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Conditioned responses of gamma and alpha motoneurons in the cat trained to conditioned avoidance
EXPERIMENTAL
NEUROLOGY
Conditioned
4, 91-105 (1961)
Responses
Motoneurons
of
in the
Conditioned JENNIFER Department
S.
BUCHWALD,
Cat
and
Trained
Alpha to
Avoidance
DONALD
of Anatomy, School of Medicine, and Veterans Administration
California,
Received
Gamma
April
BEATTY, University Hospital,
AND
EARL
of
California, Beach,
Long
ELDRED Los
Angeles,
California
25, 1961
The relationship of gamma-efferent conditioned responses to alpha-motoneuron, or behavioral, conditioned responses has been studied. Seven cats were trained to give a consistent hind-leg conditioned avoidance response, i.e., to give overt evidence of conditioned responses in alpha fibers. In terminal experiments, these animals were immobilized and gamma and alpha units from ventral roots innervating the hind leg were monitored for conditioned changes in activity. The same CS (lOOO-cycles/see tone) and US (shock to the hind paw) were used in the terminal experiments as were employed during training. In all chronically trained cats, conditioned responses of both gamma and alpha fibers were induced by the CS. When the neuromuscular block was incomplete, gamma acceleration and alpha discharge to the CS occurred concurrently with actual withdrawal of the conditioned hind leg. Acceleration of gamma-fiber discharge induced by the CS appeared with a much shorter latency than did the alpha-fiber discharge. During extinction the gamma response persisted for many trials after the alpha response was abolished. These findings support the concept that a background of conditioned proprioceptive activity, mediated by the gamma-efferent system, underlies the conditioned avoidance response.
introduction In acute experiments on naive cats, conditioned acceleration of gammaefferent fibers can be developed as early as the tenth presentation of tone (CS) paired with shock to the hind paw (US) (2). This response of the gamma motoneurons innervating muscle spindles is believed to represent true conditioning, because: (a) the response was not present initially, but developed during the pairing of CS and US and could subsequently be elicited by the CS alone; (b) the acquired response could be extinguished and then re-established with fewer CS-US trials than were originally re91
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BUCHWALD, BEATTY, AND ELDRED
quired to develop it; and (c) presentation of the unpaired CS and US in alternating sequence produced no response, whereas pairing the CS and US for the same number of trials evoked a response to the CS. The early appearance of gamma-fiber conditioned responses suggests that conditioned changes in the gamma-efferent system may facilitate development of the behavioral conditioned response. With every presentation of the CS, the conditioned gamma-fiber response would produce increased proprioceptive feedback from the muscle, This increased sensory discharge would directly impinge upon spinal motoneurons as well as project to supraspinal centers. Such spinal and supraspinal activation produced by conditioned gamma-efferent responses might significantly facilitate the acquisition and maintenance of the overt conditioned response. If behavioral conditioned responses are facilitated by a background of conditioned changes in the gamma system, these changes must be present during or preceding the discharge of alpha motoneurons producing movement in the behavioral response. This relationship of gamma- to alphaconditioned responses could not be studied in the acutely trained cat, for alpha fibers fail to develop conditioned responses in the limited time this preparation can be maintained. In the work reported here, seven cats have first been chronically trained to give a conditioned hind-leg flexion, i.e., to give overt evidence of conditioned responses in alpha fibers. Then, in terminal experiments under Flaxedil, gamma and alpha units from ventral roots innervating the hindleg were monitored for conditioned changes in activity. The use of Flaxedil was essential in these experiments because chronic techniques of gamma-unit recording are not yet available and the motor conditioned response is impaired by general anesthesia. Great care was taken to minimize discomfort of the animals. Their condition is indicated by the hind-leg flexor response elicited by the CS alone, when Flaxedil paralysis diminished sufficiently to allow movement. Before and after presentation of the CS, the animal was quiet. In all of these chronically trained cats, conditioned responses of both gamma and alpha motoneurons were induced by the CS. Conditioned acceleration of gamma-fiber discharge appeared with a much shorter latency than the alpha-fiber discharge. During extinction the gamma response persisted for many trials after the alpha response was abolished. These findings indicate that conditioned changes in the gamma-efferent system accompany and may facilitate the behavioral conditioned response.
GAMMA
AND
ALPHA
CONDITIONING
93
Methods
Seven adult cats were chronically trained to give an avoidance conditioned response. During each training session the cat, held in a hammock, was given twenty-five to fifty trials at 30- to 60-set randomly selected intertrial intervals. The conditioning stimulus, a 1S-set, lOOO-cycles/set tone generated by an audio-oscillator (Hewlett-Packard Co.), was delivered through a speaker 14 inches in front of the animal’s head. The unconditioned stimulus was a 60-cycles/set shock delivered during the last 0.5 set of the CS period through bipolar stimulating electrodes taped to the dorsum of the left hind paw. The US was withheld if a conditioned response was elicited by the CS. The animals did not resist the training situation and between trials rested quietly in the hammock. In the terminal experiments, ether anesthesia was used during the surgical procedure, which consisted of inserting tracheal and venous cannulae and exposing the lower lumbar cord. Throughout the period of recording, the animal was maintained on Flaxedil (American Cyanamid Co.) and wound edges were infused with Xylocaine (Astra Pharmaceutical Products). The cat was suspended in the training hammock in a fashion identical to that used during the chronic training sessions. Body temperature of the animal was maintained by a heater below the hammock and the oil pool formed around the spinal cord was maintained at 37-38” C by radiant heat. The laboratory was kept darkened and quiet. Ventral roots L6 and L7 were sectioned at their exit through the dura mater while the animal was under ether. Gamma-efferent and alpha units in the ventral roots were not isolated until the animal had been artificially respired for an hour. The tonic, low-amplitude discharge of gamma-efferent fibers was generally easily distinguishable from the highamplitude bursts of alpha fibers (8). Recordings were made bipolarly. Conditioning and unconditioned stimuli in the terminal experiments were the same as those used during the chronic training sessions. General anesthesia is incompatible with the appearance of gamma- and alpha-unit conditioned responses. Conditioned responses to the CS were blocked by ether for at least 1 hour after termination of this anesthetic. Nembutal, injected intravenously at a dose of 8 mg per kilogram, completely silenced tonically discharging gamma units and prevented gammaunit responses to the CS and US which were previously present.
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BUCHWALD,
BEATTY,
AND
ELDRED
Results
Each of the cats trained in daily sessionsof twenty-five to fifty presentations of tonal CS paired with shock to the hind paw began to show conditioned flexor withdrawal of the paw after one hundred to two hundred trials. By five hundred trials the animals gave responsesat a 90-100 per cent level. After the level of performance had been sustained at 90 per cent or better for five consecutive sessions,observations were made on ventral-root activity during a terminal experiment. Filaments were selected from the L6 or L7 ventral roots. These roots contribute heavily to flexor muscles (13) concerned in the conditioned response; only fibers which readily respondedto pinching the hind foot were used. Relation of Gamma- and Alpha-Fiber Responsesto the Behavioral Response. Initial presentation of the tonal CS to the chronically trained cat causedacceleration in the tonic dischargeof gammafibers and induced a burst of potentials in the alpha fibers. In two cats showing this gamma acceleration and alpha dischargeto the CS, Flaxedil paralysis was allowed to diminish so that movement could occur. In these animals the behavioral conditioned responsewas elicited by the CS. This overt response, a discrete flexion of the conditioned hind leg similar to that exhibited during training, was accompaniedby dischargeof the samegamma and alpha fibers from which responseswere recorded before the movement occurred. Thus, direct observation showed that conditioned changesin gamma-fiber activity occur during the overt conditioned avoidance response. The appearance of alpha-fiber discharge in ventral roots innervating flexor muscles at the same time that conditioned flexion of the leg occurred identifies the alpha discharge as a component of the specific conditioned movement. Consistent with this identification, alpha discharge to the CS never developed in twelve acutely trained cats given up to fifty CS-US trials under Flaxedil; neither were conditioned avoidance responsesseenin chronically trained cats in fifty trials. On the other hand, both alpha-fiber and behavioral responseswere present in cats given several hundred CS-US trials. The above points are illustrated in Fig. 1, which contrasts records from a cat acutely conditioned (trace A) with those of a chronically trained cat (B and C). In the acutely trained cat, acceleration of gamma-fiber activity developed after ten CS-US pairings, but even after thirty-five trials there was no alpha-fiber response. In contrast, the cat chronically trained over three hundred fifty trials showed a burst of high-amplitude,
GAMMA
AND
ALPHA
CONDITIONING
95
A
B
C
FIG. 1. Conditioned responses of efferent units in acutely and chronically trained cats. A, conditioned acceleration of gamma fiber to CS alone in cat acutely trained under Faxedil. Cat had received thirty-five presentations of CS (1000 cycles/set, IS-see duration tone) paired with US (overlapping 60 cycles/set, OS-set duration shock to the hind paw). Recording from filament isolated from L6 ventral root innervating hind-leg flexor muscles. Alpha fibers present in this filament fired in response to the US but not to the CS. Onset of CS indicated by artifact on record and duration by the bar below the trace. B, conditioned responses of gamma and alpha fibers in cat chronically conditioned to give a flexor withdrawal of the hind leg. This cat had been chronically trained over three hundred fifty trials to give avoidance responses consistently at the 90 per cent level; the CS and US were the same as for the acutely conditioned cat. Low-amplitude, tonically discharging spikes obtained with bipolar pickup represent gamma-fiber activity and the high-amplitude bursts, alpha-fiber activity. Acceleration of gamma-fiber discharge and a burst of alpha-fiber potentials were induced by the CS alone in this filament isolated from the L6 ventral root innervating the conditioned hind leg. The EKG activity was unaffected by the CS. C, recording from the same filament as in B, but after partial recovery from Flaxedil paralysis had occurred. Presentation of the CS induced a burst of the same alpha fibers seen in B simultaneously with an elevation of the conditioned hind leg. Movement of the leg is indicated by deflections in the baseline.
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BUCHWALD,
BEATTY,
AND
ELDRED
alpha-fiber potentials to the CS (Fig. 1B). When the level of neuromuscular block diminished, the same alpha fibers discharged as the animal withdrew his leg (C) . This movement is indicated by the gross deflections of the baseline. The remarkable stability of the conditioned avoidance response is shown in chronically trained animals by its persistence after 2 hours of ether anesthesia during surgery and several hours of complete paralysis under Flaxedil. In two cats in which paralysis was allowed to diminish, presentation of the CS produced flexion of the conditioned hind leg. This flexion was typical of the conditioned withdrawal exhibited by the cats during training. Late&es of Gamma- and Alpha-Fiber Responses. Acceleration in the tonic firing of gamma-efferent units in response to the CS consistently preceded the burst of alpha-fiber potentials recorded from the same ventral root filament. Figure 2 illustrates this gamma acceleration preceding the alpha discharge during sequential trials in one chronically trained cat. It is evident that the latency of gamma acceleration was very brief, as the second interspike interval after onset of the CS was shortened. Closer inspection reveals that there was often shortening of the interval between the last discharge prior to the onset of the CS and the first spike after the CS. This suggests that the latency of gamma acceleration was less than the duration of a single interspike interval. An attempt to measure this latency is illustrated in Fig. 3. In this experiment the responses of a single gamma fiber were followed over fifty-two trials. For each trial the durations of five interspike intervals preceding the CS were determined. The mean of these five durations was then projected from the last discharge after the CS. This predicted time for each trial is indicated by a dot on the graph. Actual time of the first discharge after the CS is shown on the graph by a cross. If the predicted and actual time of discharge coincided, acceleration of the gamma unit had not occurred. Appearance of the spike earlier than the predicted time indicated acceleration of the gamma unit. In Fig. 3, predicted and actual times of discharge coincide at about 13 msec. Thereafter, an increasing gap between the predicted and actual discharge times occurs, indicating that acceleration has taken place. The somewhat irregular discharge of gamma units makes determination of latency difficult. However, the responses of three units from two cats all show a response latency of not more than 18 msec. Alpha fibers generally did not discharge tonically, but with presentation
GAMMA
AND
ALPHA
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97
of the CS a train of spikes appeared which often outlasted the tone for several tenths of a second. The latency of the alpha-fiber responses to the CS was determined by measuring from the electrical artifact marking onset of the tone to the first alpha-fiber potential.
FIG. 2. Gamma- and alpha-fiber conditioned responses in a chronically trained cat. Presentation of the CS alone causes acceleration of tonically discharging gammaefferent fiber and initiates discharge of high amplitude alpha potentials. Onset of CS indicated by artifacts on records and duration by bar beneath. Gamma acceleration precedes the alpha discharge in each of these sequential trials (A,B,C).
Latencies of 40 to 50 msec were found in two cats; the other animals showed minimal latencies of 80 to 100 msec. The latencies of alpha-fiber discharge to the CS in one animal are shown in Fig. 4, top line. The initial latencies were about 80 msec. After repeated presentation of the CS without reinforcement the latency of the responseincreased to over 300
98
BUCHWALD,
BEATTY,
AND
ELDRED
msec. Four reinforcements of the CS by shock to the hind paw caused the latency to decrease sharply to about 50 msec, a shorter delay than recorded early in the trial series. Similar increase in latency of discharge during extinction and marked decrease following reinforcement was ob-
. PREDICTED x ACTUAL
b
Msec
lb TIME
FROM
20 CS TO INITIAL
30 DISCHARGE
40
FIG. 3. Latency of gamma-fiber acceleration to CS. Discharge of a single gamma fiber was followed during fifty-two presentations of the CS. During each trial the durations of five interspike intervals preceding the CS were measured. The mean of these durations was then projected from the last discharge preceding the CS to predict when the next discharge should occur, indicated by dots. The trials have been arranged according to the length of this predicted time of discharge. Actual time of discharge on each trial is shown by a cross. Where predicted and actual times of discharge coincide, or show random variance, no acceleration of the gamma discharge has taken place. Occurrence of the actual time of discharge before the predicted time indicates acceleration of discharge. Acceleration was present at all times greater than 18 msec.
served in the other cats. Latencies as long as 800 to 900 msec occurred during some extinction trials. Extinction of Gamma- and Alpha-Fiber Response. Both the alpha- and gamma-fiber responses could be extinguished and then re-established by
GAMMA
AND
ALPHA
CONDITIONING
99
reinforcement. However, conditioned gamma-fiber acceleration persisted long after the alpha-fiber discharge was abolished. As shown in Fig. 4, bottom line, a gradual decrease in alpha-fiber discharge rate to the CS occurred during extinction. The initial rate of 40 pulsesper seconddimin-
TRIALS
WITH
1000
c/s CS
FIG. 4. Alpha-fiber conditioned response during extinction and reinforcement. Latency of alpha discharge (top line) increased from 80 msec to over 300 msec with continued presentations of unreinforced CS. After four US reinforcements of the CS (indicated by arrows in bracket), the latency drops abruptly to 50 msec. The rate of alpha discharge (bottom line), during the same trials for which latency is plotted, represents the number of alpha potentials during the l-set interval following the first alpha potential elicited by the CS. This value diminished with repeated presentations of the unreinforced CS until the response was nearly extinguished. Between every trial indicated on the graph, a neutral, unreinforced tone was presented which may have hastened extinction to the CS. After four reinforcements of the CS, the rate of alpha discharge increased to about 20 per second. The inverse relationship between rate of alpha discharge and latency of discharge can be seen both in the general trends and in the individual trials.
ished until essentially no responsewas elicited by the CS. Following the first reinforcement, an increase in the alpha discharge rate to the CS occurred and after four reinforcements, the rate had increased to 20 per second. Oscilloscopic traces taken from this extinction series are shown
E
Tone
1.5 set
l-
FIG. 5. Extinction and re-establishment of alpha-fiber conditioned response. At the beginning of the extinction trials (A), the CS elicits acceleration of the tonically discharging gamma unit and initiates high-amplitude alpha discharge. Onset of CS is indicated by artifact on the trace and duration of CS by bar at bottom of figure. After repeated unreinforced presentations of the CS, alpha discharge was abolished leaving only the gamma-fiber acceleration (B). When the first US reinforcement was given (C), alpha potentials appeared during the 0.5-set shock period, but not during the CS period preceding the shock. On the next trial, a feeble alpha-fiber discharge occurred during the CS period preceding the second reinforcement (D). After five reinforcements the alpha-fiber response to the CS alone (E) had been reestablished.
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AND
ALPHA
CONDITIONING
101
A B C
D
E
F FIG. 6. Extinction and re-establishment of gamma-fiber conditioned response. Both acceleration of gamma-fiber discharge and alpha-fiber discharge were induced by the CS initially (A). Onset of CS indicated by artifact on the trace and duration of CS by bar at bottom of figure. After three hundred unreinforced presentations of the CS, the alpha responses were abolished from the filament shown in A and from a second fiber (B). Gamma acceleration to the CS, however, was still present (B). After two hundred more unreinforced trials the gamma-fiber response was nearly extinguished (C). The first reinforcement elicited both gamma acceleration and alpha discharge to the OS-set shock, but no response during the preceding CS period (D). In the next trial following the first reinforcement, marked acceleration in gamma activity was elicited by the CS, preceding the shock (E). Acceleration of gamma activity to the CS alone persisted after reinforcement was discontinued (F).
102
BUCHWALD,
BEATTY,
AND
ELDRED
in Fig. 5. Initially, strong alpha discharge of long duration was produced by the CS (trace A). After repeated, unreinforced presentations of the tone, the alpha response dropped out, although acceleration of gammaefferent discharge continued (B). When the first reinforcement was given (C) a burst of alpha potentials appeared during the OS-set period of shock, but not during the preceding tone. On the next trial a feeble alpha-fiber discharge occurred during the tone preceding the shock (D). After five reinforcements, the alpha-fiber response to the CS alone had been re-established (E). The CS continued to elicit the response through another series of twenty unreinforced trials. The gamma-efferent response to the CS could be extinguished only with a much larger number of trials than were needed to abolish the alpha response. In the experiment of Fig. 6, both alpha discharge and gamma acceleration were elicited by the CS (A). After three hundred unreinforced trials, the response of alpha fibers in this filament was abolished, but gamma-efferent acceleration to the CS still occurred. Trace B, taken from a second filament, shows this persisting gamma acceleration. After a series of two hundred more unreinforced trials, the gamma-fiber response was essentially extinguished (C, and left part of D). The first reinforcement elicited gamma- and alpha-fiber discharge to the shock, but none to the tone (D). In the trial following the first reinforcement, acceleration of the gamma unit to the CS appeared preceding the shock (E). After several reinforcements, marked acceleration of gamma discharge was elicited by the CS alone (F). Discussion
A background of conditioned proprioceptive activity, mediated by the gamma-efferent system, seems to underlie the conditioned avoidance response. Two sources of evidence support this concept. First, acceleration of gamma discharge during the conditioned avoidance response was directly observed when actual withdrawal of the conditioned leg occurred as Flaxedil paralysis diminished. This direct proof of participation of the gamma system in the behavioral conditioned response is supported by the recording of gamma-activation during alpha-fiber conditioned responses. The alpha-fiber response is identified as a component of the behavioral avoidance response, for only in animals previously trained to give consistent hind-leg withdrawal was alpha-fiber discharge recorded in roots innervating that leg. If the neuromuscular block were incomplete, with-
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AND
ALPHA
CONDITIONING
103
drawal of the hind-leg to the CS occurred concurrently with the conditioned bursts of alpha activity. Moreover, the response in alpha fibers resembled the behavioral response, for (a) it could be extinguished by repeated presentations of the CS alone, (6) the latency of response increased during extinction trials, and (c) the response could be re-established with a few reinforcements. A second line of evidence, which indicates that a background of conditioned proprioceptive activity underlies the behavioral conditioned response, is the greater sensitivity to conditioning procedures in the gammaefferent system than in the alpha-motor system. This greater sensitivity is shown in several ways: (a) Conditioned responses of gamma fibers developed early in training when alpha or behavioral responses were not yet attainable; (b) gamma-fiber responses to the CS persisted after total extinction of alpha-fiber responses; and (c) in any single trial, the gammafiber conditioned response occurred with a shorter latency than did the alpha-fiber response. As a consequence of this low-threshold conditioning of gamma-efferent activity, changes in proprioceptive discharge preceded alpha-conditioned responses both during training and on a single trial. The net effect of enhanced gamma-efferent activity and the resultant acceleration of proprioceptive discharge is facilitation of the alpha motoneurons. For example, the tonic excitation of motoneurons in the classic decerebrate animal is dependent upon the presence of greatly enhanced firing of gamma fibers (4). Similarly, in conditioned learning the early, low-threshold changes in gamma-efferent, muscle-spindle circuits may facilitate the appearance of the overt conditioned response. Latencies of the alpha-fiber response were adequate for the conditioned changes in proprioceptive inflow to reach the ventral horn and facilitate the alpha motoneurons. The time required for centrifugal conduction down gamma fibers, activation of spindle afferents, and conduction back to the cord is, for distal limb muscles, about 15 msec ( 10). This time added to the observed 18-msec latency of gamma-fiber acceleration indicates that alteration in proprioceptive inflow could reach ventral horn cells 33 msec after the onset of the CS. The altered afferent inflow could also project upward to the brain stem in an additional 6 msec (5). Assuming several milliseconds delay in the reticular formation, the effects of this afferent inflow might return to the lumbar cord within another 10 msec (7). Thus, in less than 50 msec-33 msec for gamma activation and proprioceptive feedback to the cord, 6 msec for conduction upward, and
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BUCHWALD,
BEATTY,
AND
ELDRED
10 msec for relay and return to the cord-conditioned gamma-efferent reinforcement of reticular formation activity could be reflected at the spinal level. The very short delay from onset of the CS to beginning of acceleration in gamma-fiber activity indicates that this response is mediated through a relatively direct pathway. In the experimental situation employed, almost 1 msec was needed for the tone to reach the ear. After sound has reached the middle ear, responses in the brain stem can appear in 2.5 to 3.0 msec (1). Additional time is required for relay in the reticular formation. From the midbrain reticular formation, fast pathways to gammamotor fibers in lumbar ventral roots have a conduction time of 8 msec (6). Thus, at least 12 msec are theoretically needed for a gamma response to tone to appear. Transmission of the conditioned gamma response along this direct route could be accommodated within the minimal latency of 18 msec indicated by our data. The early appearance of conditioned changes in reticular formation activity (14) suggests that this system could indeed contribute to the earliest conditioned changes in gamma-fiber activity. A route involving the cortex is not possible within the minimum latency at which gamma acceleration appears. One millisecond for tone conduction to the ear, 8 msec for a response to tone to appear in the auditory cortex (1, 9) and several milliseconds delay for intracortical events and conduction downward to the midbrain level must be added to the 8 msec required for fast conduction from the midbrain level to the lumbar segment (6). This route would probably require 20 msec, too long for initiation of the gamma-fiber response. In contrast, the alpha-fiber conditioned response and its behavioral counterpart, conditioned avoidance, probably are mediated by pathways involving the cortex. Several studies indicate that avoidance responses to a tonal CS conditioned in the intact cat must be relearned after complete bilateral destruction of the auditory cortex (12, 11, 3). Similarly, it has been shown that defensive conditioned responses are inhibited by focal seizures in the CS projection area (auditory or optic) produced by cortical stimulation ( 15). Only in part can the long latency of the behavioral, or alpha-conditioned response be accounted for by conduction delays in pathways involving the cortex. As calculated above, the effects of an acoustic stimulus relayed through the cortex and down to spinal levels could occur by the
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fast pathwaysin 20 msec. The minimum latency of the alpha-fiberconditioneddischarge,however,exceededthis time by 30msecor more. This additional time must be consumed in relays through the various association centers concerned with maintenance of the behavioral conditioned response. The present findings support the concept that a background of conditioned proprioceptive activity, mediated by the gamma-efferent system, underlies the conditioned avoidance response. References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10.
11. 12. 13. 14.
15.
ADES, H. W., and J. M. BROOKHART, The central auditory pathway. J. Neurophysiol. 13: 189-206, 1950. BUCHWALD, J., and E. ELDRED, Conditioned responses in the gamma efferent system. J. Nervous Mental Disease 132: 146-152, 1961. BUTLER, A., I. T. DIAMOND, and W. D. NEFF, Role of auditory cortex in discrimination of changes in frequency. 1. Neurophysiol. 20: 108-120, 1957. ELDRED, E., R. GRANIT, and P. A. MERTON, Supraspinal control of the muscle spindles and its significance. J. Physiol. London 12!2: 498-523, 1953. FRENCH, J. D., M. VERZEANO, and H. W. MAGOWN, An extralemniscal sensory system in the brain. A.M.A. Arch. Neural. Psych&. 69: 505-518, 1953. GRANIT, R., and B. HOLMGREN, Two pathways from brain stem to gamma ventral horn cells. Acta Physiol. Stand. 35: 93-108, 1955. GRANIT, R., 0. POMPEIANO, and B. WALTMAN, Fast supraspinal control of mammalian muscle spindles: extra and intrafusal co-activation. J. Physiol. London 147: 385-398, 1959. HUNT, C. C., The reflex activity of mammalian small-nerve fibers. J. Physiol. London 115: 456-469, 1951. KATSUKI, Y., T. WATANABE, and N. MARUYAMA, Activity of auditory neurons in upper levels of brain of cat. J. Neurophysiol. 22: 343-359, 1959. KUFFLER, S. W., C. C. HUNT, and J. P. QIJILLIAM, Function of medullated smallnerve fibers in mammalian ventral roots: efferent muscle spindle innervation. J. Neurophysiol. 14: 20-54, 1951. RAAB, D. W., and H. W. ADES, Cortical and midbrain mediation of a conditioned discrimination of acoustic intensities. Am. J. Psychol. 59: 59-83, 1946. ROSENZWEIG, M., Discrimination of auditory intensities in the cat. Am. J. Psychol. 52: 126-136, 1946. SHERRINGTON, C. S., Notes on the arrangement of some motor fibers in the lumbo-sacral plexus. 1. Physiol. London 13: 621-772, 1892. YOSHII, N., J. MATSUMOTO, H. OIXRA, M. SHIMOK~CHI, Y. YAMAGUCHI, and H. YAMASAKI, Conditioned reflex and electroencephalography. In “The Moscow Colloquim on Electroencephalography of Higher Nervous Activity,” H. H. Jasper and G. D. Smirnov (eds.). Electroencephalog. and Cl&. Neurophysiol. Suppl. 13: 199-210, 1960. ZUCKERMAN, E., Effect of cortical and reticular stimulation on conditioned reflex activity. J. Neurophysiol. 22: 633-643, 1959.
NEUROLOGY
Conditioned
4, 91-105 (1961)
Responses
Motoneurons
of
in the
Conditioned JENNIFER Department
S.
BUCHWALD,
Cat
and
Trained
Alpha to
Avoidance
DONALD
of Anatomy, School of Medicine, and Veterans Administration
California,
Received
Gamma
April
BEATTY, University Hospital,
AND
EARL
of
California, Beach,
Long
ELDRED Los
Angeles,
California
25, 1961
The relationship of gamma-efferent conditioned responses to alpha-motoneuron, or behavioral, conditioned responses has been studied. Seven cats were trained to give a consistent hind-leg conditioned avoidance response, i.e., to give overt evidence of conditioned responses in alpha fibers. In terminal experiments, these animals were immobilized and gamma and alpha units from ventral roots innervating the hind leg were monitored for conditioned changes in activity. The same CS (lOOO-cycles/see tone) and US (shock to the hind paw) were used in the terminal experiments as were employed during training. In all chronically trained cats, conditioned responses of both gamma and alpha fibers were induced by the CS. When the neuromuscular block was incomplete, gamma acceleration and alpha discharge to the CS occurred concurrently with actual withdrawal of the conditioned hind leg. Acceleration of gamma-fiber discharge induced by the CS appeared with a much shorter latency than did the alpha-fiber discharge. During extinction the gamma response persisted for many trials after the alpha response was abolished. These findings support the concept that a background of conditioned proprioceptive activity, mediated by the gamma-efferent system, underlies the conditioned avoidance response.
introduction In acute experiments on naive cats, conditioned acceleration of gammaefferent fibers can be developed as early as the tenth presentation of tone (CS) paired with shock to the hind paw (US) (2). This response of the gamma motoneurons innervating muscle spindles is believed to represent true conditioning, because: (a) the response was not present initially, but developed during the pairing of CS and US and could subsequently be elicited by the CS alone; (b) the acquired response could be extinguished and then re-established with fewer CS-US trials than were originally re91
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BUCHWALD, BEATTY, AND ELDRED
quired to develop it; and (c) presentation of the unpaired CS and US in alternating sequence produced no response, whereas pairing the CS and US for the same number of trials evoked a response to the CS. The early appearance of gamma-fiber conditioned responses suggests that conditioned changes in the gamma-efferent system may facilitate development of the behavioral conditioned response. With every presentation of the CS, the conditioned gamma-fiber response would produce increased proprioceptive feedback from the muscle, This increased sensory discharge would directly impinge upon spinal motoneurons as well as project to supraspinal centers. Such spinal and supraspinal activation produced by conditioned gamma-efferent responses might significantly facilitate the acquisition and maintenance of the overt conditioned response. If behavioral conditioned responses are facilitated by a background of conditioned changes in the gamma system, these changes must be present during or preceding the discharge of alpha motoneurons producing movement in the behavioral response. This relationship of gamma- to alphaconditioned responses could not be studied in the acutely trained cat, for alpha fibers fail to develop conditioned responses in the limited time this preparation can be maintained. In the work reported here, seven cats have first been chronically trained to give a conditioned hind-leg flexion, i.e., to give overt evidence of conditioned responses in alpha fibers. Then, in terminal experiments under Flaxedil, gamma and alpha units from ventral roots innervating the hindleg were monitored for conditioned changes in activity. The use of Flaxedil was essential in these experiments because chronic techniques of gamma-unit recording are not yet available and the motor conditioned response is impaired by general anesthesia. Great care was taken to minimize discomfort of the animals. Their condition is indicated by the hind-leg flexor response elicited by the CS alone, when Flaxedil paralysis diminished sufficiently to allow movement. Before and after presentation of the CS, the animal was quiet. In all of these chronically trained cats, conditioned responses of both gamma and alpha motoneurons were induced by the CS. Conditioned acceleration of gamma-fiber discharge appeared with a much shorter latency than the alpha-fiber discharge. During extinction the gamma response persisted for many trials after the alpha response was abolished. These findings indicate that conditioned changes in the gamma-efferent system accompany and may facilitate the behavioral conditioned response.
GAMMA
AND
ALPHA
CONDITIONING
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Methods
Seven adult cats were chronically trained to give an avoidance conditioned response. During each training session the cat, held in a hammock, was given twenty-five to fifty trials at 30- to 60-set randomly selected intertrial intervals. The conditioning stimulus, a 1S-set, lOOO-cycles/set tone generated by an audio-oscillator (Hewlett-Packard Co.), was delivered through a speaker 14 inches in front of the animal’s head. The unconditioned stimulus was a 60-cycles/set shock delivered during the last 0.5 set of the CS period through bipolar stimulating electrodes taped to the dorsum of the left hind paw. The US was withheld if a conditioned response was elicited by the CS. The animals did not resist the training situation and between trials rested quietly in the hammock. In the terminal experiments, ether anesthesia was used during the surgical procedure, which consisted of inserting tracheal and venous cannulae and exposing the lower lumbar cord. Throughout the period of recording, the animal was maintained on Flaxedil (American Cyanamid Co.) and wound edges were infused with Xylocaine (Astra Pharmaceutical Products). The cat was suspended in the training hammock in a fashion identical to that used during the chronic training sessions. Body temperature of the animal was maintained by a heater below the hammock and the oil pool formed around the spinal cord was maintained at 37-38” C by radiant heat. The laboratory was kept darkened and quiet. Ventral roots L6 and L7 were sectioned at their exit through the dura mater while the animal was under ether. Gamma-efferent and alpha units in the ventral roots were not isolated until the animal had been artificially respired for an hour. The tonic, low-amplitude discharge of gamma-efferent fibers was generally easily distinguishable from the highamplitude bursts of alpha fibers (8). Recordings were made bipolarly. Conditioning and unconditioned stimuli in the terminal experiments were the same as those used during the chronic training sessions. General anesthesia is incompatible with the appearance of gamma- and alpha-unit conditioned responses. Conditioned responses to the CS were blocked by ether for at least 1 hour after termination of this anesthetic. Nembutal, injected intravenously at a dose of 8 mg per kilogram, completely silenced tonically discharging gamma units and prevented gammaunit responses to the CS and US which were previously present.
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Results
Each of the cats trained in daily sessionsof twenty-five to fifty presentations of tonal CS paired with shock to the hind paw began to show conditioned flexor withdrawal of the paw after one hundred to two hundred trials. By five hundred trials the animals gave responsesat a 90-100 per cent level. After the level of performance had been sustained at 90 per cent or better for five consecutive sessions,observations were made on ventral-root activity during a terminal experiment. Filaments were selected from the L6 or L7 ventral roots. These roots contribute heavily to flexor muscles (13) concerned in the conditioned response; only fibers which readily respondedto pinching the hind foot were used. Relation of Gamma- and Alpha-Fiber Responsesto the Behavioral Response. Initial presentation of the tonal CS to the chronically trained cat causedacceleration in the tonic dischargeof gammafibers and induced a burst of potentials in the alpha fibers. In two cats showing this gamma acceleration and alpha dischargeto the CS, Flaxedil paralysis was allowed to diminish so that movement could occur. In these animals the behavioral conditioned responsewas elicited by the CS. This overt response, a discrete flexion of the conditioned hind leg similar to that exhibited during training, was accompaniedby dischargeof the samegamma and alpha fibers from which responseswere recorded before the movement occurred. Thus, direct observation showed that conditioned changesin gamma-fiber activity occur during the overt conditioned avoidance response. The appearance of alpha-fiber discharge in ventral roots innervating flexor muscles at the same time that conditioned flexion of the leg occurred identifies the alpha discharge as a component of the specific conditioned movement. Consistent with this identification, alpha discharge to the CS never developed in twelve acutely trained cats given up to fifty CS-US trials under Flaxedil; neither were conditioned avoidance responsesseenin chronically trained cats in fifty trials. On the other hand, both alpha-fiber and behavioral responseswere present in cats given several hundred CS-US trials. The above points are illustrated in Fig. 1, which contrasts records from a cat acutely conditioned (trace A) with those of a chronically trained cat (B and C). In the acutely trained cat, acceleration of gamma-fiber activity developed after ten CS-US pairings, but even after thirty-five trials there was no alpha-fiber response. In contrast, the cat chronically trained over three hundred fifty trials showed a burst of high-amplitude,
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A
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FIG. 1. Conditioned responses of efferent units in acutely and chronically trained cats. A, conditioned acceleration of gamma fiber to CS alone in cat acutely trained under Faxedil. Cat had received thirty-five presentations of CS (1000 cycles/set, IS-see duration tone) paired with US (overlapping 60 cycles/set, OS-set duration shock to the hind paw). Recording from filament isolated from L6 ventral root innervating hind-leg flexor muscles. Alpha fibers present in this filament fired in response to the US but not to the CS. Onset of CS indicated by artifact on record and duration by the bar below the trace. B, conditioned responses of gamma and alpha fibers in cat chronically conditioned to give a flexor withdrawal of the hind leg. This cat had been chronically trained over three hundred fifty trials to give avoidance responses consistently at the 90 per cent level; the CS and US were the same as for the acutely conditioned cat. Low-amplitude, tonically discharging spikes obtained with bipolar pickup represent gamma-fiber activity and the high-amplitude bursts, alpha-fiber activity. Acceleration of gamma-fiber discharge and a burst of alpha-fiber potentials were induced by the CS alone in this filament isolated from the L6 ventral root innervating the conditioned hind leg. The EKG activity was unaffected by the CS. C, recording from the same filament as in B, but after partial recovery from Flaxedil paralysis had occurred. Presentation of the CS induced a burst of the same alpha fibers seen in B simultaneously with an elevation of the conditioned hind leg. Movement of the leg is indicated by deflections in the baseline.
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alpha-fiber potentials to the CS (Fig. 1B). When the level of neuromuscular block diminished, the same alpha fibers discharged as the animal withdrew his leg (C) . This movement is indicated by the gross deflections of the baseline. The remarkable stability of the conditioned avoidance response is shown in chronically trained animals by its persistence after 2 hours of ether anesthesia during surgery and several hours of complete paralysis under Flaxedil. In two cats in which paralysis was allowed to diminish, presentation of the CS produced flexion of the conditioned hind leg. This flexion was typical of the conditioned withdrawal exhibited by the cats during training. Late&es of Gamma- and Alpha-Fiber Responses. Acceleration in the tonic firing of gamma-efferent units in response to the CS consistently preceded the burst of alpha-fiber potentials recorded from the same ventral root filament. Figure 2 illustrates this gamma acceleration preceding the alpha discharge during sequential trials in one chronically trained cat. It is evident that the latency of gamma acceleration was very brief, as the second interspike interval after onset of the CS was shortened. Closer inspection reveals that there was often shortening of the interval between the last discharge prior to the onset of the CS and the first spike after the CS. This suggests that the latency of gamma acceleration was less than the duration of a single interspike interval. An attempt to measure this latency is illustrated in Fig. 3. In this experiment the responses of a single gamma fiber were followed over fifty-two trials. For each trial the durations of five interspike intervals preceding the CS were determined. The mean of these five durations was then projected from the last discharge after the CS. This predicted time for each trial is indicated by a dot on the graph. Actual time of the first discharge after the CS is shown on the graph by a cross. If the predicted and actual time of discharge coincided, acceleration of the gamma unit had not occurred. Appearance of the spike earlier than the predicted time indicated acceleration of the gamma unit. In Fig. 3, predicted and actual times of discharge coincide at about 13 msec. Thereafter, an increasing gap between the predicted and actual discharge times occurs, indicating that acceleration has taken place. The somewhat irregular discharge of gamma units makes determination of latency difficult. However, the responses of three units from two cats all show a response latency of not more than 18 msec. Alpha fibers generally did not discharge tonically, but with presentation
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of the CS a train of spikes appeared which often outlasted the tone for several tenths of a second. The latency of the alpha-fiber responses to the CS was determined by measuring from the electrical artifact marking onset of the tone to the first alpha-fiber potential.
FIG. 2. Gamma- and alpha-fiber conditioned responses in a chronically trained cat. Presentation of the CS alone causes acceleration of tonically discharging gammaefferent fiber and initiates discharge of high amplitude alpha potentials. Onset of CS indicated by artifacts on records and duration by bar beneath. Gamma acceleration precedes the alpha discharge in each of these sequential trials (A,B,C).
Latencies of 40 to 50 msec were found in two cats; the other animals showed minimal latencies of 80 to 100 msec. The latencies of alpha-fiber discharge to the CS in one animal are shown in Fig. 4, top line. The initial latencies were about 80 msec. After repeated presentation of the CS without reinforcement the latency of the responseincreased to over 300
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msec. Four reinforcements of the CS by shock to the hind paw caused the latency to decrease sharply to about 50 msec, a shorter delay than recorded early in the trial series. Similar increase in latency of discharge during extinction and marked decrease following reinforcement was ob-
. PREDICTED x ACTUAL
b
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lb TIME
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FIG. 3. Latency of gamma-fiber acceleration to CS. Discharge of a single gamma fiber was followed during fifty-two presentations of the CS. During each trial the durations of five interspike intervals preceding the CS were measured. The mean of these durations was then projected from the last discharge preceding the CS to predict when the next discharge should occur, indicated by dots. The trials have been arranged according to the length of this predicted time of discharge. Actual time of discharge on each trial is shown by a cross. Where predicted and actual times of discharge coincide, or show random variance, no acceleration of the gamma discharge has taken place. Occurrence of the actual time of discharge before the predicted time indicates acceleration of discharge. Acceleration was present at all times greater than 18 msec.
served in the other cats. Latencies as long as 800 to 900 msec occurred during some extinction trials. Extinction of Gamma- and Alpha-Fiber Response. Both the alpha- and gamma-fiber responses could be extinguished and then re-established by
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reinforcement. However, conditioned gamma-fiber acceleration persisted long after the alpha-fiber discharge was abolished. As shown in Fig. 4, bottom line, a gradual decrease in alpha-fiber discharge rate to the CS occurred during extinction. The initial rate of 40 pulsesper seconddimin-
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FIG. 4. Alpha-fiber conditioned response during extinction and reinforcement. Latency of alpha discharge (top line) increased from 80 msec to over 300 msec with continued presentations of unreinforced CS. After four US reinforcements of the CS (indicated by arrows in bracket), the latency drops abruptly to 50 msec. The rate of alpha discharge (bottom line), during the same trials for which latency is plotted, represents the number of alpha potentials during the l-set interval following the first alpha potential elicited by the CS. This value diminished with repeated presentations of the unreinforced CS until the response was nearly extinguished. Between every trial indicated on the graph, a neutral, unreinforced tone was presented which may have hastened extinction to the CS. After four reinforcements of the CS, the rate of alpha discharge increased to about 20 per second. The inverse relationship between rate of alpha discharge and latency of discharge can be seen both in the general trends and in the individual trials.
ished until essentially no responsewas elicited by the CS. Following the first reinforcement, an increase in the alpha discharge rate to the CS occurred and after four reinforcements, the rate had increased to 20 per second. Oscilloscopic traces taken from this extinction series are shown
E
Tone
1.5 set
l-
FIG. 5. Extinction and re-establishment of alpha-fiber conditioned response. At the beginning of the extinction trials (A), the CS elicits acceleration of the tonically discharging gamma unit and initiates high-amplitude alpha discharge. Onset of CS is indicated by artifact on the trace and duration of CS by bar at bottom of figure. After repeated unreinforced presentations of the CS, alpha discharge was abolished leaving only the gamma-fiber acceleration (B). When the first US reinforcement was given (C), alpha potentials appeared during the 0.5-set shock period, but not during the CS period preceding the shock. On the next trial, a feeble alpha-fiber discharge occurred during the CS period preceding the second reinforcement (D). After five reinforcements the alpha-fiber response to the CS alone (E) had been reestablished.
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A B C
D
E
F FIG. 6. Extinction and re-establishment of gamma-fiber conditioned response. Both acceleration of gamma-fiber discharge and alpha-fiber discharge were induced by the CS initially (A). Onset of CS indicated by artifact on the trace and duration of CS by bar at bottom of figure. After three hundred unreinforced presentations of the CS, the alpha responses were abolished from the filament shown in A and from a second fiber (B). Gamma acceleration to the CS, however, was still present (B). After two hundred more unreinforced trials the gamma-fiber response was nearly extinguished (C). The first reinforcement elicited both gamma acceleration and alpha discharge to the OS-set shock, but no response during the preceding CS period (D). In the next trial following the first reinforcement, marked acceleration in gamma activity was elicited by the CS, preceding the shock (E). Acceleration of gamma activity to the CS alone persisted after reinforcement was discontinued (F).
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in Fig. 5. Initially, strong alpha discharge of long duration was produced by the CS (trace A). After repeated, unreinforced presentations of the tone, the alpha response dropped out, although acceleration of gammaefferent discharge continued (B). When the first reinforcement was given (C) a burst of alpha potentials appeared during the OS-set period of shock, but not during the preceding tone. On the next trial a feeble alpha-fiber discharge occurred during the tone preceding the shock (D). After five reinforcements, the alpha-fiber response to the CS alone had been re-established (E). The CS continued to elicit the response through another series of twenty unreinforced trials. The gamma-efferent response to the CS could be extinguished only with a much larger number of trials than were needed to abolish the alpha response. In the experiment of Fig. 6, both alpha discharge and gamma acceleration were elicited by the CS (A). After three hundred unreinforced trials, the response of alpha fibers in this filament was abolished, but gamma-efferent acceleration to the CS still occurred. Trace B, taken from a second filament, shows this persisting gamma acceleration. After a series of two hundred more unreinforced trials, the gamma-fiber response was essentially extinguished (C, and left part of D). The first reinforcement elicited gamma- and alpha-fiber discharge to the shock, but none to the tone (D). In the trial following the first reinforcement, acceleration of the gamma unit to the CS appeared preceding the shock (E). After several reinforcements, marked acceleration of gamma discharge was elicited by the CS alone (F). Discussion
A background of conditioned proprioceptive activity, mediated by the gamma-efferent system, seems to underlie the conditioned avoidance response. Two sources of evidence support this concept. First, acceleration of gamma discharge during the conditioned avoidance response was directly observed when actual withdrawal of the conditioned leg occurred as Flaxedil paralysis diminished. This direct proof of participation of the gamma system in the behavioral conditioned response is supported by the recording of gamma-activation during alpha-fiber conditioned responses. The alpha-fiber response is identified as a component of the behavioral avoidance response, for only in animals previously trained to give consistent hind-leg withdrawal was alpha-fiber discharge recorded in roots innervating that leg. If the neuromuscular block were incomplete, with-
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drawal of the hind-leg to the CS occurred concurrently with the conditioned bursts of alpha activity. Moreover, the response in alpha fibers resembled the behavioral response, for (a) it could be extinguished by repeated presentations of the CS alone, (6) the latency of response increased during extinction trials, and (c) the response could be re-established with a few reinforcements. A second line of evidence, which indicates that a background of conditioned proprioceptive activity underlies the behavioral conditioned response, is the greater sensitivity to conditioning procedures in the gammaefferent system than in the alpha-motor system. This greater sensitivity is shown in several ways: (a) Conditioned responses of gamma fibers developed early in training when alpha or behavioral responses were not yet attainable; (b) gamma-fiber responses to the CS persisted after total extinction of alpha-fiber responses; and (c) in any single trial, the gammafiber conditioned response occurred with a shorter latency than did the alpha-fiber response. As a consequence of this low-threshold conditioning of gamma-efferent activity, changes in proprioceptive discharge preceded alpha-conditioned responses both during training and on a single trial. The net effect of enhanced gamma-efferent activity and the resultant acceleration of proprioceptive discharge is facilitation of the alpha motoneurons. For example, the tonic excitation of motoneurons in the classic decerebrate animal is dependent upon the presence of greatly enhanced firing of gamma fibers (4). Similarly, in conditioned learning the early, low-threshold changes in gamma-efferent, muscle-spindle circuits may facilitate the appearance of the overt conditioned response. Latencies of the alpha-fiber response were adequate for the conditioned changes in proprioceptive inflow to reach the ventral horn and facilitate the alpha motoneurons. The time required for centrifugal conduction down gamma fibers, activation of spindle afferents, and conduction back to the cord is, for distal limb muscles, about 15 msec ( 10). This time added to the observed 18-msec latency of gamma-fiber acceleration indicates that alteration in proprioceptive inflow could reach ventral horn cells 33 msec after the onset of the CS. The altered afferent inflow could also project upward to the brain stem in an additional 6 msec (5). Assuming several milliseconds delay in the reticular formation, the effects of this afferent inflow might return to the lumbar cord within another 10 msec (7). Thus, in less than 50 msec-33 msec for gamma activation and proprioceptive feedback to the cord, 6 msec for conduction upward, and
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10 msec for relay and return to the cord-conditioned gamma-efferent reinforcement of reticular formation activity could be reflected at the spinal level. The very short delay from onset of the CS to beginning of acceleration in gamma-fiber activity indicates that this response is mediated through a relatively direct pathway. In the experimental situation employed, almost 1 msec was needed for the tone to reach the ear. After sound has reached the middle ear, responses in the brain stem can appear in 2.5 to 3.0 msec (1). Additional time is required for relay in the reticular formation. From the midbrain reticular formation, fast pathways to gammamotor fibers in lumbar ventral roots have a conduction time of 8 msec (6). Thus, at least 12 msec are theoretically needed for a gamma response to tone to appear. Transmission of the conditioned gamma response along this direct route could be accommodated within the minimal latency of 18 msec indicated by our data. The early appearance of conditioned changes in reticular formation activity (14) suggests that this system could indeed contribute to the earliest conditioned changes in gamma-fiber activity. A route involving the cortex is not possible within the minimum latency at which gamma acceleration appears. One millisecond for tone conduction to the ear, 8 msec for a response to tone to appear in the auditory cortex (1, 9) and several milliseconds delay for intracortical events and conduction downward to the midbrain level must be added to the 8 msec required for fast conduction from the midbrain level to the lumbar segment (6). This route would probably require 20 msec, too long for initiation of the gamma-fiber response. In contrast, the alpha-fiber conditioned response and its behavioral counterpart, conditioned avoidance, probably are mediated by pathways involving the cortex. Several studies indicate that avoidance responses to a tonal CS conditioned in the intact cat must be relearned after complete bilateral destruction of the auditory cortex (12, 11, 3). Similarly, it has been shown that defensive conditioned responses are inhibited by focal seizures in the CS projection area (auditory or optic) produced by cortical stimulation ( 15). Only in part can the long latency of the behavioral, or alpha-conditioned response be accounted for by conduction delays in pathways involving the cortex. As calculated above, the effects of an acoustic stimulus relayed through the cortex and down to spinal levels could occur by the
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fast pathwaysin 20 msec. The minimum latency of the alpha-fiberconditioneddischarge,however,exceededthis time by 30msecor more. This additional time must be consumed in relays through the various association centers concerned with maintenance of the behavioral conditioned response. The present findings support the concept that a background of conditioned proprioceptive activity, mediated by the gamma-efferent system, underlies the conditioned avoidance response. References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10.
11. 12. 13. 14.
15.
ADES, H. W., and J. M. BROOKHART, The central auditory pathway. J. Neurophysiol. 13: 189-206, 1950. BUCHWALD, J., and E. ELDRED, Conditioned responses in the gamma efferent system. J. Nervous Mental Disease 132: 146-152, 1961. BUTLER, A., I. T. DIAMOND, and W. D. NEFF, Role of auditory cortex in discrimination of changes in frequency. 1. Neurophysiol. 20: 108-120, 1957. ELDRED, E., R. GRANIT, and P. A. MERTON, Supraspinal control of the muscle spindles and its significance. J. Physiol. London 12!2: 498-523, 1953. FRENCH, J. D., M. VERZEANO, and H. W. MAGOWN, An extralemniscal sensory system in the brain. A.M.A. Arch. Neural. Psych&. 69: 505-518, 1953. GRANIT, R., and B. HOLMGREN, Two pathways from brain stem to gamma ventral horn cells. Acta Physiol. Stand. 35: 93-108, 1955. GRANIT, R., 0. POMPEIANO, and B. WALTMAN, Fast supraspinal control of mammalian muscle spindles: extra and intrafusal co-activation. J. Physiol. London 147: 385-398, 1959. HUNT, C. C., The reflex activity of mammalian small-nerve fibers. J. Physiol. London 115: 456-469, 1951. KATSUKI, Y., T. WATANABE, and N. MARUYAMA, Activity of auditory neurons in upper levels of brain of cat. J. Neurophysiol. 22: 343-359, 1959. KUFFLER, S. W., C. C. HUNT, and J. P. QIJILLIAM, Function of medullated smallnerve fibers in mammalian ventral roots: efferent muscle spindle innervation. J. Neurophysiol. 14: 20-54, 1951. RAAB, D. W., and H. W. ADES, Cortical and midbrain mediation of a conditioned discrimination of acoustic intensities. Am. J. Psychol. 59: 59-83, 1946. ROSENZWEIG, M., Discrimination of auditory intensities in the cat. Am. J. Psychol. 52: 126-136, 1946. SHERRINGTON, C. S., Notes on the arrangement of some motor fibers in the lumbo-sacral plexus. 1. Physiol. London 13: 621-772, 1892. YOSHII, N., J. MATSUMOTO, H. OIXRA, M. SHIMOK~CHI, Y. YAMAGUCHI, and H. YAMASAKI, Conditioned reflex and electroencephalography. In “The Moscow Colloquim on Electroencephalography of Higher Nervous Activity,” H. H. Jasper and G. D. Smirnov (eds.). Electroencephalog. and Cl&. Neurophysiol. Suppl. 13: 199-210, 1960. ZUCKERMAN, E., Effect of cortical and reticular stimulation on conditioned reflex activity. J. Neurophysiol. 22: 633-643, 1959.