Conditioned induction of paradoxical sleep in the rabbit

EXPERIMENTAL

Conditioned

NEUROLOGY

9,

470-482

Induction M.

(1964)

of Paradoxical

KAWAKAMI

Department of Anatomy and Los Angeles, and Veterans

AND

CHARLES

Sleep H.

Brain Research Institute, Administration Hospital,

Received

February

in the Rabbit

SAWYER' University Long Beach,

of California, California

10, 1964

Earlier electroencephalographic studies showed that sleep, including both spindle-slow wave and paradoxical phases, could be induced in estrogen-primed ovariectomized rabbits by low-frequency (5 cycle/set) electrical stimulation of the hypothalamus or limbic system and that the threshold varied with the hormonal status of the animal. The present investigation inquired whether electrically-induced paradoxical sleep might be conditioned to respond to a neutral pure (500 cycle/set) tone. The results in eleven out of fourteen rabbits reveal that when the threshold of the response to the unconditioned electrical stimulus is low, a conditioned response can be elicited following as few as five pairings of unconditional and conditional stimuli, to which the animal has previously become habituated. At this time a tone of somewhat different frequency (650 cycle/set) arouses the rabbit whether presented alone or paired with the unconditional stimulus. The conditioned paradoxical sleep response is rapidly lost if not frequently reinforced by pairing with the unconditional stimulus. The conditioned response is also lost when the hormonal status changes to the extent that the unconditional stimulus threshold is considerably elevated. introduction

Sleep patterns in the rabbit, as in the cat and other species studied in detail (7), include episodes of “activated” or “paradoxical” sleep as well as periods of ordinary or spindle sleep (2, 10). In 1959 Sawyer and Kawakami (20) described the paradoxical pattern under another name: a “phase of hippocampal hyperactivity” in an “EEG after-reaction” to coitus in the female rabbit. It was soon learned that this unusual response could be evoked by low-frequency electrical stimulation of the hypothalamus or various regions in the limbic system (8) and that its threshold was dependent on the hormonal status of the animal (9). As a phase in the postcoital consummatory behavior pattern signaling 1 This investigation was supported in part by National Institutes B1162. We thank Enid Busser, Arlene Koithan, and Cora Rucker and Suzanne Arlen for drawing and lettering illustrations. 470

of Health Grant for technical help

CONDITIONED

PARADOXICAL

SLEEP

471

reduction of appetitive sexual drive, paradoxical sleep appears to possess characteristics of “internal inhibition” (13, 17). With easily controlled means of inducing paradoxical sleep by electrical stimulation (8) it became of interest to determine whether the response could be conditioned to an external stimulus, such as a neutral tone, unrelated either to sexual appetite or to local subcortical electrical excitation. Neural correlates of the establishment of such temporary connections have been investigated widely during the past few years (3, 6, 15). Cortical sleep spindles in the cat, evoked by stimulating the hippocampus (23), have been conditioned, as have also spindle sleep episodes deriving from stimulation of the basal forebrain system (1, 22). In the present study, with low-frequency electrical stimulation of limbit areas as the unconditional stimulus (US) and a neutral pure tone as the conditional stimulus (CS) the results show that sleep can indeed be conditioned in the rabbit. Both spindle and activated paradoxical phases with all of their electroencephalographic (EEG) attributes can be conditioned when the threshold of the US is low. The results further reveal that when the threshold of response to the US becomes secondarily elevated the conditioned response (CR) is lost. The findings have been published in preliminary form ( 11). Materials

and

Methods

Large (4-5 kg) mature female New Zealand White rabbits were employed. Methods of housing, care and feeding were similar to those described previously (2 1) . Concentric bipolar subcortical electrodes and silver ball cortical electrodes were implanted permanently in the brains of fourteen animals by techniques also described earlier (19, 20). The rabbits were ovariectomized and primed with estrogen (estradiol benzoate in oil, 0.1 mg SC) for 2-4 days before and during the conditioning experiments because of the observed dependence of the EEG after-reaction threshold on the hormonal status of the animal (9, 10). The experiments were conducted with the rabbit in a large soundattenuated screened room with a one-way glass window. Electroencephalographic records were made with Grass inkwriting equipment, and the US was delivered by a Grass stimulator and isolation unit. The US consisted of a 30-40 set train of monophasic square-waves of 0.5msec duration, 5 cycle/set, with amperage monitored constantly with a radiopulse transformer and oscilloscope. The conditional stimulus was a pure

472

KAWAKAMI

HEAD

D.

FIG. 1. EEG estrogen-primed

record rabbit.

AND

DOWN

SAWYER

JAW

MOVEMENT

of the unconditioned response at moderate paper speed in the A, B, C are one contixiuous record and D was taken 0.5 min

CONDITIONED

PARADOXICAL

SLEEP

473

tone of 500 cycle/set produced by an oscillator and amplified electronically to a moderate intensity. Results

Characteristics of the unconditioned response (UR) including phases of both ordinary or spindle sleep and “activated” or “paradoxical” sleep are illustrated in Fig. 1. The US was applied to the septum in more than half of the subjects and to the amygdala, lateral preoptic area or central gray in the others. Typically, spindle sleep began as it did in Fig. 1, during the half-minute stimulation period, and terminated within the next few minutes by changing abruptly to the paradoxical sleep pattern. The latter was characterized by a high amplitude fast (8 cycle/set) theta rhythm such as that seenin every channel except the amygdala in Fig. ID. This accentuated theta rhythm was usually most prominent in hippocampus, central gray, or related areas and was the source of the term “hippocampal hyperactivity” used to describe this phase by Sawyer and Kawakami in 1959 (20). The paradoxical phase lasted from several secondsto a few minutes during which there was lossof tone in the muscles of the neck and ears and the only notable movements were spasmodic twitches of the eyes and jaws. Paradoxical sleep was always entered via spindle sleep. On awakening from paradoxical sleep the animal elevated its ears and head. Like all strange sounds, the 500 cycle/set CS was at first an alerting stimulus. The rabbit was hsbituated to this tone by presenting it repeatedly every few minutes during the night before it was first paired with the US. The next morning the habituated CS would permit spindle sleep but many trials failed to reveal paradoxical sleep within 10 min of the presentation of the 500 cycle/set tone. This observation indicates that positive results subsequently obtained with the application of paired stimuli were not attributable simply to “pseudoconditioning” to an habitafter C. Sleep spindles appear during the low-voltage, low-frequency stimuiation of the septum and continue into C. Paradoxical sleep, starting in C, is characterized by a rapid (8 cycle/set) theta rhythm in several channels, jaw movements and loss of tone in the neck muscles resulting in a lowering of the head. Awakening, with rapid transition to the alert EEG, is seen in D. Abbreviations in this and later figures: AMYG, amygdala; CG, central gray; DHPC, dorsal hippocampus; DMT, dorsomedial nucleus of thalamus; FC, frontal cortex; FF, fimbria of fornix; LC, limbic cortex; OB olfactory bulb; PUT, putamen; SEPT or SP, septum; VMH, ventromedial nucleus of thalamus.

FIG. ditional

2.

A.

WlRU> SnMULl

EEC records of ordinary and paradoxical stimulation. Further explanation in text.

lx 08 SP CG AMYG DHPC

sleep

(very

slow

paper

speed)

following

paired

(US

and

CS)

and

.

con-

5 “s E

F s k E

P 2

CONDITIONED

PARADOXICAL

uated stimulus. The intensity just above threshold for the next 10 min. The stimuli were had settled down from moving

T

TTT

T

of the US was determined as the current induction of paradoxical sleep within the presented in each case just after the rabbit about its cage.

T

DAY

I

T FIG. 3. Diagram the first and third are seen in Fig. 2. were considered to Further explanation

475

SLEEP

T

2

3

4

5PM

6SfJT

of sleep responses to paired and conditional stimulation during days of a 4-day experiment on the rabbit whose EEG records Any episodes of PS starting later than 10 min after stimulation be possibly spontaneous episodes and were arbitrarily excluded. in text.

In pairing the stimuli the CS (500 cycle/set habituated tone) was prefor 10 set before the US and maintained during the 30-40 set delivery of US. Figure 2 illustrates results of one of the longer experiments, further findings of which are plotted in Fig. 3. Note that the paper speed in Fig. 2 is much slower than in Fig. 1 so that a complete response is sented

DHPC

AMYG

FF

SEPT

OB

FC

B.

A.

CONT.

CONDITIONED

Fro.

4.

EEG

RESPONSE

record

of conditioned

sleep;

B is continuous

with

A. Explanation

in text.

CONDITIONED

PARADOXICAL

477

SLEEP

contained in a single horizontal grouping. In this case spindles appeared in the cortex at the end of the stimulation period and paradoxical sleep only a minute later. The latter phase, with high amplitude theta predominant in central gray and hippocampus, lasted more than a minute. Behaviorally the lowering and elevating of the ears served as the indices of onset and termination, respectively, of this phase. After the CS had been paired with the US in stimulating sleep successfully six times over the course of 4 hours the tone alone evoked the characteristic response

JULY 31 ESTROGEN,

S.C.

E=ELECTRICAL T=TOtili 500

AUG.2

AUG.3

I

8AMi

+ ++ I E 1

I

E E T T

I

8AM[

BAML

I.

++t I

E T

TT

--

+

+-+++

I

I

I

EE TT

E T

Tc

21

+

f-++-

I

I

I

I

I

+++-

41

--+ I

TTT

T:

TT Tc

T

I

++

41

ETc

+ I

121

TCTTC

NO ESTROGEN

I

I

T

17PM

T TEST! I

17PM

+41

I

I

17PM

T

‘,

I

1

17PM

c

AUG. 3,4,5,6.

+8AMI

+I

E T

AUG.6

+ I

E’

t ESTR.

TTT

E EEE T TTT

.I

EEE TTTTT

I

~PM~~PM

+++t 121

-*++*+ T

AUG.4

AUG. I

STIM. AMYGO 20pA 5W?S.=US C.RS.=CS C=CONTROL

E T

t-

--I

,a

E El!1751 TTTT

+

I

I

I

41

c

T

FIG. 5. Diagram of timing of stimuli and responses throughout an experiment on the rabbit whose EEG record is seen in Fig. 4. Explanation in text.

(Fig. 2, B). Sleep spindles appeared during the presentation of the CS and paradoxical sleep began in less than a minute thereafter. The specificity of the pitch of the CS was tested by presenting a tone of equal amplitude ‘but of 650 cycle/set. This tone alerted the sleepy animal (Fig. 2, C) and never induced sleep. Figure 3 summarizes the responses in this same rabbit during the first and third days of a 4-day experiment. The CR was extinguished each night by nonreinforcement of the repeatedly presented tone. It is apparent that on day 1 after six successful pairings of CS with US the CR was positive in three of four successive presentations of CS alone. Control

478

XAWAKAMI

AND

SAWYER

periods during which neither CS nor US was presented were uniformly negative. By the third day the CR was positive in six of seven cases with only two reinforcing positive CS-US pairings interspersed. The 6.50 cycle/set tone was presented alone four times and once paired with the US but it aroused the rabbit all five times. By the next day the US threshold had risen to more than 100 vamp and five attempts with the CS failed to induce any paradoxical sleep. Further aspects of the CR are illustrated by data from another rabbit in Figs. 4 and 5. With the appearance of sleep spindles at the presentation of CS in Fig. 4 the amplitude of activity in the olfactory bulb and septum is reduced and it is suppressed still further during paradoxical sleep. Awakening or recovery from the latter phase is attended by increased amplitude in these two channels coincident with disappearance of the high amplitude fast theta rhythm in the fornix and hippocampus. Within another half-minute the rabbit has reverted to spindle sleep. This particular CR was extracted from the record of the second full day of testing (2:47 P.M., Aug. 3). The timing of procedures in the complete experiment is summarized in Fig. 5. The rabbit received three injections of estrogen and recordings were made on four full days and one part-day. Attempts were made to extinguish the CR each night by repeating the CS every 20 min without reinforcement. On three occasions in this experiment it was possible after repeated pairings of CS with US to evoke as many as three CR without reinforcement. By the third full day (Aug. 4) even the first CS was effective and only two reinforcements were given on that day. Four days after the last injection of estrogen (Aug. 6), the US threshold had risen from 20 to 75 u-lamp and neither the CS nor the CS paired with 20 vamp was effective in evoking paradoxical sleep. However, in this case the single pairing of CS with the new US threshold was adequate to prepare the way for a successful CR. Responses similar to those illustrated above were obtained in eleven of the fourteen rabbits. In the other three the threshold of the UR was high (> 150 vamp), perhaps because of the positions of the stimulating electrodes, and no CR was obtainable. Six of the animals were subjects of experiments continuing for 3-6 days while the others were employed only 1 or 2 days. The threshold of the UR, after two injections of estrogen, usually varied in different animals from 20-100 Clamp. The latencies of the paradoxical phase varied from 1 to 8 min but were usually under 5 min. After five to nine pairings of CS with US spaced at approximately half-hourly intervals the CS was ordinarily effective for two to three

CONDITIONED

PARADOXICAL

SLEEP

479

trials without reinforcement. In the experiments lasting several days the extinguished CR ‘could usually be reestablished on subsequent days with fewer pairings of the CS with the US until the threshold of the latter had become elevated. After the rabbit had been in the experimental room 2-4 days its UR threshold usually rose whether or not estrogen was continued. A possible explanation for this phenomenon will be discussed below. As the UR threshold rose to two or more times the level at which the CR was established, the CR was usually lost. This occurred within 48 hours after the second injection of estrogen in five rabbits and in spite of continued estrogen administration in three others. In two of the positive cases the whole conditioning sequence was repeated 3 or 4 weeks later. Little improvement was observed in the rapidity of conditioning the response.The lack of residual conditioning at that time is perhaps not surprising in view of the brevity of the original conditioning program. Discussion

The US for the induction of PS in the rabbit involves the limbic forebrain (septum) and limbic midbrain (central gray) systems described by Nauta (16). Included in these systems are the areas stimulated by earlier investigators to induce sleep: e.g., the “diencephalic center” of Hess (4) the “rhinencephalic-hypothalamic” system of Kawakami and Sawyer (8) and the hippocampal region of Yoshii and Yamaguchi (23) and the “basal forebrain” system of Clemente et al. (1, 22). The last two groups were able to condition electrically-stimulated spindle sleep in the cat to respond to a neutral tone. The sleep-inducing areas listed above are closely related to and complementary to the ascending reticular system of Magoun (14), stimulation of which activates arousal mechanisms.Like other regions in the reticular system these “limbic-brainstem” areas do not receive specific afferent projections directly but only via long-latency multisynaptic connections. The conditional stimulus in the present work as well as in the studies of Yoshii and Yamaguchi (23) and Clemente et al. (1, 22) was received by the cochlea and transmitted via the auditory nerve. Whether the temporary connections which are established involve the collateral input of auditory projections to the reticular system or whether they are made at the cortical level cannot be determined from the present experiments. Cortical spindling represents a primary element in the response and

4x0

KAWAKAMI

AND

SAWYER

descending cortical reticular pathways may be instrumental in the induction of PS. However, it should be remembered that Jouvet (7) has observed PS in the cat in the absence not only of the cortex but also the rest of the brain above the pontine level. The conditioned response in our experiments was actually a sequence of spindle sleep and paradoxical sleep, as was also the unconditioned response, but the unique feature was the PS: therefore, we have used the term “conditioned PS.” The conditioning of a train of events rather than a simple response is in itself a most unusual phenomenon. Conditioned PS in the rabbit is an example of the “induced” variety PS. Episodes of the latter occur in as compared with “spontaneous” the quiet of a soundproof room usually 20 min or more after the rabbit settles down to spindle sleep. They are inhibited by afferent stimuli such as a masking noise and are relatively independent of the hormonal status of the rabbit; they are observed in males and females alike ( 12). The induced variety of PS resembles the spontaneous episodes in motor expression and EEG but differs in many aspects. In nature the induced type is observed in the female rabbit following coitus and there is evidence that pituitary hormones released as part of the copulation-ovulation sequence are instrumental in triggering PS (8). The teleological significance of PS at this period is unknown but the lowered threshold for its induction appears to be related to the facility with which the hypophysis releases luteinizing hormone (LH) ; coincidentally the arousal threshold is elevated and ,further mating interrupted until the end of the induced PS episode. A few hours later the PS threshold (as measured by electrical stimulation) is elevated and LH release appears to be terminated at this time. The threshold of induced PS is also sensitive to the level of the gonadal steroids. Testosterone and its antifertility progestogen relatives considerably elevate the threshold. Estrogen lowers the threshold for at least 2 days but on continued treatment there is a secondary elevation not unlike the effect of estrogen withdrawal, and both of these influences were observed in the present conditioning experiments. Progesterone itself exerts a similar though more rapid biphasic effect with a depression in threshold lasting only a few hours and a more prolonged elevation in threshold. The threshold changes induced by the hormones appear to be in the afferent or at least in the nonterminal elements of the PS response, since the steroids do not interfere with spontaneous PS. Certain nervous system depressants such as pentobarbital, chlorproma-

CONDITIONED

PARADOXICAL

SLEEP

481

zine, morphine and atropine appear to exert their influence on PS mechanisms at more nearly terminal sites than the steroids for these drugs inhibit the spontaneous as well as the induced variety of PS (7, 18). Of the four only pentobarbital inhibits midbrain reticular arousal mechanisms in the dosage required to block PS episodes; the other three appear to exert their influence more rostrally on diffuse thalamic projections or the limbic forebrain system. Paradoxical sleep is “deep” in the sense that a strong stimulus is required to arouse the animal but “light” in its many similarities to the aroused state: activated EEG, the type of evoked response in the cortex when the reticular formation is stimulated (Khazan and Sawyer, unpublished) and the rapid unit activity of reticular-formation (RF) neurons (5). Active suppression of afferent transmission by RF activity coupled with activation of the descending bulbar inhibitory system (14) could supply the active inhibition characteristic of this peculiar state. In his lectures on internal inhibition and sleep Pavlov (17) describes a response to “inhibitory tones” in which a dog “quickly fell into such profound sleep that even most powerful extraneous stimuli failed to awaken it.” After a period lasting from several seconds to a few minutes such an animal “quickly and spontaneously awakens and exhibits a sharp alimentary motor and salivary reaction.” Although EEG were not taken the description suggests that paradoxical sleep may have been a feature of internal inhibition in at least some of Pavlov’s conditioning experiments. The present study and the work of other recent investigators (13) have emphasized the involvement of brainstem and strbcortical structures in the expression of inhibitory as well as arousal mechanisms. References 1.

2. 3.

4.

5.

CLEMENTE, C. D., M. B. STERMAN, and W. WYRWICKA. 1963. Forebrain inhibitory mechanisms: conditioning of basal forebrain induced EEG synchronization and sleep. Exptl. Neural. 7: 404-417. FAURE, J. 1962. La phase “paradoxale” du sommeil chez le Lapin (ses relations neurohormonalesj. Rev. Neurol. 106: 190-197. HERNANDEZ-PEON, R. 1960. Neuropbysiological correlates of habituation and other manifestations of plastic inhibition. Electroencephdog. Clin. Neurophysiol. supp. 13: 101-114. HESS, W. R. 1954. The diencephalic sleep center, pp. 117-136. In “Brain Mechanisms and Consciousness.” E. D. Adrian, F. Bremer, and H. Jasper feds.]. Blackwell, Oxford. HUTTENLOCHER, P. R. 1961. Evoked and spontaneous activity in single units of medial brain stem during natural sleep and waking. J. Neurophysiol, 24: 451-468.

482 6. 7.

KAWAKAMI

9.

10. 11. 12. 13.

14. 15. 16.

17. 18.

19. 20.

21.

22.

SAWYER

JOHN, E. R. 1961. High nervous functions: Ann. Rev. Physiol. 23: 451-484. JOUVET, M. 1962. Recherches sur les structures responsibles des differentes phases du sommeil 100:

8.

AND

brain

functions

and

learning.

nerveuses et les mechanismes physiologique. Arch. Ital Biol.

125-206.

KAWAKAMI, M., and C. H. SAWYER. 1959. Induction of behavioral and electroencephalographic changes in the rabbit by hormone administration or brain stimulation, Endocrinology 65: 631-643. KAWAKAMI, M., and C. H. SAWYER. 1959. Neuroendocrine correlates of changes in brain activity thresholds by sex steroids and pituitary hormones. Endocrinology 65: 652-668. KAWAKATUI, M., and C. H. SAWYER. 1962. Effects of hormones on “paradoxical” sleep in the rabbit. Federation Proc. 21: 354, 1962. Induction of “paradoxical” sleep KAWAKAMI, M., and C. H. SAWYER. by conditional stimulation in the rabbit. Physiologist 5: 165. KHAZAN, N., M. KAWAKAMI, and C. H. SAWYER. 1963. Physiological and pharmacological aspects of paradoxical sleep. Pharmacologist 5: 266. MAGOUN, H. W. 1961. Brain mechanisms for internal inhibition, Vol. 1, pp. 6-16. In “Proceedings of the Third World Congress of Psychiatry.” Univ. of Toronto Press, Toronto. MAGOUN, H. W. 1963. “The Walking Brain,” 2nd ed. Thomas, Springfield, Illinois. MORELL, F. 1961. Electrophysiological contributions to the neural basis of learning. Physiol. Rev. 41: 443-494. NAUTA, W. J. H. 1963. Central nervous organization and the endocrine motor system, pp. S-21. In “Advances in Neuroendocrinology,” A. V. Nalbandov ted.]. Univ. of Illinois Press, Urbana, Illinois. PAVLOV, I. P. 1927. “Conditioned Reflexes,” pp. 2X-264. Oxford Univ. Press, London and New York. SAWYER, C. H. 1962. Mechanisms by which drugs and hormones activate and block release of pituitary gonadotropins, Vol. 1, pp. 27-46. In “First International Pharmacological Meeting,” R. Guillemin led.]. Pergamon Press, New York. SAWYER, C. H., J. W. EVERETT, and J. D. GREEN. 1954. The rabbit diencephalon in stereotaxic coordinates. J. Camp. Neural. 101: 801-824. SAWYER, C. H., and M. KAWAKAMI. 1959. Characteristics of behavioral and electroencephalographic stimulation in the female rabbit. Endocrinology 65: 622-630. SAWYER, C. H., and J. E. MARKEE. 1959. Estrogen facilitation of release of pituitary ovulating hormone in the rabbit in response to vaginal stimulation. Endocrinology 65: 614-621. WYRWICKA, W., M. B. STERMAN, and C. D. CLEMENTE. 1962. Conditioning of induced electroencephalographic sleep patterns in the cat. Science 137: 616-618.

23.

Yosrrn, N., and Y. YAMAGUCHI. 1960. induced by electrical stimulation of Neurologiu Med. Chir. 2: 56-62.

Conditioning of spindle dorsal hippocampus in

burst cats

discharges and dogs.