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Responses of rat nucleus submedius neurons to enkephalins applied with micropressure
252
Brain Research, 630 (1993) 252-261 Elsevier Science Publishers B.V.
BRES 19501
Responses of rat nucleus submedius neurons to enkephalins applied with micropressure Julie A. Coffield
1 and Vjekoslav
Miletic
*
Departments of Comparative Biosciences and Neurophysiology, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA
(Accepted 20 July 1993)
Key words: Opioid; Nociception; Thalamus; Analgesia; Frontal cortex; Spinal cord
The purpose of this study was to determine what effects leucine-enkephalin and D-Alae-D-Leu5-enkephalin have on both the background and naturally evoked activity of thalamic nucleus submedius neurons responsive to mechanical cutaneous stimulation. Thirty-five neurons in the nucleus submedius were fully characterized during single-unit extracellular recordings as nociceptive, low-threshold mechanoreceptive (LTM) or unresponsive. Micropressure was used to apply the opioids. Eighteen neurons were inhibited; 13 of these were nociceptive and one was LTM. Six units were activated; two of these were nociceptive and three were LTM. The remaining 11 units were unaffected. Opioid responses were tested for antagonism by naloxone in 12 neurons; eight of these responses were antagonized by naloxone. Statistical analyses indicated that the effects of enkephalins on nociceptive neurons were selective for neuronal modality. The opioids also altered the response of some nociceptive neurons to receptive field stimulation. The presence of nociceptive neurons in the nucleus submedius that are selectively inhibited by opioids provides additional support for the involvement of submedius neurons in nociception. The results of this study suggest that this involvement is more than merely transmission of nociceptive input, since the opioids may be selectively modulating the type of information that is transmitted to the cortex.
INTRODUCTION T h e nucleus s u b m e d i u s , a small b u t distinct thalamic nucleus, lies v e n t r o m e d i a l to t h e i n t e r n a l m e d u l l a r y l a m i n a o f t h e t h a l a m u s a n d is c o n s i d e r e d p a r t of the v e n t r o m e d i a l c o m p l e x 6'24 (see also n u c l e u s gelatinoSUS33). A l t h o u g h the f u n c t i o n a l significance of this nucleus is u n k n o w n , r e c e n t e v i d e n c e i m p l i c a t e s t h e nucleus s u b m e d i u s in n o c i c e p t i o n . T h e d o r s a l p a r t of t h e nucleus s u b m e d i u s in b o t h the cat a n d t h e rat receives direct a f f e r e n t i n p u t f r o m m e d u l l a r y ( t r i g e m i n a l ) a n d spinal d o r s a l h o r n n e u rons 9'13'14'43'44. M e d u l l a r y a n d spinal n e u r o n s have long b e e n p o s t u l a t e d to play an i m p o r t a n t role in n o c i c e p tion (e.g., see ref. 17 for a review). Several investigators have r e p o r t e d t h e existence of n e u r o n s in t h e nucleus responsive to t h e r m a l a n d / o r m e c h a n i c a l noxious stimuli in b o t h cat a n d rat t3AS'3°, as well as n e u r o n s a c t i v a t e d exclusively by i n n o c u o u s stimuli in t h e rat 3°. F u r t h e r m o r e , a r e c e n t b e h a v i o r a l study 37 has d e m o n -
s t r a t e d that electrolytic lesion o f the nucleus subm e d i u s results in h y p e r a l g e s i a . Earlier immunocytochemical studies from our l a b o r a t o r y 29 e s t a b l i s h e d the p r e s e n c e o f a d i s c r e t e p o p u l a t i o n of fibers in the n u c l e u s s u b m e d i u s t h a t w e r e i m m u n o r e a c t i v e for t h e e n d o g e n o u s o p i o i d p e p tide e n k e p h a l i n ( E N K ) . This c o n c e n t r a t i o n of E N K i m m u n o r e a c t i v i t y was f o u n d in t h e d o r s a l p a r t o f t h e nucleus s u b m e d i u s , o v e r l a p p i n g the r e g i o n r e p o r t e d to receive d i r e c t d o r s a l h o r n input. E n k e p h a l i n is conside r e d to b e a m a j o r n e u r o m o d u l a t o r o f n o c i c e p t i o n 2. Several studies of its action in o t h e r a r e a s of the c e n t r a l n e r v o u s system i n d i c a t e that, in g e n e r a l , t h e r e s p o n s e to o p i o i d a p p l i c a t i o n is a n a l o x o n e reversible d e c r e a s e in b o t h b a c k g r o u n d a n d noxious-stimulus e v o k e d n e u r o n a l activity 3'4'5'2°'35"36. Thus, the o p i o i d a p p e a r s to b e strategically s i t u a t e d within t h e n u c l e u s s u b m e d i u s to m o d u l a t e noxious a f f e r e n t i n p u t from t h e d o r s a l horns. T h e p u r p o s e of t h e p r e s e n t study was to e x a m i n e
* Corresponding author at the Department of Comparative Biosciences. Fax: (1) (608) 263-6573. 1 Present address: Division of Environmental Medicine and Toxicology, Department of Medicine, Thomas Jefferson University, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA.
253 how local applications of Leu-enkephalin and its more stable analog D-Ala2-D-LeuS-enkephalin (DADLE), affect the background and evoked activity of neurons in the nucleus submedius responsive to mechanical cutaneous stimulation. Preliminary results have been reported in an abstract it. MATERIALS AND METHODS Sprague-Dawley rats (Harlan, 275-300 g) were anesthetized initially with pentobarbital sodium (50 mg/kg, i.p.) and then maintained with periodic injections of ketamine (100 mg/kg, i.p.). Anesthesia was sufficiently deep to prevent arousal or movement reactions, but light enough to permit spontaneous respiration, since no paralytic agents were used and animals were not artificially ventilated. Body temperature was maintained between 36 and 37.5°C with a heating pad. The animal was placed in a stereotaxic frame and a 15-mm skin incision was made along the midline of the skull. Using stereotaxic coordinates, a midline craniotomy (3 mm diameter) was created 1.0-4.0 mm caudal to the bregma.
Electrodes Multibarrel electrode blanks were pulled on a Narishige PE-2 vertical puller (1 /~m tip) for extracellular recording and micropressure drug application. Electrode tips were then broken further under microscopic observation to a total diameter of 3-5/~m by bumping the tips against a single glass pipette. The recording barrel (20-40 MO at 12 kHz) contained 2.5% Pontamine sky blue. Iontophoretic ejection of the dye (5 /~A for 30 min) was used to mark the last recording track at the termination of an experiment3°. The drug barrels contained either Leu-ENK or DADLE (both at 30 mM in 165 mM NaC1, pH 6, ejected with 69-138 kPa for 2-5 min; 2.4 p g / 2 min) as the opioid agonists, naloxone (30 mM in 165 mM NaCl, pH 6, ejected with 69-138 kPa for 2-5 min) as the opioid antagonist and NaCl (165 mM, pH 6, 69-83 kPa) as a control for pH and non-specific pressure effects. The microelectrodes were advanced through the cortex into the medial thalamus towards the dorsal border of the nucleus submedius with a motorized microdrive. Penetrations were made in a grid pattern using stereotaxic coordinates (see nucleus gelatinosus33 ). We limited our recordings to a grid extending 2.1-2.9 mm caudal to the bregma, 0.5-1.0 mm lateral to the midline and 5.6-6.4 mm beneath the surface of the cortex. Each track was separated by at least 200 # m from any other track. Individual recording sites were also separated by 200-300/xm. At the termination of an experiment, the rat was euthanized with an overdose of barbiturate. The brain was quickly removed and fixed in 10% formalin for several days. Brains were blocked and cut either sagittally or coronally on a vibrating microtome (50-100 p.m) for recovery of electrode tracks and dye marks. Location of recorded neurons within the nucleus submedius were based on correlating electrode tracks with microdrive coordinates (see also ref. 30).
Receptive fields For these experiments, no evoked search stimuli were used and isolation of neurons depended solely on spontaneous or background activity. Previous experience has indicated that this produces an adequate sample of submedius neuronal types3°. Once a neuron was encountered, attempts to find a cutaneous receptive field were made before testing the opioid. To search for a receptive field we began with innocuous stimuli. We started at the tail and hind-feet and moved cranially until most of the body had been searched. The same procedure was repeated with noxious stimuli. To avoid receptor sensitization, weakest, least damaging, stimuli were applied first, applications of noxious stimuli were kept to a minimum and the sites of application within a receptive field varied. We did not explore intraoral receptive fields and could not access much of the ventral abdomen (given the placement of the animal in the stereotaxic
frame). It is conceivable that some of the neurons classified as unresponsive might have had receptive fields located in these unexplored areas.
Cutaneous mechanical stimulation Innocuous touch and pressure consisted of deflection of individual hairs with an artist's brush, air puffs, gentle mechanical displacement and probing of the skin (light pressure) and squeezing a fold of skin with flat forceps (innocuous squeeze). Noxious mechanical stimuli consisted of grasping a fold of skin and pinching with toothed forceps (this stimulus is highly painful when applied to the human hand). These types of stimuli are well-accepted in studies of nociceptive neurons both in the medullary and spinal dorsal horns and lateral thalamus (see refs. 1,7,17,22,42 for reviews). Thermal stimuli were not used routinely. Initially, nucleus submedius neurons were classified as low-threshold mechanoreceptive (responsive to innocuous stimuli only), wide dynamic range (WDR: responsive to both innocuous and noxious stimuli) or nociceptive specific (NS: responsive to noxious stimuli only). This type of classification for neurons of the nucleus submedius has been previously described3°. We compared the opioid responses of the NS (n = 11) and WDR (n = 10) neurons and determined that there were no obvious differences in type or magnitude of the responses between the two groups, therefore, the data from both groups were pooled into a single population classified as nociceptive.
Drug application After completion of the search for a receptive field, we examined the effects of opioids on the background activity first, followed by examination of opioid effects on evoked activity. Antagonism of opioid effects was tested with concurrent application of the opioid antagonist naloxone. In the event that a receptive field was not found, the effects of opioids on the background activity were still examined if the unit remained well isolated. For individual neuronal responses, drug applications were repeated at least twice whenever possible, as was the saline control.
Data analysis Single action potentials were recorded extracellularly with a high-input impedance amplifier, separated from background noise with a window discriminator and fed to a microcomputer for on-line histogram construction using a commercial data acquisition program (RHist). On-line analysis allowed for determination of adequate time periods between drug trials (2 min or greater) as defined by the return of a stable level of unit activity. The output from the amplifier was also digitized and stored on VHS videotape for later analysis with Systat. The continuous data points were binned and the bin sizes chosen were the smallest allowable by the resolution of the acquisition program. Bin sizes thus varied from unit to unit depending on the total amount of data being analyzed for each unit. The length of baseline collection or drug trial periods was always at least two min and often greater. Determinations of changes in firing frequency in response to a drug application were made by calculating the mean number of spikes/bin over the entire time period of drug application or baseline collection. Although in many cases this resulted in a dilution of the true maximal response to the drug, we felt that this conservative approach was necessary to prevent the possibility of misinterpreting as a drug effect a naturally occurring increase or decrease in activity resulting from the intrinsic cycling seen in some thalamic units. In general, comparisons were made between the most recent pre-drug period, the period of drug application and the post-drug period, unless otherwise noted.
Statistical analysis The data in this study are best described as repeated measures data. These data, as most biological data collected over relatively short periods of time, manifest autocorrelation among errors. We thus employed the multiple t-test approach, using the Bonferroni correction for multiple comparisons, as the most appropriate statisti-
254 cal method for the type of data collected in this study 39. The Bonferroni approach adjusts the significance level for each comparison so that the overall level of significance for the set of two comparisons is maintained at the nominal level of 0.05. The data were analyzed for statistical significance in two ways. First, for individual neuronal responses, tests of significance were done using t-tests with the Bonferroni correction for multiple comparisons (two-way). Second, the individual neuronal responses were categorized into three separate populations based on receptive field characteristics. The t-test with the Bonferroni correction was then performed on the pooled data for each population. P < 0.05 was considered significant.
RESULTS The data presented in this section are based on the analyses of only those neurons in the nucleus submedius that were recorded for sufficiently long periods of time to ensure the collection of an adequate baseline, examination for a cutaneous receptive field to mechanical stimulation and examination of the responses of background and evoked activity during and following repeated drug application. A total of 35 well-isolated submedial neurons satisfied these criteria. Twenty-one (60%) of the 35 units were classified as nociceptive and six (17%)as low-threshold mechanoreceptive (LTM). The remaining eight (23%) neurons did not respond to any of the cutaneous mechanical stimuli applied. Approximately half of these neurons demonstrated a post-stimulus afterdischarge in response to mechanical cutaneous stimulation. This post-stimulus afterdischarge ranged from modest (1-2 min) to prolonged (greater than 2 min) and is characteristic of some neurons in the nucleus submedius 15'3°. Inhibition or activation of a particular neuron was defined as a statistically significant ( P < 0.05) decrease or increase in firing rate, respectively. Statistically non-significant changes were classified as no response. We compared the effects of ENK with those of DADLE application and determined that no significant differences existed between the agonists within a given population of neurons. Therefore, the responses to both agonists have been pooled and are referred to as opioid responses. A bar graph (Fig. 1) summarizes the opioid responses for the three populations of submedial neurons as a percentage of the 35 neurons tested. Thirteen of the 21 neurons classified as nociceptive demonstrated a statistically significant decrease in activity during opioid application and were considered inhibited. Two nociceptive neurons demonstrated a significant increase in activity and were considered activated. The remaining six showed no significant changes in their activity during opioid application. Of the six neurons classified as innocuous, one was inhibited, three were activated and two were unaffected following opi-
oid application. Four of eight neurons with an unknown receptive field were inhibited, one was activated and three were unaffected by opioid application. Examples of the significant changes in neuronal activity resulting from opioid application are presented in Figs. 2-5. For each example, both a histogram of unitary spike activity and a corresponding plot comparing the mean number of spikes/bin for the different time periods of interest are shown. Since we sought to compare several different time periods within the same graph, it was necessary to use rather large bin sizes for the histograms in order to adequately demonstrate the neuronal responses. Binwidths vary for each neuron illustrated and are described in the individual figure legends.
Opioid effects on background acticity of nociceptiue neurons The background activity of 13 of 21 nociceptive neurons in the nucleus submedius was inhibited by 20-75% during opioid application. Fig. 2A,B illustrates the response of one such neuron to ENK application. The neuron was activated by noxious stimuli applied to the ipsilateral hind-limb and the response to pinch was of fairly short onset and rapid termination. Application of E N K (69 kPa i.e. 10 psi) produced a 60% mean decrease in the unit's activity when compared to the pre-ENK period ( P < 0.001) and a 30% mean decrease from the pre-stimulus baseline (i.e., bins 1-18, P < 0.001). Recovery towards a stable level of activity is '~)4~-
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Fig. 1. Summary of opioid responses. Bar graph summarizing the opioid responses for each of the three populations of nucleus submedius neurons activated by the different types of cutaneous mechanical stimulation (abscissa) as a percentage of total neurons (n = 35) tested (ordinate). The first bar (solid black shading) in each group represents the percentage of total neurons demonstrating an inhibition in background activity in the presence of the opioid. The second bar (gray stippling) in each group represents the percentage demonstrating activation. The third bar (non-shaded) represents the percentage that did not demonstrate a statistically significant change in firing rate (i.e., no response) during drug application. The number of submedius neurons included in each category is denoted by numbers above the bars.
255 evident in the post-ENK period, since the activity appears to be returning to the pre-stimulus baseline level. Fig. 2C, D compares the effect of ENK on background activity with that of a saline control (69 kPa). Although there appears to be a slight mean decrease during application of saline, this is not statistically significant (P = 0.70). However, the 42% mean decrease in activity in response to another application of ENK (69 kPa) is statistically significant (P < 0.001). Fig. 3A, B illustrates the response of another nociceptive nucleus submedius neuron to ENK application. Its receptive field was located on the contralateral hind-limb. The responses to pinch were fairly rapid in
both onset and termination (not shown). Application of ENK (69 kPa) resulted in a mean decrease of 35% in the unit's background discharge (P < 0.001). In addition, the opioid application resulted in a prolonged post-drug depression that amounted to approximately 50% of the mean pre-ENK baseline. This inhibition was greater than that seen at any other time during which this neuron's activity was recorded. The unit activity recovered close to baseline levels in approximately 20 min.
Antagonism of opioid effects by naloxone Opioid-induced changes in unit activity were tested for antagonism by naloxone in 12 of the 24 units
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Fig. 2. Opioid effects on the background discharge and comparison with saline control. A: frequency histogram illustrating the response of a nociceptive nucleus submedius neuron to ENK application (30 mM, 69 kPa). The responses to pinching of the ipsilateral hind-limb are indicated by the bars. The time period of application of ENK is indicated by the solid black shading. Arrows indicate the onset and termination of drug application. The response of this neuron to E N K was a 60% mean decrease from the unit's pre-ENK activity and a 30% mean decrease from the pre-stimulus baseline. Binwidth = 16 s; time scale = 2 min. B: plot comparing the mean no. spikes/4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001). C: frequency histogram comparing the effect of E N K on background activity with that of the saline control. Time period of saline application is represented by the cross-hatched shading. Time period of ENK application is indicated by the solid black shading. Arrows indicate the onset and termination of drug/control applications. No significant change in firing rate occurred with application of saline. ENK application resulted in a 42% mean decrease in activity. Binwidth = 15 s; time scale = 2 rain. D: plot comparing the mean no. of spikes/2 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001).
256 the nociceptive unit illustrated in Fig. 3A,B. After a sufficient period of recovery, naloxone (NAL; 69 kPa) was applied to the neuron for several minutes. No significant change from baseline was evident during this period. Next, ENK (69 kPa) was applied along with the naloxone and again no significant change was noted. The naloxone was then discontinued and ENK was applied alone. The average activity of the neuron decreased by 30% (P < 0.05). This inhibition following
demonstrating a significant opioid response. Eight of the 12 units' responses were antagonized by naloxone. Of these eight units, four were classified as nociceptive, one as innocuous and three had unknown receptive fields. Of the four units not demonstrating antagonism, three were nociceptive and one had no known receptive field. Fig. 3C,D illustrates the naloxone antagonism of the opioid induced depression in the background ac4ivity of
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Fig. 3. Antagonism of opioid effects by naloxone. A: frequency histogram illustrating the response of a nociceptive nucleus submedius neuron to ENK (30 raM, 69 kPa). This neuron was activated by pinching of the contralateral hind-limb (response not shown). Time period of ENK application is indicated by the solid black shading. Arrows indicate the onset and termination of drug application. Application of ENK resulted in a 35% mean decrease in the unit's background activity. Note the prolonged post-ENK depression in activity. The activity returned to near baseline levels in about 20 min. Binwidth = 40 s; time scale = 3 min. B: plot comparing the mean number of spikes/10 s for the different time periods indicated along the abscissa Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001). C: frequency histogram illustrating naloxone antagonism of the ENK-induced inhibition of the neuron illustrated in A,B. Naloxone (69 kPa) was applied first (NAL; cross-hatched shading). ENK (30 mM; 69 kPa) was then applied concurrently with naloxone (NAL ENK; diagonal shading). Neither of these drug applications produced significant changes in baseline firing (non-shaded areas). Naloxone application was terminated and ENK was then applied by itself (solid black shading). Application of ENK alone resulted in a 30% mean decrease in firing which was significantly different from the preceding drug period ( P < 0.05). This was followed by second application of naloxone with ENK (NAL ENK; diagonal shading). Note that in addition to reversing the ENK effect, naloxone appears to have abolished the post-ENK depression seen in Fig. 3A, B. Binwidth = 15 s; time scale = 2 min. D: plot comparing the mean number of spikes/ 10 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisk indicates statistical significance ( P < 0.05).
257 the removal of the naloxone suggests that the naloxone was blocking the effects of the opioid agonist when the they were applied together. Opioid effects on evoked activity Opioid effects on activity evoked by receptive field stimulation was additionally examined in 13 nociceptive neurons in the nucleus submedius. Opioid application suppressed the naturally evoked activity in seven of nine neurons whose background activity was inhibited. In the two remaining units there was no change in their evoked activity following opioid application. Naturally evoked responses remained unchanged also in the four nociceptive neurons that showed an increased background activity during opioid application. Fig. 4 illustrates the effect of ENK application on the evoked activity of a nociceptive neuron that was activated by pinches applied to the head region including the ears. Although this unit's response to pinch was fairly rapid in onset, it demonstrated a pronounced and repeatable post-stimulus discharge that lasted for several minutes. The application of ENK (first with 34 kPa, then with 69 kPa) resulted in approximately a 55% mean decrease in this post-stimulus afterdischarge (P < 0.001). During the maximal opioid inhibition (obtained with 69 kPa and denoted by the asterisk) the firing rate was decreased 43% from the pre-stimulus baseline (P < 0.01). When the receptive field was retested during the opioid application, the initial response to pinch remained (open circle), but the pronounced afterdischarge was absent. Following termination of drug application, recovery is evident in the immediate post-ENK period, since the level of activity becomes comparable to the pre-stimulus baseline. Additional testing of the receptive field during the postENK recovery period (filled circles) suggested that the post-stimulus afterdischarge was beginning to return. Opioid effects on low-threshold mechanoreceptive neurons Six of the 35 characterized neurons in the nucleus submedius were classified as LTM. One of these neurons was inhibited and three were activated by the opioids. Fig. 5 shows the effect of opioid application on a representative LTM neuron that had a discontinuous receptive field. The cell was activated by brushing of the upper thigh region of the contralateral hind-limb, as well as by brushing behind both ears. Application of DADLE resulted in a 31% mean increase in the background activity of the neuron (P < 0.001). Population responses Separate tests of significance (paired t-tests with Bonferroni correction) were performed on the pooled
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Fig. 4. Opioid effect on evoked activity. A: frequency histogram illustrating the effect of ENK on the evoked activity of a nociceptive nucleus submedius neuron. This neuron was activated by pinching of the entire head region including the ears. Bar indicates response to pinch applied to the ears. Note the pronounced post-stimulus afterdischarge following the period of receptive field stimulation. Time period of ENK application is indicated by the solid black shading. Arrows indicate onset and termination of drug application. Application of ENK (30 raM; first with 34 and then 69 kPa) resulted in a 55% mean decrease in the post-stimulus afterdischarge. An asterisk indicates the period of maximal opioid inhibition (with 69 kPa), which is decreased 43% from the pre-stimulus baseline. Pinching of the receptive field during ENK application (open circle) demonstrated that the initial response was still present. The post-stimulus afterdischarge appears abolished. Pinching of the receptive field during the post-ENK recovery period (filled circles) suggests that the post-stimulus afterdischarge was beginning to return. Binwidth = 16 s; time scale=2 min. B: plot comparing the mean number of spikes/4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance (P < 0.001).
opioid responses of the nociceptive and the LTM neuronal populations. The results of these tests indicated that the overall response of the nociceptive population to opioid application was a decrease in firing rate (inhibition) and this population response was statistically significant (P < 0.01). On the other hand, the overall response of the LTM population was an increase in firing rate (activation). A separate test indicated that the mean opioid response of the nociceptive population was significantly different from the mean opioid response of the LTM population (P < 0.01).
258
Distribution of responsive neurons Reconstruction of recording sites within the nucleus submedius in the present study (Fig. 6) illustrates that neurons located in the dorsal part of the nucleus submedius exhibit less variability in both their physiological classification and opioid responses compared with neurons in the ventral part of the nucleus. Thirteen of 16 neurons located within the dorsal part of the nucleus were classified as nociceptive. Eight of these 13 neurons were inhibited by opioids, two were activated and three did not respond. Only one of the six LTM neurons was located dorsally in the nucleus. Conversely, only eight of the nineteen neurons located in the ventral part of the nucleus submedius were classified as nociceptive and six of these were inhibited
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Fig. 6. Distribution of neurons and their opioid responses within the nucleus submedius. Reconstruction of the location within the nucleus submedius of physiologically characterized neurons and their responses to opioid application. Dotted line is an approximation of the division between dorsal and ventral parts of the nucleus based on the Paxinos and Watson atlas 33. N, nociceptive; L, low-threshold mechanoreceptive; ?, unresponsive; - , inhibition; + , activation; = , no response. The rostrocaudal coordinates are plotted along the abscissa, as the distance caudal to the bregma (mm) and the dorsoventral coordinates are plotted along the ordinate. Note that we referenced our depth readings to the surface of the cortex, rather than the skull, because it was easier to establish the point of electrode contact with the tissue and then zero the microdrive. Rostral is to the left, dorsal is up.
by the opioids. Five of the six LTM neurons were located ventrally in the nucleus and three of these were activated by the opioids. Since ENK immunoreactivity is concentrated in the dorsal part of the nucleus submedius 29, these data suggest that the dorsal region of the nucleus contains a neural circuit principally involved in the opioid modulation of nociception. DISCUSSION
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Fig. 5. Opioid effect on submedius neuron classified as low-threshold mechanoreceptive. A: frequency histogram illustrating the response of a nucleus submedius neuron classified as low-threshold mechanoreceptive to D A D L E (30 mM, 69 kPa). This neuron had a discontinuous receptive field. It was activated by both brushing of the contralateral upper thigh region and brushing behind both ears. Bars indicate the response to brushing behind the ears. Time period of application of D A D L E is indicated by solid black shading. Arrows indicate the onset and termination of drug application. Application of D A D L E resulted in a 31% mean increase in the unit's background activity. Binwidth = 16 s; time scale = 2 min. B: plot comparing the mean n u m b e r of s p i k e s / 4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001).
Our data demonstrate that both the background and naturally evoked activity of neurons in the rat nucleus submedius could be modified by the opioid peptides ENK and DADLE. Changes in the background neuronal activity as a result of opioid applications were varied and include inhibition (51%), activation (17%) and no response (31%). These responses (inhibition and activation) were statistically significant, and appeared specific, since they were antagonized by the opioid antagonist naloxone in most cases tested. To our knowledge, this is the first report examining the effects of opioids on rat submedius neurons and our results provide additional support for the involvement of this thalamic nucleus in nociception. These results are significant in that they demonstrate that opioid modulation of neuronal activity in the submedius is selective for nociception (vs. innocuous input) and they imply a further differential selectivity in
259 opioid action towards responses that are perhaps characteristic of motivational-affective, rather than sensory-discriminative, sensory processing (e.g., Fig. 4). These data are in general agreement with previously reported effects of opioids on nociceptive neurons examined in other regions of the thalamus 2°'35, the spinal cord 5'19'21'23'36 and the periaqueductal gray (PAG) 4. In general, studies using the iontophoretic application of enkephalins indicate that their action throughout the central nervous system is predominantly inhibition8'1°'18.
Selective effects of opioids The effects of the opioids in the nucleus submedius appear selective for neuronal modality. That is, submedius neurons that are characterized as nociceptive are inhibited by opioids, whereas LTM neurons are not. These findings are in agreement with earlier studies of iontophoretically applied morphine 5 and met-enkephalin 36 in the spinal cord which suggested that these opioid agonists selectively and reversibly inhibited nociceptive neuronal responses in the dorsal horn. These results differ somewhat from other spinal cord studies reporting both inhibitory and excitatory effects of morphine and other opioid agonists regardless of neuronal modality 16,23,34. These differences may be partly explained by studies showing that a laminar selectivity exists in the dorsal horn with respect to opiate receptors subtypes, neuronal modalities and responses obtained with different opioid agonists ~8'19. In the rat submedius, only /z-, but not 8- or K-opioid receptors have been observed 28. In addition to illustrating selective effects on different neuronal populations within the nucleus submedius, our data also demonstrate that the enkephalins can modify the responses of these neurons to receptive field stimulation. In 77% of the neurons whose background activity was inhibited by opioids, enkephalins application also suppressed the evoked activity. In three of these neurons, the opioid was especially effective at reducing the post-stimulus afterdischarge with little effect on the unit's initial response to the noxious stimulus. In contrast, all of the neurons in which opioid exposure resulted in an increase in background activity maintained their evoked activity during opioid application. Thus, nociceptive neurons in the nucleus submedius inhibited by opioids were no longer as responsive to receptive field stimulation as were neurons activated by the opioids. Finally, the post-stimulus afterdischarge present in some nociceptive neurons was selectively depressed by the opioids. We have previously reported that prolonged afterdischarges are a characteristic of submedius neurons exhibiting responses better suited to
code motivational-affective, rather than sensory-discriminative, nociceptive information (e.g., see Fig. 4C and ref. 30). The selective depression of these afterdischarges, and submedial connections to both the ventrolateral orbital cortex and the PAG ~2,44 suggest the existence of a neural circuit in the submedius involved in the modulation of motivational-affective nociceptive information ultimately reaching the cortex.
Site of opioid action in the submedius Intracellular studies have shown that enkephalins cause inhibition by hyperpolarizing cells 4'21'32'41. This hyperpolarization is often accompanied by a decrease in input resistance and North and Williams32 proposed that this hyperpolarization is mediated by an increased potassium conductance (possibly Calcium dependent). Alternatively, the enkephalins may exert their actions on calcium channels, thereby reducing the duration of the action potential without affecting the resting membrane resistance 3~. These studies suggest that enkephalins may cause inhibition either directly by driving the membrane potential away from threshold and preventing action potential initiation (postsynaptic) or indirectly by decreasing the number of calcium channel openings and thereby decreasing the amount of transmitter released (presynaptic). Although both direct (postsynaptic) and indirect (presynaptic) sites of action have been proposed for the opioid peptides, the present study does not allow us to make determinations concerning the site of opioid action on neurons in the nucleus submedius. However, several lines of evidence suggest that, at least in the dorsal part of the nucleus submedius, the opioids act directly on projection neurons. Electron microscopic analyses of the dorsal part of the nucleus submedius in rats 26'27 suggested that very few, if any, axo-axonic contacts existed in the neuropil of this region. The opioids would not affect nucleus submedius neuronal activity indirectly then (through the presynaptic inhibition of afferent input). Rather, because only axo-dendritic and axo-somatic contacts were noted, the most likely site of opioid action is directly on the neurons in the nucleus submedius. A direct site is further supported by previous data from our laboratory which demonstrated that ENK immunoreactivity in this nucleus is limited to terminals participating in axo-dendritic and axo-somatic arrangements 29. This is similar to other ultrastructural localization studies of ENK immunoreactivity in the dorsal horn 38 and the PAG 4°. Finally, Ma and Ohara 26 demonstrated that 94% of the neurons comprising the neuropil of the dorsal part of the nucleus submedius project to the prefrontal
260 cortex. Since only a small population of neurons in this dorsal region do not project to the prefrontal cortex and presynaptic action on afferent input is unlikely (due to the paucity of axo-axonic contacts), it would seem that the opioids are directly modulating the activity of submedial-cortical projection neurons.
Functional implications Our data suggest that the nucleus submedius may be an important component of opioid-mediated analgesic mechanisms selectively modulating the type of nociceptive information relayed to the cortex. One might expect that interruption of this circuit or of its opioid modulation, would result in an increase in pain induced responses and behaviors (i.e., hyperalgesia). Recent behavioral evidence supports the above concept and suggests that the nucleus submedius is not solely a medial thalamic relay center for somatosensory information. Roberts and Dong 37 demonstrate that electrolytic lesion of the medial thalamus lowers the threshold for the afterdischarge vocalization induced by tail shock in rats. This decrease in threshold occurs only when the nucleus submedius is included in the lesion site. Lesions in other parts of the medial thalamus essentially have no effect. Afterdischarge vocalization is possibly a measure of motivational-affective behavior z5 and this decrease in threshold induced by tail shock is indicative of hyperalgesia. If this nucleus were acting purely as a relay nucleus, then one would expect that lesion of the nucleus would have the opposite effect, resulting in the abolition of or at least reduction in pain-induced responses and behaviors (i.e.,
hypoalgesia ). In conclusion, the existence of nociceptive nucleus submedius neurons characterized by a pronounced post-stimulus afterdischarge responsive to opioid modulation, large receptive fields and connections with both the prefrontal cortex and the PAG, combined with the above behavioral data suggest that the nucleus submedius participates in the opioid modulation of the motivational-affective component of nociception. Acknowledgements. We thank Drs. J. Abbs, M. Behan, L. Trussell and L. Stanford for helpful discussions and critically reading the manuscript and Dr. P. Lalley for the generous use of his microelectrode puller. This study was supported in part by USPHS National Institutes of Health Grant NS26850 to V. Miletic.
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Brain Research, 630 (1993) 252-261 Elsevier Science Publishers B.V.
BRES 19501
Responses of rat nucleus submedius neurons to enkephalins applied with micropressure Julie A. Coffield
1 and Vjekoslav
Miletic
*
Departments of Comparative Biosciences and Neurophysiology, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA
(Accepted 20 July 1993)
Key words: Opioid; Nociception; Thalamus; Analgesia; Frontal cortex; Spinal cord
The purpose of this study was to determine what effects leucine-enkephalin and D-Alae-D-Leu5-enkephalin have on both the background and naturally evoked activity of thalamic nucleus submedius neurons responsive to mechanical cutaneous stimulation. Thirty-five neurons in the nucleus submedius were fully characterized during single-unit extracellular recordings as nociceptive, low-threshold mechanoreceptive (LTM) or unresponsive. Micropressure was used to apply the opioids. Eighteen neurons were inhibited; 13 of these were nociceptive and one was LTM. Six units were activated; two of these were nociceptive and three were LTM. The remaining 11 units were unaffected. Opioid responses were tested for antagonism by naloxone in 12 neurons; eight of these responses were antagonized by naloxone. Statistical analyses indicated that the effects of enkephalins on nociceptive neurons were selective for neuronal modality. The opioids also altered the response of some nociceptive neurons to receptive field stimulation. The presence of nociceptive neurons in the nucleus submedius that are selectively inhibited by opioids provides additional support for the involvement of submedius neurons in nociception. The results of this study suggest that this involvement is more than merely transmission of nociceptive input, since the opioids may be selectively modulating the type of information that is transmitted to the cortex.
INTRODUCTION T h e nucleus s u b m e d i u s , a small b u t distinct thalamic nucleus, lies v e n t r o m e d i a l to t h e i n t e r n a l m e d u l l a r y l a m i n a o f t h e t h a l a m u s a n d is c o n s i d e r e d p a r t of the v e n t r o m e d i a l c o m p l e x 6'24 (see also n u c l e u s gelatinoSUS33). A l t h o u g h the f u n c t i o n a l significance of this nucleus is u n k n o w n , r e c e n t e v i d e n c e i m p l i c a t e s t h e nucleus s u b m e d i u s in n o c i c e p t i o n . T h e d o r s a l p a r t of t h e nucleus s u b m e d i u s in b o t h the cat a n d t h e rat receives direct a f f e r e n t i n p u t f r o m m e d u l l a r y ( t r i g e m i n a l ) a n d spinal d o r s a l h o r n n e u rons 9'13'14'43'44. M e d u l l a r y a n d spinal n e u r o n s have long b e e n p o s t u l a t e d to play an i m p o r t a n t role in n o c i c e p tion (e.g., see ref. 17 for a review). Several investigators have r e p o r t e d t h e existence of n e u r o n s in t h e nucleus responsive to t h e r m a l a n d / o r m e c h a n i c a l noxious stimuli in b o t h cat a n d rat t3AS'3°, as well as n e u r o n s a c t i v a t e d exclusively by i n n o c u o u s stimuli in t h e rat 3°. F u r t h e r m o r e , a r e c e n t b e h a v i o r a l study 37 has d e m o n -
s t r a t e d that electrolytic lesion o f the nucleus subm e d i u s results in h y p e r a l g e s i a . Earlier immunocytochemical studies from our l a b o r a t o r y 29 e s t a b l i s h e d the p r e s e n c e o f a d i s c r e t e p o p u l a t i o n of fibers in the n u c l e u s s u b m e d i u s t h a t w e r e i m m u n o r e a c t i v e for t h e e n d o g e n o u s o p i o i d p e p tide e n k e p h a l i n ( E N K ) . This c o n c e n t r a t i o n of E N K i m m u n o r e a c t i v i t y was f o u n d in t h e d o r s a l p a r t o f t h e nucleus s u b m e d i u s , o v e r l a p p i n g the r e g i o n r e p o r t e d to receive d i r e c t d o r s a l h o r n input. E n k e p h a l i n is conside r e d to b e a m a j o r n e u r o m o d u l a t o r o f n o c i c e p t i o n 2. Several studies of its action in o t h e r a r e a s of the c e n t r a l n e r v o u s system i n d i c a t e that, in g e n e r a l , t h e r e s p o n s e to o p i o i d a p p l i c a t i o n is a n a l o x o n e reversible d e c r e a s e in b o t h b a c k g r o u n d a n d noxious-stimulus e v o k e d n e u r o n a l activity 3'4'5'2°'35"36. Thus, the o p i o i d a p p e a r s to b e strategically s i t u a t e d within t h e n u c l e u s s u b m e d i u s to m o d u l a t e noxious a f f e r e n t i n p u t from t h e d o r s a l horns. T h e p u r p o s e of t h e p r e s e n t study was to e x a m i n e
* Corresponding author at the Department of Comparative Biosciences. Fax: (1) (608) 263-6573. 1 Present address: Division of Environmental Medicine and Toxicology, Department of Medicine, Thomas Jefferson University, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA.
253 how local applications of Leu-enkephalin and its more stable analog D-Ala2-D-LeuS-enkephalin (DADLE), affect the background and evoked activity of neurons in the nucleus submedius responsive to mechanical cutaneous stimulation. Preliminary results have been reported in an abstract it. MATERIALS AND METHODS Sprague-Dawley rats (Harlan, 275-300 g) were anesthetized initially with pentobarbital sodium (50 mg/kg, i.p.) and then maintained with periodic injections of ketamine (100 mg/kg, i.p.). Anesthesia was sufficiently deep to prevent arousal or movement reactions, but light enough to permit spontaneous respiration, since no paralytic agents were used and animals were not artificially ventilated. Body temperature was maintained between 36 and 37.5°C with a heating pad. The animal was placed in a stereotaxic frame and a 15-mm skin incision was made along the midline of the skull. Using stereotaxic coordinates, a midline craniotomy (3 mm diameter) was created 1.0-4.0 mm caudal to the bregma.
Electrodes Multibarrel electrode blanks were pulled on a Narishige PE-2 vertical puller (1 /~m tip) for extracellular recording and micropressure drug application. Electrode tips were then broken further under microscopic observation to a total diameter of 3-5/~m by bumping the tips against a single glass pipette. The recording barrel (20-40 MO at 12 kHz) contained 2.5% Pontamine sky blue. Iontophoretic ejection of the dye (5 /~A for 30 min) was used to mark the last recording track at the termination of an experiment3°. The drug barrels contained either Leu-ENK or DADLE (both at 30 mM in 165 mM NaC1, pH 6, ejected with 69-138 kPa for 2-5 min; 2.4 p g / 2 min) as the opioid agonists, naloxone (30 mM in 165 mM NaCl, pH 6, ejected with 69-138 kPa for 2-5 min) as the opioid antagonist and NaCl (165 mM, pH 6, 69-83 kPa) as a control for pH and non-specific pressure effects. The microelectrodes were advanced through the cortex into the medial thalamus towards the dorsal border of the nucleus submedius with a motorized microdrive. Penetrations were made in a grid pattern using stereotaxic coordinates (see nucleus gelatinosus33 ). We limited our recordings to a grid extending 2.1-2.9 mm caudal to the bregma, 0.5-1.0 mm lateral to the midline and 5.6-6.4 mm beneath the surface of the cortex. Each track was separated by at least 200 # m from any other track. Individual recording sites were also separated by 200-300/xm. At the termination of an experiment, the rat was euthanized with an overdose of barbiturate. The brain was quickly removed and fixed in 10% formalin for several days. Brains were blocked and cut either sagittally or coronally on a vibrating microtome (50-100 p.m) for recovery of electrode tracks and dye marks. Location of recorded neurons within the nucleus submedius were based on correlating electrode tracks with microdrive coordinates (see also ref. 30).
Receptive fields For these experiments, no evoked search stimuli were used and isolation of neurons depended solely on spontaneous or background activity. Previous experience has indicated that this produces an adequate sample of submedius neuronal types3°. Once a neuron was encountered, attempts to find a cutaneous receptive field were made before testing the opioid. To search for a receptive field we began with innocuous stimuli. We started at the tail and hind-feet and moved cranially until most of the body had been searched. The same procedure was repeated with noxious stimuli. To avoid receptor sensitization, weakest, least damaging, stimuli were applied first, applications of noxious stimuli were kept to a minimum and the sites of application within a receptive field varied. We did not explore intraoral receptive fields and could not access much of the ventral abdomen (given the placement of the animal in the stereotaxic
frame). It is conceivable that some of the neurons classified as unresponsive might have had receptive fields located in these unexplored areas.
Cutaneous mechanical stimulation Innocuous touch and pressure consisted of deflection of individual hairs with an artist's brush, air puffs, gentle mechanical displacement and probing of the skin (light pressure) and squeezing a fold of skin with flat forceps (innocuous squeeze). Noxious mechanical stimuli consisted of grasping a fold of skin and pinching with toothed forceps (this stimulus is highly painful when applied to the human hand). These types of stimuli are well-accepted in studies of nociceptive neurons both in the medullary and spinal dorsal horns and lateral thalamus (see refs. 1,7,17,22,42 for reviews). Thermal stimuli were not used routinely. Initially, nucleus submedius neurons were classified as low-threshold mechanoreceptive (responsive to innocuous stimuli only), wide dynamic range (WDR: responsive to both innocuous and noxious stimuli) or nociceptive specific (NS: responsive to noxious stimuli only). This type of classification for neurons of the nucleus submedius has been previously described3°. We compared the opioid responses of the NS (n = 11) and WDR (n = 10) neurons and determined that there were no obvious differences in type or magnitude of the responses between the two groups, therefore, the data from both groups were pooled into a single population classified as nociceptive.
Drug application After completion of the search for a receptive field, we examined the effects of opioids on the background activity first, followed by examination of opioid effects on evoked activity. Antagonism of opioid effects was tested with concurrent application of the opioid antagonist naloxone. In the event that a receptive field was not found, the effects of opioids on the background activity were still examined if the unit remained well isolated. For individual neuronal responses, drug applications were repeated at least twice whenever possible, as was the saline control.
Data analysis Single action potentials were recorded extracellularly with a high-input impedance amplifier, separated from background noise with a window discriminator and fed to a microcomputer for on-line histogram construction using a commercial data acquisition program (RHist). On-line analysis allowed for determination of adequate time periods between drug trials (2 min or greater) as defined by the return of a stable level of unit activity. The output from the amplifier was also digitized and stored on VHS videotape for later analysis with Systat. The continuous data points were binned and the bin sizes chosen were the smallest allowable by the resolution of the acquisition program. Bin sizes thus varied from unit to unit depending on the total amount of data being analyzed for each unit. The length of baseline collection or drug trial periods was always at least two min and often greater. Determinations of changes in firing frequency in response to a drug application were made by calculating the mean number of spikes/bin over the entire time period of drug application or baseline collection. Although in many cases this resulted in a dilution of the true maximal response to the drug, we felt that this conservative approach was necessary to prevent the possibility of misinterpreting as a drug effect a naturally occurring increase or decrease in activity resulting from the intrinsic cycling seen in some thalamic units. In general, comparisons were made between the most recent pre-drug period, the period of drug application and the post-drug period, unless otherwise noted.
Statistical analysis The data in this study are best described as repeated measures data. These data, as most biological data collected over relatively short periods of time, manifest autocorrelation among errors. We thus employed the multiple t-test approach, using the Bonferroni correction for multiple comparisons, as the most appropriate statisti-
254 cal method for the type of data collected in this study 39. The Bonferroni approach adjusts the significance level for each comparison so that the overall level of significance for the set of two comparisons is maintained at the nominal level of 0.05. The data were analyzed for statistical significance in two ways. First, for individual neuronal responses, tests of significance were done using t-tests with the Bonferroni correction for multiple comparisons (two-way). Second, the individual neuronal responses were categorized into three separate populations based on receptive field characteristics. The t-test with the Bonferroni correction was then performed on the pooled data for each population. P < 0.05 was considered significant.
RESULTS The data presented in this section are based on the analyses of only those neurons in the nucleus submedius that were recorded for sufficiently long periods of time to ensure the collection of an adequate baseline, examination for a cutaneous receptive field to mechanical stimulation and examination of the responses of background and evoked activity during and following repeated drug application. A total of 35 well-isolated submedial neurons satisfied these criteria. Twenty-one (60%) of the 35 units were classified as nociceptive and six (17%)as low-threshold mechanoreceptive (LTM). The remaining eight (23%) neurons did not respond to any of the cutaneous mechanical stimuli applied. Approximately half of these neurons demonstrated a post-stimulus afterdischarge in response to mechanical cutaneous stimulation. This post-stimulus afterdischarge ranged from modest (1-2 min) to prolonged (greater than 2 min) and is characteristic of some neurons in the nucleus submedius 15'3°. Inhibition or activation of a particular neuron was defined as a statistically significant ( P < 0.05) decrease or increase in firing rate, respectively. Statistically non-significant changes were classified as no response. We compared the effects of ENK with those of DADLE application and determined that no significant differences existed between the agonists within a given population of neurons. Therefore, the responses to both agonists have been pooled and are referred to as opioid responses. A bar graph (Fig. 1) summarizes the opioid responses for the three populations of submedial neurons as a percentage of the 35 neurons tested. Thirteen of the 21 neurons classified as nociceptive demonstrated a statistically significant decrease in activity during opioid application and were considered inhibited. Two nociceptive neurons demonstrated a significant increase in activity and were considered activated. The remaining six showed no significant changes in their activity during opioid application. Of the six neurons classified as innocuous, one was inhibited, three were activated and two were unaffected following opi-
oid application. Four of eight neurons with an unknown receptive field were inhibited, one was activated and three were unaffected by opioid application. Examples of the significant changes in neuronal activity resulting from opioid application are presented in Figs. 2-5. For each example, both a histogram of unitary spike activity and a corresponding plot comparing the mean number of spikes/bin for the different time periods of interest are shown. Since we sought to compare several different time periods within the same graph, it was necessary to use rather large bin sizes for the histograms in order to adequately demonstrate the neuronal responses. Binwidths vary for each neuron illustrated and are described in the individual figure legends.
Opioid effects on background acticity of nociceptiue neurons The background activity of 13 of 21 nociceptive neurons in the nucleus submedius was inhibited by 20-75% during opioid application. Fig. 2A,B illustrates the response of one such neuron to ENK application. The neuron was activated by noxious stimuli applied to the ipsilateral hind-limb and the response to pinch was of fairly short onset and rapid termination. Application of E N K (69 kPa i.e. 10 psi) produced a 60% mean decrease in the unit's activity when compared to the pre-ENK period ( P < 0.001) and a 30% mean decrease from the pre-stimulus baseline (i.e., bins 1-18, P < 0.001). Recovery towards a stable level of activity is '~)4~-
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Fig. 1. Summary of opioid responses. Bar graph summarizing the opioid responses for each of the three populations of nucleus submedius neurons activated by the different types of cutaneous mechanical stimulation (abscissa) as a percentage of total neurons (n = 35) tested (ordinate). The first bar (solid black shading) in each group represents the percentage of total neurons demonstrating an inhibition in background activity in the presence of the opioid. The second bar (gray stippling) in each group represents the percentage demonstrating activation. The third bar (non-shaded) represents the percentage that did not demonstrate a statistically significant change in firing rate (i.e., no response) during drug application. The number of submedius neurons included in each category is denoted by numbers above the bars.
255 evident in the post-ENK period, since the activity appears to be returning to the pre-stimulus baseline level. Fig. 2C, D compares the effect of ENK on background activity with that of a saline control (69 kPa). Although there appears to be a slight mean decrease during application of saline, this is not statistically significant (P = 0.70). However, the 42% mean decrease in activity in response to another application of ENK (69 kPa) is statistically significant (P < 0.001). Fig. 3A, B illustrates the response of another nociceptive nucleus submedius neuron to ENK application. Its receptive field was located on the contralateral hind-limb. The responses to pinch were fairly rapid in
both onset and termination (not shown). Application of ENK (69 kPa) resulted in a mean decrease of 35% in the unit's background discharge (P < 0.001). In addition, the opioid application resulted in a prolonged post-drug depression that amounted to approximately 50% of the mean pre-ENK baseline. This inhibition was greater than that seen at any other time during which this neuron's activity was recorded. The unit activity recovered close to baseline levels in approximately 20 min.
Antagonism of opioid effects by naloxone Opioid-induced changes in unit activity were tested for antagonism by naloxone in 12 of the 24 units
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Fig. 2. Opioid effects on the background discharge and comparison with saline control. A: frequency histogram illustrating the response of a nociceptive nucleus submedius neuron to ENK application (30 mM, 69 kPa). The responses to pinching of the ipsilateral hind-limb are indicated by the bars. The time period of application of ENK is indicated by the solid black shading. Arrows indicate the onset and termination of drug application. The response of this neuron to E N K was a 60% mean decrease from the unit's pre-ENK activity and a 30% mean decrease from the pre-stimulus baseline. Binwidth = 16 s; time scale = 2 min. B: plot comparing the mean no. spikes/4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001). C: frequency histogram comparing the effect of E N K on background activity with that of the saline control. Time period of saline application is represented by the cross-hatched shading. Time period of ENK application is indicated by the solid black shading. Arrows indicate the onset and termination of drug/control applications. No significant change in firing rate occurred with application of saline. ENK application resulted in a 42% mean decrease in activity. Binwidth = 15 s; time scale = 2 rain. D: plot comparing the mean no. of spikes/2 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001).
256 the nociceptive unit illustrated in Fig. 3A,B. After a sufficient period of recovery, naloxone (NAL; 69 kPa) was applied to the neuron for several minutes. No significant change from baseline was evident during this period. Next, ENK (69 kPa) was applied along with the naloxone and again no significant change was noted. The naloxone was then discontinued and ENK was applied alone. The average activity of the neuron decreased by 30% (P < 0.05). This inhibition following
demonstrating a significant opioid response. Eight of the 12 units' responses were antagonized by naloxone. Of these eight units, four were classified as nociceptive, one as innocuous and three had unknown receptive fields. Of the four units not demonstrating antagonism, three were nociceptive and one had no known receptive field. Fig. 3C,D illustrates the naloxone antagonism of the opioid induced depression in the background ac4ivity of
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Fig. 3. Antagonism of opioid effects by naloxone. A: frequency histogram illustrating the response of a nociceptive nucleus submedius neuron to ENK (30 raM, 69 kPa). This neuron was activated by pinching of the contralateral hind-limb (response not shown). Time period of ENK application is indicated by the solid black shading. Arrows indicate the onset and termination of drug application. Application of ENK resulted in a 35% mean decrease in the unit's background activity. Note the prolonged post-ENK depression in activity. The activity returned to near baseline levels in about 20 min. Binwidth = 40 s; time scale = 3 min. B: plot comparing the mean number of spikes/10 s for the different time periods indicated along the abscissa Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001). C: frequency histogram illustrating naloxone antagonism of the ENK-induced inhibition of the neuron illustrated in A,B. Naloxone (69 kPa) was applied first (NAL; cross-hatched shading). ENK (30 mM; 69 kPa) was then applied concurrently with naloxone (NAL ENK; diagonal shading). Neither of these drug applications produced significant changes in baseline firing (non-shaded areas). Naloxone application was terminated and ENK was then applied by itself (solid black shading). Application of ENK alone resulted in a 30% mean decrease in firing which was significantly different from the preceding drug period ( P < 0.05). This was followed by second application of naloxone with ENK (NAL ENK; diagonal shading). Note that in addition to reversing the ENK effect, naloxone appears to have abolished the post-ENK depression seen in Fig. 3A, B. Binwidth = 15 s; time scale = 2 min. D: plot comparing the mean number of spikes/ 10 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisk indicates statistical significance ( P < 0.05).
257 the removal of the naloxone suggests that the naloxone was blocking the effects of the opioid agonist when the they were applied together. Opioid effects on evoked activity Opioid effects on activity evoked by receptive field stimulation was additionally examined in 13 nociceptive neurons in the nucleus submedius. Opioid application suppressed the naturally evoked activity in seven of nine neurons whose background activity was inhibited. In the two remaining units there was no change in their evoked activity following opioid application. Naturally evoked responses remained unchanged also in the four nociceptive neurons that showed an increased background activity during opioid application. Fig. 4 illustrates the effect of ENK application on the evoked activity of a nociceptive neuron that was activated by pinches applied to the head region including the ears. Although this unit's response to pinch was fairly rapid in onset, it demonstrated a pronounced and repeatable post-stimulus discharge that lasted for several minutes. The application of ENK (first with 34 kPa, then with 69 kPa) resulted in approximately a 55% mean decrease in this post-stimulus afterdischarge (P < 0.001). During the maximal opioid inhibition (obtained with 69 kPa and denoted by the asterisk) the firing rate was decreased 43% from the pre-stimulus baseline (P < 0.01). When the receptive field was retested during the opioid application, the initial response to pinch remained (open circle), but the pronounced afterdischarge was absent. Following termination of drug application, recovery is evident in the immediate post-ENK period, since the level of activity becomes comparable to the pre-stimulus baseline. Additional testing of the receptive field during the postENK recovery period (filled circles) suggested that the post-stimulus afterdischarge was beginning to return. Opioid effects on low-threshold mechanoreceptive neurons Six of the 35 characterized neurons in the nucleus submedius were classified as LTM. One of these neurons was inhibited and three were activated by the opioids. Fig. 5 shows the effect of opioid application on a representative LTM neuron that had a discontinuous receptive field. The cell was activated by brushing of the upper thigh region of the contralateral hind-limb, as well as by brushing behind both ears. Application of DADLE resulted in a 31% mean increase in the background activity of the neuron (P < 0.001). Population responses Separate tests of significance (paired t-tests with Bonferroni correction) were performed on the pooled
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Fig. 4. Opioid effect on evoked activity. A: frequency histogram illustrating the effect of ENK on the evoked activity of a nociceptive nucleus submedius neuron. This neuron was activated by pinching of the entire head region including the ears. Bar indicates response to pinch applied to the ears. Note the pronounced post-stimulus afterdischarge following the period of receptive field stimulation. Time period of ENK application is indicated by the solid black shading. Arrows indicate onset and termination of drug application. Application of ENK (30 raM; first with 34 and then 69 kPa) resulted in a 55% mean decrease in the post-stimulus afterdischarge. An asterisk indicates the period of maximal opioid inhibition (with 69 kPa), which is decreased 43% from the pre-stimulus baseline. Pinching of the receptive field during ENK application (open circle) demonstrated that the initial response was still present. The post-stimulus afterdischarge appears abolished. Pinching of the receptive field during the post-ENK recovery period (filled circles) suggests that the post-stimulus afterdischarge was beginning to return. Binwidth = 16 s; time scale=2 min. B: plot comparing the mean number of spikes/4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance (P < 0.001).
opioid responses of the nociceptive and the LTM neuronal populations. The results of these tests indicated that the overall response of the nociceptive population to opioid application was a decrease in firing rate (inhibition) and this population response was statistically significant (P < 0.01). On the other hand, the overall response of the LTM population was an increase in firing rate (activation). A separate test indicated that the mean opioid response of the nociceptive population was significantly different from the mean opioid response of the LTM population (P < 0.01).
258
Distribution of responsive neurons Reconstruction of recording sites within the nucleus submedius in the present study (Fig. 6) illustrates that neurons located in the dorsal part of the nucleus submedius exhibit less variability in both their physiological classification and opioid responses compared with neurons in the ventral part of the nucleus. Thirteen of 16 neurons located within the dorsal part of the nucleus were classified as nociceptive. Eight of these 13 neurons were inhibited by opioids, two were activated and three did not respond. Only one of the six LTM neurons was located dorsally in the nucleus. Conversely, only eight of the nineteen neurons located in the ventral part of the nucleus submedius were classified as nociceptive and six of these were inhibited
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by the opioids. Five of the six LTM neurons were located ventrally in the nucleus and three of these were activated by the opioids. Since ENK immunoreactivity is concentrated in the dorsal part of the nucleus submedius 29, these data suggest that the dorsal region of the nucleus contains a neural circuit principally involved in the opioid modulation of nociception. DISCUSSION
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Fig. 5. Opioid effect on submedius neuron classified as low-threshold mechanoreceptive. A: frequency histogram illustrating the response of a nucleus submedius neuron classified as low-threshold mechanoreceptive to D A D L E (30 mM, 69 kPa). This neuron had a discontinuous receptive field. It was activated by both brushing of the contralateral upper thigh region and brushing behind both ears. Bars indicate the response to brushing behind the ears. Time period of application of D A D L E is indicated by solid black shading. Arrows indicate the onset and termination of drug application. Application of D A D L E resulted in a 31% mean increase in the unit's background activity. Binwidth = 16 s; time scale = 2 min. B: plot comparing the mean n u m b e r of s p i k e s / 4 s for the different time periods indicated along the abscissa. Error bar = S.E.M. Asterisks indicate statistical significance ( P < 0.001).
Our data demonstrate that both the background and naturally evoked activity of neurons in the rat nucleus submedius could be modified by the opioid peptides ENK and DADLE. Changes in the background neuronal activity as a result of opioid applications were varied and include inhibition (51%), activation (17%) and no response (31%). These responses (inhibition and activation) were statistically significant, and appeared specific, since they were antagonized by the opioid antagonist naloxone in most cases tested. To our knowledge, this is the first report examining the effects of opioids on rat submedius neurons and our results provide additional support for the involvement of this thalamic nucleus in nociception. These results are significant in that they demonstrate that opioid modulation of neuronal activity in the submedius is selective for nociception (vs. innocuous input) and they imply a further differential selectivity in
259 opioid action towards responses that are perhaps characteristic of motivational-affective, rather than sensory-discriminative, sensory processing (e.g., Fig. 4). These data are in general agreement with previously reported effects of opioids on nociceptive neurons examined in other regions of the thalamus 2°'35, the spinal cord 5'19'21'23'36 and the periaqueductal gray (PAG) 4. In general, studies using the iontophoretic application of enkephalins indicate that their action throughout the central nervous system is predominantly inhibition8'1°'18.
Selective effects of opioids The effects of the opioids in the nucleus submedius appear selective for neuronal modality. That is, submedius neurons that are characterized as nociceptive are inhibited by opioids, whereas LTM neurons are not. These findings are in agreement with earlier studies of iontophoretically applied morphine 5 and met-enkephalin 36 in the spinal cord which suggested that these opioid agonists selectively and reversibly inhibited nociceptive neuronal responses in the dorsal horn. These results differ somewhat from other spinal cord studies reporting both inhibitory and excitatory effects of morphine and other opioid agonists regardless of neuronal modality 16,23,34. These differences may be partly explained by studies showing that a laminar selectivity exists in the dorsal horn with respect to opiate receptors subtypes, neuronal modalities and responses obtained with different opioid agonists ~8'19. In the rat submedius, only /z-, but not 8- or K-opioid receptors have been observed 28. In addition to illustrating selective effects on different neuronal populations within the nucleus submedius, our data also demonstrate that the enkephalins can modify the responses of these neurons to receptive field stimulation. In 77% of the neurons whose background activity was inhibited by opioids, enkephalins application also suppressed the evoked activity. In three of these neurons, the opioid was especially effective at reducing the post-stimulus afterdischarge with little effect on the unit's initial response to the noxious stimulus. In contrast, all of the neurons in which opioid exposure resulted in an increase in background activity maintained their evoked activity during opioid application. Thus, nociceptive neurons in the nucleus submedius inhibited by opioids were no longer as responsive to receptive field stimulation as were neurons activated by the opioids. Finally, the post-stimulus afterdischarge present in some nociceptive neurons was selectively depressed by the opioids. We have previously reported that prolonged afterdischarges are a characteristic of submedius neurons exhibiting responses better suited to
code motivational-affective, rather than sensory-discriminative, nociceptive information (e.g., see Fig. 4C and ref. 30). The selective depression of these afterdischarges, and submedial connections to both the ventrolateral orbital cortex and the PAG ~2,44 suggest the existence of a neural circuit in the submedius involved in the modulation of motivational-affective nociceptive information ultimately reaching the cortex.
Site of opioid action in the submedius Intracellular studies have shown that enkephalins cause inhibition by hyperpolarizing cells 4'21'32'41. This hyperpolarization is often accompanied by a decrease in input resistance and North and Williams32 proposed that this hyperpolarization is mediated by an increased potassium conductance (possibly Calcium dependent). Alternatively, the enkephalins may exert their actions on calcium channels, thereby reducing the duration of the action potential without affecting the resting membrane resistance 3~. These studies suggest that enkephalins may cause inhibition either directly by driving the membrane potential away from threshold and preventing action potential initiation (postsynaptic) or indirectly by decreasing the number of calcium channel openings and thereby decreasing the amount of transmitter released (presynaptic). Although both direct (postsynaptic) and indirect (presynaptic) sites of action have been proposed for the opioid peptides, the present study does not allow us to make determinations concerning the site of opioid action on neurons in the nucleus submedius. However, several lines of evidence suggest that, at least in the dorsal part of the nucleus submedius, the opioids act directly on projection neurons. Electron microscopic analyses of the dorsal part of the nucleus submedius in rats 26'27 suggested that very few, if any, axo-axonic contacts existed in the neuropil of this region. The opioids would not affect nucleus submedius neuronal activity indirectly then (through the presynaptic inhibition of afferent input). Rather, because only axo-dendritic and axo-somatic contacts were noted, the most likely site of opioid action is directly on the neurons in the nucleus submedius. A direct site is further supported by previous data from our laboratory which demonstrated that ENK immunoreactivity in this nucleus is limited to terminals participating in axo-dendritic and axo-somatic arrangements 29. This is similar to other ultrastructural localization studies of ENK immunoreactivity in the dorsal horn 38 and the PAG 4°. Finally, Ma and Ohara 26 demonstrated that 94% of the neurons comprising the neuropil of the dorsal part of the nucleus submedius project to the prefrontal
260 cortex. Since only a small population of neurons in this dorsal region do not project to the prefrontal cortex and presynaptic action on afferent input is unlikely (due to the paucity of axo-axonic contacts), it would seem that the opioids are directly modulating the activity of submedial-cortical projection neurons.
Functional implications Our data suggest that the nucleus submedius may be an important component of opioid-mediated analgesic mechanisms selectively modulating the type of nociceptive information relayed to the cortex. One might expect that interruption of this circuit or of its opioid modulation, would result in an increase in pain induced responses and behaviors (i.e., hyperalgesia). Recent behavioral evidence supports the above concept and suggests that the nucleus submedius is not solely a medial thalamic relay center for somatosensory information. Roberts and Dong 37 demonstrate that electrolytic lesion of the medial thalamus lowers the threshold for the afterdischarge vocalization induced by tail shock in rats. This decrease in threshold occurs only when the nucleus submedius is included in the lesion site. Lesions in other parts of the medial thalamus essentially have no effect. Afterdischarge vocalization is possibly a measure of motivational-affective behavior z5 and this decrease in threshold induced by tail shock is indicative of hyperalgesia. If this nucleus were acting purely as a relay nucleus, then one would expect that lesion of the nucleus would have the opposite effect, resulting in the abolition of or at least reduction in pain-induced responses and behaviors (i.e.,
hypoalgesia ). In conclusion, the existence of nociceptive nucleus submedius neurons characterized by a pronounced post-stimulus afterdischarge responsive to opioid modulation, large receptive fields and connections with both the prefrontal cortex and the PAG, combined with the above behavioral data suggest that the nucleus submedius participates in the opioid modulation of the motivational-affective component of nociception. Acknowledgements. We thank Drs. J. Abbs, M. Behan, L. Trussell and L. Stanford for helpful discussions and critically reading the manuscript and Dr. P. Lalley for the generous use of his microelectrode puller. This study was supported in part by USPHS National Institutes of Health Grant NS26850 to V. Miletic.
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