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The response of neurons of the medial pontomedullary reticular formation of rats to peripheral thermal stimuli
Brain Research, 336 (1985) 107-115
107
Elsevier BRE 10783
The Response of Neurons of the Medial Pontomedullary Reticular Formation of Rats to Peripheral Thermal Stimuli CRAIG J. FARHAM and RODNEY J. DOUGLAS
University of Cape Town, MedicalSchool, Departmentof Physiology, Observatory, 7925, Cape Town (South Africa) (Accepted September 4th, 1984)
Key words: pontomedullary reticular formation - - thermal response - - noxious stimulus
The thermal responses of 70 medial pontomeduUary neurons were studied using 0.15 °C/s ramp thermal stimuli applied to the glabrous skin on the ventral surface of the hindpaw of rats. Fifty-two neurons (74%) responded to an ipsilateral and/or contralateral thermal stimulus by increased action potential discharge rates. Eleven units (16%) responded by decreasing their discharge. The remaining 7 units (10%) were unaffected. The thresholds of 61 neurons responsive to thermal stimulation of the ipsilateral hindpaw ranged from 36.5 to 46 °C. Ninety percent of thermally responsive neurons had bilateral receptive fields. The thresholds of the ipsilateral and contralateral responses were significantly positively correlated. INTRODUCTION Neurons of the medial pontomedullary reticular formation (PMRF) respond to innocuous and noxious mechanical stimulPS,16. The aim of this investigation was to ascertain whether P M R F neurons also respond to thermal stimuli, and if so, to survey the range of thresholds of their thermal response. Slow ramp thermal stimuli were applied to the glabrous skin on the ventral surface of the hindpaw of rats, and the responses of the P M R F neurons were assessed via their action potential discharge rates. MATERIALS AND METHODS Twenty-six female hooded rats ( L o n g - E v a n s ) each weighing between 300 and 350 g were anesthetized with urethane (1.2 g/kg, i.p.). Body temperature was maintained at 36.8 + 0.5 °C using a feedback controlled heater-pad incorporating an intrarectal thermistor. The head was mounted in a stereotaxic instrument (Kopf Instruments), with the palatal bar positioned 5 mm below the level of the interaural line. A 3 mm diameter burr hole was drilled in the oc-
cipital bone. The exposed dura overlying the cerebellum was removed. The cerebellum was left intact so as to minimize brainstem movement. Extracellular recordings were derived from glasscoated tungsten microelectrodes, and on a few occasions from single-barrel glass microelectrodes. Vertical electrode penetrations were made between 1.5 and 2.0 m m posterior to the interaural line and within 1 mm of the midsagittal plane. The extracellular signal was amplified, monitored on an oscilloscope and an audio-monitor and led to a discriminator. The discriminator transformed the action potentials of the unit under investigation into a series of 0.5-ms logic pulses, which were logged by a B B C B microcomputer. Only well isolated units, with signal-tonoise ratios in excess of 3 to 1 were accepted for observation. The number of logic pulses per two-second time bin were counted, and a discharge rate histogram constructed. The electrode signal and the output of the discriminator were continuously monitored on a dual-beam oscilloscope to ensure the validity of the translation of action potentials to logic pulses. P M R F neurons were identified as units generating large (0.5-5 mV) action potentials, located within
Correspondence: R. J. Douglas. Present address: University of Cape Town, Medical School, Department of Physiology, Observatory, 7925, Cape Town, South Africa. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
108 the appropriate stereotaxic region, and responding with increased or decreased discharge rates to one or more of light tactile (fur stroke), mechanical (deep pressure) or noxious (pinch) stimuli over large receptive fields. An electrolytic microlesion (10/tA, 20 s) was produced at the bottom of each electrode penetration and sometimes at recording sites. The locations of these lesions were determined by subsequent histological examination, and the location of the recording sites confirmed.
Thermal stimulation Thermal stimuli were delivered to the glabrous skin on the ventral surface of the hindpaw ipsilateral •to, and in some cases contralateral to, the PMRF neuron under observation. The ventral surface of the hindpaw was chosen as the stimulation site because (a) this region was anticipated to be important in detecting temperature changes in the unrestrained animal, and (b) the thermal stimulator could be conveniently and firmly applied to the skin at that point. The stimulus was applied by means of an aluminum disc 25 mm in diameter and 3 mm in thickness which acted as the heat diffuser of a current-controlled power transistor. The transistor was attached to one surface of the disc while the opposite surface acted as the thermal stimulator and was firmly applied to the glabrous skin on the hindpaw. A feed-back controller sensed the temperature of the disc, and adjusted the heat dissipation of the power transistor so as to maintain the stimulator surface at a command temperature. The sensor of the feed-back controller was located in the body of the disc, and near to its center. A reference thermometer (Yellow Springs Telethermometer 43TD) was used to calibrate the sensor so that it reflected the temperature of the stimulator surface. This calibration was checked under zero heat-flux conditions by sandwiching the reference thermistor between the stimulator surface and a thermal barrier consisting of a reflector and a thermal insulator. The calibration was also checked under typical toad conditions by sandwiching the reference thermistor between the stimulator surface and the experimenter's finger. Under both these test conditions, the sensor accurately reflected the surface temperature of the stimulator as detected by the reference thermistor. The temperature profile across the surface of the disc
was checked using a Yellow Springs 'banjo' thermistor (Yellow Springs 427) and was found to be uniform over the region in contact with the foot. The area of the stimulator in contact with the foot was about 1.75 cm 2, which was about one-third of the stimulator surface. The temperature of the stimulator was monitored by the microcomputer via an analog-to-digital converter. The resolution of the digitized temperature was less than 0.1 °C. Under resting conditions the stimulator temperature was 35 °C. Thus, the glabrous skin in contact with the surface of the stimulator was maintained at that same temperature. Each experiment to test the thermal response of a single PMRF neuron was performed as follows. The activity of the neuron was logged for a control period of about 100 s, during which time the stimulator remained at 35 °C (Fig. 2). Only units showing zero, or approximately constant discharge during this control period, were observed further. At the end of the control period a 0.15 °C/s ramp was initiated. The ramp was terminated at 48 °C and the stimulator was then removed from the foot to avoid tissue damage due to prolonged exposure of the skin to elevated temperatures. The thermal threshold of the PMRF neuron was defined as the temperature of the stimulator surface at the onset of an increase or decrease in the discharge rate of the neuron and was estimated to the nearest bin of the neuronal discharge rate histogram (Fig. 2). RESULTS
Identification of PMRF neurons Seventy units were identified as typical PMRF neurons on the basis of their response to tactile, mechanical and noxious stimuli, and also on the basis of their receptive fields. Fifty-five (79%) of the 70 units responded to the tactile, mechanical or noxious stimuli by increasing their discharge rates (excitatory response), while 7 cells (10%) decreased their discharge rates (inhibitory response). The remaining 8 units (11%) showed a complex response pattern characterized by combinations of excitation and inhibition with respect to receptive field and/or modality. The receptive fields for these stimuli ranged in size from restricted to very extensive. Thirty-three per-
109 cent of the units had restricted receptive fields which included the tail and/or hindlimbs only. Extensive receptive fields (49% of units) consisted of tail and/or hindlimbs and also large areas of the trunk. Very extensive receptive fields (18% of units) included both hindlimbs and forelimbs. Some part of the hindlimbs contributed to the receptive field of every identified PMRF neuron. The tail contributed to 81%, trunk 67%, forelimbs 19% and face 20% of the receptive fields. These PMRF units were located in a region which extended caudally from the level of the superior olivary complex to the caudal border of the facial nerve nucleus (Fig. 1). The majority of the units were located in relation to the nucleus and genu of the facial nerve and within 1 mm of the midsagittal plane.
Response of PMRF neurons to peripheral thermal stimuli Sixty-three (90%) of the 70 PMRF neurons responded to thermal stimuli applied to either the ipsilateral and/or contralateral hindpaw. Both excitatory and inhibitory responses were observed. The remaining 7 units (10%) were unaffected by the thermal stimuli. The latter were all spontaneously active. Of 61 neurons that responded to ipsilateral thermal stimuli, 51 (84%) responded by increased action potential discharge rates. These excitatory responses occurred both in neurons with spontaneous background activity as well as in quiescent neurons. Ten units (16%) responded to ipsilateral stimuli by decreasing their discharge rate. There was no spatial organization of the units with respect to excitatory and inhibitory responses (Fig. 1). The PMRF neurons exhibited two types of response to the thermal ramps at temperatures beyond threshold. About half of the responses were abrupt (switch-like) changes in the discharge rate when the unit attained threshold (Fig. 2a-c). Abrupt increases or decreases were superimposed on spontaneous background activity (Fig. 2a, b) or abrupt increases occurred in quiescent neurons (Fig. 2c). Those responses which were not switch-like exhibited a discharge which increased (or decreased) monotonically with stimulus temperature in the region immediately following the threshold (Fig. 2d). In some cases the discharge frequency increased in proportion to the stimulus up to the maximum temperature applied
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(Fig. 2d), while in o t h e r cases the p r o p o r t i o n a l response r e a c h e d a p l a t e a u that was u n a f f e c t e d by further increases in t e m p e r a t u r e (Fig. 2e). T h e p r o p o r tional responses o c c u r r e d b o t h in s p o n t a n e o u s l y ac-
Fig. 2. Responses of 5 representative neurons in the pontomedullary reticular formation to a thermal stimulus applied to the glabrous skin on the ventral surface of the hindpaw. The stimulus temperature is represented as a dotted line. The discharge rate of each of the units is represented as a histogram of events per 2 s time bin (solid line), a: unit with background discharge which exhibited an abrupt increase in discharge at threshold (43.0 °C). b: unit with background discharge which exhibited an abrupt cessation of activity at 39.6 *C. c: quiescent unit which exhibited an abrupt increase in discharge at 42.7 °C. d: unit which exhibited a discharge which was approximately proportional to stimulus temperature beyond a threshold of 38.0 °C. e: unit which exhibited a proportional increase in discharge (threshold 38.6 *C) followed by a plateau.
tive (Fig. 2e) a n d q u i e s c e n t (Fig. 2d) units. T h e p a t t e r n s of discharge o c c u r r i n g m o r e t h a n a b o u t 20 s after t h r e s h o l d were often c o m p l e x (Fig. 2b, c) a n d will n o t be described here.
111 nization of the two groups; neurons with thresholds below and above 42 °C were encountered throughout the P M R F (Fig. 4).
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Fig. 3. Distribution of thresholds of 61 pontomedullary reticular formation neurons responding to thermal stimulation of the ipsilateral hindpaw. Shaded area indicates the subgroup of 10 units which exhibited inhibitory responses.
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The distribution of thresholds of 61 neurons responsive to thermal stimulation of the ipsilateral hindpaw is shown in Fig. 3. Their thresholds ranged from 36.5 to 46 °C. The distribution of thresholds of the ipsilateral responsive units was bimodal, one peak occurring at 3 6 - 4 2 °C and the other at 42-46 °C. Each group accounted for about half the thermally responsive neurons. Excitatory responses occurred in both groups, but the majority (9/10) of the inhibitory neurons had thresholds only within the first group (below 42 °C). There was no correlation between the response of the units to innocuous or noxious mechanical stimulation and their membership of the lower or higher thermal threshold group (Table I). There was also no anatomical orga-
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TABLE I Relationship between a neuron's response to innocuous or noxious mechanical stimulation and its threshold to thermal stimuli of the ipsilateral hindpaw Thermal threshold
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Fig. 4. Anatomical location of 61 pontomeduUary reticular formarion units which responded to thermal stimulation of the ipsilateral hindpaw. Those units with thresholds below 42 *(3 are indicated by closed triangles, those above 42 °C by open triangles. No spatial organization with respect to these two threshold groups is apparent.
112 TABLE II Relationship between threshold and type of response of PMRF neurons to peripheral thermalstimuli Threshold
Response type
Total
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20 28
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Total
48
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Comparison of ipsi- and contralateral thermal responses Forty-one of the 70 identified P M R F neurons were tested with both an ipsi- and a contralateral thermal stimulus. Thirty-seven (90%) of these units responded to both stimuli; two (5%) responded only to ipsilateral stimulation; the remaining two ceils responded only to stimulation of the contralateral hindpaw. Each of the neurons which responded bilaterally exhibited the same response polarity (increased or decreased discharge) to thermal stimuli applied to either hindpaw. Moreover, the thresholds to ipsi- and contralateral thermal stimulation were significantly positively correlated (r = 0.54, P < 0.001; Fig. 5).
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Fig. 5. Comparison of the ipsilateral and contralateral thresholds of 37 units in the pontomedullary reticular formation responsive to thermal stimulation of both the hindpaws. Closed circles, excitatory responses; open circles, inhibitory responses. The ipsilateral and contralateral thresholds were significantly correlated (r = 0.54 P < 0.001). The regression line was 19.2 + 0.54x.
Relationship between threshold and type of response The 61 responses to ipsilaterat stimuli and 39 responses to contralateral stimuli were pooled. Of these 100 responses, 96 could be classified as having either switch-like or proportional responses (Fig. 2). A switch response was defined as one which attained more than two-thirds of its final discharge rate within 1.5 °C above threshold. These two classes were then subdivided according to whether the threshold of the response threshold lay above or below 42 °C (Table II). When these subclasses were compared using the Z2 procedure, the null hypothesis that there was no relationship between response type and threshold was rejected at the P < 0.005 level. Thus, responses with thresholds less than 42 °C tended to be of the proportional kind, whereas those with thresholds greater than 42 °C tended to be switch-like. DISCUSSION The units observed in this study were located in the medial pontomeduUary reticular formation; in the n.r. pontis caudalis and n. r. gigantocellularis. The somatosensory responses and receptive field characteristics of these units were qualitatively similar to those obtained in this region by earlier workers 3A3AS-17. Consequently, their membership of the P M R F is confirmed on neuroanatomical and functional grounds. Our data demonstrate that 90% of P M R F neurons which respond to mechanical and/or noxious peripheral stimuli also respond to thermal stimuli applied to the glabrous skin on the ventral surface of the rat hindpaw. Moreover, of all the thermal responsive neurons tested bilaterally, 90% had thermal receptive fields which included both hindpaws. This large fraction of P M R F neurons with c o m m o n receptive field components is consistent with the receptive field redundancy noted in the P M R F with respect to other somatosensory input 15. For example, some part of the hindlimbs contributed to the mechanical receptive field of every neuron in this series. The polysensory response and redundant receptive field characteristics of P M R F neurons suggest that the P M R F is organized so as to be sensitive to permutations of the available sensory input. Only 10% of the P M R F neurons observed in this study were unaffected by the thermal stimuli era-
113 ployed. However, it is possible that even these neurons were thermally responsive but not adequately stimulated. The thresholds of these neurons might have been above the maximum stimulus temperature which was restricted to below 48 °C in order to avoid tissue damage. Alternatively, their thermal receptive fields might have excluded the hindpaws. The extensive thermal responsiveness of PMRF neurons reported here contrasts with the findings of Casey 5 who was unable to elicit thermal responses from cat medial bulboreticular units with radiant heating of the skin to 50-60 °C. Thermally responsive neurons have been detected in regions of the brainstem reticular formation more caudal 4 and more rostral 7 to that investigated in the current study. However, the thermal response characteristics of each of those groups of neurons were considerably different from the characteristics of the PMRF neurons reported here. Burton 4 observed units in cat caudal bulbar reticular formation (field F'I'L of Berman2) which responded to noxious mechanical stimuli applied in the facial region. About 65 % of those neurons also responded to thermal stimuli. Their thresholds were only in the noxious range (43 to 62 °C). Eickhoff et al. 7 detected units in the medial mesencephalon and rostral pons of rats that responded to innocuous and/or noxious mechanical stimulation. About half of those also responded only to noxious levels of radiant heat (thresholds greater than 42 °C). Both of the above reports contrast with the high percentage of thermal responsiveness and the lower range of thresholds (36.5 to 46 °C, Fig. 3) observed in the PMRF units in this study. The major sources of error in the estimation of the thermal thresholds of the PMRF neurons are due to possible differences between the temperature of the stimulator surface and the effective temperature at the receptors. It is not practical to quantify the thermal resistive-capacitive properties of the stimulator-skin-receptor complex. However, for stimuli of temperatures greater than approximately core temperature, the series resistive elements between stimulus and receptor are expected to result in the stimulator temperature being an overestimate of the effective receptor temperature. The temperature lag due to thermal equilibration effects is also expected to contribute to overestimation of the thresholds. The time constant for this
equilibration in the footpad of the cat is about 1 s (ref. 1). Thus, for the case of a positive ramp of 0.15 °C/s, the PMRF thresholds are likely to have been overestimated by a temperature difference attributable to a latency of about 3 time-constants. Thus, the error is approximately 0.45 °C. Both sources of error contribute to an overestimation of the thresholds, and so the distribution presented in Fig. 3 is expected to be displaced towards high-. er temperatures than the effective value at the receptors. However, from an operational point of view it is the surface temperature of an object (or stimulus) which is relevant to the evaluation of an animal's behavior. Therefore, either the stimulator temperature or the skin surface temperature is the appropriate measure for evaluating PMRF responses. The slow rise-time of the ramp (0.15 °C/s), the high thermal conductivity of the aluminum stimulator and the capacity of the heat source suggest that the temperature of the stimulator surface was a good estimate of the temperature of the surface of the skin applied to the stimulator. This was confirmed by the fact that a reference thermistor interposed between the experimenter's finger and the stimulator tracked the stimulus temperature exactly. The data presented here are all derived from single trial threshold estimation. In a separate series of experiments (Farham and Douglas, unpublished data) we have demonstrated that the PMRF thermal thresholds are remarkably constant over time. In these experiments, 22 ipsilateral neurons were tested with two ramp stimuli (0.15 °C/s) separated by between 8 and 21 min. There was no significant difference between the thresholds of the first and second trials (mean difference -0.14 + 0.67 °C S.E.M., paired t = 0.029, P = 0.977). More than half of the PMRF units had thermal thresholds below 42 °C (Fig. 3) and were therefore not primarily activated by noxious thermal stimuli. The input to these neurons might be derived indirectly from warm receptors or from receptors responding differentially to warm and noxious thermal stimuli. The distribution of the thermal thresholds of PMRF neurons is bimodal (Fig. 3), and therefore it is likely that the PMRF itself contains two populations of thermally responsive neurons. Since the two peaks of the distribution have threshold ranges (36.5 to 42 °C and 42 to 46 °C) that conform to the maximum
114 discharge ranges for warm and noxious peripheral thermoreceptorsll, ~2, it is possible that thermal input converging onto a given PMRF neuron is restricted to that derived from either of those two receptor types. Sixty-four percent of the responses with thresholds below 42 °C were characterized by discharges which increased or decreased monotonically with increasing stimulus temperature (Table II). This monotonic relationship between discharge and the intensity of the thermal stimulus provides support for indirect warm receptor input to PMRF neurons 6. However, it is not known whether warm receptors are present in the glabrous skin of the rat hindpaw. Zimmermann and Handwerker18 were unable to find warm receptors in footpads of cats, but Lamotte and Campbell 12 have observed these receptors in the glabrous skin of monkey hand. On the other hand, the thermal input to the PMRF neurons may be derived from a single population of nociceptors with a wide range of temperature thresholds. Lynn and Carpenter TM reported that C-fiber polymodal nociceptors located in the hairy skin on the dorsal and medial aspects of the rat hindpaw responded to thermal stimuli between 36 and 59 °C. The distribution of the thresholds was unimodal, with a mean of 47 + 5.8 °C. While most of these receptors had thermal thresholds in the noxious range, about 20% had thresholds below 42 °C and so provided thermal input in the warm range. If a similar population of polymodal nociceptors is present in the glabrous skin of the rat hindpaw, then the thermal input from these receptors would be sufficient to explain both the low temperature thresholds observed in many PMRF units, as well as those in the noxious range. Since the temperature thresholds of polymodal nociceptors are unimodal, the bimodal distribution of PMRF thresholds would be due to selective convergence of input in either the warm or the noxious ranges. Whatever the receptor types providing the thermal input, the correlation of ipsilateral and contralateral thermal thresholds of PMRF neurons (Fig. 5) is evidence for selective convergence at either the spinal or brainstem level. The selectivity appears to be restricted to thermal responsiveness, since there was no correlation between a PMRF neuron's response to innocuous or noxious mechanical stimulation and its response to innocuous or noxious thermal stimula-
tion (Table I). However, this lack of correlation could be a property derived from the receptor characteristics, since a similar lack of correlation between the thermal and mechanical thresholds was noted by Lynn and Carpenter t4 in the C-fiber polymodal nociceptors. Most of the PMRF units had bilateral thermal receptive fields. This spatial convergence could also occur at either the spinal or brainstem level. Fields et al. s,9 reported that although there was considerable convergence of somatosensory input at the level of the spinoreticular neurons, the majority of those neurons had ipsilateral receptive fields. The authors did not examine the thermal responses of the spinoreticular neurons. However, if it is supposed that thermal input is likely to be handled in a manner similar to other somatosensory input, then the large fraction of bilateral PMRF thermal receptive fields observed in this study was probably due to convergence at the PMRF rather than at the spinal level. The spatial convergence of somatosensory input at the PMRF level is implied by the bilaterality of the spinoreticular projections: about two-thirds of spinoreticular neurons project to the ipsilateral nucleus gigantocellularis, while the remainder project to the contralateral n. gigantocellularis 9. The functional role of the PMRF neurons is ambiguous. The activity of these neurons might be related to sensory processing, arousal, reflex integration or motor control 10. Since the majority of these neurons had thermal receptive fields which included at least both hindpaws in each case, their role in discriminating stimulus location cannot be significant. Moreover, half of the PMRF neurons exhibited abrupt 'switching' responses (Table II) which suggest that at least those neurons are concerned with the detection of the stimulus rather than with resolving its intensity. Thus, the thermal responses of these neurons are likely to be relevant in an integrative context rather than one of sensory discrimination. ACKNOWLEDGEMENTS The authors gratefully acknowledge the electronic designs and technical assistance of Jan Pepler. This research was supported by the South African Medical Research Council.
115 REFERENCES 1 Beck, P. W., Handwerker, H. O. and Zimmermann, M., Nervous outflow from the cat's foot during noxious radiant heat stimulation, Brain Research, 67 (1974) 373-386. 2 Berman, A. L., The Brain Stem of the Cat, The University of Wisconsin Press, Madison, WI, 1968, 175 pp. 3 Bowsher, D., Mallart, A., Petit, D. and Albe-Fessard, D., A bulbar relay to the centre median, J. Neurophysiol., 32 (1968) 288-300. 4 Burton, H., Somatic sensory properties of caudal bulbar reticular neurons in the cat (Felis domestica), Brain Research, 11 (1968) 357-372. 5 Casey, K. L., Somatic stimuli, spinal pathways, and size of cutaneous fibres influencing unit activity in the medial medullary reticular formation, Exp. Neurol., 25 (1969) 35-56. 6 Dubner, R. and Bennett, G. J., Spinal and trigeminal mechanisms of nociception, Ann. Rev. Neurosci., 6 (1983) 381-418. 7 Eickhoff, R., Handwerker, H. O., McQueen, D. S. and Schick, E., Noxious and tactile input to medial structures of midbrain and pons in the rat, Pain, 5 (1978) 99-113. 8 Fields, H. L., Partridge, L. D. and Winter, D. L., Somatic and visceral receptive field properties of fibres in ventral quadrant white matter of the cat spinal cord, J. Neurophysiol., 33 (1970) 827-837. 9 Fields, H. L., Wagner, G. M. and Anderson, S. D., Some
10 11 12
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14
15
16 17 18
properties of spinal neurons projecting to the medial brainstem reticular formation, Exp. Neurol., 47 (1975) 118-134. Hobson, J. A. and Brazier, M. A. B., The Reticular Formation Revisited, Raven Press, New York, 1980. Iggo, A., Cutaneous thermoreccptors in primates and subprimates, J. Physiol. (Lond.), 200 (1969) 403-430. Lamotte, R. H. and Campbell, J. N., Comparison of the responses of warm and nociceptivc C-fiber afferents in monkey with human judgements of thermal pain, J. Neurophysiol., 41 (1978) 509-528. LeBlanc, H. J. and Gatipon, G. B., Medial bulborcticular response to peripherally applied noxious stimuli, Exp. Neurol., 42 (1974) 264-273. Lynn, B. and Carpenter, S. E., Primary afferent units from the hairy skin of the rat hind limb, Brain Research, 238 (1982) 29-43. Segundo, J. P., Takenaka, T. and Encabo, B., Somatic sensory responses of bulbar reticular neurons, J. NeurophysioL, 30 (1967) 1221-1238. Siegel, J. M., Behavioural functions of the reticular formation, Brain Res. Rev., 1 (1979) 69-105. Wolstencroft, J. H., Reticulospinal neurones, J. Physiol. (Lond.), 174 (1964) 91-108. Zimmermann, M. and Handwerker, H. O., Total afferent inflow and dorsal horn activity upon radiant heat stimulation to the cat's footpad, Advanc. Neurol., 4 (1974) 29-33.
107
Elsevier BRE 10783
The Response of Neurons of the Medial Pontomedullary Reticular Formation of Rats to Peripheral Thermal Stimuli CRAIG J. FARHAM and RODNEY J. DOUGLAS
University of Cape Town, MedicalSchool, Departmentof Physiology, Observatory, 7925, Cape Town (South Africa) (Accepted September 4th, 1984)
Key words: pontomedullary reticular formation - - thermal response - - noxious stimulus
The thermal responses of 70 medial pontomeduUary neurons were studied using 0.15 °C/s ramp thermal stimuli applied to the glabrous skin on the ventral surface of the hindpaw of rats. Fifty-two neurons (74%) responded to an ipsilateral and/or contralateral thermal stimulus by increased action potential discharge rates. Eleven units (16%) responded by decreasing their discharge. The remaining 7 units (10%) were unaffected. The thresholds of 61 neurons responsive to thermal stimulation of the ipsilateral hindpaw ranged from 36.5 to 46 °C. Ninety percent of thermally responsive neurons had bilateral receptive fields. The thresholds of the ipsilateral and contralateral responses were significantly positively correlated. INTRODUCTION Neurons of the medial pontomedullary reticular formation (PMRF) respond to innocuous and noxious mechanical stimulPS,16. The aim of this investigation was to ascertain whether P M R F neurons also respond to thermal stimuli, and if so, to survey the range of thresholds of their thermal response. Slow ramp thermal stimuli were applied to the glabrous skin on the ventral surface of the hindpaw of rats, and the responses of the P M R F neurons were assessed via their action potential discharge rates. MATERIALS AND METHODS Twenty-six female hooded rats ( L o n g - E v a n s ) each weighing between 300 and 350 g were anesthetized with urethane (1.2 g/kg, i.p.). Body temperature was maintained at 36.8 + 0.5 °C using a feedback controlled heater-pad incorporating an intrarectal thermistor. The head was mounted in a stereotaxic instrument (Kopf Instruments), with the palatal bar positioned 5 mm below the level of the interaural line. A 3 mm diameter burr hole was drilled in the oc-
cipital bone. The exposed dura overlying the cerebellum was removed. The cerebellum was left intact so as to minimize brainstem movement. Extracellular recordings were derived from glasscoated tungsten microelectrodes, and on a few occasions from single-barrel glass microelectrodes. Vertical electrode penetrations were made between 1.5 and 2.0 m m posterior to the interaural line and within 1 mm of the midsagittal plane. The extracellular signal was amplified, monitored on an oscilloscope and an audio-monitor and led to a discriminator. The discriminator transformed the action potentials of the unit under investigation into a series of 0.5-ms logic pulses, which were logged by a B B C B microcomputer. Only well isolated units, with signal-tonoise ratios in excess of 3 to 1 were accepted for observation. The number of logic pulses per two-second time bin were counted, and a discharge rate histogram constructed. The electrode signal and the output of the discriminator were continuously monitored on a dual-beam oscilloscope to ensure the validity of the translation of action potentials to logic pulses. P M R F neurons were identified as units generating large (0.5-5 mV) action potentials, located within
Correspondence: R. J. Douglas. Present address: University of Cape Town, Medical School, Department of Physiology, Observatory, 7925, Cape Town, South Africa. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
108 the appropriate stereotaxic region, and responding with increased or decreased discharge rates to one or more of light tactile (fur stroke), mechanical (deep pressure) or noxious (pinch) stimuli over large receptive fields. An electrolytic microlesion (10/tA, 20 s) was produced at the bottom of each electrode penetration and sometimes at recording sites. The locations of these lesions were determined by subsequent histological examination, and the location of the recording sites confirmed.
Thermal stimulation Thermal stimuli were delivered to the glabrous skin on the ventral surface of the hindpaw ipsilateral •to, and in some cases contralateral to, the PMRF neuron under observation. The ventral surface of the hindpaw was chosen as the stimulation site because (a) this region was anticipated to be important in detecting temperature changes in the unrestrained animal, and (b) the thermal stimulator could be conveniently and firmly applied to the skin at that point. The stimulus was applied by means of an aluminum disc 25 mm in diameter and 3 mm in thickness which acted as the heat diffuser of a current-controlled power transistor. The transistor was attached to one surface of the disc while the opposite surface acted as the thermal stimulator and was firmly applied to the glabrous skin on the hindpaw. A feed-back controller sensed the temperature of the disc, and adjusted the heat dissipation of the power transistor so as to maintain the stimulator surface at a command temperature. The sensor of the feed-back controller was located in the body of the disc, and near to its center. A reference thermometer (Yellow Springs Telethermometer 43TD) was used to calibrate the sensor so that it reflected the temperature of the stimulator surface. This calibration was checked under zero heat-flux conditions by sandwiching the reference thermistor between the stimulator surface and a thermal barrier consisting of a reflector and a thermal insulator. The calibration was also checked under typical toad conditions by sandwiching the reference thermistor between the stimulator surface and the experimenter's finger. Under both these test conditions, the sensor accurately reflected the surface temperature of the stimulator as detected by the reference thermistor. The temperature profile across the surface of the disc
was checked using a Yellow Springs 'banjo' thermistor (Yellow Springs 427) and was found to be uniform over the region in contact with the foot. The area of the stimulator in contact with the foot was about 1.75 cm 2, which was about one-third of the stimulator surface. The temperature of the stimulator was monitored by the microcomputer via an analog-to-digital converter. The resolution of the digitized temperature was less than 0.1 °C. Under resting conditions the stimulator temperature was 35 °C. Thus, the glabrous skin in contact with the surface of the stimulator was maintained at that same temperature. Each experiment to test the thermal response of a single PMRF neuron was performed as follows. The activity of the neuron was logged for a control period of about 100 s, during which time the stimulator remained at 35 °C (Fig. 2). Only units showing zero, or approximately constant discharge during this control period, were observed further. At the end of the control period a 0.15 °C/s ramp was initiated. The ramp was terminated at 48 °C and the stimulator was then removed from the foot to avoid tissue damage due to prolonged exposure of the skin to elevated temperatures. The thermal threshold of the PMRF neuron was defined as the temperature of the stimulator surface at the onset of an increase or decrease in the discharge rate of the neuron and was estimated to the nearest bin of the neuronal discharge rate histogram (Fig. 2). RESULTS
Identification of PMRF neurons Seventy units were identified as typical PMRF neurons on the basis of their response to tactile, mechanical and noxious stimuli, and also on the basis of their receptive fields. Fifty-five (79%) of the 70 units responded to the tactile, mechanical or noxious stimuli by increasing their discharge rates (excitatory response), while 7 cells (10%) decreased their discharge rates (inhibitory response). The remaining 8 units (11%) showed a complex response pattern characterized by combinations of excitation and inhibition with respect to receptive field and/or modality. The receptive fields for these stimuli ranged in size from restricted to very extensive. Thirty-three per-
109 cent of the units had restricted receptive fields which included the tail and/or hindlimbs only. Extensive receptive fields (49% of units) consisted of tail and/or hindlimbs and also large areas of the trunk. Very extensive receptive fields (18% of units) included both hindlimbs and forelimbs. Some part of the hindlimbs contributed to the receptive field of every identified PMRF neuron. The tail contributed to 81%, trunk 67%, forelimbs 19% and face 20% of the receptive fields. These PMRF units were located in a region which extended caudally from the level of the superior olivary complex to the caudal border of the facial nerve nucleus (Fig. 1). The majority of the units were located in relation to the nucleus and genu of the facial nerve and within 1 mm of the midsagittal plane.
Response of PMRF neurons to peripheral thermal stimuli Sixty-three (90%) of the 70 PMRF neurons responded to thermal stimuli applied to either the ipsilateral and/or contralateral hindpaw. Both excitatory and inhibitory responses were observed. The remaining 7 units (10%) were unaffected by the thermal stimuli. The latter were all spontaneously active. Of 61 neurons that responded to ipsilateral thermal stimuli, 51 (84%) responded by increased action potential discharge rates. These excitatory responses occurred both in neurons with spontaneous background activity as well as in quiescent neurons. Ten units (16%) responded to ipsilateral stimuli by decreasing their discharge rate. There was no spatial organization of the units with respect to excitatory and inhibitory responses (Fig. 1). The PMRF neurons exhibited two types of response to the thermal ramps at temperatures beyond threshold. About half of the responses were abrupt (switch-like) changes in the discharge rate when the unit attained threshold (Fig. 2a-c). Abrupt increases or decreases were superimposed on spontaneous background activity (Fig. 2a, b) or abrupt increases occurred in quiescent neurons (Fig. 2c). Those responses which were not switch-like exhibited a discharge which increased (or decreased) monotonically with stimulus temperature in the region immediately following the threshold (Fig. 2d). In some cases the discharge frequency increased in proportion to the stimulus up to the maximum temperature applied
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Fig. 2. Responses of 5 representative neurons in the pontomedullary reticular formation to a thermal stimulus applied to the glabrous skin on the ventral surface of the hindpaw. The stimulus temperature is represented as a dotted line. The discharge rate of each of the units is represented as a histogram of events per 2 s time bin (solid line), a: unit with background discharge which exhibited an abrupt increase in discharge at threshold (43.0 °C). b: unit with background discharge which exhibited an abrupt cessation of activity at 39.6 *C. c: quiescent unit which exhibited an abrupt increase in discharge at 42.7 °C. d: unit which exhibited a discharge which was approximately proportional to stimulus temperature beyond a threshold of 38.0 °C. e: unit which exhibited a proportional increase in discharge (threshold 38.6 *C) followed by a plateau.
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111 nization of the two groups; neurons with thresholds below and above 42 °C were encountered throughout the P M R F (Fig. 4).
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The distribution of thresholds of 61 neurons responsive to thermal stimulation of the ipsilateral hindpaw is shown in Fig. 3. Their thresholds ranged from 36.5 to 46 °C. The distribution of thresholds of the ipsilateral responsive units was bimodal, one peak occurring at 3 6 - 4 2 °C and the other at 42-46 °C. Each group accounted for about half the thermally responsive neurons. Excitatory responses occurred in both groups, but the majority (9/10) of the inhibitory neurons had thresholds only within the first group (below 42 °C). There was no correlation between the response of the units to innocuous or noxious mechanical stimulation and their membership of the lower or higher thermal threshold group (Table I). There was also no anatomical orga-
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112 TABLE II Relationship between threshold and type of response of PMRF neurons to peripheral thermalstimuli Threshold
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Comparison of ipsi- and contralateral thermal responses Forty-one of the 70 identified P M R F neurons were tested with both an ipsi- and a contralateral thermal stimulus. Thirty-seven (90%) of these units responded to both stimuli; two (5%) responded only to ipsilateral stimulation; the remaining two ceils responded only to stimulation of the contralateral hindpaw. Each of the neurons which responded bilaterally exhibited the same response polarity (increased or decreased discharge) to thermal stimuli applied to either hindpaw. Moreover, the thresholds to ipsi- and contralateral thermal stimulation were significantly positively correlated (r = 0.54, P < 0.001; Fig. 5).
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Fig. 5. Comparison of the ipsilateral and contralateral thresholds of 37 units in the pontomedullary reticular formation responsive to thermal stimulation of both the hindpaws. Closed circles, excitatory responses; open circles, inhibitory responses. The ipsilateral and contralateral thresholds were significantly correlated (r = 0.54 P < 0.001). The regression line was 19.2 + 0.54x.
Relationship between threshold and type of response The 61 responses to ipsilaterat stimuli and 39 responses to contralateral stimuli were pooled. Of these 100 responses, 96 could be classified as having either switch-like or proportional responses (Fig. 2). A switch response was defined as one which attained more than two-thirds of its final discharge rate within 1.5 °C above threshold. These two classes were then subdivided according to whether the threshold of the response threshold lay above or below 42 °C (Table II). When these subclasses were compared using the Z2 procedure, the null hypothesis that there was no relationship between response type and threshold was rejected at the P < 0.005 level. Thus, responses with thresholds less than 42 °C tended to be of the proportional kind, whereas those with thresholds greater than 42 °C tended to be switch-like. DISCUSSION The units observed in this study were located in the medial pontomeduUary reticular formation; in the n.r. pontis caudalis and n. r. gigantocellularis. The somatosensory responses and receptive field characteristics of these units were qualitatively similar to those obtained in this region by earlier workers 3A3AS-17. Consequently, their membership of the P M R F is confirmed on neuroanatomical and functional grounds. Our data demonstrate that 90% of P M R F neurons which respond to mechanical and/or noxious peripheral stimuli also respond to thermal stimuli applied to the glabrous skin on the ventral surface of the rat hindpaw. Moreover, of all the thermal responsive neurons tested bilaterally, 90% had thermal receptive fields which included both hindpaws. This large fraction of P M R F neurons with c o m m o n receptive field components is consistent with the receptive field redundancy noted in the P M R F with respect to other somatosensory input 15. For example, some part of the hindlimbs contributed to the mechanical receptive field of every neuron in this series. The polysensory response and redundant receptive field characteristics of P M R F neurons suggest that the P M R F is organized so as to be sensitive to permutations of the available sensory input. Only 10% of the P M R F neurons observed in this study were unaffected by the thermal stimuli era-
113 ployed. However, it is possible that even these neurons were thermally responsive but not adequately stimulated. The thresholds of these neurons might have been above the maximum stimulus temperature which was restricted to below 48 °C in order to avoid tissue damage. Alternatively, their thermal receptive fields might have excluded the hindpaws. The extensive thermal responsiveness of PMRF neurons reported here contrasts with the findings of Casey 5 who was unable to elicit thermal responses from cat medial bulboreticular units with radiant heating of the skin to 50-60 °C. Thermally responsive neurons have been detected in regions of the brainstem reticular formation more caudal 4 and more rostral 7 to that investigated in the current study. However, the thermal response characteristics of each of those groups of neurons were considerably different from the characteristics of the PMRF neurons reported here. Burton 4 observed units in cat caudal bulbar reticular formation (field F'I'L of Berman2) which responded to noxious mechanical stimuli applied in the facial region. About 65 % of those neurons also responded to thermal stimuli. Their thresholds were only in the noxious range (43 to 62 °C). Eickhoff et al. 7 detected units in the medial mesencephalon and rostral pons of rats that responded to innocuous and/or noxious mechanical stimulation. About half of those also responded only to noxious levels of radiant heat (thresholds greater than 42 °C). Both of the above reports contrast with the high percentage of thermal responsiveness and the lower range of thresholds (36.5 to 46 °C, Fig. 3) observed in the PMRF units in this study. The major sources of error in the estimation of the thermal thresholds of the PMRF neurons are due to possible differences between the temperature of the stimulator surface and the effective temperature at the receptors. It is not practical to quantify the thermal resistive-capacitive properties of the stimulator-skin-receptor complex. However, for stimuli of temperatures greater than approximately core temperature, the series resistive elements between stimulus and receptor are expected to result in the stimulator temperature being an overestimate of the effective receptor temperature. The temperature lag due to thermal equilibration effects is also expected to contribute to overestimation of the thresholds. The time constant for this
equilibration in the footpad of the cat is about 1 s (ref. 1). Thus, for the case of a positive ramp of 0.15 °C/s, the PMRF thresholds are likely to have been overestimated by a temperature difference attributable to a latency of about 3 time-constants. Thus, the error is approximately 0.45 °C. Both sources of error contribute to an overestimation of the thresholds, and so the distribution presented in Fig. 3 is expected to be displaced towards high-. er temperatures than the effective value at the receptors. However, from an operational point of view it is the surface temperature of an object (or stimulus) which is relevant to the evaluation of an animal's behavior. Therefore, either the stimulator temperature or the skin surface temperature is the appropriate measure for evaluating PMRF responses. The slow rise-time of the ramp (0.15 °C/s), the high thermal conductivity of the aluminum stimulator and the capacity of the heat source suggest that the temperature of the stimulator surface was a good estimate of the temperature of the surface of the skin applied to the stimulator. This was confirmed by the fact that a reference thermistor interposed between the experimenter's finger and the stimulator tracked the stimulus temperature exactly. The data presented here are all derived from single trial threshold estimation. In a separate series of experiments (Farham and Douglas, unpublished data) we have demonstrated that the PMRF thermal thresholds are remarkably constant over time. In these experiments, 22 ipsilateral neurons were tested with two ramp stimuli (0.15 °C/s) separated by between 8 and 21 min. There was no significant difference between the thresholds of the first and second trials (mean difference -0.14 + 0.67 °C S.E.M., paired t = 0.029, P = 0.977). More than half of the PMRF units had thermal thresholds below 42 °C (Fig. 3) and were therefore not primarily activated by noxious thermal stimuli. The input to these neurons might be derived indirectly from warm receptors or from receptors responding differentially to warm and noxious thermal stimuli. The distribution of the thermal thresholds of PMRF neurons is bimodal (Fig. 3), and therefore it is likely that the PMRF itself contains two populations of thermally responsive neurons. Since the two peaks of the distribution have threshold ranges (36.5 to 42 °C and 42 to 46 °C) that conform to the maximum
114 discharge ranges for warm and noxious peripheral thermoreceptorsll, ~2, it is possible that thermal input converging onto a given PMRF neuron is restricted to that derived from either of those two receptor types. Sixty-four percent of the responses with thresholds below 42 °C were characterized by discharges which increased or decreased monotonically with increasing stimulus temperature (Table II). This monotonic relationship between discharge and the intensity of the thermal stimulus provides support for indirect warm receptor input to PMRF neurons 6. However, it is not known whether warm receptors are present in the glabrous skin of the rat hindpaw. Zimmermann and Handwerker18 were unable to find warm receptors in footpads of cats, but Lamotte and Campbell 12 have observed these receptors in the glabrous skin of monkey hand. On the other hand, the thermal input to the PMRF neurons may be derived from a single population of nociceptors with a wide range of temperature thresholds. Lynn and Carpenter TM reported that C-fiber polymodal nociceptors located in the hairy skin on the dorsal and medial aspects of the rat hindpaw responded to thermal stimuli between 36 and 59 °C. The distribution of the thresholds was unimodal, with a mean of 47 + 5.8 °C. While most of these receptors had thermal thresholds in the noxious range, about 20% had thresholds below 42 °C and so provided thermal input in the warm range. If a similar population of polymodal nociceptors is present in the glabrous skin of the rat hindpaw, then the thermal input from these receptors would be sufficient to explain both the low temperature thresholds observed in many PMRF units, as well as those in the noxious range. Since the temperature thresholds of polymodal nociceptors are unimodal, the bimodal distribution of PMRF thresholds would be due to selective convergence of input in either the warm or the noxious ranges. Whatever the receptor types providing the thermal input, the correlation of ipsilateral and contralateral thermal thresholds of PMRF neurons (Fig. 5) is evidence for selective convergence at either the spinal or brainstem level. The selectivity appears to be restricted to thermal responsiveness, since there was no correlation between a PMRF neuron's response to innocuous or noxious mechanical stimulation and its response to innocuous or noxious thermal stimula-
tion (Table I). However, this lack of correlation could be a property derived from the receptor characteristics, since a similar lack of correlation between the thermal and mechanical thresholds was noted by Lynn and Carpenter t4 in the C-fiber polymodal nociceptors. Most of the PMRF units had bilateral thermal receptive fields. This spatial convergence could also occur at either the spinal or brainstem level. Fields et al. s,9 reported that although there was considerable convergence of somatosensory input at the level of the spinoreticular neurons, the majority of those neurons had ipsilateral receptive fields. The authors did not examine the thermal responses of the spinoreticular neurons. However, if it is supposed that thermal input is likely to be handled in a manner similar to other somatosensory input, then the large fraction of bilateral PMRF thermal receptive fields observed in this study was probably due to convergence at the PMRF rather than at the spinal level. The spatial convergence of somatosensory input at the PMRF level is implied by the bilaterality of the spinoreticular projections: about two-thirds of spinoreticular neurons project to the ipsilateral nucleus gigantocellularis, while the remainder project to the contralateral n. gigantocellularis 9. The functional role of the PMRF neurons is ambiguous. The activity of these neurons might be related to sensory processing, arousal, reflex integration or motor control 10. Since the majority of these neurons had thermal receptive fields which included at least both hindpaws in each case, their role in discriminating stimulus location cannot be significant. Moreover, half of the PMRF neurons exhibited abrupt 'switching' responses (Table II) which suggest that at least those neurons are concerned with the detection of the stimulus rather than with resolving its intensity. Thus, the thermal responses of these neurons are likely to be relevant in an integrative context rather than one of sensory discrimination. ACKNOWLEDGEMENTS The authors gratefully acknowledge the electronic designs and technical assistance of Jan Pepler. This research was supported by the South African Medical Research Council.
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