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Neurons in the rostral mesencephalic reticular formation of the cat responding specifically to noxious mechanical stimulation
EXPERIMENTAL
Neurons
NEUROLOGY
(1976)
51,628-636
in the Rostra1 Mesencephalic Reticular Formation of the Cat Responding Specifically to Noxious Mechanical Stimulation l D. W.
YOUNG
Max-Ptanck-lnstitut
AND
fiir
K.-M.
GOTTSCHALDT
Biophysikatische Chemie, Am West Germany
FaPberg
2,
34 Giittingen, Received
December
11,1975;
revision
received
March
2,1976
In a microelectrode study, we investigated response and discharge properties of neurons which were activated exclusively by noxious mechanical ‘stimuli applied to wide aneas of the body surface. Both excitatory and inhibitory response patterns were observed. Histological controls revealed the location of these neurons in the ventromdial reticular formation of the midbrain. 14 is suggested that these ,resqonses were recorded from morphologically distinct neural elements and that they are specifically involved in central pain mechanisms.
INTRODUCTION It is now well established that the reticular formation of the brain stem receives afferent input from heterogeneous sensory sources. Recording single neuron activity, several workers (2, 6-8, 29) have described responses to stimuli of different sensory modalities in individual neurons. Using stimulation of cutaneous receptors or peripheral nerves, particular attention has been paid to responsesevoked in the reticular formation of the medulla (7, 11-13, 19, 23, 29, 30), of the midbrain (3, 4, 8, 14, 15, 25), and of the diencephalon (1, 17, 22). There is general agreement among these various studies that neurons in the reticular formation which respond to somatic stimuli have large peripheral receptive fields, often bilaterally located, with a high proportion responding to both nonnoxious and noxious stimuli. However, it seemsthat there is less clarity about the existence and proportion of reticular units responding exclusively to noxious natural 1 This work was supported by the Sonderforschungsbereich 33 of the Deutsche Forschungsgemeinschaft. We thank Dr. D. Bowsher for hi,s valuable comments on this study. 628 Copyright All rights
1916 by Academic Press, IllC. o9 reproduction in any form reserved.
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stimulation as reported by some authors (3, 11, 12, 23). The present paper describes some physiological and morphological observations of neurons in the rostra1 mesencephalic reticular formation responding specifically to noxious mechanical stimulation in the periphery. METHODS The data were obtained from 12 cats weighing 2.4 + 0.3 kg. Intravenous infusion of Nembutal was used for anesthesia. In two nonoperated animals the dosage for achieving and maintaining a stable level of anesthesia was determined. Narcosis was initiated by ethylchloride and ether inhalation followed by an intravenous injection of 12 to 18 mg/kg Nembutal. This provided a degree of anesthesia at which the animals never showed signs of discomfort either spontaneously or after noxious mechanical stimulation. By a continuous infusion of 3 mg/kg/hr of Nembutal, the level of the anesthesia was maintained and appeared to remain stable during an experimental period of no longer than 16 hr, resulting in a cumulative dose of 50 to 60 mg/kg of Nembutal, depending on the length of the experiment. In all experiments, blood pressure, measured directly through an intraarterial cannula, was at least 140 mm Hg. End-tidal COz concentration was required to be 3 to 4% ; and in four experiments, where breathing was unsatisfactory, artificial respiration was induced following immobilization with gallamine triethiodide. In two of the immobolized animals prostigmine was given at the end of the experiment. In both cases we did not observe spontaneous movements or other signs of discomfort in the animal, with or without the application of noxious stimuli, after spontaneous breathing had been resumed. Body temperature was maintained between 36 and 38°C. The animals were placed in a standard Horsley-Clarke stereotaxic frame and craniotomies were performed on the left parietal skull at coordinates AP : 0 to + 10, ML : 0.5 to 8 mm. After removal of the dura the craniotomy was closed by 37% agar jelly. Nonnoxious mechanical stimuli were considered to be light touch by a fine probe, moderate pressure, brushing of hairs, and joint movements. Noxious stimuli, all distinctly painful when applied to the experimenters, were strong squeezing of skin or muscles, pinching with forceps, or contact with a water-heated brass rod. Crude visual (light on and off) and acoustic stimuli (claps and taps to the ear bars of the head holder) were also used. Electrical stimuli were delivered through a bipolar stainlesssteel electrode inserted into the skin. For recording, fine, glass-insulated and electrolytically sharpened tungsten microelectrodes were used, with a tip diameter of 1 to 2 pm. The tungsten wire was exposed enough to yield impedances of only 0.5 to 1 MQ.
630
YOUNG
AND
GOTTSCHALDT
With such electrodes the dorsoventral and mediolateral extent of the left midbrain was systematically explored at different rostrocaudal levels. In four experiments insl-X-coated steel electrodes were used and recording points were marked using the method of Green (20). The animals were perfused with 4% formaldehyde containing 1% potassium ferrocyanide. The midbrain was cut on a freezing microtome and 40-pm sections were stained with cresyl violet. Individual sections were compared with a stereotaxic atlas of the cat’s brain stem (5) to determine locations of recording sites. RESULTS Both cell and fiber multiunit discharges were encountered in the midbrain using the fine-tip, low-impedance electrodes described. The electrodes were placed stereotaxically in the coronal plane at different rostrocaudal levels forming a grid of several hundred recording positions spaced 0.25 or 0.5 mm apart. During such mapping procedures discharges in 43 neurons were observed which differed in several respects from the activity recorded at other positions in a given penetration with the same electrode. These
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FIG. 1 (a to f) : Response patternsof six different neurons after noxious mechanical stimuli. Time course of the stimuli indicated by bars below the responses. Thin lines represent time bases of I sec. (g) spontaneous activity, (h) and (i) responses of one neuron to noxious stimuli at two different sites. Time base: 400 msec. (k to n) responses to single elecniqal @ipplj at various sitesof the body. Time base: 10 msec.
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units were characterized by an unusually large spike amplitude and excellent isolation from activity in other neural elements (Fig. 1). Often the same neuron could be held over distances of up to 400 pm and, typically, the same neuron could be encountered again after the electrode was advanced for several millimeters and retracted to the original recording position. However, only rarely was a neuron lost accompanied by cellular injury discharges. All except one unit showed continuous spontaneous activity at rates of between 3 and 15 impulses/set. None of the units changed its spontaneous discharge rate when nonnoxious, tactile, visual, or acoustic stimuli were applied but all of them displayed clear responses to mechanical stimuli which we would consider to be painful in the awake animal. A notable characteristic of all these neurons was the marked tonicity of the responses during maintained noxious stimulation and in all cases strong squeezing was the most effective stimulus, while pinching with forceps or noxious heat had little or no effect. All 43 neurons exhibited a large convergent input in having receptive fields on both sides of the body, often on all four limbs, the trunk, tail, and face (37 out of 43 units = 86%). Five units (11.6%) responded to noxious stimulation of the face and forelimbs only and one unit to stimulation of the contralateral hindlimb only (the other hindlimb was rendered progressively moribund by the indwelling arterial catheter). Irrespective of the amount of convergence, different response patterns could be distinguished, ranging from simple excitation to simple inhibition, as demonstrated in Figs. la-f. Simple excitation (Fig. la) was seen in 23 neurons and contrasted to simple inhibition of the spontaneous activity (Fig. Id) seen in seven units. Similarly, responses in four units, showing excitation during the stimulus and a subsequent inhibitory or silent period (Fig. lb), could be contrasted to discharge patterns in seven units with inhibition during the stimulus and a subsequent excitation (Figs. le, f). More complex response patterns were also seen (Fig. lc) with an initial inhibitory period followed by an increasing frequency of discharge during stimulus application and a marked “off)’ excitation after stimulus removal. Both excitatory and inhibitory responses occurred in individual experiments and in the record shown in Fig. If the spontaneous activity of one neuron was inhibited during a noxious mechanical stimulus while another neuron was excited. The response pattern of a given unit was always constant and independent of the site of stimulation. The records of Figs. lg-i illustrate an almost equal excitatory response to stimuli at two different sites. In other units slight quantitative differences were seen in the responses to stimuli given at various loci and, in general, more rostra1 parts of the body were more effective than caudal parts.
632
YOUNG
AND
GOTTSCHALDT
FIG. 2 (A to E) : Drawings made from histological sections indicating the loeation of five lesion points in the midbrain tegmentum at different ,rostrocaudal levels. (A, B, C) and (D, E) from one experiment, respectively. (F) : reproduction of a mapping grid in the midbrain (Horsley-Clarke (plane +6) indicating the relative location of recording points of single neurons responding specifically to noxious stimuli (stars), and of multiunit activity evoked by nonnoxious tactile :stimuli (circles). All recordings were made with the same electrode. Abbreviations: MC&medial geniculate body, SC-superior colliculus, SN-substantia nigra, R-red nucleus, PP-pes pedunculi, Py-pyramidal tract, Pi-pineal body, Po-posterior complex of the thalamus, PAG-periaqueductal gray, CTF-central tegmental field, and 3Noculomotor nerve.
Although no quantitative analysis was made, in most units it was evident that the responseswere proportional to the stimulus intensity. In inhibitory responses, transitions from slight reductions of the spontaneous activity to complete cessation were observed with increasing intensity of the noxious stimulus ; similarly, graded responses were observed in neurons showing excitation. In addition, simultaneous noxious stimulation of two or three limbs increased the response of an individual unit, suggesting extensive spatial summation. Latencies to electrical stimuli varied between 8 and 30 msec, depending in individual neurons also on the site of stimulation. In Figs. lk-n the shortest latency response was elicited by stimulation of the tip of the tongue and the longest latency by stimulation of the contralateral forepaw. In the latter caseit was not quite clear whether the small spikes preceding
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the large action potentials were not afferent impulses which failed to trigger the discharge in the recorded neuron. In units inhibited by noxious stimulation, electrical stimulation caused a cessation of the spontaneous activity lasting up to 100 msec after a single stimulus. According to stereotaxic measurements, all neurons were found in a region extending mediolaterally from 1.3 to 4.0 mm and rostrocaudally from AP +6.5 to +2 mm. At levels further rostra1 no responses of the kind described for the anterior midbrain were found. If in a single experiment several “pain” neurons were recorded in a given coronal plane, they tended to occur at almost the same depths of penetration measured from the surface of the skull (stars in Fig. 2f) and only rarely was more than one “pain” neuron encountered in a single dorsoventral penetration. In two experiments the recording positions were marked electrolytically and located histologically. Figures 2a-e indicate the location of five lesion points in different rostrocaudal planes of the midbrain in two cats. All units were contained within the central tegmental field, an imprecisely defined region of the mesencephalic reticular formation which is delineated dorsomedially by the periaqueductal grey and ventrolaterally by the red nucleus and the substania nigra. DISCUSSION The histological findings as well as the results of systematic mapping indicate that throughout the rostrocaudal extent of the midbrain reticular formation there may exist a sheet of neurons which are specifically activated by noxious stimuli. It has been reported (2, 27) that reticular neurons are particularly sensitive to pentobarbital anesthesia. It is possible that the specificity of our neurons was mimicked by the exclusion of nonnoxious inputs to the neurons in question. However, the anesthetic drug did not suppress the input to neighboring neurons which responded to nonnoxious somatosensory stimuli (open circles in Fig. 2f) and neither was the great convergence onto these cells reduced. The occurrence of long tonic responses and their proportionality to stimulus intensity suggests that the system was not significantly affeoted, in a qualitative sense, by the anesthesia and the results did not differ whether or not the animals were immobilized. Previous authors (3, 8) reported neurons in the mesencephalic reticular formation responding to both noxious and nonnoxious stimuli. With the recording techniques used in our experiments such neurons were not observed as single unit discharges. Therefore, we suggest that the unit activity which we did observe with our electrodes was derived from neurons having different properties. Physiologically these neurons responded to modality but not place-specific stimuli of a noxious character. It might be possible that in some cases we were recording from fibers
634
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of passage ascending from the nucleus gigantocellularis of the medulla towards the medial diencephalon (7). On the other hand, the large, well isolated impulses, the remarkable recording stability, and the reaquisition of seemingly the same neuron after movements of the electrode for 2 or 3 mm is not typical of fiber recordings, especially with a low impedance electrode. Also, recording with the same electrode in the medial lemniscus, fiber responses invariably consisted of small-amplitude, multiunit activity. Consequently, we postulate that the responses were obtained from neurons in the centra1 tegmental field which have a dense and extensive dendritic tree or terminal axonal arborization from whitin which the impulses were recorded. Large cells with several long dendrites were seen in the neighborhood of lesion points in our histologic material and elsewhere in the reticular formation by other investigators (9, 28, 29, 31). The comparatively short latency responses to electrical stimulation suggest that natural stimuli activated nociceptors with myelinated fibers, as described by Burgess and Per1 (10) and Per1 (26). The afferent input to the recorded neurons in the midbrain is probably transmitted via the anterolateral spinal pathways. Anatomical and electrophysiological studies have demonstrated projections from these pathways to the midbrain tegmentum (12, 21, 24, 25) and previous physiological and behavioral work has already implicated the mesencephalic reticular formation in pain mechanisms (3, 15, 16, 18, 19). Our results suggest that the action of these pain mechanisms may involve activity in a morphologically and physioIogically distinct population of mesencephalic neurons which respond specifically, and in a quantitatively related manner to noxious, natural stimuli. The similarity of our observations to those reported by Casey (12) and Le Blanc and Gatipon (23) in the bulboreticular formation and by Becker et al. (3) in the caudal mesencephalon indicates that a neuronal system activated primarily by noxious stimuli extends from the medulla rostrally at least up to the mesodiencephalic junction. REFERENCES 1. ALB~FESSARD, D., and L. KRUGER. 1962. Duality of unit discharges from cat centrum medianum in response to n,atural and electrical stimulation. J. Newophysiol. 25 : l-20. 2. AMASSIAN, V. E., and R. V. DEVITO. 1954. Unit activity in reticular formation and nearby structures. I. Neurophysiol. 17: 575-603. 3. BECKER, D. P., H. GLUCK, F. E. NULSEN, and J. A. JANE. 1969. An inquiry into the neurophysiological basis for pain. J. Neurosurg. 30: 1-13. 4. BELL, C., G. SIERRA, N. BUENDIA, and J. P. SEGUNDO. 1964. Sensory properties of neurons in the mesencophalic reticular formation. J. Newophysiol. 27: 961-987. 5. BERMAN, A. L. 1968. “The Brain Stem of the Cat.” The University of Wisconsin Press, Madison.
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6. BOWSHER, D. 1970. Place and modality analysis in caudal reticular formation. J. Physiol. (Lordon) 209: 473486. 7. BOWSHER, D., A. MALLART, D. PETIT, and D. ALB~FESSARD. 1968. A bulbar relay to the centre median. .I. Nez~roplzysiol. 31: 288-300. 8. BOWSHER, D., and D. PETIT. 1970. Pl,ace and modality analysis in nucleus of posterior comissure. J. Physiol. (Lo~do~t) 206 : 663-675. 9. BOWSHER, D., and J. WESTMAN. 1971. Ultrastructural characteristics of the caudal and rostra1 brain stem reti’cular formation. Brain Rcs. 28: 443-457. 10. BURGESS, P. R., and E. R. PERL. 1967. Myelinated afferent fibr,es responding specifically to noxious stimulation of the skin. J. Physiol. (Lopzdon) 190 : 541-562. 11. BURTON, H. 1968. Somatic sensory properties of caudal bulbar reticular neurons in the cat (Felis domestica). Brake Rcs. 11 : 357-372. 12. CASEY, K. L. 1969. Somatic stimuli, spinal pathways, and size of cutaneous fibres influencing unit activity in the medial medullary reticular formation. Exp. Neural. 25 : 35-56. 13. CASEY, K. L. 1971. Somatosensory responses of bulboreticular units in awake cat: relation to escapeqoducing stimuli. Scie~rce 173 : 77-80. 14. COLLINS, W. E., and J. L. O’LEARY. 1954. Study of ,a somatic evoked response of midbrain reticular substance. Elcctrorwcph. Ch. Ncnrophysiol. 6 : 619-628. 15. COLLINS, W. F., and C. T. RANDT. 1960. Midbrain evoked responses relating to peripheral unmyelinated or ‘C’ fibers in cat. J. NczLrophysiol. 23: 47-53. 16. DELGADO, J. M. R. 1955. Cerebral structures involved in transmission and elaboration of noxious stimulation. Z. N~l~~o~Zqsiol. 18: 261-275. 17. FELTZ, P., G. KRAUTHAMER, and D. ALB&FESSARD. 1967. Neurons of th,e medial diencephalon. I. Somatosensory responses and caudate inhibition. J. Ncrtropkysiol. 30 : 55-80. 18. FIELDS, H. L., G. M. WAGNER, and S. D. ANDERSON. 1975. Some properties of spinal neurons projecting to the medial brainstem reticular formation. Exp. NeuroZ. 47 : 118-134. 19. FULLER, J. H. 1975. Brain stem reticular units: Some properties of the course and origin of the ascending trajectory. Braitt Rcs. 83: 349-367. 20. GREEN, J. W. 1958. A simple microelectrode for recording from the central nervous system. Nature (Loxdolz) 182 : 962. 21. KERR, F. W. L. 1975. The ventral .spinothalamic tract and other ascending systems of the ventral funiculus of the spinal cord. .Z. Co~itp. Nczrvo/. 159: 335-356. 22. KRUGER, L., and D. ALBB-FESSARD. 1960. Distribution of responses to somatic afferent stimuli in the diencephalon of the cat under chloralose anesthesia. Exp. Ntwrol. 2 : 442-467. 23. LEBLANC, H. J., and G. B. GATIPON. 1974. Medial bulboreticular res,ponse to peri’pherally applied noxious stimuli. Exp. Nezwol. 42: 264273. 24. MEHLER, W. R., M. E. FEFERMAN, and W. J. H. NAUTA. 1960. Ascending axon degeneration following anterolateral cordotomy. An experimental study in the monkey. Brain 83 : 718-750. 25. MORIN, F. 1953. Afferent projections to the midbrain tegmentum and their spinal course. Awtcr. J. Physiol. 12 : 85-99. 26. PERL, E. R. 1968. Myelinated afferent fibers innervating the prim,ate skin and their response to noxious stimuli. Z. PltysioE. (Lorzdo?l) 197: 593-615. 27. SCHEIBEL, M. E., and A. B. SCHISIBEL. 1965. The response of reticular units to repetitive stimuli. Arch. Ital. Biol. 103 : 279-299.
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M. E., and A. B. SCHEIBEL. 1965. Periodic sensory nonr’esponsiveness in reticular neurons. A&z. Ital. Biol. 103 : 300-316. 29. SCHEIBEL, M. E., A. B. SCHEIBEL, A. MOLLICA, and G. MORUZZI. 1955. Convergence and interaction of afferent im~pulses on #single units of reticular formation. 1. Neurophysiol. 18 : 309-331. 30. SEGUNDO, J. P., T. TAKENAKA, and H. ENCABO. 1967. Somatic sensory properties of bulbar reticular neurons. J. Neurophysiol. 30: 1221-1238. 31. WESTMAN, J., and D. BOWSHER. 1971. Fine structure of the centre-m8edian-parafascicular complex in the cat. Brain Res. 30: 331337.
28. SCHEIBEL,
Neurons
NEUROLOGY
(1976)
51,628-636
in the Rostra1 Mesencephalic Reticular Formation of the Cat Responding Specifically to Noxious Mechanical Stimulation l D. W.
YOUNG
Max-Ptanck-lnstitut
AND
fiir
K.-M.
GOTTSCHALDT
Biophysikatische Chemie, Am West Germany
FaPberg
2,
34 Giittingen, Received
December
11,1975;
revision
received
March
2,1976
In a microelectrode study, we investigated response and discharge properties of neurons which were activated exclusively by noxious mechanical ‘stimuli applied to wide aneas of the body surface. Both excitatory and inhibitory response patterns were observed. Histological controls revealed the location of these neurons in the ventromdial reticular formation of the midbrain. 14 is suggested that these ,resqonses were recorded from morphologically distinct neural elements and that they are specifically involved in central pain mechanisms.
INTRODUCTION It is now well established that the reticular formation of the brain stem receives afferent input from heterogeneous sensory sources. Recording single neuron activity, several workers (2, 6-8, 29) have described responses to stimuli of different sensory modalities in individual neurons. Using stimulation of cutaneous receptors or peripheral nerves, particular attention has been paid to responsesevoked in the reticular formation of the medulla (7, 11-13, 19, 23, 29, 30), of the midbrain (3, 4, 8, 14, 15, 25), and of the diencephalon (1, 17, 22). There is general agreement among these various studies that neurons in the reticular formation which respond to somatic stimuli have large peripheral receptive fields, often bilaterally located, with a high proportion responding to both nonnoxious and noxious stimuli. However, it seemsthat there is less clarity about the existence and proportion of reticular units responding exclusively to noxious natural 1 This work was supported by the Sonderforschungsbereich 33 of the Deutsche Forschungsgemeinschaft. We thank Dr. D. Bowsher for hi,s valuable comments on this study. 628 Copyright All rights
1916 by Academic Press, IllC. o9 reproduction in any form reserved.
MESENCEPHALIC
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stimulation as reported by some authors (3, 11, 12, 23). The present paper describes some physiological and morphological observations of neurons in the rostra1 mesencephalic reticular formation responding specifically to noxious mechanical stimulation in the periphery. METHODS The data were obtained from 12 cats weighing 2.4 + 0.3 kg. Intravenous infusion of Nembutal was used for anesthesia. In two nonoperated animals the dosage for achieving and maintaining a stable level of anesthesia was determined. Narcosis was initiated by ethylchloride and ether inhalation followed by an intravenous injection of 12 to 18 mg/kg Nembutal. This provided a degree of anesthesia at which the animals never showed signs of discomfort either spontaneously or after noxious mechanical stimulation. By a continuous infusion of 3 mg/kg/hr of Nembutal, the level of the anesthesia was maintained and appeared to remain stable during an experimental period of no longer than 16 hr, resulting in a cumulative dose of 50 to 60 mg/kg of Nembutal, depending on the length of the experiment. In all experiments, blood pressure, measured directly through an intraarterial cannula, was at least 140 mm Hg. End-tidal COz concentration was required to be 3 to 4% ; and in four experiments, where breathing was unsatisfactory, artificial respiration was induced following immobilization with gallamine triethiodide. In two of the immobolized animals prostigmine was given at the end of the experiment. In both cases we did not observe spontaneous movements or other signs of discomfort in the animal, with or without the application of noxious stimuli, after spontaneous breathing had been resumed. Body temperature was maintained between 36 and 38°C. The animals were placed in a standard Horsley-Clarke stereotaxic frame and craniotomies were performed on the left parietal skull at coordinates AP : 0 to + 10, ML : 0.5 to 8 mm. After removal of the dura the craniotomy was closed by 37% agar jelly. Nonnoxious mechanical stimuli were considered to be light touch by a fine probe, moderate pressure, brushing of hairs, and joint movements. Noxious stimuli, all distinctly painful when applied to the experimenters, were strong squeezing of skin or muscles, pinching with forceps, or contact with a water-heated brass rod. Crude visual (light on and off) and acoustic stimuli (claps and taps to the ear bars of the head holder) were also used. Electrical stimuli were delivered through a bipolar stainlesssteel electrode inserted into the skin. For recording, fine, glass-insulated and electrolytically sharpened tungsten microelectrodes were used, with a tip diameter of 1 to 2 pm. The tungsten wire was exposed enough to yield impedances of only 0.5 to 1 MQ.
630
YOUNG
AND
GOTTSCHALDT
With such electrodes the dorsoventral and mediolateral extent of the left midbrain was systematically explored at different rostrocaudal levels. In four experiments insl-X-coated steel electrodes were used and recording points were marked using the method of Green (20). The animals were perfused with 4% formaldehyde containing 1% potassium ferrocyanide. The midbrain was cut on a freezing microtome and 40-pm sections were stained with cresyl violet. Individual sections were compared with a stereotaxic atlas of the cat’s brain stem (5) to determine locations of recording sites. RESULTS Both cell and fiber multiunit discharges were encountered in the midbrain using the fine-tip, low-impedance electrodes described. The electrodes were placed stereotaxically in the coronal plane at different rostrocaudal levels forming a grid of several hundred recording positions spaced 0.25 or 0.5 mm apart. During such mapping procedures discharges in 43 neurons were observed which differed in several respects from the activity recorded at other positions in a given penetration with the same electrode. These
-
FIG. 1 (a to f) : Response patternsof six different neurons after noxious mechanical stimuli. Time course of the stimuli indicated by bars below the responses. Thin lines represent time bases of I sec. (g) spontaneous activity, (h) and (i) responses of one neuron to noxious stimuli at two different sites. Time base: 400 msec. (k to n) responses to single elecniqal @ipplj at various sitesof the body. Time base: 10 msec.
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units were characterized by an unusually large spike amplitude and excellent isolation from activity in other neural elements (Fig. 1). Often the same neuron could be held over distances of up to 400 pm and, typically, the same neuron could be encountered again after the electrode was advanced for several millimeters and retracted to the original recording position. However, only rarely was a neuron lost accompanied by cellular injury discharges. All except one unit showed continuous spontaneous activity at rates of between 3 and 15 impulses/set. None of the units changed its spontaneous discharge rate when nonnoxious, tactile, visual, or acoustic stimuli were applied but all of them displayed clear responses to mechanical stimuli which we would consider to be painful in the awake animal. A notable characteristic of all these neurons was the marked tonicity of the responses during maintained noxious stimulation and in all cases strong squeezing was the most effective stimulus, while pinching with forceps or noxious heat had little or no effect. All 43 neurons exhibited a large convergent input in having receptive fields on both sides of the body, often on all four limbs, the trunk, tail, and face (37 out of 43 units = 86%). Five units (11.6%) responded to noxious stimulation of the face and forelimbs only and one unit to stimulation of the contralateral hindlimb only (the other hindlimb was rendered progressively moribund by the indwelling arterial catheter). Irrespective of the amount of convergence, different response patterns could be distinguished, ranging from simple excitation to simple inhibition, as demonstrated in Figs. la-f. Simple excitation (Fig. la) was seen in 23 neurons and contrasted to simple inhibition of the spontaneous activity (Fig. Id) seen in seven units. Similarly, responses in four units, showing excitation during the stimulus and a subsequent inhibitory or silent period (Fig. lb), could be contrasted to discharge patterns in seven units with inhibition during the stimulus and a subsequent excitation (Figs. le, f). More complex response patterns were also seen (Fig. lc) with an initial inhibitory period followed by an increasing frequency of discharge during stimulus application and a marked “off)’ excitation after stimulus removal. Both excitatory and inhibitory responses occurred in individual experiments and in the record shown in Fig. If the spontaneous activity of one neuron was inhibited during a noxious mechanical stimulus while another neuron was excited. The response pattern of a given unit was always constant and independent of the site of stimulation. The records of Figs. lg-i illustrate an almost equal excitatory response to stimuli at two different sites. In other units slight quantitative differences were seen in the responses to stimuli given at various loci and, in general, more rostra1 parts of the body were more effective than caudal parts.
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FIG. 2 (A to E) : Drawings made from histological sections indicating the loeation of five lesion points in the midbrain tegmentum at different ,rostrocaudal levels. (A, B, C) and (D, E) from one experiment, respectively. (F) : reproduction of a mapping grid in the midbrain (Horsley-Clarke (plane +6) indicating the relative location of recording points of single neurons responding specifically to noxious stimuli (stars), and of multiunit activity evoked by nonnoxious tactile :stimuli (circles). All recordings were made with the same electrode. Abbreviations: MC&medial geniculate body, SC-superior colliculus, SN-substantia nigra, R-red nucleus, PP-pes pedunculi, Py-pyramidal tract, Pi-pineal body, Po-posterior complex of the thalamus, PAG-periaqueductal gray, CTF-central tegmental field, and 3Noculomotor nerve.
Although no quantitative analysis was made, in most units it was evident that the responseswere proportional to the stimulus intensity. In inhibitory responses, transitions from slight reductions of the spontaneous activity to complete cessation were observed with increasing intensity of the noxious stimulus ; similarly, graded responses were observed in neurons showing excitation. In addition, simultaneous noxious stimulation of two or three limbs increased the response of an individual unit, suggesting extensive spatial summation. Latencies to electrical stimuli varied between 8 and 30 msec, depending in individual neurons also on the site of stimulation. In Figs. lk-n the shortest latency response was elicited by stimulation of the tip of the tongue and the longest latency by stimulation of the contralateral forepaw. In the latter caseit was not quite clear whether the small spikes preceding
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the large action potentials were not afferent impulses which failed to trigger the discharge in the recorded neuron. In units inhibited by noxious stimulation, electrical stimulation caused a cessation of the spontaneous activity lasting up to 100 msec after a single stimulus. According to stereotaxic measurements, all neurons were found in a region extending mediolaterally from 1.3 to 4.0 mm and rostrocaudally from AP +6.5 to +2 mm. At levels further rostra1 no responses of the kind described for the anterior midbrain were found. If in a single experiment several “pain” neurons were recorded in a given coronal plane, they tended to occur at almost the same depths of penetration measured from the surface of the skull (stars in Fig. 2f) and only rarely was more than one “pain” neuron encountered in a single dorsoventral penetration. In two experiments the recording positions were marked electrolytically and located histologically. Figures 2a-e indicate the location of five lesion points in different rostrocaudal planes of the midbrain in two cats. All units were contained within the central tegmental field, an imprecisely defined region of the mesencephalic reticular formation which is delineated dorsomedially by the periaqueductal grey and ventrolaterally by the red nucleus and the substania nigra. DISCUSSION The histological findings as well as the results of systematic mapping indicate that throughout the rostrocaudal extent of the midbrain reticular formation there may exist a sheet of neurons which are specifically activated by noxious stimuli. It has been reported (2, 27) that reticular neurons are particularly sensitive to pentobarbital anesthesia. It is possible that the specificity of our neurons was mimicked by the exclusion of nonnoxious inputs to the neurons in question. However, the anesthetic drug did not suppress the input to neighboring neurons which responded to nonnoxious somatosensory stimuli (open circles in Fig. 2f) and neither was the great convergence onto these cells reduced. The occurrence of long tonic responses and their proportionality to stimulus intensity suggests that the system was not significantly affeoted, in a qualitative sense, by the anesthesia and the results did not differ whether or not the animals were immobilized. Previous authors (3, 8) reported neurons in the mesencephalic reticular formation responding to both noxious and nonnoxious stimuli. With the recording techniques used in our experiments such neurons were not observed as single unit discharges. Therefore, we suggest that the unit activity which we did observe with our electrodes was derived from neurons having different properties. Physiologically these neurons responded to modality but not place-specific stimuli of a noxious character. It might be possible that in some cases we were recording from fibers
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of passage ascending from the nucleus gigantocellularis of the medulla towards the medial diencephalon (7). On the other hand, the large, well isolated impulses, the remarkable recording stability, and the reaquisition of seemingly the same neuron after movements of the electrode for 2 or 3 mm is not typical of fiber recordings, especially with a low impedance electrode. Also, recording with the same electrode in the medial lemniscus, fiber responses invariably consisted of small-amplitude, multiunit activity. Consequently, we postulate that the responses were obtained from neurons in the centra1 tegmental field which have a dense and extensive dendritic tree or terminal axonal arborization from whitin which the impulses were recorded. Large cells with several long dendrites were seen in the neighborhood of lesion points in our histologic material and elsewhere in the reticular formation by other investigators (9, 28, 29, 31). The comparatively short latency responses to electrical stimulation suggest that natural stimuli activated nociceptors with myelinated fibers, as described by Burgess and Per1 (10) and Per1 (26). The afferent input to the recorded neurons in the midbrain is probably transmitted via the anterolateral spinal pathways. Anatomical and electrophysiological studies have demonstrated projections from these pathways to the midbrain tegmentum (12, 21, 24, 25) and previous physiological and behavioral work has already implicated the mesencephalic reticular formation in pain mechanisms (3, 15, 16, 18, 19). Our results suggest that the action of these pain mechanisms may involve activity in a morphologically and physioIogically distinct population of mesencephalic neurons which respond specifically, and in a quantitatively related manner to noxious, natural stimuli. The similarity of our observations to those reported by Casey (12) and Le Blanc and Gatipon (23) in the bulboreticular formation and by Becker et al. (3) in the caudal mesencephalon indicates that a neuronal system activated primarily by noxious stimuli extends from the medulla rostrally at least up to the mesodiencephalic junction. REFERENCES 1. ALB~FESSARD, D., and L. KRUGER. 1962. Duality of unit discharges from cat centrum medianum in response to n,atural and electrical stimulation. J. Newophysiol. 25 : l-20. 2. AMASSIAN, V. E., and R. V. DEVITO. 1954. Unit activity in reticular formation and nearby structures. I. Neurophysiol. 17: 575-603. 3. BECKER, D. P., H. GLUCK, F. E. NULSEN, and J. A. JANE. 1969. An inquiry into the neurophysiological basis for pain. J. Neurosurg. 30: 1-13. 4. BELL, C., G. SIERRA, N. BUENDIA, and J. P. SEGUNDO. 1964. Sensory properties of neurons in the mesencophalic reticular formation. J. Newophysiol. 27: 961-987. 5. BERMAN, A. L. 1968. “The Brain Stem of the Cat.” The University of Wisconsin Press, Madison.
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6. BOWSHER, D. 1970. Place and modality analysis in caudal reticular formation. J. Physiol. (Lordon) 209: 473486. 7. BOWSHER, D., A. MALLART, D. PETIT, and D. ALB~FESSARD. 1968. A bulbar relay to the centre median. .I. Nez~roplzysiol. 31: 288-300. 8. BOWSHER, D., and D. PETIT. 1970. Pl,ace and modality analysis in nucleus of posterior comissure. J. Physiol. (Lo~do~t) 206 : 663-675. 9. BOWSHER, D., and J. WESTMAN. 1971. Ultrastructural characteristics of the caudal and rostra1 brain stem reti’cular formation. Brain Rcs. 28: 443-457. 10. BURGESS, P. R., and E. R. PERL. 1967. Myelinated afferent fibr,es responding specifically to noxious stimulation of the skin. J. Physiol. (Lopzdon) 190 : 541-562. 11. BURTON, H. 1968. Somatic sensory properties of caudal bulbar reticular neurons in the cat (Felis domestica). Brake Rcs. 11 : 357-372. 12. CASEY, K. L. 1969. Somatic stimuli, spinal pathways, and size of cutaneous fibres influencing unit activity in the medial medullary reticular formation. Exp. Neural. 25 : 35-56. 13. CASEY, K. L. 1971. Somatosensory responses of bulboreticular units in awake cat: relation to escapeqoducing stimuli. Scie~rce 173 : 77-80. 14. COLLINS, W. E., and J. L. O’LEARY. 1954. Study of ,a somatic evoked response of midbrain reticular substance. Elcctrorwcph. Ch. Ncnrophysiol. 6 : 619-628. 15. COLLINS, W. F., and C. T. RANDT. 1960. Midbrain evoked responses relating to peripheral unmyelinated or ‘C’ fibers in cat. J. NczLrophysiol. 23: 47-53. 16. DELGADO, J. M. R. 1955. Cerebral structures involved in transmission and elaboration of noxious stimulation. Z. N~l~~o~Zqsiol. 18: 261-275. 17. FELTZ, P., G. KRAUTHAMER, and D. ALB&FESSARD. 1967. Neurons of th,e medial diencephalon. I. Somatosensory responses and caudate inhibition. J. Ncrtropkysiol. 30 : 55-80. 18. FIELDS, H. L., G. M. WAGNER, and S. D. ANDERSON. 1975. Some properties of spinal neurons projecting to the medial brainstem reticular formation. Exp. NeuroZ. 47 : 118-134. 19. FULLER, J. H. 1975. Brain stem reticular units: Some properties of the course and origin of the ascending trajectory. Braitt Rcs. 83: 349-367. 20. GREEN, J. W. 1958. A simple microelectrode for recording from the central nervous system. Nature (Loxdolz) 182 : 962. 21. KERR, F. W. L. 1975. The ventral .spinothalamic tract and other ascending systems of the ventral funiculus of the spinal cord. .Z. Co~itp. Nczrvo/. 159: 335-356. 22. KRUGER, L., and D. ALBB-FESSARD. 1960. Distribution of responses to somatic afferent stimuli in the diencephalon of the cat under chloralose anesthesia. Exp. Ntwrol. 2 : 442-467. 23. LEBLANC, H. J., and G. B. GATIPON. 1974. Medial bulboreticular res,ponse to peri’pherally applied noxious stimuli. Exp. Nezwol. 42: 264273. 24. MEHLER, W. R., M. E. FEFERMAN, and W. J. H. NAUTA. 1960. Ascending axon degeneration following anterolateral cordotomy. An experimental study in the monkey. Brain 83 : 718-750. 25. MORIN, F. 1953. Afferent projections to the midbrain tegmentum and their spinal course. Awtcr. J. Physiol. 12 : 85-99. 26. PERL, E. R. 1968. Myelinated afferent fibers innervating the prim,ate skin and their response to noxious stimuli. Z. PltysioE. (Lorzdo?l) 197: 593-615. 27. SCHEIBEL, M. E., and A. B. SCHISIBEL. 1965. The response of reticular units to repetitive stimuli. Arch. Ital. Biol. 103 : 279-299.
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M. E., and A. B. SCHEIBEL. 1965. Periodic sensory nonr’esponsiveness in reticular neurons. A&z. Ital. Biol. 103 : 300-316. 29. SCHEIBEL, M. E., A. B. SCHEIBEL, A. MOLLICA, and G. MORUZZI. 1955. Convergence and interaction of afferent im~pulses on #single units of reticular formation. 1. Neurophysiol. 18 : 309-331. 30. SEGUNDO, J. P., T. TAKENAKA, and H. ENCABO. 1967. Somatic sensory properties of bulbar reticular neurons. J. Neurophysiol. 30: 1221-1238. 31. WESTMAN, J., and D. BOWSHER. 1971. Fine structure of the centre-m8edian-parafascicular complex in the cat. Brain Res. 30: 331337.
28. SCHEIBEL,