Journal of Comparative and Physiological Psychology 1975, Vol. 89, No. 4, 371-378
Correlation of Overt Escape Behavior, Multiunit Thalamic Activity, and Midbrain Lemniscal Stimulation in Rats Steven E. Brauth Division of Biology, California of Technology
Institute
Edgar E. Coons New York University
An acute and a chronic experiment were conducted in order to assess the extent to which ventral thalamic multiunit activity could account for the specific form of bar-pressing escape behavior in rats stimulated by trains of midbrain medial lemniscal pulse pairs. A paradigm used by Kestenbaum, Deutsch, and Coons was utilized in which the intra-pair interval of the train was varied. Results in anesthetized and freely moving animals indicated that midbrain lemniscal stimulation produces both an excitatory short-latency thalamic response showing the property of temporal facilitation and a long-lasting inhibitory process consistent with results of studies using anesthetized cats. The overall electrophysiological response, however, correlated significantly with the behavioral response function.
Although rats can be trained to escape from electrical stimulation of many points in the brain stem (M. E. Olds & J. Olds, 1963; J. Olds & Peretz, 1960), no studies have as yet been able to identify those neural centers responsible for integrating the motivating aspects of a pain-producing stimulus. As such, it is not clear at \\hat level in the central nervous system afferent signals carrying the pain message are pooled and the decision is made to emit an escape response. In search of an answer to this question, Kestenbaum, Deutsch, and Coons (1970) have used a technique in -which the form of the overt escape response is used to infer the properties of neurons mediating electrically elicited escape behavior. In their study, rats were shaped to press a bar to turn off midbrain medial lemniscal and spinothalamic tract stimulation for 4-sec periods. The experiment involved measuring the effect of changing the pattern of stimulation on bar-
pressing rates. Specifically, trains of pulse pairs were presented to medial lemniscal and spinothalamic fibers in the dorsolateral tegmentum of the midbrain. The first pulse in each pair \\as called the "C" or conditioning pulse, and the second was called the "T" or test pulse. In the Kestenbaum, Deutsch, and Coons experiment, the C-T interval was varied while the C-C interval was held constant. In this way, the poststimulus excitability cycle of the systems under investigation could be studied by assessing changes in the magnitude of the overt escape response. Two effects are observed when the C-T interval is varied. The first effect is a sharp increase in response rate as the C-T interval is increased from 0 to 2.5 msec. This effect is inferred by the authors to reflect the emergence of fibers near the tip of the electrode from refractoriness. The second effect is a gradual decrease in response rate as the This report is based on the doctoral dissertation C-T interval is further increased from 2.5 to of Steven E. Brauth at New York University. 50 msec. This effect is inferred by the auThe research was supported by National Institute thors to constitute evidence of the existence of Mental Health Predoctoral Fellowship MH- of temporal summation of neurotransmitter 43775, awarded to Steven E. Brauth, and by Biomedical Sciences Support Grant FR-07062 from at a key synapse somewhere in the pain the General Research Support Branch, National pathway. The present study was undertaken Institutes of Health, awarded to Edgar E. Coons. to investigate this effect further. Requests for reprints should be sent to Steven Since medial lemniscal fibers project to E. Brauth, Department of Psychiatry and Beneurons in the ventral thalamus, t\\ o experihavioral Science, State University of New York, ments were designed in which stimulating Stony Brook, New York 11790. 371
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STEVEN E. BRAUTH AND EDGAR E. COONS
electrodes were implanted at those midbrain cording); and 1.8 anterior, —1.0 horizontal, 1.7 lemniscal sites studied by Kestenbaum, lateral (stimulating). activity was amplified in two stages. Deutsch, and Coons (1970) and recording TheThalamic first stage was a small transducer which electrodes were implanted at points in the could be attached to the electrode leads on the ventrobasal complex. In one experiment, an animal's head. This transducer consisted of a acute preparation was used (anesthesia). matched pair of field-effect transistors in the configuration and provided a low Five animals were studied, in which the source-follower impedance output with a gain of approximately effects of trains of pulse pairs on thalamic unity. Voltage amplification was accomplished by activity could be assessed. In the second means of a Princeton applied research preamplifier experiment, a chronic preparation was used Model CR-4 with the filters set between 300 Hz so that both thalamic activity and overt and 10 kHz. Brain activity was then displayed on a Tektronix 502A oscilloscope, the cathode folescape behavior could be studied in a freely lower output of which was fed into a Tandberg moving lemniscally stimulated animal. In audio tape recorder in order to make permanent this way, a contribution of the ventral records. The design of the behavioral task for the thalamus to the total response of the orchronic study was identical to that of Kestenganism could be measured. Undoubtedly, baum, Deutsch, and Coons (1970). Lemniscal stimulation of the lemniscus in the midbrain stimulation was presented for 1-min periods activates other brain regions besides the during which the C-T interval was held constant. thalamus, and any of these might be in- During this period, each bar press turned the volved in mediating electrically elicited stimulation off for 4 sec. At the end of the stimulaperiod, the animal was given a 1-min rest escape responses. The net response of the tion period. The C-T interval was then changed and animal will reflect the effects of all of these the stimulation turned on. Number of presses per regions, and by monitoring ventral thalamic stimulation period was determined for each animal multiunit activity, the specific role of the at each C-T interval. Three animals were run C-C intervals of 100 msec and four with thalamus in elaborating the pain message with C-C intervals of 50 msec. (One animal was run can be determined. As such, the purpose of under both conditions.) In the 50-msec C—C condithis study was to determine the extent to tion, C-T intervals of 3.3, 5, 6, 10, 15, 20, and 25 which thalamic output could account for the msec were used. In the 100-msec C-C condition, specific form of the behavioral response as C-T intervals of 3.3, 6, 10, 15, 20, 30, and 50 msec were used. In the acute study, all animals were the C-T interval was varied. run under the 100-msec C-C condition. In addiMETHOD The recording technique used was based on methods developed by Olds and his co-workers (J. Olds, Disterhoft, Segal, Kornblith, & Hirsh, 1972). Implants of 57.5-^m-diam. nichrome wire were positioned in the ventroposteromedial thalamic nucleus, in regions where units could be driven by lightly shaking the contralateral vibrissae. Coordinates used were 3.5 mm anterior to lambda, 1.5-2 mm lateral, and 5.8-6.3 mm deep with the skull flat. In this region, large evoked unit responses can be obtained by gently bending individual vibrissae on the contralateral side and allowing them to snap back. This test was used to position the recording electrode in the thalamus. Medial lemniscal electrodes were then lowered into the midbrain at sites studied by Kestenbaum, Deutsch, and Coons (1970) and sealed at positions from which cells monitored by the thalamic electrode could be driven. Histological results for the six chronic animals confirmed the locations of the stimulating and recording electrodes. The actual placements correspond to the following coordinates on the KQnig and Klippel (1963) atlas: —3.0 anterior, — .4 horizontal, 1.7 lateral (re-
tion, more than one position in the thalamus could be studied in a single rat in this preparation, and a total of 14 recording points were analyzed in the acute study. As in the Kestenbaum, Deutsch, and Coons study, the C-T intervals were randomized in both studies so that the effects of order were completely counterbalanced. Preliminary Investigations Preliminary investigations in both the acute and chronic preparations revealed that lemniscal stimulation has two effects on thalamic neurons. The first effect is a short-latency excitatory effect which shows evidence of facilitation. As the C-T interval is decreased from 50 to 3.3 msec, the short-latency responses increase in strength. This effect is shown in the upper four panels in Figure 1. In this case, single pulse pairs were presented to the lemniscus, and the thalamic response was monitored. In the upper left-hand panel, a C~T interval of 50 msec was used. The pulses produced little driving at the 300-juA current level. In the next three panels, the C-T interval was reduced from 20 to 10 to 3.3 msec. Both a large population multiunit response and driving of one cell were facilitated by shortening the C-T interval. For
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FIGUBE 1. Upper four panels: thalamic responses to single presentations of pulse pairs delivered to the medial lemnisous in the midbrain. (A 200-msec sweep was used. The intra-pair (C-T) intervals are 50, 20,10, and 3.3 msec, in the upper left, upper right, middle left, andmiddle right panels, respectively.) Lower two panels: five superimposed 1-sec and .5-seo sweeps following the presentation of a single pulse in an anesthetized (left) and freely moving (right) preparation, respectively. (The upper portion of the lower right panel presents a single sweep (200 msec) showing the thalamic response to a train of evenly spaced pulses. Current levels were set at 300 ^A in all cases.)
STEVEN E. BRAUTH AND EDGAR E. COONS
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SECOND EPOCH
3
6
10
15
20 C-T
INTERVAL (MSEC)
FIGUEE 2. Multiunit response strength in each epoch plotted as a function of the C-T interval for a control animal (anesthetized preparation). (Thalamic response disappears following sacrifice from overdose of anesthetic [1-cc diabutol, ip].) example, when the C-T interval = 10 msec (middle left), driving of one spike from an underlying cell is evident. At a C-T interval of 3.3 msec (middle right), two occurrences of the spike are driven. The second effect is shown in the lower lefthand frame of Figure 1. This panel was made by superimposing five 1-sec sweeps after presentation of a single pulse. As can be seen, a long-lasting inhibitory response is also released in the thalamus when the lemniscus is stimulated. In the acute preparation, this effect lasts 200-300 msec. As can also be seen from this panel, unit firing as well as the population response exhibits this effect. The lower right-hand frame of Figure 1 shows the same phenomenon in the chronic preparation. For the unanesthetized preparation, five superimposed .5-sec sweeps reveal a long-lasting inhibitory process whose time course appears shortened to 150-200 msec. Both unit firing and population multiunit activity exhibit the effect. A combination of both excitatory and inhibitory effects is obtained when a train of pulses is presented to the medial lemniscus. This is shown in the lower right-hand frame of Figure 1. Each pulse in the train invades the inhibition released by previous pulses and gives rise to a new excitation process. In this way, pulses in a train, which are separated by a time interval greater than the period of facilitation noted previously, invade the inhibitory process of previous pulses and give rise to new excitatory effects. Thus, activity appears to wax and wane in the intervals between successive pulses in an evenly spaced train of stimuli.
Quantification of Thalamic Activity Since population neuronal responses are evoked in the thalamus by medial lemniscal stimulation and inasmuch as single cells are extremely difficult
to hold over the period of days needed to run a chronic experiment, both the acute and chronic studies described in this article involved neural measures obtained by integrating multiunit neuronal activity. These measures were based on activity immediately after the T pulse, i.e., second pulse, in each pair of pulses in the train delivered to the lemniscus. The effect of changes in the C-T interval on this thalamic activity could then be assessed in anesthetized and freely moving rats. In order to quantify multiunit neuronal activity, a signal-averaging technique was used, based on the work of Arduini and Pinnio (1962), Kaufman and Price (1967), and Michaels and Kaufman (1969). These researchers used a method for handling high-frequency neuronal population responses in which the rms voltage level of recorded compound spike activity was taken as an index of the number of units discharging at each instant in time. This is based on the fact that neurons produce spikes in an all-or-none fashion so that the instantaneous power output of the brain point sampled by the recording electrode would be proportional to the number of cells firing. This method was considered applicable since spike heights and frequencies did not vary significantly between thalamic points and since the power output recorded by the electrode largely reflected changes in the population multiunit activity. In this study, a PDP-8 computer was used to obtain averaged measures of rms activity level for three time epochs after the presentation of the T pulse. When the C-C interval was set at 100 msec, three epochs of 12.8 msec were used. When the C-C interval was set at 50 msec, three epochs of 6.4 msec were used. These values were chosen so that approximately half of the C-C interval would be analyzed. Since the maximum C-T interval in each condition was half of the C-C interval, this method assured that activity values computed for all C-T intervals would be based on neural activity occurring during an equal time period. In addition, the first 3.2 msec of activity after the T pulse were discarded in order to avoid any possibility of contaminating the first epoch measure with aftereffects of the stimulus. Thus, the latency of the first epoch was 3.2 msec after the presentation of the T pulse. Average response measures in each epoch were then based on a mean of 1,800 sweeps per C-T interval. In the chronic study, these averages were made while the animal was bar pressing so that a direct comparison of electrophysiological and behavioral responses could be made. As an additional control for these techniques, the entire experimental procedure was repeated for two animals in the acute and chronic preparations, respectively, after they had been sacrificed with an overdose of the anesthetic (1-cc diabutol ip). As can be seen in Figure 2, the results of the analysis for each epoch are not due to artifacts of the stimulus or the averaging process, since the
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AVERAGE RESPONSE CURVES PRESERVING INTENSITY LEVEL 50
CHRONIC STUDY ( C - C = 50)
40 30
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0, 3
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z o CHRONIC STUDY (C-C = 100)
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T H I R D EPOCH
i ^0 r> J. 30
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'
u a:
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UJ 0.
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FIQUKE 3. Percentage of multiunit response in each epoch for the chronic (freely moving) and acute (anesthetized) preparations. (Method for obtaining the percentized composites was the same as in Kestenbaum, Deutsch, and Coons, 1970.) response is at a low level and basically flat in the dead animal.
RESULTS Averaged electrophysiological data for the chronic and acute (anesthetized) experiments are shown in Figure 3. Both the facilitatory and inhibitory effects of lemniscal stimulation are apparent in these graphs of integrated thalamic activity. As expected, short-latency driving, as represented by first epoch activity, is smaller in magnitude than
second and third epoch activity except for the very shortest C-T intervals (3.3-6 msec). This is due to the fact that presentation of the T pulse invades the inhibition generated by previous pulses unless the C-T interval is small enough to permit a facilitative interaction between the C and T pulses. Subsequent higher activity levels in the second and third epochs represent the release of new excitation. These facilitative and inhibitory effects
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were apparent in the multiunit records of represents the entrance and emergence of individual subjects. Inhibition was released ventrobasal neurons from a period of inat all thalamic points in all animals. Al- hibition. In addition, at the very shortest though all thalamic points were excited by intervals (3.3-6 msec), facilitation of the the lemniscal electrode, some showed more excitatory effects of the C and T pulses takes pronounced short-latency driving than place, and first epoch activity rises above the others, suggesting that points of maximal level of thalamic responding in the second excitation exist for a given placement of the and third epochs. stimulating electrode. Although the responses in the second and In the chronic preparation, thalamic ac- third epochs take on a more complicated tivity driven by a train of lemniscal pulse oscillatory pattern representing the interacpairs is characterized by a short-latency tion of both excitatory and inhibitory influ(first epoch) response which takes on a ences, two effects are very clear. For C-T basically U-shaped form. At C-T intervals intervals of 3.3-6 msec, second and third of 3.3-15 msec, first epoch response strength epoch activity is low because of high redecreases as C-T interval increases. As C-T sponding in the first epoch. For longer C-T interval is increased from 15 to 50 msec, intervals, however, the T pulse invades the however, first epoch activity also increases. inhibition of previous pulses, and first epoch In the case of animals run under the 50-msec. activity is low. New excitation released by condition, this upturn is not seen until the the T pulse then results in the increased second epoch. However, the epochs were activity levels seen in the later episodes. half size in this condition, and as such, first Differences between the acute and chronic and second epochs covered the same time preparations are consistent •« ith the observaperiod in the 50-msec C-C condition as the tion that the time course of thalamic inhibifirst epoch alone in the 100-msec C-C condi- tion is longer in the anesthetized state. Thus, tion. first epoch activity for the acute study folUBoth the fall and the rise of short-latency lows a U-shaped course with the C-T trough activity as C-T interval is increased from occurring at 30 msec instead of 15 msec, as in 3.3 to 50 msec are consistent with the ob- the chronic study. Second and third epoch servation that lemniscal stimulation gen- responses in the anesthetized preparation are orates short-latency excitation followed by also above first epoch responses, showing the long-lasting inhibition in ventrobasal neu- rebound effect generated \\hen lemniscal rons. The U-shaped first epoch response thus pulses invade thalamic inhibition released by previous pulses. BEHAVIORAL COMPOSITE (PRESSES PER MINUTE) Behavioral response data are shown in Figure 4. The pattern of C-T responding generally resembles the results obtained by Kestenbaum, Deutsch, and Coons (1970) except for a marked drop in response level as the C-T interval is shortened from 6 to 3.3 msec and an intermediate peak in the response curve at a C-T interval of 20 msec. There is also a slight increase in response as the C-T interval is increased from 30 to 50 msec so that the overall pattern of responding is slightly U-shaped. The form of the C - T INTERVAL ( M S E C ) behavioral response thus shows a general FIQUKE 4. Behavioral response functions for resemblance to the form of the first epoch animals in the chronic study with C-C intervals response obtained in the chronic study. of 100 msec or 50 msec. (Percentized composites A product-moment r was computed for the were obtained using the same method as Kestensix chronic animals between averaged thalabaum, Deutsch, and Coons, 1970.
RESPONSES TO MIDBRAIN LEMNISCAL STIMULATION
mic activity and behavioral response strength. A value of +.39 was obtained between presses per minute and first epoch activity, which is significant at the 1 % level. Correlations with second and third epoch activity were +.19 and +.20, which are not significant. DISCUSSION The results of the analysis of the multiunit recordings are consistent with studies done on anesthetized decorticate cats (Andersen & Andersson, 1968), in which intracellular recordings were made from ventrobasal relay neurons. In these studies, peripheral lemniscal stimulation, ventrobasal stimulation, and antidromic thalamic stimulation were used. Results showed the existence of long-lasting inhibitory-postsynaptic-potential-produced periods of hyperpolarization after lemniscal stimulation. In addition, these researchers attribute these processes to the operation of numerous multisynaptic recurrent postsynaptic inhibitory loops possessing lateral inhibitory features as well. Lemniscal stimulation would therefore be expected to result in widespread, long-lasting inhibition throughout the ventral thalamus, as was found in this study. Andersen and Andersson have also found evidence of resetting properties for thalamic rhythmicity so that an antidromic or sufficiently strong orthodromic stimulus occurring during the inhibitory phase of the ventrobasal cycle can either shorten or prolong the time course of emergence from inhibition. These effects were observed in this study insofar as second and third epoch responses were often higher in intensity than first epoch responses and their cycles of inhibition and recovery shifted. The expressed purpose of this study was to assess the correspondence between overt behavior and thalamic activity in lemniscally stimulated animals in order to determine the extent to which the ventrobasal thalamic nuclei act as a final integrating center for the motivating aspects of pain-producing stimuli. As has been noted, a significant resemblance does exist between the form of the behavioral response and short-latency thala-
377
mic activity following the T pulse, when the C-T interval is varied. This correspondence, however, does not indicate that the ventral thalamus is the only relay point in the pain pathway involved in integrating the motivating aspects of the stimulation, insofar as the correlation for first epoch activity is not perfect and the correlations for second and third epoch activity are not significant. The evidence therefore suggests that additional elaboration of the pain message is carried on outside of the ventrobasal complex. This means that although the thalamus alone does not make the decision to perform an escape response, it is involved to a significant degree in transmitting information about the motivating aspects of pain-producing stimuli. In summary, the following conclusions can be drawn from the shape of the averaged electrophysiological response graphs and averaged behavioral response graphs: 1. The U-shaped electrophysiological function exhibited in the first epoch, although significantly correlated with the shape of the behavioral response function, does not indicate that the pain message is fully elaborated at the level of the ventral thalamus, insofar as the correlation is imperfect, Low correlations of the behavioral function with second and third epoch responses further reinforce this conclusion. 2. The low level of responding at the very shortest C-T intervals in the second and third epochs, combined with high first epoch responding at these intervals, provides evidence of an additional excitatory effect which shows the property of facilitation. This phenomenon has not been noted in previous studies. 3. Agreement between the results of the multiunit integration technique used in this study and the results of prior experiments involving intraccllular recording is good enough to validate this technique for work involving freely moving animals in which chronic intracellular recording would be difficult if not impossible. This, fortunately, means that an interpretable measure of both excitatory and inhibitory neuronal population responses can be obtained in conscious,
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freely moving animals while studies are made of underlying motivational systems. REFERENCES Andersen, P., & Andersson, S. Physiological basis of the alpha rhythm. New York: AppletonCentury-Crofts, 1968. Arduini, A., & Pinnio, L. A method for the quantification of tonic activity in the nervous system. Archives Italiennes de Biologia, 1962, 700, 415422. Kaufman, L., & Price, R. The detection of cortical spike activity at the human scalp. IEEE Transactions on Bio-Medical Engineering, 1967, 14, 84. Kestenbaum, R. 8., Deutsch, J. A., & Coons, E. E. Behavioral measurement of neural post-stimulation excitability cycle: Pain cells in the brain of the rat. Science, 1970, 167, 393.
KOnig, J. F. R., & Klippel, R. A. The rat brain: A stereotaxic atlas. Baltimore: Williams & Wilkins, 1963. Michaels, J. A., & Kaufman, L. High frequency visual evoked responses: A product of neuronal spiking. Experimental Brain Research, 1969, IB, 255-258. Olds, J., Disterhoft, J. F., Segal, M., Kornblith, C. L., & Hirsh, R. Learning centers of rat brain mapped by measuring latencies of conditioned unit responses. Journal of Neurophysiology, 1972, 35, 202-219. Olds, J., & Peretz, B. A motivational analysis of the reticular activating system. Electroencephalography and Clinical Neurophysiology, 1960, 18, 445-454. Olds, M. E., & Olds, J. Approach-avoidance analysis of rat diencephalon. Journal of Comparative Neurology, 1963, 170, 259-295. (Received May 3, 1974)
Correlation of Overt Escape Behavior, Multiunit Thalamic Activity, and Midbrain Lemniscal Stimulation in Rats Steven E. Brauth Division of Biology, California of Technology
Institute
Edgar E. Coons New York University
An acute and a chronic experiment were conducted in order to assess the extent to which ventral thalamic multiunit activity could account for the specific form of bar-pressing escape behavior in rats stimulated by trains of midbrain medial lemniscal pulse pairs. A paradigm used by Kestenbaum, Deutsch, and Coons was utilized in which the intra-pair interval of the train was varied. Results in anesthetized and freely moving animals indicated that midbrain lemniscal stimulation produces both an excitatory short-latency thalamic response showing the property of temporal facilitation and a long-lasting inhibitory process consistent with results of studies using anesthetized cats. The overall electrophysiological response, however, correlated significantly with the behavioral response function.
Although rats can be trained to escape from electrical stimulation of many points in the brain stem (M. E. Olds & J. Olds, 1963; J. Olds & Peretz, 1960), no studies have as yet been able to identify those neural centers responsible for integrating the motivating aspects of a pain-producing stimulus. As such, it is not clear at \\hat level in the central nervous system afferent signals carrying the pain message are pooled and the decision is made to emit an escape response. In search of an answer to this question, Kestenbaum, Deutsch, and Coons (1970) have used a technique in -which the form of the overt escape response is used to infer the properties of neurons mediating electrically elicited escape behavior. In their study, rats were shaped to press a bar to turn off midbrain medial lemniscal and spinothalamic tract stimulation for 4-sec periods. The experiment involved measuring the effect of changing the pattern of stimulation on bar-
pressing rates. Specifically, trains of pulse pairs were presented to medial lemniscal and spinothalamic fibers in the dorsolateral tegmentum of the midbrain. The first pulse in each pair \\as called the "C" or conditioning pulse, and the second was called the "T" or test pulse. In the Kestenbaum, Deutsch, and Coons experiment, the C-T interval was varied while the C-C interval was held constant. In this way, the poststimulus excitability cycle of the systems under investigation could be studied by assessing changes in the magnitude of the overt escape response. Two effects are observed when the C-T interval is varied. The first effect is a sharp increase in response rate as the C-T interval is increased from 0 to 2.5 msec. This effect is inferred by the authors to reflect the emergence of fibers near the tip of the electrode from refractoriness. The second effect is a gradual decrease in response rate as the This report is based on the doctoral dissertation C-T interval is further increased from 2.5 to of Steven E. Brauth at New York University. 50 msec. This effect is inferred by the auThe research was supported by National Institute thors to constitute evidence of the existence of Mental Health Predoctoral Fellowship MH- of temporal summation of neurotransmitter 43775, awarded to Steven E. Brauth, and by Biomedical Sciences Support Grant FR-07062 from at a key synapse somewhere in the pain the General Research Support Branch, National pathway. The present study was undertaken Institutes of Health, awarded to Edgar E. Coons. to investigate this effect further. Requests for reprints should be sent to Steven Since medial lemniscal fibers project to E. Brauth, Department of Psychiatry and Beneurons in the ventral thalamus, t\\ o experihavioral Science, State University of New York, ments were designed in which stimulating Stony Brook, New York 11790. 371
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STEVEN E. BRAUTH AND EDGAR E. COONS
electrodes were implanted at those midbrain cording); and 1.8 anterior, —1.0 horizontal, 1.7 lemniscal sites studied by Kestenbaum, lateral (stimulating). activity was amplified in two stages. Deutsch, and Coons (1970) and recording TheThalamic first stage was a small transducer which electrodes were implanted at points in the could be attached to the electrode leads on the ventrobasal complex. In one experiment, an animal's head. This transducer consisted of a acute preparation was used (anesthesia). matched pair of field-effect transistors in the configuration and provided a low Five animals were studied, in which the source-follower impedance output with a gain of approximately effects of trains of pulse pairs on thalamic unity. Voltage amplification was accomplished by activity could be assessed. In the second means of a Princeton applied research preamplifier experiment, a chronic preparation was used Model CR-4 with the filters set between 300 Hz so that both thalamic activity and overt and 10 kHz. Brain activity was then displayed on a Tektronix 502A oscilloscope, the cathode folescape behavior could be studied in a freely lower output of which was fed into a Tandberg moving lemniscally stimulated animal. In audio tape recorder in order to make permanent this way, a contribution of the ventral records. The design of the behavioral task for the thalamus to the total response of the orchronic study was identical to that of Kestenganism could be measured. Undoubtedly, baum, Deutsch, and Coons (1970). Lemniscal stimulation of the lemniscus in the midbrain stimulation was presented for 1-min periods activates other brain regions besides the during which the C-T interval was held constant. thalamus, and any of these might be in- During this period, each bar press turned the volved in mediating electrically elicited stimulation off for 4 sec. At the end of the stimulaperiod, the animal was given a 1-min rest escape responses. The net response of the tion period. The C-T interval was then changed and animal will reflect the effects of all of these the stimulation turned on. Number of presses per regions, and by monitoring ventral thalamic stimulation period was determined for each animal multiunit activity, the specific role of the at each C-T interval. Three animals were run C-C intervals of 100 msec and four with thalamus in elaborating the pain message with C-C intervals of 50 msec. (One animal was run can be determined. As such, the purpose of under both conditions.) In the 50-msec C—C condithis study was to determine the extent to tion, C-T intervals of 3.3, 5, 6, 10, 15, 20, and 25 which thalamic output could account for the msec were used. In the 100-msec C-C condition, specific form of the behavioral response as C-T intervals of 3.3, 6, 10, 15, 20, 30, and 50 msec were used. In the acute study, all animals were the C-T interval was varied. run under the 100-msec C-C condition. In addiMETHOD The recording technique used was based on methods developed by Olds and his co-workers (J. Olds, Disterhoft, Segal, Kornblith, & Hirsh, 1972). Implants of 57.5-^m-diam. nichrome wire were positioned in the ventroposteromedial thalamic nucleus, in regions where units could be driven by lightly shaking the contralateral vibrissae. Coordinates used were 3.5 mm anterior to lambda, 1.5-2 mm lateral, and 5.8-6.3 mm deep with the skull flat. In this region, large evoked unit responses can be obtained by gently bending individual vibrissae on the contralateral side and allowing them to snap back. This test was used to position the recording electrode in the thalamus. Medial lemniscal electrodes were then lowered into the midbrain at sites studied by Kestenbaum, Deutsch, and Coons (1970) and sealed at positions from which cells monitored by the thalamic electrode could be driven. Histological results for the six chronic animals confirmed the locations of the stimulating and recording electrodes. The actual placements correspond to the following coordinates on the KQnig and Klippel (1963) atlas: —3.0 anterior, — .4 horizontal, 1.7 lateral (re-
tion, more than one position in the thalamus could be studied in a single rat in this preparation, and a total of 14 recording points were analyzed in the acute study. As in the Kestenbaum, Deutsch, and Coons study, the C-T intervals were randomized in both studies so that the effects of order were completely counterbalanced. Preliminary Investigations Preliminary investigations in both the acute and chronic preparations revealed that lemniscal stimulation has two effects on thalamic neurons. The first effect is a short-latency excitatory effect which shows evidence of facilitation. As the C-T interval is decreased from 50 to 3.3 msec, the short-latency responses increase in strength. This effect is shown in the upper four panels in Figure 1. In this case, single pulse pairs were presented to the lemniscus, and the thalamic response was monitored. In the upper left-hand panel, a C~T interval of 50 msec was used. The pulses produced little driving at the 300-juA current level. In the next three panels, the C-T interval was reduced from 20 to 10 to 3.3 msec. Both a large population multiunit response and driving of one cell were facilitated by shortening the C-T interval. For
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FIGUBE 1. Upper four panels: thalamic responses to single presentations of pulse pairs delivered to the medial lemnisous in the midbrain. (A 200-msec sweep was used. The intra-pair (C-T) intervals are 50, 20,10, and 3.3 msec, in the upper left, upper right, middle left, andmiddle right panels, respectively.) Lower two panels: five superimposed 1-sec and .5-seo sweeps following the presentation of a single pulse in an anesthetized (left) and freely moving (right) preparation, respectively. (The upper portion of the lower right panel presents a single sweep (200 msec) showing the thalamic response to a train of evenly spaced pulses. Current levels were set at 300 ^A in all cases.)
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FIGUEE 2. Multiunit response strength in each epoch plotted as a function of the C-T interval for a control animal (anesthetized preparation). (Thalamic response disappears following sacrifice from overdose of anesthetic [1-cc diabutol, ip].) example, when the C-T interval = 10 msec (middle left), driving of one spike from an underlying cell is evident. At a C-T interval of 3.3 msec (middle right), two occurrences of the spike are driven. The second effect is shown in the lower lefthand frame of Figure 1. This panel was made by superimposing five 1-sec sweeps after presentation of a single pulse. As can be seen, a long-lasting inhibitory response is also released in the thalamus when the lemniscus is stimulated. In the acute preparation, this effect lasts 200-300 msec. As can also be seen from this panel, unit firing as well as the population response exhibits this effect. The lower right-hand frame of Figure 1 shows the same phenomenon in the chronic preparation. For the unanesthetized preparation, five superimposed .5-sec sweeps reveal a long-lasting inhibitory process whose time course appears shortened to 150-200 msec. Both unit firing and population multiunit activity exhibit the effect. A combination of both excitatory and inhibitory effects is obtained when a train of pulses is presented to the medial lemniscus. This is shown in the lower right-hand frame of Figure 1. Each pulse in the train invades the inhibition released by previous pulses and gives rise to a new excitation process. In this way, pulses in a train, which are separated by a time interval greater than the period of facilitation noted previously, invade the inhibitory process of previous pulses and give rise to new excitatory effects. Thus, activity appears to wax and wane in the intervals between successive pulses in an evenly spaced train of stimuli.
Quantification of Thalamic Activity Since population neuronal responses are evoked in the thalamus by medial lemniscal stimulation and inasmuch as single cells are extremely difficult
to hold over the period of days needed to run a chronic experiment, both the acute and chronic studies described in this article involved neural measures obtained by integrating multiunit neuronal activity. These measures were based on activity immediately after the T pulse, i.e., second pulse, in each pair of pulses in the train delivered to the lemniscus. The effect of changes in the C-T interval on this thalamic activity could then be assessed in anesthetized and freely moving rats. In order to quantify multiunit neuronal activity, a signal-averaging technique was used, based on the work of Arduini and Pinnio (1962), Kaufman and Price (1967), and Michaels and Kaufman (1969). These researchers used a method for handling high-frequency neuronal population responses in which the rms voltage level of recorded compound spike activity was taken as an index of the number of units discharging at each instant in time. This is based on the fact that neurons produce spikes in an all-or-none fashion so that the instantaneous power output of the brain point sampled by the recording electrode would be proportional to the number of cells firing. This method was considered applicable since spike heights and frequencies did not vary significantly between thalamic points and since the power output recorded by the electrode largely reflected changes in the population multiunit activity. In this study, a PDP-8 computer was used to obtain averaged measures of rms activity level for three time epochs after the presentation of the T pulse. When the C-C interval was set at 100 msec, three epochs of 12.8 msec were used. When the C-C interval was set at 50 msec, three epochs of 6.4 msec were used. These values were chosen so that approximately half of the C-C interval would be analyzed. Since the maximum C-T interval in each condition was half of the C-C interval, this method assured that activity values computed for all C-T intervals would be based on neural activity occurring during an equal time period. In addition, the first 3.2 msec of activity after the T pulse were discarded in order to avoid any possibility of contaminating the first epoch measure with aftereffects of the stimulus. Thus, the latency of the first epoch was 3.2 msec after the presentation of the T pulse. Average response measures in each epoch were then based on a mean of 1,800 sweeps per C-T interval. In the chronic study, these averages were made while the animal was bar pressing so that a direct comparison of electrophysiological and behavioral responses could be made. As an additional control for these techniques, the entire experimental procedure was repeated for two animals in the acute and chronic preparations, respectively, after they had been sacrificed with an overdose of the anesthetic (1-cc diabutol ip). As can be seen in Figure 2, the results of the analysis for each epoch are not due to artifacts of the stimulus or the averaging process, since the
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FIQUKE 3. Percentage of multiunit response in each epoch for the chronic (freely moving) and acute (anesthetized) preparations. (Method for obtaining the percentized composites was the same as in Kestenbaum, Deutsch, and Coons, 1970.) response is at a low level and basically flat in the dead animal.
RESULTS Averaged electrophysiological data for the chronic and acute (anesthetized) experiments are shown in Figure 3. Both the facilitatory and inhibitory effects of lemniscal stimulation are apparent in these graphs of integrated thalamic activity. As expected, short-latency driving, as represented by first epoch activity, is smaller in magnitude than
second and third epoch activity except for the very shortest C-T intervals (3.3-6 msec). This is due to the fact that presentation of the T pulse invades the inhibition generated by previous pulses unless the C-T interval is small enough to permit a facilitative interaction between the C and T pulses. Subsequent higher activity levels in the second and third epochs represent the release of new excitation. These facilitative and inhibitory effects
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were apparent in the multiunit records of represents the entrance and emergence of individual subjects. Inhibition was released ventrobasal neurons from a period of inat all thalamic points in all animals. Al- hibition. In addition, at the very shortest though all thalamic points were excited by intervals (3.3-6 msec), facilitation of the the lemniscal electrode, some showed more excitatory effects of the C and T pulses takes pronounced short-latency driving than place, and first epoch activity rises above the others, suggesting that points of maximal level of thalamic responding in the second excitation exist for a given placement of the and third epochs. stimulating electrode. Although the responses in the second and In the chronic preparation, thalamic ac- third epochs take on a more complicated tivity driven by a train of lemniscal pulse oscillatory pattern representing the interacpairs is characterized by a short-latency tion of both excitatory and inhibitory influ(first epoch) response which takes on a ences, two effects are very clear. For C-T basically U-shaped form. At C-T intervals intervals of 3.3-6 msec, second and third of 3.3-15 msec, first epoch response strength epoch activity is low because of high redecreases as C-T interval increases. As C-T sponding in the first epoch. For longer C-T interval is increased from 15 to 50 msec, intervals, however, the T pulse invades the however, first epoch activity also increases. inhibition of previous pulses, and first epoch In the case of animals run under the 50-msec. activity is low. New excitation released by condition, this upturn is not seen until the the T pulse then results in the increased second epoch. However, the epochs were activity levels seen in the later episodes. half size in this condition, and as such, first Differences between the acute and chronic and second epochs covered the same time preparations are consistent •« ith the observaperiod in the 50-msec C-C condition as the tion that the time course of thalamic inhibifirst epoch alone in the 100-msec C-C condi- tion is longer in the anesthetized state. Thus, tion. first epoch activity for the acute study folUBoth the fall and the rise of short-latency lows a U-shaped course with the C-T trough activity as C-T interval is increased from occurring at 30 msec instead of 15 msec, as in 3.3 to 50 msec are consistent with the ob- the chronic study. Second and third epoch servation that lemniscal stimulation gen- responses in the anesthetized preparation are orates short-latency excitation followed by also above first epoch responses, showing the long-lasting inhibition in ventrobasal neu- rebound effect generated \\hen lemniscal rons. The U-shaped first epoch response thus pulses invade thalamic inhibition released by previous pulses. BEHAVIORAL COMPOSITE (PRESSES PER MINUTE) Behavioral response data are shown in Figure 4. The pattern of C-T responding generally resembles the results obtained by Kestenbaum, Deutsch, and Coons (1970) except for a marked drop in response level as the C-T interval is shortened from 6 to 3.3 msec and an intermediate peak in the response curve at a C-T interval of 20 msec. There is also a slight increase in response as the C-T interval is increased from 30 to 50 msec so that the overall pattern of responding is slightly U-shaped. The form of the C - T INTERVAL ( M S E C ) behavioral response thus shows a general FIQUKE 4. Behavioral response functions for resemblance to the form of the first epoch animals in the chronic study with C-C intervals response obtained in the chronic study. of 100 msec or 50 msec. (Percentized composites A product-moment r was computed for the were obtained using the same method as Kestensix chronic animals between averaged thalabaum, Deutsch, and Coons, 1970.
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mic activity and behavioral response strength. A value of +.39 was obtained between presses per minute and first epoch activity, which is significant at the 1 % level. Correlations with second and third epoch activity were +.19 and +.20, which are not significant. DISCUSSION The results of the analysis of the multiunit recordings are consistent with studies done on anesthetized decorticate cats (Andersen & Andersson, 1968), in which intracellular recordings were made from ventrobasal relay neurons. In these studies, peripheral lemniscal stimulation, ventrobasal stimulation, and antidromic thalamic stimulation were used. Results showed the existence of long-lasting inhibitory-postsynaptic-potential-produced periods of hyperpolarization after lemniscal stimulation. In addition, these researchers attribute these processes to the operation of numerous multisynaptic recurrent postsynaptic inhibitory loops possessing lateral inhibitory features as well. Lemniscal stimulation would therefore be expected to result in widespread, long-lasting inhibition throughout the ventral thalamus, as was found in this study. Andersen and Andersson have also found evidence of resetting properties for thalamic rhythmicity so that an antidromic or sufficiently strong orthodromic stimulus occurring during the inhibitory phase of the ventrobasal cycle can either shorten or prolong the time course of emergence from inhibition. These effects were observed in this study insofar as second and third epoch responses were often higher in intensity than first epoch responses and their cycles of inhibition and recovery shifted. The expressed purpose of this study was to assess the correspondence between overt behavior and thalamic activity in lemniscally stimulated animals in order to determine the extent to which the ventrobasal thalamic nuclei act as a final integrating center for the motivating aspects of pain-producing stimuli. As has been noted, a significant resemblance does exist between the form of the behavioral response and short-latency thala-
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mic activity following the T pulse, when the C-T interval is varied. This correspondence, however, does not indicate that the ventral thalamus is the only relay point in the pain pathway involved in integrating the motivating aspects of the stimulation, insofar as the correlation for first epoch activity is not perfect and the correlations for second and third epoch activity are not significant. The evidence therefore suggests that additional elaboration of the pain message is carried on outside of the ventrobasal complex. This means that although the thalamus alone does not make the decision to perform an escape response, it is involved to a significant degree in transmitting information about the motivating aspects of pain-producing stimuli. In summary, the following conclusions can be drawn from the shape of the averaged electrophysiological response graphs and averaged behavioral response graphs: 1. The U-shaped electrophysiological function exhibited in the first epoch, although significantly correlated with the shape of the behavioral response function, does not indicate that the pain message is fully elaborated at the level of the ventral thalamus, insofar as the correlation is imperfect, Low correlations of the behavioral function with second and third epoch responses further reinforce this conclusion. 2. The low level of responding at the very shortest C-T intervals in the second and third epochs, combined with high first epoch responding at these intervals, provides evidence of an additional excitatory effect which shows the property of facilitation. This phenomenon has not been noted in previous studies. 3. Agreement between the results of the multiunit integration technique used in this study and the results of prior experiments involving intraccllular recording is good enough to validate this technique for work involving freely moving animals in which chronic intracellular recording would be difficult if not impossible. This, fortunately, means that an interpretable measure of both excitatory and inhibitory neuronal population responses can be obtained in conscious,
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freely moving animals while studies are made of underlying motivational systems. REFERENCES Andersen, P., & Andersson, S. Physiological basis of the alpha rhythm. New York: AppletonCentury-Crofts, 1968. Arduini, A., & Pinnio, L. A method for the quantification of tonic activity in the nervous system. Archives Italiennes de Biologia, 1962, 700, 415422. Kaufman, L., & Price, R. The detection of cortical spike activity at the human scalp. IEEE Transactions on Bio-Medical Engineering, 1967, 14, 84. Kestenbaum, R. 8., Deutsch, J. A., & Coons, E. E. Behavioral measurement of neural post-stimulation excitability cycle: Pain cells in the brain of the rat. Science, 1970, 167, 393.
KOnig, J. F. R., & Klippel, R. A. The rat brain: A stereotaxic atlas. Baltimore: Williams & Wilkins, 1963. Michaels, J. A., & Kaufman, L. High frequency visual evoked responses: A product of neuronal spiking. Experimental Brain Research, 1969, IB, 255-258. Olds, J., Disterhoft, J. F., Segal, M., Kornblith, C. L., & Hirsh, R. Learning centers of rat brain mapped by measuring latencies of conditioned unit responses. Journal of Neurophysiology, 1972, 35, 202-219. Olds, J., & Peretz, B. A motivational analysis of the reticular activating system. Electroencephalography and Clinical Neurophysiology, 1960, 18, 445-454. Olds, M. E., & Olds, J. Approach-avoidance analysis of rat diencephalon. Journal of Comparative Neurology, 1963, 170, 259-295. (Received May 3, 1974)
