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Changes in cortical and tegmental evoked responses to sensory stimuli reinforced by electrical stimulation of the area of recording
249
BRAIN RESEARCH
C H A N G E S IN CORTICAL A N D T E G M E N T A L EVOKED RESPONSES TO SENSORY STIMULI R E I N F O R C E D BY E L E C T R I C A L S T I M U L A T I O N OF T H E AREA OF R E C O R D I N G
E. CAVIEDES* AND J. BURES
Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Accepted October 30th, 1969)
INTRODUCTION
Although in the last 15 years electrophysiology has revealed many electrical correlates of behavioral conditioning, it has not succeeded in identifying the regions responsible for the formation of new connections. Both conditioned stimulus (CS) and unconditioned stimulus (UCS) activate large neural populations extensively overlapping in wide brain areas, any part of which may become the site of the actual switching process. As the electrical concomitants of conditioning are usually diffuse, it is almost impossible to decide which of them correspond to the primary plastic change and which are only its consequence or byproducts. The plastic change can be located with more precision if electrical stimulation of a restricted brain region serves as UCS4,6,7,10,13. Most experiments of this type 5 use electrical stimuli eliciting overt motor, vegetative or emotional responses, and the ample feedback afferentation prevents straightforward interpretation of results. When local electrical responses are used instead of behavioral reactions, stimulation of any brain region can be used as U CS. Bureg and Bure~ov~ 1,z and Gerbrandt et al. 9 observed, in unanesthetized curarized rats, the appearance of single unit responses to initially ineffective sensory stimuli, the application of which was reinforced by electrophoretic injection of K + ions or by polarization through the recording microelectrode. Positive results were obtained only in 10-15 ~ of the examined neurons in the cortex, thalamus, hippocampus and reticular formationZ. The low incidence of plastic reactions indicates that stimulation of a single neuron may be an inadequate UCS and that the success of conditioning is proportional to the size of the neural population activated by the UCS. The aim of the present paper is to test this assumption by recording responses evoked by the sensory CS in cortical or tegmental areas subjected to direct electrical stimulation (UCS).
* Visiting scientist from the Institute of Physiology, Department of Sciences, University of Chile, Valparaiso, Chile. Brain Research, 19 (1970) 249-261
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METHODS
A total of 49 experiments was performed on 29 hooded rats, aged 3-4 months. Under pentothal anesthesia (40 mg/kg) electrodes were implanted into either the cerebral cortex or the tegmentum. Cortical electrodes were 0.2 mm silver wires, which were inserted through 0.5 mm trephine openings 1.0 mm deep into the parietal cortex. The distance between the two stimulating electrodes was 1.5 mm. The recording electrodes were placed roughly in the line midperpendicular to the stimulating electrodes, again with a 1.5-2.0 mm interelectrode distance. Tegmental electrodes formed an assembly of 4 glass-insulated silver wires (0.2 mm diameter, 0.5 mm bare tip) with similar geometry of stimulating and recording electrodes. The electrode assembly was inserted through a T-shaped trephine opening into the region of the stratum griseum centrale. The stereotaxic coordinates of stimulating electrodes were AP7, L0.5, V6; AP7, L2, V6; and of the recording electrodes were AP5.5, L1, V6; AP4, L1, V6; all according to the atlas of Fifkov/t and MarSala s. A silver wire suture stitched through the edge of the occipital bone served as the ground lead. Two selftapping anchor-screws were placed in the parietal and occipital bones. The lead-off wires were soldered to a subminiature 5 pin socket which was fixed to the skull with acrylate. The experiments usually started 3-5 days after surgery and continued for up to 4 weeks. The animal was put into a tall narrow glass vessel (10 cm × 15 cm × 36 cm), the floor of which was covered with adsorbent cotton. The small size of the container considerably reduced the activity of the animal without causing noticeable discomfort. The glass vessel was placed in an electrically and acoustically screened box containing the preamplifier and output unit of the stimulator. The rat was connected through a microdot cable with the output unit of the stimulator and with the preamplifier (Tektronix 122). A loudspeaker placed directly above the glass container (average distance from the animal's head: 28 cm) was used as source of acoustic clicks, generated by another stimulator. The auditory conditioned stimuli were of constant intensity, approximately 70 dB above the human threshold. A preset counter generated the intertrial intervals as well as the CS-UCS delay. The scheme of the stimulating and recording setup is shown in Fig. 1 (A), which also shows the different stimulus sequences used (B, C and D). In most experiments the electrical stimulus was a 100 msec train of 10 #sec, 100/sec pulses, the amplitude of which was adjusted to a level just supramaximal for eliciting a local response. The mesencephalic stimulus of this intensity evoked a slight generalized jerk in all animals. The output unit of the stimulator was isolated from the ground and~0connectedlwithlthe main stimulator through a low capacity inductive coupling. The bipolarly recorded evoked responses were displayed on a CRO and fed into an on-line average response computer is, the sweep of which was triggered by the first click. With 1023 addresses and ! msec or 700/zsec per address, the sweep duration was slightly'over 1 sec or 700 msec respectively. Usually 10-100 sweeps were summated to form an average. The average waveforms were plotted with an X - Y recorder.
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EVOKED POTENTIALSAND CONDITIONING
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A typical experiment started with a 1 h habituation period to the isolated acoustic CS followed by 30 min of conditioning and 60 min of extinction. Periods of conditioning followed by extinction were repeated up to 3 times in one session.
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Pseudoconditioning (random application of CS and UCS) was tested after the last extinction in some experiments. Each rat was used in 2-3 sessions only. Statistical evaluation of results was based on comparison of amplitudes of the most prominent component of the average evoked potential (AEP), usually occurring with a latency of 30-40 msec. In conditioning and extinction experiments the amplitude of the last habituation AEP was used as 100 ~ and the amplitudes of the first and last conditioning and first and last extinction averages were expressed accordingly. When the initial habituation to the isolated presentation of the CS was evaluated, the AEP to the first click in the first block of responses was taken as 1 0 0 ~ and the other responses and their changes during habituation were expressed in percentages. The percentage values obtained in individual experiments were averaged and standard errors of means were computed in the conventional way. Localization of the subcortical electrodes was verified in Nissl stained serial sections. It usually corresponded to the target coordinates with an error not exceeding 0.5 mm. RESULTS Preliminary e x p e r i m e n t s
The aim of the first series of experiments was to find an optimal paradigm. The results are summarized in Fig. 2, the upper part of which represents a group of
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Fig. 2. Changes of cortical AEPs during conditioning in unanesthetized curarized (above) and freely moving (below) rats. Empty columns -- mean values of AEPs. Vertical bars denote the SEM values. Shaded columns = incidence of modified AEPs. H, last habituation block; C1, first conditioning block; CL, last conditioning block; El, first extinction block; EL, last extinction block. For details see text. Brain Research, 19 (1970) 249-261
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EVOKED POTENTIALS AND CONDITIONING
9 experiments performed on curarized rats (D-tubocurarine 0.1-0.2 mg/100 g). A liminal electric shock to the forepaw was used as CS, whereas a single electrical stimulus (pulse of l0 #sec duration) directly applied to the contralateral parietal cortex was used as UCS (Fig. 1B). The CS-UCS delay was 200 msec and the intertrial interval 5 sec. Each empty column in the figure represents the mean amplitude of the AEP based on 40 individual responses, expressed in percentages of the amplitude of the last habituation AEP, which was taken as 100 % (Fig. 2, H). Practically no changes were seen (109% 4- 17) during the first 40 CS-UCS pairings (Fig. 2, C1). Even the small increase (137% ± 24) of the AEP amplitude in the last part of the 30 min conditioning period was not statistically significant (Fig. 2, C~,). The AEP amplitude decreased during the first block of extinction trials (117% -4- 20, Fig. 2, El), and after an hour the level of the last habituation was attained again (95 % ± 21, Fig. 2, El,). The shaded columns of the same figure represent the quali-
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Fig. 3. Modification of cortical AEPs during conditioning in a freely moving rat (A, AEPs based on 100 individual responses) and in a curarized rat (B, AEPs based on 40 individual responses). The numbers denote time (in minutes) during which the respective AEPs were obtained. H, habituation; C, conditioning; E, extinction. Note changes of the AEP waveform in B when the cortical stimulus was increased after the first hour of reinforcement (increased direct cortical response).
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tative changes, either in amplitude or in the shape of the AEPs, expressed in percentages of the total number of experiments. Fig. 3B shows an example of such an experiment, demonstrating changes in amplitude, as well as some changes in the AEP waveform during more prolonged conditioning and first extinction trials. The lower part of Fig. 2 represents the results of 8 experiments performed in freely-moving rats with implanted electrodes in the left parietal cortex. A single click was used as CS and a train of pulses (100 msec, 10 #sec, 100/sec) directly applied to the region of recording, as UCS (Fig. 1C). The intertrial interval was 5 sec and the CS-UCS delay 200 msec. Practically the same results were obtained as in the first group: no significant changes in AEP amplitude were observed during the first block of 100 pairings of CS-UCS (106 ~ ~ 14). A slight AEP increase during the last period of the 30 rain conditioning (125 ~ zk 12) approached statistical significance (P = 0.05) and could also be seen during the first block of extinction (128 ~ zk 22). After an hour of extinction the AEP amplitude reached habituation level again (103 ~ ~ 11). The qualitative changes (shaded columns) were better expressed using this paradigm, however. Fig. 3A shows an example of this group of experiments. Train o f clicks as CS
The clearest results were obtained with another conditioning paradigm: a train of 4 clicks applied at 200 msec intervals was reinforced by a 100 msec train of electrical pulses (10 #sec, 100/sec) immediately following the last click and directly applied to the area of recording. The intertrial interval was 30 sec (Fig. 1D). Fig. 4 summarizes the results of 18 experiments (Fig. 4, Cx) performed in 5 unanesthetized rats with implanted electrodes in the left sensorimotor cortex (SMC) and of 10 experiments (Fig. 4, Tg) carried out in 10 unanesthetized rats with implanted electrodes placed stereotaxically in the left tegmental area. The results are represented by empty and shaded columns in the same way as in Fig. 2, but the mean AEP amplitudes to each acoustic stimulus are expressed in percentage of the corresponding response in the last habituation block. As the AEPs to the 4th click were masked during conditioning by the stimulation artifact, they had to be omitted in the results (C1 and CL). Changes in amplitude of the AEP to the first click occurred in the SMC (133 ~ :k 13) as well as in the tegmentum (203 ~ :k 36), in the first block of reinforced trials, but they were manifested better in the latter structure. In the cortex these changes practically disappeared (102 ~ -k 9) during 30 min of reinforcement, whereas in the tegmentum they remained high (186 ~ ~:~ 33). At the beginning of extinction the amplitudes of AEPs in the SMC sometimes increased again but in general they remained at the habituation level throughout this period. In the tegmentum the AEP amplitude decreased only a little (169 ~ i 32) during the first block of extinction trials and reached the level of the last habituation amplitude after an hour of extinction. Pseudoconditioning caused no significant increase in the AEP to the first click (94 :~ 14). The changes found for the 2nd, 3rd and 4th clicks, both in the SMC and the tegmentum were essentially similar but less prominent and only exceptionally statistically significant. Brain Research, 19 (1970) 249-261
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EVOKED POTENTIALS AND CONDITIONING
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Conditioned motor responses were seen in approximately 50 % of the experiments in which direct stimulation of the tegmentum was used as UCS. This conditioned motor reaction appeared during the conditioning period and was also present in the first 6-10 extinction trials. It consisted of jerking movements of the head Brain Research, 19 (1970) 249-261
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Z. CAVIEDES AND J. BURES
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Fig. 5. Modification of cortical AEPs (based on 20 individual responses) during conditioning. P, pseudoconditioning. Other description as for Fig. 3. For details see text.
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Fig. 6. Modification of tegmental AEPs (based on 10 individual responses) during conditioning. Other description as for Figs. 3 and 5. Note modifications of both AEP amplitude and shape.
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EVOKED P O T E N T I A L S A N D C O N D I T I O N I N G
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Fig. 7. Changes of cortical (Cx) and tegmental (Tg)AEPs during conditioning. All values are expressed in percentages of the AEP to the first click in the last habituation block. Vertical bars indicate SEM. coincident with each individual click. Qualitative changes of the AEPs were also better expressed in the tegmentum than in the SMC (shaded columns in Fig. 4). Fig. 5 shows an example of the above experiments with recording and stimulating electrodes implanted in the SMC; 20 individual responses were averaged in each of the following periods: habituation, first and last conditioning period, first and last extinction period and a pseudoconditioning test. Changes in AEP amplitude are most clear-cut in the first reinforcement period. Similar changes were also seen at the onset of extinction and in the pseudoconditioning control, their magnitude, however, being practically indistinguishable from the conditioning results. A typical experiment with tegmental recording and stimulating electrodes is shown in Fig. 6 (averages of 10 evoked responses). Conditioning induced bigger changes in AEP amplitude in the tegmentum than in the cortex. The effect of pseudoconditioning was clearly smaller than the effect of time-locked reinforcement. The above results are summarized in Fig. 7 showing the relationships between the AEPs to the 4 clicks during the different phases of the experiments. The curves represent the evolution of the AEP amplitude during the period of the last habituation, first and last conditioning, and the first and last extinction. The values are expressed as percentages of the amplitude of the AEP to the first click ( = 100~) during the last period of habituation. Initial habituation
The initial habituation to the isolated presentation of the CS was evaluated by using the data of the experiments performed with the train of clicks, in the SMC and tegmentum. Fig. 8 summarizes the results; the heavy line represents the amplitude Brain Research, 19 (1970) 249-261
258
E. CAVIEDESAND J. BURES
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Fig. 8. Changes of cortical (Cx) and tegmental (Tg) AEPs at the onset (0) and after 30 and 60 rain of habituation. All values are expressed in percentage of the AEP to the first click in the first block of habituation trials.
of the cortical AEPs for each click at the beginning, in the middle and at the end of the 60 min period of habituation. The mean values of the AEP amplitude are expressed in percentages of the AEP to the first click in the first block of responses. The rapid decrease of the A E P amplitude during the first 30 rain (60 presentations of the acoustic stimulus) of habituation (65 % 4- 11 for the first click) slowed down during the second half hour of habituation (last habituation value 54 % 4- 6). The same results were obtained with the AEPs to other clicks. During a second application (after 4-5 days interval) of identical acoustic stimuli to the same group of animals, the rats maintained a certain degree of habituation. The dashed line in Fig. 8 corresponds to results obtained in 6 rats with the implanted electrodes in the tegmentum. The initial habituation is exhibited much less than in the SMC: the average amplitudes of the first click response reached 93% -k 12 and 84% 4- 12 after 30 and 60 min of habituation respectively. Neither of these decrements was statistically significant. DISCUSSION Although the results of the present study confirm the possibility of using brain stimulation as UCS, they indicate that the efficiency of the conditioning procedure depends on the location of the stimulating electrodes. With approximately the same intensity of stimuli, and consequently with the same size of neural populations affected by the UCS, cortical stimulation led to a significant increment of the AEP to the CS only at the beginning of conditioning, and the effect was considerably less regular and smaller than with tegmental stimulation. With continuing reinforcement the initial increase of cortical AEPs disappeared and no significant changes could be observed during extinction. As the cortical response to the UCS did not attenuate during the 30 min of reinforcement, the decrement of the acoustic response must be sought in processes occurring outside the area of stimulation. The time course of the Brain Research, 19 (1970) 249-261
EVOKED POTENTIALS AND CONDITIONING
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initial change points to the possibility that it is due to a transitory, rapidly habituating emotional state caused by cortical stimulation or to orientation of the animal's attention to sensory stimuli associated with this state. The results of the pseudoconditioning experiments favor this explanation. The amplitude of the AEP in the tegmental reticular formation followed a time course corresponding much better to a conditioning paradigm: it was approximately doubled during the first block of reinforced trials and remained at this increased level as long as reinforcement was applied. The AEPs remained high in the first block of extinction trials, but at the end of extinction they were the same as at the beginning of the experiment. Pseudoconditioning controls indicated that non-specific factors were responsible for only a part of the observed effects and that time coincidence of CS and UCS is essential. In approximately 50~o of experiments with tegmental stimulation a conditioned motor reaction appeared during reinforcement and disappeared during extinction. Comparison of AEP changes in experiments with and without the conditioned motor response indicates that the latter is not reflected in the electrical recording and that its presence or absence is not essential for the success of the EEG conditioning. On the other hand the conditioned motor response was never seen without the coincident AEP change, which seems to appear sooner and more regularly than the motor effect. The difference between the efficiency of cortical and tegmental stimuli is probably due to their reinforcing properties. Whereas according to Olds16,17 and Doty and Giurgea 6 cortical stimulation usually has neither rewarding nor punishing effects, tegmental stimuli elicit reactions resembling pain or fear z and the rat readily learns to escape them. A difference between motivated and non-motivated learning was reported by Doty 4. A monkey learned a conditioned reaction to an electrical CS of 0.55 mA, if stimulations of cerebral cortex were used both as CS and UCS; when painful skin stimulation replaced the electrical brain UCS, the threshold of the CS in the same cortical area dropped to 0.2 mA. The failure to obtain conditioning with cortical stimulation in the present study contrasts with earlier experiments, in which plastic reactions were obtained in 10-15 of examined cortical neurons, when microelectrophoretic stimulation of the recorded nerve cell was used as UCSL The assumption that the efficiency of conditioning will increase proportionally to the size of the populations activated was not confirmed. This negative evidence cannot be considered as quite conclusive, however, since the reactions evoked by CS and UCS were too different in the two studies. The present results support the assumption12, ~4 that the amplitude of the late component of acoustically evoked potentials in central auditory structures and in the mesencephalic reticular formation is related to the occurrence of fear. Besides this general emotional factor, more specific associative mechanisms seem to account for a part of the observed changes. On the basis of the present experiments it is impossible to decide, however, whether the plastic changes underlying the establishment of new connections occur in the area of stimulation or in some remote regions. The possible value of electrical brain stimulation for locating the plastic change depends entirely Brain Research, 19 (1970) 249-261
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on the answer to the above question. The experimental evidence (Caviedes and Buret, unpublished results) indicates that responses to acoustic stimuli reinforced by tegmental stimulation are modified not only in the stimulated region but also in the thalamus and sensorimotor cortex. Thus with strong stimulation of a biologically important center as UCS the plastic change can occur far away from the point of stimulation and cannot be located more easily than when using sensory stimuli for both CS and UCS. Further work is needed to define conditions indicating the occurrence of the plastic process in the area directly affected by the brain stimulus. The plastic change was most clearly expressed for the first click of the train, whereas the responses to the second and third clicks were influenced less regularly. The response to the fourth click, which coincided with the onset of the UCS, and that could not be recorded during reinforcement, showed in the first extinction block the same relative increase as the response to the first click. It seems that the train is perceived as a single stimulus which favors reactions to the first and last click. The preliminary experiments also indicated that single clicks and single electrical shocks are less suitable for successful conditioning than trains of stimuli. Similarly, conditioning seems to be more efficient in freely moving rats than in curarized animals. The habituation observations essentially confirm results of earlier studies according to which the late components of cortical evoked potentials decrease fast at the beginning of the repetitive application of stimuli and much more slowly later 11,15. Although the arousal level was not systematically checked the AEP decrement cannot be due to increasing drowsiness, since AEP amplitudes are higher in sleep than in alerted conditions ~5. According to the same authors no habituation occurs in sleeping animals. The present recordings were made far from the specific acoustic pathways, but the latencies of the most prominent waves were the same as the latencies of the late components of the auditory evoked responses in the acoustic cortex or in the inferior colliculus in rats 14. The present finding that decrement of AEP is pronounced in the cortex but absent from the tegmentum is consistent, therefore, with the failure of HalP 1 to obtain habituation at the lower levels of the acoustic pathway (inferior colliculus, cochlear nucleus). SUMMARY
Plastic changes underlying classical conditioning were studied in unanesthetized curarized (N ~ 9) or freely moving (N = 23) rats, by reinforcing acoustic or somesthetic stimuli (conditioned stimulation, CS) with electrical stimulation of the sensorimotor cortex or tegmentum (unconditioned stimulation, UCS). Responses evoked by the CS were recorded in the regions exposed to the electrical UCS during 60 min habituation, 30 min CS-UCS pairing and 60 min extinction. During habituation the averaged evoked potentials (AEP) decreased to 54 ~ of the initial amplitude in the cortex but only to 84 ~ in the tegmentum. Conditioning proved to be more efficient when freely moving animals were used instead of curarized rats, and trains of sensory signals (4 clicks spaced 200 msec apart) and of electrical shocks (ten t0 #sec pulses Brain Research, 19 (1970) 249-261
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at 10 msec intervals) instead o f single stimuli. A s s o c i a t i o n o f local brain stimulation increased the cortical and t e g m e n t a l A E P s to 130 and 200 ~o o f the h a b i t u a t i o n level respectively. D u r i n g extinction cortical A E P s r e t u r n e d to the h a b i t u a t i o n level earlier than the t e g m e n t a l ones. P s e u d o c o n d i t i o n i n g did not cause a significant increase o f the tegmental AEPs. As the greater efficiency o f t e g m e n t a l stimulation seems to be due to its aversiveness, the A E P changes are p r o b a b l y not limited to the stimulated region but rather represent a local manifestation o f a m o r e general effect.
REFERENCES 1 BUREt,J., AND BURE~OVA,O., Plasticity at the single neuron level, Lectures and Symposia, XXIII Int. Physiol. Congr., Tokyo, 1965, pp. 359-364. 2 BUREt, J., AND BURE~OVA,O., Plastic changes of unit activity based on reinforcing properties of extracellular stimulation of single neurons, J. Neurophysiol., 30 (1967) 98-113. 3 DELGADO,J. M. R., ROSVOLD, H. E., AND LOONEY,E., Evoking conditioned fear by electrical stimulation of subcortical structures in the monkey brain, J. comp. physiol. Psychol., 49 (1956) 373-380. 4 DOTY,R. W., Conditioned reflexes formed and evoked by brain stimulation. In D. E. SHEER(Ed.), Electrical Stimulation of the Brain, Univ. Texas Press, Houston, 1961, p. 397. 5 DOTY, R. W., Electrical stimulation of the brain in behavioral context, Ann. Rev. Psychol., 20 (1969) 289-320. 6 DOTY,R. W., AND GIURGEA,C., Conditioned reflexes establislaed by coupling electrical excitation of two cortical areas. In A, FESSARD,R. W. GERARDAND J. KONORSKI(Eds.), Brain Mechanism and Learning, Blackwell, London, 1961, p. 133. 7 DOTY, R. W., RUTLEDGE,L. T., JR., AND LARSEN,R. M., Conditioned reflexes established to electrical stimulation of cat cerebral cortex, J. Neurophysiol., 19 (1956) 401415. 8 FIFKOVA,E., AND MARSALA,J., Stereotaxic atlases for cat, rabbit and rat. In J. BLrRE~,M. PETRA~, ANDJ. ZACHAR(Eds.), Electrophysiological Methods in Biological Research, Academia, Publishing House of the Czechoslovak Academy of Sciences, Prague, 1967, p. 653. 9 GERBRANDT,L. K., SKREBITSKY,V. G., BURESOV~,,O., AND BURES, J., Plastic changes of unit activity induced by tactile stimuli followed by electrical stimulation of single hippocampal and reticular neurons, Neuropsychologia, 6 (1968) 3-10. 10 GIURGEA,C., ET RAICIULESCU,N., t~tude 61ectroencdphalographique du r6flexe conditionnel l'excitation 61ectrique corticale directe, First Int. Congr. Neurol. Sci., 3 (1957) 156-176. 11 HALL,R. D., Habituation of evoked potentials in the rat under conditions of behavioral control, Electroenceph. din. Neurophysiol., 24 (1968) 155-165. 12 HALL, R. D., AND MARK, R. G., Fear and the modification of acoustically evoked potentials during conditioning, J. Neurophysiol., 30 (1967) 893-910. 13 LOUCKS,R. B., Preliminary report of a technique for stimulation or destruction of tissues beneath the integument and the establishing of conditioned reactions with faradization of cerebral cortex, J. comp. Psychol., 16 (1933) 439-444. 14 MARK,R. G., AND HALL, R. D., Acoustically evoked potentials in the rat during conditioning, J. Neurophysiol., 30 (1967) 875-892. 15 MARSH,J. T., AND WORDEN, E. G., Auditory potentials during acoustic habituation: cochlear nucleus, cerebellum and auditory cortex, Electroenceph. clin. Neurophysiol., 17 (1964) 685-692. 16 OLDS, J., Hypothalamic substrates of reward, Physiol. Roy., 42 (1962) 554-604. 17 OLDS,J., AND MILNER,P., Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain, J. comp. physiol. Psychol., 47 (1954) 419427. 18 TOMA, V., AND KREKULE,L, A special purpose digital computer for on-line processing of the physiological data, Physiol. bohemoslov., 18 (1969) 373.
Brain Research, 19 (1970) 249-261
BRAIN RESEARCH
C H A N G E S IN CORTICAL A N D T E G M E N T A L EVOKED RESPONSES TO SENSORY STIMULI R E I N F O R C E D BY E L E C T R I C A L S T I M U L A T I O N OF T H E AREA OF R E C O R D I N G
E. CAVIEDES* AND J. BURES
Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Accepted October 30th, 1969)
INTRODUCTION
Although in the last 15 years electrophysiology has revealed many electrical correlates of behavioral conditioning, it has not succeeded in identifying the regions responsible for the formation of new connections. Both conditioned stimulus (CS) and unconditioned stimulus (UCS) activate large neural populations extensively overlapping in wide brain areas, any part of which may become the site of the actual switching process. As the electrical concomitants of conditioning are usually diffuse, it is almost impossible to decide which of them correspond to the primary plastic change and which are only its consequence or byproducts. The plastic change can be located with more precision if electrical stimulation of a restricted brain region serves as UCS4,6,7,10,13. Most experiments of this type 5 use electrical stimuli eliciting overt motor, vegetative or emotional responses, and the ample feedback afferentation prevents straightforward interpretation of results. When local electrical responses are used instead of behavioral reactions, stimulation of any brain region can be used as U CS. Bureg and Bure~ov~ 1,z and Gerbrandt et al. 9 observed, in unanesthetized curarized rats, the appearance of single unit responses to initially ineffective sensory stimuli, the application of which was reinforced by electrophoretic injection of K + ions or by polarization through the recording microelectrode. Positive results were obtained only in 10-15 ~ of the examined neurons in the cortex, thalamus, hippocampus and reticular formationZ. The low incidence of plastic reactions indicates that stimulation of a single neuron may be an inadequate UCS and that the success of conditioning is proportional to the size of the neural population activated by the UCS. The aim of the present paper is to test this assumption by recording responses evoked by the sensory CS in cortical or tegmental areas subjected to direct electrical stimulation (UCS).
* Visiting scientist from the Institute of Physiology, Department of Sciences, University of Chile, Valparaiso, Chile. Brain Research, 19 (1970) 249-261
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E. CAVIEDES AND J. BURES
METHODS
A total of 49 experiments was performed on 29 hooded rats, aged 3-4 months. Under pentothal anesthesia (40 mg/kg) electrodes were implanted into either the cerebral cortex or the tegmentum. Cortical electrodes were 0.2 mm silver wires, which were inserted through 0.5 mm trephine openings 1.0 mm deep into the parietal cortex. The distance between the two stimulating electrodes was 1.5 mm. The recording electrodes were placed roughly in the line midperpendicular to the stimulating electrodes, again with a 1.5-2.0 mm interelectrode distance. Tegmental electrodes formed an assembly of 4 glass-insulated silver wires (0.2 mm diameter, 0.5 mm bare tip) with similar geometry of stimulating and recording electrodes. The electrode assembly was inserted through a T-shaped trephine opening into the region of the stratum griseum centrale. The stereotaxic coordinates of stimulating electrodes were AP7, L0.5, V6; AP7, L2, V6; and of the recording electrodes were AP5.5, L1, V6; AP4, L1, V6; all according to the atlas of Fifkov/t and MarSala s. A silver wire suture stitched through the edge of the occipital bone served as the ground lead. Two selftapping anchor-screws were placed in the parietal and occipital bones. The lead-off wires were soldered to a subminiature 5 pin socket which was fixed to the skull with acrylate. The experiments usually started 3-5 days after surgery and continued for up to 4 weeks. The animal was put into a tall narrow glass vessel (10 cm × 15 cm × 36 cm), the floor of which was covered with adsorbent cotton. The small size of the container considerably reduced the activity of the animal without causing noticeable discomfort. The glass vessel was placed in an electrically and acoustically screened box containing the preamplifier and output unit of the stimulator. The rat was connected through a microdot cable with the output unit of the stimulator and with the preamplifier (Tektronix 122). A loudspeaker placed directly above the glass container (average distance from the animal's head: 28 cm) was used as source of acoustic clicks, generated by another stimulator. The auditory conditioned stimuli were of constant intensity, approximately 70 dB above the human threshold. A preset counter generated the intertrial intervals as well as the CS-UCS delay. The scheme of the stimulating and recording setup is shown in Fig. 1 (A), which also shows the different stimulus sequences used (B, C and D). In most experiments the electrical stimulus was a 100 msec train of 10 #sec, 100/sec pulses, the amplitude of which was adjusted to a level just supramaximal for eliciting a local response. The mesencephalic stimulus of this intensity evoked a slight generalized jerk in all animals. The output unit of the stimulator was isolated from the ground and~0connectedlwithlthe main stimulator through a low capacity inductive coupling. The bipolarly recorded evoked responses were displayed on a CRO and fed into an on-line average response computer is, the sweep of which was triggered by the first click. With 1023 addresses and ! msec or 700/zsec per address, the sweep duration was slightly'over 1 sec or 700 msec respectively. Usually 10-100 sweeps were summated to form an average. The average waveforms were plotted with an X - Y recorder.
Brain Research, 19 (1970) 249-261
251
EVOKED POTENTIALSAND CONDITIONING
A
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Fig. 1. Scheme of the experimental setup (A) and of the stimulus sequences used (B, C, D), SS, somatosensory stimulus; AS, acoustic stimulus; BS, brain stimulation; ITI, intertrial interval. For details see text.
A typical experiment started with a 1 h habituation period to the isolated acoustic CS followed by 30 min of conditioning and 60 min of extinction. Periods of conditioning followed by extinction were repeated up to 3 times in one session.
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Pseudoconditioning (random application of CS and UCS) was tested after the last extinction in some experiments. Each rat was used in 2-3 sessions only. Statistical evaluation of results was based on comparison of amplitudes of the most prominent component of the average evoked potential (AEP), usually occurring with a latency of 30-40 msec. In conditioning and extinction experiments the amplitude of the last habituation AEP was used as 100 ~ and the amplitudes of the first and last conditioning and first and last extinction averages were expressed accordingly. When the initial habituation to the isolated presentation of the CS was evaluated, the AEP to the first click in the first block of responses was taken as 1 0 0 ~ and the other responses and their changes during habituation were expressed in percentages. The percentage values obtained in individual experiments were averaged and standard errors of means were computed in the conventional way. Localization of the subcortical electrodes was verified in Nissl stained serial sections. It usually corresponded to the target coordinates with an error not exceeding 0.5 mm. RESULTS Preliminary e x p e r i m e n t s
The aim of the first series of experiments was to find an optimal paradigm. The results are summarized in Fig. 2, the upper part of which represents a group of
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Fig. 2. Changes of cortical AEPs during conditioning in unanesthetized curarized (above) and freely moving (below) rats. Empty columns -- mean values of AEPs. Vertical bars denote the SEM values. Shaded columns = incidence of modified AEPs. H, last habituation block; C1, first conditioning block; CL, last conditioning block; El, first extinction block; EL, last extinction block. For details see text. Brain Research, 19 (1970) 249-261
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EVOKED POTENTIALS AND CONDITIONING
9 experiments performed on curarized rats (D-tubocurarine 0.1-0.2 mg/100 g). A liminal electric shock to the forepaw was used as CS, whereas a single electrical stimulus (pulse of l0 #sec duration) directly applied to the contralateral parietal cortex was used as UCS (Fig. 1B). The CS-UCS delay was 200 msec and the intertrial interval 5 sec. Each empty column in the figure represents the mean amplitude of the AEP based on 40 individual responses, expressed in percentages of the amplitude of the last habituation AEP, which was taken as 100 % (Fig. 2, H). Practically no changes were seen (109% 4- 17) during the first 40 CS-UCS pairings (Fig. 2, C1). Even the small increase (137% ± 24) of the AEP amplitude in the last part of the 30 min conditioning period was not statistically significant (Fig. 2, C~,). The AEP amplitude decreased during the first block of extinction trials (117% -4- 20, Fig. 2, El), and after an hour the level of the last habituation was attained again (95 % ± 21, Fig. 2, El,). The shaded columns of the same figure represent the quali-
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Fig. 3. Modification of cortical AEPs during conditioning in a freely moving rat (A, AEPs based on 100 individual responses) and in a curarized rat (B, AEPs based on 40 individual responses). The numbers denote time (in minutes) during which the respective AEPs were obtained. H, habituation; C, conditioning; E, extinction. Note changes of the AEP waveform in B when the cortical stimulus was increased after the first hour of reinforcement (increased direct cortical response).
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tative changes, either in amplitude or in the shape of the AEPs, expressed in percentages of the total number of experiments. Fig. 3B shows an example of such an experiment, demonstrating changes in amplitude, as well as some changes in the AEP waveform during more prolonged conditioning and first extinction trials. The lower part of Fig. 2 represents the results of 8 experiments performed in freely-moving rats with implanted electrodes in the left parietal cortex. A single click was used as CS and a train of pulses (100 msec, 10 #sec, 100/sec) directly applied to the region of recording, as UCS (Fig. 1C). The intertrial interval was 5 sec and the CS-UCS delay 200 msec. Practically the same results were obtained as in the first group: no significant changes in AEP amplitude were observed during the first block of 100 pairings of CS-UCS (106 ~ ~ 14). A slight AEP increase during the last period of the 30 rain conditioning (125 ~ zk 12) approached statistical significance (P = 0.05) and could also be seen during the first block of extinction (128 ~ zk 22). After an hour of extinction the AEP amplitude reached habituation level again (103 ~ ~ 11). The qualitative changes (shaded columns) were better expressed using this paradigm, however. Fig. 3A shows an example of this group of experiments. Train o f clicks as CS
The clearest results were obtained with another conditioning paradigm: a train of 4 clicks applied at 200 msec intervals was reinforced by a 100 msec train of electrical pulses (10 #sec, 100/sec) immediately following the last click and directly applied to the area of recording. The intertrial interval was 30 sec (Fig. 1D). Fig. 4 summarizes the results of 18 experiments (Fig. 4, Cx) performed in 5 unanesthetized rats with implanted electrodes in the left sensorimotor cortex (SMC) and of 10 experiments (Fig. 4, Tg) carried out in 10 unanesthetized rats with implanted electrodes placed stereotaxically in the left tegmental area. The results are represented by empty and shaded columns in the same way as in Fig. 2, but the mean AEP amplitudes to each acoustic stimulus are expressed in percentage of the corresponding response in the last habituation block. As the AEPs to the 4th click were masked during conditioning by the stimulation artifact, they had to be omitted in the results (C1 and CL). Changes in amplitude of the AEP to the first click occurred in the SMC (133 ~ :k 13) as well as in the tegmentum (203 ~ :k 36), in the first block of reinforced trials, but they were manifested better in the latter structure. In the cortex these changes practically disappeared (102 ~ -k 9) during 30 min of reinforcement, whereas in the tegmentum they remained high (186 ~ ~:~ 33). At the beginning of extinction the amplitudes of AEPs in the SMC sometimes increased again but in general they remained at the habituation level throughout this period. In the tegmentum the AEP amplitude decreased only a little (169 ~ i 32) during the first block of extinction trials and reached the level of the last habituation amplitude after an hour of extinction. Pseudoconditioning caused no significant increase in the AEP to the first click (94 :~ 14). The changes found for the 2nd, 3rd and 4th clicks, both in the SMC and the tegmentum were essentially similar but less prominent and only exceptionally statistically significant. Brain Research, 19 (1970) 249-261
255
EVOKED POTENTIALS AND CONDITIONING
7-9
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Fig. 4. Changes of cortical (Cx) and tegmental (Tg) AEPs during conditioning. Other description as for Fig. 2.
Conditioned motor responses were seen in approximately 50 % of the experiments in which direct stimulation of the tegmentum was used as UCS. This conditioned motor reaction appeared during the conditioning period and was also present in the first 6-10 extinction trials. It consisted of jerking movements of the head Brain Research, 19 (1970) 249-261
256
Z. CAVIEDES AND J. BURES
E 0-10 i H 0-10
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Fig. 5. Modification of cortical AEPs (based on 20 individual responses) during conditioning. P, pseudoconditioning. Other description as for Fig. 3. For details see text.
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Fig. 6. Modification of tegmental AEPs (based on 10 individual responses) during conditioning. Other description as for Figs. 3 and 5. Note modifications of both AEP amplitude and shape.
Brain Research, 19 (1970)249-261
257
EVOKED P O T E N T I A L S A N D C O N D I T I O N I N G
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Fig. 7. Changes of cortical (Cx) and tegmental (Tg)AEPs during conditioning. All values are expressed in percentages of the AEP to the first click in the last habituation block. Vertical bars indicate SEM. coincident with each individual click. Qualitative changes of the AEPs were also better expressed in the tegmentum than in the SMC (shaded columns in Fig. 4). Fig. 5 shows an example of the above experiments with recording and stimulating electrodes implanted in the SMC; 20 individual responses were averaged in each of the following periods: habituation, first and last conditioning period, first and last extinction period and a pseudoconditioning test. Changes in AEP amplitude are most clear-cut in the first reinforcement period. Similar changes were also seen at the onset of extinction and in the pseudoconditioning control, their magnitude, however, being practically indistinguishable from the conditioning results. A typical experiment with tegmental recording and stimulating electrodes is shown in Fig. 6 (averages of 10 evoked responses). Conditioning induced bigger changes in AEP amplitude in the tegmentum than in the cortex. The effect of pseudoconditioning was clearly smaller than the effect of time-locked reinforcement. The above results are summarized in Fig. 7 showing the relationships between the AEPs to the 4 clicks during the different phases of the experiments. The curves represent the evolution of the AEP amplitude during the period of the last habituation, first and last conditioning, and the first and last extinction. The values are expressed as percentages of the amplitude of the AEP to the first click ( = 100~) during the last period of habituation. Initial habituation
The initial habituation to the isolated presentation of the CS was evaluated by using the data of the experiments performed with the train of clicks, in the SMC and tegmentum. Fig. 8 summarizes the results; the heavy line represents the amplitude Brain Research, 19 (1970) 249-261
258
E. CAVIEDESAND J. BURES
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Fig. 8. Changes of cortical (Cx) and tegmental (Tg) AEPs at the onset (0) and after 30 and 60 rain of habituation. All values are expressed in percentage of the AEP to the first click in the first block of habituation trials.
of the cortical AEPs for each click at the beginning, in the middle and at the end of the 60 min period of habituation. The mean values of the AEP amplitude are expressed in percentages of the AEP to the first click in the first block of responses. The rapid decrease of the A E P amplitude during the first 30 rain (60 presentations of the acoustic stimulus) of habituation (65 % 4- 11 for the first click) slowed down during the second half hour of habituation (last habituation value 54 % 4- 6). The same results were obtained with the AEPs to other clicks. During a second application (after 4-5 days interval) of identical acoustic stimuli to the same group of animals, the rats maintained a certain degree of habituation. The dashed line in Fig. 8 corresponds to results obtained in 6 rats with the implanted electrodes in the tegmentum. The initial habituation is exhibited much less than in the SMC: the average amplitudes of the first click response reached 93% -k 12 and 84% 4- 12 after 30 and 60 min of habituation respectively. Neither of these decrements was statistically significant. DISCUSSION Although the results of the present study confirm the possibility of using brain stimulation as UCS, they indicate that the efficiency of the conditioning procedure depends on the location of the stimulating electrodes. With approximately the same intensity of stimuli, and consequently with the same size of neural populations affected by the UCS, cortical stimulation led to a significant increment of the AEP to the CS only at the beginning of conditioning, and the effect was considerably less regular and smaller than with tegmental stimulation. With continuing reinforcement the initial increase of cortical AEPs disappeared and no significant changes could be observed during extinction. As the cortical response to the UCS did not attenuate during the 30 min of reinforcement, the decrement of the acoustic response must be sought in processes occurring outside the area of stimulation. The time course of the Brain Research, 19 (1970) 249-261
EVOKED POTENTIALS AND CONDITIONING
259
initial change points to the possibility that it is due to a transitory, rapidly habituating emotional state caused by cortical stimulation or to orientation of the animal's attention to sensory stimuli associated with this state. The results of the pseudoconditioning experiments favor this explanation. The amplitude of the AEP in the tegmental reticular formation followed a time course corresponding much better to a conditioning paradigm: it was approximately doubled during the first block of reinforced trials and remained at this increased level as long as reinforcement was applied. The AEPs remained high in the first block of extinction trials, but at the end of extinction they were the same as at the beginning of the experiment. Pseudoconditioning controls indicated that non-specific factors were responsible for only a part of the observed effects and that time coincidence of CS and UCS is essential. In approximately 50~o of experiments with tegmental stimulation a conditioned motor reaction appeared during reinforcement and disappeared during extinction. Comparison of AEP changes in experiments with and without the conditioned motor response indicates that the latter is not reflected in the electrical recording and that its presence or absence is not essential for the success of the EEG conditioning. On the other hand the conditioned motor response was never seen without the coincident AEP change, which seems to appear sooner and more regularly than the motor effect. The difference between the efficiency of cortical and tegmental stimuli is probably due to their reinforcing properties. Whereas according to Olds16,17 and Doty and Giurgea 6 cortical stimulation usually has neither rewarding nor punishing effects, tegmental stimuli elicit reactions resembling pain or fear z and the rat readily learns to escape them. A difference between motivated and non-motivated learning was reported by Doty 4. A monkey learned a conditioned reaction to an electrical CS of 0.55 mA, if stimulations of cerebral cortex were used both as CS and UCS; when painful skin stimulation replaced the electrical brain UCS, the threshold of the CS in the same cortical area dropped to 0.2 mA. The failure to obtain conditioning with cortical stimulation in the present study contrasts with earlier experiments, in which plastic reactions were obtained in 10-15 of examined cortical neurons, when microelectrophoretic stimulation of the recorded nerve cell was used as UCSL The assumption that the efficiency of conditioning will increase proportionally to the size of the populations activated was not confirmed. This negative evidence cannot be considered as quite conclusive, however, since the reactions evoked by CS and UCS were too different in the two studies. The present results support the assumption12, ~4 that the amplitude of the late component of acoustically evoked potentials in central auditory structures and in the mesencephalic reticular formation is related to the occurrence of fear. Besides this general emotional factor, more specific associative mechanisms seem to account for a part of the observed changes. On the basis of the present experiments it is impossible to decide, however, whether the plastic changes underlying the establishment of new connections occur in the area of stimulation or in some remote regions. The possible value of electrical brain stimulation for locating the plastic change depends entirely Brain Research, 19 (1970) 249-261
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on the answer to the above question. The experimental evidence (Caviedes and Buret, unpublished results) indicates that responses to acoustic stimuli reinforced by tegmental stimulation are modified not only in the stimulated region but also in the thalamus and sensorimotor cortex. Thus with strong stimulation of a biologically important center as UCS the plastic change can occur far away from the point of stimulation and cannot be located more easily than when using sensory stimuli for both CS and UCS. Further work is needed to define conditions indicating the occurrence of the plastic process in the area directly affected by the brain stimulus. The plastic change was most clearly expressed for the first click of the train, whereas the responses to the second and third clicks were influenced less regularly. The response to the fourth click, which coincided with the onset of the UCS, and that could not be recorded during reinforcement, showed in the first extinction block the same relative increase as the response to the first click. It seems that the train is perceived as a single stimulus which favors reactions to the first and last click. The preliminary experiments also indicated that single clicks and single electrical shocks are less suitable for successful conditioning than trains of stimuli. Similarly, conditioning seems to be more efficient in freely moving rats than in curarized animals. The habituation observations essentially confirm results of earlier studies according to which the late components of cortical evoked potentials decrease fast at the beginning of the repetitive application of stimuli and much more slowly later 11,15. Although the arousal level was not systematically checked the AEP decrement cannot be due to increasing drowsiness, since AEP amplitudes are higher in sleep than in alerted conditions ~5. According to the same authors no habituation occurs in sleeping animals. The present recordings were made far from the specific acoustic pathways, but the latencies of the most prominent waves were the same as the latencies of the late components of the auditory evoked responses in the acoustic cortex or in the inferior colliculus in rats 14. The present finding that decrement of AEP is pronounced in the cortex but absent from the tegmentum is consistent, therefore, with the failure of HalP 1 to obtain habituation at the lower levels of the acoustic pathway (inferior colliculus, cochlear nucleus). SUMMARY
Plastic changes underlying classical conditioning were studied in unanesthetized curarized (N ~ 9) or freely moving (N = 23) rats, by reinforcing acoustic or somesthetic stimuli (conditioned stimulation, CS) with electrical stimulation of the sensorimotor cortex or tegmentum (unconditioned stimulation, UCS). Responses evoked by the CS were recorded in the regions exposed to the electrical UCS during 60 min habituation, 30 min CS-UCS pairing and 60 min extinction. During habituation the averaged evoked potentials (AEP) decreased to 54 ~ of the initial amplitude in the cortex but only to 84 ~ in the tegmentum. Conditioning proved to be more efficient when freely moving animals were used instead of curarized rats, and trains of sensory signals (4 clicks spaced 200 msec apart) and of electrical shocks (ten t0 #sec pulses Brain Research, 19 (1970) 249-261
EVOKED POTENTIALS AND CONDITIONING
261
at 10 msec intervals) instead o f single stimuli. A s s o c i a t i o n o f local brain stimulation increased the cortical and t e g m e n t a l A E P s to 130 and 200 ~o o f the h a b i t u a t i o n level respectively. D u r i n g extinction cortical A E P s r e t u r n e d to the h a b i t u a t i o n level earlier than the t e g m e n t a l ones. P s e u d o c o n d i t i o n i n g did not cause a significant increase o f the tegmental AEPs. As the greater efficiency o f t e g m e n t a l stimulation seems to be due to its aversiveness, the A E P changes are p r o b a b l y not limited to the stimulated region but rather represent a local manifestation o f a m o r e general effect.
REFERENCES 1 BUREt,J., AND BURE~OVA,O., Plasticity at the single neuron level, Lectures and Symposia, XXIII Int. Physiol. Congr., Tokyo, 1965, pp. 359-364. 2 BUREt, J., AND BURE~OVA,O., Plastic changes of unit activity based on reinforcing properties of extracellular stimulation of single neurons, J. Neurophysiol., 30 (1967) 98-113. 3 DELGADO,J. M. R., ROSVOLD, H. E., AND LOONEY,E., Evoking conditioned fear by electrical stimulation of subcortical structures in the monkey brain, J. comp. physiol. Psychol., 49 (1956) 373-380. 4 DOTY,R. W., Conditioned reflexes formed and evoked by brain stimulation. In D. E. SHEER(Ed.), Electrical Stimulation of the Brain, Univ. Texas Press, Houston, 1961, p. 397. 5 DOTY, R. W., Electrical stimulation of the brain in behavioral context, Ann. Rev. Psychol., 20 (1969) 289-320. 6 DOTY,R. W., AND GIURGEA,C., Conditioned reflexes establislaed by coupling electrical excitation of two cortical areas. In A, FESSARD,R. W. GERARDAND J. KONORSKI(Eds.), Brain Mechanism and Learning, Blackwell, London, 1961, p. 133. 7 DOTY, R. W., RUTLEDGE,L. T., JR., AND LARSEN,R. M., Conditioned reflexes established to electrical stimulation of cat cerebral cortex, J. Neurophysiol., 19 (1956) 401415. 8 FIFKOVA,E., AND MARSALA,J., Stereotaxic atlases for cat, rabbit and rat. In J. BLrRE~,M. PETRA~, ANDJ. ZACHAR(Eds.), Electrophysiological Methods in Biological Research, Academia, Publishing House of the Czechoslovak Academy of Sciences, Prague, 1967, p. 653. 9 GERBRANDT,L. K., SKREBITSKY,V. G., BURESOV~,,O., AND BURES, J., Plastic changes of unit activity induced by tactile stimuli followed by electrical stimulation of single hippocampal and reticular neurons, Neuropsychologia, 6 (1968) 3-10. 10 GIURGEA,C., ET RAICIULESCU,N., t~tude 61ectroencdphalographique du r6flexe conditionnel l'excitation 61ectrique corticale directe, First Int. Congr. Neurol. Sci., 3 (1957) 156-176. 11 HALL,R. D., Habituation of evoked potentials in the rat under conditions of behavioral control, Electroenceph. din. Neurophysiol., 24 (1968) 155-165. 12 HALL, R. D., AND MARK, R. G., Fear and the modification of acoustically evoked potentials during conditioning, J. Neurophysiol., 30 (1967) 893-910. 13 LOUCKS,R. B., Preliminary report of a technique for stimulation or destruction of tissues beneath the integument and the establishing of conditioned reactions with faradization of cerebral cortex, J. comp. Psychol., 16 (1933) 439-444. 14 MARK,R. G., AND HALL, R. D., Acoustically evoked potentials in the rat during conditioning, J. Neurophysiol., 30 (1967) 875-892. 15 MARSH,J. T., AND WORDEN, E. G., Auditory potentials during acoustic habituation: cochlear nucleus, cerebellum and auditory cortex, Electroenceph. clin. Neurophysiol., 17 (1964) 685-692. 16 OLDS, J., Hypothalamic substrates of reward, Physiol. Roy., 42 (1962) 554-604. 17 OLDS,J., AND MILNER,P., Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain, J. comp. physiol. Psychol., 47 (1954) 419427. 18 TOMA, V., AND KREKULE,L, A special purpose digital computer for on-line processing of the physiological data, Physiol. bohemoslov., 18 (1969) 373.
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