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Medial hypothalamic stimulation suppresses nociceptive spinal dorsal horn neurons but not the tail-flick reflex in the rat
Brain Research, 438 (1988) 137-144 Elsevier
137
BRE 13180
Medial hypothalamic stimulation suppresses nociceptive spinal dorsal horn neurons but not the tail-flick reflex in the rat* E.S. Culhane and E. Carstens Department of Animal Physiology, Universi~ of California at Davis, Davis, CA 95616 (U.S.A.) (Accepted 23 June 1987)
Key words: Medial hypothalamus; Dorsal horn neuron; Heat-evoked response; Tail-flick reflex; Descending inhibition; Stimulation-produced analgesia; Rat
This study investigated the potentia~ analgesic effects of medial hypothalamic stimulation (HS) on a measure of nocifensive behavior (tail-flick test (TF)) in awake rats, and potential inhibitory effects of identical HS on spinal dorsal horn neuronal responses to noxious skin heating in the same animals anesthetized with sodium pentobarbital. Sixty-five male Sprague-Dawley rats implanted with a bipolar stimulation electrode in histologically verified medial hypothalamic sites were tested behaviorally for TF suppression during HS (100 ms trains at 100 Hz. 3/s, 100-1100 liA) in 2-4 consecutive weekly test sessions. Thirty-three of these rats were then used in electrophysiological experiments to record responses of 36 dorsal horn units to noxious skin heating (48-54 °C, 10 s/2 rain) of the hindfoot pad in the absence of and during HS. Behaviorally, 31/65 rats had no TF suppression at the highest HS intensity tested (1100 !tA), 24/65 rats exhibited aversive behavior or motor activity which disallowed reliable TF testing, and only 10/65 rats showed TF suppression in at least one test session. In electrophysiologicai experiments, the heat-evoked responses of 25/36 dorsal horn units were inhibited to at least 50% of control ~uring HS. The responses of 11 units remained at 65-100% of the control responses during HS of up to 1lOOltA. In rats demonstrating T~ ~~~-~ression. 4/7 units were inhibited. In rats with no TF suppression, 10/15 units were inhibited, and in rats showing aversive behavior. 11/14 units were inhibited by HS. These data indicate that although HS suppresses spinal nociceptive neurons, it does not cause reimt~ie "IT suppression in unanesthetized rats and bring into question the often-held assumption that stimulation-evoked descending inhibition of spinal nociceptive neurons implies behavioral analgesia.
key !'7-9. A d d i t i o n a l l y , lesions in the m e d i a l hypot h a l a m u s produce h y p e r a l g e s i a 22"35. F u r t h e r m o r e ,
INTRODUCTION N u m e r o u s studies have d e m o n s t r a t e d the exist-
the h y p o t h a l a m u s has an extensive d e s c e n d i n g input
ence of e n d o g e n o u s p a i n control s y s t e m s in h u m a n s
to the m i d b r a i n P A G , suggesting that these areas m a y be functionally l i n k e d 2"-'4.
and in a n i m a l s . It is well e s t a b l i s h e d that electrical m e d u l l a r y n u c l e u s r a p h e m a g n u s a n d the m i d b r a i n
It has b e e n suggested that inhibition of nociceptive dorsal horn n e u r o n s m a y be a m e c h a n i s m for S P A ~8"
periaqueductal
26. If so, there should be a correlation b e t w e e n S P A
stimulation in m e d i a l b r a i n s t e m areas, such as the gray
(PAG),
produces
analgesia
( s t i m u l a t i o n - p r o d u c e d analgesia ( S P A ) ) a n d inhibits
and s t i m u l a t i o n - e v o k e d
the responses of dorsal horn n e u r o n s to noxious stimuli 4a7-18"36. Electrical stimulation in m o r e rostral
neurons
structures i n c l u d i n g the p e r i v e n t r i c u l a r gray and hyp o t h a l a m u s has also b e e n s h o w n to support S P A in h u m a n s -l'-s--'~, m o n k e y s .... and r~lts 13"lt''ltL-t':°-'- as
in the
same
inhibition of nociceptive animal.
The
few
studies
addressing this have i n d e e d shown a correlation between SPA and i n h i b i t i o n from ~he P A G 3"26, alt h o u g h it was c o m m e n t e d that inhibition was also
well as to inhibit e v o k e d responses o f spinal cord dor-
e v o k e d from m o r e lateral reticular areas that were aversive when s t i m u l a t e d in the awake a n i m a l 26.
sal horn cells to noxious heat in the rat, cat and mon-
T h e aims of the p r e s e n t investigation were to es-
An abstract of this research has been previously published~-'. Correspondence." E. Carstens. Department of Animal Physiology. University of California at Davis. Davis. CA t~5f'lf,. U.S.A. *
138 tablish: (1) if stimulation in the medial hypothalamus suppresses the nociceptive tail-flick reflex in the rat; and (2) if there is a correlation between the occurrence of tail-flick suppression and inhibition of dorsal horn neuronal heat-evoked responses produced by identical hypothalamic stimulation in the same rat. MATERIALS AND METHODS
Animals Sixty-five adult male Sprague-Dawley rats (Simonsen Labs., Gilroy, CA) were stereotaxically implanted with a bipolar stimulation electrode in medial hypothalamic (and periventricular) areas under sodium pentobarbital anesthesia (65 mg/kg) supplemented with Metofane (Pitman-Moore) as needed. Each electrode was constructed from two twisted strands of teflon-coated stainless-steel wire (approximately 0.5 mm diameter, Medwire) soldered into two female Amphenol pins. Dental acrylic was used to insulate the assembly and to anchor it to 4 screws inserted into the skull. Following a 5-7-day recovery period, each rat underwent a series of 5 daily 1-h training sessions in which they were habituated to the Plexiglas restrainers used during behavioral testing. All rats were housed individually on a 12:12 h light/ dark cycle with food and water available ad libitum.
Behavioral testing A modification of the tail-flick (TF) test ~4was used to assess nociceptive responses on a behavioral level beginning 2 weeks after electrode implantation and continuing for 2-4 consecutive weekly test sessions. Prior to any hypothalamic stimulation (HS), baseline (BL) TF latencies were measured (average of 3 stable trials, 2 min inter-trial intervals). Bulb voltages on the TF apparatus were adjusted to obtain BL latencies of approximately 3.0-3.5 s and were thereafter held constant throughout the experiment. A method of ascending limits was then used to determine the threshold current for TF suppression. HS (100 ms trains at 100 Hz, 3/s, 100-1100/~A) intensity was increased by 200/~A in each successive TF trial. HS began 10 s before the initiation of the TF trial and was terminated immediately after the trial. TF suppression was defined as two consecutive TF latencies of 8.0 s during HS with the TF required to return to within 0.5 s of the BL latency between the two stimu-
lation trials. If TF suppression occurred, the current intensity was lowered half a step (1001~A) to obtain a more accurate threshold for TF suppression. Testing was terminated if any one of the following conditions occurred: (1) TF suppression; (2) HS intensity reached 1100/~A with no TF suppression; or (3) aversive or motor effects disallowed reliable TF testing. Two to 4 consecutive weekly test sessions were employed to test the reliability of TF suppression or any other behavior observed. During each test session, each rat was subjectively rated on a 4-point scale based on behaviors observed during HS at the highest intensity used in that session. Behavior ratings were as follows: (1) no apparent reaction or only slight alerting to HS; (2) slight motor responses such as eye and nose twitching or more organized behaviors such as pawing and teeth chattering; (3) mild motor responses involving the head and/or the whole body yet which were not so severe as to disallow a reliable TF test; (4) vocalization or profound motor and escape responses which disallowed reliable TF testing. Although behavioral studies typically employ biphasic stimulati~,n parameters, we chose to use monophasic stimulation for two reasons: (1) to be able to directly compare these results with our previous electrophysiologicai studiesS; and (2) we have previously found that there was no difference in the incidence of SPA evoked by mono- compared to biphasic stimulation 33. These parameters produced an estimated current spread of no more than 1 ram. Current intensities were monitored using a Tektronix P6021 current probe, and both the current and voltage across the electrode were simultaneously displayed on an oscilloscope.
Electrophysiological testing Thirty-three rats tested behaviorally were subsequently tested electrophysiologically. Under sodium pentobarbital anesthesia (65 mg/kg i.p.), the jugular vein was cannulated for continuous infusion of pentobarbital (10 mg/kg/h) throughout the experiment. The lumbosacral spinal cord was exposed by laminectomy and bathed in saline placed in an agar pool formed over the exposed cord. A tungsten microelectrode was inserted into the spinal cord to record the responses of dorsal horn units (n = 36) to skin heating (48-54 °C, 10 s/2 min) oi the ipsilateral ventral
139 hindfoot both during and in the absence of HS (25 s duration starting 10 s before onset of heat). In most experiments contact heat was delivered via a feedback controlled peltier thermode from a constant adapting temperature of 30 °C. In 9 experiments radiant heat was applied with a quartz-halogen lamp (adapting temperature 30 °C) which was controlled by feedback from a thermistor in contact with the skin. No differences were observed in the responses of the dorsal horn units to either form of heat in this or in a previous study m. To histologically verify all stimulation (Fig. 1) and recording sites, electrolytic lesions were made under deep sodium pentobarbital anesthesia and each animal was then perfused transcardially with 0.9% saline followed by 10% formalin containing appro×i-
mately 1% potassium ferricyanide. RESULTS
Behavior Behaviorally, 31/41 rats had no TF suppression at the highest HS intensity tested (1100/xA, Fig. 1A, A ) , 24/65 rats exhibited aversive behavior or intense motor activity which disallowed reliable TF testing (Fig. 1A, O), and only 10/65 rats showed TF suppression in at least one test session (Fig. 1A~ -#). Of the 10 rats displaying TF suppression, 6 showed it only during the first of two weekly test sessions, 3 showed it in two consecutive test sessions but not in a third test session, and only one animal showed TF suppression in 4 out of 4 weekly test ses-
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Fig. 1. Histologically verified HS sites are compiled on representative brain sections. A: 65 HS sites were tested behaviorally. Twentyfour sites evoked aversive or motor behavior which disallowed reliable TF testing (©), 31 sites elicited no TF suppression (A), and 10 sites elicited TF suppression in at least one test session (~'). B: 33 HS sites tested bel~av~orally were subsequently tested dectrophysiologically. Heat-evoked responses of 25 units were inhibited to at least 50% of their control responses during HS (open symbols). Responses of 11 units during HS up to 1100/~A remained at 65-100% of the control response (closed symbols). Circles indicate sites producing aversive/motor behavior during behavioral testing, triangles indicate sites not producing TF suppression up to 1100!~A HS, and stars indicate sites producing TF suppression in at least one behavioral test session.
140 sions. The mean currents producing TF suppression and aversion were 637.5 + 275.4 and 647.8 + 257.1 pA, respectively. Most of the observed motor activity or aversive behavior was HS dependent. There was no significant difference between the mean behavior scores for test sessions one and two (2.8 + 1.07 and 2.98 + 0.98, respectively, paired t-test, P > 0.05). Fifty-eight percent of the animals exhibited the same behaviors in two consecutive test sessions, while 21% had less severe motor effects the second week, and 21% had more severe motor effects the second week. In 5 animals, motor effects such as pawing, chattering, tail rigidity, and occasionally shaking (similar to wet dog shakes) continued for 2-5 min after brain stimulation was terminated. Although EEGs were not recorded, these behaviors indicate some seizure activity. They were never associated with TF suppression, and they typically occurred only during the second weekly test session, although one animal exhibited them in two consecutive test sessions. All of the stim'alation sites producing these effects were in the medial thalamus (nucleus-reuniens and rhomboid nucleus).
Eiectrophysiology Thirty-three rats tested behaviorally were subsequently tested electrophysiologically. Thirty-one of the 36 heat-responsive units tested were of the wide dynamic range type 36, responding to light touch, pressure and noxious pinch stimuli. The remaining 5 units were characterized as nociceptive specific, responding only to noxious pinching and heating. Receptive fields generally included the toes and footpads but often included the whole foot and heel area. Histologically localized recording sites were in laminae II-V of the dorsal horn 23. Heat-evoked responses of 25/36 dorsal horn units were inhibited by HS to at least 50% of their control responses (Fig. 1B, open symbols), and all but 3 of these units were inhibited to <35% of the control response. The mean current needed to produce 50% inhibition was 558.8 + 282.0 pA. Responses of the remaining 11 units (10 wide dynamic range, one nociceptive specific) remained at 65-100% of the control responses during HS of up to l l00 p A (Fig. 1B, closed symbols). Table I summarizes the effects of HS on dorsal
TABLE I Thirty-three rats were behaviorally assessed during HS and categorized as having TF suppression (TFS). not having TF suppression, or as exhibiting aversive behavior. The same rats were then tested electrophysiologically and each dorsal horn unit's response to noxious heat during HS (identical to behavioral HS) was categorized as inhibited (0-50% of control) or not inhibited (65-100% of control). There was no correlation between the behavioral response during HS and whether or not a unit was inhibited.
Electrophysiology
65-100% of control 0-50% of control
Behavior TFS
No TFS
Aversive
3 4
5 10
3 I1
horn neuronal heat-evoked responses of those animals also tested behaviorally. In rats showing TF suppression, 4/7 units were inhibited to >50% by identical HS (Fig. 1B, ,A-). In rats with no TF suppression, 10/15 dorsal horn units were inhibited (Fig. 1B, A), and in rats showing aversive behavior, 11/14 units were inhibited (Fig. 1B, (3). Nine of these 11 units were inhibited at currents less than the currents producing aversion in the behavioral testing. There was no correlation between the behavioral response during HS and whether or not a unit was inhibited (g2 with Pirie and Hamden correction, P > 0.50). Figs. 2 and 3 summarize behavioral and electrophysiological data from two representative rats. Behaviorally, rat 4 (Fig. 2) showed no TF suppression during HS up to 1100pA in test session 1, while in test session 2, the rat showed aversive behavior at HS intensities greater than 500 p A which prevented further TF trials (Fig. 2A). The heat-evoked responses recorded electrophysiologically in this rat were inhibited to 50% at 435 p A (Fig. 2B). As the peristimulus-time histograms (PSTHs) in Fig. 2C show, the responses to 50 °C are reduced during HS (lower row) compared to control responses in the absence of HS (upper row). Fig. 2D shows the stimulation site on a representative histological section. Rat 5 (Fig. 3) showed TF suppression at 300pA in test session 1 but exhibited aversive behavior during 300 p A of HS in test session 2 (Fig. 3A). The heat-evoked responses of the dorsal horn unit were reduced to 50% of control at 135 p A (Fig. 3B). Again, the PSTHs (Fig. 3C) clearly show the strong inhibition of the unit's response during HS (lower row) as compared to control
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HS INTENSITY (uA) Fig. 2. Representative behavioral and electrophysiological data obtained from the same rat (rat 4). A: graph plots TF latency against intensity of HS. There was no TF suppression during HS in test session l (O), while in test sessioa 2 (O) the rat exhibited aversive behavior which disallowed further trials. B: graph plots unit response to 50 °C heat during HS (expressed as % of control) against HS intensity. Fifty percent inhibition of the unit's response was reached at 435/~A (based on regression analysis). C: peristimulus-time histograms (PSTHs; bin width 1 s) of the unit's response to heat (indicated by black bar). Upper row shows control responses in the absence of HS and the lower row shows responses during 25 s of HS (denoted by brackets below PSTHs) at the indicated current intensities. Note increase in degree of inhibition as HS intensity is increased. D: stimulation site (O) is shown on representative histological section.
levels (upper row). The stimulation site is shown in the representative histological section (Fig. 3D). DISCUSSION This study provides evidence that HS-evoked inhibition of dorsal horn neuronal responses to noxious skin heating does not imply behavioral analgesia. Oliveras et al. 26 previously reported a strong relationship between SPA from the midbrain P A G and dor-
sal raphe nucleus, and inhibition of dorsal horn neuronal responses to noxious (pinch) stimuli in the cat. Sites not supporting SPA, however, were also effective in inhibiting dorsal horn unit responses to noxious pinch, indicating that inhibition is a more widespread phenomenon than analgesia. The present study extends those findings and raises several important questions on the interpretation of analgesia and descending inhibition. Many previous electrophysiologicai studies of des-
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Fig. 3. Representative behavioral and electrophysiological data from the same rat (rat 5). A: graph as in Fig. 2A. In test session 1 (©), 300/~A HS produced TF suppression. However, in test session 2 (0), the rat showed aversive behavior at 300 ~A HS which disallowed reliable TF testing. B: graph as in Fig. 2B plotting response of unit to 54 °C heat during HS against HS intensity. Based on regression analysis, 135 pA of HS produced 50% inhibition. C: PSTHs (as in Fig. 2C) of the unit's response to 54 °C (black bars), in the absence of HS (CON; upper row) and during HS at the indicated intensities (lower row). D: stimulation site (,b) is shown on representative histological section.
cending modulation of spinal transmission have utilized cats or monkeys, while most behavioral studies on SPA use rats. To adequately assess the relationship between SPA and inhibition, it is necessary to study both in the same animal while stimulating identical brain sites. In this study, we used the spinally mediated tail-flick reflex as a behavioral test of analgesia but examined neurons with nociceptive input from the foot in the electrophysiological experiments. It would obviously be advantageous to use identical noxious stimuli in both testing situations. However, using standard electrophysiological methods, it has been found that units with receptive
fields on the tail do not reliably respond to noxious heating which elicits the TF reflex in an unanesthetized animal (Guinan et al., unpublished observation; Dickenson, personal communication). We are therefore currently developing a behavioral test which utilizes the hindlimb flexion withdrawal reflex evoked by noxious heat applied to the hindfoot. This will hopefully allow for a more direct comparison of behavioral and electrophysiological data. Although we found that HS did not produce reliable SPA, others 5J3'25'32 have reported that H S produces analgesia as measured by tests involving supraspinally organized nocifensive behaviors sue'-
143 cape. However, it has been suggested that such complex behaviors might be more readily disrupted by brain stimulation than segmentally organized nocifensive reflexes such as the TF 2°. The present behavioral experiments showing a lack of reliable SPA used only the TF test. We did not examine more complex nocifensive behaviors, partly due to the aversive reactions frequently observed during HS. In many cases, HS caused such intense motor effects that assessment of analgesia was difficult or impossible. In 11 such rats a TF trial was performed immediately following 20 s of HS to assess possible SPA in the absence of stimulation-dependent motor activity. There was no TF suppression in any of these rats following HS at up to 1100/~A. Since higher stimulation intensities are generally required for post-stimulation as compared to during-stimulation analgesia 6, it is possible that TF suppression may have occurred at intensities greater than 1100/zA. The aversive and motor effects of HS, however, made testing at higher intensities undesirable. Others have also reported that stimulation throughout the PAG at intensities sufficient to produce ~analgesia' frequently produces aversive side ~ffec~ 15"27. Prado and Roberts 27 reported a positive correlation between aversive responses and the incidence of SPA suggesting that SPA may be secondary to stress associated with brain stimulation. This was not the case in the present study, however, since rats which were tested for TF suppression, but showed intense motor reactions during HS, were not analgesic. In addition, rats exhibiting TF suppression showed only alerting or slight motor responses during HS. It is possible that the hypothalamus can modulate nociception via its connections with the PAG 2"25, but that analgesia was masked by the aversive side effects of HS. That there was a low incidence of analgesia in those rats not exhibiting aver~ive behavior d~!ring HS argues against this possibility. Considering the diverse functions and neural connections of the hypothalamus, it is not surprising that HS would elicit a variety of responses in the awake rat. One possible explanation for the low incidence and unreliability of TF suppression in this study might be the development of a lesion at the stimulation site. The absence of behavioral hyperalgesia 22"35 and the high incidence of inhibition in the electrophysiological tests argue against this, but we cannot rule out this possibility in the less common cases in which HS did not inhibit dorsal horn units. That HS intensities nec-
essary to inhibit dorsal horn units were higher in this study compared to our previous report (mean current to produce 50% inhibition = 191 + 90 ~A) a is most likely due to differences in current densities resulting from the use of twisted strands of thicker wire in this study compared to the concentric bipolar semi-microelectrodes used in the previous study. A second explanation for the low incidence of TF suppression in this study is that the lack of behavioral antinociception is real but that the electrophysiological evidence of antinociception is artifactual due to anesthetic effects. Several recent studies have shown that barbiturate anesthesia can unmask neural activity that is not normally observed in the awake animal. Sandkuhler and Gebhart 34 found that animals lightly anesthetized with pentobarbital had significantly shorter TF latencies and more vigorous responses than the same animal in the awake state. Collins ll has reported that the proportion of wide dynamic range cells in the dorsal horn is higher in barbiturate-anesthetized cats than in the same unanesthetized cats. In addition, dorsal horn units often responded to noxious thermal stimuli in the anesthetized animal, but not in the same awake, drug-free animal ~. These reports, together with our finding that the majority of dorsal horn units in anesthetized animals are inhibited at currents which do not produce behavioral inhibition (i.e. TF suppression), suggest that barbiturates not only unmask the responses of wide dynamic range units, but may facilitate their inhibition by brain stimulation. These data suggest that HS-induced inhibition of nociceptive dorsal horn units in anesthetized animals does not imply behavioral analgesia and that care must be taken in the interpretation of descending inhibition of "nociceptive" wide dynamic range cells in relation to mechanisms of analgesia. If descending inhibitory control of noxious input at the spinal level does not necessarily imply behavioral analgesia, the true functional significance of descending inhibition and of 'nociceptive' wide dynamic range dorsal horn neurons needs to be elucidated.
ACKNOWLEDGEMENTS The authors wish to thank Dr. L.R. Watkins for her helpful comments on the manuscript. Supported by Grants NS20037 and NS19330 from the National Institutes of Health.
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Medial hypothalamic stimulation suppresses nociceptive spinal dorsal horn neurons but not the tail-flick reflex in the rat* E.S. Culhane and E. Carstens Department of Animal Physiology, Universi~ of California at Davis, Davis, CA 95616 (U.S.A.) (Accepted 23 June 1987)
Key words: Medial hypothalamus; Dorsal horn neuron; Heat-evoked response; Tail-flick reflex; Descending inhibition; Stimulation-produced analgesia; Rat
This study investigated the potentia~ analgesic effects of medial hypothalamic stimulation (HS) on a measure of nocifensive behavior (tail-flick test (TF)) in awake rats, and potential inhibitory effects of identical HS on spinal dorsal horn neuronal responses to noxious skin heating in the same animals anesthetized with sodium pentobarbital. Sixty-five male Sprague-Dawley rats implanted with a bipolar stimulation electrode in histologically verified medial hypothalamic sites were tested behaviorally for TF suppression during HS (100 ms trains at 100 Hz. 3/s, 100-1100 liA) in 2-4 consecutive weekly test sessions. Thirty-three of these rats were then used in electrophysiological experiments to record responses of 36 dorsal horn units to noxious skin heating (48-54 °C, 10 s/2 rain) of the hindfoot pad in the absence of and during HS. Behaviorally, 31/65 rats had no TF suppression at the highest HS intensity tested (1100 !tA), 24/65 rats exhibited aversive behavior or motor activity which disallowed reliable TF testing, and only 10/65 rats showed TF suppression in at least one test session. In electrophysiologicai experiments, the heat-evoked responses of 25/36 dorsal horn units were inhibited to at least 50% of control ~uring HS. The responses of 11 units remained at 65-100% of the control responses during HS of up to 1lOOltA. In rats demonstrating T~ ~~~-~ression. 4/7 units were inhibited. In rats with no TF suppression, 10/15 units were inhibited, and in rats showing aversive behavior. 11/14 units were inhibited by HS. These data indicate that although HS suppresses spinal nociceptive neurons, it does not cause reimt~ie "IT suppression in unanesthetized rats and bring into question the often-held assumption that stimulation-evoked descending inhibition of spinal nociceptive neurons implies behavioral analgesia.
key !'7-9. A d d i t i o n a l l y , lesions in the m e d i a l hypot h a l a m u s produce h y p e r a l g e s i a 22"35. F u r t h e r m o r e ,
INTRODUCTION N u m e r o u s studies have d e m o n s t r a t e d the exist-
the h y p o t h a l a m u s has an extensive d e s c e n d i n g input
ence of e n d o g e n o u s p a i n control s y s t e m s in h u m a n s
to the m i d b r a i n P A G , suggesting that these areas m a y be functionally l i n k e d 2"-'4.
and in a n i m a l s . It is well e s t a b l i s h e d that electrical m e d u l l a r y n u c l e u s r a p h e m a g n u s a n d the m i d b r a i n
It has b e e n suggested that inhibition of nociceptive dorsal horn n e u r o n s m a y be a m e c h a n i s m for S P A ~8"
periaqueductal
26. If so, there should be a correlation b e t w e e n S P A
stimulation in m e d i a l b r a i n s t e m areas, such as the gray
(PAG),
produces
analgesia
( s t i m u l a t i o n - p r o d u c e d analgesia ( S P A ) ) a n d inhibits
and s t i m u l a t i o n - e v o k e d
the responses of dorsal horn n e u r o n s to noxious stimuli 4a7-18"36. Electrical stimulation in m o r e rostral
neurons
structures i n c l u d i n g the p e r i v e n t r i c u l a r gray and hyp o t h a l a m u s has also b e e n s h o w n to support S P A in h u m a n s -l'-s--'~, m o n k e y s .... and r~lts 13"lt''ltL-t':°-'- as
in the
same
inhibition of nociceptive animal.
The
few
studies
addressing this have i n d e e d shown a correlation between SPA and i n h i b i t i o n from ~he P A G 3"26, alt h o u g h it was c o m m e n t e d that inhibition was also
well as to inhibit e v o k e d responses o f spinal cord dor-
e v o k e d from m o r e lateral reticular areas that were aversive when s t i m u l a t e d in the awake a n i m a l 26.
sal horn cells to noxious heat in the rat, cat and mon-
T h e aims of the p r e s e n t investigation were to es-
An abstract of this research has been previously published~-'. Correspondence." E. Carstens. Department of Animal Physiology. University of California at Davis. Davis. CA t~5f'lf,. U.S.A. *
138 tablish: (1) if stimulation in the medial hypothalamus suppresses the nociceptive tail-flick reflex in the rat; and (2) if there is a correlation between the occurrence of tail-flick suppression and inhibition of dorsal horn neuronal heat-evoked responses produced by identical hypothalamic stimulation in the same rat. MATERIALS AND METHODS
Animals Sixty-five adult male Sprague-Dawley rats (Simonsen Labs., Gilroy, CA) were stereotaxically implanted with a bipolar stimulation electrode in medial hypothalamic (and periventricular) areas under sodium pentobarbital anesthesia (65 mg/kg) supplemented with Metofane (Pitman-Moore) as needed. Each electrode was constructed from two twisted strands of teflon-coated stainless-steel wire (approximately 0.5 mm diameter, Medwire) soldered into two female Amphenol pins. Dental acrylic was used to insulate the assembly and to anchor it to 4 screws inserted into the skull. Following a 5-7-day recovery period, each rat underwent a series of 5 daily 1-h training sessions in which they were habituated to the Plexiglas restrainers used during behavioral testing. All rats were housed individually on a 12:12 h light/ dark cycle with food and water available ad libitum.
Behavioral testing A modification of the tail-flick (TF) test ~4was used to assess nociceptive responses on a behavioral level beginning 2 weeks after electrode implantation and continuing for 2-4 consecutive weekly test sessions. Prior to any hypothalamic stimulation (HS), baseline (BL) TF latencies were measured (average of 3 stable trials, 2 min inter-trial intervals). Bulb voltages on the TF apparatus were adjusted to obtain BL latencies of approximately 3.0-3.5 s and were thereafter held constant throughout the experiment. A method of ascending limits was then used to determine the threshold current for TF suppression. HS (100 ms trains at 100 Hz, 3/s, 100-1100/~A) intensity was increased by 200/~A in each successive TF trial. HS began 10 s before the initiation of the TF trial and was terminated immediately after the trial. TF suppression was defined as two consecutive TF latencies of 8.0 s during HS with the TF required to return to within 0.5 s of the BL latency between the two stimu-
lation trials. If TF suppression occurred, the current intensity was lowered half a step (1001~A) to obtain a more accurate threshold for TF suppression. Testing was terminated if any one of the following conditions occurred: (1) TF suppression; (2) HS intensity reached 1100/~A with no TF suppression; or (3) aversive or motor effects disallowed reliable TF testing. Two to 4 consecutive weekly test sessions were employed to test the reliability of TF suppression or any other behavior observed. During each test session, each rat was subjectively rated on a 4-point scale based on behaviors observed during HS at the highest intensity used in that session. Behavior ratings were as follows: (1) no apparent reaction or only slight alerting to HS; (2) slight motor responses such as eye and nose twitching or more organized behaviors such as pawing and teeth chattering; (3) mild motor responses involving the head and/or the whole body yet which were not so severe as to disallow a reliable TF test; (4) vocalization or profound motor and escape responses which disallowed reliable TF testing. Although behavioral studies typically employ biphasic stimulati~,n parameters, we chose to use monophasic stimulation for two reasons: (1) to be able to directly compare these results with our previous electrophysiologicai studiesS; and (2) we have previously found that there was no difference in the incidence of SPA evoked by mono- compared to biphasic stimulation 33. These parameters produced an estimated current spread of no more than 1 ram. Current intensities were monitored using a Tektronix P6021 current probe, and both the current and voltage across the electrode were simultaneously displayed on an oscilloscope.
Electrophysiological testing Thirty-three rats tested behaviorally were subsequently tested electrophysiologically. Under sodium pentobarbital anesthesia (65 mg/kg i.p.), the jugular vein was cannulated for continuous infusion of pentobarbital (10 mg/kg/h) throughout the experiment. The lumbosacral spinal cord was exposed by laminectomy and bathed in saline placed in an agar pool formed over the exposed cord. A tungsten microelectrode was inserted into the spinal cord to record the responses of dorsal horn units (n = 36) to skin heating (48-54 °C, 10 s/2 min) oi the ipsilateral ventral
139 hindfoot both during and in the absence of HS (25 s duration starting 10 s before onset of heat). In most experiments contact heat was delivered via a feedback controlled peltier thermode from a constant adapting temperature of 30 °C. In 9 experiments radiant heat was applied with a quartz-halogen lamp (adapting temperature 30 °C) which was controlled by feedback from a thermistor in contact with the skin. No differences were observed in the responses of the dorsal horn units to either form of heat in this or in a previous study m. To histologically verify all stimulation (Fig. 1) and recording sites, electrolytic lesions were made under deep sodium pentobarbital anesthesia and each animal was then perfused transcardially with 0.9% saline followed by 10% formalin containing appro×i-
mately 1% potassium ferricyanide. RESULTS
Behavior Behaviorally, 31/41 rats had no TF suppression at the highest HS intensity tested (1100/xA, Fig. 1A, A ) , 24/65 rats exhibited aversive behavior or intense motor activity which disallowed reliable TF testing (Fig. 1A, O), and only 10/65 rats showed TF suppression in at least one test session (Fig. 1A~ -#). Of the 10 rats displaying TF suppression, 6 showed it only during the first of two weekly test sessions, 3 showed it in two consecutive test sessions but not in a third test session, and only one animal showed TF suppression in 4 out of 4 weekly test ses-
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Fig. 1. Histologically verified HS sites are compiled on representative brain sections. A: 65 HS sites were tested behaviorally. Twentyfour sites evoked aversive or motor behavior which disallowed reliable TF testing (©), 31 sites elicited no TF suppression (A), and 10 sites elicited TF suppression in at least one test session (~'). B: 33 HS sites tested bel~av~orally were subsequently tested dectrophysiologically. Heat-evoked responses of 25 units were inhibited to at least 50% of their control responses during HS (open symbols). Responses of 11 units during HS up to 1100/~A remained at 65-100% of the control response (closed symbols). Circles indicate sites producing aversive/motor behavior during behavioral testing, triangles indicate sites not producing TF suppression up to 1100!~A HS, and stars indicate sites producing TF suppression in at least one behavioral test session.
140 sions. The mean currents producing TF suppression and aversion were 637.5 + 275.4 and 647.8 + 257.1 pA, respectively. Most of the observed motor activity or aversive behavior was HS dependent. There was no significant difference between the mean behavior scores for test sessions one and two (2.8 + 1.07 and 2.98 + 0.98, respectively, paired t-test, P > 0.05). Fifty-eight percent of the animals exhibited the same behaviors in two consecutive test sessions, while 21% had less severe motor effects the second week, and 21% had more severe motor effects the second week. In 5 animals, motor effects such as pawing, chattering, tail rigidity, and occasionally shaking (similar to wet dog shakes) continued for 2-5 min after brain stimulation was terminated. Although EEGs were not recorded, these behaviors indicate some seizure activity. They were never associated with TF suppression, and they typically occurred only during the second weekly test session, although one animal exhibited them in two consecutive test sessions. All of the stim'alation sites producing these effects were in the medial thalamus (nucleus-reuniens and rhomboid nucleus).
Eiectrophysiology Thirty-three rats tested behaviorally were subsequently tested electrophysiologically. Thirty-one of the 36 heat-responsive units tested were of the wide dynamic range type 36, responding to light touch, pressure and noxious pinch stimuli. The remaining 5 units were characterized as nociceptive specific, responding only to noxious pinching and heating. Receptive fields generally included the toes and footpads but often included the whole foot and heel area. Histologically localized recording sites were in laminae II-V of the dorsal horn 23. Heat-evoked responses of 25/36 dorsal horn units were inhibited by HS to at least 50% of their control responses (Fig. 1B, open symbols), and all but 3 of these units were inhibited to <35% of the control response. The mean current needed to produce 50% inhibition was 558.8 + 282.0 pA. Responses of the remaining 11 units (10 wide dynamic range, one nociceptive specific) remained at 65-100% of the control responses during HS of up to l l00 p A (Fig. 1B, closed symbols). Table I summarizes the effects of HS on dorsal
TABLE I Thirty-three rats were behaviorally assessed during HS and categorized as having TF suppression (TFS). not having TF suppression, or as exhibiting aversive behavior. The same rats were then tested electrophysiologically and each dorsal horn unit's response to noxious heat during HS (identical to behavioral HS) was categorized as inhibited (0-50% of control) or not inhibited (65-100% of control). There was no correlation between the behavioral response during HS and whether or not a unit was inhibited.
Electrophysiology
65-100% of control 0-50% of control
Behavior TFS
No TFS
Aversive
3 4
5 10
3 I1
horn neuronal heat-evoked responses of those animals also tested behaviorally. In rats showing TF suppression, 4/7 units were inhibited to >50% by identical HS (Fig. 1B, ,A-). In rats with no TF suppression, 10/15 dorsal horn units were inhibited (Fig. 1B, A), and in rats showing aversive behavior, 11/14 units were inhibited (Fig. 1B, (3). Nine of these 11 units were inhibited at currents less than the currents producing aversion in the behavioral testing. There was no correlation between the behavioral response during HS and whether or not a unit was inhibited (g2 with Pirie and Hamden correction, P > 0.50). Figs. 2 and 3 summarize behavioral and electrophysiological data from two representative rats. Behaviorally, rat 4 (Fig. 2) showed no TF suppression during HS up to 1100pA in test session 1, while in test session 2, the rat showed aversive behavior at HS intensities greater than 500 p A which prevented further TF trials (Fig. 2A). The heat-evoked responses recorded electrophysiologically in this rat were inhibited to 50% at 435 p A (Fig. 2B). As the peristimulus-time histograms (PSTHs) in Fig. 2C show, the responses to 50 °C are reduced during HS (lower row) compared to control responses in the absence of HS (upper row). Fig. 2D shows the stimulation site on a representative histological section. Rat 5 (Fig. 3) showed TF suppression at 300pA in test session 1 but exhibited aversive behavior during 300 p A of HS in test session 2 (Fig. 3A). The heat-evoked responses of the dorsal horn unit were reduced to 50% of control at 135 p A (Fig. 3B). Again, the PSTHs (Fig. 3C) clearly show the strong inhibition of the unit's response during HS (lower row) as compared to control
141
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HS INTENSITY (uA) Fig. 2. Representative behavioral and electrophysiological data obtained from the same rat (rat 4). A: graph plots TF latency against intensity of HS. There was no TF suppression during HS in test session l (O), while in test sessioa 2 (O) the rat exhibited aversive behavior which disallowed further trials. B: graph plots unit response to 50 °C heat during HS (expressed as % of control) against HS intensity. Fifty percent inhibition of the unit's response was reached at 435/~A (based on regression analysis). C: peristimulus-time histograms (PSTHs; bin width 1 s) of the unit's response to heat (indicated by black bar). Upper row shows control responses in the absence of HS and the lower row shows responses during 25 s of HS (denoted by brackets below PSTHs) at the indicated current intensities. Note increase in degree of inhibition as HS intensity is increased. D: stimulation site (O) is shown on representative histological section.
levels (upper row). The stimulation site is shown in the representative histological section (Fig. 3D). DISCUSSION This study provides evidence that HS-evoked inhibition of dorsal horn neuronal responses to noxious skin heating does not imply behavioral analgesia. Oliveras et al. 26 previously reported a strong relationship between SPA from the midbrain P A G and dor-
sal raphe nucleus, and inhibition of dorsal horn neuronal responses to noxious (pinch) stimuli in the cat. Sites not supporting SPA, however, were also effective in inhibiting dorsal horn unit responses to noxious pinch, indicating that inhibition is a more widespread phenomenon than analgesia. The present study extends those findings and raises several important questions on the interpretation of analgesia and descending inhibition. Many previous electrophysiologicai studies of des-
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Fig. 3. Representative behavioral and electrophysiological data from the same rat (rat 5). A: graph as in Fig. 2A. In test session 1 (©), 300/~A HS produced TF suppression. However, in test session 2 (0), the rat showed aversive behavior at 300 ~A HS which disallowed reliable TF testing. B: graph as in Fig. 2B plotting response of unit to 54 °C heat during HS against HS intensity. Based on regression analysis, 135 pA of HS produced 50% inhibition. C: PSTHs (as in Fig. 2C) of the unit's response to 54 °C (black bars), in the absence of HS (CON; upper row) and during HS at the indicated intensities (lower row). D: stimulation site (,b) is shown on representative histological section.
cending modulation of spinal transmission have utilized cats or monkeys, while most behavioral studies on SPA use rats. To adequately assess the relationship between SPA and inhibition, it is necessary to study both in the same animal while stimulating identical brain sites. In this study, we used the spinally mediated tail-flick reflex as a behavioral test of analgesia but examined neurons with nociceptive input from the foot in the electrophysiological experiments. It would obviously be advantageous to use identical noxious stimuli in both testing situations. However, using standard electrophysiological methods, it has been found that units with receptive
fields on the tail do not reliably respond to noxious heating which elicits the TF reflex in an unanesthetized animal (Guinan et al., unpublished observation; Dickenson, personal communication). We are therefore currently developing a behavioral test which utilizes the hindlimb flexion withdrawal reflex evoked by noxious heat applied to the hindfoot. This will hopefully allow for a more direct comparison of behavioral and electrophysiological data. Although we found that HS did not produce reliable SPA, others 5J3'25'32 have reported that H S produces analgesia as measured by tests involving supraspinally organized nocifensive behaviors sue'-
143 cape. However, it has been suggested that such complex behaviors might be more readily disrupted by brain stimulation than segmentally organized nocifensive reflexes such as the TF 2°. The present behavioral experiments showing a lack of reliable SPA used only the TF test. We did not examine more complex nocifensive behaviors, partly due to the aversive reactions frequently observed during HS. In many cases, HS caused such intense motor effects that assessment of analgesia was difficult or impossible. In 11 such rats a TF trial was performed immediately following 20 s of HS to assess possible SPA in the absence of stimulation-dependent motor activity. There was no TF suppression in any of these rats following HS at up to 1100/~A. Since higher stimulation intensities are generally required for post-stimulation as compared to during-stimulation analgesia 6, it is possible that TF suppression may have occurred at intensities greater than 1100/zA. The aversive and motor effects of HS, however, made testing at higher intensities undesirable. Others have also reported that stimulation throughout the PAG at intensities sufficient to produce ~analgesia' frequently produces aversive side ~ffec~ 15"27. Prado and Roberts 27 reported a positive correlation between aversive responses and the incidence of SPA suggesting that SPA may be secondary to stress associated with brain stimulation. This was not the case in the present study, however, since rats which were tested for TF suppression, but showed intense motor reactions during HS, were not analgesic. In addition, rats exhibiting TF suppression showed only alerting or slight motor responses during HS. It is possible that the hypothalamus can modulate nociception via its connections with the PAG 2"25, but that analgesia was masked by the aversive side effects of HS. That there was a low incidence of analgesia in those rats not exhibiting aver~ive behavior d~!ring HS argues against this possibility. Considering the diverse functions and neural connections of the hypothalamus, it is not surprising that HS would elicit a variety of responses in the awake rat. One possible explanation for the low incidence and unreliability of TF suppression in this study might be the development of a lesion at the stimulation site. The absence of behavioral hyperalgesia 22"35 and the high incidence of inhibition in the electrophysiological tests argue against this, but we cannot rule out this possibility in the less common cases in which HS did not inhibit dorsal horn units. That HS intensities nec-
essary to inhibit dorsal horn units were higher in this study compared to our previous report (mean current to produce 50% inhibition = 191 + 90 ~A) a is most likely due to differences in current densities resulting from the use of twisted strands of thicker wire in this study compared to the concentric bipolar semi-microelectrodes used in the previous study. A second explanation for the low incidence of TF suppression in this study is that the lack of behavioral antinociception is real but that the electrophysiological evidence of antinociception is artifactual due to anesthetic effects. Several recent studies have shown that barbiturate anesthesia can unmask neural activity that is not normally observed in the awake animal. Sandkuhler and Gebhart 34 found that animals lightly anesthetized with pentobarbital had significantly shorter TF latencies and more vigorous responses than the same animal in the awake state. Collins ll has reported that the proportion of wide dynamic range cells in the dorsal horn is higher in barbiturate-anesthetized cats than in the same unanesthetized cats. In addition, dorsal horn units often responded to noxious thermal stimuli in the anesthetized animal, but not in the same awake, drug-free animal ~. These reports, together with our finding that the majority of dorsal horn units in anesthetized animals are inhibited at currents which do not produce behavioral inhibition (i.e. TF suppression), suggest that barbiturates not only unmask the responses of wide dynamic range units, but may facilitate their inhibition by brain stimulation. These data suggest that HS-induced inhibition of nociceptive dorsal horn units in anesthetized animals does not imply behavioral analgesia and that care must be taken in the interpretation of descending inhibition of "nociceptive" wide dynamic range cells in relation to mechanisms of analgesia. If descending inhibitory control of noxious input at the spinal level does not necessarily imply behavioral analgesia, the true functional significance of descending inhibition and of 'nociceptive' wide dynamic range dorsal horn neurons needs to be elucidated.
ACKNOWLEDGEMENTS The authors wish to thank Dr. L.R. Watkins for her helpful comments on the manuscript. Supported by Grants NS20037 and NS19330 from the National Institutes of Health.
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