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Response patterns to noxious and non-noxious stimuli in rostral trigeminal relay nuclei
Brain Research, 97 (1975) 47-60 ~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
47
RESPONSE PATTERNS TO NOXIOUS A N D NON-NOXIOUS STIMULI I N ROSTRAL T R I G E M I N A L RELAY N U C L E I
GHASSAN F. KHAYYAT, Y O U N G J. YU A•D ROBERT B. KING
Department of Netuvsurgery, State University of New York, Upstate Medical Center, Syracuse, N.Y. (U.S.A.) (Accepted April 7th, 1975)
SUMMARY
Poststimulus time histogram analysis of second-order neuron responses in rostral trigeminal relay nuclei of cat demonstrated characteristic firing patterns after noxious (tooth pulp) and non-noxious (tooth tap) stimuli. The response to noxious stimulation was prolonged and frequently bimodal while the response to nonnoxious stimulation was brief. The same neurons were fired by electrical stimuli applied directly to nucleus caudalis but with longer latencies suggesting a contributory role of nucleus caudalis to the characteristic prolonged bimodal response pattern to noxious stimuli. Interacting noxious and non-noxious stimuli using conditiontest sequences demonstrated further stimulus mode-related changes in firing patterns. Electrical conditioning stimuli in nucleus caudalis reduced some responses while strychnine sulfate applied into nucleus caudalis augmented the responses evoked in rostral nuclei by both noxious and non-noxious peripheral stimuli. Nucleus caudalis appeared to contain elements which may modulate activity in rostral trigeminal nuclei by either augmenting or reducing specific firing patterns of second-order neurons in rostral relay nuclei.
INTRODUCTION
The functional subdivision of the trigeminal brain stem nuclear complex into main sensory nucleus for the relay of tactile stimuli and nucleus caudalis essential for perception of pain and temperature stimuli has been based on the observation that trigeminal tractotomy at the medullary obex relieved patients of facial pain with relative preservation of touch on the ipsilateral side of the face 19,24. This observation has been interpreted as indicating direct relay of noxious stimuli at nucleus caudalis and of non-noxious stimuli primarily at the main sensory nucleus.
48 Such a model implies a dissociation of sensory events into modality specili~ relays and projection systems. A number of single unit studies in trigeminal primary relay nuclei, however, do not support an exclusive rostrocaudal segregation ofneuron~ responding to tactile and noxious stimuli s,9,1",'):3. Trigeminal neurons responding to tactile stimuli are located throughout the trigeminal nuclear complex and neurons responding to stimuli presumed to be noxious are not restricted to a specific uanse verse level in the trigeminal nuclei:~, s,.~,'-'zL Furthermore, neurons responding to motthan one stimulus modality have been identified throughout the extent of the trigeminal nuclear complex ~, ~2,~:~. Such observations are difficult to reconcile with a direct relay at nucleus caudalis as the sole prerequisite for the perception of noxious facial stimuli. To resolve this controversy some investigators ~a suggested that a painful stimulus produced 'many impulses by many cells' in the system and, therefore, isolating nucleus caudalis as by a tractotomy decreased the number of afferent fibers and thus reduced the intensity of activity leaving the primary nuclei. Denny-Brown and Yanagisawa '~ have recently suggested that there is a 'critical central threshold' of activity in the nucleus of the descending tract of the fifth nerve which must be exceeded in order to produce a conscious or reflex response in the organism. They further concluded that this central threshold is higher for pain than for other non-noxious stimuli and thus pain is a 'quantitative aspect of common sensation'. Others 14,1:~ have identified neurons in nucleus caudalis specilically responding to noxious stimuli suggesting a specilic trigeminal pain pathway dependent on relays in nucleus caudalis -~ thus eliminating the need for polymodal cells. Response pattern characteristics of trigeminal polymodal neurons to a variety of peripheral stimuli have not been described. Studies of dorsal horn cells in the spinal cord, however, have suggested at least that there is a difference in the duration of firing of neurons consequent to different peripheral stimulus modalities e'z. We, therefore, have undertaken this study of second-order neuron responses in rostral trigeminal nuclei of cat to noxious and non-noxious stimuli to determine if these polymodal neurons have specific response patterns characteristic of the stimulus modality. The role of nucleus caudalis in modulating such response patterns in rostral nuclei was also examined, since earlier physiologic studies suggested that the excitability of neural elements in rostral nuclei may be modulated by neuron influences from nucleus caudalis ~,~s.-':','-'~L
METHODS
Experiments were performed on 84 adult cats weighing between 2.6 and 3.8 kg. Tracheostomy and cannulation of the femoral artery and vein were performed under brief ether inhalation. Anesthesia was continued with intravenous alpha-chloralose (80 mg/kg). The head was fixed in a stereotaxic apparatus and respiration maintained artificially. Animals were immobilized with gallamine triethiodide (Flaxedil) 5 mg/kg every 2 h. Arterial blood pressure was continuously monitored and maintained
49 at 80-110 m m Hg. End expiratory CO2 was maintained between 5 ~o and 6~o. A heating pad maintained rectal temperature at 37-38 °C. The posterior fossa was exposed through a suboccipital craniectomy and the cerebellum and brain stem were covered with artificial CSF at 37 °C. In some animals parts of the cerebellum were removed for more direct access to the brain stem. Bipolar electrical stimulation of the dental pulp was used as a noxious stimulus. Two holes were drilled into the dentine of the proximal part of the upper canine tooth. Two 30-gauge insulated silver wire electrodes with 1 mm exposed tips were inserted into the holes and secured by dental cement with the anode positioned distally. The interelectrode resistance varied between 75 and 100 kg). Single rectangular electrical pulses (0.1 msec duration) of variable voltage were delivered to the tooth pulp by a type 2533 Devices isolated stimulator. Bipolar electrical stimulation of dental pulp has been considered to be a noxious stimulus 1,1°,1v. As a non-noxious stimulus, a light tap applied to the surface of the same tooth was delivered with a Type 102 Ling linear solenoid unit, driven by a Model A112 Wavetek waveform generator. The waveform generator was set to deliver a pulse that produced variable displacement of a 3-cm long stylet attached to the solenoid unit. The free end of this styler was positioned 0.5-1.0 mm away from the surface of the tooth so that with each displacement the tooth was tapped. The impact of this tap on the tooth, in a labiolingual or an anteroposterior direction, was measured as tooth tip displacement in #m using a mechanical transducer attached to the tooth. The same range of stimulus intensities used in cats was tried on human subjects and the only sensation they reported was light touch. Glass micropipettes filled with 3 M KCI or NaCl with 2-7 M ~ resistance were used for extracellular recordings. Brain stem areas at coordinates of 4-10 mm rostral and 2.5-5.5 mm lateral to the obex, corresponding to the territory of the main sensory nucleus and nucleus oralis of the fifth nerve s, were penetrated in a direction perpendicular to the surface of the medulla. Only neurons fulfilling all the following criteria for second-order neurons were studied: first, a response latency of 2.5 3,5 msec4,6; second, a small discrete receptive field for touch or pressure stimulil2: and third, confirmation of the electrode tip position within the area of the main sensory and oralis nuclei on serial sections of the brain stem, after perfusing animals arterially with 10 ~ formalin at the end of the experiment while the glass micropipette was still in situ. Brain stems were removed intact and laid in 10~Voformalin for several days before sections were cut and stained with PAS-gallocyanin. The stimulus threshold for each second-order neuron was determined for electrical dental pulp and light touch stimulation. Gradually increasing stimulus intensities were then used to relate the average number of spikes/10 stimuli to the strength of stimuli until the response was maximal. A stimulus intensity was then chosen midway between threshold and maximal response for both test and conditioning stimulus intensity. Ten trials at this intensity were delivered at 5-sec intervals for control prior to conditioning sequences. Further study on the same units was then conducted by delivering an electrical dental pulp or a touch stimulus (conditioning stimulus) followed by a second electrical
50 dental pulp or touch stimulus (test stimulus) at time intervals of 50--1200 msec. This conditioning-testing (C-T) technique was used for all pairings of the dental pulp and touch stimuli, i.e., touch-pulp, pulp-pulp, pulp-touch, touch-touch. In 5 experiments bipolar David K o p f SNEX-200 steel electrodes with 0.25 mm tip separation were inserted into the ipsilateral nucleus caudalis at coordinates of 3-5 mm below and 3 4 mm lateral to the obex at a depth of 1.5-2 mm for elecmcal stimulation while recording second-order neuron responses to dental pulp and to light touch in rostral trigeminal nuclei. Single rectangular pulses of 0. t msec duration and variable voltage were delivered through a Type 2533 Devices isolated stimulator to caudalis. The condition-test sequence was then examined, applying a conditiomng stimulus at nucleus caudalis followed by a light touch or a dental pulp test stimulus at C - T intervals from 50-1000 msec. In 6 experiments 0.036 ml of a 3,~; solution of strychnine sull:ate was infused into the ipsilateral nucleus caudalis via a glass micropipette over a 2-min period. The micropipette tip was located at a depth of 1.5 mm belowthe surface of the medulla, 3.5 mm lateral and 3 mm below the obex. The response of second-order neurons in rostral trigeminal nuclei was examined separately to both light touch and electrical dental pulp stimuli before and after the strychnine infusion. Single unit activity was amplified by a Model P5111 Grass AC preamplitier and cathode follower and displayed on a Tektronix Rm 565 dual beam oscilloscope. Data and stimulus control signals were recorded on magnetic tape by a Model 4700 Sangamo FM tape recorder for further analysis. Data from magnetic tape were led through an analog-digital converter unit and then ted to a computer for analysis. The computer was programmed to count the number of action potentials occurring at equal time intervals (3-msec bins) starting from the time at which the stimulus was applied. This data was printed out as a poststimulus time histogram (PSTH) accumulating the number of action potentials/unit time for the sum of 10 stimuli. RESULTS
Eighty-five neurons located at coordinates within the region of the main sensory and oralis nuclei s satisfied the criteria for second-order neurons. All units were fired by a bipolar electrical dental pulp stimulus to the ipsilateral upper canine tooth as well as light touch application to the same tooth. Other neurons activated with dental pulp stimuli at latencies of 4-8 msec were encountered with electrode penetrations in regions less than 4 m m lateral to the obex. Those neurons had large receptive fields extending widely over the face, body segments and limbs in an ipsilateral or a bilateral distribution justifying their exclusion from the neuron population under study.
(A) Patterns of response to dental pulp and light touch stimuli The threshold to dental pulp stimulation varied from one unit to another between 0.5 and 3.0 V of a 0.1 msec rectangular pulse. The threshold to touch also varied between 50 and 100 tzm of displacement of the tooth tip. At threshold, neurons
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Fig. l, Relationship of stimulus intensity to firing response of a representative neuron for both dental pulp and touch stimuli. Inserts show poststimulus time histograms of the responses at threshold and maximal stimulation intensities for both modalities. Threshold intensity fired the neuron in 50% of multiple trials. fired one or two action potentials in 50 % of multiple trials in response to either dental pulp or light touch. As the electrical dental pulp stimulus intensity was gradually increased above threshold there was an increase in the number of action potentials per stimulus and in the duration of firing which stabilized as the maximal response was reached at intensities that varied between 15 and 20 times threshold. For touch, there was an increase in response as the tooth displacement was carried above threshold. A maximal response was noted at 3-5 times threshold (Fig. 1) Unit response characteristics following dental pulp and tooth touch stimuli were then determined at stimulus intensities that gave a response midway between threshold and maximum. In all neurons studied with above threshold stimuli, the response to electrical dental pulp stimulation consisted of a significantly greater number of action potentials and a longer firing duration than the response to light touch stimulation. In 31 units where a PSTH study was available it was noted that the response to electrical dental pulp stimulation was characterized by a high frequency response in the second and fourth time bins. Beyond these bins there were differences in the appearance of the PSTH across units that justified subcategorizing unit responses into 3 types (Fig. 2). In 13 units the dental pulp P S T H showed a gradual decrease in activity per bin beyond the second and third bin (Fig. 2a). In 15 units a later rise in frequency of response per bin was noted beyond the fourth and fifth bin with a bimodal appearance of the PSTH (Fig. 2b). In 3 units a double burst response was apparent in the P S T H (Fig. 2c). In all units the corresponding PSTH for touch showed a brief burst of activity restricted to the early bins of this histogram (Fig. 2).
(B) Neuron response to electrical stimulation of nucleus caudalis In 12 second-order neurons in the rostral trigeminal nuclei with a stable latency and response to dental pulp and light touch stimulation of the upper canine tooth
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Fig. 2. Oscilloscopic tracing and poststimulus time histogram analysis of second-order neuron responses in rostral trigeminal nuclei to dental pulp and touch stimuli in 3 representative units a, b and c. Arrows in the touch response indicate the time at which the stimulus was applied. These data represent unit firing patterns obtained midway between threshold and maximal intensities. the response to bipolar electrical stimulation of the nucleus caudalis was also studied. Each of these neurons was fired by the nucleus caudalis stimulus as well as by dental pulp and light touch stimuli. Fig. 3 shows an extracellular tracing of one such unit with PSTH analysis. The caudalis stimulus threshold varied across units between 0.1 and 0.5 V of a 0.l-msec rectangular pulse. At stimulus intensities of 1.5-2 times threshold the neurons fired consistently with each stimulus at a stable latency varying between 6 and 8 msec from unit to unit.
(C) Effect of strychnine & nucleus caudalis on response patterns to dental pulp and touch stimuli in rostral nuclei In 6 second-order neurons responding to dental pulp and light touch stimulation, strychnine sulfate in a solution of 3 ~ was infused into nucleus caudalis after control stimuli were delivered. One minute after starting the strychnine sulfate injection at a rate of 0.018 ml/min there was an increase in the number of spikes and duration of activity per response to both dental pulp and touch stimulation throughout the succeeding recording period. Responses before and after strychnine are illustrated in Fig. 4. The increased unit firing occurred~almost exclusively beyond the third time bin following the stimulus.
(D) Effect of light touch conditioning on response pattern to dental pulp stimulatio~z In 25 units that were held long enough for study, the dental pulp stimulus was preceded by light touch as a conditioning stimulus at C - T intervals from 50 to 1200
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Fig. 3. Responses of a second-order neuron in rostral trigeminal nucleus to dental pulp, touch and electrical stimulation applied directly into nucleus caudalis.
msec. Ten trials of conditioning testing sequences were performed for each C - T interval. In all 25 units there was a drop in the number of action potentials per response and a shortening of the firing time. Fig. 5A shows PSTH of one representative unit when conditioned by light touch and tested with dental pulp stimuli at different C - T intervals. Maximal discharge decrement was noted at intervals of 50 100 msec. The average total number of spikes at these intervals was 37 % of the control (mean of 25 units) (Fig. 6). The drop in activity was more pronounced in the late time bins of the PSTH than in the early bins. As the C - T intervals were increased beyond 150 msec there was gradual increase in response until full recovery to control by 800 1000 msec.
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Fig. 4. A representative unit illustrating response characteristics to dental pulp and touch stimuli before and after application of 0.036 ml of 3 % solution of strychnine sulfate to nucleus caudalis. The increased unit firing occurs almost exclusively in bins beyond the first 9 msec following the stimulus.
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Fig. 5. Poststimulus time histogram analysis of neuron responses to dental pulp and touch paired as conditioning and test stimuli in different combinations at variable C-T time intervals. (A) Dental pulp-touch. (B) Dental pulp-dental pulp. ((7) Dental pulp-touch: in I only suppression of activity was noted and in II early suppression was followed by augmentation of response at longer ( T intervals. (D) Touch~ touch,
( E) Ef/'ect of dental pulp conditioning on re,sponse pattern to dental pulp stimulus On the same 25 units, the response to dental pulp stimulation conditioned by another dental pulp stimulus applied to the same tooth was studied. Fig. 5B shows a unit conditioned with dental pulp stimulation before it was fired by another dental pulp stimulus, This pairing o f stimuli resulted in a decrease o f activity of all neurons with a drop in the number o f spikes to a mean o f 60 ~o o f control at intervals o f 50 100 msec (Fig. 6). As in the touch-dental pulp pairing there was more significant decrement o f activity in the late bins o f the firing sequence with a decrease in the total duration o f firing. Full recovery o f response to control level was reached at intervals o f 800-1000 msec.
(F) Effect of dental pulp conditioning on response pattern to light touch stimulus This analysis was available on 15 units. In 10 neurons conditioned by a dental
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Fig. 6. Average number of spikes per 10 responses of neurons as percentage of control in 4 possible conditioning-testing combinations at variable intervals. Each point on the curves denotes the average of all units on which the conditioning-testing technique was applied. The insert shows a plot of a dental pulp-touch sequence with the same coordinates for the average of 5 units not included in the main curve where there was an increased response. p u l p stimulus there was only a d e c r e m e n t in response to light t o u c h in b o t h the mean n u m b e r o f spikes a n d firing time c o m p a r e d to control. This effect was m a x i m a l at 50 msec with a m e a n o f 73 % o f c o n t r o l (Fig. 6). Fig. 5C~ shows a P S T H study o f one such unit at m a x i m a l decrease in response c o m p a r e d to control. In 5 o u t o f 15 neurons in the dental p u l p - t o u c h p a i r i n g (Fig. 5CII) there was an initial decrease in total activity ( m a x i m a l at 100 msec) with a significant increase in n u m b e r o f spikes and d u r a t i o n o f firing b e y o n d 100 msec.
(G) Effect of light touch conditioning on response pattern to #ght touch stimulus In 10 units analyzed for light t o u c h response c o n d i t i o n e d by a light touch stimulus to the same t o o t h there was a significant decrease o f activity in response to the test stimulus reaching an average o f 15% o f c o n t r o l at C - T intervals o f 50-100 msec (Fig. 6) with shortening o f discharge d u r a t i o n . Typically all unit responses g r a d u a l l y a p p r o a c h e d c o n t r o l p a t t e r n as the C - T intervals were increased to 1000 msec. Fig. 5D illustrates a P S T H study o f one such unit with light t o u c h - l i g h t touch interaction.
(H) Effect of nucleus caudalis conditioning stimuli on re,sponse pattern to dental pulp and light touch stimulation The effect o f an electrical c o n d i t i o n i n g stimulus a p p l i e d to nucleus c a u d a l i s on responses o f second o r d e r n e u r o n s in the m a i n sensory a n d oralis nuclei to peripheral stimuli was d e t e r m i n e d in 5 units r e s p o n d i n g to dental p u l p a n d touch stimuli. W h e n a dental p u l p o r a t o u c h stimulus was preceded by a caudalis stimulus at intervals varying f r o m 50 to 1000 msec, a d e c r e m e n t in response was n o t e d for b o t h dental p u l p a n d t o u c h which was m a x i m a l at intervals o f 50-100 msec. G r a d u a l return to c o n t r o l response was n o t e d as the interval increased b e y o n d 100 msec. Fig. 7 illustrates this p h e n o m e n o n where dental p u l p a n d t o u c h were p r e c e d e d by a
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Fig, 7. Poststimulus time histogram analysis of neuron response to dental pulp and touch stimuli conditioned by electrical stimulation in nucleus caudalis.
caudalis conditioning stimulus. Fig. 8 relates the average number of spikes of 5 units for 10 responses compared to control at various C - T intervals. DISCUSSION
Polymodal cells have been defined as those that fire in response to a variety of stimuli. No earlier report, however, has described distinct patterns of firing specifically related to noxious and non-noxious stimuli in such cells. Our observations indicate that second order neurons in rostral trigeminal nuclei feature different firing patterns following dental pulp and touch stimuli. We suggest, therefore, that these units were not indiscriminately polymodal. Their pattern of response reflected in a characteristic way the stimulus modality. To determine the origin of the later component in the dental pulp response sequence we considered that it may be related to influences from nucleus caudalis. Earlier studies have indicated that nucleus caudalis maintined a tonic hyperpolarizing influence on primary afferent fiber preterminats in the main sensory and oralis nuclei t~, 18,g0 and that a dental pulp stimulus produced hyperpolarization of preterminals in these rostral trigeminal nuclei via ascending relays from the nucleus caudalis2'L Those studies, however, indicated that this excitability change (PAD) in the preterminals was first recorded more than 100 msec after a dental pulp stimulus. If such a mechanism enhanced synaptic transmission following a single stimulus it would not account for the second burst or the increased frequency in the late bins of the PSTH of second-order neurons which appeared within 15-20 msec after the stimulus. An alternate explanation, therefore, was sought. An ascending intranuclear trigeminal pathway from nucleus caudalis to rostral
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Fig. 8. Effects of electrical stimuli applied to nucleus caudalis as conditioning stimuli on neuron response in rostral nuclei to dental pulp or touch stimuli applied at variable time intervals. The average number of spikes per 10 responses is plotted for each time interval as a percentage of 10 control responses. Each point on the curve is a mean of 5 units. nuclei has been described 21. Activity in nucleus caudalis may be relayed to rostral nuclei by this anatomically defined ascending system. Second-order neurons in rostral nuclei that respond to both dental pulp and touch stimuli also fire after electrical stimulation of nucleus caudalis but always with a longer latency (6-8 msec). The bimodal response pattern evoked by a dental pulp stimulus may therefore represent two separate inputs converging on the same neurons: (1) a direct monosynaptic input from the dental pulp to rostral nuclei accounting for the early activity in the response sequence and (2) a delayed input via nucleus caudalis which contributes the late response that may be manifest either as a distinct burst in the late bins of the histogram or as a prolonged extension of activity into the same late bins. Such a mechanism could apply only following dental pulp stimuli since there is no suggestion of late activity following touch stimuli. Nucleus caudalis in this context may contribute to processing information about a noxious stimulus not only by direct relay of activity to more central relays but also by modulating the firing patterns of second-order neurons in rostral trigeminal relay nuclei. Previous studies have indicated that strychnine increases neuronal activity by preferentially blocking inhibitory postsynaptic potentials 2,v. Strychnine applied to nucleus caudalis has been reported to cause an increase in the tonic hyperpolarizing influence of this nucleus16, 20 on primary afferent terminals in the rostral trigeminal nuclei. Young and King 25 demonstrated that the hyperpolarizing effect on primary afferent preterminals in nucleus oralis evoked by a dental pulp stimulus was increased with topical application of strychnine over nucleus caudalis. Our results with strychnine infusion into nucleus cauda!is have indicated that the response of second-order neurons to a test stimulus in main sensory and oralis nuclei was augmented following both dental pulp and touch stimulation. In this circumstance we suggest that strychnine increased neuronal excitability of elements of nucleus caudalis that converge on second-order neurons in rostral relay nuclei producing an exaggerated response to both dental pulp and touch stimuli. The observation that a light touch stimulus in the strychnine preparation produced a prolonged high frequency response compar-
58 ed to control is of interest because psychophysical studies employing strychnine application over nucleus caudalis ~1 in cats resulted in marked overreaction to gentle stroking of the fur of the face suggesting a pain-like state as judged by adversive motor behavior. Our observations suggest that one mechanism (but not necessarily tile only mechanism) by which nucleus caudalis may contribute to this overreaction to otherwise non-noxious stimuli is by augmenting the response of second-order neurons m rostral trigeminal nuclei, Changes in excitability of primary afferent terminals in rostral trigeminal nuclei e:, show that a non-noxious vibratory stimulus applied to the face produced primary afferent depolarization {PAD) while a noxious dental pulp stimulus produced a low magnitude PAl) a t short condition test intervals of 5-100 msec and prhnary afferent hyperpolarization ( PA H) at C - T intervals of 100-300 msec ~a. Our observations of the effect of noxious and non-noxious conditioning stimuli on the response of second-order neurons imply that the activity of these neurons does not fully reflect the results one might predict from those studies. Light touch conditioning stimulation decreased the response to a test dental pulp or touch stimulus. A noxious conditioning stimulus preceding a touch test stimulus showed early decreased firing in all the units whereas late augmentation as predicted from preterminal exitability changes, occurred only in 5 units. Noxious conditioning stimuli as a rule produced an early reduction with no late increase in firing following a noxious test stimulus. -lhese observations confirm an earlier study ''G on the effects of conditioning noxious and non-noxious stimuli on the level of activity of second-order neurons. The reason for the absence of a lilcilitated response to dental pulp stimuli when the system was conditioned by dental pulp stimulation is not certain. The fact that the pretermmal excitability studies examined only primary afferent presynaptic events and not other elements impinging on the second-order neurons may be a major reason l'or this discrepancy. The reduced unit response to a test dental pulp stimulus preceded by either a touch or a dental pulp conditioning stimulus was most apparent in late bins in the PSTH sequence. Since activity in these late bins depend primarily on relays through nucleus caudalis, we suggest that each of these conditioning stimuli was primarily effective in reducing that component of the discharge pattern that was relayed via nucleus caudalis. The stimuli for this study were chosen because of psychophysical characteristics. On this basis we cannot be absolutely certain that the tooth pulp stimulus did not activate any neural elements in the periodontal membrane. We suggest nonetheless that such a contaminating mixture of afferent elements was not a major limiting feature of the data for 3 reasons which are illustrated in Figs. 5 and 6. First, a touch conditioning stimulus consistently caused more depression of the test stimuli than did a pulp conditioning stimulus. Second, the maximal effect of a conditioning stimulus noted at 50 msec in Fig. 5A decreased activity in both early and late time bins of the pulp histogram, whereas a pulp conditioning stimulus caused a predominant decrease of activity in the late bins at the same C - T interval of 50 msec (Fig. 5B). If there had been a major spread of the conditioning stimulus to the periodontal membrane we
59 would have expected at least as much depression as following touch, if the same afferent systems were being activated. Third, a touch conditioning stimulus always decreased the responsiveness to a test stimulus whereas a pulp conditioning stimulus augmented the response to touch stimuli in the population of units shown in the inset of Fig. 6. Hence, we suggest that stimulus spread from pulp stimulation to afferent fibers in the periodontal membrane was not a major determining factor in the events we have observed. The effect of conditioning stimulation of nucleus caudalis on activity in more rostral nuclei was reported earlier TM, where an increase in the excitability of rostral primary afferent terminals was noted at C - T intervals beginning at 15 msec and lasting up to 800 msec. Our present study of the effect of a caudalis conditioning stimulus on the response of second-order neurons in rostral trigeminal nuclei showed similar C - T time characteristics. The effective C-T intervals were similar whether the test stimuli were non-noxious, noxious or applied directly to nucleus caudalis. All 3 types of conditioning stimuli may therefore depend on relays in the nucleus caudalis. Earlier studies 2~ suggested the presence of interneurons in nucleus caudalis responsible for mediating PAD in rostral nuclei in response to a non-noxious vibratory stimulus to account for marked decreases in PAD observed after tractotomy at the level of the obex. Insofar as an electrical stimulus at nucleus caudalis may produce PAD but also fire second-order neurons in rostral relay nuclei, we suggest that nucleus caudalis contains neural elements which may either augment or suppress the firing characteristics of more rostral second-order neurons. Thus, under normal circumstances, nucleus caudalis contributes to, and is an essential element for, the perception oftrigeminal noxious stimuli and may also be effective in the modulation of non-noxious stimuli. Under abnormal conditions it may contribute to the conversion of a tactile stimulus to a painful experience. Our observations suggest that modality specificity of second-order neurons in rostral trigeminal nuclei may be represented by changing patterns of unit firing. Variations of interdependent firing patterns of these neurons subserving general sensibility may determine in part, at least, the discrimination and modulation of perceptions induced by a given peripheral stimulus. Some secondary projections from second-order neurons may be modality specific. To what extent these two constructs may be interdependent is yet uncertain. They need not be mutually exclusive. A CKNOWLEDGEMENTS
We wish to thank David L. Blank, Ph. D. for his assistance throughout the research project, and Miss Judie Seubert and Miss Helena Braunfeld for technical assistance. This work was supported in part by Training Grant No. 5-T01 NS-05605 from the National Institute of Neurological Diseases and Stroke, Research Grant 5-S01RR-05402 from the Division of Research Resources, U.S.P.H.S. and Special Research Biotechnology Branch Grant RR-00353, National Institutes of Health.
60 REFERENCES I ANDERSON, D. J., HANNUM, A. G., AND MATTHEWS, B., Sensory mechanisms in nmnlmaiian teeth and their supportive structures, Physiol. Rev., 50 (1970) 171-195: 2 BRADLEY,K., EASTON, D. M., AND ECCLES, J. C., An investigation of primary or direct inhibition, J. Physh~l. (Lond.), 122 (1953) 474-488. 3 DARIAN-SMJTH,1., Neural mechanisms of facial sensation, Int. Rev. Neurobiol., 9 (1966) 301 3!;)5. 4 DARIAN-SMJTH, I., AND YOKOIA, T., Corticofugal effects on different neuron types within the cat's brain stem activated by tactile stimulation of the lace, J. Neurophysiol., 29 (1966) 185-206. 5 DENNY-BROWN, D., AND YANAGISAWA,N., The function of the decending root of the fifth neJve, Brabz, 96 (1973) 783-814. 6 DUBNER, R., Interaction of peripheral and central input in the main sensory trigeminal nucteus of the cat, Exp. Nemvl., 17 (.1967) 186-202. 7 ECCLES, J. C., The Physiolog) of Synapses, Springer, Berlin, 1959, pp. 191,220-238. 8 EISENMAN, J., LANDGREN, S., AND NOVIN, D., Function organization in the main sensory trigeminal nucleus and in the rostral sub-division of the nucleus of the spinal trigeminal tract in the cat, Acta physioL, ~wand., 59, Suppl. 214 (1963) 1-44. 9 GORDON, G., LANDt;REN~ S., AND SEED, W. A., The functional characteristics of sing'.e cells in the caudal part of the spinal nucleus of the trigeminal nerve of the cat, J. Physiol. (l~md.), 158 (1961) 544-558. 10 HENG CHIN, J., ANX) [)OMJ~O, E. F., Effects of morphine on brain potentials evoked by stimulation of the tooth pulp of the dog, J. Pharmacol. exp. Ther., 132 (1961) 74-86. 11 KING, R. B., AND BARNETT, J. C., Studies of trigeminal nerve potentials. Over-reaction to tactile facial stimulation in acute laboratory preparations, J. Neurosurg., 14 (1957) 617-627. 12 KRUGER, L., AND MICHEL, F., Reinterpretation of the representation of pain based on physiological excitation of single neurons in the trigeminal sensory complex, Exp. NeuroL, 5 (1962) 157--178. 13 MELZACI<, R., AND WALL, P. D., Pain mechanisms: A new theory, Science, 150 (1965) 971 979. 14 Mosso, J. A., AND KRUGER, L., Spinal trigeminal neurons excited by noxious and thermal stimuli, Brain Research, 38 (1972) 206-210. 15 Mosso, J. A., AND KRUGER, L., Receptor categories represented in spinal trigeminal nucleus caudalis, J. NeurophysioL, 36 (1973) 472-488. 16 SCmETTA, C. J., AND KING, R. B., Hyperpolarizing influence of trigeminal nucleus caudalis on primary afferent preterminals in trigeminal nucleus oralis, J. Neurophysiol., 32 (1969) 229-238. 17 SCOTT, D., JR., The tooth as an experimental preparation for the study of pain, &led. biol. lllus., 18 (1968) 118 121. 18 SESSLE, B. J., AND GREENWOOO, F., Influence of trigeminal nucleus caudalis on the responses of cat trigeminal brain stem neurons with orofacial mechanoreceptive fields, Brain Research, 67 (1974) 330~333. 19 SJ~JQVIST, O., Studies on pain conduction in the trigeminal nerve. A contribution to the surgical treatment of facial pain, Aeta psychiat, scattd., Suppl. 17 (1938) 1-39. 20 STeWAR~, D. H., JR., SC'mETTA, C. J., AND KING, R, B., Presynaptic inhibition in the trigeminal relay nuclei, J. NeurophysioL, 30 (1967) 135-153. 21 TIWARt, R., AND KING, R. B., Fiber projections from trigeminal nucleus caudalis in primates (squirrel monkey and baboon), J. comp. Neurol., 158 (1974) 191-206. 22 WALL, P, D., AND CRONLY-I')ILt,ON, J., Pain, itch, and vibration, Arch. N~,urol. ~Chk'.), 2 (1960) 365-375. 23 WALL, P. D., AND TAtm, A., Four aspects of trigeminal nucleus and a paradox, .I. Neurophysi,~l., 25 (1962) 110--126. 24 WHITE, J. L., AND SWEET, W. H., Pain and the Neurosurgeon, Thomas, Springfield, lll., 1969, pp. 232--243. 25 YOUNG, R. lZ'., AND KING, R. B., Excitability changes in trigeminal primary afferent fibers in response to noxious and non-noxious stimuli, J. Neurophysiol., 35 (1972) 87 95, 26 Yu, Y. J., AND KING, R. B., Trigeminat main sensory nucleus polymodal unit responses to noxious and nonnoxious stimuli, Brain Research, 71 (1974) 65-71.
47
RESPONSE PATTERNS TO NOXIOUS A N D NON-NOXIOUS STIMULI I N ROSTRAL T R I G E M I N A L RELAY N U C L E I
GHASSAN F. KHAYYAT, Y O U N G J. YU A•D ROBERT B. KING
Department of Netuvsurgery, State University of New York, Upstate Medical Center, Syracuse, N.Y. (U.S.A.) (Accepted April 7th, 1975)
SUMMARY
Poststimulus time histogram analysis of second-order neuron responses in rostral trigeminal relay nuclei of cat demonstrated characteristic firing patterns after noxious (tooth pulp) and non-noxious (tooth tap) stimuli. The response to noxious stimulation was prolonged and frequently bimodal while the response to nonnoxious stimulation was brief. The same neurons were fired by electrical stimuli applied directly to nucleus caudalis but with longer latencies suggesting a contributory role of nucleus caudalis to the characteristic prolonged bimodal response pattern to noxious stimuli. Interacting noxious and non-noxious stimuli using conditiontest sequences demonstrated further stimulus mode-related changes in firing patterns. Electrical conditioning stimuli in nucleus caudalis reduced some responses while strychnine sulfate applied into nucleus caudalis augmented the responses evoked in rostral nuclei by both noxious and non-noxious peripheral stimuli. Nucleus caudalis appeared to contain elements which may modulate activity in rostral trigeminal nuclei by either augmenting or reducing specific firing patterns of second-order neurons in rostral relay nuclei.
INTRODUCTION
The functional subdivision of the trigeminal brain stem nuclear complex into main sensory nucleus for the relay of tactile stimuli and nucleus caudalis essential for perception of pain and temperature stimuli has been based on the observation that trigeminal tractotomy at the medullary obex relieved patients of facial pain with relative preservation of touch on the ipsilateral side of the face 19,24. This observation has been interpreted as indicating direct relay of noxious stimuli at nucleus caudalis and of non-noxious stimuli primarily at the main sensory nucleus.
48 Such a model implies a dissociation of sensory events into modality specili~ relays and projection systems. A number of single unit studies in trigeminal primary relay nuclei, however, do not support an exclusive rostrocaudal segregation ofneuron~ responding to tactile and noxious stimuli s,9,1",'):3. Trigeminal neurons responding to tactile stimuli are located throughout the trigeminal nuclear complex and neurons responding to stimuli presumed to be noxious are not restricted to a specific uanse verse level in the trigeminal nuclei:~, s,.~,'-'zL Furthermore, neurons responding to motthan one stimulus modality have been identified throughout the extent of the trigeminal nuclear complex ~, ~2,~:~. Such observations are difficult to reconcile with a direct relay at nucleus caudalis as the sole prerequisite for the perception of noxious facial stimuli. To resolve this controversy some investigators ~a suggested that a painful stimulus produced 'many impulses by many cells' in the system and, therefore, isolating nucleus caudalis as by a tractotomy decreased the number of afferent fibers and thus reduced the intensity of activity leaving the primary nuclei. Denny-Brown and Yanagisawa '~ have recently suggested that there is a 'critical central threshold' of activity in the nucleus of the descending tract of the fifth nerve which must be exceeded in order to produce a conscious or reflex response in the organism. They further concluded that this central threshold is higher for pain than for other non-noxious stimuli and thus pain is a 'quantitative aspect of common sensation'. Others 14,1:~ have identified neurons in nucleus caudalis specilically responding to noxious stimuli suggesting a specilic trigeminal pain pathway dependent on relays in nucleus caudalis -~ thus eliminating the need for polymodal cells. Response pattern characteristics of trigeminal polymodal neurons to a variety of peripheral stimuli have not been described. Studies of dorsal horn cells in the spinal cord, however, have suggested at least that there is a difference in the duration of firing of neurons consequent to different peripheral stimulus modalities e'z. We, therefore, have undertaken this study of second-order neuron responses in rostral trigeminal nuclei of cat to noxious and non-noxious stimuli to determine if these polymodal neurons have specific response patterns characteristic of the stimulus modality. The role of nucleus caudalis in modulating such response patterns in rostral nuclei was also examined, since earlier physiologic studies suggested that the excitability of neural elements in rostral nuclei may be modulated by neuron influences from nucleus caudalis ~,~s.-':','-'~L
METHODS
Experiments were performed on 84 adult cats weighing between 2.6 and 3.8 kg. Tracheostomy and cannulation of the femoral artery and vein were performed under brief ether inhalation. Anesthesia was continued with intravenous alpha-chloralose (80 mg/kg). The head was fixed in a stereotaxic apparatus and respiration maintained artificially. Animals were immobilized with gallamine triethiodide (Flaxedil) 5 mg/kg every 2 h. Arterial blood pressure was continuously monitored and maintained
49 at 80-110 m m Hg. End expiratory CO2 was maintained between 5 ~o and 6~o. A heating pad maintained rectal temperature at 37-38 °C. The posterior fossa was exposed through a suboccipital craniectomy and the cerebellum and brain stem were covered with artificial CSF at 37 °C. In some animals parts of the cerebellum were removed for more direct access to the brain stem. Bipolar electrical stimulation of the dental pulp was used as a noxious stimulus. Two holes were drilled into the dentine of the proximal part of the upper canine tooth. Two 30-gauge insulated silver wire electrodes with 1 mm exposed tips were inserted into the holes and secured by dental cement with the anode positioned distally. The interelectrode resistance varied between 75 and 100 kg). Single rectangular electrical pulses (0.1 msec duration) of variable voltage were delivered to the tooth pulp by a type 2533 Devices isolated stimulator. Bipolar electrical stimulation of dental pulp has been considered to be a noxious stimulus 1,1°,1v. As a non-noxious stimulus, a light tap applied to the surface of the same tooth was delivered with a Type 102 Ling linear solenoid unit, driven by a Model A112 Wavetek waveform generator. The waveform generator was set to deliver a pulse that produced variable displacement of a 3-cm long stylet attached to the solenoid unit. The free end of this styler was positioned 0.5-1.0 mm away from the surface of the tooth so that with each displacement the tooth was tapped. The impact of this tap on the tooth, in a labiolingual or an anteroposterior direction, was measured as tooth tip displacement in #m using a mechanical transducer attached to the tooth. The same range of stimulus intensities used in cats was tried on human subjects and the only sensation they reported was light touch. Glass micropipettes filled with 3 M KCI or NaCl with 2-7 M ~ resistance were used for extracellular recordings. Brain stem areas at coordinates of 4-10 mm rostral and 2.5-5.5 mm lateral to the obex, corresponding to the territory of the main sensory nucleus and nucleus oralis of the fifth nerve s, were penetrated in a direction perpendicular to the surface of the medulla. Only neurons fulfilling all the following criteria for second-order neurons were studied: first, a response latency of 2.5 3,5 msec4,6; second, a small discrete receptive field for touch or pressure stimulil2: and third, confirmation of the electrode tip position within the area of the main sensory and oralis nuclei on serial sections of the brain stem, after perfusing animals arterially with 10 ~ formalin at the end of the experiment while the glass micropipette was still in situ. Brain stems were removed intact and laid in 10~Voformalin for several days before sections were cut and stained with PAS-gallocyanin. The stimulus threshold for each second-order neuron was determined for electrical dental pulp and light touch stimulation. Gradually increasing stimulus intensities were then used to relate the average number of spikes/10 stimuli to the strength of stimuli until the response was maximal. A stimulus intensity was then chosen midway between threshold and maximal response for both test and conditioning stimulus intensity. Ten trials at this intensity were delivered at 5-sec intervals for control prior to conditioning sequences. Further study on the same units was then conducted by delivering an electrical dental pulp or a touch stimulus (conditioning stimulus) followed by a second electrical
50 dental pulp or touch stimulus (test stimulus) at time intervals of 50--1200 msec. This conditioning-testing (C-T) technique was used for all pairings of the dental pulp and touch stimuli, i.e., touch-pulp, pulp-pulp, pulp-touch, touch-touch. In 5 experiments bipolar David K o p f SNEX-200 steel electrodes with 0.25 mm tip separation were inserted into the ipsilateral nucleus caudalis at coordinates of 3-5 mm below and 3 4 mm lateral to the obex at a depth of 1.5-2 mm for elecmcal stimulation while recording second-order neuron responses to dental pulp and to light touch in rostral trigeminal nuclei. Single rectangular pulses of 0. t msec duration and variable voltage were delivered through a Type 2533 Devices isolated stimulator to caudalis. The condition-test sequence was then examined, applying a conditiomng stimulus at nucleus caudalis followed by a light touch or a dental pulp test stimulus at C - T intervals from 50-1000 msec. In 6 experiments 0.036 ml of a 3,~; solution of strychnine sull:ate was infused into the ipsilateral nucleus caudalis via a glass micropipette over a 2-min period. The micropipette tip was located at a depth of 1.5 mm belowthe surface of the medulla, 3.5 mm lateral and 3 mm below the obex. The response of second-order neurons in rostral trigeminal nuclei was examined separately to both light touch and electrical dental pulp stimuli before and after the strychnine infusion. Single unit activity was amplified by a Model P5111 Grass AC preamplitier and cathode follower and displayed on a Tektronix Rm 565 dual beam oscilloscope. Data and stimulus control signals were recorded on magnetic tape by a Model 4700 Sangamo FM tape recorder for further analysis. Data from magnetic tape were led through an analog-digital converter unit and then ted to a computer for analysis. The computer was programmed to count the number of action potentials occurring at equal time intervals (3-msec bins) starting from the time at which the stimulus was applied. This data was printed out as a poststimulus time histogram (PSTH) accumulating the number of action potentials/unit time for the sum of 10 stimuli. RESULTS
Eighty-five neurons located at coordinates within the region of the main sensory and oralis nuclei s satisfied the criteria for second-order neurons. All units were fired by a bipolar electrical dental pulp stimulus to the ipsilateral upper canine tooth as well as light touch application to the same tooth. Other neurons activated with dental pulp stimuli at latencies of 4-8 msec were encountered with electrode penetrations in regions less than 4 m m lateral to the obex. Those neurons had large receptive fields extending widely over the face, body segments and limbs in an ipsilateral or a bilateral distribution justifying their exclusion from the neuron population under study.
(A) Patterns of response to dental pulp and light touch stimuli The threshold to dental pulp stimulation varied from one unit to another between 0.5 and 3.0 V of a 0.1 msec rectangular pulse. The threshold to touch also varied between 50 and 100 tzm of displacement of the tooth tip. At threshold, neurons
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Fig. l, Relationship of stimulus intensity to firing response of a representative neuron for both dental pulp and touch stimuli. Inserts show poststimulus time histograms of the responses at threshold and maximal stimulation intensities for both modalities. Threshold intensity fired the neuron in 50% of multiple trials. fired one or two action potentials in 50 % of multiple trials in response to either dental pulp or light touch. As the electrical dental pulp stimulus intensity was gradually increased above threshold there was an increase in the number of action potentials per stimulus and in the duration of firing which stabilized as the maximal response was reached at intensities that varied between 15 and 20 times threshold. For touch, there was an increase in response as the tooth displacement was carried above threshold. A maximal response was noted at 3-5 times threshold (Fig. 1) Unit response characteristics following dental pulp and tooth touch stimuli were then determined at stimulus intensities that gave a response midway between threshold and maximum. In all neurons studied with above threshold stimuli, the response to electrical dental pulp stimulation consisted of a significantly greater number of action potentials and a longer firing duration than the response to light touch stimulation. In 31 units where a PSTH study was available it was noted that the response to electrical dental pulp stimulation was characterized by a high frequency response in the second and fourth time bins. Beyond these bins there were differences in the appearance of the PSTH across units that justified subcategorizing unit responses into 3 types (Fig. 2). In 13 units the dental pulp P S T H showed a gradual decrease in activity per bin beyond the second and third bin (Fig. 2a). In 15 units a later rise in frequency of response per bin was noted beyond the fourth and fifth bin with a bimodal appearance of the PSTH (Fig. 2b). In 3 units a double burst response was apparent in the P S T H (Fig. 2c). In all units the corresponding PSTH for touch showed a brief burst of activity restricted to the early bins of this histogram (Fig. 2).
(B) Neuron response to electrical stimulation of nucleus caudalis In 12 second-order neurons in the rostral trigeminal nuclei with a stable latency and response to dental pulp and light touch stimulation of the upper canine tooth
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Fig. 2. Oscilloscopic tracing and poststimulus time histogram analysis of second-order neuron responses in rostral trigeminal nuclei to dental pulp and touch stimuli in 3 representative units a, b and c. Arrows in the touch response indicate the time at which the stimulus was applied. These data represent unit firing patterns obtained midway between threshold and maximal intensities. the response to bipolar electrical stimulation of the nucleus caudalis was also studied. Each of these neurons was fired by the nucleus caudalis stimulus as well as by dental pulp and light touch stimuli. Fig. 3 shows an extracellular tracing of one such unit with PSTH analysis. The caudalis stimulus threshold varied across units between 0.1 and 0.5 V of a 0.l-msec rectangular pulse. At stimulus intensities of 1.5-2 times threshold the neurons fired consistently with each stimulus at a stable latency varying between 6 and 8 msec from unit to unit.
(C) Effect of strychnine & nucleus caudalis on response patterns to dental pulp and touch stimuli in rostral nuclei In 6 second-order neurons responding to dental pulp and light touch stimulation, strychnine sulfate in a solution of 3 ~ was infused into nucleus caudalis after control stimuli were delivered. One minute after starting the strychnine sulfate injection at a rate of 0.018 ml/min there was an increase in the number of spikes and duration of activity per response to both dental pulp and touch stimulation throughout the succeeding recording period. Responses before and after strychnine are illustrated in Fig. 4. The increased unit firing occurred~almost exclusively beyond the third time bin following the stimulus.
(D) Effect of light touch conditioning on response pattern to dental pulp stimulatio~z In 25 units that were held long enough for study, the dental pulp stimulus was preceded by light touch as a conditioning stimulus at C - T intervals from 50 to 1200
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msec. Ten trials of conditioning testing sequences were performed for each C - T interval. In all 25 units there was a drop in the number of action potentials per response and a shortening of the firing time. Fig. 5A shows PSTH of one representative unit when conditioned by light touch and tested with dental pulp stimuli at different C - T intervals. Maximal discharge decrement was noted at intervals of 50 100 msec. The average total number of spikes at these intervals was 37 % of the control (mean of 25 units) (Fig. 6). The drop in activity was more pronounced in the late time bins of the PSTH than in the early bins. As the C - T intervals were increased beyond 150 msec there was gradual increase in response until full recovery to control by 800 1000 msec.
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Fig. 4. A representative unit illustrating response characteristics to dental pulp and touch stimuli before and after application of 0.036 ml of 3 % solution of strychnine sulfate to nucleus caudalis. The increased unit firing occurs almost exclusively in bins beyond the first 9 msec following the stimulus.
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Fig. 5. Poststimulus time histogram analysis of neuron responses to dental pulp and touch paired as conditioning and test stimuli in different combinations at variable C-T time intervals. (A) Dental pulp-touch. (B) Dental pulp-dental pulp. ((7) Dental pulp-touch: in I only suppression of activity was noted and in II early suppression was followed by augmentation of response at longer ( T intervals. (D) Touch~ touch,
( E) Ef/'ect of dental pulp conditioning on re,sponse pattern to dental pulp stimulus On the same 25 units, the response to dental pulp stimulation conditioned by another dental pulp stimulus applied to the same tooth was studied. Fig. 5B shows a unit conditioned with dental pulp stimulation before it was fired by another dental pulp stimulus, This pairing o f stimuli resulted in a decrease o f activity of all neurons with a drop in the number o f spikes to a mean o f 60 ~o o f control at intervals o f 50 100 msec (Fig. 6). As in the touch-dental pulp pairing there was more significant decrement o f activity in the late bins o f the firing sequence with a decrease in the total duration o f firing. Full recovery o f response to control level was reached at intervals o f 800-1000 msec.
(F) Effect of dental pulp conditioning on response pattern to light touch stimulus This analysis was available on 15 units. In 10 neurons conditioned by a dental
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Fig. 6. Average number of spikes per 10 responses of neurons as percentage of control in 4 possible conditioning-testing combinations at variable intervals. Each point on the curves denotes the average of all units on which the conditioning-testing technique was applied. The insert shows a plot of a dental pulp-touch sequence with the same coordinates for the average of 5 units not included in the main curve where there was an increased response. p u l p stimulus there was only a d e c r e m e n t in response to light t o u c h in b o t h the mean n u m b e r o f spikes a n d firing time c o m p a r e d to control. This effect was m a x i m a l at 50 msec with a m e a n o f 73 % o f c o n t r o l (Fig. 6). Fig. 5C~ shows a P S T H study o f one such unit at m a x i m a l decrease in response c o m p a r e d to control. In 5 o u t o f 15 neurons in the dental p u l p - t o u c h p a i r i n g (Fig. 5CII) there was an initial decrease in total activity ( m a x i m a l at 100 msec) with a significant increase in n u m b e r o f spikes and d u r a t i o n o f firing b e y o n d 100 msec.
(G) Effect of light touch conditioning on response pattern to #ght touch stimulus In 10 units analyzed for light t o u c h response c o n d i t i o n e d by a light touch stimulus to the same t o o t h there was a significant decrease o f activity in response to the test stimulus reaching an average o f 15% o f c o n t r o l at C - T intervals o f 50-100 msec (Fig. 6) with shortening o f discharge d u r a t i o n . Typically all unit responses g r a d u a l l y a p p r o a c h e d c o n t r o l p a t t e r n as the C - T intervals were increased to 1000 msec. Fig. 5D illustrates a P S T H study o f one such unit with light t o u c h - l i g h t touch interaction.
(H) Effect of nucleus caudalis conditioning stimuli on re,sponse pattern to dental pulp and light touch stimulation The effect o f an electrical c o n d i t i o n i n g stimulus a p p l i e d to nucleus c a u d a l i s on responses o f second o r d e r n e u r o n s in the m a i n sensory a n d oralis nuclei to peripheral stimuli was d e t e r m i n e d in 5 units r e s p o n d i n g to dental p u l p a n d touch stimuli. W h e n a dental p u l p o r a t o u c h stimulus was preceded by a caudalis stimulus at intervals varying f r o m 50 to 1000 msec, a d e c r e m e n t in response was n o t e d for b o t h dental p u l p a n d t o u c h which was m a x i m a l at intervals o f 50-100 msec. G r a d u a l return to c o n t r o l response was n o t e d as the interval increased b e y o n d 100 msec. Fig. 7 illustrates this p h e n o m e n o n where dental p u l p a n d t o u c h were p r e c e d e d by a
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caudalis conditioning stimulus. Fig. 8 relates the average number of spikes of 5 units for 10 responses compared to control at various C - T intervals. DISCUSSION
Polymodal cells have been defined as those that fire in response to a variety of stimuli. No earlier report, however, has described distinct patterns of firing specifically related to noxious and non-noxious stimuli in such cells. Our observations indicate that second order neurons in rostral trigeminal nuclei feature different firing patterns following dental pulp and touch stimuli. We suggest, therefore, that these units were not indiscriminately polymodal. Their pattern of response reflected in a characteristic way the stimulus modality. To determine the origin of the later component in the dental pulp response sequence we considered that it may be related to influences from nucleus caudalis. Earlier studies have indicated that nucleus caudalis maintined a tonic hyperpolarizing influence on primary afferent fiber preterminats in the main sensory and oralis nuclei t~, 18,g0 and that a dental pulp stimulus produced hyperpolarization of preterminals in these rostral trigeminal nuclei via ascending relays from the nucleus caudalis2'L Those studies, however, indicated that this excitability change (PAD) in the preterminals was first recorded more than 100 msec after a dental pulp stimulus. If such a mechanism enhanced synaptic transmission following a single stimulus it would not account for the second burst or the increased frequency in the late bins of the PSTH of second-order neurons which appeared within 15-20 msec after the stimulus. An alternate explanation, therefore, was sought. An ascending intranuclear trigeminal pathway from nucleus caudalis to rostral
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Fig. 8. Effects of electrical stimuli applied to nucleus caudalis as conditioning stimuli on neuron response in rostral nuclei to dental pulp or touch stimuli applied at variable time intervals. The average number of spikes per 10 responses is plotted for each time interval as a percentage of 10 control responses. Each point on the curve is a mean of 5 units. nuclei has been described 21. Activity in nucleus caudalis may be relayed to rostral nuclei by this anatomically defined ascending system. Second-order neurons in rostral nuclei that respond to both dental pulp and touch stimuli also fire after electrical stimulation of nucleus caudalis but always with a longer latency (6-8 msec). The bimodal response pattern evoked by a dental pulp stimulus may therefore represent two separate inputs converging on the same neurons: (1) a direct monosynaptic input from the dental pulp to rostral nuclei accounting for the early activity in the response sequence and (2) a delayed input via nucleus caudalis which contributes the late response that may be manifest either as a distinct burst in the late bins of the histogram or as a prolonged extension of activity into the same late bins. Such a mechanism could apply only following dental pulp stimuli since there is no suggestion of late activity following touch stimuli. Nucleus caudalis in this context may contribute to processing information about a noxious stimulus not only by direct relay of activity to more central relays but also by modulating the firing patterns of second-order neurons in rostral trigeminal relay nuclei. Previous studies have indicated that strychnine increases neuronal activity by preferentially blocking inhibitory postsynaptic potentials 2,v. Strychnine applied to nucleus caudalis has been reported to cause an increase in the tonic hyperpolarizing influence of this nucleus16, 20 on primary afferent terminals in the rostral trigeminal nuclei. Young and King 25 demonstrated that the hyperpolarizing effect on primary afferent preterminals in nucleus oralis evoked by a dental pulp stimulus was increased with topical application of strychnine over nucleus caudalis. Our results with strychnine infusion into nucleus cauda!is have indicated that the response of second-order neurons to a test stimulus in main sensory and oralis nuclei was augmented following both dental pulp and touch stimulation. In this circumstance we suggest that strychnine increased neuronal excitability of elements of nucleus caudalis that converge on second-order neurons in rostral relay nuclei producing an exaggerated response to both dental pulp and touch stimuli. The observation that a light touch stimulus in the strychnine preparation produced a prolonged high frequency response compar-
58 ed to control is of interest because psychophysical studies employing strychnine application over nucleus caudalis ~1 in cats resulted in marked overreaction to gentle stroking of the fur of the face suggesting a pain-like state as judged by adversive motor behavior. Our observations suggest that one mechanism (but not necessarily tile only mechanism) by which nucleus caudalis may contribute to this overreaction to otherwise non-noxious stimuli is by augmenting the response of second-order neurons m rostral trigeminal nuclei, Changes in excitability of primary afferent terminals in rostral trigeminal nuclei e:, show that a non-noxious vibratory stimulus applied to the face produced primary afferent depolarization {PAD) while a noxious dental pulp stimulus produced a low magnitude PAl) a t short condition test intervals of 5-100 msec and prhnary afferent hyperpolarization ( PA H) at C - T intervals of 100-300 msec ~a. Our observations of the effect of noxious and non-noxious conditioning stimuli on the response of second-order neurons imply that the activity of these neurons does not fully reflect the results one might predict from those studies. Light touch conditioning stimulation decreased the response to a test dental pulp or touch stimulus. A noxious conditioning stimulus preceding a touch test stimulus showed early decreased firing in all the units whereas late augmentation as predicted from preterminal exitability changes, occurred only in 5 units. Noxious conditioning stimuli as a rule produced an early reduction with no late increase in firing following a noxious test stimulus. -lhese observations confirm an earlier study ''G on the effects of conditioning noxious and non-noxious stimuli on the level of activity of second-order neurons. The reason for the absence of a lilcilitated response to dental pulp stimuli when the system was conditioned by dental pulp stimulation is not certain. The fact that the pretermmal excitability studies examined only primary afferent presynaptic events and not other elements impinging on the second-order neurons may be a major reason l'or this discrepancy. The reduced unit response to a test dental pulp stimulus preceded by either a touch or a dental pulp conditioning stimulus was most apparent in late bins in the PSTH sequence. Since activity in these late bins depend primarily on relays through nucleus caudalis, we suggest that each of these conditioning stimuli was primarily effective in reducing that component of the discharge pattern that was relayed via nucleus caudalis. The stimuli for this study were chosen because of psychophysical characteristics. On this basis we cannot be absolutely certain that the tooth pulp stimulus did not activate any neural elements in the periodontal membrane. We suggest nonetheless that such a contaminating mixture of afferent elements was not a major limiting feature of the data for 3 reasons which are illustrated in Figs. 5 and 6. First, a touch conditioning stimulus consistently caused more depression of the test stimuli than did a pulp conditioning stimulus. Second, the maximal effect of a conditioning stimulus noted at 50 msec in Fig. 5A decreased activity in both early and late time bins of the pulp histogram, whereas a pulp conditioning stimulus caused a predominant decrease of activity in the late bins at the same C - T interval of 50 msec (Fig. 5B). If there had been a major spread of the conditioning stimulus to the periodontal membrane we
59 would have expected at least as much depression as following touch, if the same afferent systems were being activated. Third, a touch conditioning stimulus always decreased the responsiveness to a test stimulus whereas a pulp conditioning stimulus augmented the response to touch stimuli in the population of units shown in the inset of Fig. 6. Hence, we suggest that stimulus spread from pulp stimulation to afferent fibers in the periodontal membrane was not a major determining factor in the events we have observed. The effect of conditioning stimulation of nucleus caudalis on activity in more rostral nuclei was reported earlier TM, where an increase in the excitability of rostral primary afferent terminals was noted at C - T intervals beginning at 15 msec and lasting up to 800 msec. Our present study of the effect of a caudalis conditioning stimulus on the response of second-order neurons in rostral trigeminal nuclei showed similar C - T time characteristics. The effective C-T intervals were similar whether the test stimuli were non-noxious, noxious or applied directly to nucleus caudalis. All 3 types of conditioning stimuli may therefore depend on relays in the nucleus caudalis. Earlier studies 2~ suggested the presence of interneurons in nucleus caudalis responsible for mediating PAD in rostral nuclei in response to a non-noxious vibratory stimulus to account for marked decreases in PAD observed after tractotomy at the level of the obex. Insofar as an electrical stimulus at nucleus caudalis may produce PAD but also fire second-order neurons in rostral relay nuclei, we suggest that nucleus caudalis contains neural elements which may either augment or suppress the firing characteristics of more rostral second-order neurons. Thus, under normal circumstances, nucleus caudalis contributes to, and is an essential element for, the perception oftrigeminal noxious stimuli and may also be effective in the modulation of non-noxious stimuli. Under abnormal conditions it may contribute to the conversion of a tactile stimulus to a painful experience. Our observations suggest that modality specificity of second-order neurons in rostral trigeminal nuclei may be represented by changing patterns of unit firing. Variations of interdependent firing patterns of these neurons subserving general sensibility may determine in part, at least, the discrimination and modulation of perceptions induced by a given peripheral stimulus. Some secondary projections from second-order neurons may be modality specific. To what extent these two constructs may be interdependent is yet uncertain. They need not be mutually exclusive. A CKNOWLEDGEMENTS
We wish to thank David L. Blank, Ph. D. for his assistance throughout the research project, and Miss Judie Seubert and Miss Helena Braunfeld for technical assistance. This work was supported in part by Training Grant No. 5-T01 NS-05605 from the National Institute of Neurological Diseases and Stroke, Research Grant 5-S01RR-05402 from the Division of Research Resources, U.S.P.H.S. and Special Research Biotechnology Branch Grant RR-00353, National Institutes of Health.
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