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Deficits in conditioned movement and visual discrimination following rubral area lesions in the rat
Physiology and Behavior. Vol. 5, pp. 893-898. Pergamon Press, 1970. Printed in Great Britain
Deficits in Conditioned Movement and Visual Discrimination Following Rubral Area Lesions in the Raf ALLAN
M. S M I T H ~'a
Department o f Psychology, McGill University, Montreal, Quebec, Canada (Received 16 J a n u a r y 1970)
SMITH,A. M. Deficits in conditioned movement and visual discrimination following rubral area lesions in the rat. PHYSIOL. BEHAV. 5 (8) 893-898, 1970.--Bilateral electrolytic lesions in the region of the red nucleus impaired the ability of 15 hooded rats to perform a conditioned avoidance response. The deficit is interpreted as implying a failure to initiate conditioned movement rather than direct impairment of motor organization or ability to learn. A smaller sample of the same group trained to perform simultaneous black-white and stripe discriminations showed deficits only in the acquisition of the stripe discrimination. A second experiment found that rats with rubral area lesions showed impaired learning and slower running speed in a food motivated successive brightness discrimination task. A third experiment found that rats with rubral area lesions were significantly slower to initiate movement on both pre and post shock days in one-trial passive avoidance training. Rubral area lesions Initiation of conditioned movement Successive brightness discrimination Passive avoidance
Simultaneous brightness and pattern discrimination
no accompanying paresis and the quality of motor performance is not impaired. In general these studies suggest that following red nucleus lesions the performance of voluntary movement responses is impaired. The first series of experiments reported here attempted to determine if the deficits in conditioned movement and visual descrimination following red nucleus lesions are independent of each other. A second experiment attempted to evaluate the effects on behavior of rubral lesions when appetitive rather than aversive incentives are used, and finally passive avoidance conditioning was undertaken to compare the effects of rubral lesions on both the initiation and inhibition of movement.
RECENT studies have indicated that the red nucleus may be important for visually guided movement reponses [6, 7, 12, 13]. Single units of the red nucleus in the anesthetized cat yield phasic responses to various kinds of sensory input including responses to photic stimulation [6]. Thompson et al. [13] report that rats with bilateral red nucleus lesions fail to retain a visually cued avoidance response and also that relearning a brightness discrimination is significantly impaired. In subsequent studies, it has been found that retention of pattern discrimination is more greatly impaired than brightness discrimination by red nucleus lesions [12]. It should be mentioned, however, that these studies involved a variety of lesions and only a few animals in each study had lesions of the red nucleus. Using a larger sample, McNew [8] found that rats with red nucleus lesions are more impaired than normal rats in the performance of size and pattern discrimination and are also significantly worse than rats with dorsal mesencephalic lesions. Behavioral anomalies have been noted in monkeys that were being prepared for anatomical studies [2, 3]. The most striking of these anomalies, according to Carpenter, is hypokinesis or disinclination to move. He states that although there is a severe depression of voluntary movement, there is
METHOD
Subjects The subjects were 76 male and female hooded rats of the Royal Victoria Hospital stock weighing between 200 and 400 g. Since no difference in behavior was observed between male and female animals, the subjects are referred to simply as rats.
1The studies reported here formed part of a dissertation submitted by the author to the Faculty of Graduate Studies and Research, McGill University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. A preliminary report of some of these data was presented to the annual meeting of the Canadian Psychological Association, June 1968. 2This research was supported by a predoctoral fellowship from the National Research Council of Canada. The author wishes to thank Dr. Muriel Stern for her advice and criticism of this research. 8Present address--D6partement de Neurophysiologie G6n6rale, Institut de Neurophysiologie et Psychophysiologie, C. N. R. S., 31 chemin Joseph Aiguier, Marseille (9me), France. 893
894
SMITI~
Surgical Procedures
Procedure
Bilateral electrolytic lesions in the region of the red nucleus were achieved under general anesthesia with sodium pentobarbital (Nembutal). A twisted nichrome wire electrode, insulated, except at the tip, was lowered into place and a cathodal current of 2.0 mA for 10 sec was used to produce the lesions. Since the insertion of the electrode alone caused profuse bleeding, sham operations also included insertion of the electrode to a depth just dorsal to the red nucleus. The scalp wound was closed with Michel clips, and methetharamide (Mikedimide) and penicillin were administered to facilitate recovery.
Each rat was placed in the white compartment facing away from the door and was given five sec to exit into the black compartment, after which the floor of the white compartment was electrified. Twenty trials a day were given and for those animals with brain lesions that did not attain the criterion of 20 consecutive avoidances by the sixth day or 120th trial, the preshock interval was increased to t5 sec. Avoidance training was discontinued for all animals that had not reached criterion within 200 trials. Results
Postoperative Syndrome
The effect of bilateral rubral lesions was extremely severe. Most often the rats were aphagic and adipsic. Motor dysfunctions of various kinds were observed frequently. Tortieollis was common and probably due to an asymmetry of bilateral damage. Transient hypotonus and paresis were observed in the limbs of most of the animals. No evidence of tremor was ever observed, but frequently some animals displayed wild uncontrolled running that closely resembled audiogenic seizures. The seizures never occurred beyond the third day after the operation. All animals were given three weeks in which to recover after which they appeared to eat and drink normally and general locomotion was coordinated. No animal that showed any signs of motor dysfunction was included in any experiment.
EXPERIMENT I - A
Using 32 rats, 15 with red nucleus lesions and 17 sham operation control animals, it was decided to examine the effects of rubral lesions first on a simple one-way avoidance task. Apparatus
The avoidance apparatus consisted of a box 12 × 18 × 18 in. with two compartments of equal size separated by a sliding guillotine door. One compartment was painted white and the other, black. The floor of the apparatus was a stainless steel grid that could be electrified on one side.
The most evident differences between the group with red nucleus lesions and the sham operation control group occurred in the number of trials required to reach criterion and the number of failures to avoid shock. As shown in Table l, a student's t-test revealed that rats with red nucleus lesions required a significantly greater number of trials to reach criterion and made significantly more errors than the sham operation control subjects. There was no overlap in the scores for the two groups. Increasing the preshock avoidance time from five sec to 15 sec, after 120 trials, improved the performance of the braindamaged rats. The addition of 10 see to the preshock period increased successful avoidances from an average of 37 per cent during the five-see training period (trials 1-120) to an average of 63 per cent avoidances for the 15-sec training period (trials 1 2 1 - 2 0 0 or criterion) and when the scores are transformed into arcsin values the difference is statistically significant (t - - 2.20. 28, df, p < 0.05). A comparison of the mean times taken in seconds to complete an avoidance response in the last 10 trials (taken to represent optimal performance) indicated that rats with red nucleus lesions were significantly slower to avoid than normal rats (t - - 4.55, 18 dr, p < 0.01). Moreover, a comparison of the mean times required to complete escape responses on the last escape trial indicated that the control animals were slower to escape shock than the rats with red nucleus lesions (t - - 2.37, 18 dr, p < 0.05). It should be stressed, however, that the rats with rubral lesions received a mean of 90 more escape trials than the control subjects and the difference in escape time is undoubtedly due to prolonged practice received by the rats with red nucleus lesions. The difference in escape
TABLE 1 TRIALS TO CRITERION AND ERRORS TO CRITERION IN RATS LEARNING AN ACTIVE SHOCK AVOIDANCE RESPONSE AS A FUNCTION OF RED NUCLEUS LESIONS
Subjects
Trials to Criterion
Errors to Criterion
Red nucleus Damaged N = 15
Range = 131-200 Mean = 182.6
Range = 25-196 Mean = 101.2
Sham operation N = 17
Range = 26-52 Mean = 36.6
Range = 3-21 Mean = 10.2
t = 21.19 d f = 30
t = 7.00 df = 30 p < O.Ol
p < o.01
RUBRAL AREA LESIONS AND CONDITIONED MOVEMENT time is presented simply to indicate that the brain damaged rats were not unable to run. 1-B
EXPERIMENT
It was decided to test a sample of the same animals that had shown deficits in avoidance training, in a shock-motivated visual discrimination apparatus similar to the one described by Bauer and Cooper [1 ] and shown in Fig. I.
Pretraining Procedures Since the usual avoidance pretraining would have been ineffective for rats with red nucleus lesions, all animals were pretrained to escape from the apparatus. Each of 13 rats with rubral lesions and 17 sham operation control animals was placed in the start box with the sliding door open. Simultaneously, the floor was electrified and the start box door was closed behind the animal to prevent retreat. The shock was
I(
17" GOAL
~1 BOX
DOORS
~~
Grid
36~ ~
(2)
895 card was alternated from side to side in a predetermined random sequence. An error was counted if a rat placed two paws in the threshold area of the incorrect door and each rat had to correct the error to escape from the apparatus. If a rat failed to leave the start box in five sec, or failed to leave the choice area in 25 see, shock was applied for one sec. every five sec. It was noted on each trial if shock was needed to move the animal from the start box. Each day 10 trials were given and training was terminated after nine correct responses in any 10 trials. Immediately after reaching the acquisition criterion of the black-white discrimination problem, a smaller sample of eight rats with rubral lesions and 10 animals with sham lesions were trained in the same apparatus to discriminate between horizontal and vertical black and white stripes to a criterion of nine correct responses in any 10 trials.
Results There were no significant differences between rats with red nucleus lesions and sham lesion control animals with regard to either the number of trials required to reach the learning criterion or the number of errors made on the brightness discrimination task. On the pattern discrimination task, however, the brain-damaged animals required a significantly greater number of trials to reach criterion and made more errors. The moan trials to criterion for both tasks are shown in Fig. 2. Rats with red nucleus lesions required a mean of 79.5 trials to reach criterion and made a mean of 26.7 errors whereas control animals only required a mean of 33.6 trials (t = 6.27, 16 dr, p < 0.01) and made a mean of 15.5 errors (t = 2.64, 16df, p < 0.05). Despite the additional trials of avoidance training inherent in this discrimination conditioning, little improvement in performance was noted. Rats with red nucleus lesions successfully avoided shock on an average of only 32 per cent of the trials whereas sham operation control animals avoided shock on an average of 75 per cent of the trials.
MEAN TRIALSTO CRITERION SIMULTANEOUSBRIGHTNESSAND PATTERN DISCRIMINATION
i i q~'SL~G PLAN VIEW
~
~ - - ~ - ~ STARTBOX /
/
8O
VISUAl DISCRIMINATIONBOX
FIG. l. Diagram of visual discrimination apparatus. continued until the rat escaped through one of the appropriate doors. The exit doors were initially left open and were shut gradually until the animal learned to open them. The last door used was then locked and the rat was trained to seek out the alternate door. When a rat made three consecutive exits in less than 10 sec, pretraining was concluded.
Discrimination Training On the day following pretraining, training of black-white discrimination began. A black or white card attached to the exit doors served as the cue to the unlocked door. The threshold to the negative door (either area three or four in Fig. 1) was electrified and the door itself was locked. The cue
z
o
n" hi in"
60
u 40 o <_J
F-
N=8
]
SHAM OPERATION CONTROLS
]
REDNUCLEUS ABLATED
N:L'3
N=I7 N=IO
2O
DISCRIMINATION FIG. 2. Mean trials to learning criterion for rats with red nucleus lesions, and sham operation control rats on simultaneous brighmess and pattern discrimination.
896
g M l ! I{
EXPERIMENT
2
This experiment was designed to determine if the behavioral deficits following red nucleus lesions appeared when an appetitive motivation was used and also to obtain more information about the nature of the perceptual difficulties suggested by the first experiment.
Procedures Bilateral red nucleus lesions were performed on 10 rats and sham operations were conducted on eight additional animals. Following recovery all animals were food deprived for 23 hr each day for one week prior to the experiment. The animals were given free access to food and water one hour each day and during the last three days of this period feeding took place on an elevated T maze (5 in. wide with a stem of 30 in. and branches of 15 in. each).
in the learning scores for the three position response tasks, The mean trials to criterion for the three position response tasks and the successive brightness discrimination task are shown in Table 2. A two-way analysis of variance for repeated measures corrected for unequal sample size [16] was computed for the mean running times for each task. The results indicate that although the rtmning times become shorter with practice, the rats with red nucleus lesions require considerably more time to complete a trial (F 4.53, p 0.05). This change in running speed for both the braindamaged and the normal rats is sbo~n in t:ig, 3.
RUNNING TIME ON ELEVATED T MAZE
30'
Pretraining
x
Each animal was pretrained on a series of position response problems on the elevated T maze. All rats were taught first to go to the right-side goal box for food. The criterion for learning was set at 18 correct responses in any 20 trials and 20 trials a days were given. The criterion for learning remained the same throughout. An incorrect response was scored when a rat entered the wrong goal box. Following the acquisition of the first position response, the animals were trained to go to the left side and finally to go back to the right side again.
Discrimination Training This procedure consisted of training the rats to perform a successive black-white discrimination in which a 4 × 7 in. black or white card located directly behind the choice point indicated the presence of food in either the right or left goal box.
O'}
\ \
8
\
20"
.,¢ SHAM OPERATION CONTROLS
/I X \
e - - e RED NUCLEUS A B L A T E D
/ \
Z
/ \
laJ
\
," \ \
t-0 Z Z Z ne
/
/
/
/
/
k@/
I0"
Restdts
O
Animals with red nucleus lesions required significantly more trials to reach the learning criterion and made significantly more errors than control animals in learning the successive black-white discrimination although there were no differences
RIGHT
LEFT TASK
RIGHT
FIG. 3. Running time on the elevated T maze for the three position tasks and the successive brightness discrimination;
TABLE 2 t-TESTS OF THE MEAN TRIALS TO CRrrERION FOR THREE POSITION TASKS AND SUCCESSIVE BRIGHTNESS DISCRIMINATION
Red nucleus Damaged N = 10 Sham operation N=8
DISC.
Right Position Response
Left Position Response
Right Position Response
"i~=31.5
X=35.8
X=27.0
R-
: 29.5
X : 31.6
~ = 32.3
,'~ = 55.2
t = 0.25
t = 0.56
t = 1.01
t = 2.94
d f = 16
dr= 16
d f = 16
N.S.
N.S.
N.S.
Successive Brightness Discrimination 106.0
d./': 16 p = 0.01
BILATERAL LESIONS
Typical
lesion
RED IN
NUCLEUS
THE
RAT
Composite
lesion
FIG. 4. The sections on the right show the maximal extent of the bilateral red nucleus lesions made from tracings of individual lesions superimposed on sections from the atlas of Pelligrino and Cushman [IO].
RUBRAL
AREA
LESIONS AND
EXPERIMENT
CONDITIONED
MOVEMENT
3
A consistent observation throughout these experiments is that rats with red nucleus lesions are extremely slow to initiate movement in response to appropriate conditioned stimuli. It might therefore be expected that rats with rubral lesions would be superior at conditioned inhibition of movement or passive avoidance. Apparatus
The passive avoidance apparatus consisted of a 9 x 12 in. compartment surrounded on three sides by 18 in. walls. Adjacent to the fourth side was a 9 x 12 in. grid area that could be electrified with 0.8 mA of unscrambled shock. Procedure
Naive rats, eight with rubral lesions and IO control animals that had received sham operations, were trained on the acquisition of a passive avoidance response. On the first day of training the rat was placed in the safe area, and his latency to step down to the grid area was recorded. After each animal had placed all four feet on the grid, continuous shock was given until the animal returned to the safe area. For two consecutive days following the passive avoidance training the animals were placed in the safe area to measure the increase in latency to step down. Each animal was given up to 1800 set in which to step down and if an animal failed to do so the latency was recorded as 1800 sec. Results
Rats with red nucleus lesions took longer to step down on the first day as well as on the two postshock days. Following the foot shock on the first day both groups had increased step-down latencies. A two-way analysis of variance for repeated measures corrected for unequal groups indicated that there was a significant difference between groups on all three days (F = 5.72, p < 0.05) and a significant increase in latency to step down for both groups on the two postshock days (F = 12.40, p < 0.01). The results of this experiment suggest that rats with red nucleus lesions do not initiate movement readily to novel stimuli but are superior to normal rats at conditioned inhibition of movement or one-trial passive avoidance.
DISCUSSION OF RUBRAL
AREA LESIONS
The close proximity of the red nucleus to other structures in the ventral mesencephalon of the rat made it impossible to confme the lesions to the red nucleus alone. None of the rats used in these experiments had discrete bilateral lesions confined only to the red nucleus. The histological criterion for inclusion in all these experiments was simply the evidence of bilateral rubral damage and therefore, the red nucleus was the only structure consistently damaged in all cases. A composite drawing showing the maximal extent of the rubral area lesions and an example of a typical lesion are shown in Fig. 4. The most frequent tendency was for the ablations to extend ventrally and rostrally. Several cases that were excluded from the tabulation of the results because the lesions did not appear to encroach on the red nucleus, did however, show a deficit in avoidance conditioning but with a much lower frequency. The coincidence of damage to structures ventral to the red nucleus and the exhibition of the avoidance deficit demonstrates the weaker
897
relationship. Only one third of nine cases with medial lemniscus damage showed the avoidance deficit and four animals from a sample of nine with damage to the substantia nigra demonstrated the deficit. A smaller number of animals with more rostra1 lesions indicated that two of three animals with damage to the habenulo-interpeduncular tract and one with damage to the ventral tegmental area of Tsai failed to learn the avoidance response easily. All cases in which bilateral red nucleus lesions were present failed to achieve the learning criterion of the avoidance response after 100 trials. The fact that several animals exhibited similar deficits in avoidance conditioning but did not have red nucleus lesions raises certain questions about the neural systems that are critical for this behavior. Many investigators have now reported avoidance conditioning deficits following lesions in this region of the ventral mesencephalon [4, 5, 12, 131 but the neural mechanism subserving this behavior remain obscure. Data from acute neurophysiological experiments suggest at least two possible explanations. Massion and Croize [7] have demonstrated that unilateral red nucleus lesions reduce the tonic facilitatory influence of the cerebellum on the ipsilateral ventral lateral nucleus of the thalamus by interrupting the fibers of the brachium conjunctivum passing through the red nucleus. An additional effect of such lesions might be to disorganize the relationship between the ventral lateral nucleus and the motor cortex and therefore producing a deficit in voluntary movement. Alternatively, Tsukahara et al. [I43 have recently presented evidence for the existence of a collateral branch of the corticospinal pathway which runs through the medullary and penduncular pyramids to termination in or near the red nucleus in the cat. This pathway conveys a reciprocal inhibition between the corticospinal tract and the rubrospinal tract. In the opinion of these authors, the function of this mechanism might be to mediate the control of spinal targets between the cerebral cortex and the cerebellum. Assuming that such a pathway exists in the rat, interruption of these fibers by ventral mesencephalic lesions in this and similar studies may help explain why lesions to the red nucleus produce deficits in conditioned movement as well as why lesions ventral to the nucleus also have this effect.
GENERAL
DISCUSSION
In the rat the most striking effect of red nucleus lesions is apparent in the animals’ failure to initiate rapid conditioned movements. It appears that lesions of the red nucleus do not interfere with the motor organization of running behavior in general, but do influence running behavior that is elicited as a learned response to a conditioned stimulus. Running behavior that is associated with a response to an unconditioned stimulus remains unimpaired. Rats with rubral lesions made a significantly increased percentage of successful avoidances when the onset of shock was delayed an additional 10 set although they remained consistently slower than normal rats in the execution of avoidance responses. A further observation that rats with red nucleus lesions made some escape responses more rapidly than normal rats shows that they were not too ataxic to run. Together, these results seem to suggest that the rats with rubral lesions learned the appropriate response but were incapable of initiating rapid learned responses.
hhll’l
898
The prolonged latency to initiate exploratory movements in the passive avoidance experiment and the long duration of the running times on the elevated T maze also suggest that disinclination to initiate movement characterizes the responses of rats with red nucleus lesions to appetitive as well as aversive stimuli. In general, the findings are consistent with Carpenter’s [2] observations on the effect of rubral lesions in the rhesus monkey and are remarkably similar to Vanderwolf’s [15] description of rats with medial thalamic lesions. In addition to a failure to initiate rapid conditioned movements rats with rubral lesions in the present study also exhibited impaired learning of certain visual discrimination tasks. The rate of learning a simultaneous black-white discrimination was unimpaired although rats with red nucleus lesions required more trials to learn both a simultaneous pattern discrimination and a successive black-white discrimination. Despite the fact that these data are consistent with the earlier studies of Thompson et nl. [12] and McNew [S], the further discovery that rats with rubrdl lesions take significantly longer than normal rats to learn a successive black-white discrimination makes interpretation of the results more difficult. There are at least two possible explanations for these data. The first and perhaps the simpler explanation is that damage to the oculomotor nerve fibers that course through the red nucleus results in such poor ocular control that the rat is incapable of pattern discrimination. An unpublished study conducted in collaboration with Mr. John Best attempted to evaluate this hypothesis by making third nerve nucleus lesions. Rats with oculomotor nucleus lesions learned both the black-
II
white discrimination and the pattern discrlminatton at the same rate as normal rats. The result failed to support thr proposition that the oculomotor nerve damage in rats with rubral lesions causes the difficulty in pattern perception. Furthermore, it is difficult to understand how poor ocular control would interfere with the learning of a successive brightness discrimination. It would seem that no simple perceptual dysfunction can account for these data. An alternate explanation is suggested by the studies of Schneider [I l] who makes a distinction between two mechanisms of visual processing. The first is the capacity to identify a stimulus pattern and the second is the capacity to orient to a stimulus in space. Schneider has shown that this latter capacity is a function of the optic tectum and there are reported
connections
between
the superior
colliculus
and the
red nucleus [9]. A comparison of the simultaneous discrimination tasks with the successive discrimination task reveals that with simultaneous discrimination the positive stimulus indicates by virtue of its position, the direction of approach for the animal. In the successive discrimination learning, however, the locus of the discriminative stimulus gives no indication about the direction of approach. When both the discrimination and the response are simple, as with simultaneous black-white discrimination and avoidance learning, the only effect of rubral lesions is apparent in the increased latency of response initiation. Alternatively, when either the stimulus complexity is increased as in the simultaneous pattern discrimination or the response becomes more complex, as in the successive black-white discrimination, a deficit in learning appears.
REFERENCES 1.
Bauer, J. H. and R. M. Cooper. Effects of posterior cortical
8. McNew, J. Role of the red nucleus in visually guided behaviors
J. camp.
in the rat. J. camp. physiol. Psychol. 65: 282-289, 1968. 9. Papez, J. W. and W. A. Stotler. Connections of the red nucleus.
lesions on performance
of a brightness discrirt&ation.
ohvsiol. Psvchol. 58: 84-92.
1964.
2. Carpenter; M. B. A study of the red nucleus of the rhesus monkey. Anatomic degenerations and physiological effects from localized lesions in the red nucleus. J. camp. Neural. 109: 195-249, 1956. 3. Carpenter, M. B. and J. Pines. The rubro-bulbar tract: anatomical relationships, course and terminations in the rhesus monkey. Anat. Rec. 128: 171-185, 1957. 4. Hatton, G. Retention of discrimination and avoidanbe habits following lesions to the interpeduncular nucleus. J. camp. physiol. Psychol. 59: 331-334,
5. Krieckhause.
1965.
E. Decrements in avoidance behavior followinn mamillothalamic tractotomy in rats and subsequent recover; with d-amphetamine. J. camp. physiol. Psychol. 60: 31-35, 1965. 6. Massion, J. and D. Albe-Fessard. Dualit des voies sensorielles afferentes contralant l’activit6 du noyeau rouge. Electroenceph. clin. Neurophysiol. 15: 435-459, 1963. 7. Massion, J. and B. Croize. Tonic facilitatory action of the cerebellum on the ventrolaterai nucleus. In: The Cerebellum in Health and Disease, edited by W. S. Fields and W. D. Wills, Jr. St. Louis, Missouri: Green, 1969, 332-338.
Archs. Neural. Psychiat. 44: 776-791,
1940.
10. Pellegrino, L. and A. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century Crofts, 1967. 11. Schneider, G. E. Contrasting visuomotor functions of the tectum and cortex in the golden hamster. Psycho/. Forsch. 31: 52-62, 1967. 12. Thompson, R., 1. Lukaszewska, A. Schweigerdt and J. McNew. Retention of visual and kinesthetic discriminations in rats following pretectodiencephalic and ventral mesencephalic damage. J. camp. physiol. Psychol. 63: 458-468, 1967. 13. Thompson, R., I. Rich and S. Langer. Lesions studies on the functional significance of the posterior-mesencephalic tract. J. camp. Neural. 123: 29-44, 1964. 14. Tsukahara, N., D. R. G. Fuller and V. B Brooks. Collateral pyramidal influences on the corticorubrospinal system. J. Neurophysiol. 31: 461-484,
1968.
15. Vanderwolf, C. H. Medial thalamic functions in voluntary behavior. Can. J. Psychol. 16: 318-330, 1962. 16. Winer, B. J. Statistical Principles in Experimental Design. New York : McGraw-Hill, 1962.
Deficits in Conditioned Movement and Visual Discrimination Following Rubral Area Lesions in the Raf ALLAN
M. S M I T H ~'a
Department o f Psychology, McGill University, Montreal, Quebec, Canada (Received 16 J a n u a r y 1970)
SMITH,A. M. Deficits in conditioned movement and visual discrimination following rubral area lesions in the rat. PHYSIOL. BEHAV. 5 (8) 893-898, 1970.--Bilateral electrolytic lesions in the region of the red nucleus impaired the ability of 15 hooded rats to perform a conditioned avoidance response. The deficit is interpreted as implying a failure to initiate conditioned movement rather than direct impairment of motor organization or ability to learn. A smaller sample of the same group trained to perform simultaneous black-white and stripe discriminations showed deficits only in the acquisition of the stripe discrimination. A second experiment found that rats with rubral area lesions showed impaired learning and slower running speed in a food motivated successive brightness discrimination task. A third experiment found that rats with rubral area lesions were significantly slower to initiate movement on both pre and post shock days in one-trial passive avoidance training. Rubral area lesions Initiation of conditioned movement Successive brightness discrimination Passive avoidance
Simultaneous brightness and pattern discrimination
no accompanying paresis and the quality of motor performance is not impaired. In general these studies suggest that following red nucleus lesions the performance of voluntary movement responses is impaired. The first series of experiments reported here attempted to determine if the deficits in conditioned movement and visual descrimination following red nucleus lesions are independent of each other. A second experiment attempted to evaluate the effects on behavior of rubral lesions when appetitive rather than aversive incentives are used, and finally passive avoidance conditioning was undertaken to compare the effects of rubral lesions on both the initiation and inhibition of movement.
RECENT studies have indicated that the red nucleus may be important for visually guided movement reponses [6, 7, 12, 13]. Single units of the red nucleus in the anesthetized cat yield phasic responses to various kinds of sensory input including responses to photic stimulation [6]. Thompson et al. [13] report that rats with bilateral red nucleus lesions fail to retain a visually cued avoidance response and also that relearning a brightness discrimination is significantly impaired. In subsequent studies, it has been found that retention of pattern discrimination is more greatly impaired than brightness discrimination by red nucleus lesions [12]. It should be mentioned, however, that these studies involved a variety of lesions and only a few animals in each study had lesions of the red nucleus. Using a larger sample, McNew [8] found that rats with red nucleus lesions are more impaired than normal rats in the performance of size and pattern discrimination and are also significantly worse than rats with dorsal mesencephalic lesions. Behavioral anomalies have been noted in monkeys that were being prepared for anatomical studies [2, 3]. The most striking of these anomalies, according to Carpenter, is hypokinesis or disinclination to move. He states that although there is a severe depression of voluntary movement, there is
METHOD
Subjects The subjects were 76 male and female hooded rats of the Royal Victoria Hospital stock weighing between 200 and 400 g. Since no difference in behavior was observed between male and female animals, the subjects are referred to simply as rats.
1The studies reported here formed part of a dissertation submitted by the author to the Faculty of Graduate Studies and Research, McGill University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. A preliminary report of some of these data was presented to the annual meeting of the Canadian Psychological Association, June 1968. 2This research was supported by a predoctoral fellowship from the National Research Council of Canada. The author wishes to thank Dr. Muriel Stern for her advice and criticism of this research. 8Present address--D6partement de Neurophysiologie G6n6rale, Institut de Neurophysiologie et Psychophysiologie, C. N. R. S., 31 chemin Joseph Aiguier, Marseille (9me), France. 893
894
SMITI~
Surgical Procedures
Procedure
Bilateral electrolytic lesions in the region of the red nucleus were achieved under general anesthesia with sodium pentobarbital (Nembutal). A twisted nichrome wire electrode, insulated, except at the tip, was lowered into place and a cathodal current of 2.0 mA for 10 sec was used to produce the lesions. Since the insertion of the electrode alone caused profuse bleeding, sham operations also included insertion of the electrode to a depth just dorsal to the red nucleus. The scalp wound was closed with Michel clips, and methetharamide (Mikedimide) and penicillin were administered to facilitate recovery.
Each rat was placed in the white compartment facing away from the door and was given five sec to exit into the black compartment, after which the floor of the white compartment was electrified. Twenty trials a day were given and for those animals with brain lesions that did not attain the criterion of 20 consecutive avoidances by the sixth day or 120th trial, the preshock interval was increased to t5 sec. Avoidance training was discontinued for all animals that had not reached criterion within 200 trials. Results
Postoperative Syndrome
The effect of bilateral rubral lesions was extremely severe. Most often the rats were aphagic and adipsic. Motor dysfunctions of various kinds were observed frequently. Tortieollis was common and probably due to an asymmetry of bilateral damage. Transient hypotonus and paresis were observed in the limbs of most of the animals. No evidence of tremor was ever observed, but frequently some animals displayed wild uncontrolled running that closely resembled audiogenic seizures. The seizures never occurred beyond the third day after the operation. All animals were given three weeks in which to recover after which they appeared to eat and drink normally and general locomotion was coordinated. No animal that showed any signs of motor dysfunction was included in any experiment.
EXPERIMENT I - A
Using 32 rats, 15 with red nucleus lesions and 17 sham operation control animals, it was decided to examine the effects of rubral lesions first on a simple one-way avoidance task. Apparatus
The avoidance apparatus consisted of a box 12 × 18 × 18 in. with two compartments of equal size separated by a sliding guillotine door. One compartment was painted white and the other, black. The floor of the apparatus was a stainless steel grid that could be electrified on one side.
The most evident differences between the group with red nucleus lesions and the sham operation control group occurred in the number of trials required to reach criterion and the number of failures to avoid shock. As shown in Table l, a student's t-test revealed that rats with red nucleus lesions required a significantly greater number of trials to reach criterion and made significantly more errors than the sham operation control subjects. There was no overlap in the scores for the two groups. Increasing the preshock avoidance time from five sec to 15 sec, after 120 trials, improved the performance of the braindamaged rats. The addition of 10 see to the preshock period increased successful avoidances from an average of 37 per cent during the five-see training period (trials 1-120) to an average of 63 per cent avoidances for the 15-sec training period (trials 1 2 1 - 2 0 0 or criterion) and when the scores are transformed into arcsin values the difference is statistically significant (t - - 2.20. 28, df, p < 0.05). A comparison of the mean times taken in seconds to complete an avoidance response in the last 10 trials (taken to represent optimal performance) indicated that rats with red nucleus lesions were significantly slower to avoid than normal rats (t - - 4.55, 18 dr, p < 0.01). Moreover, a comparison of the mean times required to complete escape responses on the last escape trial indicated that the control animals were slower to escape shock than the rats with red nucleus lesions (t - - 2.37, 18 dr, p < 0.05). It should be stressed, however, that the rats with rubral lesions received a mean of 90 more escape trials than the control subjects and the difference in escape time is undoubtedly due to prolonged practice received by the rats with red nucleus lesions. The difference in escape
TABLE 1 TRIALS TO CRITERION AND ERRORS TO CRITERION IN RATS LEARNING AN ACTIVE SHOCK AVOIDANCE RESPONSE AS A FUNCTION OF RED NUCLEUS LESIONS
Subjects
Trials to Criterion
Errors to Criterion
Red nucleus Damaged N = 15
Range = 131-200 Mean = 182.6
Range = 25-196 Mean = 101.2
Sham operation N = 17
Range = 26-52 Mean = 36.6
Range = 3-21 Mean = 10.2
t = 21.19 d f = 30
t = 7.00 df = 30 p < O.Ol
p < o.01
RUBRAL AREA LESIONS AND CONDITIONED MOVEMENT time is presented simply to indicate that the brain damaged rats were not unable to run. 1-B
EXPERIMENT
It was decided to test a sample of the same animals that had shown deficits in avoidance training, in a shock-motivated visual discrimination apparatus similar to the one described by Bauer and Cooper [1 ] and shown in Fig. I.
Pretraining Procedures Since the usual avoidance pretraining would have been ineffective for rats with red nucleus lesions, all animals were pretrained to escape from the apparatus. Each of 13 rats with rubral lesions and 17 sham operation control animals was placed in the start box with the sliding door open. Simultaneously, the floor was electrified and the start box door was closed behind the animal to prevent retreat. The shock was
I(
17" GOAL
~1 BOX
DOORS
~~
Grid
36~ ~
(2)
895 card was alternated from side to side in a predetermined random sequence. An error was counted if a rat placed two paws in the threshold area of the incorrect door and each rat had to correct the error to escape from the apparatus. If a rat failed to leave the start box in five sec, or failed to leave the choice area in 25 see, shock was applied for one sec. every five sec. It was noted on each trial if shock was needed to move the animal from the start box. Each day 10 trials were given and training was terminated after nine correct responses in any 10 trials. Immediately after reaching the acquisition criterion of the black-white discrimination problem, a smaller sample of eight rats with rubral lesions and 10 animals with sham lesions were trained in the same apparatus to discriminate between horizontal and vertical black and white stripes to a criterion of nine correct responses in any 10 trials.
Results There were no significant differences between rats with red nucleus lesions and sham lesion control animals with regard to either the number of trials required to reach the learning criterion or the number of errors made on the brightness discrimination task. On the pattern discrimination task, however, the brain-damaged animals required a significantly greater number of trials to reach criterion and made more errors. The moan trials to criterion for both tasks are shown in Fig. 2. Rats with red nucleus lesions required a mean of 79.5 trials to reach criterion and made a mean of 26.7 errors whereas control animals only required a mean of 33.6 trials (t = 6.27, 16 dr, p < 0.01) and made a mean of 15.5 errors (t = 2.64, 16df, p < 0.05). Despite the additional trials of avoidance training inherent in this discrimination conditioning, little improvement in performance was noted. Rats with red nucleus lesions successfully avoided shock on an average of only 32 per cent of the trials whereas sham operation control animals avoided shock on an average of 75 per cent of the trials.
MEAN TRIALSTO CRITERION SIMULTANEOUSBRIGHTNESSAND PATTERN DISCRIMINATION
i i q~'SL~G PLAN VIEW
~
~ - - ~ - ~ STARTBOX /
/
8O
VISUAl DISCRIMINATIONBOX
FIG. l. Diagram of visual discrimination apparatus. continued until the rat escaped through one of the appropriate doors. The exit doors were initially left open and were shut gradually until the animal learned to open them. The last door used was then locked and the rat was trained to seek out the alternate door. When a rat made three consecutive exits in less than 10 sec, pretraining was concluded.
Discrimination Training On the day following pretraining, training of black-white discrimination began. A black or white card attached to the exit doors served as the cue to the unlocked door. The threshold to the negative door (either area three or four in Fig. 1) was electrified and the door itself was locked. The cue
z
o
n" hi in"
60
u 40 o <_J
F-
N=8
]
SHAM OPERATION CONTROLS
]
REDNUCLEUS ABLATED
N:L'3
N=I7 N=IO
2O
DISCRIMINATION FIG. 2. Mean trials to learning criterion for rats with red nucleus lesions, and sham operation control rats on simultaneous brighmess and pattern discrimination.
896
g M l ! I{
EXPERIMENT
2
This experiment was designed to determine if the behavioral deficits following red nucleus lesions appeared when an appetitive motivation was used and also to obtain more information about the nature of the perceptual difficulties suggested by the first experiment.
Procedures Bilateral red nucleus lesions were performed on 10 rats and sham operations were conducted on eight additional animals. Following recovery all animals were food deprived for 23 hr each day for one week prior to the experiment. The animals were given free access to food and water one hour each day and during the last three days of this period feeding took place on an elevated T maze (5 in. wide with a stem of 30 in. and branches of 15 in. each).
in the learning scores for the three position response tasks, The mean trials to criterion for the three position response tasks and the successive brightness discrimination task are shown in Table 2. A two-way analysis of variance for repeated measures corrected for unequal sample size [16] was computed for the mean running times for each task. The results indicate that although the rtmning times become shorter with practice, the rats with red nucleus lesions require considerably more time to complete a trial (F 4.53, p 0.05). This change in running speed for both the braindamaged and the normal rats is sbo~n in t:ig, 3.
RUNNING TIME ON ELEVATED T MAZE
30'
Pretraining
x
Each animal was pretrained on a series of position response problems on the elevated T maze. All rats were taught first to go to the right-side goal box for food. The criterion for learning was set at 18 correct responses in any 20 trials and 20 trials a days were given. The criterion for learning remained the same throughout. An incorrect response was scored when a rat entered the wrong goal box. Following the acquisition of the first position response, the animals were trained to go to the left side and finally to go back to the right side again.
Discrimination Training This procedure consisted of training the rats to perform a successive black-white discrimination in which a 4 × 7 in. black or white card located directly behind the choice point indicated the presence of food in either the right or left goal box.
O'}
\ \
8
\
20"
.,¢ SHAM OPERATION CONTROLS
/I X \
e - - e RED NUCLEUS A B L A T E D
/ \
Z
/ \
laJ
\
," \ \
t-0 Z Z Z ne
/
/
/
/
/
k@/
I0"
Restdts
O
Animals with red nucleus lesions required significantly more trials to reach the learning criterion and made significantly more errors than control animals in learning the successive black-white discrimination although there were no differences
RIGHT
LEFT TASK
RIGHT
FIG. 3. Running time on the elevated T maze for the three position tasks and the successive brightness discrimination;
TABLE 2 t-TESTS OF THE MEAN TRIALS TO CRrrERION FOR THREE POSITION TASKS AND SUCCESSIVE BRIGHTNESS DISCRIMINATION
Red nucleus Damaged N = 10 Sham operation N=8
DISC.
Right Position Response
Left Position Response
Right Position Response
"i~=31.5
X=35.8
X=27.0
R-
: 29.5
X : 31.6
~ = 32.3
,'~ = 55.2
t = 0.25
t = 0.56
t = 1.01
t = 2.94
d f = 16
dr= 16
d f = 16
N.S.
N.S.
N.S.
Successive Brightness Discrimination 106.0
d./': 16 p = 0.01
BILATERAL LESIONS
Typical
lesion
RED IN
NUCLEUS
THE
RAT
Composite
lesion
FIG. 4. The sections on the right show the maximal extent of the bilateral red nucleus lesions made from tracings of individual lesions superimposed on sections from the atlas of Pelligrino and Cushman [IO].
RUBRAL
AREA
LESIONS AND
EXPERIMENT
CONDITIONED
MOVEMENT
3
A consistent observation throughout these experiments is that rats with red nucleus lesions are extremely slow to initiate movement in response to appropriate conditioned stimuli. It might therefore be expected that rats with rubral lesions would be superior at conditioned inhibition of movement or passive avoidance. Apparatus
The passive avoidance apparatus consisted of a 9 x 12 in. compartment surrounded on three sides by 18 in. walls. Adjacent to the fourth side was a 9 x 12 in. grid area that could be electrified with 0.8 mA of unscrambled shock. Procedure
Naive rats, eight with rubral lesions and IO control animals that had received sham operations, were trained on the acquisition of a passive avoidance response. On the first day of training the rat was placed in the safe area, and his latency to step down to the grid area was recorded. After each animal had placed all four feet on the grid, continuous shock was given until the animal returned to the safe area. For two consecutive days following the passive avoidance training the animals were placed in the safe area to measure the increase in latency to step down. Each animal was given up to 1800 set in which to step down and if an animal failed to do so the latency was recorded as 1800 sec. Results
Rats with red nucleus lesions took longer to step down on the first day as well as on the two postshock days. Following the foot shock on the first day both groups had increased step-down latencies. A two-way analysis of variance for repeated measures corrected for unequal groups indicated that there was a significant difference between groups on all three days (F = 5.72, p < 0.05) and a significant increase in latency to step down for both groups on the two postshock days (F = 12.40, p < 0.01). The results of this experiment suggest that rats with red nucleus lesions do not initiate movement readily to novel stimuli but are superior to normal rats at conditioned inhibition of movement or one-trial passive avoidance.
DISCUSSION OF RUBRAL
AREA LESIONS
The close proximity of the red nucleus to other structures in the ventral mesencephalon of the rat made it impossible to confme the lesions to the red nucleus alone. None of the rats used in these experiments had discrete bilateral lesions confined only to the red nucleus. The histological criterion for inclusion in all these experiments was simply the evidence of bilateral rubral damage and therefore, the red nucleus was the only structure consistently damaged in all cases. A composite drawing showing the maximal extent of the rubral area lesions and an example of a typical lesion are shown in Fig. 4. The most frequent tendency was for the ablations to extend ventrally and rostrally. Several cases that were excluded from the tabulation of the results because the lesions did not appear to encroach on the red nucleus, did however, show a deficit in avoidance conditioning but with a much lower frequency. The coincidence of damage to structures ventral to the red nucleus and the exhibition of the avoidance deficit demonstrates the weaker
897
relationship. Only one third of nine cases with medial lemniscus damage showed the avoidance deficit and four animals from a sample of nine with damage to the substantia nigra demonstrated the deficit. A smaller number of animals with more rostra1 lesions indicated that two of three animals with damage to the habenulo-interpeduncular tract and one with damage to the ventral tegmental area of Tsai failed to learn the avoidance response easily. All cases in which bilateral red nucleus lesions were present failed to achieve the learning criterion of the avoidance response after 100 trials. The fact that several animals exhibited similar deficits in avoidance conditioning but did not have red nucleus lesions raises certain questions about the neural systems that are critical for this behavior. Many investigators have now reported avoidance conditioning deficits following lesions in this region of the ventral mesencephalon [4, 5, 12, 131 but the neural mechanism subserving this behavior remain obscure. Data from acute neurophysiological experiments suggest at least two possible explanations. Massion and Croize [7] have demonstrated that unilateral red nucleus lesions reduce the tonic facilitatory influence of the cerebellum on the ipsilateral ventral lateral nucleus of the thalamus by interrupting the fibers of the brachium conjunctivum passing through the red nucleus. An additional effect of such lesions might be to disorganize the relationship between the ventral lateral nucleus and the motor cortex and therefore producing a deficit in voluntary movement. Alternatively, Tsukahara et al. [I43 have recently presented evidence for the existence of a collateral branch of the corticospinal pathway which runs through the medullary and penduncular pyramids to termination in or near the red nucleus in the cat. This pathway conveys a reciprocal inhibition between the corticospinal tract and the rubrospinal tract. In the opinion of these authors, the function of this mechanism might be to mediate the control of spinal targets between the cerebral cortex and the cerebellum. Assuming that such a pathway exists in the rat, interruption of these fibers by ventral mesencephalic lesions in this and similar studies may help explain why lesions to the red nucleus produce deficits in conditioned movement as well as why lesions ventral to the nucleus also have this effect.
GENERAL
DISCUSSION
In the rat the most striking effect of red nucleus lesions is apparent in the animals’ failure to initiate rapid conditioned movements. It appears that lesions of the red nucleus do not interfere with the motor organization of running behavior in general, but do influence running behavior that is elicited as a learned response to a conditioned stimulus. Running behavior that is associated with a response to an unconditioned stimulus remains unimpaired. Rats with rubral lesions made a significantly increased percentage of successful avoidances when the onset of shock was delayed an additional 10 set although they remained consistently slower than normal rats in the execution of avoidance responses. A further observation that rats with red nucleus lesions made some escape responses more rapidly than normal rats shows that they were not too ataxic to run. Together, these results seem to suggest that the rats with rubral lesions learned the appropriate response but were incapable of initiating rapid learned responses.
hhll’l
898
The prolonged latency to initiate exploratory movements in the passive avoidance experiment and the long duration of the running times on the elevated T maze also suggest that disinclination to initiate movement characterizes the responses of rats with red nucleus lesions to appetitive as well as aversive stimuli. In general, the findings are consistent with Carpenter’s [2] observations on the effect of rubral lesions in the rhesus monkey and are remarkably similar to Vanderwolf’s [15] description of rats with medial thalamic lesions. In addition to a failure to initiate rapid conditioned movements rats with rubral lesions in the present study also exhibited impaired learning of certain visual discrimination tasks. The rate of learning a simultaneous black-white discrimination was unimpaired although rats with red nucleus lesions required more trials to learn both a simultaneous pattern discrimination and a successive black-white discrimination. Despite the fact that these data are consistent with the earlier studies of Thompson et nl. [12] and McNew [S], the further discovery that rats with rubrdl lesions take significantly longer than normal rats to learn a successive black-white discrimination makes interpretation of the results more difficult. There are at least two possible explanations for these data. The first and perhaps the simpler explanation is that damage to the oculomotor nerve fibers that course through the red nucleus results in such poor ocular control that the rat is incapable of pattern discrimination. An unpublished study conducted in collaboration with Mr. John Best attempted to evaluate this hypothesis by making third nerve nucleus lesions. Rats with oculomotor nucleus lesions learned both the black-
II
white discrimination and the pattern discrlminatton at the same rate as normal rats. The result failed to support thr proposition that the oculomotor nerve damage in rats with rubral lesions causes the difficulty in pattern perception. Furthermore, it is difficult to understand how poor ocular control would interfere with the learning of a successive brightness discrimination. It would seem that no simple perceptual dysfunction can account for these data. An alternate explanation is suggested by the studies of Schneider [I l] who makes a distinction between two mechanisms of visual processing. The first is the capacity to identify a stimulus pattern and the second is the capacity to orient to a stimulus in space. Schneider has shown that this latter capacity is a function of the optic tectum and there are reported
connections
between
the superior
colliculus
and the
red nucleus [9]. A comparison of the simultaneous discrimination tasks with the successive discrimination task reveals that with simultaneous discrimination the positive stimulus indicates by virtue of its position, the direction of approach for the animal. In the successive discrimination learning, however, the locus of the discriminative stimulus gives no indication about the direction of approach. When both the discrimination and the response are simple, as with simultaneous black-white discrimination and avoidance learning, the only effect of rubral lesions is apparent in the increased latency of response initiation. Alternatively, when either the stimulus complexity is increased as in the simultaneous pattern discrimination or the response becomes more complex, as in the successive black-white discrimination, a deficit in learning appears.
REFERENCES 1.
Bauer, J. H. and R. M. Cooper. Effects of posterior cortical
8. McNew, J. Role of the red nucleus in visually guided behaviors
J. camp.
in the rat. J. camp. physiol. Psychol. 65: 282-289, 1968. 9. Papez, J. W. and W. A. Stotler. Connections of the red nucleus.
lesions on performance
of a brightness discrirt&ation.
ohvsiol. Psvchol. 58: 84-92.
1964.
2. Carpenter; M. B. A study of the red nucleus of the rhesus monkey. Anatomic degenerations and physiological effects from localized lesions in the red nucleus. J. camp. Neural. 109: 195-249, 1956. 3. Carpenter, M. B. and J. Pines. The rubro-bulbar tract: anatomical relationships, course and terminations in the rhesus monkey. Anat. Rec. 128: 171-185, 1957. 4. Hatton, G. Retention of discrimination and avoidanbe habits following lesions to the interpeduncular nucleus. J. camp. physiol. Psychol. 59: 331-334,
5. Krieckhause.
1965.
E. Decrements in avoidance behavior followinn mamillothalamic tractotomy in rats and subsequent recover; with d-amphetamine. J. camp. physiol. Psychol. 60: 31-35, 1965. 6. Massion, J. and D. Albe-Fessard. Dualit des voies sensorielles afferentes contralant l’activit6 du noyeau rouge. Electroenceph. clin. Neurophysiol. 15: 435-459, 1963. 7. Massion, J. and B. Croize. Tonic facilitatory action of the cerebellum on the ventrolaterai nucleus. In: The Cerebellum in Health and Disease, edited by W. S. Fields and W. D. Wills, Jr. St. Louis, Missouri: Green, 1969, 332-338.
Archs. Neural. Psychiat. 44: 776-791,
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10. Pellegrino, L. and A. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century Crofts, 1967. 11. Schneider, G. E. Contrasting visuomotor functions of the tectum and cortex in the golden hamster. Psycho/. Forsch. 31: 52-62, 1967. 12. Thompson, R., 1. Lukaszewska, A. Schweigerdt and J. McNew. Retention of visual and kinesthetic discriminations in rats following pretectodiencephalic and ventral mesencephalic damage. J. camp. physiol. Psychol. 63: 458-468, 1967. 13. Thompson, R., I. Rich and S. Langer. Lesions studies on the functional significance of the posterior-mesencephalic tract. J. camp. Neural. 123: 29-44, 1964. 14. Tsukahara, N., D. R. G. Fuller and V. B Brooks. Collateral pyramidal influences on the corticorubrospinal system. J. Neurophysiol. 31: 461-484,
1968.
15. Vanderwolf, C. H. Medial thalamic functions in voluntary behavior. Can. J. Psychol. 16: 318-330, 1962. 16. Winer, B. J. Statistical Principles in Experimental Design. New York : McGraw-Hill, 1962.