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Habituation of the flexor reflex: inhibitory build-up or synaptic depression?
Brain Research, 53 (1973) 451-454 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
451
Habituation of the flexor reflex: inhibitory build'up or synaptic depression? J. A. PEARSON AND J. F. MACDONALD Department of Physiology, University of British Columbia, Vancouver, B.C. (Canada)
(Accepted January 10th, 1973)
In recent years the attention of many neurophysiologists and psychologists has been devoted towards understanding the nature of mechanisms involved in behavioural plasticity. It is understandable therefore that the phenomenon of habituation has been under considerable scrutiny. Habituation is perhaps the most elementary form of behavioural change, and can be defined as a progressive diminution of response to a repeatedly applied stimulus. Although the characteristics of habituation are fairly well understood 11, controversy exists as to the synaptic mechanisms responsible for the gradual diminution of response3,4,10,13,14. Such mechanisms are usually discussed under two categories: synaptic depression or inhibitory build-up la. In the former case, there is assumed to be a depression of efficacy of excitatory synapses at one or more sites on the pathway under consideration. Such a depression could be due to (a) a gradual diminution in the amount o f excitatory transmitter released by each stimulus, or (b) a progressive desensitization of the postsynaptic membrane. The alternative theory a~sumes that there is inhibition of activity in the habituating pathway. It is further a~sumed that the inhibitory neurone is activated by neurones other than those constituting the habituating pathway. Studies of the gill withdrawal reflex of Aplysia 1,6,9 and the flexor withdrawal reflex of mammals have yielded much important information on the characteristics of habituation 13. In the former case habituation is due to depression of a monosynaptic connection and does not depend on an inhibitory input from an extrinsic source. This conclusion can be made with a fair degree of certainty in view of the fact that the wiring diagram of the neuronal substrate of the reflex is well understood. A similar claim cannot be made for the mammalian flexor withdrawal reflex. Evidence has been presented both for and against the hypothesis that an extrinsic inhibitory input is responsible for habituation of this reflex. From the results of experiments on transfer of habituation, Wickelgren 14 has argued that a build-up of postsynaptic inhibition, rather than synaptic depression, is likely to be the mechanism underlying flexor reflex habituation. This conclusion has not received universal support2,3,L The most frequently quoted piece of evidence against the inhibitory build-up theory is the finding of Spencer et aL lo that in spinal cats habituation can still be
452
SHORT COMMUNICATIONS
demonstrated after administration of strychnine and/or picrotoxin. In order to achieve response levels comparable to those obtained before administration of the drug, the stimulus intensity was reduced. Under these circumstances the degree of habituation was similar to that obtained in the control situation. In view of the well known fact that rate and degree of habituation is increased when stimuli of low intensity are used ]], we consider that reassessment of their data is appropriate. Moreover, inspection of the published recordings of these workers reveals that in situations where the stimulus intensity was returned to that used to obtain 'control' habituation, strychnine and picrotoxin clearly impaired the diminution of response to repeated stimulation. It therefore seemed pertinent to reinvestigate the effect of administration of strychnine on habituation of the flexor reflex. Experiments were carried out on unanaesthetized rats of the Wistar strain (weight range 240-285 g). One day prior to the experiment a polyethylene catheter was inserted into the right jugular vein, E M G electrodes were implanted into the caudal head of the right biceps femoris muscle and stimulating electrodes were inserted into the skin of the ipsilat~ral hind paw. These procedures were carried out using ether anaesthesia. After the rats recovered from the anaesthetic they were placed in Bollman-type restraining cages in which they remained until completion of the experiments. E M G discharge, integrated over the 250 msec period immediately following
5.0
4.0
-
-~
~ Strychnine
o
o Control
3.0o
ca 2 . O c~
u_ I.O
O.O
I-Io
I~oi-IIo Stimulus
1201-210
3oi-31o
Number
Fig. 1. The effect of intravenous infusion of strychnine (30/~g/kg/min) on habituation of the flexor reflex. Mean ( ± S.E.) flexor reflex responses are shown.
SHORT COMMUNICATIONS
453
each stimulus was used as the index of flexor reflex response. The experimental procedures are described in greater detail elsewhere 7,a. Either strychnine sulphate in a dose of 30 #g/kg/min (10 rats) or saline (9 rats) was administered as a continuous infusion at a rate of 3 ml/h. The infusion was started 20 min before the first stimulus and was continued throughout the experiment. The results are shown in Fig. 1. It can be seen that whereas strychnine did not cause any enhancement of the responses to the initial stimuli as compared to the control rats (t ----0.97; P > 0. l) it did result in an impairment of habituation to subsequent stimuli. We suggest that this finding lends support to the hypothesis that postsynaptic inhibition is at least partly responsible for habituation of the flexor reflex in the intact animal. These experiments do not of course give any information as to the site of action, or the source of activation of the inhibitory neurones. Other workers have demonstrated habituation of activity of interneurones situated in the dorsal laminae of the lumbar spinal corda, 14. Wall la has suggested that flexor habituation may be the result of failure of transmission between cells situated in lamina 4 and those in lamina 5 of Rexed. Moreover, after blocking impulses descending from higher centres, Wall 12 was no longer able to demonstrate habituation of activity in lamina 5 cells. It is possible therefore that in intact animals, habituation of the flexor reflex is at least in part due to a strychnine sensitive inhibition of cells in lamina 5 and that this inhibitory system can be influenced by supra-spinal centres. This work was supported by a grant to J.A.P. from the Medical Research Council of Canada, Grant No. MA-3561. We are grateful to Mrs. Karen White for technical assistance, and to Mr. K. Henze for preparation of the illustration.
1 CASTELLUCCI,V., PINSKER, H., KUPFERMANN,F., AND KANDEL, E., Neuronal mechanisms of
habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1745-1748. 2 GLANZMAN,O. L., GROVES,P. M., AND THOMPSON,R. F., Stimulus generalization of habituation
in spinal interneurons, Physiol. Behav., 8 (1972) 155-158. 3 GROVES,P. M., AND THOMPSON, R. E., Habituation: A dual-process theory, PsychoL Rev., 77 (1970) 419-450. 4 HORN,G., Neuronal mechanisms of habituation, Nature (Lond.), 215 (1967) 707-711. 5 HORN,G., Changes in neuronal activity and their relationship to behaviour. In G. HORNANDR. A. HINDE(Eds.), Short-Term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 567-606. 6 KUPFERMANN,L, PINSKER,H., CASTELLUCI,V., ANDKANDEL,E., Neuronal correlates of habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1743-1745. 7 PEARSON,J. A., ANDKRAJINA,V. P. J., Effects of occlusion of the thoracic aorta on habituation of the flexor withdrawal reflex in the rat, Exp. Neurol., 35 (1972) 39-49. 8 PEARSON,J. A., AND WENKSTERN, B., Habituation and sensitization of the flexor withdrawal reflex, Brain Research, 43 (1972) 107-118. 9 PINSKER,H., KUPFERMANN,I., CASTELLUCCI,V., AND KANDEL,E., Habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1740-1742. 10 SPENCER,W. A., THOMPSON,R. F., AND NEILSON,D. R., Decrement of ventral root electrotonus
454
SHORT COMMUNICATIONS
and intracellularly recorded PSPs produced by iterated cutaneous afferent volleys, J. Neuro-
physiol., 29 (1966) 253-274. 11 THOMeSON,R. F., AND SPENCER, W. A., Habituation: A model phenomenon for the study of neural substrates of behavior, Psychol. Rev., 173 (1966) 1643. 12 WALL, P. D., The laminar organization of dorsal horn and effects of descending impulses, J. Physiol. (Lond.), 188 (1967) 403-423. 13 WALL,P. D., Habituation and post-tetanic potentiation in the spinal cord. In G. HORN AND R. A. HINDE (Eds.), Short-Term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 181-210. 14 WICKELGREN,B. G.~ Habituation of spinal interneurones, J. Neurophysiol., 30 (1967) 1424-1438.
451
Habituation of the flexor reflex: inhibitory build'up or synaptic depression? J. A. PEARSON AND J. F. MACDONALD Department of Physiology, University of British Columbia, Vancouver, B.C. (Canada)
(Accepted January 10th, 1973)
In recent years the attention of many neurophysiologists and psychologists has been devoted towards understanding the nature of mechanisms involved in behavioural plasticity. It is understandable therefore that the phenomenon of habituation has been under considerable scrutiny. Habituation is perhaps the most elementary form of behavioural change, and can be defined as a progressive diminution of response to a repeatedly applied stimulus. Although the characteristics of habituation are fairly well understood 11, controversy exists as to the synaptic mechanisms responsible for the gradual diminution of response3,4,10,13,14. Such mechanisms are usually discussed under two categories: synaptic depression or inhibitory build-up la. In the former case, there is assumed to be a depression of efficacy of excitatory synapses at one or more sites on the pathway under consideration. Such a depression could be due to (a) a gradual diminution in the amount o f excitatory transmitter released by each stimulus, or (b) a progressive desensitization of the postsynaptic membrane. The alternative theory a~sumes that there is inhibition of activity in the habituating pathway. It is further a~sumed that the inhibitory neurone is activated by neurones other than those constituting the habituating pathway. Studies of the gill withdrawal reflex of Aplysia 1,6,9 and the flexor withdrawal reflex of mammals have yielded much important information on the characteristics of habituation 13. In the former case habituation is due to depression of a monosynaptic connection and does not depend on an inhibitory input from an extrinsic source. This conclusion can be made with a fair degree of certainty in view of the fact that the wiring diagram of the neuronal substrate of the reflex is well understood. A similar claim cannot be made for the mammalian flexor withdrawal reflex. Evidence has been presented both for and against the hypothesis that an extrinsic inhibitory input is responsible for habituation of this reflex. From the results of experiments on transfer of habituation, Wickelgren 14 has argued that a build-up of postsynaptic inhibition, rather than synaptic depression, is likely to be the mechanism underlying flexor reflex habituation. This conclusion has not received universal support2,3,L The most frequently quoted piece of evidence against the inhibitory build-up theory is the finding of Spencer et aL lo that in spinal cats habituation can still be
452
SHORT COMMUNICATIONS
demonstrated after administration of strychnine and/or picrotoxin. In order to achieve response levels comparable to those obtained before administration of the drug, the stimulus intensity was reduced. Under these circumstances the degree of habituation was similar to that obtained in the control situation. In view of the well known fact that rate and degree of habituation is increased when stimuli of low intensity are used ]], we consider that reassessment of their data is appropriate. Moreover, inspection of the published recordings of these workers reveals that in situations where the stimulus intensity was returned to that used to obtain 'control' habituation, strychnine and picrotoxin clearly impaired the diminution of response to repeated stimulation. It therefore seemed pertinent to reinvestigate the effect of administration of strychnine on habituation of the flexor reflex. Experiments were carried out on unanaesthetized rats of the Wistar strain (weight range 240-285 g). One day prior to the experiment a polyethylene catheter was inserted into the right jugular vein, E M G electrodes were implanted into the caudal head of the right biceps femoris muscle and stimulating electrodes were inserted into the skin of the ipsilat~ral hind paw. These procedures were carried out using ether anaesthesia. After the rats recovered from the anaesthetic they were placed in Bollman-type restraining cages in which they remained until completion of the experiments. E M G discharge, integrated over the 250 msec period immediately following
5.0
4.0
-
-~
~ Strychnine
o
o Control
3.0o
ca 2 . O c~
u_ I.O
O.O
I-Io
I~oi-IIo Stimulus
1201-210
3oi-31o
Number
Fig. 1. The effect of intravenous infusion of strychnine (30/~g/kg/min) on habituation of the flexor reflex. Mean ( ± S.E.) flexor reflex responses are shown.
SHORT COMMUNICATIONS
453
each stimulus was used as the index of flexor reflex response. The experimental procedures are described in greater detail elsewhere 7,a. Either strychnine sulphate in a dose of 30 #g/kg/min (10 rats) or saline (9 rats) was administered as a continuous infusion at a rate of 3 ml/h. The infusion was started 20 min before the first stimulus and was continued throughout the experiment. The results are shown in Fig. 1. It can be seen that whereas strychnine did not cause any enhancement of the responses to the initial stimuli as compared to the control rats (t ----0.97; P > 0. l) it did result in an impairment of habituation to subsequent stimuli. We suggest that this finding lends support to the hypothesis that postsynaptic inhibition is at least partly responsible for habituation of the flexor reflex in the intact animal. These experiments do not of course give any information as to the site of action, or the source of activation of the inhibitory neurones. Other workers have demonstrated habituation of activity of interneurones situated in the dorsal laminae of the lumbar spinal corda, 14. Wall la has suggested that flexor habituation may be the result of failure of transmission between cells situated in lamina 4 and those in lamina 5 of Rexed. Moreover, after blocking impulses descending from higher centres, Wall 12 was no longer able to demonstrate habituation of activity in lamina 5 cells. It is possible therefore that in intact animals, habituation of the flexor reflex is at least in part due to a strychnine sensitive inhibition of cells in lamina 5 and that this inhibitory system can be influenced by supra-spinal centres. This work was supported by a grant to J.A.P. from the Medical Research Council of Canada, Grant No. MA-3561. We are grateful to Mrs. Karen White for technical assistance, and to Mr. K. Henze for preparation of the illustration.
1 CASTELLUCCI,V., PINSKER, H., KUPFERMANN,F., AND KANDEL, E., Neuronal mechanisms of
habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1745-1748. 2 GLANZMAN,O. L., GROVES,P. M., AND THOMPSON,R. F., Stimulus generalization of habituation
in spinal interneurons, Physiol. Behav., 8 (1972) 155-158. 3 GROVES,P. M., AND THOMPSON, R. E., Habituation: A dual-process theory, PsychoL Rev., 77 (1970) 419-450. 4 HORN,G., Neuronal mechanisms of habituation, Nature (Lond.), 215 (1967) 707-711. 5 HORN,G., Changes in neuronal activity and their relationship to behaviour. In G. HORNANDR. A. HINDE(Eds.), Short-Term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 567-606. 6 KUPFERMANN,L, PINSKER,H., CASTELLUCI,V., ANDKANDEL,E., Neuronal correlates of habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1743-1745. 7 PEARSON,J. A., ANDKRAJINA,V. P. J., Effects of occlusion of the thoracic aorta on habituation of the flexor withdrawal reflex in the rat, Exp. Neurol., 35 (1972) 39-49. 8 PEARSON,J. A., AND WENKSTERN, B., Habituation and sensitization of the flexor withdrawal reflex, Brain Research, 43 (1972) 107-118. 9 PINSKER,H., KUPFERMANN,I., CASTELLUCCI,V., AND KANDEL,E., Habituation and dishabituation of the gill withdrawal reflex in Aplysia, Science, 167 (1970) 1740-1742. 10 SPENCER,W. A., THOMPSON,R. F., AND NEILSON,D. R., Decrement of ventral root electrotonus
454
SHORT COMMUNICATIONS
and intracellularly recorded PSPs produced by iterated cutaneous afferent volleys, J. Neuro-
physiol., 29 (1966) 253-274. 11 THOMeSON,R. F., AND SPENCER, W. A., Habituation: A model phenomenon for the study of neural substrates of behavior, Psychol. Rev., 173 (1966) 1643. 12 WALL, P. D., The laminar organization of dorsal horn and effects of descending impulses, J. Physiol. (Lond.), 188 (1967) 403-423. 13 WALL,P. D., Habituation and post-tetanic potentiation in the spinal cord. In G. HORN AND R. A. HINDE (Eds.), Short-Term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 181-210. 14 WICKELGREN,B. G.~ Habituation of spinal interneurones, J. Neurophysiol., 30 (1967) 1424-1438.