Ontogenesis of ponto-geniculo-occipital activity in the lateral geniculate nucleus of the kitten

EXPERIMENTAL

NEUROLOGY

43, 242-260

(1974)

Ontogenesis of Ponto-Geniculo-Occipital Activity in the Lateral Geniculate Nucleus of the Kitten CONSTANCE

BOWE-ANDERS,

Departwnt

JOELLE ADRIEN,

of Psychiatry, Bronx, Received

AND HOWARD

Montejiore Hospital New York 10467 December

P. ROFFWARG

l

and Medical Ceflter,

27,1973

In the adult cat, just before the onset of and throughout paradoxical sleep, sharp, monophasic waves appear in the pontine reticular formation, and are transmitted to the lateral geniculate nucleus and occipital cortex. This report describes a study of the appearance and maturation of these ponto-geniculooccipital waves, or PGO “spikes,” in the lateral geniculate nucleus of the developing kitten. Thirty-nine implantations of the lateral geniculate nucleus were carried out in 34 kittens (ages 8-75 days). Electrodes were also placed for the recording of electrocorticograms, neck muscle activity, and eye movements. Despite the high proportion of paradoxical sleep, and the copious amount of peripheral phasic activity in the neonatal kitten, no PGO activity was found prior to Day 15. The average age of emergence of the activity was Day 21. The PGO waves, at initial appearance, were low in frequency as well as amplitude. However, during a 3- to 6-day sequence, the spikes increased in both frequency and amplitude, and an association developed progressively with the increasingly well-defined periods of paradoxical sleep. Adult PGO spike frequencies were reached by Day 35, though the temporal characteristics of their discharge continued to change for several weeks thereafter. The 3-week postbirth latency to the first appearance of the PGO spikes, as well as the ensuing rise in their recorded frequency and amplitude, seem to be basic characteristics of PGO development. Further, the ontogenesis of spontaneous PGO activity in the lateral geniculate nucleus of the kitten during paradoxical sleep parallels the pace of maturation of the sleep-wake pattern and of the neuroanatomical and neurochemical pathways that function in the mediation of the stages of sleep. 1 This investigation was supported in part by Project Grant MH-I3269 and by Career Research Scientist Award MH-18739 (H.P.R.) from the National Institute of Mental Health. A fellowship from the French Government (Service des fichanges Gulturels, Scientifiques et Techniques) supported Joelle Adrien. We acknowledge the help of Dr. Ruth Kaslow in the breeding and care of our animals, and Marie-Louise Elbert who prepared the histological sections. C. Bowe-Anders’ present address (for reprint requests) is: 114 Morris Avenue, Buffalo, New York 14214. 242 Copyright 0 All

rights

1974 by of reprodurtio?

Academic n1 my

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INTRODUCTION Sharp, monophasic waves, called ponto-geniculo-occipital (PGO) waves, have been described in the pons, lateral geniculate nucleus, and occipital cortex during paradoxical sleep in the adult cat (5, 6, 20, 27, 28). These PGO “spikes” constitute a major source of nonretinal input to the lateral geniculate nucleus from the pontine reticular formation (3, 25) and are associated in the fully mature animal with phasic somatic phenomena of paradoxical sleep, particularly rapid eye movements. PGO spikes are generally believed to be a manifestation of the activity of the fundamental triggering mechanism of paradoxical sleep. It has been suggested that the frequency of the PC0 activity is an index of the “intensity” of that state (13). Our primary purpose in this study was to establish the timing of appearance and the quantitative characteristics of PGO activity observed in the lateral geniculate nucleus of the developing kitten. We wished to describe the neurophysiological attributes of the PGO waves, and their relationship to the sleep-wake cycle during the course of the first several weeks of life. The PC0 waves recorded from the lateral geniculate in the adult cat have a duration of 70-100 msec and an amplitude of 100-300 pv (400 pv with an optimal placement of the recording electrode). The spikes occur at a fairly predictable rate during paradoxical sleep (60 +/min) and during a given 24-hr period (14,000 -+30OO/day) (5, 19). The amount of PGO activity and its association with episodes of paradoxical sleep may be altered by the deprivation as well as the recovery of paradoxical sleep, and by persistent exposure to drugs such as reserpine and p-chloro-phenylalanine (5, 11, 13, 17). A s a result of administration of p-chloro-phenylalanine, spiking loses its periodic distribution and appears continually without respect to state. Surgical lesions also alter the occurrence of PGO activity. Destruction of pontine catecholaminergic neurons in the nucleus locus coeruleus (IS), or of serotonergic neurons in the raph6 nuclei (19, 26). drastically alters the frequency and distribution of PGO spikes. Section of the optic nerves bi-laterally also modifies the appearance and duration of PGO activity in paradoxical sleep (4). Earlier work has demonstrated that, immediately following birth, the kitten shows an extremely high proportion of paradoxical sleep, approaching 70-80 % of total recording time (21, 22). Peripheral indicators of paradoxical sleep, such as rapid eye movement, body and facial twitches, appear to be more intense in the newborn kitten than in the adult cat (7, 8, 21, 22). However, within the first month of life, the polygraphic characteristics of the sleep states gradually assume the typical adult forms. Accordingly, study of the kitten affords an opportunity to examine development of PGO activity in relation to the sleep electrocorticogram (EEG)

244

BOWE-ANDERS,

ADRIEN

AND

ROFFWARG

and to motor behavior before, during, and after the time when variations in these parameters are sufficiently congruent to define changes in “state.” METHOD Electrodes were implanted in 34 kittens, ranging in age from 8 to 75 days, that were anesthetized with pentobarbital supplemented by ether when necessary. Electrodes were later reimplanted in the opposite lateral geniculate nucleus in five kittens resulting in a total of 39 implantations. For the EEG, the electrodes consisted of two or three stainless-steel jeweler’s screws placed over the parietal and frontal cortex, as well as a transcortical placement over the occipital cortex ; for the electromyogram (EMG), no. 36 Teflon-coated nichrome wires inserted into the nuchal muscles, and for the electrooculogram (EOG), two or three solder balls inserted subcutaneously about the eyes. In addition, tripolar electrodes of no. 30 nichrome wire were implanted stereotaxically in the lateral geniculate nucleus according to coordinates developed in our laboratory (2). Placement of electrodes into the lateral geniculate nucleus was confirmed during the operation by means of photic stimulation of the contralateral eye, Each electrode was individually cemented to the skull, attached to flexible wire, and connected to a section of Amphenol stripping at the base of the neck. This procedure permitted some growth of the skull without the damage that might result from pull between a fixed plug and the individual electrode placement. After the operation, which lasted 2-3 hours, the animal was returned to its litter. The mother cats, permanent members of our cat colony, readily accepted their kittens. Care was taken to bandage the head region with a gauze cap to avoid tangling or pulling of the electrode wires. Behavioral observations and weight gain failed to distinguish experimental animals from their unoperated littermates after the first postoperative day. After 24 hr of recovery, experimental animals were taken from their litter after a suckling (nursing) period, and recordings were made for 60120 min on a Grass Model 6 electroencephalograph. Photic stimulation was periodically delivered during the recording days to assess any changes in the evoked response which might indicate movement of the lateral geniculate nucleus electrodes. At the conclusion of the sequence of recording days (usually 4-8 days in the younger kittens and somewhat longer in the older ones) the animals were perfused with 4% formaldehyde for histological examination and verification of electrode positions. Each recording included EMG, EOG, and EEG (frontal or parietal lead referred to occipital) tracings as well as several lateral geniculate nucleus derivations. We scored the sleep state by 30-set epochs. The state occupy-

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ing the greatest proportion of the 30 set determined the score for that epoch. For the definition of sleep states, we adopted the criteria of JouvetMounier (21, 22). In the younger kittens, paradoxical sleep and quiet sleep were distinguished; in kittens older than 3 weeks, paradoxical sleep and slow wave sleep were designated. The sleep stages were identified in reference to the status of several physiological parameters : paradoxical sleep by neck muscle atonia, presence of rapid eye movements, body twitches, and a relatively low voltage, fast EEG; quiet sleep by the presence of muscle tone, absenceof eye movements, body twitches, and an EEG similar to that in paradoxical sleep. The EEG criteria for quiet sleepwere difficult to score, particularly before the third week. Sometimes, the EEG displayed a mixed pattern, i.e., low voltage, fast EEG activity interspersed with slow waves, but frequently in the younger animals the EEG during quiet sleep closely resembled the pattern in paradoxical sleep. However, with the appearance in quiet sleep of clear slow waves at the end of the third week, this state could be scored as the slow wave sleep pattern of the adult. We found it necessary to add to the paradoxical sleep and quiet sleepslow wave sleep states of Jouvet-Mounier a transitional state that was applied to certain epochs, often during shifts in state, when the parameters for distinguishing the sleep stages were not uniform for identification of either the stage of paradoxical or quiet (slow wave) sleep. Such an interval, for example, might be observed at the end of a period of slow wave sleep when muscle tone had diminished, a low-voltage, fast EEG had replaced slow waves, but eye movements had not yet appeared. In younger kittens, transitional sleep was often scored at the onset of sleep, before the initial paradoxical sleepperiod. In this study, the identification and cumulative counting of monophasic waves in the lateral geniculate nucleus determined the nature of the critical dependent variable. Accordingly, it was necessary to establish rigorous minimal requirements for PGO waves. We defined them as repetitive wave forms of short duration (about 100 msec) having a higher amplitude than background activity (over 20 ,UVuntil 35 days of age and over 40 pv thereafter). Spike frequency per 30-set epoch was calculated for each state. We anticipateed difficulty in discriminating PGO activity in the kitten from the brief episodes of EMG artifact caused by the numerous body twitches during paradoxical sleep. However, even during bursts of EMG artifact, we were generally able to differentiate spikes in the lateral geniculate nucleus by virtue of their distinctive frequency, duration, and wave form. Scrutiny of unquestionable EMG artifact on the polygraphic tracing, at the time of directly observed body movements, left little doubt of the reliability of the distinction between PGO and EMG discharge.

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ADRIEN

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Nevertheless, because the possibility remained that spikes and movement artifact might be confused on the channels used for counting the lateral geniculate nucleus spikes, the spike tallies were interrupted when EMG artifact appeared on any of the channels devoted to the cortical and subcortical derivations. Once we had established a familiarity with the characteristics of the lateral geniculate nucleus spikes in the 3- to 4-week-old kittens, we were more sensitive to the earliest representations of PGO activity in the first tracings. The prior recordings of these animals were then reviewed in search of the initial emergence of the spike potentials. RESULTS In the 34 kittens employed in this study, a total of 39 lateral geniculate nucleus implantations were successfully carried out. The results of the investigation are summarized in Table 1. The chief findings were : (i) PGO waves were not evidenced in the lateral geniculate nucleus prior to the fifteenth postnatal day in any kitten. (ii) The mean age of first spike appearance in the lateral geniculate nucleus fell between Days 18 and 23, the middle of the third to the beginning of the fourth week of life. (iii) A period of “maturation” of the PGO activity in the lateral geniculate nucleus, characterized by an increase in frequency and amplitude over a 3- to 6-day interval, typically followed the initial onset of the PGO waves. Spiking was recorded from 26 of the 39 electrodes. In 23 of the 26 cases, the electrodes were documented to be in the lateral geniculate nucleus (Table 1, Group A). In three others (Table 1, Group C) , the location in the lateral geniculate nucleus was presumptive. Of the 13 implantations that failed to exhibit spiking, four were histologically verified within the lateral geniculate nucleus (Table 1, Group A), and four were shown to be misplaced (Table 1, Group B) ; five others remained questionable in terms of placement because of the lack of histological verification (Table 1, Group C). In the four failures, in which spiking was not observed despite histological proof that the electrodes were in the lateral geniculate nucleus (Table 1, Group A), three may be accounted for, in retrospect, on the basis of inadequate maturation (presumably in the lateral geniculate nucleus) at the time of recording; that is, for technical reasons (see below, footnote) registration was restricted to a limited number of days and terminated before PGO spiking had emerged at the recording site. The oldest of these three animals was operated upon on Day 20 (Table 1, Group A), and was monitored for 5 days. By means of a reimplantation into the opposite lateral geniculate nucleus in this animal on Day 26, spikes were demonstrated to make their first appearance on Day 28. The fourth animal

PONTO-GENICULO-OCCII'ITAL TABLE AGE

Group

A

Day of implant

1

AT\VHICH ELECTKODES WEKE IMPLANTEI~ANDATLVHICH PGO SPIKESAPPEARED~ Electrodes

in LGN

Day of first appearance of PGO spikes

Group

B

Electrodes

Day of implant

8 11

247

ACTIVITY

spikes 1.5” 23”,c 176 19c

1

2()“,’ 248 No spikes 286 22 22 22 24b 30 24b 23 24 25

1.5 20 20 23

Group

1

1

not in LGN

Day of first appearance of PGO spikes No No No No

spikes spikes spikes spikes

C Electrode placement not established”

Day of implant

8 10 20 14 16 17 22 28

Day of first appearance of PGO spikes No spikes INO spikes No spikes 1W No spikes No spikes 23 29

1

1

CLThe initial recording day was always the day after implantation. Groups are categorized according to the placement of LGN electrodes. [ ] Kitten implanted a second time on the opposite side. b Cohort of animals in which the onset of PGO spiking in the LGN was preceded by a recording period of 1 or more days in which no spikes were monitored. c Cohort of animals implanted on or before Day 18 all of which subsequently developed PGO activity. d Eight animals implanted between Day 24 and 75, all of which demonstrated LGN spikes on the first postoperative day. e Lack of certainty about electrode placement (Group C) resulted from either failure to locate the electrode tract on histological examination, or to a decision not to autopsy an animal and to continue observation.

in this group (Day 15, reimplant) was recorded into the fourth week without the emergence of spikes. Accordingly, this cat is the only one in our series of confirmed lateral geniculate placements that never exhibited PGO

248

BOWE-ANDERS,

ADRIEN

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activity, though undoubtedly monitored well into the period of expectation of PGO activity (see Group A, after Day 21). Lower Age Limit of PGO Spike Dewelopnzent in Lateral Geniculate Nucleus. Electrode implantations performed during approximately the second week of life (Day 8 through 15) resulted in a considerable incidence of morbidity and occasional death owing to the risks of anesthesia and surgery in this period. A successful procedure additionally had to overcome the difficulty of adequate fixation of the electrodes to a soft and porous calvarium. Consequently, our data in the second week are limited. Nevertheless, the findings are entirely consistent with the findings in the animals with later implantations, and suggest strongly that PGO spiking does not appear in the lateral geniculate nucleus much before Day 15. This conclusion is based on observations of several key animals with lateral geniculate nucleus-verified electrodes. In this group, the two earliest implants (Day 8 and 11) had not developed PGO spiking by Days 14 and 15, respectively, when lesions were made at the electrode sites.’ Two additional animals had electrodes placed on Days 14 and 15 but did not develop the first signs of PGO activity until Days 17 and 23. On the other hand, the possibility of spiking in the lateral geniculate nucleus preceding Day 15 is suggested by two other animals in which electrodes were implanted on Days 14 and 16. Inasmuch as spikes were found at initial recording on the first postoperative day in these two, it is possible that PGO activity was extant sooner. In summary, the findings point to Day 15 as the lower limit of the maturational period in which PGO activity becomes manifest in the lateral geniculate nucleus. However, our data are by no means definitive. The possibility cannot be excluded that if a large group of animals were available for monitoring from the ‘beginning of the second week, PGO waves might have been demonstrated in some at a slightly younger age. Mean Age of Development of PGO Spikes. In the course of the study, the impression developed that consistent and rapid PGO spike manifestation was almost nil in the second week, intermediary in the third week, and nearly complete after the start of the fourth week. Almost half of the electrode implantations attempted in this study were carried out after the commencement of the fourth week with the aim of accumulating data on age and frequency relationships, changes in spike distribution, and the *A decision to wait longer before making lesions at the implantation positions in these two animals,in order to discoverwhether spikes may have developed later, would have run the possibility that the original electrode positions, which were correlatedto the absenceof PGO spikes at Days 14 and 15, might shift position due to brain and skull growth. The fact that the electrodes were confirmed to be in the lateral geniculate nucleus on Days 14 and 15, provided critical, albeit negative, information concerning the factor of age relative to lateral geniculate nucleus spiking.

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interaction of spiking and stage of sleep. The wide variation in age of implantation made it possible to examine in only a small group of animals the important issue of when, in the development of the kitten, PGO spike activity usually becomes observable at the thalamic level. Moreover, it was apparent from the results in the young animals that the onset of PGO spiking in the lateral geniculate nucleus was variable, and that no one day in the course of development could be specified for the emergence of spikes in all animals. Accordingly, for the purpose of generating a sound approximation in terms of the question of the typical time of first appearance of lateral geniculate nucleus spiking, several approaches were taken to the data, We were able to select two groups of animals from which to calculate mean times of PGO onset, recognizing the limitations imposed upon the comparability of the data owing to the small size of the groups and the differences in their average age of implantation. The first group was composed of seven animals (Table 1, footnote b) which demonstrated PGO spikes 2 or more days after implantation, Because at least 1 day of registration after implantation was devoid of evidence of PGO activity, it is likely that in these kittens we had the opportunity to distinguish the precise day of development of spiking in the lateral geniculate nucleus. The mean day of onset was Day 23 (range 17-28). However, there is some indication that this mean is skewed toward a late onset. In Fig. 1, which displays PC0 spike frequency in paradoxical sleep in relation to age in the 14 kittens showing the largest signal (spike amplitude) to noise (background amplitude) ratio, an index of optimal electrode placement in the lateral geniculate nucleus, a rapid surge of PGO frequency may be noted to occur in many instances during the early and middle portion of the third week. This indicates, of course, in addition, that spiking had commenced even earlier. In view of the data contained in Fig. 1, we utilized a second group of kittens to acquire another data base relative to the mean onset of PGO activity. A cohort was selected of our youngest animals at the time of electrode placement, those six animals in which electrodes had been implanted on or before Day 18, provided that they ultimately developed lateral geniculate nucleus spiking (Table 1, footnote c). The mean age of PGO spike onset in this grouping was Day 18.5 (range 15-23). However, because three of the six animals exhibited spiking on the very first day of monitoring, it is again reasonable to assume that they actually may have developed spiking earlier, and that the true mean day of spike onset for this cohort might be slightly earlier. Also, three of these six animals were part of the first subset in which the day of emergence of spiking was

250

BOWE-ANDERS,

0 1;//

15

ADRIEN

I 25

AND

I 35

ROFFWARG

’

I 55

I 65

I 75

Age in Days FIG. I. Evolution of the frequency of PGO activity in the Iateraf geniculate nucleus in 14 kittens over several days of recording. Each dot represents the mean of all frequencies recorded in paradoxical sleep (P,S) on that day in each animal Note that there is a phase of rapid increase in frequency following the initial observation of PGO waves, and a leveling off of frequency values around Day 35. The animals that were selected for this illustration showed the best discrimination of spike amplitude to background in their LGN tracings.

ascertainable. The mean day of PGO onset for the net combined group of nine kittens was Day 21. Taking these several approachestogether, we conclude that PGO spiking in the kitten generally makes its initial appearance in the lateral geniculate nucleus between the middle of the third week and the beginning of the fourth week after birth. As previously pointed out, however, PGO spiking was observed in our population as early as Day 15. A further consideration is whether some of this variability derived from maturational differences among the animals. We examined this issue by studying a group of four animals from the samelitter. The onset of PGO activity varied from Day 19 to Day 24 in these kittens. They were com-

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pared in terms of birth weight, subsequent weight gains, age of eye opening, observable behavioral differences, and sleep parameters. No simple correlations with age of spike onset could be identified. Maturation of the PGO Pattern and Relationship to Paradoxical Sleep. The PGO activity recorded in the lateral geniculate nucleus, when it was observed at or close to its commencement, irrespective of the day of

initiation,

showed a characteristic developmental evolution in terms of

frequency and amplitude over the course of the 3- to 6-day period after spike onset. Figures 2 and 3 illustrate this maturational pattern. Figure 2 shows tracings from a kitten in which electrodes were implanted on Day 21 and recorded from on Days 22,124, and 27. On Day 22 (A, top) the record exemplifies the difficulty in identifying PGO activity in the young animals. Within a 5-day period (sections B and C), however, clear PGO waves were evident and increased in amplitude to 50-100 pv. Figure 3 illustrates

FIG. 2. Ontogenesis of PGO activity in the lateral geniculate nucleus during paradoxical sleep in the fourth week of life. Recordings taken over a 5day period illustrate that in this kitten spiking is difficult to identify on Day 22 (section A), clearly present on Day 24 (section B), and increased in frequency and amplitude on Day 27 (section C). Note on Day 22 two artifactual waves in the lateral geniculate ruckus tracing half-way through the epoch. Calibration : 50 pv ; 2 sec.

40 cO”

oco--.‘i

. . ...”

. .. . h

0

0

0 0

oo” 0 00

O

0

00

.

O0

00

” 0

0

O

0 . ..““.“W.“.-m.ow

FIG. 3. Development of the association of PGO activity with paradoxical sleep (same kitten as geniculate nucleus spikes per 30-set epoch are plotted at three different ages for the individual sleep sitional sleep, 0 = paradoxical sleep. No relationship of spike frequency to sleep state is observed frequency is evident during paradoxical sleep on Day 24 (section B), and continues to increase to of spiking in quiet sleep follows a reverse trend in this kitten.

501

0 0

00

ooo”0

0

n

0

0 Oo

Day 27

0

c

oo”

0

0

A

..“WL

in Fig. 2). Frequencies of lateral states : l = quiet sleep, n = tranon Day 22 (section A) ; increased Day 27 (section C). The frequency

o”

o%OO

6

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the corresponding sequence of changes in the frequency of spiking in the same animal. Furthermore, it demonstrates the inchoate contingency observetl IJetween sllil
19

A

302826242220-

Day

24

30

I

31

19

20

19

20

IO8-

24

r-

20, I IO

51

I

23

26 t

t 6

Day

22

d 24

27

22

Recording

Frc. 4. State-related changes in mean frequency of PGO activity. Section A (left) shows the group mean rates of lateral geniculate nucleus spiking per 30-set epoch for a group of five kittens on their initial and final recording days. A developmental increase in spike activity occurs clearly in paradoxical sleep. Section B (right) illustrates the presence of this trend in the individual data when four of the kittens, each varying in initial recording day, were subjected to the same analysis. (QS = quiet sleep, TS = transitional sleep and PS = paradoxical sleep.)

254

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frequency in five kittens, all of which had serial recordings spanning a period of 4-8 days. The mean spike frequency for the three sleep states was calculated for each of the animals. Figure 4A compares the mean activity of the group in the initial recordings to that in the final recordings. Whereas in quiet sleep, frequency remained fairly constant, in transitional sleep it increased slightly, and in paradoxical sleep increased 3-fold. In Figure 4B, the increases in spike frequency in paradoxical sleep in the individual animals are roughly equivalent despite variation in terms of the day of PGO commencement, indicating a similar pace of the individual maturational courses. Lateral geniculate spiking in the early stages of development did not appear to be highly coordinated in time with the discharges of peripheral phasic phenomena such as eye movements and muscle twitches. By 1 month of age, however, the PGO activity recorded at the lateral geniculate nucleus seemed to approach the adult level of association with eye movements. A sample of representative PGO recordings obtained at various ages in the kittens is presented in Fig. 5. Increases in amplitude followed closely upon the initial emergence of PGO activity. Although the plot of spike frequencies during paradoxical sleep, shown in Fig. 1, reveals a tendency for mean frequency to plateau at about Day 35, there were continuing alterations in the spike distribution, eventually producing the cluster pattern of discharge observed in the adult (Fig. 5, compare Day 42 and Day SO). Alternative Explanations for ‘Naturation” of PGO Activity. We were concerned that some factor other than physiological development may be responsible for the absence of PGO spikes before Day 15, and for the sequence of enhancement in frequency and amplitude that followed their initial appearance in the lateral geniculate nucleus. One possible alternative cause of such phenomena might be an electrode that originally was placed relatively distant from the source of spiking, and that later moved to a more optimal position due to growth of the brain. We are inclined to reject on two counts such a phenomenon as the likely mechanism responsible for our findings. First, a latency period, between birth and the onset of spiking in the lateral geniculate nucleus, followed by a sequence of PGO “maturation,” was observed in numerous animals in our study. It would be unreasonable to suppose that a process, such as electrode displacement into the lateral geniculate nucleus, was more than an infrequent possibility. Moreover, changes in the spike pattern almost always occurred in the direction of increased amplitude and frequency. If electrode movement were the critical variable, it should lead to the expectation that a comparable number of electrode displacements out of the lateral geniculate nucleus would be observed, leading to a decrease in amplitude and frequency. This was never found in all the kittens we

PONTO-GENICULO-OCCIPITAL

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2.55

A

B

C

FIG. 5. Maturation of lateral geniculate nucleus spike activity during paradoxical sleep with age. Recordings are taken from kittens at different ages illustrating the gradual development of PGO activity from the end of the third week (section A) through the fifth week (section B), and reaching full maturation by the third month (section C). The different direction of the spikes in the 80-day-old kitten is due simply to a reversal of pen polarity in that recording. Calibration : 50 pv ; 2 sec.

followed, some for many weeks. A second argument against an electrode movement hypothesis is that, in the face of progressive maturation of the PGO spike pattern, no gross changes were found in the evoked response to light. Another conceivable etiology of a spurious developmental progression from immature to mature spiking might be a process of “recovery” of spike activity, after a temporary cessation resulting from the physiological trauma of implantation. Such a “recovery” phenomenon would be suggested if a general delay in spiking had been observed for several days after each implantation, or, if when spikes appeared, they always augmented in frequency and amplitude from “immature” levels. However, in the case of 19 animals, spiking was exhibited on the very first recording opportunity, 1 day after the implant. Moreover, in a substantial number of these animals, this spiking was of the mature type on the first day of recording. Figure 6

256

BOWE-ANDERS,

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ROFFWARC

LGN ; FIG. 6. PGO activity in the same kitten recorded from two lateral geniculate nucleus implantations, each one at a different age. Adjusting for calibration changes, it may be seen that the PGO spikes on Day 24 (section A) are less frequent and lower in amplitude than those occurring on Day 30 (section B). Calibration: 50 pv; 2 sec.

displays tracings taken from a kitten in which electrodes were implanted in two occasions (Days 23 and 29) in opposite lateral geniculate nuclei. The recordings, both taken 1 day postoperatively on Days 24 and 30, showed relatively mature spike activity. There were slight differences in amplitude, Day 30 somewhat increased over Day 24. The full tracings revealed distinct differences in frequency of spiking during paradoxical sleep. The frequency on Day 24 was 14 spikes per 30 set and on Day 30 it was 28 spikes per 30 sec. It appears that the critical variable in determining frequency and amplitude is the time from birth, and not the interval after implantation. Accordingly, a “recovery” hypothesis was not supported. DISCUSSION It was not an unreasonable expectation, in view of the pervasive nature of paradoxical sleep in the newborn kitten and the intensity of peripherally observed and recorded phasic activity, that we would find in neonatal kittens a high, possibly higher than in the adult, frequency of PGO activity in the lateral geniculate nucleus. The fact is that in no instance were PGO spikes recordable at that site before the beginning of the third week after birth. That a 2- to 3-week latency to emergence of the characteristic PGO wave form in the lateral geniculate nucleus describesthe normative developmental state of affairs is supported by the finding that a gradual maturation of the PC0 activity is observed during a 3- to 6-day sequenceimmediately after its initial appearance. Moreover, it is now clear that the frequent

PONTO-GENICULO-OCCIPITAL

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episodes of peripheral phasic activity, which are observed in the neonatal period, occur in the absence of PGO spiking at the thalamic level. These data do not rule out the possibility that PGO activity is present, though difficult to record, in very localized regions of the lateral geniculate nucleus before the time that the techniques available to us are able to demonstrate it. It should also be cautioned that the failure of PGO activity per se to appear above the brain stem before the third week of life does not mean that neuronal activity in the pontine triggering areas for paradoxical sleep is not, in some form, being transmitted to neurons in the thalamus and even in the cortex, though perhaps not in the pattern requisite to the formation of PGO waves. The findings from another study in this laboratory (l), that unit activity in the lateral geniculate nucleus does not begin to take on adult frequency and distribution characteristics until the end of the second week of life, render it unlikely that characteristic PGO waves will be found in the lateral geniculate nucleus much before this time. Moreover, the absence of characteristic PGO activity in paradoxical sleep at the level of the lateral geniculate nucleus during the first 2-3 weeks should not be interpreted as signifying that PGO activity is also absent in the brain stem. We have provisional evidence from studies of the extraocular muscles in very young kittens that PGO spikes in the lateral rectus muscle, which probably reflect pontine activity (32)) are already recordable as early as the third day after birth (2). The absence of PGO waves in the lateral geniculate nucleus at this time simply indicates a dissociation, or, rather, an as yet incomplete link between the pontine activity (and its correlated phasic behavioral activity) and the visual relay in the thalamus. The development of PGO activity in the lateral geniculate nucleus at about the third week of life is part of a series of other electrophysiological changes that, by the fourth and fifth week, brings the appearance of the sleep polygram close to that of the adult, though complete maturation of the temporal characteristics of sleep-wake and sleep stage periodicities is not finally complete until the sixth month (9). A number of these changes have been observed by other workers. In regard to sleep parameters and behavior, it was noted by Jouvet-Mounier (21, 22), and demonstrated again in our study, that slow wave activity begins to appear in the EEG during quiet sleep at about Day 21 and, in the next several days, quiet sleep gradually organizes into typical periods of slow wave sleep. At about the same time the amount of paradoxical sleep and the intensity of the peripheral phasic phenomena diminish. Correspondingly, the proportion of time spent in the awake state rapidly increases, and play and exploratory behavior appear. These alterations in the sleep-wake cycle, most specifically

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the reduction in the amount of paradoxical sleep, are generally correlated with the appearance of PGO spikes in the lateral geniculate nucleus. Other investigators have studied the importance of the first weeks of life relative to development of the CNS, particularly in the visual system. The fourth and fifth week have been described by Hubel and Wiesel (16) as a “critical period” for the development of binocular vision in the kitten. The effects of short periods of monocular deprivation of vision are the most severe when carried out at this time. Beginning at about the third week of life, firing patterns of neurons of the lateral geniculate nucleus start to show an association to the sleep stages in the kitten that is similar to the state relationships in the adult (1). Norton (30) has reported that maturation of directional selectivity in the superior colliculus takes place at about Day 20. At about this same time, development of evoked responses in the lateral geniculate nucleus and occipital cortex, as well as EEG sensitivity to drug administration, have become manifest (24, 29, 33). Chase has examined brain stem reflexes elicited during the various sleep stages in young kittens (7, 8) and has reported that the monosynaptic reflex response shows no change in amplitude when the animal shifts into paradoxical sleep, whereas it diminishes at the point of this change in the adult. The first signs of diminution of the response begin after the second week. Accordingly, the maturation of a discrete, state-specific, inhibitory system seems to parallel the appearance of PGO activity in the lateral geniculate nucleus. PGO activity has been demonstrated to ascend from the pons to the lateral geniculate nucleus in the cat, traveling over a still undefined pathway in the tegmentum (3, 15, 20, 25). The lateral geniculate nucleus contains serotonin (5-hydroxytryptamine) terminals from cell bodies presumably arising in the raphe nuclei (14). It may simply be coincidental that before PGO spikes are observed in the lateral geniculate nucleus, an early rise in serotonin concentration is observed in the pons of the kitten (23). This is followed by an increase in the thalamic concentration beginning on Day 14 (31). P revious work has already contributed evidence that serotonin may play a role in inhibitory neural systems in the lateral geniculate nucleus, functioning possibly as an inhibitory transmitter ( 10, 14, 34). Pertinent to these data are the suggestions by Dement (12) that serotonin, which has an increased turnover rate during paradoxical sleep, may function as a “drive inhibitor,” that it functions to contain PGO activity and thereby aids in confining this phasic brain stem activation to the periods of paradoxical sleep. Our data indirectly support this view inasmuch as the appearance of spikes in the thalamus generally coordinates with a restriction of the amount of paradoxical sleep. It was beyond the scope of this investigation to study the time of onset of PGO activity in each kitten in relation to the point at which reduction and consolidation of paradoxical

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sleep begin. Investigation of the closeness of the link between these two phenomena may shed light on the relationship of phasic and tonic systems in paradoxical sleep. Final assessment of the functional significance of PGO activity in the adult animal must await full understanding of its early developmental functions and of the mechanismsinvolved in its integration. The constellation of electrophysiological and behavioral changes, noted above, which occur during the third and fourth week, do not lend themselves as yet to clear assignment in terms of cause and effect vis A vis PGO spiking. Additional studies of the precise time, pattern, and intensity of the earlier appearance of PGO activity in the pons, and the timing of its eventual appearance at the cortical level are being undertaken. Further studies should attempt also to alter the developmental time course of PGO waves by means of manipulation of the neurophysiological and neurochemical factors on which they depend. REFERENCES J., and H. ROFFWARG. 1974. The development of unit nctivity in the lateral geniculate nucleus of the kitten. Ex/J. Nc~vol. 43 : 261-275. ADRIEN, J. 1973. Ontogeneses des activites electriques du Noyau Geniculate Lateral chez le chat. These de 3” Cycle. UniversitC de Lyon, France. BIZZI, E., and D. C. BROOKS. 1963. Functional connections between pontine reticular formation and lateral geniculate nucleus during deep sleep, Arch. Ital. Biol. 101: 666-680. RROOKS, D. C. 1967. Effect of bilateral optic nerve section on visual system monophasic wave activity in the cat. Elcctroencrphalogr. Clis. Nezcrophysiol, 23: 134-141. BROOKS, D. C. 1967. Localization of lateral geniculate nucleus monophasic waves associated with paradoxical sleep in the cat. Elcctroencephalogr. Clin. Newo-

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