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The development of unit activity in the lateral geniculate nucleus of the kitten
XPERIMENTAL
43, 261-275
NEUROLOGY
The Development
JOELLE
Departwrest
(1974)
of Unit Activity in the Lateral Nucleus of the Kitten ADRIEN
AND
HOWARD
P.
ROFFWARG
of Psychiatry, Montefiore Hospital atzd Medical E&stein College of Mcdicise, Bronx, Nezc York Received
Dccewber
Geniculate
l
Center 10467
artd Albert
27,1973
The spontaneous unit activity in the lateral geniculate nucleus of 15 kittens has been analyzed in terms of the sleep-wake state. Lateral geniculate units in animals ranging from 2 days to 5 weeks of age were recorded under chronic conditions by means of a hydraulically movable glass micropipet. During the first postnatal month, the mean frequency of firing increased progressively in the awake state and in paradoxical sleep but was not modified in quiet or slow wave sleep. However, as early as the third postnatal week, the pattern of modulation of the frequency of unit discharge in response to changes in state reached an organization close to that seen in the adult cat. The most dramatic alteration in the cellular discharge configuration was represented by the introduction of the “burst-pause” pattern in slow wave sleep. Serial interspike interval distributions demonstrated the appearance of this pattern at the end of the second week. The organization of unit firing in the l-month-old animal was qualitatively similar to that in the adult, though quantitatively less intense, The pattern of development of unit firing is discussed in light of the appearance of other important developmental features such as cortical synchronization, ponto-geniculo-occipital spikes, and maturation of certain monamine-containing cells in the central nervous system. Some structural and neurochemical factors in development are mentioned that may be responsible for the ontogenetic organization of unit discharges in the lateral geniculate nucleus. 1 This study was made possible by a fellowship from the French Government (Service des Bchanges Culturels, Scientifiques et Techniques). It was also supported in part by Career Research Scientist Award MH-18739 and Project Grant MH-13269 (H. P. R.) from the National Institute of Mental Health. We acknowledge the generous help of Dr. Herbert Weiner who made unit recording equipment available and Dr. Vahe Amassian for allowing us to use his computer facilities. We also thank Marie-Louise Elbert for the histological preparations, and Constance Bowe-Anders for her generous assistance with the experimental animals. Dr. Adrien’s present address (for reprint requests) is: Laboratoire de Physiologie, C. H. U. PitiP-SalpOtri&e, 91 Boulevard De l’H6pita1, 75- Paris 13”, France. 261 Copyright All rights
0 1974 by Academic af reproduction in any
Press, Inc. fnrm reserved.
262
ADRIEN
AND
ROFFWARG
INTRODUCTION Studies of the sleep of animals that are born very immature, such at the cat or the rat, have revealed that during the first few days after birth only two states of vigilance may be observed, the awake state, and sleep with jerks, or active sleep. At a later point, a stage of quiet sleep emerges. This stage is called slow wave sleep when the cortex begins to invest the electrocorticogram (EEG) with slow waves during the periods of quiet sleep. These findings were based exclusively on behavioral observations and the usual EEG, electrooculographic (EOG) and electromyographic (EMG) parameters of sleep recording (3, 7, 14). Little is known of the electrophysiology of the newborn animal’s CNS under chronic conditions of recording. Acute preparations have been studied in regard to evoked responses (16, 20, 22, 24). However, chronic recording of spontaneous activity of the brain in the newborn has been explored only at the cortical level (7, 8, 13, 14). Considering that the cerebral cortex matures later than the thalamus and the pons (19)) we felt it worthwhile, with respect to the need for information about the developmental neurophysiology of sleep, to examine the changes in spontaneous activity at a subcortical level as well. The work represented in this paper focuses on the ontogenesis of spontaneous unit activity in the neurons of lateral geniculate nucleus under chronic conditions in the cat during the first 5 weeks of life. A related report has described the first appearance of macroelectrode-recorded pontogeniculo-occipital activity in the lateral geniculate nucleus and its subsequent maturational changes (3). A temporal relationship between unit discharge and the ponto-geniculo-occipital spike, which has been found in the lateral geniculate nucleus of the adult cat, has not yet been investigated in the kitten (2, 21, 23). METHODS Iw@lantation and Apparatus. Fifteen kittens received implanted electrodes under ether or pentobarbital anesthesia (25-35 mg/kg) in the period from 2-36 days after birth. Transcortical, periocular, and neck muscle electrodes were placed according to the technique described (3). For the recording of units, an Evarts-type microdrive (6) was adapted to small animals and modified for extracellular recording utilizing glass micropipets. The micropipets were pulled to an internal diameter of 0.5-2.0 pm at the tip. The electrolyte was 3 M KCl. Figure 1 is a schematic display of the components of the device and their interrelationships. They comprise: a stainless-steel chamber of 4 mm i.d. ; a guide that fits exactly inside the chamber insuring the lateral stability of the electrode, which is a glass micropipet; a cylinder,
Fro. 1. Schematic cross section of the microdrive. micronipet. and supporting apparatus. 1. _ wire spring; 5. cylinder;. 6. watermetallic chamber ; 2 guide; 3 glass micropipet ; 4. tungsten tight piston; 7. flexible wire. The chamber and cylinder have a combined height of 20 mm. Represented alongside the diagram are action potentials recorded from a chronically implanted electrode in a lateral geniculate unit in a g-day-old kitten. Calibration: 50 msec, 2 mv.
264
ADRIEN
AND
ROFFWARG
containing a piston, that adapts onto the top of the chamber; the piston can be driven down by a micromanipulator placed at some distance from the animal through the medium of an intervening hydraulic system; the glass micropipet, attached to the piston by means of a stiff tungsten wire is fixed to the lower part of the piston and, because of a slight bend, it acts like a spring clamp within the micropipet ; the wire provides vertical stability to the electrode as well as electrical contact with the electrolyte. Stereotaxic coordinates for the lateral geniculate nucleus of the kitten at different ages, which were developed in this laboratory (l), were used for the implantations. The chamber of the recording device was placed over the skull directly above the lateral geniculate nucleus. Before the chamber was cemented into its permanent position, a micropipet was inserted into it and units that could be specifically activated by photic stimulation were monitored. This procedure provided electrophysiologic confirmation of the placement of the chamber at least in the visual pathways, and, hopefully, in the lateral geniculate nucleus. At this point, the chamber was permanently cemented, the electrode removed and the chamber capped. The implant was protected by a head helmet made of gauze and surgical tape. At the completion of the surgery, the animals were returned to their littermates. Weights were noted every day and compared to those of control littermates. All animals recovered normal weight within 2-3 postoperative days. Recording Conditions. Recordings were carried out for l-3 weeks after surgery (Table 1). Recording sessions lasted from 2 to 6 hr. At the beginning of each session, the animal was wrapped in a towel and placed in a dimly illuminated cage. If older than 12 days, the kitten was partially restrained at the time of registration. This was effected by attaching a stud, which had been embedded in the cement of the implant at the time of surgery, to a bar fixed to the recording cage, thereby reducing head mo:-emerit. As judged from the latency to the onset of sleep of the restrained animals, this technique did not disturb sleep. It provided excellent stability for the recording of single cells and made it possible to “hold” a single unit for more than an hour, even in several animals younger than 3 weeks of age. In the older kittens, whose skulls are ossified, the recording of single cells was frequently extended for as long as 2 hr. The EEG, EOG, EMG, and the output of a Schmitt trigger wzre monitored on a Grass Model 6 electroencephalograph. The unit activity as well as the other parameters were also recorded directly onto tape for subsequent computer analysis. Each cell that was encountered was tested for a specific short latency response to photic stimulation. If this criterion was met, the unit was recorded during one or more sleep-wake cycles. After l-3 postoperative weeks the recording sites were marked with micro-
LATERAL
GENICULATE
KUCLEUS
TABLE AGES
OF 1.5 KITTENS
CROSSES
AT THE TIME
DENOTE
THE
1
THAT
DAYS
UNIT
WHEN
Age
1
2
265
DATA
KECORDIN~~S
WERE
COLLECTED.
WEKE
THE
MADE
(weeks)
3
4
5
6
xxxxxxx xxxxxxx
xxxxxxx
xxxxxxx
xxxxxx
xxxxxxx
x
xxxxxxx xxxxxx
xxxx xxxx xxxxxx xxxxxx
xxxxxxx
xxxxxxx
xxxxxx
xxx
Kittens
K9 KS
K7 K15 K4 KS Kl K16 K3 K17 Kll K10 K12 K2 K13
xxxxxx xxxxx xx
xxxx x
xxxxxxx
xxx XX
xxxxxxx xxxx
xxxx xxxxxxx xxxx
lesions, and the brains were perfused through the carotid artery with 4% formaldehyde for the purpose of histological verification of the microelectrode tract. Co~zpztter Analysis. Data were analyzed only from lateral geniculate cells that gave satisfactory signal to noise ratios and showed no signs of injury discharge. The polygraphic record of the other electrophysiological parameters was carefully sampled so that a recording from each cell was available from an incontrovertible segment of the awake state, quiet sleep or slow wave sleep, and active or paradoxical sleep. The polygraphic and behavioral criteria for designation of these states have been described (3). The spontaneous unit activity of each neuron that was selected for analysis was fed to a DCA 160 computer for separate examination of the mean frequency and interspike interval distribution from each state. Modifications of the discharge pattern in the different states of vigilance were followed by means of these parameters during the first month of postnatal life. No analyses were performed on unit discharge recordings from the mixed stage of transitional sleep (3). RESULTS Data were obtained from l-5 kittens ranging from 2 days to 5 weeks of age. Table 1 summarizes the collection of data among the 15 animals.
266
ADRIEN
AND
ROFFWARG
Because of the effect of skull growth on electrode placement, no single animal contributed data over the entire age span. Rather, individual animals provided small segments of data within the approximately 5-week period of study. A total of 150 lateral geniculate units were recorded. Analysis was performed on the 60 cells that were held through one or more sleep-wake cycles and at least two of the three states. The mean recording time for a cell was 16.25 min. Evolution of the Mean Frequency with Age. As mentioned above, the mean frequency of unit discharge was calculated from the most clearly defined samples of recording from each state. The durations of the segments subjected to analysis ranged from l-10 min, with mean intervals of 3 min in the awake state, 3 min in quiet sleep and slow wave sleep and 4.5 min in the awake state and paradoxical sleep. In terms of state relationships, the frequency of discharge generally increased with age in the awake state (Y = 0.71, p < 0.005) as well in paradoxical sleep (r = 0.85, p < 0.005). On the contrary, mean discharge in slow wave sleep did not significantly increase during the first postnatal month, though a slight trend was visible. (Note that data for quiet sleep are not reported for the first week of life because of the sparseness of quiet sleep until the second week (14). In Fig. 3, the frequency of discharge by week is plotted for each of the 32 units observed through all three states. It can be seen that the “V” shape frequency plot, characteristic of the adult lateral geniculate discharge across the three states (23), appeared clearly by the third postnatal week. As in the adult cat (23), however, some cells did not show this pattern. To summarize these data, photoresponsive cells in the lateral geniculate nucleus showed a significant, almost linear, increase in discharge frequency in the awake state and in paradoxical sleep. No statistically significant change was observed in slow wave sleep during the first postnatal month. However, as early as the third postnatal week, modulation of the discharge rate from one state to another had already assumed the adult pattern. Evolution of the Interspike Interval Distribution with Age. This analysis was performed on both a 4-msec and a 20-msec time base. Two examples are given in Fig. 4. Figure 4A, displays histograms of the interspike interval distribution of a representative cell in each state at 2 weeks of age. At this time, qualitative differences among the three states of vigilance were clearly observable. By 1 month (Fig. 4B), the histograms were qualitatively similar to those of the adult cat (IS). In the awake state, unit discharge was rather regular. The mode of the distribution was at 8-12 msec with an amplitude of 19%. There were only a small number of intervals longer than 1 set duration. In slow wave sleep, the distribution was more unsymmetrical. A peak of
LATERAL
GENICULATE
0
10
NUCLEUS
20 66E
267
30 (days)
FIG. 2. Evolution of the mean frequency of unit discharge in the three states during the first month of life. Awake state : triangles ; quiet sleep and slow wave sleep : open circles; awake state and paradoxical sleep: filled circles. Each data symbol represents the mean frequency of all units recorded on a given day. The variation about the mean, though not displayed in these graphs, is not different from what is found in the adult
(22). short, 48 msec, intervals at an amplitude of 28% was combined with a substantial quantity (approximately 10%) of longer intervals. This pattern was very distinctive and is known as the “burst-pause” pattern of discharge. In paradoxical sleep discharge was intermediary between the awake state and slow wave sleep; namely, there were slightly more short, 8- to 1Zmsec intervals than in the awake state (23%)) but no long pauses as in slow wave sleep.
I ,
II
I
15
25
30
&
\ \
St’
sak
S S
FIG. 3. Mean frequency of unit discharge for all cells recorded through the awake state, slow wave sleep, quiet and paradoxical sleep during the first 5 weeks of life. In the first week, there is neither enough time spent in quiet nor in the awake state without movement, to provide sufficent data for a graph.
4th Wnk
sws
sleep, sleep
I i
LATERAL
GENICULATE
269
NUCLEUS
The results of the interspike interval distribution analyses for all cells are represented in Figs. 5 and 6. Figure 5 portrays the proportion of short intervals in the three states from the first to the fifth week of age. Short
! 20
A
B
14 days
31 days
20,
w
w 10
10 n-599
ll4765 0
I
20-
QS 10.
n=wr 0
m-
PS
PS
II=6867
FIG. 4. Interspike interval distributions for single cells in each of the three states. A. a 14-day-old kitten, and B. a 31-day-old kitten. Time base: 4 msec. The longest interspike interval portrayed in these histograms is 200 msec.
270
ADRIEN
AND
ROFFWARG
60-
0 0 0
0
40-
0
0 0
20-
cl 0 *
0
, 0”
. . l
. .
.
.
10
.
.
.
0
.
:
. .
20
30
AGE (days) FIG. 5. Evolution of the proportion of short interspike intervals with age in the three states. Awake state: triangles; quiet sleep and slow wave sleep: open circles; awake state and paradoxical sleep: filled circles. Data are averaged for each day.
LATERAL
GENICULATE
NUCLEUS
271
.
01
O-20
40-80
modal intervelr fmsec.) FIG. 6. Evolution of the modes of the interspike interval distributions with age. For each cell in each state, the value of the mode is plotted against its amplitude. Awake state : triangles ; quiet sleep and slow wave sleep : open circles ; awake state and paradoxical sleep : filled circles.
intervals were arbitrarily defined as having less than 20-msec duration because in the very young kitten the units have low firing rates (Fig. 2). In the awake state, the proportion of short intervals showed a progressive rise from the second to the fifth week of age. This phenomenon was principally due to an increase in frequency (Fig. 2), and the distribution of inter-spike intervals, though constricted, persisted in a more symmetrical shape than in slow wave sleep and paradoxical sleep (Fig. 4). In slow wave sleep, an evolution with age toward a greater proportion of short intervals parallelled that observed in the awake state. However, in the case of slow wave sleep, the increase in the ratio of short intervals was Ilot due to an incrrase in frerluencv of clisciiargc (Fig. 2). Rather, it
272
ADRIEN
AND
ROFFWARG
derived exclusively from a qualitative change in the pattern of the discharge, specifically a progressive appearance of high-frequency bursts. This is reflected in Fig. 4 which portrays the development of greater polarization in the distribution of interspike intervals in slow wave sleep. In paradoxical sleep the evolution of the discharge pattern was also very similar to that of the awake state. During the first week, however, the proportion of short intervals was higher, averaging about 15%. This proportion of short intervals in the first week was rather high considering the very low frequency of discharge during that period. The combination of the two factors actually defined a unique discharge pattern comprised of groupings (not bursts) of five to ten spikes, separated by periods of silence, often exceeding 10 sec. This is not a “burst-pause” pattern, but rather describes an absence of tonic discharge. A similar lack of tonic discharge in the cortex has also been found at this age (9). Figure 6 demonstrates the change in the mode of the interspike interval distribution of each cell in each state as maturation proceeds : In the awake state, the interspike interval distribution modes of the cells showed a large dispersion in the second week. By the fifth week they localized toward the vertical axis and had a mean amplitude of 30%. These data reveal that through the second week the discharge was quite random (see Fig. 4A, W) . A shift to shorter frequencies took place in the latter 3 weeks. The early random discharge may be explained by insufficient maturation in sensory systems and the consequent relative sensory isolation of the neonatal kitten. Mukhametov (18) has found that in lateral geniculate neurons of the adult cat, interspike interval distributions were more symmetrical during quiet waking than during alert waking. This may indicate that the only waking state of attention available to the young kitten corresponds to the nonattentive awake state of the adult. Our data indicate that a shift toward greater alertness in the awake kitten took place after the second week of life. In slow wave sleep, there was a change with development in the arrangement of the modes progressively from a predominantly horizontal, to a diagonal, and then to a vertical configuration. The 0- to ZO-msec modes had a mean amplitude of 4.5% in slow wave sleep at the fifth week, which was much higher than the corresponding amplitude in the awake state. In paradoxical sleep, alteration of the modal pattern was similar to that in the awake state, all the modes falling in the 0- to ZO-msec range by the fifth week. However, dispersion was less before the third week than in the awake state. The average amplitude of the 0- to ZO-msec modes at the fifth week was 4.5% as in slow wave sleep. In the summary, the interspike interval distributions changed progressively in the awake state, slow wave sleep, and paradoxical sleep toward higher modes in the shorter intervals during the f&t postnatal month.
LATERAL
GENICULATE
NUCLEUS
273
However, in light of the stable discharge frequency in slow wave sleep, we can conclude that the proportional increase in short intervals in this state was a function of a qualitative rearrangement of the unit activity into a “burst-pause” organization. The development of this capacity to alternate excitation and inhibition is probably a good indicator of the degree of electrophysiological maturation of the cells of the lateral geniculate nucleus. DISCUSSION The evolution of unit discharge in the lateral geniculate nucleus of the kitten in the first month after birth has been described. It was marked in part by a general increase in mean frequency during the course of maturation. This increase was seen chiefly in the awake state and in paradoxical sleep. Similar findings had been obtained in the cortex by Huttenlecher (9)) though the state of the animal was not systematically taken into consideration in regard to the frequency of data from the cortex. In comparison to unit activity in the cortex, unit activity in the lateral geniculate nucleus may be monitored by the second postnatal day, whereas it does not appear until the fourth day in the cortex. Furthermore, the mean frequency of discharge in the first month of life is higher in the lateral geniculate nucleus at any given point in time, a phenomenon that persists into adulthood (6, 23). At the end of the first month of life, the unit discharge rate in the lateral geniculate nucleus of the kitten is still lower than it is in the adult. However, the organization of unit activity has assumed the adult pattern relatively early, by the end of the second postnatal week. At this time the proportion of short and long interspike intervals approximates the pattern seen in equivalent states of the CNS in the adult (23). The organization, at 2 weeks, of the “burst-pause” pattern of discharge during behavioral quiet sleep may represent the most prominent qualitative step in the maturation of spontaneous unit activity in the lateral geniculate nucleus. It should be noted that at this juncture in the maturation of the nervous system, serotonergic terminals have matured, particularly at the level of the raphC nuclei (15). By this time, too, periods of quiet sleep have lengthened (14). Synchronization of the EEG, and appearance of the monophasic waves in the lateral geniculate nucleus have yet to emerge (3, 14). but will occur shortly. It may be that the specific distribution of unit activity during paradoxical sleep begins to coordinate with the timing of ponto-geniculateoccipital bursts once they appear. This is suggested by data from adult animals (2, 21, 23). The specific neural routes that serve as the condt~its of brainstem stimulation to the lateral geniculate nuclms during sleep, and that inay in part determine the nature of the unit discharge nf k~teral geniculate nucleus cells,
274
ADRIEN
AND
ROFFWARG
have not yet been established. Nevertheless, among the probable neuroanatomical loci that bear on the ultimate organization of neuronal discharge in the lateral geniculate nucleus, are maturation of the ultrastructure of the lateral geniculate nucleus as well as of the optic tract terminals and of the ponto-geniculate pathways. We are currently investigating several of these possibilities by attempting to effect modifications in the rate and quality of maturation of the lateral geniculate cells and of the synapses playing upon them. On the one hand, we are dark-rearing animals and, alternatively placing lesions in the pontine regions that seem to be critical for paradoxical sleep and slow wave sleep (11, 12). As we pointed out, our data reveal that state-specific, adult-like unit firing patterns were not organized at the thalamic level until the beginning of the third postnatal week. Furthermore, we have also reported that pontogeniculo-occipital spikes did not appear in the lateral geniculate nucleus during sleep until just about this time (3). However, these findings do not contravene the evidence that the centers in the brain stem subserving paradoxical sleep are functioning at birth. According to the best electrophysiological and behavioral indicators that are available in the immature animal (14), the sleep of the neonatal kitten is comprised virtually entirely of awake state, the precursor of paradoxical state (11). The absence of recordable quiet sleep and slow wave sleep at this time goes along with the finding that the raphe nuclei are not yet biochemically developed (5, 15). In addition, in this laboratory we have been able to record pontogeniculo-occipital activity reflected in the extraocular muscles during awake state as early as the third day of extrauterine life. These muscle spikes have been shown in the adult to represent probably a direct outflow of the spike activity in the pontine reticular formation (17). Accordingly, the polygraphic data, combined with the histochemical demonstration of very early maturation of catecholamine-containing neurons in the pons (5, 15), suggests that though there is a lag in the maturation of distinct electrophysiological signs of paradoxical sleep (ponto-geniculooccipital waves) at the level of the thalamus, the ponto-geniculo-occipitalgenerating apparatus of paradoxical sleep in the pons is in evidence weeks before. In order to better understand the nature and mechanisms of the awake state, as well as the genesis of slow wave sleep in the newborn organism, additional studies must be performed at the pontine level. REFERENCES 1. ADRIEN, J. 1973. Ontogenbe des activites electriques du Noyau chez le chat. Th&e de 3” Cycle, UniversitC de Lyon, France. 2. BIZZI, E. 1966. Discharge pattern of single geniculate neurons eye movements of sleep. J. Nezwophysiol. 29: 1087-1095.
Genicule during
Lateral the
rapid
LATERAL
GENICULATE
NUCLEUS
27.5
3. ROWE-ANDERS, C., H. P. ROFFWARC., and J. ADRIF.N. 1973. Ontogenesis of pontogeniculo-occipital activity in the lateral geniculate nucleus of the kitten. E.rp. Nffitvl., in press. 4. BROOKS, D. C. 1968. Waves associated with eye movemeuts in the awake and sleeping act. Electrocncrpl~nloyr. Clin. Neurophysiol. 24 : 532-541. 5. DAHLSTROM, A., and K. FUXE,. 1964. Evidence for the existence of monoaminecontaining neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta Physiol. Sca?zd. 62: Suppl. 232: l-55. 6. EVARTS, E. V. 1960. Effects of sleep and waking on spontaneous and evoked discharge of single units in the visual cortex. Fed. PYOC. 19:828-837. 7. GARMA, L., and R. VERLEY. 1967. ActivitCs cellulaires corticales etudiees par electrodes implantees chez le lapin nouveau-nP. J. Physiol. Paris. 59: 357-376. 8. HUBEL, D. H. 1959. Single unit activity in striate cortex of unrestrained cats. J. Plzysiol. Lovldow. 147 : 22&238. 9. HUTTENLOCHER, P. R. 1967. Development of cortical neuronal activity in the neonatal cat. Exp. Nezrrol. 17 : 247-262. 10. JEANKEROD, M., and K. SAKAI. 1970. Occipital and geniculate potentials related to eye movements in the unanesthetized cat. Brain Res. 19 : 361-377. 11. JOUVET, M. 1967. Neurophysiology of the states of sleep. Physiol. Rea. 47 : 117-177. 12. JOUVET, M. 1969. Biogenic amines and the states of sleep. Science 1’64: 32-41. 13. JOUVET, D., J. 1,. VALAXT, and M. JOUVET, 1961. fitude polygraphique du sommeil du chaton. C. R. Sot. Biol. 155: 1660-1664. 14. JOUVET-MOUNIER, D., L. ASTIC, and D. LACOTE. 1970. Ontogenesis of the states of sleep in rat, cat, and guinea-pig during the first post-natal month. Develop. Psychobiol. 2 : 216239. 15. LOUP, M., and J. CADILHAC. 1970. Le developpement des neurones ?I monoamines du cetveau chez le chaton. C. R. Sot. Biol. 164: 1582-1587. 16. MARTY, R., and J. SCIIERRER. 1964. CritPres de maturation des systemes afferents corticaux, pp. 222-236. In “Progress In Brain Research,” Vol. 4, D. P. Pu;pura, and J. P. Schade (Eds.). Elsevier, Amsterdam. 17. MICHEL, F., M. JEANKEROD, J. MOURET, A. RECHTSCHAFFEN, and M. JOUVET. 1963. Sur les mecanismes de 1’ activite de pointes au niveau du syst&me visuel au tours de la phase paradoxale du sommeil. C. R. Sot. Biol. 157 : 103-105. 18. MUKHAMETOV, L. M., G. RIZZOLATI, and A. SEITUN. 1970. An analysis of the spontaneous activity of lateral geniculate neurons and optic tract fibers in freemoving cats. Arch. Ital. Biol. 108: 325-347. 19. NOBACK, C. R., and D. P. PURPURA. 1961. Post-natal ontogenesis of neurons in cat neo-cortex. .I. Cowp. Ncwol. 117: 291-307. 20. PURPURA, D. P. 1961. Morphophysiological basis of elementary evoked response pattern in the neo-cortex of the newborn cat. Ann. iv. I’. Acad. Sri. 92 : 604-654. 21. RECUTSCHAFFEN, A., F. MICHEL, and J. METZ. 1972. Relationship between extraocular and PGO activity in the cat. Psyckopll~~vsiol. 9: 128. (abstract) 2.Z. ROSE, G. H., and D. B. LINDSLEY. 196s. Development of visually evoked potentials in kittens : specific and non-specific responses. J. NrzrropAysiol. 31: 607623. 23. SAKAKURA, H. 1968. Spontaneous and evoked unitary activities of cat lateral geniculate neurons in sleep and wakefulness. Jap. J. Pkysiol. 18: 23-42. 24. SCHERRER, J., and D. OECOIYOMOS. 1954. Responses corticales somethesiques du mammifere nouveau& comparPes g celles de l’adulte. Btxd. Neo-Natales. 3: 199-216.
43, 261-275
NEUROLOGY
The Development
JOELLE
Departwrest
(1974)
of Unit Activity in the Lateral Nucleus of the Kitten ADRIEN
AND
HOWARD
P.
ROFFWARG
of Psychiatry, Montefiore Hospital atzd Medical E&stein College of Mcdicise, Bronx, Nezc York Received
Dccewber
Geniculate
l
Center 10467
artd Albert
27,1973
The spontaneous unit activity in the lateral geniculate nucleus of 15 kittens has been analyzed in terms of the sleep-wake state. Lateral geniculate units in animals ranging from 2 days to 5 weeks of age were recorded under chronic conditions by means of a hydraulically movable glass micropipet. During the first postnatal month, the mean frequency of firing increased progressively in the awake state and in paradoxical sleep but was not modified in quiet or slow wave sleep. However, as early as the third postnatal week, the pattern of modulation of the frequency of unit discharge in response to changes in state reached an organization close to that seen in the adult cat. The most dramatic alteration in the cellular discharge configuration was represented by the introduction of the “burst-pause” pattern in slow wave sleep. Serial interspike interval distributions demonstrated the appearance of this pattern at the end of the second week. The organization of unit firing in the l-month-old animal was qualitatively similar to that in the adult, though quantitatively less intense, The pattern of development of unit firing is discussed in light of the appearance of other important developmental features such as cortical synchronization, ponto-geniculo-occipital spikes, and maturation of certain monamine-containing cells in the central nervous system. Some structural and neurochemical factors in development are mentioned that may be responsible for the ontogenetic organization of unit discharges in the lateral geniculate nucleus. 1 This study was made possible by a fellowship from the French Government (Service des Bchanges Culturels, Scientifiques et Techniques). It was also supported in part by Career Research Scientist Award MH-18739 and Project Grant MH-13269 (H. P. R.) from the National Institute of Mental Health. We acknowledge the generous help of Dr. Herbert Weiner who made unit recording equipment available and Dr. Vahe Amassian for allowing us to use his computer facilities. We also thank Marie-Louise Elbert for the histological preparations, and Constance Bowe-Anders for her generous assistance with the experimental animals. Dr. Adrien’s present address (for reprint requests) is: Laboratoire de Physiologie, C. H. U. PitiP-SalpOtri&e, 91 Boulevard De l’H6pita1, 75- Paris 13”, France. 261 Copyright All rights
0 1974 by Academic af reproduction in any
Press, Inc. fnrm reserved.
262
ADRIEN
AND
ROFFWARG
INTRODUCTION Studies of the sleep of animals that are born very immature, such at the cat or the rat, have revealed that during the first few days after birth only two states of vigilance may be observed, the awake state, and sleep with jerks, or active sleep. At a later point, a stage of quiet sleep emerges. This stage is called slow wave sleep when the cortex begins to invest the electrocorticogram (EEG) with slow waves during the periods of quiet sleep. These findings were based exclusively on behavioral observations and the usual EEG, electrooculographic (EOG) and electromyographic (EMG) parameters of sleep recording (3, 7, 14). Little is known of the electrophysiology of the newborn animal’s CNS under chronic conditions of recording. Acute preparations have been studied in regard to evoked responses (16, 20, 22, 24). However, chronic recording of spontaneous activity of the brain in the newborn has been explored only at the cortical level (7, 8, 13, 14). Considering that the cerebral cortex matures later than the thalamus and the pons (19)) we felt it worthwhile, with respect to the need for information about the developmental neurophysiology of sleep, to examine the changes in spontaneous activity at a subcortical level as well. The work represented in this paper focuses on the ontogenesis of spontaneous unit activity in the neurons of lateral geniculate nucleus under chronic conditions in the cat during the first 5 weeks of life. A related report has described the first appearance of macroelectrode-recorded pontogeniculo-occipital activity in the lateral geniculate nucleus and its subsequent maturational changes (3). A temporal relationship between unit discharge and the ponto-geniculo-occipital spike, which has been found in the lateral geniculate nucleus of the adult cat, has not yet been investigated in the kitten (2, 21, 23). METHODS Iw@lantation and Apparatus. Fifteen kittens received implanted electrodes under ether or pentobarbital anesthesia (25-35 mg/kg) in the period from 2-36 days after birth. Transcortical, periocular, and neck muscle electrodes were placed according to the technique described (3). For the recording of units, an Evarts-type microdrive (6) was adapted to small animals and modified for extracellular recording utilizing glass micropipets. The micropipets were pulled to an internal diameter of 0.5-2.0 pm at the tip. The electrolyte was 3 M KCl. Figure 1 is a schematic display of the components of the device and their interrelationships. They comprise: a stainless-steel chamber of 4 mm i.d. ; a guide that fits exactly inside the chamber insuring the lateral stability of the electrode, which is a glass micropipet; a cylinder,
Fro. 1. Schematic cross section of the microdrive. micronipet. and supporting apparatus. 1. _ wire spring; 5. cylinder;. 6. watermetallic chamber ; 2 guide; 3 glass micropipet ; 4. tungsten tight piston; 7. flexible wire. The chamber and cylinder have a combined height of 20 mm. Represented alongside the diagram are action potentials recorded from a chronically implanted electrode in a lateral geniculate unit in a g-day-old kitten. Calibration: 50 msec, 2 mv.
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containing a piston, that adapts onto the top of the chamber; the piston can be driven down by a micromanipulator placed at some distance from the animal through the medium of an intervening hydraulic system; the glass micropipet, attached to the piston by means of a stiff tungsten wire is fixed to the lower part of the piston and, because of a slight bend, it acts like a spring clamp within the micropipet ; the wire provides vertical stability to the electrode as well as electrical contact with the electrolyte. Stereotaxic coordinates for the lateral geniculate nucleus of the kitten at different ages, which were developed in this laboratory (l), were used for the implantations. The chamber of the recording device was placed over the skull directly above the lateral geniculate nucleus. Before the chamber was cemented into its permanent position, a micropipet was inserted into it and units that could be specifically activated by photic stimulation were monitored. This procedure provided electrophysiologic confirmation of the placement of the chamber at least in the visual pathways, and, hopefully, in the lateral geniculate nucleus. At this point, the chamber was permanently cemented, the electrode removed and the chamber capped. The implant was protected by a head helmet made of gauze and surgical tape. At the completion of the surgery, the animals were returned to their littermates. Weights were noted every day and compared to those of control littermates. All animals recovered normal weight within 2-3 postoperative days. Recording Conditions. Recordings were carried out for l-3 weeks after surgery (Table 1). Recording sessions lasted from 2 to 6 hr. At the beginning of each session, the animal was wrapped in a towel and placed in a dimly illuminated cage. If older than 12 days, the kitten was partially restrained at the time of registration. This was effected by attaching a stud, which had been embedded in the cement of the implant at the time of surgery, to a bar fixed to the recording cage, thereby reducing head mo:-emerit. As judged from the latency to the onset of sleep of the restrained animals, this technique did not disturb sleep. It provided excellent stability for the recording of single cells and made it possible to “hold” a single unit for more than an hour, even in several animals younger than 3 weeks of age. In the older kittens, whose skulls are ossified, the recording of single cells was frequently extended for as long as 2 hr. The EEG, EOG, EMG, and the output of a Schmitt trigger wzre monitored on a Grass Model 6 electroencephalograph. The unit activity as well as the other parameters were also recorded directly onto tape for subsequent computer analysis. Each cell that was encountered was tested for a specific short latency response to photic stimulation. If this criterion was met, the unit was recorded during one or more sleep-wake cycles. After l-3 postoperative weeks the recording sites were marked with micro-
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lesions, and the brains were perfused through the carotid artery with 4% formaldehyde for the purpose of histological verification of the microelectrode tract. Co~zpztter Analysis. Data were analyzed only from lateral geniculate cells that gave satisfactory signal to noise ratios and showed no signs of injury discharge. The polygraphic record of the other electrophysiological parameters was carefully sampled so that a recording from each cell was available from an incontrovertible segment of the awake state, quiet sleep or slow wave sleep, and active or paradoxical sleep. The polygraphic and behavioral criteria for designation of these states have been described (3). The spontaneous unit activity of each neuron that was selected for analysis was fed to a DCA 160 computer for separate examination of the mean frequency and interspike interval distribution from each state. Modifications of the discharge pattern in the different states of vigilance were followed by means of these parameters during the first month of postnatal life. No analyses were performed on unit discharge recordings from the mixed stage of transitional sleep (3). RESULTS Data were obtained from l-5 kittens ranging from 2 days to 5 weeks of age. Table 1 summarizes the collection of data among the 15 animals.
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Because of the effect of skull growth on electrode placement, no single animal contributed data over the entire age span. Rather, individual animals provided small segments of data within the approximately 5-week period of study. A total of 150 lateral geniculate units were recorded. Analysis was performed on the 60 cells that were held through one or more sleep-wake cycles and at least two of the three states. The mean recording time for a cell was 16.25 min. Evolution of the Mean Frequency with Age. As mentioned above, the mean frequency of unit discharge was calculated from the most clearly defined samples of recording from each state. The durations of the segments subjected to analysis ranged from l-10 min, with mean intervals of 3 min in the awake state, 3 min in quiet sleep and slow wave sleep and 4.5 min in the awake state and paradoxical sleep. In terms of state relationships, the frequency of discharge generally increased with age in the awake state (Y = 0.71, p < 0.005) as well in paradoxical sleep (r = 0.85, p < 0.005). On the contrary, mean discharge in slow wave sleep did not significantly increase during the first postnatal month, though a slight trend was visible. (Note that data for quiet sleep are not reported for the first week of life because of the sparseness of quiet sleep until the second week (14). In Fig. 3, the frequency of discharge by week is plotted for each of the 32 units observed through all three states. It can be seen that the “V” shape frequency plot, characteristic of the adult lateral geniculate discharge across the three states (23), appeared clearly by the third postnatal week. As in the adult cat (23), however, some cells did not show this pattern. To summarize these data, photoresponsive cells in the lateral geniculate nucleus showed a significant, almost linear, increase in discharge frequency in the awake state and in paradoxical sleep. No statistically significant change was observed in slow wave sleep during the first postnatal month. However, as early as the third postnatal week, modulation of the discharge rate from one state to another had already assumed the adult pattern. Evolution of the Interspike Interval Distribution with Age. This analysis was performed on both a 4-msec and a 20-msec time base. Two examples are given in Fig. 4. Figure 4A, displays histograms of the interspike interval distribution of a representative cell in each state at 2 weeks of age. At this time, qualitative differences among the three states of vigilance were clearly observable. By 1 month (Fig. 4B), the histograms were qualitatively similar to those of the adult cat (IS). In the awake state, unit discharge was rather regular. The mode of the distribution was at 8-12 msec with an amplitude of 19%. There were only a small number of intervals longer than 1 set duration. In slow wave sleep, the distribution was more unsymmetrical. A peak of
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FIG. 2. Evolution of the mean frequency of unit discharge in the three states during the first month of life. Awake state : triangles ; quiet sleep and slow wave sleep : open circles; awake state and paradoxical sleep: filled circles. Each data symbol represents the mean frequency of all units recorded on a given day. The variation about the mean, though not displayed in these graphs, is not different from what is found in the adult
(22). short, 48 msec, intervals at an amplitude of 28% was combined with a substantial quantity (approximately 10%) of longer intervals. This pattern was very distinctive and is known as the “burst-pause” pattern of discharge. In paradoxical sleep discharge was intermediary between the awake state and slow wave sleep; namely, there were slightly more short, 8- to 1Zmsec intervals than in the awake state (23%)) but no long pauses as in slow wave sleep.
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FIG. 3. Mean frequency of unit discharge for all cells recorded through the awake state, slow wave sleep, quiet and paradoxical sleep during the first 5 weeks of life. In the first week, there is neither enough time spent in quiet nor in the awake state without movement, to provide sufficent data for a graph.
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The results of the interspike interval distribution analyses for all cells are represented in Figs. 5 and 6. Figure 5 portrays the proportion of short intervals in the three states from the first to the fifth week of age. Short
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FIG. 4. Interspike interval distributions for single cells in each of the three states. A. a 14-day-old kitten, and B. a 31-day-old kitten. Time base: 4 msec. The longest interspike interval portrayed in these histograms is 200 msec.
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AGE (days) FIG. 5. Evolution of the proportion of short interspike intervals with age in the three states. Awake state: triangles; quiet sleep and slow wave sleep: open circles; awake state and paradoxical sleep: filled circles. Data are averaged for each day.
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modal intervelr fmsec.) FIG. 6. Evolution of the modes of the interspike interval distributions with age. For each cell in each state, the value of the mode is plotted against its amplitude. Awake state : triangles ; quiet sleep and slow wave sleep : open circles ; awake state and paradoxical sleep : filled circles.
intervals were arbitrarily defined as having less than 20-msec duration because in the very young kitten the units have low firing rates (Fig. 2). In the awake state, the proportion of short intervals showed a progressive rise from the second to the fifth week of age. This phenomenon was principally due to an increase in frequency (Fig. 2), and the distribution of inter-spike intervals, though constricted, persisted in a more symmetrical shape than in slow wave sleep and paradoxical sleep (Fig. 4). In slow wave sleep, an evolution with age toward a greater proportion of short intervals parallelled that observed in the awake state. However, in the case of slow wave sleep, the increase in the ratio of short intervals was Ilot due to an incrrase in frerluencv of clisciiargc (Fig. 2). Rather, it
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derived exclusively from a qualitative change in the pattern of the discharge, specifically a progressive appearance of high-frequency bursts. This is reflected in Fig. 4 which portrays the development of greater polarization in the distribution of interspike intervals in slow wave sleep. In paradoxical sleep the evolution of the discharge pattern was also very similar to that of the awake state. During the first week, however, the proportion of short intervals was higher, averaging about 15%. This proportion of short intervals in the first week was rather high considering the very low frequency of discharge during that period. The combination of the two factors actually defined a unique discharge pattern comprised of groupings (not bursts) of five to ten spikes, separated by periods of silence, often exceeding 10 sec. This is not a “burst-pause” pattern, but rather describes an absence of tonic discharge. A similar lack of tonic discharge in the cortex has also been found at this age (9). Figure 6 demonstrates the change in the mode of the interspike interval distribution of each cell in each state as maturation proceeds : In the awake state, the interspike interval distribution modes of the cells showed a large dispersion in the second week. By the fifth week they localized toward the vertical axis and had a mean amplitude of 30%. These data reveal that through the second week the discharge was quite random (see Fig. 4A, W) . A shift to shorter frequencies took place in the latter 3 weeks. The early random discharge may be explained by insufficient maturation in sensory systems and the consequent relative sensory isolation of the neonatal kitten. Mukhametov (18) has found that in lateral geniculate neurons of the adult cat, interspike interval distributions were more symmetrical during quiet waking than during alert waking. This may indicate that the only waking state of attention available to the young kitten corresponds to the nonattentive awake state of the adult. Our data indicate that a shift toward greater alertness in the awake kitten took place after the second week of life. In slow wave sleep, there was a change with development in the arrangement of the modes progressively from a predominantly horizontal, to a diagonal, and then to a vertical configuration. The 0- to ZO-msec modes had a mean amplitude of 4.5% in slow wave sleep at the fifth week, which was much higher than the corresponding amplitude in the awake state. In paradoxical sleep, alteration of the modal pattern was similar to that in the awake state, all the modes falling in the 0- to ZO-msec range by the fifth week. However, dispersion was less before the third week than in the awake state. The average amplitude of the 0- to ZO-msec modes at the fifth week was 4.5% as in slow wave sleep. In the summary, the interspike interval distributions changed progressively in the awake state, slow wave sleep, and paradoxical sleep toward higher modes in the shorter intervals during the f&t postnatal month.
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However, in light of the stable discharge frequency in slow wave sleep, we can conclude that the proportional increase in short intervals in this state was a function of a qualitative rearrangement of the unit activity into a “burst-pause” organization. The development of this capacity to alternate excitation and inhibition is probably a good indicator of the degree of electrophysiological maturation of the cells of the lateral geniculate nucleus. DISCUSSION The evolution of unit discharge in the lateral geniculate nucleus of the kitten in the first month after birth has been described. It was marked in part by a general increase in mean frequency during the course of maturation. This increase was seen chiefly in the awake state and in paradoxical sleep. Similar findings had been obtained in the cortex by Huttenlecher (9)) though the state of the animal was not systematically taken into consideration in regard to the frequency of data from the cortex. In comparison to unit activity in the cortex, unit activity in the lateral geniculate nucleus may be monitored by the second postnatal day, whereas it does not appear until the fourth day in the cortex. Furthermore, the mean frequency of discharge in the first month of life is higher in the lateral geniculate nucleus at any given point in time, a phenomenon that persists into adulthood (6, 23). At the end of the first month of life, the unit discharge rate in the lateral geniculate nucleus of the kitten is still lower than it is in the adult. However, the organization of unit activity has assumed the adult pattern relatively early, by the end of the second postnatal week. At this time the proportion of short and long interspike intervals approximates the pattern seen in equivalent states of the CNS in the adult (23). The organization, at 2 weeks, of the “burst-pause” pattern of discharge during behavioral quiet sleep may represent the most prominent qualitative step in the maturation of spontaneous unit activity in the lateral geniculate nucleus. It should be noted that at this juncture in the maturation of the nervous system, serotonergic terminals have matured, particularly at the level of the raphC nuclei (15). By this time, too, periods of quiet sleep have lengthened (14). Synchronization of the EEG, and appearance of the monophasic waves in the lateral geniculate nucleus have yet to emerge (3, 14). but will occur shortly. It may be that the specific distribution of unit activity during paradoxical sleep begins to coordinate with the timing of ponto-geniculateoccipital bursts once they appear. This is suggested by data from adult animals (2, 21, 23). The specific neural routes that serve as the condt~its of brainstem stimulation to the lateral geniculate nuclms during sleep, and that inay in part determine the nature of the unit discharge nf k~teral geniculate nucleus cells,
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have not yet been established. Nevertheless, among the probable neuroanatomical loci that bear on the ultimate organization of neuronal discharge in the lateral geniculate nucleus, are maturation of the ultrastructure of the lateral geniculate nucleus as well as of the optic tract terminals and of the ponto-geniculate pathways. We are currently investigating several of these possibilities by attempting to effect modifications in the rate and quality of maturation of the lateral geniculate cells and of the synapses playing upon them. On the one hand, we are dark-rearing animals and, alternatively placing lesions in the pontine regions that seem to be critical for paradoxical sleep and slow wave sleep (11, 12). As we pointed out, our data reveal that state-specific, adult-like unit firing patterns were not organized at the thalamic level until the beginning of the third postnatal week. Furthermore, we have also reported that pontogeniculo-occipital spikes did not appear in the lateral geniculate nucleus during sleep until just about this time (3). However, these findings do not contravene the evidence that the centers in the brain stem subserving paradoxical sleep are functioning at birth. According to the best electrophysiological and behavioral indicators that are available in the immature animal (14), the sleep of the neonatal kitten is comprised virtually entirely of awake state, the precursor of paradoxical state (11). The absence of recordable quiet sleep and slow wave sleep at this time goes along with the finding that the raphe nuclei are not yet biochemically developed (5, 15). In addition, in this laboratory we have been able to record pontogeniculo-occipital activity reflected in the extraocular muscles during awake state as early as the third day of extrauterine life. These muscle spikes have been shown in the adult to represent probably a direct outflow of the spike activity in the pontine reticular formation (17). Accordingly, the polygraphic data, combined with the histochemical demonstration of very early maturation of catecholamine-containing neurons in the pons (5, 15), suggests that though there is a lag in the maturation of distinct electrophysiological signs of paradoxical sleep (ponto-geniculooccipital waves) at the level of the thalamus, the ponto-geniculo-occipitalgenerating apparatus of paradoxical sleep in the pons is in evidence weeks before. In order to better understand the nature and mechanisms of the awake state, as well as the genesis of slow wave sleep in the newborn organism, additional studies must be performed at the pontine level. REFERENCES 1. ADRIEN, J. 1973. Ontogenbe des activites electriques du Noyau chez le chat. Th&e de 3” Cycle, UniversitC de Lyon, France. 2. BIZZI, E. 1966. Discharge pattern of single geniculate neurons eye movements of sleep. J. Nezwophysiol. 29: 1087-1095.
Genicule during
Lateral the
rapid
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3. ROWE-ANDERS, C., H. P. ROFFWARC., and J. ADRIF.N. 1973. Ontogenesis of pontogeniculo-occipital activity in the lateral geniculate nucleus of the kitten. E.rp. Nffitvl., in press. 4. BROOKS, D. C. 1968. Waves associated with eye movemeuts in the awake and sleeping act. Electrocncrpl~nloyr. Clin. Neurophysiol. 24 : 532-541. 5. DAHLSTROM, A., and K. FUXE,. 1964. Evidence for the existence of monoaminecontaining neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta Physiol. Sca?zd. 62: Suppl. 232: l-55. 6. EVARTS, E. V. 1960. Effects of sleep and waking on spontaneous and evoked discharge of single units in the visual cortex. Fed. PYOC. 19:828-837. 7. GARMA, L., and R. VERLEY. 1967. ActivitCs cellulaires corticales etudiees par electrodes implantees chez le lapin nouveau-nP. J. Physiol. Paris. 59: 357-376. 8. HUBEL, D. H. 1959. Single unit activity in striate cortex of unrestrained cats. J. Plzysiol. Lovldow. 147 : 22&238. 9. HUTTENLOCHER, P. R. 1967. Development of cortical neuronal activity in the neonatal cat. Exp. Nezrrol. 17 : 247-262. 10. JEANKEROD, M., and K. SAKAI. 1970. Occipital and geniculate potentials related to eye movements in the unanesthetized cat. Brain Res. 19 : 361-377. 11. JOUVET, M. 1967. Neurophysiology of the states of sleep. Physiol. Rea. 47 : 117-177. 12. JOUVET, M. 1969. Biogenic amines and the states of sleep. Science 1’64: 32-41. 13. JOUVET, D., J. 1,. VALAXT, and M. JOUVET, 1961. fitude polygraphique du sommeil du chaton. C. R. Sot. Biol. 155: 1660-1664. 14. JOUVET-MOUNIER, D., L. ASTIC, and D. LACOTE. 1970. Ontogenesis of the states of sleep in rat, cat, and guinea-pig during the first post-natal month. Develop. Psychobiol. 2 : 216239. 15. LOUP, M., and J. CADILHAC. 1970. Le developpement des neurones ?I monoamines du cetveau chez le chaton. C. R. Sot. Biol. 164: 1582-1587. 16. MARTY, R., and J. SCIIERRER. 1964. CritPres de maturation des systemes afferents corticaux, pp. 222-236. In “Progress In Brain Research,” Vol. 4, D. P. Pu;pura, and J. P. Schade (Eds.). Elsevier, Amsterdam. 17. MICHEL, F., M. JEANKEROD, J. MOURET, A. RECHTSCHAFFEN, and M. JOUVET. 1963. Sur les mecanismes de 1’ activite de pointes au niveau du syst&me visuel au tours de la phase paradoxale du sommeil. C. R. Sot. Biol. 157 : 103-105. 18. MUKHAMETOV, L. M., G. RIZZOLATI, and A. SEITUN. 1970. An analysis of the spontaneous activity of lateral geniculate neurons and optic tract fibers in freemoving cats. Arch. Ital. Biol. 108: 325-347. 19. NOBACK, C. R., and D. P. PURPURA. 1961. Post-natal ontogenesis of neurons in cat neo-cortex. .I. Cowp. Ncwol. 117: 291-307. 20. PURPURA, D. P. 1961. Morphophysiological basis of elementary evoked response pattern in the neo-cortex of the newborn cat. Ann. iv. I’. Acad. Sri. 92 : 604-654. 21. RECUTSCHAFFEN, A., F. MICHEL, and J. METZ. 1972. Relationship between extraocular and PGO activity in the cat. Psyckopll~~vsiol. 9: 128. (abstract) 2.Z. ROSE, G. H., and D. B. LINDSLEY. 196s. Development of visually evoked potentials in kittens : specific and non-specific responses. J. NrzrropAysiol. 31: 607623. 23. SAKAKURA, H. 1968. Spontaneous and evoked unitary activities of cat lateral geniculate neurons in sleep and wakefulness. Jap. J. Pkysiol. 18: 23-42. 24. SCHERRER, J., and D. OECOIYOMOS. 1954. Responses corticales somethesiques du mammifere nouveau& comparPes g celles de l’adulte. Btxd. Neo-Natales. 3: 199-216.