Brain Research, 525 (1990) 189-197 Elsevier
189
BRES 15770
Plasticity of some spinal dorsal horn neurons as revealed by pentobarbital-induced disinhibition J.G. Collins, K. Ren, Y. Saito, H. Iwasaki and J. Tang Department of Anesthesiology, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Accepted 20 February 1990)
Key words: Plasticity; Spinal dorsal horn; Pentobarbital; Tonic inhibition
Extracellular activity was recorded from single spinal dorsal horn neurons in physiologically intact, awake, drug-free cats before and after the intravenous administration of 20 mg/kg pentobarbital (Pb). Pb produced a series of changes in response properties that reflect a significant moment-to-moment plasticity of some spinal dorsal horn neurons. Pb administration unmasked the ability of some low-threshold (LT) neurons to respond to noxious mechanical or thermal stimuli resulting in their being reclassified as wide dynamic range (WDR) neurons. Pb also appeared to unmask an afterdischarge in some neurons following noxious mechanical stimulation. In addition, some neurons appeared to be better able to signal changes in the intensity of mechanical stimulation after Pb. Neuronal receptive fields for low-threshold stimulation were reduced in many instances but enlargement was also observed. The responses of some neurons to peripheral stimulation were unchanged by Pb. We hypothesize that the relatively low doses of Pb used in the study reduced tonic inhibition of some spinal dorsal neurons although the observed effects could have been produced by excitation. INTRODUCTION As our appreciation of the sensory processing capability of the spinal dorsal horn improves, it is becoming increasingly apparent that a system that had been thought of by many as static in its ability to handle information contains at least some components that, as a result of tonic modulatory systems, are capable of dynamic changes in their response to stimuli of varying intensity. Several laboratories, using acute preparations, have clearly demonstrated that some spinal dorsal horn neurons are under tonic influences that appear to preferentially inhibit neuronal responsitivity to noxious peripheral stimulation (e.g. refs. 2, 13, 25). The type of neuron that is capable of responding to both non-noxious and noxious peripheral stimulation has been at the center of a controversy not only concerning its nomenclature (multireceptive, wide dynamic range, type 1, type 2) but also more importantly concerning its physiologic significance. At the two extremes of the argument it has been proposed that these wide dynamic range ( W D R ) neurons are either sufficient for the signalling of pain in humans 19, 22 or are at best, under pathological conditions, associated with some form of abnormal somesthetic processing 21.
During initial studies using the physiologically intact, awake, drug-free preparation, we found that the population of W D R neurons from which we were able to record was much smaller than expected based upon similar sampling procedures used in acute preparations 6. As part of a series of studies to control for possible sources of this difference, we examined the effect of pentobarbital (Pb) on dorsal horn neurons. Administration of Pb to animals in which low numbers of W D R neurons had been encountered when they were drug-free allowed us to observe a larger population of W D R neurons that more closely approximated the expected percentages 8. We hypothesized that the presence of barbiturate anesthesia was having an impact upon tonically active inhibitory systems that were present in the intact drug-free preparation. We assumed that barbiturate induced removal of inhibition on noxiously evoked activity allowed us to encounter more neurons that responded to both noxious as well as innocuous activity ( W D R neurons), a process similar to that observed by others (e.g. ref. 25) when a reversible cold block was employed in acute preparations. This study was carried out to extend initial observations that Pb anesthesia unmasked response profiles of some spinal dorsal horn neurons.
Correspondence: J.G. Collins, Department of Anesthesiology, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
190 MATERIALS AND METHODS This protocol was approved by the Yale Animal Care and Use Committee. Experiments were performed on female cats ranging in weight from 2.6 to 3.9 kg that had been prepared for chronic neurophysiologic recording. Animal preparation as described elsewhere v included a period of adaptation during which the animals were acclimated to sitting in a restraint box. When an animal could sit quietly in the restraint box for a period of 1 h, it was judged ready for surgical implantation of the recording chamber and external jugular vein catheter. Following induction with i.m. ketamine (15 mg/kg + 0.1 mg atropine) general anesthesia was maintained (halothane, nitrous oxide, oxygen) during all surgical procedures which were carried out using sterile technique. A midline incision was employed to expose the vertebral column from L v through L~. The L4 spinous process and underlying bone were removed to form a window that was 12 mm long by 6 mm wide. A stainless-steel recording chamber was affixed to the vertebral column with a similar sized opening aligned over the window that had been made in the vertebral column. Muscle and skin were closed around the recording chamber so that the top of the chamber protruded from the skin on the back of the animal and provided access through the intact dura for penetration by tungsten microelectrodes. A catheter was placed in the external jugular vein and tunneled subcutaneously to the head where it was externalized on a dental acrylic pedestal. Prophylactic antibiotic treatment was routinely employed. The animals were allowed a minimum of 2 weeks of recovery following surgical implantation of the recording chamber before electrophysiological studies were initiated. A recording session began by placing the animal in the restraint box which had an opening that allowed the recording chamber to protrude from the top. A microdrive assembly was attached to the recording chamber. Major blood vessels on the surface of the spinal cord were mapped at the time of chamber implantation and included in a map that was maintained for all penetration sites. Dural thickening, while a problem, was reduced by the application of Panlog a steroid antibiotic preparation (nystatin-neomycin sulfate-thiostrepton-triancinolone acetonide) to the exposed dura at the end of each recording session '~. Following attachment of the microdrive assembly, a tungsten microelectrode with a tip diameter of 5-10 f~m (10 meg tested at 1000 k) was manually positioned so that its tip could be seen to be in contact with the surface of the dura. From that point in time, electrode manipulation was conducted with a micromanipulator and involved the slow advancement of the microelectrode through the dura. Penetration of the dura produced no apparent discomfort for the animal. However, if either the tip of the electrode had been damaged or the dura had thickened to the point that penetration was difficult and there was significant compression of the dura, movement by the animal, vocalization, or a change in pupillary size was interpreted as animal discomfort and the electrode was immediately withdrawn. Following successful penetration of the dura, the electrode was advanced until extracellular activity could be recorded from a single neuron with a receptive field located on a body part distal to the recording chamber. Following adequate isolation of a single neuron, control studies were conducted in which the neuronal response to a series of stimuli of increasing intensity including airpuff, brushing, rubbing, squeezing with the fingers, pinching with forceps and the presentation of either contact thermal or radiant heat stimuli (maximum 51 °C) were evaluated. Noxious stimuli (pinch and thermal) were terminated when the animal reflexly withdrew. Receptive field sensitivity to light probing or brushing was determined and the borders of the receptive field were marked for later quantification. Stimuli within the noxious range were maintained at relatively low levels and separated in time by at least 2 rain to reduce the likelihood of receptor sensitization. Following characterization of a neuron's response profile in the physiologically intact awake, drug-free animal, pentobarbital (Pb 20 mg/kg) was administered rapidly i.v. Rapid administration was used to avoid the excitatory phase which can sometimes be seen when low
levels of a general anesthetic are present. Even with rapid administration it was not uncommon for brief excitation or cough to cause loss of isolation. Rapid Pb administration did result in a short period of apnea that resolved spontaneously and never required treatment. Following barbiturate administration, response profiles were evaluated at various time points depending upon the rate of recovery of the animal from the general anesthetic. The goal of each experiment was to achieve a minimum of 30 rain of neuronal evaluation after barbiturate administration. In some experiments an additional dose of Pb (10 mg/kg) was administered at various times after the initial dose. For those neurons for which the entire border of the receptive field could be determined both before and after drug administration, the marked areas on the skin of the animal were traced onto drawing paper and, with the assistance of a computer-driven digitizing system, the areas of the various receptive fields were compared before and after drug administration. Amplitude discrimination was used to separate the neuron under study from baseline. Typical signal-to-noise ratios were 4 or 5:1. Neuronal activity was recorded on magnetic tape and analyzed off line. The response profile of each neuron, both before and after drug administration was evaluated for changes in activity elicited by the various forms of stimuli. Neurons that showed no increased response to stimuli approaching the noxious range, either mechanical or thermal, were considered to be low-threshold neurons. If a neuron demonstrated an increased responsivity to mechanical and/or thermal stimuli approaching the noxious range, such that the maximum activation occurred when noxious stimuli were presented and that activity was above the level produced by non-noxious stimuli, those neurons were considered to be wide dynamic range neurons. Since control studies were carried out in awake, drug-free, intact animals noxious stimuli were not employed as search stimuli. In addition to evaluating the presence or absence of a response to the various forms of stimulation, the nature of that response was also evaluated. We were interested in determining if any changes in signal processing were likely to be associated with barbiturate administration. RESULTS T h e r e s u l t s r e p o r t e d in this s t u d y w e r e o b t a i n e d f r o m 193 e x p e r i m e n t s c o n d u c t e d in 10 a n i m a l s . D u r i n g 111 o f t h o s e e x p e r i m e n t s , a d e q u a t e i s o l a t i o n o f a single s p i n a l d o r s a l h o r n n e u r o n w a s o b t a i n e d . O f 111 n e u r o n s t h a t w e r e o b s e r v e d d u r i n g c o n t r o l s t u d i e s , it w a s p o s s i b l e t o c o n d u c t 45 c o m p l e t e d r u g s t u d i e s . S o m e a n i m a l s u s e d in this s t u d y w e r e also e m p l o y e d f o r o t h e r s t u d i e s in w h i c h d r u g effects o n s p i n a l d o r s a l h o r n
neurons
were ob-
served. A minimum of 2 days separated pentobarbital a d m i n i s t r a t i o n in a n y o n e a n i m a l .
Pentobarbital-induced unmasking of neuronal responses to noxious stimuli T h e m a i n p u r p o s e o f this s t u d y w a s t o e x t e n d initial observations suggesting that intravenous administration of P b c o u l d u n m a s k t h e r e s p o n s e o f s o m e s p i n a l d o r s a l horn neurons to noxious stimulation. Table I summarizes the
classification
of
the
various
neurons
for
which
c o m p l e t e d r u g s t u d i e s w e r e a v a i l a b l e . W e s e e in T a b l e I t h a t p r i o r t o P b a d m i n i s t r a t i o n , 36 o f t h e 45 n e u r o n s w e r e considered to be low threshold because they either failed to r e s p o n d to p i n c h o r t h e r m a l s t i m u l i at a p o i n t w h e n t h e
191 TABLE I
animal reflexly withdrew from the stimulus or because their response to those stimuli was no greater than that to non-noxious stimuli. In addition, 9 neurons were considered to have a wide dynamic range response profile prior to drug administration. Following Pb administration, 13 (36%) of the LT neurons were reclassified. Two of them appeared to become high threshold since their response to non-noxious stimulation was eliminated, although they continued to respond to noxious stimuli. Eleven neurons that were considered to be low threshold prior to Pb administration were now reclassified as W D R neurons. Fig. 1 presents an example of the kind of change induced by Pb that resulted in a neuron being reclassified as a W D R neuron. During control the neuron shown in Fig. 1 did not respond to 10 s, 47 °C thermal stimulus even though the animal withdrew at the end of that period of time. Following the 20 mg/kg dose of Pb, however, the same stimulus produced a significant response that was still seen after an additional 10 mg/kg of Pb was administered intravenously. Changes of this type (i.e. an enhanced response) were also seen following pinch stimuli (Figs. 2 and 3) and were the reasons for neurons being reclassified as W D R rather than LT neurons. In addition to extending our initial observation that intravenous Pb is capable of unmasking the response of some spinal dorsal horn neurons to noxious stimulation, we also observed an afterdischarge following noxious mechanical stimulation that appeared to be unmasked following Pb administration. In 10 of the 45 neurons that were completely studied, 20 mg/kg of Pb was associated with an afterdischarge following mechanical pinch that was not seen during control studies. All of the neurons in
CONTROL
Classification of neuronal response profiles
Numbers in parentheses indicate percent of total sample. Baseline
30 min after 20 mg/kg Pb i. v.
LT 36 (80%) WDR9 (20%) HT0 (0%)
23 (51%) 20 (45%) 2 (4%)
Total 45
45
which the afterdischarge was observed were considered to be W D R neurons at that time. For the neuron depicted in Fig. 2, during control stimulation, pinching of the receptive field until such time as the animal withdrew from the stimulus resulted in a brisk response to the stimulus with an immediate end of that response when the stimulus was terminated. However, presentation of a similar stimulus 30 min after the intravenous administration of 20 mg/kg of pentobarbital produced a slight enhancement of the response to pinch, but in addition to that produced a significant afterdischarge that lasted longer than the response to the pinch itself. In one neuron in which Pb administration was followed by an afterdischarge an additional dose of Pb (10 mg/kg) eliminated the afterdischarge even though the response to pinch was still present. This would suggest the possibility of a biphasic, dose-dependent effect on the afterdischarge. Near the end of this study, we began utilizing forceps instrumented with strain gauges that allowed us to record the relative change in pinch intensity that occurred during stimulus presentation. When we began using this new stimulator, we presented the pinch stimuli more slowly so
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Fig. 1. Pb unmasking of a neuronal response to thermal stimulation. During control, a 10-s 47 °C radiant heat stimulus did not cause activation of this neuron even though it did elicit animal withdrawal. However, following initial Pb administration there was a clear response to same thermal stimulus. An additional 10 mg/kg of Pb was administered at 30 min but had no further effect on the thermally evoked activity.
192
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Fig. 2. Pb unmasking of pinch-evoked afterdischarge. During control, pinch evoked a brisk discharge of this neuron that ended abruptly when the stimulus was terminated (animal withdrawal occurred at stim off). Following Pb administration a similar pinch evoked a greater peak discharge but also was followed by an afterdischarge that was not observed during control.
that a slow ramp could be evaluated and in doing so observed an additional effect of pentobarbital on the neuronal activity of 3 of the 4 neurons that were tested in this way and that were changed from LT to W D R neurons. This change can be best described as an alteration in the ability of the neurons to signal the changing intensity of the stimulus and is shown in Fig. 3. During control, pinch stimulation comparable to that shown in the inset produced activation of this neuron that
was approximately the same amplitude over time with the exception of an Off response when the stimulus was terminated. However, following the i.v. administration of 20 mg/kg of pentobarbital, we observed not only an increased response to the pinch stimulus, but also a much closer correspondence between the changing intensity of the stimulus and the changing of neuronal firing frequency. This neuron also demonstrated an afterdischarge that was not observed during control studies.
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Fig. 3. Pb-induced change in signalling of changing stimulus intensity. The inset is an example of a typical stimulus used during this experiment. It depicts the changing output voltage from strain gauges monitored on a pair of forceps. In the histogram depicting neuronal activity, the initial peak results from initial contact with the receptive field (area of contact was 3 mm in diameter). There was rapid adaptation to the control contact at which time pinch was initiated. During control, although the pinch intensity was increasing over time the firing frequency of this neuron was relatively constant. In contrast, following Pb administration not only was the peak response to pinch enhanced but the slope of the firing frequency during the stimulus approximated that of the changing pinch intensity.
193
Barbiturate effects on neuronal responses to low-intensity stimulation The most pronounced effect of Pb on low-intensity stimulation was the significant change in receptive field size that was observed in those neurons in which receptive fields could be accurately mapped (n = 17). Nine of those 17 neurons had a greater than 20% reduction in receptive field area within 10 min after the intravenous administration of 20 mg/kg of Pb. Five neurons had an increase in receptive field area that was greater than 20% at that same point in time. Three of the receptive fields that were mapped showed no change in area. Fig. 4 demonstrates the profound effect that intravenous Pb had on some receptive fields. The baseline receptive field area of the cell shown in Fig. 4A was 241 cm 2. However, 20 min after the i.v. administration of 20 mg/kg of Pb, the receptive field had been reduced to approximately 10% of control. With time, the receptive field tended to recover, such that at 45 min it had returned to 82% of control. Notice that the 'hot spot' (area of the receptive field that was most sensitive to mechanical stimulation) during control (marked by the X) was totally insensitive to low intensity stimulation 20 min after Pb administration. The receptive field of the neuron shown in Fig. 4B (control area was 54.9 cm 2) demonstrated a biphasic change in area. The receptive field area sensitive to light touch in Fig. 4B was decreased such that 10 rain after 20 mg/kg of Pb it was only 71% of control. However, 40 min after the initial administration of Pb the receptive field area had increased to 170% of the control value. A n
additional 10 mg/kg of Pb was then administered resulting in a subsequent reduction in the receptive field area to 58% of control. In contrast to Fig. 4A, the cell depicted in Fig. 4B maintained its 'hot spot' within the center of the receptive field even though the receptive field area had been reduced. The change in low-threshold receptive field size was not associated with a particular neuron type. Some neurons remained LT after Pb but had an increase (2), decrease (2) or no change in receptive field size (1). Some LT neurons became W D R s with either an increase (1) or decrease in receptive field size (3) and some neurons remained W D R s with an increase (2) decrease (3) or no change in receptive field size (1). The effect of Pb on neuronal response profiles appeared to be very complex. Fig. 5 (top) demonstrates that, at times, i.v. Pb significantly suppressed the brush response. Movement of a 3/4 in. wide camel-hair brush across the receptive field elicited a predictable response by the neuron as shown in the top left of Fig. 5. However, following 20 mg/kg of Pb most of the brush strokes failed
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Fig. 4. Pb-induced change in low-threshold mechanical receptive field area. Both receptive fields were located on the hip. A: 20-min after 20 mg/kg Pb, the receptive field area sensitive to light touch was reduced to 10% of control (24.1 cm2). 45 min after Pb administration the area had returned to 82% of control. Notice that the 'hot spot' marked by the X that was near the center of the receptive field during control, was insensitive to low-intensity stimulation 20 min after Pb administration. B: 10 min after Pb this cell also had a reduced receptive field (71% of control) but 40 min after Pb the receptive field had increased in size to 170% of control. However, an additional 10 mg/kg of Pb (55 min) caused the receptive field to shrink to 58% of control (31.8 cm2).
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Fig. 5. Top: Pb suppression of a neuron's response to receptive field brushing. Arrows below histograms indicate gentle brushing of the receptive field with a 3/4 in. wide camel hair brush. During control (left) a reproducible response was elicited. Pb administration almost completely suppressed the brush-evoked response. Bottom. Response of the same neuron shown above to squeeze. Arrows indicate initial low-threshold contact with the receptive field. In spite of the fact that this neuron's response to brushing was suppressed by Pb the response to low-intensity contact was, if anything, enhanced by Pb.
194 CONTROL
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Fig. 6. Apparent biphasic effect of Pb on heat-evoked activity. During control, heating the receptive field (45 °C, 8 s) elicited a response. Pb administration (20 mg/kg) enhanced that response (15 min) but an additional 10 mg/kg of Pb reduced activity to that seen during control.
to elicit a response and in those few in which a response was elicited, it was minimal in nature. Although the response of this particular neuron to brushing was significantly suppressed by Pb, the response of the same neuron to another form of low-intensity stimulation was relatively unchanged as shown in the bottom of Fig. 5. Squeezing of the receptive field involved, first contacting the receptive field. In the bottom panels of Fig. 5, the arrows indicate initial contact with the receptive field and as is evident in that figure the response to the initial contact, which adapted rapidly, was, if anything, enhanced by administration of 20 mg/kg of Pb. This is in spite of the fact that this is the same neuron as shown in the top of Fig. 5 in which the response to brushing was almost totally suppressed by the same dose of Pb. As stated earlier, we began employing the instrumented forceps near the end of the study. As such, we are not able to make this comparison for all cells studied. Additional evidence of the complex nature of the Pb effects is well demonstrated in Fig. 6. During control studies, the 45 °C heat stimulus for 8 s produced activation of this neuron. Following 20 mg/kg of Pb, the same stimulus produced an increase in the overall activity of this neuron. However, an additional dose of 10 mg/kg of Pb appeared to produce a suppression of the enhanced activity to a level equivalent to that seen during control. As indicated in Table I, although there were neurons that had their response profiles changed by the administration of Pb, there were also many neurons that were unaffected by drug administration. Twenty-three LT and 9 W D R neurons retained their classification following Pb administration. Rates of spontaneous activity among the neurons in
this study were quite low. Many neurons had no spontaneous activity within 30-s periods of time that were analyzed. Most neurons with spontaneous activity had rates below 0.5 impulses/s. Pb did not produce a consistent increase in spontaneous activity even for those neurons in which responses to noxious stimuli were enhanced. DISCUSSION
The present results clearly indicate that there is a proportion of low-threshold neurons, as defined in the preparation used in these studies, that are capable of responding to higher intensities of stimulation. We have interpreted the results of this study to indicate that tonic inhibition blocks that response in the awake, drug-free animal. The application of 20 mg/kg of Pb appears to disrupt that tonic inhibition and allows the noxiously evoked activity of those neurons to be observed. In this study, 11 of the 45 neurons that were completely evaluated had their classifications changed from LT to W D R following the administration of Pb. The percentage of neurons that underwent such a change, 24%, is similar to the percentage we have recently reported to be changed in a similar fashion by the systemic administration of the non-specific serotonin antagonist methysergide 23. Although approximately one-fourth of the LT neurons encountered in this preparation appear to be unmasked by disinhibition, we have not yet been able to identify any response features that would allow us to predict which of the neurons would be so effected. An alternative explanation of the results of this study is that pentobarbital is having a direct excitatory effect on the neurons that were changed. It is our contention that
195 disinhibition rather than excitation is the most reasonable explanation but the most important consideration is the effect of the unmasking, whatever the process may be that is responsible for it. The unmasking demonstrates the moment-to-moment plasticity of some spinal dorsal horn neurons. A major reason for choosing disinhibition rather than excitation is the lack of effect of Pb on spontaneous activity. If the Pb effects were due to excitation, we would have expected to see increased levels of 'spontaneous activity' after drug administration. Perhaps a more compelling reason to favor disinhibition over excitation is seen in Figs. 2 and 3. If Pb was causing excitation of the neurons under study, we would expect the overall level of activity to be increased not just the activity elicited by certain intensities of stimulation. In Figs. 2 and 3 the response to higher levels of intensity are enhanced by Pb although responses to lower levels of intensity remain relatively unchanged. Low levels of general anesthesia are often associated with behavioral excitation 5. This excitation has been assumed to be due to an initial greater effect of general anesthetics on inhibitory rather than excitatory systems. In this study, it is likely that a low level of anesthesia resulted in disinhibition of tonic inhibitory systems that would normally modulate afferent input to spinal dorsal horn neurons. Although Pb administration clearly is capable of unmasking the response profiles of some neurons to noxious stimulation this is not a phenomenon that is observed only following Pb administration. It also has been seen recently in our laboratory following administration of the non-specific serotonin antagonist methysergide 23. The ability of Pb to unmask an afterdischarge in some spinal dorsal horn neurons following noxious pinch provides additional evidence of tonic inhibition in this preparation. The question arises as to the source of this afterdischarge. Yaksh 26 has recently demonstrated that the administration of glycine or GABA antagonists is capable of producing a significant hyperalgesic state with what appears to be spontaneous pain in rats. Although emphasis is usually placed upon tonic inhibitory systems, it is possible that a normal balance is maintained between tonic inhibitory and excitatory impulses at the level of the spinal dorsal horn. In the presence of Pb anesthesia, with what we assume to be a disinhibition of inhibitory systems, the presentation of a noxious stimulus may be capable of activating or enhancing tonic excitatory systems resulting in an afterdischarge. This may also be an explanation for spontaneous activity that has been seen following spinal cord transection or reversible cold-block of the spinal cord in acute studies. Since afterdischarge has been suggested to be a cause of pain
outlasting a stimulus, removal of tonic inhibition may be a physiologic mechanism by which pain signalling is enhanced. The use of forceps instrumented with strain gauges, not only provided a much better quantification of stimulus presentation, but also provided an opportunity to observe an additional change induced by barbiturate, a change that has been replicated and quantified in studies using methysergide administration 23. That change is shown in Fig. 3 where we see that barbiturate administration appears to alter the ability of this neuron to signal changing intensity of stimulation. If we assume that a frequency code is responsible for signalling changes in stimulus intensity, then after barbiturate administration this cell clearly is much more capable of signalling the changing intensity of the pinch stimulus than it was during control. One of the more striking features of the effects of Pb on spinal dorsal horn neuron was the profound change in receptive field size of some neurons. Although a reduction in receptive field area was most frequently observed, there were instances in which initial dosing with Pb resulted in an increase in receptive field area. Others have demonstrated changes in receptive field size in acute preparations following the administration of various anesthetic agents (e.g. refs. 1, 4, 11). The present studies suggest two counter-balancing mechanisms that may have an important role to play in regulating somatic sensation. It has been suggested that one possible way that the nervous system interprets afferent input to the level of the spinal cord is by monitoring the level of activity within a key central pool of neurons (see ref. 12 for discussion). The ability of a general anesthetic like Pb to significantly reduce the receptive field size for lowthreshold input of some spinal dorsal horn neurons may be part of a mechanism by which afferent input to the central nervous system is reduced resulting ultimately in the state referred to as anesthesia. At the other extreme, we see evidence for the ability of a barbiturate anesthetic to increase receptive field size as well. The difference between decreased and increased receptive field size is likely to be a dose-dependent phenomenon. As we saw in Fig. 4B, higher doses of Pb were capable of decreasing receptive field size. However, as time progressed (i.e. the total amount of Pb in the system was decreased) the receptive field size greatly increased. We hypothesize that as the level of general anesthesia is reduced, disinhibition allows maximum afferent input to the individual dorsal horn neurons. This would suggest that in the preparation used here there is tonic inhibition not only of noxiously evoked activity, but also of non-noxiously evoked activity to some spinal dorsal horn neurons. In keeping with the hypothesis that
196 general anesthesia may result from a reduction in receptive field size, the hyperactivity associated with emergence from most general anesthetics may result in part from a significant afferent barrage as a result of many m o r e neurons being activated than normally would be by an a d e q u a t e somatosensory stimulus. Viewed in a b r o a d perspective, the results of this study d e m o n s t r a t e that in the animal model that was used, there is tonic modulation of some spinal dorsal horn neurons. That modulation is capable of significantly changing the afferent input that normally excites these neurons. This tonic m o d u l a t i o n is not effective on all neurons or at least is not altered by barbiturate administration. A question that is frequently asked is if W D R neurons studied in anesthetized animals are artifactual. This study, and others in awake animals (e.g. refs. 3, 15, 16, 24), clearly d e m o n s t r a t e that W D R neurons exist in intact, awake, drug-free animals. Although the W D R neurons may have a different physiological function than those present in the awake animals, it is clear that W D R neurons do exist in the dorsal horn of intact animals. It is possible that both extremes of the argument 19'21'22 about the role of W D R neurons may be correct. A s the animal's status changes (attention, stress, pain) different W D R neurons may be called upon to signal events in the periphery. Two recent studies may suggest some physiological significance for the 'unmasking' observed in the present study. H y l d e n et al. ]7 have recently r e p o r t e d an elegant study that d e m o n s t r a t e s changes in spinal lamina I
projection neurons within 6 h of a d j u v a n t - i n d u c e d inflammation. Most notable was the significant increase in receptive field area of the lamina I neurons (noxious stimuli) at a time when no similar changes were observed in primary afferents. The neurophysioiogic changes were observed at a time that c o r r e s p o n d e d with behavioral hyperalgesia. This is one of the best examples of central changes in afferent processing as p r o p o s e d by H a r d y 40 years ago 14 and d e m o n s t r a t e d recently by others (e.g. refs. 10, 20). A recent behavioral study by Yaksh ~6 suggests how such a change could be produced. In rats, antagonism of known inhibitory systems in the spinal cord ( G A B A , glycine) p r o d u c e d an i m m e d i a t e and intense nocifensive response, if c o m p o n e n t s of the spinal dorsal horn are normally inhibited from responding to noxious stimuli, removal of that inhibition could provide 'new channels' of communication about pain. Chemical disinhibition in the Yaksh study caused 'pain behavior' and in this study u n m a s k e d neural response to noxious stimuli. It is likely that a similar unmasking process may have occurred in the H y l d e n study p r o d u c e d by noxious afferent input and resulting in changed neuronal responsivity to noxious stimuli. The unmasking seen in this study may be evidence of the physiologic process that allows the CNS to b e c o m e , among o t h e r things, sensitized to pain.
Acknowledgements. The outstanding technical assistance and animal care provided by Ann Hinds and Tami Morgan are gratefully acknowledged. This research was supported in part by NIH GM29065 and NS23033.
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