- Email: [email protected]
Control of sensory transmission by electrical stimulation within the caudal raphe nuclei of the cat
EXPERIMENTAL
NEUROLOGY
72,570-581
(1981)
Control of Sensory Transmission by Electrical Stimulation within the Caudal Raphe Nuclei of the Cat PAUL S. BLUM’ Department
of Physiology,
Received
Jefferson Philadelphia,
September
Medical College, Thomas Pennsylvania 19107
8, 1980: revision
received
November
Jefferson
University
11, 1980
These experiments used evoked potential techniques to define further the role of neurons in the caudal raphe nuclei (CRN) in the control of sensory transmission. In each experiment, single-pulse, low-intensity stimuli were tested for the ability to change the amplitude of potentials in the spinothalamic tract (ST) and medial lemniscus (ML) evoked by superficial radial nerve stimulation. It was shown that the effect of CRN stimulation was directed specifically to neurons of the ST because only a small and variable effect was seen on the sensory-evoked potential in the ML. Also, CRN sites were located in two discontinuous regions where electrical stimulation was most effective in inhibiting sensory-evoked activity in the ST. Finally, it was shown that the effect on inhibition of the ST included activity carried by both large-diameter and small-diameter primary afferent fibers.
INTRODUCTION Electrical stimulation within the nucleus raphe magnus, nucleus raphe pallidus, and nucleus raphe obscurus (caudal raphe nuclei; CRN) can control activity in sensory pathways. Oliveras et al. (12) for example, demonstrated that stimulation within the CRN can inhibit the response of spinothalamic tract (ST) neurons in the dorsal horn of the cat to peripheral stimulation, The inhibition of ST neurons by CRN stimulation was confirmed in numerous studies, including those by Fields et al. (IO), Willis et al. (I 5), and McCreery et al. (11). Those investigators showed that Abbreviations: CRN-caudal raphe nuclei, C-T-condition-test, ML-medial lemniscus, ST-spinothalamic tract, SR-superficial radial VL-ventrolateral quadrant of the spinal cord. ’ Gratitude is expressed to Dr. Jewel1 L. Osterholm, Department of Neurosurgery, Jefferson Medical College for the use of a Nicolet MED-80 data system and to Drs. David Whitehorn and Lillian M. Pubols who read preliminary versions of this manuscript. 570 0014-4886/81/060570-12$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
ELECTRICAL
STIMULATION
OF
RAPHE
NUCLEI
571
stimulation-produced inhibition of ST neurons was most pronounced when stimulus sites were in the CRN, compared with sites dorsal to or ventral to the CRN or in the nearby reticular formation. Three important questions remain, however, regarding the interaction between neural elements of the CRN and the sensory pathways. First, is the CRN homogeneous in its influence on spinal neurons or are there sites within the CRN that are more effective than others in controlling transmission in sensory pathways? Previous investigations that found the most effective medullary sites for inhibition of ST neurons to be in the CRN ( 11, 15) were carried out by selecting stimulus sites only along a single electrode tract placed in the middle region of the CRN (11, 15). The rostral+audal distribution of stimulus sites was not investigated. Second, is activity in the CRN capable of modulating transmission of the dorsal column-medial lemniscal system? This question is particularly important because stimulation within the medullary reticular formation, and at sites less than 2 mm from the CRN, can inhibit transmission through the cuneate nucleus (7,8). Third, is the control of sensory transmission by CRN neurons directed to specific components of the ST? Experiments by Fields et al. (10) showed that the descending projection from the CRN is directed specifically to ST neurons that are activated by nociceptors. Other data, however, showed that inhibitory activity originating from the raphe nuclei was directed to all ST neurons, regardless of their sensitivity to peripheral stimuli ( 11, 15). The experiments described here used evoked potential techniques to answer these three questions. First, it was found that stimulation at two separate regions of the CRN was most effective in inhibiting evoked activity in the ST. Second, stimulation within the CRN which inhibited activity in the ST did not reduce evoked responses in the medial lemniscus (ML). Third, the inhibition of the evoked response in the ST was present when activity was carried only on large-diameter primary afferent fibers, as well as when smaller fibers were involved. A preliminary report of this research has been published (3). METHODS Adult cats were anesthetized with alpha-chloralose (60 mg/kg, i.v.) and cannulas were placed in the trachea, a femoral vein, and a femoral artery. Mean arterial blood pressure was monitored with the intraarterial cannula and experiments were terminated if it decreased to less than 80 mm Hg for more than 10 min. Body temperature was monitored rectally and maintained above 37°C with a thermostatically controlled heating pad. At the time recordings were begun, the animal was paralyzed with gallamine
572
PAUL S. BLUM
triethiodide and artificially respired. End-tidal CO2 was monitored and maintained between 3.5 and 5.0% by adjusting the volume of respiration. Recordingfrom Sensory Pathways. The arrangement is shown in Fig. 1A for recording sensory-evoked, slow-wave potentials in the M L and ventrolateral quadrant of the spinal cord (VL). A wire electrode was used to stimulate the superficial radial (SR) nerve at the elbow and the compound action potential was recorded from a distal site. A bipolar concentric electrode (center contact diameter = 0.2 mm; outer contact diameter = 0.5 mm) was placed stereotaxically in the M L in the midbrain and a second bipolar concentric electrode (center contact diameter = 0.1 mm; outer con#,.
ML
R VL
_+1 SR NERVE
0.
E.
FIG. l. A--diagram of electrode arrangement for recording (R) sensory-evoked potentials in the ventrolateral quadrant of the spinal cord (VL) and the medial lemniscus (ML) after stimulation (S) of the superficial radial (SR) nerve. A recording also was made of the compound action potential in the SR nerve. B--recordings from the ML (top trace) and VL (bottom trace) after SR stimulation. Calibrations: 10 ms; top trace, 50 ~V; bottom trace, 5 ,V. Dot indicates stimulus onset. C--recordings as above after hemisection of the spinal cord contralateral to the SR nerve, caudal to the VL recording electrode. D---camera lucida drawing of placement of ML electrode. Calibration: 1 mm. SC, superior colliculus, SN, substantia nigra. E--camera lucida drawing of placement of VL electrode. Calibration: 2 mm. Tip of electrode is within circle at bottom of electrode track. DC, dorsal columns, VH, ventral horns of spinal cord.
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
573
tact diameter = 0.25 mm) was placed in the ventrolateral quadrant of the spinal cord at C2. Both electrodes were contralateral to the SR electrode. The recording electrodes were adjusted in the vertical direction so that maximum-amplitude, short-latency potentials (between 5 and 10 ms) were recorded in response to SR stimulation (Fig. 1B). In each experiment, stimulation of the SR nerve was at two or three intensities. Recordings are shown in Fig. 2 of the compound action potential and averaged SR-evoked potentials in the ML and VL at three intensities of SR stimulation. The highest stimulus intensity (8 V) shows a largeamplitude wave produced by activity in A-alpha and A-beta fibers (Aalpha activity), and activity in A-delta fibers. The lowest stimulus intensity (2 V) is just above threshold for A-alpha activity. The middle record was at an intermediate stimulus intensity. Stimulation of the Caudal Raphe Nuclei. Stimulation within the CRN was tested for the ability to change the amplitude of SR-evoked ML and VL potentials. In each experiment, as many as nine sites in the CRN were stimulated. Stimulation was carried out using an array of three bipolar concentric electrodes (center contact diameter = 0.1 mm; outer contact diameter = 0.25 mm; center contact negative). The stimulus sites were spread evenly in an area caudal to the ports, rostra1 to the obex, and dorsal to the trapezoid body and pyramidal tract. All tracts studied were less than 0.5 mm from the midline. Single-pulse, square-wave stimuli were given at
FIG. 2. Compound action potential recorded from the SR nerve (top trace) and averaged evoked potentials recorded from the ML (middle trace) and VL (bottom trace) after stimulation of the SR nerve at three intensities. Arrow at third record of top trace indicates onset of activity caused by A-delta fibers.
574
PAUL S. BLUM
these sites, 0.2 ms in duration. A “low” and a “high” intensity of brain stem stimulus was used at each site in each experiment. The low intensity was set at one-half the value for the high intensity and for all experiments the low intensity value varied from 25 to 150 PA (Table 1). Procedures for Measuring the Effect of Caudal Raphe Nuclei Stimulation on Sensory Transmission. Figure 3 shows the procedure used to determine whether or not stimulation at a site in the CRN could change the amplitude of SR-evoked potentials in the ML and VL. This was a condition-test design. The following experimental sequence was carried out for each combination of SR stimulus intensity (high, medium, or low) and CRN stimulus intensity (high or low). Stimulation of the SR nerve at one of the stimulus intensities produced the test response in the VL and ML (see Fig. 2). The amplitude of averaged evoked potentials in the VL and ML after the test stimulation was measured four times during an experimental sequence. Then, a conditioning stimulus at one CRN site, and at one intensity was paired with the test stimulus. Three intervals between condition and test stimulation (C-T intervals) were chosen: 40, 80, and 150 ms. Changes caused by the conditioning stimulus were calculated on each C-T interval by determining the percentage difference between the amplitude of the conditioned response and the average amplitude of the test responses before and after the trial. The percentage change values are plotted for the values from one set of conditions in Fig. 3B. Reduced amplitude of the test response produced by the conditioning stimulus was defined as inhibition and an increased amplitude was defined as enhancement. The area of these C-T curves were calculated (Fig. 3B) and this area value was taken as a single number that would indicate the type of TABLE
1
Summary of Sites in the Caudal Raphe Nuclei (CRN) Where Stimulation Produced Inhibition (-) or Enhancement (+) of Superficial Radial Nerve-Evoked Activity in the Ventrolateral Quadrant of the Spinal Cord (VL) and the Medial Lemniscus (ML)
Expt
“Low” intensity CRN stimulation 6.4
I 2 3 4 5 6
125 25 125 I50 50 100
Number of sites in CRN
VL
ML
-
+
-
+
8 6 8 7 8 8
5 I 3 I 4 2
0 0 0 0 0 1
0 0 0 2 1 0
0 0 0 0 0 1
45
16
1
3
1
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
575
C
FIG. 3. Method used to compute the effect of caudal raphe nuclei (CRN) stimulation on the amplitude of sensory-evoked potentials in the ventrolateral quadrant of the spinal cord (VL) and medial lemniscus (ML). A-sample peak heights during test (T) stimulus and changes produced by a single shock conditioning stimulus (C-T) within the CRN. Intervals between conditioning and test stimulus were set at 40.80, and 150 ms. B-plot of above data showing percentage change in amplitude of VL potential at three afferent C-T intervals. Areas in this curve (shaded region) were computed for the effect at each combination of CRN stimulus site, CRN stimulus intensities, and superficial radial nerve stimulus intensities. Cdata from one experiment. Each bar represents the mean * SE for the areas of the C-T curves after stimulation at each of eight stimulus sites in the CRN. Effect on VL potentials shown on left, ML potentials on right. Means of control curves are marked “C.” The CR stimulus intensities for this experiment were single square-wave stimuli, 0.2 ms duration, 125 and 250 PA. Insert shows the location of electrode tracks in the CRN and the position of eight stimulus sites for this experiment. PT, pyramidal tract; PX, PT decussation; TB, trapezoid body.
effect (inhibition or enhancement) and the magnitude of the effect. Thus, there was one area value for the effect produced at each combination of brain stem stimulus site, brain stem stimulus intensity, and SR stimulus intensity. Control curves were calculated by computing the percentage change of adjacent test responses in each experimental sequence. The re-
576
PAUL
S. BLUM
maining data to be described are expressed in terms of the area of the CT curve. It is possible that the short-latency potentials recorded from the ML and VL sites in response to SR stimulation are not an accurate representation of the activity in the two ascending tracts. The recording at the ML electrode may include activity from the ST because these two tracts are close to one another in the rostra1 brain stem. The recording in the ventrolateral quadrant of the spinal cord could contain activity in several tracts, both ascending and descending. To examine these possibilities, recordings were made after a hemisection of the spinal cord at C4, ipsilateral to the recording electrode (Fig. 1C). This lesion should eliminate all activity in the ST from reaching the ML and VL recording site, while not affecting activity in the dorsal column-medial lemniscal system. After the lesion, there was no change in the amplitude or latency of the SR-evoked potential in the ML, indicating that the recording electrode was not positioned to record activity from the ST in the midbrain. In contrast, the portion of the response between 5 and 35 ms was eliminated in the VL recording after spinal hemisection. This suggests that the short-latency response was caused by activity in ascending tracts including the ST. The longer-latency response probably was a result of polysynaptic activity and was not considered in the analysis below. Histologic Verification of the Electrode Position. After data collection, electrolytic lesions were made at the site of ML and VL recordings, and at the bottom of the electrode tracts in the CRN. The animal then was perfused intracardially with saline followed by 10% Formalin. Frozen sections were cut at 60 to 80 pm and stained either with cresyl violet or 1~x01 fast blue to reconstruct the position of electrode tracts in the brain stem and spinal cord. Anatomic landmarks were identified with reference to the atlases of Berman (1) and Snider and Niemer ( 13), and using the cytoarchitectural description of Taber et al. (14). RESULTS Within each experiment, electrical stimuli were delivered to as many as eight CRN sites. The effect produced by stimulation at each stimulus site was analyzed by averaging the area of the C-T curves resulting from stimulation at that site. There were from four to six C-T curves from each site from the different combinations of SR stimulus intensity and CRN stimulus intensities. The areas of the ML and VL C-T curves and the control curves from one experiment are shown in Fig. 3C. Stimulation at a CRN site was defined as effective in changing the SR-evoked response in the ML or VL if the average change in area was more than two standard
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
577
error units from the average of the control curves. In the illustrated example, electricaLstimulation at three sites (numbers 4, 5, and 7) produced inhibition of the VL potential whereas there were no sites where electrical stimulation produced enhancement. No effect of either type was seen on the SR-evoked ML potential. Table 1 summarizes the results from six experiments with 45 CRN stimulus sites. Stimulation at 16 sites produced inhibition of the SR-evoked potential in the VL, and stimulation at one site produced enhancement. The SR-evoked response in the ML was inhibited by stimulation at three sites and enhanced by stimulation at one site. Distribution of Effective Inhibition-Producing Sites. Stimulation at sites within two regions of the CRN were most effective in producing inhibition of SR-evoked potentials in the VL. This is seen in the experiment illustrated in Fig. 3C where the two inhibition-producing sites (sites 5 and 7) are in the middle region of the nucleus, and a third (site 4) is in a more rostra1 location. The distribution of all 16 inhibition-producing sites from the six experiments was visualized by first normalizing the data points within each experiment as a percentage of maximum inhibitory effect for that experiment. For example, in Fig. 3C, site 5 showed the maximum effect (100%) and stimulation at sites 4 and 7 produced 85 and 60% of maximum effect. The percentage values for 15 inhibition-producing sites then were plotted in the proper anatomic locus on a midsagittal diagram of the medulla and contour lines were drawn surrounding regions where there was 100, 50, and 25% of maximum inhibition. In Figs. 4A and B the distribution is shown separately for the magnitude of inhibition produced by stimulation at the high- and low-intensity brain stem stimulation. At the higher stimulus intensity (Fig. 4A) the inhibition-producing region is larger, compared with lower intensity stimulation (Fig. 4B). Two discontinuous regions are evident in Fig. 4B. In contrast to the orderly distribution of sites where there was inhibition of the VL compound action potential, the three sites where electrical stimulation produced inhibition of the ML potential were located throughout the CRN. One site was near the trapezoid body, another site was in the most caudal part of the nucleus near the obex, and the third site was in an intermediate position. Inhibition of Activity Evoked Only by Large-Diameter Afferent Fibers. Figure 5 shows the data sorted according to intensity of SR stimulation. At each intensity of SR stimulation, the data produced by stimulation at inhibition-producing sites is separated from other sites. Inhibition of the SR-evoked VL potential occurred at all intensities of SR stimulation, including the lowest. At the low intensity SR stimulation, A-alpha fibers alone carried afferent activity (Fig. 2).
578
PAUL S. BLUM
HIGH INTENSITY
Be*
Low INI FIG. 4. Midsagittal diagram of the brain stem showing regions where electrical stimulation was most effective in inhibiting the sensory-evoked potential in the ventrolateral quadrant of the spinal cord. Contours enclose regions where 100, 50, and 25% of maximum effect were located for each experiment. The data are separated to the effect produced after stimulation at “high” intensity A and “low” intensity B, as described in the test. Abbreviations as in Fig. 3c.
DISCUSSION The objective of these experiments was to define further neurons in the CRN in the control of sensory transmission. potentials were recorded in the VL and ML after stimulation
0
3000 0 %
the role of Slow-wave of the SR
INHIBITKIN-FRCCWCMO sm
q OTHERSITES 2000
t 1000 ii Y z
I
0 SR sllMULus
INrENsrrf
-1000
FIG. 5. Effect of superficial radial nerve (SR) stimulus intensity on inhibition of sensoryevoked potentials in the ventrolateral quadrant of the spinal cord (VL). Bars show the mean area of the C-T curve (*SE) for the low (1). medium (2). and high (3) intensity of SR stimulation. Inhibition-producing sites are those in the CRN where stimulation inhibited sensory-evoked activity in the VL.
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
579
nerve at several intensities. Changes in magnitude of these potentials were examined when conditioned by low-intensity stimulation within the CRN. In each experiment, stimuli were delivered to sites throughout the CRN. The results indicate that neurons in two separate regions of the CRN are involved in inhibition of sensory-evoked activity in the ST (Fig. 4). Only a small and inconsistent effect was seen on activity in the ML, suggesting that neurons in the CRN do not control activity in the dorsal column nuclei. Inhibition was present at low-intensity SR stimulation, when only afferent activity was carried on A-alpha fibers. This suggests that inhibition of the ST is not directed exclusively to activity produced by nociceptors, because nociceptors are innervated exclusively by A-delta and C fibers (2, 6). The use of evoked potential data has obvious limitations. The exact nature and sites of termination of fibers contributing to the evoked response is unknown. We are confident that the SR-evoked ML potential was an indication of activity in the dorsal column-medial lemniscal system because. cord hemisection contralateral to the stimulus site did not influence the recording (Figs. 1B, C). The potential recorded in the ventrolateral cord in response to SR stimulation was more difficult to characterize. After spinal cord hemisection caudal to the recording site, the initial 35 ms of the response that follows SR stimulation is eliminated, suggesting that it is produced by activity in ascending pathways (Figs. lB, C). This was the portion of the response analyzed during these experiments. It is recognized that not all activity that was recorded reaches the thalamus, because pathways in this region of the spinal cord also terminate in the cerebellum (9) and reticular formation (5). The fact that ascending pathways in the VL are functionally quite similar (4) make this criticism less important. Support for the use of the ventral lateral cord recording also comes from experiments underway in this laboratory in which changes in sensoryevoked activity were measured from recording sites in the thalamus after CRN stimulation (Blum and Yen, unpublished observations). These experiments followed a similar protocol as described in this paper; however, sensory-evoked potentials were recorded from the ventral basal thalamus after a complete dorsal column transection was made at C2. The change in SR-evoked responses produced by CRN stimulation is quantitively similar when the VL response from these experiments is compared with the thalamic response in the more recent experiments. This suggests that a similar neuronal circuit from the CRN influences potentials that are recorded in the ventral lateral cord and the thalamus. It is possible that the uneven distribution of effective stimulus sites after CRN stimulation was the result of uneven distribution of neurons, stimulation of axons of passage, or stimulus spread to nearby regions. These factors could not be analyzed directly with the present techniques, but they
580
PAULSBLUM
need not be ignored completely. There were no obvious differences in the density of axons or cell bodies when the effective inhibition-producing sites were compared with other sites in the CRN. There was no indication that inhibition-producing sites clustered around any of the prominent midline fiber pathways, such as the medial longitudinal fasciculus or the pyramidal tract. Comparisons of the distance between effective inhibition-producing sites and nearby noneffective sites (Fig. 3C) show that the effective stimulus spread for the CRN electrode was no more than 1 to 2 mm. These data are consistent with the conclusion that the activity of neurons in two regions of the CRN is primarily responsible for control of activity in the ST. A consistent finding in all studies that have investigated the effect of electrical stimulation within the CRN is that this stimulus inhibits sensory transmission in the ST. In the present experiments, further information was obtained on the nature of the control of sensory transmission by CRN neurons. It was shown that the effect is directed specifically to neurons of the ST because only a small and variable effect was seen on the sensoryevoked potential in the ML. It also was shown that there is a regional distribution of neurons in the CRN such that sites most effective in producing inhibition are situated preferentially in two discontinuous regions of these nuclei. Finally, it was shown that the effect on sensory transmission includes activity produced by large-diameter primary afferent fibers. REFERENCES I. BERMAN, A. L. 1968. The Brain Stem offhe Car. Univ. of Wisconsin Press, Madison. 2. BESSON,P., AND E. R. PERL. 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimulation. J. Neurophysiol. 32: 1025-1043. 3. BLUM, P. 1979. Control of sensory transmission by electrical stimulation within the caudal raphe nuclei of the cat. Sot. Neurosci. Abstr. 5: 703. 4. BOWSHER, D. 1957. Termination of the central pain pathway in man: the conscious appreciation of pain. Bruin 80: 606-622. 5. BRODAL. A. 1949. Spinal afferents to the lateral reticular nucleus of the medulla oblongata in the cat. An experimental study. J. Comp. Neural. 91: 259-295. 6. BURGESS,P. R., AND E. R. PERL. 1967. Myelinated afferents responding specifically to noxious stimulation of the skin. J. Physiol. (London) 190: 541-562. 7. CESA-BIANCHI, M. G., M. MANCIA, AND M. L. SOTGIU. 1968. Depolarizations of afferent fibers to the Gall and Burdach nuclei introduced by stimulation of the brainstem. Exp. Brain Rex 5: l-5. 8. CESA-BIANCHI, M. G., AND M. L. SOTGIU. 1969. Control by brainstem reticular formation of sensory transmission in Burdach nucleus. Brain Rex 13: 129-139. 9. ECCLES, J. C., M. ITO, AND J. SZENTH~GOTHAI. 1967. The Cerebellum as a Neuronal Machine. Springer, New York. 10. FIELDS, H. L., A. J. BASBAUM, C. H. CLANTON, AND S. D. ANDERSON. 1977. Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons. Bruin Res. 126: 441-453. 1 I. MCCREERY, D. B.. J. R. BLOEDEL, AND E. G. HAINES. 1979. Effects of stimulating in
ELECTRICAL
12.
13. 14.
15.
STIMULATION
OF RAPHE NUCLEI
581
raphe nuclei and in reticular formation in response of spinothalamic neurons to mechanical stimuli. J. Neurophysiof. 42: 166-182. OLIVERAS, J. L.. Y. HOSOLIUCHI, F. REDJBMI, G. G.UILBAUD, AND J. M. BESSON. 1977. Opiate antagonist, naloxone. strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior). Bruin Res. 120: 221-229. SNIDER, R. S., AND W. T. NIEMER. 1961. A Stereotaxic Atlas of the Cut Brain. Univ. of Chicago Press, Chicago. TABER, E., A. BRODAL, AND F. WALBERG. 1960. The raphe nuclei of the brain stem in the cat. 1. Normal topography and cytoarchitecture and general discussion. J. Camp. Neurol. 114: 161-187. WILLIS, W. D., L. H. HABER, AND R. F. MARTIN. 1977. Inhibition of spinothalamic tract cells and interneurons by brain stem stimulation in the monkey. J. Neurophysiol. 401968-98 I.
NEUROLOGY
72,570-581
(1981)
Control of Sensory Transmission by Electrical Stimulation within the Caudal Raphe Nuclei of the Cat PAUL S. BLUM’ Department
of Physiology,
Received
Jefferson Philadelphia,
September
Medical College, Thomas Pennsylvania 19107
8, 1980: revision
received
November
Jefferson
University
11, 1980
These experiments used evoked potential techniques to define further the role of neurons in the caudal raphe nuclei (CRN) in the control of sensory transmission. In each experiment, single-pulse, low-intensity stimuli were tested for the ability to change the amplitude of potentials in the spinothalamic tract (ST) and medial lemniscus (ML) evoked by superficial radial nerve stimulation. It was shown that the effect of CRN stimulation was directed specifically to neurons of the ST because only a small and variable effect was seen on the sensory-evoked potential in the ML. Also, CRN sites were located in two discontinuous regions where electrical stimulation was most effective in inhibiting sensory-evoked activity in the ST. Finally, it was shown that the effect on inhibition of the ST included activity carried by both large-diameter and small-diameter primary afferent fibers.
INTRODUCTION Electrical stimulation within the nucleus raphe magnus, nucleus raphe pallidus, and nucleus raphe obscurus (caudal raphe nuclei; CRN) can control activity in sensory pathways. Oliveras et al. (12) for example, demonstrated that stimulation within the CRN can inhibit the response of spinothalamic tract (ST) neurons in the dorsal horn of the cat to peripheral stimulation, The inhibition of ST neurons by CRN stimulation was confirmed in numerous studies, including those by Fields et al. (IO), Willis et al. (I 5), and McCreery et al. (11). Those investigators showed that Abbreviations: CRN-caudal raphe nuclei, C-T-condition-test, ML-medial lemniscus, ST-spinothalamic tract, SR-superficial radial VL-ventrolateral quadrant of the spinal cord. ’ Gratitude is expressed to Dr. Jewel1 L. Osterholm, Department of Neurosurgery, Jefferson Medical College for the use of a Nicolet MED-80 data system and to Drs. David Whitehorn and Lillian M. Pubols who read preliminary versions of this manuscript. 570 0014-4886/81/060570-12$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
ELECTRICAL
STIMULATION
OF
RAPHE
NUCLEI
571
stimulation-produced inhibition of ST neurons was most pronounced when stimulus sites were in the CRN, compared with sites dorsal to or ventral to the CRN or in the nearby reticular formation. Three important questions remain, however, regarding the interaction between neural elements of the CRN and the sensory pathways. First, is the CRN homogeneous in its influence on spinal neurons or are there sites within the CRN that are more effective than others in controlling transmission in sensory pathways? Previous investigations that found the most effective medullary sites for inhibition of ST neurons to be in the CRN ( 11, 15) were carried out by selecting stimulus sites only along a single electrode tract placed in the middle region of the CRN (11, 15). The rostral+audal distribution of stimulus sites was not investigated. Second, is activity in the CRN capable of modulating transmission of the dorsal column-medial lemniscal system? This question is particularly important because stimulation within the medullary reticular formation, and at sites less than 2 mm from the CRN, can inhibit transmission through the cuneate nucleus (7,8). Third, is the control of sensory transmission by CRN neurons directed to specific components of the ST? Experiments by Fields et al. (10) showed that the descending projection from the CRN is directed specifically to ST neurons that are activated by nociceptors. Other data, however, showed that inhibitory activity originating from the raphe nuclei was directed to all ST neurons, regardless of their sensitivity to peripheral stimuli ( 11, 15). The experiments described here used evoked potential techniques to answer these three questions. First, it was found that stimulation at two separate regions of the CRN was most effective in inhibiting evoked activity in the ST. Second, stimulation within the CRN which inhibited activity in the ST did not reduce evoked responses in the medial lemniscus (ML). Third, the inhibition of the evoked response in the ST was present when activity was carried only on large-diameter primary afferent fibers, as well as when smaller fibers were involved. A preliminary report of this research has been published (3). METHODS Adult cats were anesthetized with alpha-chloralose (60 mg/kg, i.v.) and cannulas were placed in the trachea, a femoral vein, and a femoral artery. Mean arterial blood pressure was monitored with the intraarterial cannula and experiments were terminated if it decreased to less than 80 mm Hg for more than 10 min. Body temperature was monitored rectally and maintained above 37°C with a thermostatically controlled heating pad. At the time recordings were begun, the animal was paralyzed with gallamine
572
PAUL S. BLUM
triethiodide and artificially respired. End-tidal CO2 was monitored and maintained between 3.5 and 5.0% by adjusting the volume of respiration. Recordingfrom Sensory Pathways. The arrangement is shown in Fig. 1A for recording sensory-evoked, slow-wave potentials in the M L and ventrolateral quadrant of the spinal cord (VL). A wire electrode was used to stimulate the superficial radial (SR) nerve at the elbow and the compound action potential was recorded from a distal site. A bipolar concentric electrode (center contact diameter = 0.2 mm; outer contact diameter = 0.5 mm) was placed stereotaxically in the M L in the midbrain and a second bipolar concentric electrode (center contact diameter = 0.1 mm; outer con#,.
ML
R VL
_+1 SR NERVE
0.
E.
FIG. l. A--diagram of electrode arrangement for recording (R) sensory-evoked potentials in the ventrolateral quadrant of the spinal cord (VL) and the medial lemniscus (ML) after stimulation (S) of the superficial radial (SR) nerve. A recording also was made of the compound action potential in the SR nerve. B--recordings from the ML (top trace) and VL (bottom trace) after SR stimulation. Calibrations: 10 ms; top trace, 50 ~V; bottom trace, 5 ,V. Dot indicates stimulus onset. C--recordings as above after hemisection of the spinal cord contralateral to the SR nerve, caudal to the VL recording electrode. D---camera lucida drawing of placement of ML electrode. Calibration: 1 mm. SC, superior colliculus, SN, substantia nigra. E--camera lucida drawing of placement of VL electrode. Calibration: 2 mm. Tip of electrode is within circle at bottom of electrode track. DC, dorsal columns, VH, ventral horns of spinal cord.
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
573
tact diameter = 0.25 mm) was placed in the ventrolateral quadrant of the spinal cord at C2. Both electrodes were contralateral to the SR electrode. The recording electrodes were adjusted in the vertical direction so that maximum-amplitude, short-latency potentials (between 5 and 10 ms) were recorded in response to SR stimulation (Fig. 1B). In each experiment, stimulation of the SR nerve was at two or three intensities. Recordings are shown in Fig. 2 of the compound action potential and averaged SR-evoked potentials in the ML and VL at three intensities of SR stimulation. The highest stimulus intensity (8 V) shows a largeamplitude wave produced by activity in A-alpha and A-beta fibers (Aalpha activity), and activity in A-delta fibers. The lowest stimulus intensity (2 V) is just above threshold for A-alpha activity. The middle record was at an intermediate stimulus intensity. Stimulation of the Caudal Raphe Nuclei. Stimulation within the CRN was tested for the ability to change the amplitude of SR-evoked ML and VL potentials. In each experiment, as many as nine sites in the CRN were stimulated. Stimulation was carried out using an array of three bipolar concentric electrodes (center contact diameter = 0.1 mm; outer contact diameter = 0.25 mm; center contact negative). The stimulus sites were spread evenly in an area caudal to the ports, rostra1 to the obex, and dorsal to the trapezoid body and pyramidal tract. All tracts studied were less than 0.5 mm from the midline. Single-pulse, square-wave stimuli were given at
FIG. 2. Compound action potential recorded from the SR nerve (top trace) and averaged evoked potentials recorded from the ML (middle trace) and VL (bottom trace) after stimulation of the SR nerve at three intensities. Arrow at third record of top trace indicates onset of activity caused by A-delta fibers.
574
PAUL S. BLUM
these sites, 0.2 ms in duration. A “low” and a “high” intensity of brain stem stimulus was used at each site in each experiment. The low intensity was set at one-half the value for the high intensity and for all experiments the low intensity value varied from 25 to 150 PA (Table 1). Procedures for Measuring the Effect of Caudal Raphe Nuclei Stimulation on Sensory Transmission. Figure 3 shows the procedure used to determine whether or not stimulation at a site in the CRN could change the amplitude of SR-evoked potentials in the ML and VL. This was a condition-test design. The following experimental sequence was carried out for each combination of SR stimulus intensity (high, medium, or low) and CRN stimulus intensity (high or low). Stimulation of the SR nerve at one of the stimulus intensities produced the test response in the VL and ML (see Fig. 2). The amplitude of averaged evoked potentials in the VL and ML after the test stimulation was measured four times during an experimental sequence. Then, a conditioning stimulus at one CRN site, and at one intensity was paired with the test stimulus. Three intervals between condition and test stimulation (C-T intervals) were chosen: 40, 80, and 150 ms. Changes caused by the conditioning stimulus were calculated on each C-T interval by determining the percentage difference between the amplitude of the conditioned response and the average amplitude of the test responses before and after the trial. The percentage change values are plotted for the values from one set of conditions in Fig. 3B. Reduced amplitude of the test response produced by the conditioning stimulus was defined as inhibition and an increased amplitude was defined as enhancement. The area of these C-T curves were calculated (Fig. 3B) and this area value was taken as a single number that would indicate the type of TABLE
1
Summary of Sites in the Caudal Raphe Nuclei (CRN) Where Stimulation Produced Inhibition (-) or Enhancement (+) of Superficial Radial Nerve-Evoked Activity in the Ventrolateral Quadrant of the Spinal Cord (VL) and the Medial Lemniscus (ML)
Expt
“Low” intensity CRN stimulation 6.4
I 2 3 4 5 6
125 25 125 I50 50 100
Number of sites in CRN
VL
ML
-
+
-
+
8 6 8 7 8 8
5 I 3 I 4 2
0 0 0 0 0 1
0 0 0 2 1 0
0 0 0 0 0 1
45
16
1
3
1
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
575
C
FIG. 3. Method used to compute the effect of caudal raphe nuclei (CRN) stimulation on the amplitude of sensory-evoked potentials in the ventrolateral quadrant of the spinal cord (VL) and medial lemniscus (ML). A-sample peak heights during test (T) stimulus and changes produced by a single shock conditioning stimulus (C-T) within the CRN. Intervals between conditioning and test stimulus were set at 40.80, and 150 ms. B-plot of above data showing percentage change in amplitude of VL potential at three afferent C-T intervals. Areas in this curve (shaded region) were computed for the effect at each combination of CRN stimulus site, CRN stimulus intensities, and superficial radial nerve stimulus intensities. Cdata from one experiment. Each bar represents the mean * SE for the areas of the C-T curves after stimulation at each of eight stimulus sites in the CRN. Effect on VL potentials shown on left, ML potentials on right. Means of control curves are marked “C.” The CR stimulus intensities for this experiment were single square-wave stimuli, 0.2 ms duration, 125 and 250 PA. Insert shows the location of electrode tracks in the CRN and the position of eight stimulus sites for this experiment. PT, pyramidal tract; PX, PT decussation; TB, trapezoid body.
effect (inhibition or enhancement) and the magnitude of the effect. Thus, there was one area value for the effect produced at each combination of brain stem stimulus site, brain stem stimulus intensity, and SR stimulus intensity. Control curves were calculated by computing the percentage change of adjacent test responses in each experimental sequence. The re-
576
PAUL
S. BLUM
maining data to be described are expressed in terms of the area of the CT curve. It is possible that the short-latency potentials recorded from the ML and VL sites in response to SR stimulation are not an accurate representation of the activity in the two ascending tracts. The recording at the ML electrode may include activity from the ST because these two tracts are close to one another in the rostra1 brain stem. The recording in the ventrolateral quadrant of the spinal cord could contain activity in several tracts, both ascending and descending. To examine these possibilities, recordings were made after a hemisection of the spinal cord at C4, ipsilateral to the recording electrode (Fig. 1C). This lesion should eliminate all activity in the ST from reaching the ML and VL recording site, while not affecting activity in the dorsal column-medial lemniscal system. After the lesion, there was no change in the amplitude or latency of the SR-evoked potential in the ML, indicating that the recording electrode was not positioned to record activity from the ST in the midbrain. In contrast, the portion of the response between 5 and 35 ms was eliminated in the VL recording after spinal hemisection. This suggests that the short-latency response was caused by activity in ascending tracts including the ST. The longer-latency response probably was a result of polysynaptic activity and was not considered in the analysis below. Histologic Verification of the Electrode Position. After data collection, electrolytic lesions were made at the site of ML and VL recordings, and at the bottom of the electrode tracts in the CRN. The animal then was perfused intracardially with saline followed by 10% Formalin. Frozen sections were cut at 60 to 80 pm and stained either with cresyl violet or 1~x01 fast blue to reconstruct the position of electrode tracts in the brain stem and spinal cord. Anatomic landmarks were identified with reference to the atlases of Berman (1) and Snider and Niemer ( 13), and using the cytoarchitectural description of Taber et al. (14). RESULTS Within each experiment, electrical stimuli were delivered to as many as eight CRN sites. The effect produced by stimulation at each stimulus site was analyzed by averaging the area of the C-T curves resulting from stimulation at that site. There were from four to six C-T curves from each site from the different combinations of SR stimulus intensity and CRN stimulus intensities. The areas of the ML and VL C-T curves and the control curves from one experiment are shown in Fig. 3C. Stimulation at a CRN site was defined as effective in changing the SR-evoked response in the ML or VL if the average change in area was more than two standard
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
577
error units from the average of the control curves. In the illustrated example, electricaLstimulation at three sites (numbers 4, 5, and 7) produced inhibition of the VL potential whereas there were no sites where electrical stimulation produced enhancement. No effect of either type was seen on the SR-evoked ML potential. Table 1 summarizes the results from six experiments with 45 CRN stimulus sites. Stimulation at 16 sites produced inhibition of the SR-evoked potential in the VL, and stimulation at one site produced enhancement. The SR-evoked response in the ML was inhibited by stimulation at three sites and enhanced by stimulation at one site. Distribution of Effective Inhibition-Producing Sites. Stimulation at sites within two regions of the CRN were most effective in producing inhibition of SR-evoked potentials in the VL. This is seen in the experiment illustrated in Fig. 3C where the two inhibition-producing sites (sites 5 and 7) are in the middle region of the nucleus, and a third (site 4) is in a more rostra1 location. The distribution of all 16 inhibition-producing sites from the six experiments was visualized by first normalizing the data points within each experiment as a percentage of maximum inhibitory effect for that experiment. For example, in Fig. 3C, site 5 showed the maximum effect (100%) and stimulation at sites 4 and 7 produced 85 and 60% of maximum effect. The percentage values for 15 inhibition-producing sites then were plotted in the proper anatomic locus on a midsagittal diagram of the medulla and contour lines were drawn surrounding regions where there was 100, 50, and 25% of maximum inhibition. In Figs. 4A and B the distribution is shown separately for the magnitude of inhibition produced by stimulation at the high- and low-intensity brain stem stimulation. At the higher stimulus intensity (Fig. 4A) the inhibition-producing region is larger, compared with lower intensity stimulation (Fig. 4B). Two discontinuous regions are evident in Fig. 4B. In contrast to the orderly distribution of sites where there was inhibition of the VL compound action potential, the three sites where electrical stimulation produced inhibition of the ML potential were located throughout the CRN. One site was near the trapezoid body, another site was in the most caudal part of the nucleus near the obex, and the third site was in an intermediate position. Inhibition of Activity Evoked Only by Large-Diameter Afferent Fibers. Figure 5 shows the data sorted according to intensity of SR stimulation. At each intensity of SR stimulation, the data produced by stimulation at inhibition-producing sites is separated from other sites. Inhibition of the SR-evoked VL potential occurred at all intensities of SR stimulation, including the lowest. At the low intensity SR stimulation, A-alpha fibers alone carried afferent activity (Fig. 2).
578
PAUL S. BLUM
HIGH INTENSITY
Be*
Low INI FIG. 4. Midsagittal diagram of the brain stem showing regions where electrical stimulation was most effective in inhibiting the sensory-evoked potential in the ventrolateral quadrant of the spinal cord. Contours enclose regions where 100, 50, and 25% of maximum effect were located for each experiment. The data are separated to the effect produced after stimulation at “high” intensity A and “low” intensity B, as described in the test. Abbreviations as in Fig. 3c.
DISCUSSION The objective of these experiments was to define further neurons in the CRN in the control of sensory transmission. potentials were recorded in the VL and ML after stimulation
0
3000 0 %
the role of Slow-wave of the SR
INHIBITKIN-FRCCWCMO sm
q OTHERSITES 2000
t 1000 ii Y z
I
0 SR sllMULus
INrENsrrf
-1000
FIG. 5. Effect of superficial radial nerve (SR) stimulus intensity on inhibition of sensoryevoked potentials in the ventrolateral quadrant of the spinal cord (VL). Bars show the mean area of the C-T curve (*SE) for the low (1). medium (2). and high (3) intensity of SR stimulation. Inhibition-producing sites are those in the CRN where stimulation inhibited sensory-evoked activity in the VL.
ELECTRICAL
STIMULATION
OF RAPHE NUCLEI
579
nerve at several intensities. Changes in magnitude of these potentials were examined when conditioned by low-intensity stimulation within the CRN. In each experiment, stimuli were delivered to sites throughout the CRN. The results indicate that neurons in two separate regions of the CRN are involved in inhibition of sensory-evoked activity in the ST (Fig. 4). Only a small and inconsistent effect was seen on activity in the ML, suggesting that neurons in the CRN do not control activity in the dorsal column nuclei. Inhibition was present at low-intensity SR stimulation, when only afferent activity was carried on A-alpha fibers. This suggests that inhibition of the ST is not directed exclusively to activity produced by nociceptors, because nociceptors are innervated exclusively by A-delta and C fibers (2, 6). The use of evoked potential data has obvious limitations. The exact nature and sites of termination of fibers contributing to the evoked response is unknown. We are confident that the SR-evoked ML potential was an indication of activity in the dorsal column-medial lemniscal system because. cord hemisection contralateral to the stimulus site did not influence the recording (Figs. 1B, C). The potential recorded in the ventrolateral cord in response to SR stimulation was more difficult to characterize. After spinal cord hemisection caudal to the recording site, the initial 35 ms of the response that follows SR stimulation is eliminated, suggesting that it is produced by activity in ascending pathways (Figs. lB, C). This was the portion of the response analyzed during these experiments. It is recognized that not all activity that was recorded reaches the thalamus, because pathways in this region of the spinal cord also terminate in the cerebellum (9) and reticular formation (5). The fact that ascending pathways in the VL are functionally quite similar (4) make this criticism less important. Support for the use of the ventral lateral cord recording also comes from experiments underway in this laboratory in which changes in sensoryevoked activity were measured from recording sites in the thalamus after CRN stimulation (Blum and Yen, unpublished observations). These experiments followed a similar protocol as described in this paper; however, sensory-evoked potentials were recorded from the ventral basal thalamus after a complete dorsal column transection was made at C2. The change in SR-evoked responses produced by CRN stimulation is quantitively similar when the VL response from these experiments is compared with the thalamic response in the more recent experiments. This suggests that a similar neuronal circuit from the CRN influences potentials that are recorded in the ventral lateral cord and the thalamus. It is possible that the uneven distribution of effective stimulus sites after CRN stimulation was the result of uneven distribution of neurons, stimulation of axons of passage, or stimulus spread to nearby regions. These factors could not be analyzed directly with the present techniques, but they
580
PAULSBLUM
need not be ignored completely. There were no obvious differences in the density of axons or cell bodies when the effective inhibition-producing sites were compared with other sites in the CRN. There was no indication that inhibition-producing sites clustered around any of the prominent midline fiber pathways, such as the medial longitudinal fasciculus or the pyramidal tract. Comparisons of the distance between effective inhibition-producing sites and nearby noneffective sites (Fig. 3C) show that the effective stimulus spread for the CRN electrode was no more than 1 to 2 mm. These data are consistent with the conclusion that the activity of neurons in two regions of the CRN is primarily responsible for control of activity in the ST. A consistent finding in all studies that have investigated the effect of electrical stimulation within the CRN is that this stimulus inhibits sensory transmission in the ST. In the present experiments, further information was obtained on the nature of the control of sensory transmission by CRN neurons. It was shown that the effect is directed specifically to neurons of the ST because only a small and variable effect was seen on the sensoryevoked potential in the ML. It also was shown that there is a regional distribution of neurons in the CRN such that sites most effective in producing inhibition are situated preferentially in two discontinuous regions of these nuclei. Finally, it was shown that the effect on sensory transmission includes activity produced by large-diameter primary afferent fibers. REFERENCES I. BERMAN, A. L. 1968. The Brain Stem offhe Car. Univ. of Wisconsin Press, Madison. 2. BESSON,P., AND E. R. PERL. 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimulation. J. Neurophysiol. 32: 1025-1043. 3. BLUM, P. 1979. Control of sensory transmission by electrical stimulation within the caudal raphe nuclei of the cat. Sot. Neurosci. Abstr. 5: 703. 4. BOWSHER, D. 1957. Termination of the central pain pathway in man: the conscious appreciation of pain. Bruin 80: 606-622. 5. BRODAL. A. 1949. Spinal afferents to the lateral reticular nucleus of the medulla oblongata in the cat. An experimental study. J. Comp. Neural. 91: 259-295. 6. BURGESS,P. R., AND E. R. PERL. 1967. Myelinated afferents responding specifically to noxious stimulation of the skin. J. Physiol. (London) 190: 541-562. 7. CESA-BIANCHI, M. G., M. MANCIA, AND M. L. SOTGIU. 1968. Depolarizations of afferent fibers to the Gall and Burdach nuclei introduced by stimulation of the brainstem. Exp. Brain Rex 5: l-5. 8. CESA-BIANCHI, M. G., AND M. L. SOTGIU. 1969. Control by brainstem reticular formation of sensory transmission in Burdach nucleus. Brain Rex 13: 129-139. 9. ECCLES, J. C., M. ITO, AND J. SZENTH~GOTHAI. 1967. The Cerebellum as a Neuronal Machine. Springer, New York. 10. FIELDS, H. L., A. J. BASBAUM, C. H. CLANTON, AND S. D. ANDERSON. 1977. Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons. Bruin Res. 126: 441-453. 1 I. MCCREERY, D. B.. J. R. BLOEDEL, AND E. G. HAINES. 1979. Effects of stimulating in
ELECTRICAL
12.
13. 14.
15.
STIMULATION
OF RAPHE NUCLEI
581
raphe nuclei and in reticular formation in response of spinothalamic neurons to mechanical stimuli. J. Neurophysiof. 42: 166-182. OLIVERAS, J. L.. Y. HOSOLIUCHI, F. REDJBMI, G. G.UILBAUD, AND J. M. BESSON. 1977. Opiate antagonist, naloxone. strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior). Bruin Res. 120: 221-229. SNIDER, R. S., AND W. T. NIEMER. 1961. A Stereotaxic Atlas of the Cut Brain. Univ. of Chicago Press, Chicago. TABER, E., A. BRODAL, AND F. WALBERG. 1960. The raphe nuclei of the brain stem in the cat. 1. Normal topography and cytoarchitecture and general discussion. J. Camp. Neurol. 114: 161-187. WILLIS, W. D., L. H. HABER, AND R. F. MARTIN. 1977. Inhibition of spinothalamic tract cells and interneurons by brain stem stimulation in the monkey. J. Neurophysiol. 401968-98 I.