Physiology and Behavior, Vol. 14, pp. 7-11. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Caudate Nucleus Lesion Selectively Increases Paradoxical Sleep Episodes in the Rat
M. CORSI-CABRERA, J. GRINBERG-ZYLBERBAUM AND L. S. ARDITTI

Laboratory o f Psychophysiological Research, Psychology College, Universidad Andhauc Apartado Postal 10-844 Mdxico 10, D. F., Mdxico

(Received 26 June 1974)

CORSI-CABRERA, M., J. GRINBERG-ZYLBERBAUM AND L. S. ARDITTI. Caudate nucleus lesion selectively increases paradoxical sleep episodes in the rat. PHYSIOL. BEHAV. 14(1) 7-11, 1975. - The EMG, ECG and the behavioral activity of seven rats were recorded for three days previous to and 24 hours after lesiordng both caudate-putamen complexes. Following the lesion, the paradoxical sleep stage lengthened by about 248%, without noticeable changes in the slow wave sleep or in the waking stages. The greatest increase was obtained with lesions placed in the mediomedial portion of the caudate-putamen complex. In other six rats submitted to the above procedure except that the lesions were placed in the anterior cerebral cortex, and also in five more rats implanted in both caudate-putamen complexes but not lesioned, no change was noted in the waking, paradoxical sleep or slow wave sleep stages. Caudate nucleus Paradoxical sleep Electrolytic lesion

EVIDENCE for an influence of the caudate nucleus (CN) on the regulation of the electrical activity of the cerebral cortex has been reported within the past few years. Its lesion produces an increase in the size of the cortical potentials evoked by thalamic stimulation [3], while the opposite effect follows its stimulation [4]. Furthermore, stimulation of the dorsolateral portion of the head of the CN inhibits the secondary components of the cortical potentials evoked by visual, auditory and somatic stimulation without altering their primary components [7]. These facts indicate an inhibitory influence of the caudate that possibly counteracts the thalamic and reticular formation activities [ 2]. On the other hand, it has been shown that high frequency stimulation of the CN produces cortical desynchronization and wakefulness while slow frequency stimulation of the same structure produces cortical synchronization and slow waves very similar to those recorded during sleep [ 1 ]. However there is no information concerning a possible role of the CN in the sleep process though its inhibitory influence on the electrical activity of the brain suggests this possibility. In order to investigate whether suppression of this inhibitory action would modify some of the stages of the waking/sleep cycle, the following experiments were performed. METHOD

months old, were used. All were kept for at least 3 months in a semi-soundproof room, with an automatic control which turned the lights on from 9 a.m. until 2 p.m. daily, in order to induce a uniform waking-sleep cycle. At the end of this period the rats were randomly divided into the following three groups: A, caudate-putamen group (7 rats); B, cortex group (6 rats); C, control group (5 rats).

Procedure
Under pentobarbital anesthesia (40 mg/kg) and using the stereotaxic technique, all the rats were implanted as follows: a midline longitudinal incision was made on the skin of the skull and after separating the periosteum, a screw was inserted 1.0 mm in each of two small holes drilled in the left side 3.0 mm lateral to the midline, one at the lambda level, and the other between this and the bregrna level. By means of these screws the electrocorticographic activity (ECG) was recorded. Two stainless steel surgical clasps were clamped in the left nuchal muscle in order to record electromiographic activity (EMG). Bipolar electrodes made of stainless steel wire 0.3 mm in diameter and insulated except at the tip, were introduced in each side through small holes drilled at the level of the bregma, 3.0 mm from the midline. Their tip was placed 4.5 mm below the dura into the caudate-putamen complex, in the rats of Groups A and C, and only 1.0 mm into the anterior cerebral cortex in the animals of Group B. The electrodes were cemented with acrylic to the exposed skull and the skin incision sutured. Penicillin (40,000 u/kg) was given

Animals
A total of 18 adult albino rats of either sex, 4 to 6

8 intramuscularly to each rat at the end of the surgical procedure. At least 3 to 4 days were allowed for complete recovery, and then the rats were introduced into a sound-proof, illuminated room and placed on a small elevated platform where they could move freely without jumping to the floor or escaping. By means of wires the four muscle and posterior cerebral cortex electrodes were attached to a special rotary connector and to a recording polygraph. ECG and EMG were recorded continuously from 10 a.m. until 2 p.m. every day for 6 consecutive days. In order to measure the mean voltage and frequency of the ECG and EMG activities, 10 random samples of 5 sec each, were taken, for each stage of the sleep-waking cycle. The voltage was measured from the crest to the valley of each wave, the frequency was quantified counting how many crests per sec appeared in each sample. On the third day, after completing the recording time, the CN was lesioned on both sides in the rats of Group A and the anterior Cerebral cortex in the rats of Group B by passing 3 mA direct current for 60 sec through each of the corresponding electrodes. After 24 hr, the rats were introduced in the sound-proof room, in order to continue with the recordings. No lesion was made in the rats of Group C. Waking, slow wave sleep and paradoxical sleep were identified by means of the polygraphic record and with the

CORSI-CABRERA, E T A L . aid of a closed circuit TV set that allowed the close observation of the overt behavior of the animals. This permitted determination of the number and length of each of the above mentioned stages for each rat during the two threeday periods of the test. These determinations were done using the whole 4-hour recording without sampling it. These data were compared for each group by means of a Student's test [8]. The correlation coefficient [8] between the extent of the lesions made in the caudate-putamen complex and the changes which it provoked on the sleep waking cycle, was also calculated. At the end of the experiment, the animals were deeply anesthetized and their brains were perfused with 10% Formalin solution, preceded by 50 ml of a 0.85% saline solution. The brains were kept for one week in 40% Formalin solution after which tranverse frozen sections of 50 ~ width were made. These sections were used as negatives to make photographic prints [5], which with the aid of a planimeter permitted locating and measuring the size of the lesions. RESULTS During wakefulness, the rats of the three groups showed a desynchronized ECG activity with predominant frequencies of 8 to 10 Hz and amplitudes of 50 to 150 ++V.

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FIG. 1. The four stages of the waking-sleep cycle of the rat. Typical electrocorticographic (ECG) and electromiographic (EMG) records during A attentive wakefulness. B relaxed wakefulness. C slow wave sleep and D paradoxical sleep. Note the decrease in the EMG activity from A to D and the similarity of the ECG activity in attentive wakefulness and paradoxical sleep.

CAUDATE NUCLEUS AND PARADOXICAL SLEEP

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FIG. 2. Each bar represents the relative duration (percent) of time that the three groups of rats spent in wakefulness, slow sleep and paradoxical sleep during the last three days of recording. The percent was calculated by taking as 100% (0 horizontal line) the total time that the rats spent in the three stages during the first three days of recording. Only the increase (148%) in the paradoxical sleep stage of the eaudate-putamen complex lesioned group of rats (white bars) was statisticaUy significant (t = 5.47, p = 0.01).

The EMG activity was of high amplitude, average 50 to 125 uV, and varied considerably in close correlation with the rat's motor activity. When the rat directed its attention to something in its environment, the ECG activity became synchronized and regular, with a frequency of 7 Hz (theta rhythm) (Fig. 1, A and B). It is interesting to note that these characteristics are different in terms of frequency and rhythm from the ones obtained in humans and in cats [9,10]. During slow wave sleep, no movements were observed in the rat and the ECG showed a great increase in amplitude (average 350 uV) and the typical slow wave bursts intermingled with slow waves of 6 to 8 Hz. The EMG voltage decreased considerably, average 50 to 18 uV (Fig. 1, C). The paradoxical sleep stage appeared among slow wave sleep episodes, and was signaled by a typical behavioral component, i.e. a change of the posture of the rat which nodded its head and curled up. At the same time the EMG activity disappeared entirely, becoming an isopotential line. The ECG showed the appearance of a synchronized theta rhythm of exactly 7 Hz very similar to, but more regular than the one recorded during the attentive behavior (Fig. 1, D). No noticeable changes in the frequency and morphology of the EMG and the ECG were noticed after the caudateputamen or the cerebral cortex lesions, and though the total time that each rat spent in the waking stage decreased

during the last three days of recording (Fig. 2), the differences with the figures obtained during the first three days, were not statistically significant (Group A: t = 2.45, p = 0.10; Group B: t = 1.88, p = 0.10 and Group C: t = 0.24, p = 0.20) (see Table 1). Again, the total time that the three groups of rats spent in slow wave sleep stage increased during the last three days of recording (Fig. 2), but the differences, in relation with the first three days, were not statistically significant either (Group A: t = 1.54, p = 0.20; Group B: t = 2.50, p = 0.10 and Group C: t = 0.20, p = 0.90) (see Table 1). Similarly there were no significant differences in the number and the length of waking and slow wave periods exhibited by the rats of the three groups, during the six days of recording (see Table 1). In contrast, the total time that the rats of Group A spent in the paradoxical sleep stage increased from an average of 381.16 sec to 958.37 sec following the caudate-putamen lesion (Fig. 2) and this considerable increase was statistically significant (t = 5.47, p = 0.01) (see Table 1). It was also noted an increase from the first day to the third day following the lesion, this increment was statistically significant (from 6.38% during the first day, to 9.56% during the third day, t = 3.81, p = 0.01). This change was fundamentally due to an increase in the number of periods of paradoxical sleep in these rats (from an average of 4 before

10 TABLE1

CORSI-CABRERA, E T A L .

PERCENTAGE OF THE TOTAL TIME AND THE NUMBER OF EPISODES THAT THE CEREBRAL CORTEX, CAUDATE PUTAMEN NUCLEUS AND CONTROL GROUPS OF RATS SPENT IN EACH STAGE OF THE SLEEP-WAKING CYCLE, IN THE FIRST AND LAST THREE DAYS OF RECORDING

Wakefulness first 3 days Mean S.D. last 3 days Mean S.D. Mean

Slow wave sleep first 3 days S.D. last 3 days Mean S.D. Mean

Paradoxical sleep first 3 days S.D. last 3 days Mean S.D.

Control Percent Number of episodes Caudate-Putamen lesion Percent Number of episodes Cerebral cortex lesion Percent Number of episodes 44.93 28.59 5.54 6.30 43.73 33.70 8.46 1.88 55.58 30.79 6.29 6.30 56.57 32.49 14.99 1.20 3.06 5.39 .85 3.65 4.31 5.03 1.41 2.27 49.36 28.71 43.41 4.08 38.90 30.27 9.34 8.72 44.46 29.66 5.11 5.09 49.81 35.88 6.77 10.51 3.16" 4.19" 1.40 1.65 7.86* 11.43" 2.46 4.05 36.64 21.36 10.94 3.71 32.36 19.84 5.77 4.80 52.40 25.61 8.32 2.88 59.51 26.25 5.79 2.28 7.33 8.50 3.23 4.28 8.16 7.97 2.65 3.55

*is the 0.01 level of statistical significance between differences.

C.11

........................

C.N. 18
FIG. 3. Histological sections showing the cortical lesions in two rats (C.C. 1 and 2) and the largest caudate-putamen complex lesions in other two rats. (C.N. 18 and 20).

CAUDATE NUCLEUS AND PARADOXICAL SLEEP to 11 after the lesion, t = 4.30, p = 0.01) and only partly as a result of an increase in the length of each episode (t = 2.49, p = 0.05) (see Table 1). This means that the increment in the frequency of the episodes is not correlated with a shorter duration of each of them because both suffered increment. This result is more significant since the increase in duration and number of the paradoxical sleep periods in the rats with a cerebral cortex lesion and in the nonlesioned rats was not statistically significant (Group B: t = 0.62, p = 0.60; Group C: t = 0.23, p = 0.90) (see Table 1). The histological analysis showed that the most pronounced increase in the paradoxical sleep stage was obtained with lesions placed in the medio-medial portion of the caudate-putamen complex (Fig. 3). Similar mediomedial lesions were obtained in 4 of the 7 rats of the group; the other three had dorsomedial lesions. Furthermore, the correlation coefficient between the size of the lesions and the increase in the paradoxical sleep stage was not significant (r = .03), indicating that in the production of the observed changes, the location was more important than the extent of the lesion, because in the medio-medial lesioned rats the increments were more pronounced. DISCUSSION These findings underline the important participation of the caudate nucleus in the sleep processes, which appears fundamentally and selectively related to the paradoxical sleep stage. This selective participation is very obvious

11 because neither the waking stage nor the slow wave sleep stage undergo modifications after the caudate lesions. The fact that the paradoxical sleep stage increment was not reflected in a correspondent decrement in the other stages, is understandable if we consider that the total time that the rats spent in paradoxical sleep is minimal compared to the time spent in the other stages. Furthermore, the observed changes consisted fundamentally in an increase in the number of periods of paradoxical sleep and only partly in an increase of the length of each period, This result indicates that perhaps the caudate does not act on the mechanisms which maintain the paradoxical sleep, rather it possibly exerts an inhibitory control on the mechanisms that trigger the onset of the paradoxical stages [6]. The medio-medial portion of the caudate nucleus could be pointed out as the site of this inhibitory control, because the greatest changes were observed after lesioning this region. The fact that no significant changes were observed in the implanted but not lesioned rats and in the cerebral cortex lesioned rats, indicates that the result obtained in the caudate-putamen complex lesioned group are not due to the surgical procedure nor to a nonspecific lesion, nor to an habituation to the stereotype either. ACKNOWLEDGEMENTS The authors express their gratitude to Prof. Dr. Alberto Guevara Rojas for his critical analysis of the manuscript and to Mr. Louis Chauvet for his help in its redaction.

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