Il. A mixed ANOVA of the proportion of rheobase current at each pulse duration for the NTS and NRV groups revealed a significant main effect of pulse duration only. F (8, 80) = 46.0. There was no effect of site, indicating that the proportion of rheobase current at each pulse duration was the same for NTS and NRV sites. The chronaxie is defined as the pulse duration required to produce a given effect at twice the rheobase current [36]. The chronaxies for sites within NTS or NRV were both approximately 170 psec (Fig. 11). The mean arterial blood pressure response following 10 set of electrical stimulation was examined across pulse durations between the NTS and NRV. Electrical stimulation in either the NTS or NRV resulted in increases in mean arterial blood pressure ranging between 2 and 23%. but a mixed-design ANOVA revealed no significant differences in the pressor response as a function of

Fig 5. Three-dimensional shaded-surface reconstructions of the stimulation sites for the data shown in Figs. 1-4. Each section shows the mean and standard deviation (in the x, y and z. planes) of each of 5 ventral placements along 1 of 4 lateral tracks (depicted by crosses). The 4 rostrocaudal groups of tracks are shown in each section, moving from most caudal (top panel) to most rostra1 (bottom panel). Structures that arc outlined within the reconstruction are the area postrema. the NTS, the left hypoglossal nucleus. and the left LRN.

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either pulse duration, F (8,80)= 0.8or stimulation site, F (1, 80) = 0.2. Stimulation in these regions produced virtually no change in heart rate and an ANOVA indicated no significant differences in the change in heart rate produced by stimulaticn in either the NTS or NRV.

I

Discussion

P 13.30

Dorsal .---w-w_ Ventral

NRV NRV

P

LRN

13.80 Lotsral

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! Medial NTS

The first experiment illustrated that inhibition of the tail-flick reflex could be produced by electrical stimulation in most sites tested within the caudal medulla including the NTS, NRV, NRO, NRGC, NRPGC, and LRN. This outcome is not surprising in light of the fact that multiple and separate cell groups send projections to the spinal cord through the caudal medulla, e.g., nucleus raphe magnus (NRM) and locus coeruleus (LC) [5,6,12,19]. There are also many cell groups in the caudal medulla that have been directly implicated in the production of antinociception and are known to project to regions that support antinociception. For example, cells in the NTS project to structures such as the NRM, PAG, and LC [7,8,34], all of which are established to support both stimulation-produced antinociception (SPA) and inhibition of spinal dorsal horn neuronal responses to noxious stimuli [3,12,14,15,21,30,47,49]. The NRV also projects to the LC, lateral parabrac~a~ (PB) nucleus and to other regions

P 14.30 Lateral

REGIONAL NTS

THRESHOLDS

! Medial NTS 150

3

Dorsal NRV ________ Ventral NRV

P

14.80

_lmm

Fig. 6. Schematic representation of the divisions of NTS and NRV used for the regional analysis and stimulation sites within each subdivision. NTS placements (circles) were divided into medial and lateral divisions at each of 4 rostrocaudal levels. NRV placements (triangles) were divided into dorsai and ventral subdi~sions, regardless of rostrocaudal placement.

125 100

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75

z g +

50

h I: izl I D ix]

medial NTS lateral NE dorsal NW ventral NRV

25

0 -

0.5

mm

0.0

mm

0.5

mm

1.0 mm

011 levels NRV

Fig. 7. Threshold intensities for inhibition of the tail-flick reflex at placements within each of 8 subdivisions of NTS and 2 subdi~sions of NRV.

Medial NTS 100. 80 60 if I bp

40 20 0

-20

I 1

a.25

Proportion

of Threshold

Lateral 100

60

0.75

0.50

1.00

Intensity

NTS

ously reported to not support SPA [la]. On the other hand, stimulation in or near the hypoglossal nucleus consistently resulted in overt movements. These outcomes are generally consistent with other reports of antinociception, but differ from those reported by Morgan et al. [31] and Lewis et al. 1261 in two regards. First, they reported that sites of stimulation either lateral or ventral to the NTS required greater intensities to produce inhibition of the tail-flick reflex or failed to produce antinociception following either electrical stimulation or microinjections of glutamate. Clearly, the present experiments indicate that such sites outside NTS do support inhibition of the tail-flick reflex and sites ventral to the NTS, in the NRV, consistently show low threshold intensities. Second.

1

60 i

NRV Toil-Flick

40

100

7.0 .

80

O-20

I

60

nw

1 0.25

Proportion

0.50

of Threshold

0.75

1 .oo

;

intensity

40

i 20 i 0

Fig. 8. Subthreshold W MPE values for sites in medial (top panel) or lateral (bottom panel) NTS at each of 4 rostrocaudal groups. The & MPE is shown as a function of the proportion of threshold intensity. where 1.0 was the intensity that produced either in~bitjon of the tail-flick reflex or motor responses.

~-20

1 /_

0.25 Proportion

0.50 of Threshold

0.75

1 .oo

Intensity

NRV

within the reticular formation, such as the NRGC 1521 which has also been implicated in the descending modulation of nociceptive spinal transmission (491. Although most sites tended to support inhibition of the tail-flick reflex in the present studies, there were marked differences in thresholds of electrical stimulation required to produce such inhibition. Specifically, dorsal sites, such as the NTS, generally required significantly greater intensities of stimulation to produce inhibition of the tail-flick reflex than more ventral sites such as the NRV, NRGC, or LRN. Midline sites that showed the lowest threshold intensities for inhibition were located within the NRO, a site previ-

Blood Pressure 50

40 30 I &

20

a M

IO

*/*

0 -10

-2Of

___flI_ 0.25 Proportion

0.50 of Threshold

0.75

I .oo

Intensity

Fig. 9. Top panel: the % MPE, values for sites within dorsal or ventral NRV as a proportion of threshold intensity. Bottom panel: similar functions for the percent change in blood pressure after 10 set of brain stimulation.

115

Morgan et al. also reported a failure to produce any motor responses or avoidance behaviors during stimulation in any sites tested in or around the NTS in either awake or lightly anesthetized rats [31]. In the present experiment, strong overt movements were produced in many animals during stimulation in the vicinity of the hypoglossal nucleus, including some sites in the medial NTS. These responses usually involved ipsilateral contraction of both limbs, as well as tonic flexion of the tail toward the side of stimulation. Previous reports have indicated that movements elicited by hypoglossal afferent stimulation are limited to facial, esophageal, and oral musculature [55]. However, these differences in the magnitude of movements may be due to differences in stimulation parameters or experimental preparation, including the level of anesthesia [55]. Alternatively, the motor responses seen in the present experiments may be due to stimulation of cells or pathways in the dorsomedial medulla adjacent to the hypoglossal Medial 50

NTS

Rostrol-Coudal Group:

-

.

-20

0.25 Proportion

0.50

0.75

of Threshold

Lateral

-0.5

1.oo

Intensity

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50 40 30

k

20.

Q bp

10

0 ----

----------_

-10. -20

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0.75

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Intensity

Fig. 10. The percent change in blood pressure for sites in medial (top panel) or lateral (bottom panel) NTS at each of 4 rostrocaudal groups as a function of the proportion of threshold intensity.

nucleus, such as the medial longitudinal fasciculus. Regional analyses of the NTS and NRV revealed that significantly lower current thresholds were required to inhibit the tail-flick reflex from sites in the NRV compared to the NTS. These data are in agreement with studies of glutamate microinjections into these regions showing that inhibition of the tail-flick reflex could be produced with lower doses/volumes of glutamate when microinjected in the NRV than in the NTS 1421. Since relatively high intensities of electrical stimulation in the NTS are required to produce inhibition of the tail-flick reflex, together with the fact that there are few spinopetal projections from this region, it seems plausible to assert that the inhibition is mediated via projections from the NTS to other brain regions or by antidromic activation of sites that project to NTS. Some of the possible projections from the NTS that may be important in the production of antinociception are those to the NRM, LRN, and LC [7,8,34]. These regions provide spinopetal pathways that mediate antinociception [2,12-14,16,21] and the NRM and the caudal LRN were shown previously to be necessary for the inhibition of the tail-flick reflex produced by electrical stimulation of cervical vagal afferents that terminate in the caudal NTS [37]. The NRO was also shown to be necessary for the antinociceptive effects of vagal afferent stimulation, although it is not known if either cell bodies or fibers of passage in any of these regions are the substrates for the antinociception [37]. The NRV contains cell bodies that project directly to the spinal cord and previous work has indicated that brain regions that appear to be final pathways for the inhibition of spinopetal nociceptive transmission tend to demonstrate lower stimulation thresholds for the production of antinociception [30]. Therefore, the lower threshold intensities for sites in NRV as compared to NTS may indicate possible direct spinopetal pathways. However, it is also possible that the differences between thresholds for the NTS and the NRV are due to either cytoarchitectural differences or to the stimulation of ascending/descending fiber pathways projecting through these regions. The

125 !j

100

D z I

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: L. iff

50

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NTS

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I

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.,--.,. 300

400

a

500

600

700

, a00 PYr

Pulse Duration

(ps)

Imm

Fig. Il. Top panel: threshold intensity fur inhibition of the tail-flick reflex as a function of pulse duration for sites in either the NTS or NRV. Bottom panel: threshold intensity at each of 9 pulse durations expressed as a proportion of the rheobase current for stimulation sites in NTS or NRV. Right panel: stimulation sites in NTS and NRV used ior strength-duration experiments.

ventral region of NRV had the lowest intensity thresholds for producing inhibition of the tail-flick reflex and contains serotonergic immunoreactivity [51] perhaps reflecting bulbospinal projections from the NRM [5]. Two differences were found between the use of electrical stimulation in NTS in the present studies and previous studies using glutamate microinjections in NTS. First, contrary to the depressor effects on blood pressure that are seen with microinjections of ~utamate into NTS [42], electrical stimulation of all regions of the caudal NTS that were tested in the present experiment produced a pressor response that was intensity-dependent.

Second, electrical stimulation in more rostra1 regions of the NTS produced inhibition of the tailflick reflex, while glutamate microinjections into these regions failed to produce similar inhibitory effects although still evoking depressor responses [42]. Several explanations for these two differences are feasible. First, the antinociceptive effects of stimulation in the rostra1 NTS, as well as the pressor response produced by electrical stimulation throughout the NTS, may be due to an overriding activation of fibers passing through or projecting to the NTS {e.g., vagal fibers) as opposed to activation of cells in the NTS. Indeed, high frequency stimulation of vagal afferents (100 Hz -

117

as used in the present experiments) results in pressor effects rather than the depressor effects that are observed with frequencies of stimulation lower than 50 Hz [17]. However, the chronaxie of 170 psec derived from stimulation in NTS indicates that the antinociceptive effects were not exclusively due to the stimulation of fibers in the region [36]. The ability to produce antinociception in the more rostra1 sites could also be due to the stimulation of axon collaterals of cells from more caudal regions of NTS or other sites that project to the NTS [lo] or to the stimulation of cell bodies that are not sensitive to glutamate [20,24]. However, since the chronaxie was derived for the antinociceptive effects, it is not known what the contribution of cells or fibers of passage may be to the pressor response seen with stimulation in this region. Another possible interpretation would be that the effects of stimulation in sites within NTS are due to the activation of cells or fibers outside of the nucleus, due to the high stimulation intensities required. However, the antinociceptive effects were obtained with lower thresholds of stimulation than were reported by other laboratories [26,31], and at these current intensities the current spread is not likely to exceed the boundaries of NTS [36]. These differences in the st~ulus-response functions for the antinociceptive and cardiovascular effects may also indicate that the mechanisms for their production are divergent. It now seems plausible to assert that these responses are mediated either by distinct populations of cells or by distinct processes within the same group of cells after initial input to the NTS. The findings that the antinociceptive effects of some treatments can be prevented with selective lesions of the spinal cord while leaving the reflexive cardiovascular responses intact supports this view [40,45]. In this study, the recruitment indices for inhibition of the tail-flick reflex during stimulation of either the lateral NTS or the dorsal NRV were large. These values are comparable to those observed with either inhibition of the tail-flick reflex produced by electrical stimulation of the vagus (441 or inhibition of spinal dorsal horn neurons produced by either electrical stimulation of the vagus [45] or the NTS [46]. This abrupt onset for

antinociception is somewhat analogous to the “quantal” nature of analgesia produced by i.v. or S.C. administration of morphine [25]. Levine and colleagues reported that the onset of analgesia was very abrupt and appeared to be an all-or-none effect. Further, they contend that the production of quanta1 changes in the latency of a nociceptive reflex is a reliable index of analgesic efficacy for pharmacological compounds and may reflect characteristics of the neural systems that mediate analgesia. On the other hand, it is not possible to rule out a more gradual change in inhibition as a function of stimulation intensity if more sensitive measures of the reflex arc, e.g., EMG activity, had been used. The NTS also receives ascending projections from the spinal cord 1291.The physiological role(s) of these pathways has not been established, but it has been postulated that they may serve as a substrate for the integration of visceral and somatic reflexes [29]. Yen and Blum [53] have used single unit recording techniques to demonstrate convergence of afferent information arising from baroreceptors and noxious somatic stimuli in the medullary reticular formation of the cat. This implies the existence of an integrative network for reflexive changes to these types of stimuli at the level of a single neuron and emphasizes the need to examine neural substrates at an integrative systems level.

The authors are grateful for the technical assistance of Michael Burcham, Mary G. Roose, and Angela McAllister, and the input of Drs. G.F. Gebhart and W.T. Talman. We would also like to thank the staff at the Iowa Image Analysis Facility for their patient and helpful assistance. This work was conducted as a portion of the doctoral dissertation of S. Aicher at the University of Iowa, Department of Psychology and portions of this work have been previously reported [2]. This work was supported by a Neurobeha~oral Sciences Training Fellowship to SA (5-T-32MH15172-12) and by Grants NS-22966 and NS24958 to AR.

11X

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