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Acquisition of a conditioned eyeblink response during reversible denervation of orbicularis oculi muscles in the cat
414
Brain Research, 64 (1973) 414-418 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Acquisition of a conditioned eyeblink response during reversible d,enervation of orbicularis oculi muscles in I~hecat
TERRY J. CROW AND CHARLES D. WOODY Laboratory of Neurophysiology, UCLA Mental Retardation Center, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted September 7th, 1973)
Movement2, 5,9,12 and afferent feedback la are presumably not necessary features for the acquisition of classically conditioned reflexes. However, experimental evidence concerning the need for the integrity of peripheral m o t o r pathways for the development of conditioned m o t o r responses to aversive stimuli has not produced consistent results. Light and G a n t t 9 reported that crushing the ventral roots innervating one hindlimb did not impair the acquisition of a conditioned leg flexion response to hindlimb shock. In contrast to these findings, Kellogg et al. 6 reported that acquisition of a conditioned hindlimb flexion response to shock was impaired when training sessions were presented during the period of paralysis induced by a ventral root crush. More recently, Beck and Doty 2 found that cats acquired flexion conditioned responses (CRs) to shock when trained during hindlimb paralysis induced by a ventral root crush in conjunction with a drug induced cataleptic state. Similar evaluations of the necessity of intact m o t o r pathways and associated movement for the acquisition of non-aversive conditioned reflexes have not been investigated. The criterion for denervation and the subsequent recovery of nerve function in the previous studies was an absence of gross movement in the impaired limb as contrasted with E M G activity adequately recorded from the denervated muscles. Recent evidence has demonstrated that microstimulation of units in m o t o r cortex evokes E M G activity in target muscles1,16,1L Thus, the possibility of subliminal myokinetic responses occurring during training that could confound the interpretation of behavioral tests following recovery of nerve function has not been previously ruled out. We have examined the role of the peripheral m o t o r pathway in classical conditioning of non-aversive facial reflexes using the more sensitive electromyographic measure of m o t o r responses. Five adult female cats were used in the experiment. E M G responses to auditory stimuli and tap to the glabella 11 were recorded by two pairs of Grass E2B subdermal electrodes inserted into the orbicularis oculi muscles bilaterally and led to differential amplifiers with a low frequency attenuation of 100 Hz. E M G responses of fixed latency evoked by the tap and auditory stimuli were separated i
415
SHORT COMMUNICATIONS
from spontaneous background activity by examining successive sweeps superimposed on a storage oscilloscope. The crush lesions of the facial nerves were performed as follows. The cats were anesthetized with sodium pentobarbital (Nembutal 35 mg/kg i.p.) and incisions were made in the skin overlying the zygomatic arch. The zygomatic orbital branches of the facial nerves were separated from superficial fascia by the use of blunt dissection and crushed with fine hemostats. Immediately following the release of the hemostats the effectiveness of the crush was initially assessed by touching the corneas with a cotton swab and tapping the facial musculature with blunt forceps. In all cases this procedure did not elicit an eyeblink. The upper and lower lids were then sutured together to prevent the development o f corneal ulcerations during the recovery period. Following recovery from anesthesia the cats received 13 training sessions, one on the day following surgery and two on each of 6 subsequent days. Each training session consisted of 150 trials of the click conditioned stimulus (CS) paired with glabella tap. The 70 dB CS consisted o f a 1 msec square wave delivered to a loudspeaker in front of the cats. The glabella tap (unconditioned stimulus, UCS) was generated by an electromagnetic tapper connected to a screw implanted in the glabella. The CS-UCS interval was 400 msec and the inter-trial interval was 10 sec. Further details on the conditioning paradigm and procedure have been described previously ]7. During training, E M G responses to the tap were recorded before each session and immediately following the last training session to insure that responses in
A
15ov 25 uV
B
125.V
t
50~V 150~V I
lOmsec Fig. 1. E M G activity evoked by glabella tap recorded from left (upper) and right (lower) orbicularis oculi muscles (3 superimposed sweeps). A: pre-operatively; B: at the conclusion of training; and C : following the termination of behavioral testing. Delivery of stimulus shown by arrows.
416
SHORT COMMUNICATIONS
A 125~,V m
10 msec
80o
60-
_1_'
c o .= c o u
g
40-
20.
nerve crush
normal control
Fig. 2. A: EMG activity (3 superimposed sweeps) evoked by the 70 dB click recorded from left (upper) and right (lower) orbicularis oculi muscles following the recovery of facial nerve function. B: mean percent conditioned responses and standard error of the mean for the nerve crush group and normal controls.
orbicularis oculi connoting recovery of the crushed nerve did not occur during the training period. E M G activity recorded from orbicularis oculi muscles before surgery and at the termination of training (Fig. 1) showed an absence of a response to glabella tap following the crush lesions of the facial nerves. After training was terminated the cats were tested for recovery of nerve function by tapping the facial musculature with blunt forceps and noting any observable movement. The reappearance of the blink response was further substantiated by recording E M G responses evoked by tapping the glabella. The unconditioned blink responses to glabella tap following recovery were as large and of the same latency as those recorded prior to surgery (Fig. 1). After recovery of nerve function (averaging 24 days following the crush lesion), a test session was conducted to assess the level of acquisition of the conditioned response following the 13, pre-recovery training sessions. In this test session the cats received 10 unreinforced presentations of the CS followed by 10 paired presentations of the CS and UCS. The number of conditioned responses for the unreinforced trials and reinforced trials were computed for each cat. Examples of E M G responses to the click CS recorded at the conclusion of behavioral testing are shown in Fig. 2A. The average number of conditioned responses over the group of cats for the 10 unreinforced presentations of the CS and the 10 reinforced presentations of the CS and UCS were 6.8 and 6.4 respectively. F o r purposes of analysis the two blocks of 10 trials were grouped together and the average percentage of conditioned responses
SHORT COMMUNICATIONS
417
for the 20 trials was computed. The average percentage of conditioned responses and standard error of the mean for the nerve crush group (n = 5) and a group of normal controls (n = 5) with 13 training sessions are presented in Fig. 2B. The mean percentage of conditioned responses did not differ significantly between the nerve crush group and the control group (t test; P > 0.50). Acquisition of a conditioned eyeblink response to a click CS does not require movement or the integrity of the peripheral motor pathway. These findings support and extend previous investigations suggesting that movement is not a necessary condition for the acquisition of classically conditioned reflexes to aversive stimuli 2,5,9. The possibility that proprioceptive feedback is a necessary cue for acquisition can also be ruled out on the grounds that no movement occurred in response to the UCS as confirmed by E M G activity measured during the training period. Recent evidence has shown that antidromic activation of facial motoneurons via electrical stimulation of the facial nerve is a sufficient condition for acquisition of an eyeblink CR (see ref. 3). Such results suggest that the facial nucleus is involved in conditioning of this type. This might appear to be in conflict with the present findings. Clearly the question is raised as to the functional integrity of the facial nucleus following crush lesions of the facial nerve. Electron microscopic studies in rats, 4 days following axotomy of facial motoneurons, have shown that microglia processes have separated or displaced synaptic terminals from the soma and proximal surface membranes in the facial nucleus 4. A similar microglial reaction in facial motoneurons has been reported for crush lesions of the facial nerves in mice although the glial processes do not extend over as extensive an area of the soma and dendrites as was the case following axotomy 15. Additional reports by Torvik and Skjorten 14 have demonstrated that during the first 4 days following crush lesions of the facial nerves in mice, the changes in facial motoneurons are identical to the changes resulting from axotomy, although the crush lesions resulted in complete cell regeneration. However, other electrophysiological studies of facial motoneurons undergoing chromatolysis in cats have shown that EPSPs can still be evoked at the facial nucleus 6-10 days following axotomy by stimulation of trigeminal afferents 1°. The evoked EPSPs are of smaller amplitude and longer time-to-peak as compared with rapid EPSPs recorded before the nerve transection. Additional electrophysiological evidence from chromatolysed spinal motoneurons 7-20 days following ventral root transection in cats suggests that the diminished EPSPs may reflect a lack of excitatory synaptic input to the cell body s which may be compensated for by partial responses originating in the dendrites 7. Thus it is possible in the present experiments that either the facial nucleus was sufficiently intact to be functionally involved in mediating acquisition of the conditioned eyeblink during the period of motor paralysis following the crush lesions, or that trigeminal activation is sufficient for acquisition. This research was supported by U.S. Public Health Service Grants HD-05958, HD-04612 and California Department of Mental Hygiene.
418
SHORT COMMUNICATIONS
1 ASANUMA, H., STONEY, JR., S. D., AND ABZUG, C., Relationship between afferent input and motor outflow in cat motorsensory cortex, J. Neurophysiol., 31 (1968) 670-681. 2 BECK, E. C., AND DOTY, R. W., Conditioned flexion reflexes acquired during combined catalepsy and de-efferentation, J. comp. physiol. Psychol., 50 (1957) 211-216. 3 BLACK-CLEWORTH,P., WOODY, C. n., AND NIEMANN, J., Acquistion of a classically conditioned eye blink by pairing click-CS and electrical stimulation of facial nerve, Soc. Neurol., 3 (1973) Abstr. 118. 4 BLINZINGER,K., AND KREUTZBERG, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 5 HARLOW, H. F., AND BROMER, J. A., Acquisition of new responses during inactivation of the motor, premotor, and somesthetic cortex in the monkey, d. gen. PsychoL, 26 (1942) 299-313. 6 KELLOGG,W. N., SCOTT,V. B., DAVIS, R. C., AND WOLF,I. S., Is movement necessary for learning? J. comp. Psychol., 29 (1940) 43-74. 7 KUNO, M., AND LLIN.~S, R., Enhancement of synaptic transmission by dendritic potentials in chromatolysed motoneurones of the cat, d. Physiol. (Lond.), 210 (1970) 807-821. 8 KUNO, M., AND LLIN~,S, R., Alterations of synaptic action in chromatolysed motoneurones of the cat, J. Physiol. (Lond.), 210 (1970) 823-838. 9 LIGHT, J. S., AND GANTT, W. H., Essential part of reflex arc for establishment of conditioned reflex. Formation of conditioned reflex after exclusion of motor peripheral end, J. comp. Psychol., 21 (1936) 19-36. 10 Lux, H. D., AND WINTER, P., Studies on EPSPs in normal and retrograde reacting facial motoneurones, Proc. lnt. Union Physiol. Sci., 7 (1968) Abst. 818. 11 RUSHWORTH, G., Observations on blink reflexes, J. Neurol. Neltrosurg. Psychiat., 25 (1962) 93-108. 12 SOLOMON,R. L., AND TURNER, L. H., Discriminative classical conditioning in dogs paralyzed by curare can later control discriminative avoidance responses in the normal state, Psychol. Rev., 69 (1962) 202-219. 13 TAUB, E., AND BERMAN, A. J., Movement and learning in the absence of sensory feedback. In S. J. FREEDMAN(Ed.), The Neuropsychology of Spatially Oriented Behavior, Dorsey Press, Homewood, II1., 1960, pp. 173-192. 14 TORVIK,m., AND SKJORTEN,F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions. I. Changes in nerve cell cytoplasm, Acta Neuropath. (BerL), 17 (1971) 248-264. 15 TORVIK,A., AND SKJORTEN,F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions. II. changes in the glial cells, Acta Neuropath. (Berl.), 17 (1971) 265-282. 16 WOODY, C. D., AND ENGEL, JR., J., Changes in unit activity and thresholds to electrical microstimulation at coronal-pericruciate cortex of cat with classical conditioning of different facial movements, J. NeurophysioL, 35 (1972) 230-241. 17 WOODY, C. D., VASSILEVSKY,N. N., AND ENGEL, JR., J., Conditioned eye blink: unit activity at coronal-precruciate cortex of the cat, J. Neurophysiol., 33 (1970) 851-864.
Brain Research, 64 (1973) 414-418 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Acquisition of a conditioned eyeblink response during reversible d,enervation of orbicularis oculi muscles in I~hecat
TERRY J. CROW AND CHARLES D. WOODY Laboratory of Neurophysiology, UCLA Mental Retardation Center, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted September 7th, 1973)
Movement2, 5,9,12 and afferent feedback la are presumably not necessary features for the acquisition of classically conditioned reflexes. However, experimental evidence concerning the need for the integrity of peripheral m o t o r pathways for the development of conditioned m o t o r responses to aversive stimuli has not produced consistent results. Light and G a n t t 9 reported that crushing the ventral roots innervating one hindlimb did not impair the acquisition of a conditioned leg flexion response to hindlimb shock. In contrast to these findings, Kellogg et al. 6 reported that acquisition of a conditioned hindlimb flexion response to shock was impaired when training sessions were presented during the period of paralysis induced by a ventral root crush. More recently, Beck and Doty 2 found that cats acquired flexion conditioned responses (CRs) to shock when trained during hindlimb paralysis induced by a ventral root crush in conjunction with a drug induced cataleptic state. Similar evaluations of the necessity of intact m o t o r pathways and associated movement for the acquisition of non-aversive conditioned reflexes have not been investigated. The criterion for denervation and the subsequent recovery of nerve function in the previous studies was an absence of gross movement in the impaired limb as contrasted with E M G activity adequately recorded from the denervated muscles. Recent evidence has demonstrated that microstimulation of units in m o t o r cortex evokes E M G activity in target muscles1,16,1L Thus, the possibility of subliminal myokinetic responses occurring during training that could confound the interpretation of behavioral tests following recovery of nerve function has not been previously ruled out. We have examined the role of the peripheral m o t o r pathway in classical conditioning of non-aversive facial reflexes using the more sensitive electromyographic measure of m o t o r responses. Five adult female cats were used in the experiment. E M G responses to auditory stimuli and tap to the glabella 11 were recorded by two pairs of Grass E2B subdermal electrodes inserted into the orbicularis oculi muscles bilaterally and led to differential amplifiers with a low frequency attenuation of 100 Hz. E M G responses of fixed latency evoked by the tap and auditory stimuli were separated i
415
SHORT COMMUNICATIONS
from spontaneous background activity by examining successive sweeps superimposed on a storage oscilloscope. The crush lesions of the facial nerves were performed as follows. The cats were anesthetized with sodium pentobarbital (Nembutal 35 mg/kg i.p.) and incisions were made in the skin overlying the zygomatic arch. The zygomatic orbital branches of the facial nerves were separated from superficial fascia by the use of blunt dissection and crushed with fine hemostats. Immediately following the release of the hemostats the effectiveness of the crush was initially assessed by touching the corneas with a cotton swab and tapping the facial musculature with blunt forceps. In all cases this procedure did not elicit an eyeblink. The upper and lower lids were then sutured together to prevent the development o f corneal ulcerations during the recovery period. Following recovery from anesthesia the cats received 13 training sessions, one on the day following surgery and two on each of 6 subsequent days. Each training session consisted of 150 trials of the click conditioned stimulus (CS) paired with glabella tap. The 70 dB CS consisted o f a 1 msec square wave delivered to a loudspeaker in front of the cats. The glabella tap (unconditioned stimulus, UCS) was generated by an electromagnetic tapper connected to a screw implanted in the glabella. The CS-UCS interval was 400 msec and the inter-trial interval was 10 sec. Further details on the conditioning paradigm and procedure have been described previously ]7. During training, E M G responses to the tap were recorded before each session and immediately following the last training session to insure that responses in
A
15ov 25 uV
B
125.V
t
50~V 150~V I
lOmsec Fig. 1. E M G activity evoked by glabella tap recorded from left (upper) and right (lower) orbicularis oculi muscles (3 superimposed sweeps). A: pre-operatively; B: at the conclusion of training; and C : following the termination of behavioral testing. Delivery of stimulus shown by arrows.
416
SHORT COMMUNICATIONS
A 125~,V m
10 msec
80o
60-
_1_'
c o .= c o u
g
40-
20.
nerve crush
normal control
Fig. 2. A: EMG activity (3 superimposed sweeps) evoked by the 70 dB click recorded from left (upper) and right (lower) orbicularis oculi muscles following the recovery of facial nerve function. B: mean percent conditioned responses and standard error of the mean for the nerve crush group and normal controls.
orbicularis oculi connoting recovery of the crushed nerve did not occur during the training period. E M G activity recorded from orbicularis oculi muscles before surgery and at the termination of training (Fig. 1) showed an absence of a response to glabella tap following the crush lesions of the facial nerves. After training was terminated the cats were tested for recovery of nerve function by tapping the facial musculature with blunt forceps and noting any observable movement. The reappearance of the blink response was further substantiated by recording E M G responses evoked by tapping the glabella. The unconditioned blink responses to glabella tap following recovery were as large and of the same latency as those recorded prior to surgery (Fig. 1). After recovery of nerve function (averaging 24 days following the crush lesion), a test session was conducted to assess the level of acquisition of the conditioned response following the 13, pre-recovery training sessions. In this test session the cats received 10 unreinforced presentations of the CS followed by 10 paired presentations of the CS and UCS. The number of conditioned responses for the unreinforced trials and reinforced trials were computed for each cat. Examples of E M G responses to the click CS recorded at the conclusion of behavioral testing are shown in Fig. 2A. The average number of conditioned responses over the group of cats for the 10 unreinforced presentations of the CS and the 10 reinforced presentations of the CS and UCS were 6.8 and 6.4 respectively. F o r purposes of analysis the two blocks of 10 trials were grouped together and the average percentage of conditioned responses
SHORT COMMUNICATIONS
417
for the 20 trials was computed. The average percentage of conditioned responses and standard error of the mean for the nerve crush group (n = 5) and a group of normal controls (n = 5) with 13 training sessions are presented in Fig. 2B. The mean percentage of conditioned responses did not differ significantly between the nerve crush group and the control group (t test; P > 0.50). Acquisition of a conditioned eyeblink response to a click CS does not require movement or the integrity of the peripheral motor pathway. These findings support and extend previous investigations suggesting that movement is not a necessary condition for the acquisition of classically conditioned reflexes to aversive stimuli 2,5,9. The possibility that proprioceptive feedback is a necessary cue for acquisition can also be ruled out on the grounds that no movement occurred in response to the UCS as confirmed by E M G activity measured during the training period. Recent evidence has shown that antidromic activation of facial motoneurons via electrical stimulation of the facial nerve is a sufficient condition for acquisition of an eyeblink CR (see ref. 3). Such results suggest that the facial nucleus is involved in conditioning of this type. This might appear to be in conflict with the present findings. Clearly the question is raised as to the functional integrity of the facial nucleus following crush lesions of the facial nerve. Electron microscopic studies in rats, 4 days following axotomy of facial motoneurons, have shown that microglia processes have separated or displaced synaptic terminals from the soma and proximal surface membranes in the facial nucleus 4. A similar microglial reaction in facial motoneurons has been reported for crush lesions of the facial nerves in mice although the glial processes do not extend over as extensive an area of the soma and dendrites as was the case following axotomy 15. Additional reports by Torvik and Skjorten 14 have demonstrated that during the first 4 days following crush lesions of the facial nerves in mice, the changes in facial motoneurons are identical to the changes resulting from axotomy, although the crush lesions resulted in complete cell regeneration. However, other electrophysiological studies of facial motoneurons undergoing chromatolysis in cats have shown that EPSPs can still be evoked at the facial nucleus 6-10 days following axotomy by stimulation of trigeminal afferents 1°. The evoked EPSPs are of smaller amplitude and longer time-to-peak as compared with rapid EPSPs recorded before the nerve transection. Additional electrophysiological evidence from chromatolysed spinal motoneurons 7-20 days following ventral root transection in cats suggests that the diminished EPSPs may reflect a lack of excitatory synaptic input to the cell body s which may be compensated for by partial responses originating in the dendrites 7. Thus it is possible in the present experiments that either the facial nucleus was sufficiently intact to be functionally involved in mediating acquisition of the conditioned eyeblink during the period of motor paralysis following the crush lesions, or that trigeminal activation is sufficient for acquisition. This research was supported by U.S. Public Health Service Grants HD-05958, HD-04612 and California Department of Mental Hygiene.
418
SHORT COMMUNICATIONS
1 ASANUMA, H., STONEY, JR., S. D., AND ABZUG, C., Relationship between afferent input and motor outflow in cat motorsensory cortex, J. Neurophysiol., 31 (1968) 670-681. 2 BECK, E. C., AND DOTY, R. W., Conditioned flexion reflexes acquired during combined catalepsy and de-efferentation, J. comp. physiol. Psychol., 50 (1957) 211-216. 3 BLACK-CLEWORTH,P., WOODY, C. n., AND NIEMANN, J., Acquistion of a classically conditioned eye blink by pairing click-CS and electrical stimulation of facial nerve, Soc. Neurol., 3 (1973) Abstr. 118. 4 BLINZINGER,K., AND KREUTZBERG, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 5 HARLOW, H. F., AND BROMER, J. A., Acquisition of new responses during inactivation of the motor, premotor, and somesthetic cortex in the monkey, d. gen. PsychoL, 26 (1942) 299-313. 6 KELLOGG,W. N., SCOTT,V. B., DAVIS, R. C., AND WOLF,I. S., Is movement necessary for learning? J. comp. Psychol., 29 (1940) 43-74. 7 KUNO, M., AND LLIN.~S, R., Enhancement of synaptic transmission by dendritic potentials in chromatolysed motoneurones of the cat, d. Physiol. (Lond.), 210 (1970) 807-821. 8 KUNO, M., AND LLIN~,S, R., Alterations of synaptic action in chromatolysed motoneurones of the cat, J. Physiol. (Lond.), 210 (1970) 823-838. 9 LIGHT, J. S., AND GANTT, W. H., Essential part of reflex arc for establishment of conditioned reflex. Formation of conditioned reflex after exclusion of motor peripheral end, J. comp. Psychol., 21 (1936) 19-36. 10 Lux, H. D., AND WINTER, P., Studies on EPSPs in normal and retrograde reacting facial motoneurones, Proc. lnt. Union Physiol. Sci., 7 (1968) Abst. 818. 11 RUSHWORTH, G., Observations on blink reflexes, J. Neurol. Neltrosurg. Psychiat., 25 (1962) 93-108. 12 SOLOMON,R. L., AND TURNER, L. H., Discriminative classical conditioning in dogs paralyzed by curare can later control discriminative avoidance responses in the normal state, Psychol. Rev., 69 (1962) 202-219. 13 TAUB, E., AND BERMAN, A. J., Movement and learning in the absence of sensory feedback. In S. J. FREEDMAN(Ed.), The Neuropsychology of Spatially Oriented Behavior, Dorsey Press, Homewood, II1., 1960, pp. 173-192. 14 TORVIK,m., AND SKJORTEN,F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions. I. Changes in nerve cell cytoplasm, Acta Neuropath. (BerL), 17 (1971) 248-264. 15 TORVIK,A., AND SKJORTEN,F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions. II. changes in the glial cells, Acta Neuropath. (Berl.), 17 (1971) 265-282. 16 WOODY, C. D., AND ENGEL, JR., J., Changes in unit activity and thresholds to electrical microstimulation at coronal-pericruciate cortex of cat with classical conditioning of different facial movements, J. NeurophysioL, 35 (1972) 230-241. 17 WOODY, C. D., VASSILEVSKY,N. N., AND ENGEL, JR., J., Conditioned eye blink: unit activity at coronal-precruciate cortex of the cat, J. Neurophysiol., 33 (1970) 851-864.