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J 33.. ...£,\.a‘..v@Ssll.vtr: " LIBRARY ' Michigan State is- University A. ‘19“: .‘J 35 .u-s This is to certify that the thesis entitled The Developmental Significance of Low VOltage, Fast wave Sleep for the Stimulus Input Requirements, Regulative Mechanisms, and DevelOpment of the Central Nervous System in a Species of Deermouse, Peromyscus Maniculatus Bairdi presented by Merrill M. Mitler has been accepted towards fulfillment of the requirements for Ph.D. degree mm EM X final- Major professor Date ,OMLaq 1220 C/ { / 0-169 ABSTRACT THE DEVELOPMENTAL SIGNIFICANCE OF _ LOW VOLTAGE, FAST WAVE SLEEP FOR THE STIMULUS INPUT REQUIREMENTS, REGULATIVE MECHANISMS, AND DEVEOPMENT OF THE CENTRAL NERVOUS SYSTEM IN A SPECIES OF DEERMOUSE, PEROMYSCUS MANICULATUS BAIRDI By Merrill M. Mitler Four studies were conducted on various aspects of low voltage, fast wave sleep in a species of deermouse, E. m. M. In Study I electrocorticographic (ECoG) and electromyographic (EMG) records were time-sampled from each of three adult mice (over 53 days of age) when motionless with eyes closed. Sixty intervals of five seconds each.were categorized, by standard qualitative criteria, into three arousal states: Low Voltage, Fast Wave Sleep (LVF), High Vbltage, Slow wave Sleep (HVS), or waking (W). Independently, each electr0physiological index was scored quantitatively either by counting large changes in ECoG potential or by rating intensity of EMG activity. The mean percent of total sleep Judged as LVF was 37.1%. Analyses indicated, in general, that the count and rating patterns were strongly associated with the initial qualitative Judgments of arousal state and, in particular, that low EMG activity was concomitant with LVF. It was hypothesized from these results and from previous studies that LVF could be reduced by curtailing low levels of muscle tonus. Study II tested such a hypothesis with three more adult mice. Sixty S-second ECoG and EMG records were similarly sampled.(when §fs- eyes were closed), categorized, and independently quantified. Each § was perched over a shock-grid on a pedestal too small to permit total loss of muscle tonus. These records showed only HVS and W‘but in ratios slightly altered from those Observed for the first three animals. Comparing both studies suggested that, by preventing low muscle tonus, the pedestal-over-shock-grid can radically reduce the proportion of LVF in the sleep of _13. m. @2511..- Tb construct some estimate of the develOpmental decrease in LVF, Study III repeated the procedures of Study I on Juvenile mice. SubJects were weaned.at 17 days of age and recording began at 20 days of age. Analyses indicated that the mean percentage of total sleep Judged.as LVF for Juveniles was 19.1%. Studies I and III indicated a 25.0% decline in LVF proportions between 20 and 53 days of age. Study IV explored the effects of long-term LVF deprivation on Juvenile and adult mice. Six litters of four animals each were selected at either 20 or 53 days of age. Littermates were randomly assigned to one of four lh-day treatment conditions to fill a 2 x h, age x treatment design with three §s per cell. The LVF-deprivation con- dition involved spending 1% days in the pedestal-over-shock cage described in Study II. The remaining three conditions ran simultaneously with the former and.were controls for the effects of shock arousal, non-specific sleep loss, and isolation, respectively. After treatment, the animals were placed in activity recording cages for 21 days. Dependent variables included.measures of body weight taken before treatment, after treatment, and after the entire procedure, brain weight, brain-to-body weight ratios, various measures of circadian activity, and measures of regularity of activity for the 21 days post- treatment. MaJor findings indicated that animals undergoing LVF- deprivation from 20 to 3h days of age were more active than control animals, while animals deprived.of LVF from S3 to 67 days of age were less active than control animals. There were no differences among treatments in regularity of activity. Adult control animals were more active than Juvenile control animals. Body weight and brain weight differences did not reveal any biological correlates to such activity differences. Results were discussed in terms of input requirements for CNS deveIOpment and/or maintenance. Directions for future research were suggested. THE DEVELOPMENTAL SIGNIFICANCE OF LOW VOLTAGE,iFAST WAVE SLEEP FOR THE STIMULUS INPUT REQUIREMENTS, REGULATIVE MECHANISMS, AND DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM IN A SPECIES OF DEERMOUSE, PEROMYSCUS MANICULATUS BAIRDI By Merrill M Mitler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology I970 ACKNOWLEDGEMENTS I wish to thank the members of my thesis committee: Drs. Martin Balaban, Hiram Fitzgerald, Lauren Harris, and Lester WOlterink for their helpful criticisms of my work. Special thanks are due Dr. Ralph Levine, chairman of my thesis committee, for his advice and encouragement throughout the course of my research. I also wish to thank Dr. thn King for supplying subJects, Dr. James Halters for his advice on polygraph recording technique, Dr. Glenn Hatton for providing surgical equipment, and Earl walker and Mike Botner for their help with reliability checks on the polygraph data. TABLE OF CONTENTS CHAPTER PAGE An Overview .............................. 1 Introduction ............................. l6 Experimentation .......................... 22 Study I: Sleep Analysis ................ 22 Study II: LVF Deprivation .............. 29 Study III: Sleep Analysis in Young 2.13. bairdi ......... 36 Study IV: DeveIOpmental LVF mprivation OOIIOOOOOOOOOOOOO. 39 References O...0......OOOOOOOOOOOOOOOOOOOO 63 ii Table 1 Table 2 'Ihble 3 Table A Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Thble ll TABLES Mean number of large changes in ECoG potential (LCs) per S-second interval as a function of Judged arousal category for animals 1, 2, and 3. Number of S-second intervals as a Joint function of Judged arousal category and EMG rating for animals 1, 2, and 3. Mean number of large changes in ECoG potential per S-second interval as a function of Judged arousal category for animals h, 5, and 6. Number of S-second intervals as a Joint function of Judged arousal category and EMG rating for animals h, 5, and 6. Number of 5-second intervals Judged as fitting the various arousal categories for animals 7, 8, and 9. Mean weights in grams Observed for Juvenile and Adult animals as a function of condition, age group, and.weighing. Mean brain weight in grams as a function of condition and age group. Mean brain weight to body weight ratio as a function of condition and age group. Activity means (standard deviations in paren- theses) expressed in frequency of movement per half-hour for the E vs. I + S + Y contrasts, as a function of age group and 5—day interval Mean number of significant periods as a function of condition, age group, and 5-day interval. Mean rhythmicity index as a function of condition, age group, and age at computation. iii PAGE 28 3O 33 3h 38 “9 52 55 57 FIGURES PAGE Figure 1 Sample tracings typically seen in low-voltage fast-wave sleep (LVF), high-voltage slow-wave sleep (HVS), and waking (w) 25 Figure 2 Mean activity per half-hour for Juvenile and adult animals as a function of condition. ... 51 iv cams I, AN OVERVIEW Research 22.§$S§E While there is much early research on sleep (e.g. Landis, 1927; Kleitman, 1929), most authorities consider Aserinsky's discovery of periods of rapid eye movement during sleep in adult humans (Aserinsky and Kleitman, 1953) as the beginning of modern sleep research. Aserinsky noted that his subJects moved their eyes rapidly beneath closed eyelids for short periods of time interspersed throughout longer periods of sleep with no rapid eye movement. During the eye movement periods, Aserinsky observed concomitant increases in body movement, respiratory activity, and heart rate. These historic Observations countered thinking of that time by showing that sleep was not Just one phenomenon and that a sleeping individual was not physiologically inactive but was periodically very active indeed. The last 15 years have seen the development of new methods for more complete study of sleeping behavior. Current electrOphysiological recording techniques permit distinctions in human sleep between sleep with eye movements and four stages of sleep without eye movements. (Dement and Kleitman, 1957b, Luce 1966). For purposes of the present discussion, however, only two maJor subdivisions are considered. The sleep of many mammals, including human sleep, can be par- titioned by these two behaviorally distinct states. Based on elec- trocorticographic (ECoG) or electroencephalographic (EEG) records and electromyographic (EMG) records of muscle activity, one state is characterized'by large slow changes in electrocortical potential and'by little or no muscle movement with.moderate levels of muscle tonus in the face and neck. This state corresponds to the periods of no rapid eye movements and quiet, even respiration which Aserinsky Observed. various terms are associated.with this sleep state several of which include: 'no-rapid-eye-movement-sleep' (NREM-sleep) (Aserinsky, and Kleitman, 1953: Dement, 1960), 'quiet sleep' or 'telencephalic sleep' (JOuvet, 1960), and 'High VOltage, Slow wave Sleep' (HVS), (Meier and.Berger, 1965). The second state, when it appears, follows the former in.most mammals and is characterized by: small, rapid changes in electrocortical potential similar to records of awake subjects, a radical loss of neck.muscle tonus, cardiac irregularity, and uneven respiration. This state corresponds to Aserinsky's rapid eye movement periods. A number of terms have also been applied to this stage of sleep. Those most often used.are: 'rapidseye-movement-sleep' (REM- sleep) (Aserinsky and ICLeitman, 1953; Dement, 1965), 'parodoxical- sleep' or 'rhombencephalic-sleep' (JOuvet, 1960), and 'Low VOltage, Fast Wave Sleep' (LVF) (Meier and Berger, 1965). This stage of sleep appears to be concomitant with dreaming (Dement and Kleitman, 1957a) and thus is also called dreaming sleep or D-state (Hartmann, 1966) as well. Tb avoid confusion from.many different terms used in the sleep literature, throughout this paper Meier and Berger's (1965) terminology will be used exclusively regardless of actual terminology in the literature cited. Their terms, 'High VOltage, Slow wave Sleep (HVS) and 'Low Vbltage, Fast wave Sleep' (LVF) seem the most Opera— tional and closest to most polygraphic data language. Of the two states LVF has received more attention by far. Several important characteristics of LVF have been reported. First, EEG records of LVF are almost indistinguishable from waking EEG records without the aid of additional physiological data. In human subjects, several nights of LVF deprivation are usually followed.by temporary increases in the LVF-HVS ratio over a baseline measure taken prior to deprivation (Dement, 1960; Witken and Lewis, 1967). This "rebound effect" after LVF depri- vation suggests that the maintenance of homeostasis requires certain, minimal amounts of LVF. Ephron and Carrington (1966) elaborate on this homeostatic interpretation of LVF deprivation effects. Other data on humans suggest that individual differences in base-rate LVF-HVS ratios are related to reported quality of sleep (Monroe, 1967). The biological underpinnings of such ratio variability have not been isolated. One of the most striking prOperties of the human LVF-HVS sleep cycle is its radical change with age. The percentage of total sleep spent in LVF dr0ps from over 50% in infancy to less than 1h% in old age (Roffwarg, Muzio, and Dement, 1966). Whatever the reasons for such a developmental decline in this ratio, these findings suggest that the importance of LVF is inversely related to level of develop- ment in the human. As questions about sleep function arose, investigators with an ever growing variety of research interests and theoretical orienta- tions have studied an ever growing variety of species. Such diversity of research has brought interesting findings to the literature. JOuvet and his associates have reported extensive data on sleep in the domestic cat (JOuvet, 1960; JOuvet and.Mounier, 1960; JOuvet, 1968). Their behavioral research indicates that cats clearly have both LVF and HVS and that reliable rebound effects result from selective LVF depriva- tion. They also noted considerable individual variation in symptomatic reaction to such selective deprivation. Dement (1965) reported similar findings. Hartmann (1966) concluded from comparative research on sleep development that, as in humans, LVF-HVS ratios decrease with age in rats and cats. Jouveteuounier's data (1968) further support this conclusion. She also found a developmental decline in LVF-HVS ratios for guinea pigs and different rates of sleep pattern develop- ment among guinea pigs, rats, and cats. Such differences, she suggests, may have been related to the Obvious interspecific differences in maturity at birth and to species-specific rates of maturation. Research with Rhesus monkeys (Meier and Berger 1965) also indicates a develop- mental LVF-HVS decline following temporary increases in LVF from'birth to seven days of age. With greater freedom for experimental manipulation, sleep research with animals has prObed some of the mechanisms controlling the LVF-HVS cycle. Jouvet (1960) reported the isolation of an area in the caudal pontine reticular nucleus in the vicinity of the locus coeruleus as a center initiating LVF in cats. Data for other mammals suggest, however, that the amygdala and the hippocampus are intimately in- volved in LVF activity, if not in its initiation (Adey gt 21,, 1963; Brown and Shryne, 196k). Regardless of the number and location of control centers, LVF appears to be related to the build-up of a chemical mediator, perhaps some neurosecretion. Dement and his associates (1965) noted that normal cats behaved as if they had been deprived of LVF following intra-spinal injections of cerebrospinal fluid drawn from LVF deprived animals. If the donor animals were permitted several nights of uninterrupted sleep, similar injections had no effect on normal recipients. Jouvet and his associates have more extensively explored the neurochemical parameters associated with the deposition of HVS and.LNF and have related various biogenic amines to HVS and LVF regulation (see Jouvet, 1967 and 1968 for further elaboration). Aside from such groundawork research on LVF control, deprivation research with humans and animals has offered few clues as to the function of LVF or the reason for its decline relative to HVS in developing mammals. Symptoms of LVF deprivation are only suggestive of possible directions for further research. Humans deprived of LVF tend to become anxious and/or excitable (Dement, 1960). LVF deprived cats can become hyper-sexual and irritable. Some data suggest that LVF deprivation may cause changes in EEG evoked potentials (Dewson gt 31., 1965; Luce, 1966). Several hypotheses concerning LVF function have been offered. 'The Phylogenetic Theory' (Snyder, 1966) holds that LVF evolved in.mammals as a system to maintain vigilance necessary for survival from attack. Such a theory has several prOblems. First, it would be very difficult to devise ecologically valid experiments to test for differential survival prObabilities for animals in whom LVF is blocked.and.for normal control animals. Second, as Berger (1966) points out, Snyder's theory does not satisfactorily explain why prey animals such as rabbits have less LVF than do predators such as cats. Third, it cannot explain why a special state should evolve for vigilance maintenance when rapid and.motionless arousal from HVS would.work as well. 'The Pr0gramming Hypothesis' (Dewan, 1969) suggests that LVF acts as a reprogramming mechanism to integrate new experiences with older memories. This theory may have some merit in view of the findings of personality changes and disturbances of attention noted in LVF-deprived humans. However rigorous and long-term.tests of such a theory would be difficult with humans. Furthenmore, any test of the Programming Hypothesis with non-human subjects is limited to animal learning paradigms. Such paradigms would seem to over-restrict Dewan's notions of "experiences" and."memories". 'The Oculomotor Innervation Hypothesis' (Berger, 1969) pro- poses that LNF provides a mechanism for establishing neuromuscular pathways necessary for binocularly coordinated eye movements. This theory seems promising but faces two severe prOblems. First, it fails to adequately account for massive neural activity during LVF in areas unrelated to the visual or oculomotor system. Second, while among various species there seems to be a positive relation between the degree of stereopsis (thus the degree of partial decussa- tion of optic fibers) and the proportion of LVF, Berger's theory fails to adequately explain why there should be any LVF at all in animals without stereopsis such as the hen. A further prOblem for Berger's hypothesis stems from.Allison and van Twyver's (1970) findings of no LVF in echidnas, primitive egg-laying mammals which have, at least, partial binocular vision. 'The Hemeostatic Theory' (Ephron and Carrington 1966) suggests that LVF serves to balance HVS by periodically reinstating cortical tonus during sleep. This theory seems general enough to account for the massive neural activity during LVF. It also explains the periodic characteristics of sleep stages by implying negative feedback control mechanisms for maintaining minimal levels of cortical activity during sleep. Hewever, Ephron and Carrington's theory does not easily account for developmental changes in LVF proportion. Roffward,.Muzio, and Dement's (1966) 'Supplemental Input Hypothesis' deal with such developmental changes in LVF. Ephron and Carrington's homeostatic approach is incorporated into a broader theoretical position. The 'Supplemental Input Hypothesis' holds that LVF prepares the central nervous system (CNS) to cope with exogenous stimulation. These workers note that neural activity resulting from.LVF is similar to that re- sulting from.sensory input during waking. They suggest that this endogenous stimulation may promote CNS development and that the characteristically early myelination of the visual tract may be related to corresponding neural activity during LVF. Snyder (1965) points out that LVF involves massive activity in other cortical centers as well, noting that were it not for an efferent inhibitory mechanimm, the frenzied.activity in motor areas during LVF would result in equally frenzied.muscle activity throughout the body. Indeed some studies have shown that the locus coeruleus of the pons may be a key component in such an efferent inhibitory'mechanism. (Jouvet and Mounier, 1960). I Each of the theories on LVF function requires further substan- tion. However, in testing each position, it would seem.difficult to pit one theory against another. For example, to test Snyder's Phylogenetic Theory, the sleep of animals representing different points in mammalian evolution should be examined. However, as Hodos and Campbell (1969) point out, the concept of a phylogentic scale is now untenable given our clearer understanding of evolution, and contempory creatures simply cannot be considered as differentially evolved in any direct sense. Thus a test of Snyder's position would be very difficult, but almost impossible if the test must simultaneously test Dewan's programming hypothesis or Berger's oculomotor control hypothesis. Rather, each theory must be examined separately and each evaluated on its ability to handle various research findings. The present research is an attempt to further examine the Supplemental Input Hypothesis of Roffwarg and.his associates. These workers essentially argue that LVF provides endogenous stimulation needed for the development and/or maintenance of the CNS during periods of reduced exogenous input. On further consideration we will see that such a hypothesis follows logically from several related hypotheses about the homeostatic relation between endogenous and exogenous input. If valid, the supplemental stimulation hypothesis could explain some interesting findings. Dement (1965) reported that cats, having been deprived of LVF for as many as 30 days, had difficulty learning a simple Ykmaze under food reinforcement. One cat could not reach criterion after 5,000 trials. Dement suggested that such difficulty arose because the animals were unable to control their maze-running speeds and thus could not attend to relevant cues. Dement did not report the ages of his animals. One explanation for these findings may be that learning difficulties arose from poorly developed and/or poorly maintained neural mechanisms resulting from insufficient input during sleep. For animals having such deficits, maze-learning would be severely impaired. A search of the literature disclosed no systematic studies on long term.and/or developmental effects of reduced input during sleep (i.e. long-term.or developmental effects of LVF deprivation) however, there is abundant literature on the importance of various types of input during waking for normal development (see weinstein gt _a_l_., 1968). A brief discussion of this related topic seems crucial here. Selected.Research.gg Early Stimulation and Development The importance of stimulation quantity and.quality during waking has been well established in short-term.and developmental research. Data on the effects of impoverished environments of one sort or another point to the importance of varied stimulation during waking hours and have important implications for sleep research. Research by D. o. Hebb's group at McGill (e.g. Bexton, Heron, and Scott, 1951+) suggests that after a few days of low and homogeneous input, humans have difficulty with problem.solving and tend to provide their own stimulation in the form.of visual hallucinations. Working from the opposite tack by examining the effects of increased input, Bennett, Diamond, Krech, and.Bosenzweig (l96h) noted reliable increases in mass and changes in chemical constituents in the brains of rats reared in an "enriched" environment when com- pared to isolated control animals. These data suggest that genetic background is also an important factor, but with approPriate controls, cortical mass seems to depend directly on the degree of variability in the rearing environment. Interestingly, subcortical brain mass, 10 they report, bore a slight but significant inverse relation to variability in environment. Bennett and.associates, however, demonstrated rather convincingly that the results were not purely developmental. Very similar trends in brain weights obtained when the procedures were repeated with adult rats. Other developmental studies on early experience suggest that deficits in accommodation to novelty in dogs (Thompson and.Melzack, 1956), growth and adult size in rats (Levine, 1960), and normal feeding, filial, and sexual behavior in monkeys (Harlow, 1959, Harlow and Harlow, 1962) and in various Species(Beach and Jaynes, 1951+) can be related to degree and kind of environmental stimulation. Finally, the most striking of the stimulation deprivation de- monstrations involve the visual system. Experiments by Riesen (1950) indicate that oculomotor coordination, retinal development, and/or retinal maintenance are related to early visual input. Riesen noted that chimpanzees reared in darkness for over 16 months developed pallor of the optic disk, suggesting that some minimal amount of light stimulation is required for neurological development of the eye. Other deprivation studies, also employing binocular visual deprivation, have failed to find anatomical changes at more central levels of the visual systems of rabbits (Goodman, 1932) and chimpanzees (Chow, 1955). More recently, however, Wiesel and Hubel (1963a) have noted severe anatomical, and some functional deficiency in lateral geniculate cells of kittens monocularly ll deprived of light from birth to 3 months of age. These deficien- cies were reduced if the kittens were allowed some binocular visual input before shmilar deprivation, and were not Obtained in similarly deprived adult cats. Extending this type of monocular deprivation research, Wiesel and Hubel (1963b) noted only functional deficien- cies in the cat striate cortex. These deficiencies were charac- terized by a radically decreased proportion of cells responsive to stimulation of the deprived eye in comparison to the proportion of cells responsive to stimulation of the undeprived eye. In normal cats most striate cells respond to stimuli presented to either eye. Nevertheless, Wiesel and Hubel did find intact striate cells which could not be driven by either eye, indicating that such cells were connected only to the deprived eye. These connections should have degenerated.with disuse. The fact that the cells themselves did not degenerate implies that some form of non-visual input prevented their strephy. Wiesel and Hubel recognize the discrepancy between their findings of atrophy beyond the retina after visual deprivation and earlier failures to find such atrophy. They offer two possible explanations. On one hand, the differences may stem from Species- specific differences in the effects of visual deprivation. 0n the other hand, the discrepancy may be explained by the greater ease in detecting changes in cell structure with the monocular depriva- tion method, since this method permits both deprived and undeprived cells to be seen on the same slide. 12 A third possibility exists, however. Previous failures to find atrOphy after‘binocular deprivation.may be due to a growth of non-visual connections to visual cells during some 'critical' period of neurophysiological development. This third hypothesis implies that such non-visual connections do not develop to any great extent if some visual input remains as is the case with monocular deprivation. The finding of intact cells in the striate cortex with no visual input supports this third line of thinking. Wiesel and Hubel, moreover, suggest that because the degree of atrophy found in the lateral geniculate depends on the age at which deprivation begins, there must exist a critical period beyond which visual deprivation has no effect on lateral geniculate cells. During this period however, the animal must be more sensi- tive to sensory deprivation and can more easily make new nervous connections to adapt to variations in sensory input. Linking the above discussion with the Supplemental Input Hypothesis are some suggestive data supporting the existence of such non-visual input to the visual cortex during LVF periods. Pertinent here are the extensive investigations of JOuvet and his co-workers (see JOuvet, 1967, p. 1&3) on periodic ponto- geniculo-occipital activity. These workers have noted that activity appears in the form of monOphasic, high-voltage spikes occurring in groups of five to six. These spikes characteristically begin Just before and continue throughout LVF periods in cats. 13 These findings suggest that an ascending pontine extraretinal projection provides input to the visual cortex during LVF. Jouvet's conclusions mesh well with Evarts' (1962) findings of higher activity levels in visual cortex neurons during LVF as compared.with levels during HVF. Perhaps some developmental elaboration of this source of non-visual input could explain other failures to find atrophy beyond the retina. Therefore, it seems fair to include non-visual input during LVF as a possible reason for the discrepancy between Hubel and Wiesel's findings of atrophy and other failures to disclose any anatomical change following early visual deprivation. In sum, stimulation in certain minimal quantities throughout development, at least during waking, seems a prerequisite for normal anatomical and behavioral development. And there is some evidence that endogenous input during sleep may also be necessary for normal development. However, the developmental significance of stimulation during LVF has not been systematically studied. Since LVF involves massive stimulation throughout the cortex, this source of input may be crucial, Just as Roffwarg and his co-workers postulate. More broadly now, cutting across all sensory mechanisms, if LVF serves as an important source of endogenous input and if endogenous input supplements waking exogenous stimulation, then in a homeostatic system reducing exogenous input should lead to 1h some sort of enhanced endogenous input. Several studies are in support of such a hypothesis. Wood (1962) noted that adult humans who had.spent the day in social isolation tended to spend more time in LVF than unisolated controls. Other supporting data are the findings of hallucinations during waking after reduced and homo- geneous input (Bexton, Heron, and Scott, 195h; Heron, 1957). Keeping in mind that only subcortical areas have been shown necessary for LVF, the Bennett §_t_ 51,, (l96h) findings of increased subcortical mass after decreased environmental stimulation have further relevance. All suggest an inverse relation between exOgenous and endogenous input. But to assess the developmental significance of LVF, the supplemental stimulation hypothesis of Roffwarg and his associates must be placed in proper conceptual perspective. 15 CHAPTER II, INTRODUCTION waard.§_Broader Conceptual Base The discussion in the preceding chapter logically implied several important and successively more general hypotheses. First is that, generally, reciprocity exists between exogenous and endo- genous input. Roffwarg and his associates hypothesize such a relation only in connection with LVF. WOod (1962) offers some supportive evidence. More broadly, however, the McGill research on the effects of input reduction suggests that supplemental en- dogenous input may occur in hallucination-like waking experience as well as during sleep. These data in combination with other stimulus deprivation data suggest an even more general hypothesis: Hemeostasis requires that the CNS regulate input. Stimulus dep- rivation studies suggest that certain minimal amounts of input are required. It follows, then, that the CNS must establish a com- plementary or inverse relation between sources of input in an attempt to meet homeostatic requirements. That the CNS maintains homeostasis can hardly be questioned, as it contains the control site of every vegetive function in the body. That the role of the CNS as a regulator of stimulus input is subsumed by its role of maintaining homeostasis follows directly. The brain stem reticular formation has been implicated in states of arousal (3,3, variations in attention) so often that the formation 16 is commonly referred to as the reticular activating system (Hilgard and Bower, 1966; Hernandez-Peon, 1961). Another striking manifes- tation of how the CNS regulates input is the characteristic pattern- ing of an organismfs daily activity (circadian rhythm). Even without regularly occurring external signals (Zeitgebers) many animals show cycles composed of periods of relative activity and inactivity, the cycle usually being slightly more or less than 2h hours by some constant (Aschoff, 1963; Barker, 1958; Altman, 1966). Other researchers under various conditions have demonstrated reliable rhythms much shorter than 2h hours (Wolterink e_t_ pp, 1968). Some of these shorter periods in the range of 3.8 to h.2 hours, for example, may represent simple harmonics of the often Observed biolog- ical periods from 23.5 to 21+.5 hours (Marler and Hamilton, 1967). On the other hand, such shorter periods may represent real biological rhythms, associated with various neural networks or with the cyclic processing of food and water (Levine, R., personal communication). Such rhythms could then combine and interact with external Zeitgerbers to form.the '1ess biologically relevant' circadian activity patterns (WOlterink, L., personal communication). Nevertheless, whether the shorter (3 g h hour) or the longer (23 - 25 hour) periods are considered true biological rhythms, most studies suggest that daily activity is regulated. Thus the amount of exogenous input which the animal experiences by inter- acting with its environment is also regulated. The present 17 researcher's position is that this input control is endogenous in nature and stems directly from CNS mechanisms. However, Marler and Hamilton (1967) along with others (2.5. Brown, 1959) point out that in testing for endogenous control it is extremely difficult to maintain constancy in all factors of the external environment. Even in the best designs, periodic variations in non-visual electromagnetic radiation, for example, are rarely eliminated. Therefore it is difficult to prove that biological rhythms are completely endogenous. PrObability, however, points to some endogenous control of periodic activity. Thus we may tentatively conclude that in many normal organisms activity patterns and hence stimulus input are under a measure of endogenous regulation. That is, the CNS governs the duration and circadian positioning of input. The hypothesis of Roffwarg and his associates now can be placed in its prOper, theoretical perspective: 1) The CNS maintains homeostasis. 2) Certain levels of stimulus input are required for homeostasis. 3) The CNS regulates input in maintaining required levels of input. A) Uhder reduced exogenous input, endogenous input tends to increase. 5) LVF is an important source of stimulation and serves to supplement an organism's exOgenous input during periods of relative inactivity. 6) Such supplemental input is relatively more important for young than for old organisms. 18 In this context, data exist in support of the first four hypotheses and the last two hypotheses are experimentally testable. Methodological Considerations and Research Orientation On the basis of the preceding hypotheses, the present research is an attempt to assess the general role of LVF in CNS development. The subjects were deermice of the species Peromyscus maniculatus bad—xvii. Since 2. 13. Mi are hardy and easily bred, this Species seemed an excellent beginning subject population for such research. First, extensive literature exists on the biology of the Peromyscus genus (King, 1968). Second, the genus, having many related Species with identifiable geographic backgrounds, offers different wild populations for assessing genetic factors as well as interspecific differences in sleep patterns. Third, the relatively rapid.matura- tion rate of Peromyscus species can shorten the time required for developmental research on LVF function. Fourth, while there is abundant behavioral and physiological literature on P. m, bairdi, no one has yet analyzed sleep in any detail in this group of animals. Therefore, the aim of the first study in this series was to demonstrate and quantitatively verify the existence of HVF and LVF in P. m, bairdi. Once this was accomplished, the second study was designed to test an inexpensive LVF deprivation technique which could operate within the demand characteristics of P. m, bairdi and could be used to distinguish the effects of 19 LVF deprivation from other typically confounding factors stemming from.time and.method.of arousal. Such controlled.deprivation is crucial for developmental and/or long-term research on LVF function. The deprivation technique, like the "pedestal-over-water" technique (see Jouvet, 1960) did not require ECoG or EEG monitoring. Instead it relied on rapid loss of muscle tonus at the onset of an LVF period. A cat or rat can‘be selectively deprived of LVF if it is placed on a pedestal above water. The pedestal must be large enough to sit on and sleep on, but too small to permit the loss of muscle tonus concomitant with LVF. However, continual immersion of g. m. bairdi in water increases dangers of respiratory infection. Because of such dangers and because of the difficulty in controlling for confounding effects of the water'bath, a shock-grid was substi- tuted beneath the pedestal. The purpose of Study III was to provide a rough estimate of the relative proportions of LVF and HVF in Juvenile _P: _m'. bairdi. These data, in combination with those of Study I, would permit an estimate of the developmental decline in LVF for _P_. _m_. bairdi. Thus Studies I, II, and III provide necessary groundwork for the develOpmentally oriented fourth study in this series. Before introducing Study IV, several methodological points should be emphasized. First, LVF is imbedded in HVS, a physiolog- ical state apparently necessary for physical recouperation (Dement, 1967); thus careful control procedures are required for 20 deprivation research. Second, the hypotheses in the preceding section do not specify CNS mechanisms, but suggest that the development of the CNS in general depends on LVF for input. Thus it would seem premature to single out any one sensory system for*analysis. More general indices of CNS development and integrity are required. With these considerations in mind, the general approach to Study IV involved deprivation of LVF for an extended period of ' time. The effect of such deprivation on Juvenile and adult animals were examined with respect to: (1) activity levels, (2) regularity of activity patterns under various environmental conditions, and (3) brain and body weights. Such dependent variables were selected.because they seemed to reflect the general status of input requirements, regulative function, and CNS development. 21 CHAPTER III, EXPERIMENTATION subjects for Studies I, II, III, and IV. Subjects for the present research were 33 P. m. bairdi. These mice are commonly referred to as deermice and'belong to the genus Peromyscus. All subject were bred in the Peromyscus colony at Michigan State university's Biology Research Center. These mice are normally weaned between 21-28 days of age, reach sexual maturity by 33-h0 days of age, are fully grown by 50-60 days of age, and can live under laboratory conditions for over two years. See Layne (1968) for a complete discussion of ontogeny and King (1968) for a complete treatment of the biology of Peromyscus. Study I: Sleep Analysis The purpose of Study I was to demonstrate and quantitatively verify the existence of HVF and LVF in P. E. 133231. To achieve this aim, traditional recording techniques were coupled with a time sampling procedure. Behavioral time sampling techniques, when applied wisely, can replace expensive and often wasteful continuous Observation of behavior (Bombardieri and Johnson, 1969; Bendat and Piersol, 1966) or easily summarize continuous records (Goldberg 33 91., 1961+). Traditional qualitative pattern analysis was related to quantitative methods of scoring sleep records. A high association between traditional categorization of sleep states and quantitative patterns in the data was expected. The time-sample size was chosen to extract the maximum.amount of information about 22 this association from a minimum amount of data. Statistical analyses of such time-samples permitted inferences with specific error prObabilities about the complete time-behavior spectrum from which the sample data were Obtained. Method General_Procedure. Three adult (over 53 days of age) animals of the P. m. bairdi species were selected from the psycholog mouse colony at Michigan State University. Each animal was examined individually, each examination being regarded as a separate replication. Surgery. The surgery, performed under ether anesthetic, con- sisted of chronically fixing three steel skull screws with connec- ted nichrome wire, two over the left parietal cortex and one over the right parietal cortex, to the skull so that the screw tips were in contact with the dura mater beneath. In addition, two nichrome wire sutures were placed in the flesh over the neck muscles. The connecting wires from the screws and nichrome wire sutures were connected to the female components of one 3-socket and one 2-socket amphenol plug. External portions of the screws and wires were insulated.with clear nail-polish and the two female components were built into a dental cement head.platform. Recording Format. Two channels of a 6-channe1 Grass Model III electroencephalograph were used. For one channel the two screws over the left parietal area served as electrodes for push-pull type ECoG records and connections to the third screw 23 grounded the subject. The second channel recorded EMG super- imposed on a record of heart activity from the suture electrodes in the flesh of the neck. Recording Procedure. Just after surgery each animal, under anesthetic, was connected with male-component plugs and.p1aced in a recording cage. Purina Mouse Breeder's Chow and tap water were provided on an ad libitum.basis. During 2h hours of recovery and 2h hours acclimation to the recording apparatus, sample records were taken and gain controls were adjusted to the most convenient gain setting for each preparation. After calibration, paper speed was set at 60 mm. per second. Then five l-minute parallel ECoG and EMG records were taken throughout the next 2h hours, sampling from periods when the animal appeared.motionless with eyes closed. Results and Discussion Categorization of Records. ECoG and EMG records for every 5-second interval (60 intervals for each animal) were categorized on the qualitative bases of general amplitude and frequency of ECoG waves and concomitant muscle activity into one of the following arousal states: LVF, HVS, and wakingl' (W). (See Figure l.) 1‘ It should.be noted that while 60 5-second intervals were analyzed for each S, in no animal were all intervals categorized as either HVS or LVF even though each l-minute record began when the animal was motionless with its eyes closed. The noise of the recorder sometimes caused arousal and.movement, thus making "waking" a reasonable and necessary category. ' 2h .Aav mower: was .Am>mv ammam o>msiaoam omenao>nnwan .Am>qv amoam o>m3upmmm ommpao>lkoa cw comm hHHsothv mmoflomap mamsmm .H madman >> m): is. H .090. ¢\F III 37% as t\lJI\\I\\l(()\)lll\\)k\ll)l( vaRUmu u.>._ 25 With pilot data as well as with the present records, it was not difficult to categorize each 5-second interval into one of the three arousal states. The next aim was to select and validate one criterion for ECoG records and one for EMG records that could be used to quantitatively differentiate LVF, HVS, and W. Quantification of Records. After qualitative judgements were made for each 5-second interval, they were set aside. Then for each interval, the parallel EMG record was covered.while the ECoeras independently analyzed for number of relatively large changes in potential (LCs) per 5-second period. Since the precise placement and depth of electrodes differed among Se, and since relative amplitudes and frequencies are commonly used to differen- tiate sleep stages (J0uvet, 1960), the criteria of an LC were reset for each §fs ECoG record. This method seemed compatible with the standards used in computer analysis (Rosenblith, 1962) and.with the criteria used in making qualitative judgements by experts in the field (Jeannerod, M. and Jouvet-Mournier, D., personal communication). A rating procedure was used for quantifying muscle tonus levels. For each interval, with parallel ECoG records covered, the EMG was independently rated on a three point scale for muscle tonicity: '1' for low tonus, '2' for moderate, and '3' for high tonus (and/or muscle movements). Since EMG records on animals as small as P, m, bairdi are often subject to cardiac 26 interference, it was necessary to rate EMG tonicity by scoring only those pen deflections that occurred.between heart beats. Reliability_9f Quantification. The same data were scored in a simdlar fashion by two naive laboratory assistants. The mean inter-scorer reliabilities were 0.91% (SD = 0.070) and 0.872 (SD = 0.109) for ECoG and EMG records respectively. The somewhat lower reliability for EMG ratings seemed to be due to the restricted range of ratings, because at least 80% of the time all scorers were in complete agreement and in remaining cases two scorers agreed, the third being no more than one rank away. validity of Quantification. Analyses for this section were aimed at relating the LVF, HVS, and W categorizations to quanti— fiable criteria. If the qualitative categories do have measurable differences, the scoring procedures should reflect these states in the relation between muscle tone ratings and.number of LCs. LVF periods should have a mean tonus rating near 1.00 (1,2. low tonus) associated with few LCs; HVS periods, mean rating near 2.00 associated with many LCs and W, mean rating near 3.00 associated with few LCs. Furthermore, such relationships should be strong enough to emerge clearly from the small time sample. Table 1 presents the mean number of LCs per 5-second interval for each §_as a function of arousal state. As can be seen HVS was quantitatively distinguished from LVF and W. However, to quantitatively discriminate between LVF and W, information about muscle tonus level was required. 27 Table 1 Mean number of large changes in ECoG potential (LCs) per 5-second interval as a function of judged arousal category for animals 1, 2, and 3. Animal (male) 3 (female) LVF 18.70 Nz20 Squ 19.1’4 SD:10. 79 1+.81 N227 511:3 . 77 HVS 51+. 18 N238 SD=l3 .89 (47.148 H9 80:16.28 21 . 85 N=20 313:8 . 96 Mean number LCs 28 ' 18.00 N22 SD=5.66 3.30 30:3.20 1.h6 N=l3 SD:1.50 in: § 21.72 df=2/58 ; p <.0001 88.19 df=2/58 p<.0001 6h.h3 df=2/58 p < .0001 Each cell in Table 2 contains the number of 5—second inter- vals (the joint frequency) for every combination of arousal state and EMG rating. The'X? test was used to assess the association between these two variables. The values of y? for SS 1, 2, and 3 were 89.5, 65.2, and 91.3 respectively, (df = h, p<<.0001). Combining these three independent replications of the same experiment disclosed an over-all'x? of 2h6.0 (df = 12, p (.0001), and suggested that a valid relationship exists between tonus level and arousal state. LVF, then, may be distinguished by few LCs and low tonus levels and.W, by few LCs and high tonus levels. These data suggest that qualitative distinction among arousal states can also be made on quantitative bases. The data further indicate that P. m. bairdi have sleep patterns similar to those of many mammals studied by other investigators. Finally, the mean percent of sleep time judged as LVF was Obtained by dividing the number of LVF intervals by the number of LVF and HVS intervals for each animal and then averaging those ratios. This procedure disclosed a mean LVF percentage of 37.1%. Study II: LVF Deprivation The purpose of this study was to test an inexpensive and automated device for selectively depriving P. m, bairdi_of LVF. In Study 1, an important finding was the almost complete concomitancy of LVF and low muscle tonus. This attempt to decrease time in LVF depended upon that high association. 29 Table 2 Number of 5-Second intervals as a joint function of judged arousal category and EMG rating for animals 1, 2, and 3. Animal Arousal Category (male) 3 (male) 3 (female) LVF mean rating;l.00 HVS mean rating:2.00 W mean rating:2 . 50 LVF mean rating:l.1h HVS mean rating=1.96 W mean rating:2.65 LVF mean rating=l.15 HVS mean rating:l.95 W mean rating:2.92 30 EMG rating 1 2 3 .---.. TM”-.- 20 0 0 ; i. 0 38 0 g _7 ._ .1 , k 5- 0 1 1 Q l i L 6 g 1 . 0 E i 1 '~ 28 o E i I 0 . 11 13 s i +-- g 23 h g 0 i 1 g 19 § 0 i i i E i 0 ; 1 g 12 The sampling procedures of Study I revealed that of total time sampled, LVF averaged 30% (range 11.7% to h5.0%). The percentage of time spent at muscle tonus level '1' averaged 28.3% (range 11.7% to 100%). And 91.0% of all LVF observed occurred in periods with tonus level '1’. From these findings, it seems reasonable to pre- dict that if an animal were perched on a sufficiently small pedestal above a shock-grid, LVF with preceding or concomitant loss in muscle tonus would be significantly reduced. Method General Procedure. Three more adult animals (two females) of the _I_’. m. bairdi species were selected and again studied indivi- dually with each animal conceptualized as a separate replication. Surgery and recording format were identical to that of Study I. Recording Procedure. After 2h hours recovery time in their home cages, each animal was trained to avoid shock by perching on successively smaller pedestals above an electrified grid cage floor. (This training usually required about 10 minutes). The smallest pedestal was just large enough to rest on without loss of muscle tonus. The grid.mechanism, triggered by a weight operated switch, was designed to give a 250 milliampere shock as long as no weight was on the pedestal. A Rustrak even recorder marked times off the pedestal. After training, each animal was connected to the recording device and again placed in the grid cage. Food pellets and.water were suspended within reach from the pedestal. During an additional 2h hours acclimation to the pedestal situation, gain and calibration 31 adjustments were made. Then five l-minute records were sampled 'throughout the third day when the animal was motionless with eyes closed on the pedestal. Each animal was then etherized and returned to the grid cage with the shocking device turned off. An additional l-minute set of records was taken to provide a sample of low EMG activity for comparison purposes. Results and Discussion The ECoG and EMG records were categorized and independently scored.as in Study I. The mean inter-scorer reliabilities were 0.928 (SD = 0.05M) and 0.918 (SD = 0.077) for ECoG and EMG records respectively, and the percent of pair agreement on EMG records was 98%. Table 3 presents the mean number of LCs per 5-second interval for SS h-6. While no LVF was noted, Table 3 indicates that the quantitative distinction between HVS and W was as clear as in Study I. Table h presents the number of 5-second intervals meeting joint criteria for each arousal-state x EMG-rating cell. Both the qualitative and quantitative data indicate that no LVF occurred in Study II. Tb assess possible differences between the time-behavior spectra sampled in Study I and Study II a Mann-Whitney U-test (Siegel, 1956)was made on LVF time-percentages observed for each animal. These percentages were computed.with respect to 32 Table 3 Mean number of large changes in ECoG potential per 5-second interval as a function of judged arousal category for animals h, 5, and.6. Animal Mean number LCs p 1-1 LVF HVS w 1%..” ______ 1.1 . . a _ .1 76.h8 h0.35 ; 6.37 1+ N=0 N=29 11:31 df:36.658 (male) SD=20.6h SD=8.1h p < .001 ‘ *7“ “ “ ’ l 18.16 10.55 10.00 5 N2=0 N:3l N:29 df=61.2h (female): SD=3.1h SD=2.75 p < .001 hh.97 . 26.76 7.37 6 N20 N=35 11:25 dr=5b..01 f (female) i ‘7 SD=9.1+8 jg SD=9.39 p <.001 8Corrected for non-homogeneity of variance (Hays, 1963). 33 Table h Number of 5-second intervals as a joint function of judged.arousal category and EMG rating for animals h, 5, and 6. Arousal Animal Category EMG rating 1 2 3 : LVF E i i i 7 mean 3 0 . 0 . O 3‘ 1 rating:-- Q ' 5 l HVS % : ‘ % ‘ h mean 3 0 29 ‘ 0 ? i (male) rating:2 s g | ; w z . mean 0 § 28 ; 3 . rating:2.10 ‘ § . 3 i LVF ; 3 mean 0 . 0 ' 0 § rating:-- ' g 5 7 l HVS i i ; g 5 mean 2 f 23 E 6 1 (female) rating:2.l3 ' g 4 mean : 0 g 8 21 { rating:2.72 ; § 3 i LVF i E i mean ; 0 g 0 E 0 ; ratins:-- 3 2 3 i ? HVS ; i 6 mean ; 0 ; 35 3 0 § 3 (female) rating:2.00 ; g ? g 1 W I g 1 I mean ; 0 ; 12 i 13 rating:2.52 ’ : 2 3h total time sampled for each S which resulted in scores of 0.0%, 0.0%, 0.0%, 11.7%, 33.3%, and 100% for animals h, 5, 6, 2, l, and 3, respectively (U :10. p‘<.05). Since the U-test is rObust and makes few a 25125; assumptions about the data, these results strongly suggest that the time-behavior spectra were different. The same Uevalue and p-1evel obtained when percent of time in tonus level '1' was analyzed. Thus the prediction that LVF can be significantly reduced by reducing time spent at low muscle t0nus levels is clearly supported. At this point an additional question arose. Did the data indi- cate any change in the ratio of W to HVS as a function of selective LVF deprivation? If the probabilities of the three arousal states are mutually independent, then deprivation of one state should not alter the ratio of the remaining two. unfortunately, only a post hgg_analysis was possible on this issue. An additional Uestatistic was computed on the W#HVS time ratios 0.053, 0.65, 0.72, 0.83, 0.9M, and 1.07 for animals 1, 3, 6, 2, 5, and h, respectively (U = 1. p< .10). In terms of averaged percentage changes, W and HVS went from 31% and 69% respectively in Study I to h7% and 55% in Study II. Thus the methods employed in Study II appeared to alter the relative proportion of W to HVS (at least for the second day on the pedestal). Such a difference suggests that with this deprivation technique, the prObabilities of the various ECoG states were not mutually independent in _P. m. bairdi. More generally, these findings suggest that with any 35 system purporting to selectively deprive one state of arousal, the possibility of artifactually changing relative proportions among remaining statesshould‘be analyzed and controlled for, or at least taken into consideration. One further point should be noted. During recording no animal fell off the pedestal. The absence of any falls from the pedestal during recording periods might be explained in terms of a conditional maintenance of muscle tonus. During the 2% hours in the grid cage prior to recording, the animals may have become sensitive to physiolog- ical circumstances which precede any loss of muscle tonus. (Event recorder tapes marking 'times off the pedestal mechanism' indicated that each animal left the pedestal several times). Such physiological circumstances may have served as the CS(s) for arousal and avoidance of shock (UCS). Conditional LVF onset has been demonstrated in rabbits (Kawakami and Sawyer, 1964). Perhaps temporary conditional LVF suppression occurred in our deermice too. Nevertheless, during less formal tests of the pedestal mechanism in long-term LVF deprivation, animals motionless with eyes closed were often seen nodding their heads, just before tumbling from their perch to the grid below. They then opened their eyes, behaved in a disoriented fashion, and finally hopped back to the pedestal thus avoiding further shock. Study III: Sleep Analysis _ip Young _P_. m. bairdi. The data of Study I indicate that for adult 2. m. bairdi, the percents of total sleep sampled judged as LVF were 3h.5%, l9.h%, 36 and 57.5% for animals 1-3 respectively, yielding an average of 37.1% for adults. Before conducting the main developmental study in this series, it was thought useful to have some contrasting data on LVF percentages in young _P_. m. M. The purpose of this study was to gather such data for 20-day-old animals. Method Each of three _P. m. M (two males) was weaned at 17 days of age. For the next two days the procedure exactly followed that of Study I, surgery taking place at 18 days and recordings, at 20 days of age. Results and Discussion Since the quantification methods of Studies I and II upheld the accuracy of the qualitative categorization procedure, only the qualitative analysis was performed on these data. Each of the 60 5-second intervals was categorized as in Study I. The results are summarized in Table 5. The percents of total sleep time judged as LVF were: 3h.0%, 5h.2%, and 60.0% for animals 7-9 respectively, yielding an average of h9.h% for 20-day-old animals. These data mesh well with the much more extensive findings for the rat, cat, and guinea pig (J0uvet-Mounier, 1968; JCuvet- Mounier gp_§l,, 1970). The present findings suggest that in in p, baipdi, as in rats and cats, LVF comprises more total sleep time in young animals than in adults. The present data, limited though they are, indicate an average LVF decrease of 25% between 20 and 53 days of age. 37 Table 5 Number of 5-second intervals judged as fitting the various arousal categories for animals 7,’ 8, and 9. Animal LVF HVS W I I _ I 7 f 19 i 37 -, h . (female) . I I I l g i . E 8 g 32 ; 27 r 1 3 (male) ‘ § . 3 9 30 20 , 10 ? (male) I 2 I 38 Conclusion The time-sampling data of Study I indicated that P. m. M can serve as a useful species for Sleep analysis. Time-sampled data in Study II suggest that, with P. m. bairdi, a pedestal-over-shock method can be used to significantly reduce time spent in LVF. The results of Study III suggest that, as with a variety of other animals, LVF percentages decrease with age in _P. m. pair-ii. Combined these data comprise sufficient ground work for an exploratory study on the developmental significance of LVF. Study IV: Developmental LVF Deprivation The aim of this study was to explore developmentally some of the relations among LVF, CNS development, circadian activity patterns, and regulative function in P. m. M. The experimental design evolved from several methodological considerations. First, there may be a variety of factors operating during LVF deprivation. Such factors stem from the fact that LVF occurs only during certain phases of circadian activity. Thus any LVF-contingent arousal system also has the effect of disrupting these phases. The type and frequency of arousal may also have effects distinct from those of LVF-deprivation. The pedestal-over-shock method of LVF deprivation employed in the present study has been described in Study II (see also Mitler and Levine, 1970). rue pedestal-operated system.has many advantages over the pedestal-above-water method in that other grid cages may be wired in parallel to the deprivation 39 cage and activated by the animal on the pedestal. Such a yoked control cage was employed here. This condition was aimed at controlling for the confounding effects already mentioned as well as for the effects of restricted movement experienced.by the animal confined to the pedestal. Second, if the reciprocal relation between endogenous and exogenous input hypothesized in Chapter I does Operate, then LVF deprived animals, in order to compensate for loses in that endo- genous source of input, may increase exogenous input at the expense of some HVS. To control for this possible sleep sacrifice, an unselective sleep deprivation condition was included. Third, since all three of the above conditions involved isolation, an isolated.but unmanipulated control condition was added. Finally, to insure that the data would be clearly interpretable, the various experimental treatments were performed on both juvenile and adult mice. Studies I and III indicated a developmental change in LVF proportion. Those data and the logical requirement that any developmental study Should provide juvenile and adult comparisons led to the inclusion of both juvenile and adult groups. A cursory review of many studies on the effects of early environment indi- cated that such an adult group is often omitted.(g.g. Thompson and Melzack, 1956; King, 1969). The omission of data on adults un- necessarily Obscures the distinction between environmental effects and age x environmental interaction effects. ho Method Juvenile Group. This group consisted of three animals in each of four conditions. The conditions were filled simultaneously by randomly assigning each of four littermates, one to a condition, with the restriction that LVF-deprived animals and their yoked controls were of the same sex. Both sexes were represented in each treatment condition. Each litter was weaned at 18 days of age and treatment began on day 20 with the weighing of each animal. Throughout the following lh-day treatment period, a 12 hour light-l2 hour dark schedule prevailed, the light onset being at 7 a.m. local time. Purina Mause Breeder's Chow and tap water were provided on an 29 libitum basis. Animals in the experimental or LVF deprivation (E) condition spent all 1h days in the deprivation cage. Training procedure for shock avoidance and operating characteristics of the cage were described in Study II. The animals remained in the pedestal cage continuously except for approximately five minutes daily in a transfer bucket while the apparatus was cleaned. Animals in the yoked control (Y) condition were housed in a small cage with approximately the same free movement area as the animal restricted to the pedestal. The animal was removed from this cage approximately five minutes per day for cage clean- ing. The cage's electrically gridded floor was connected in #1 parallel with the grid of the deprivation cage, so that a shock was delivered to each cage when no weight was on the pedestal. Animals in the sleep deprivation (S) condition were housed in a 6-inch deep, 5-inch wide, and 7-inch long plexiglass cage suspen- ded over a clock-triggered, motor-driven disc four feet in diameter. The cage had no floor and.was situated 1/8 inch over the outer portion of the disc with the 7-inch side parallel to the direction of rota- tion. This arrangement forced the animal to live on a treadmill floor moving 5 feet per minute (measured from cage center) for 19 of each 24 hours. Pilot work revealed that young _P_. :3. p333; could endure no more than 19 hours per day of floor movement. The five hour interval without floor movement was timed so that the onset of darkness exactly split the interval. Animals in the isolated (I) condition spent their in days undisturbed in laboratory plexiglass cages. After the reapective treatments, each animal was again weighed and transferred for the following 21 days to an activity recording cage. Each cage was equipped.with a jiggle-switch.which sent records of gross motor movement with lateral force equal to or greater than 0.35 grams to a Rustrak event recorder. Thus a con- tinuous record of activity was Obtained for each animal. The first seven days had the same 12 hour light-12 hour dark lighting schedule as during the treatment phase. During the second seven days, free running activity was recorded in continuous dark- ness. For the last seven days the same light schedule was reinstituted. h2 Following the circadian rhythm measurements each animal was weighed, killed, and its brain, rostral to the pyramidal decussa- tion, was removed and.weighed.immediately. Adult Group. This group consisted of three animals per condition. The entire procedure for these animals was exactly the same as for the juvenile group, except that after weaning, the litter was separated by sex and left undisturbed.until 53 days of age. It was not possible to match adult litters to juvenile litters with respect to sex, however, the frequency distribution of adult male and female mice over the four treatments was identical to that for the juvenile group. Results and Discussion Dependent variables. Body weight was measured three times throughout the 35 day procedure: on day l, on day 1h (post- treatment), and on day 35 (post-procedure). Brain weight was taken immediately after the last body weighing, and the brain to body weight ratio was computed using the last body weight. The activity records were scored'by a naive laboratory technician and analyzed by computer. Five successive days from each 7-day interval were selected for analysis. For each five days, the mean number of movements per half-hour period was computed. In addition, a Fourier analysis was performed on the activity scores, and.major cycles of activity were counted.by extracting from the output those period lengths with a signal-to- noise ratio greater than 2.51I (p < .01 against the null hypothesis h2.1 of no periodic activity). Finally, a crude rhythmicity index was devised.which could be used to measure the predictability of each animal's daily activity pattern. For each 2h hours in each 5-day interval, the total activity score for hours 7 a.m. to 7 p.m. was subtracted.from.the total activity score for hours 7 p.m, to 7 a.m. This process yielded five difference scores for each 5-day period. The rhythmicity index was obtained by extracting the fourth2 root of the variance of those five difference scores for each 5-day period. With this index, the smaller the value, the more regular was the activity pattern. This procedure produced three variables for each five day interval: mean activity, number of significant periods, and rhythmicity index. Thus the overall procedure involved 1h dependent variables per animal: three body weights, one brain weight, one brain to body weight ratio, three mean activity scores, three counts of significant periods, and three rhythmicity indices. Data Analysis. With 1% dependent variables and the high likelihood of non-independence among them, a multivariate analysis of variance (Mbrrison, 1967) was performed by computer on the entire data matrix. The program also provided univariate F-statistics corrected for non-independence and for the inflated 2' Since the variances were typically very large, and since only relative magnitude was of interest, the fourth root of the variance (square root of the standard deviation) was employed to facilitate tabulation and analysis. h3 prObability of alpha-error associated with repeated univariate analyses. Each variable was regarded as a separate dependent variable for the purposes of this analysis. Analysis Design. Since the treatment conditions were run simultaneously with littermates, a matched sample design seemed appropriate. Such a design involved analyzing treatments as repeated measures in a 2 x h age group by treatment factorial design with three replications per cell. While males and females were equally distributed throughout the design, the sex effect could not be formally analyzed with this small number of subjects. The design of the analysis produced one contrast test for the age-at-treatment effect (hereafter referred to as the "age effect"), three pair-wise contrasts for treatment and, three pair-wise contrasts for age x treatment. These contrasts successively compared: S :3. I, Y ya. I + S, and E yg. 11+ S + Y. Such §_p£ig£i tests permitted precise examination of all differences between conditions. Missing Data. Five data points were missind due to the death of a juvenile animal in the E condition. The animal apparently died of starvation in the morning of the 28th day of the pro- cedure. The animal's record indicated it was highly active throughout its last 1h day. During the 2h hour dark condition it was provided as much food as counterparts in other conditions. Yet on the morning of day 28, after first light onset for the hh final 7-day period, all had surplus food but the E animal. The animal seemed extremely weak and died during examination. Its missing weights were estimated with linear projections based on the body and brain weights observed for the other juvenile animals in the E condition. The first five days of that second 7-day period were used for analysis so as to minimize the con- tamination of activity records by effects of the animal's food shortage. This procedure seemed apprOpriate since 2, p, 222591 starve to death in 2h - 36 hours after removal of food and 72 hours were allowed prior to the animal's death. With respect to activity score, period count, and rhythmicity index for the third 5-day interval, those measures on the dead animal for the second 5-day interval were used. This decision seemed fair, if not conservative, since there was an overall tendency for activity to increase, number of periods to remain constant, and rhythmicity index values to increase over the three intervals. weight Measures. Table 6 presents mean body weights for each of the three weighings as a function of condition, age group, and age at weighing. There were no significant treatment or age x treatment effects for any of the weights (all EP‘(3°96: all ps>>0.12). Juveniles, however, showed reduced weight gain from the first to the second weighing in the E and Y conditions relative to the S and I conditions. The age effects for the first and second weighings were significant (F = 51.68 and 30.8h, dfs = l/h, p<:0.002 and 0.006 respectively). These age differences were trivial and, since the juveniles grew, the weight differences #5 mam.c cmm.a ‘ amm.a 6mm.a _ ass.o aca.o mom.a amo.a an we.ma m>.ms A ms.ma Ho.ma I ms.ma mm.aa mm.ma mm.sa m m mm mm 1 mm mm mm mm mm mm Aaascv om< omm.o HwO.H Omo.H N®N.H _ Nmm.H :mN.H mmm.a ::®.H mm mo.mH N:.ma N>.ma m>.HH mN.MH mm.oa HN.NH :m.OH M m as em so am so am so am Assaev use Nmm.d m:m.0 W mHP.N :mw.o sam.o- me.o m:m.o mmO.H _ mm ae.sa sa.m _ be.ma mm.m ss.sa mm.m . mm.ma om.m , a H mm om _ mm om mm om mm om Anxscv has pamp< 0Haooaoh_ 9H56< oaaoo>oh paoo< oaaoo>oh paso< oaaso>ohw oopsHomH po>Hamoo mooam uoxow H Hopoosaammxm M moanwfioz .wsanwfios pom smooam owe sooapfioooo mo soapooOM m on massage pase< use mafiso>oh Mom e0>homoo macaw ow enemas: one: 6 sense disappeared by the time of the final weighing (F = 3.15, df = l/h, p) 0.15). Aside from the suggestive reduced weight gain for juveniles in the E and Y conditions, the non-significant treatment and age x treatment effects indicate that growth and body maintenance were not appreciably affected.by any treatment. Table 7 presents mean brain weights as a function of condition and age group. Neither the treatment or age x treatment effects were significant (all §s< 1.1I0, all dfs = l/h, all ps > 0.28). The age effect was noteworthy(§ = 5.80, df = 1/h, p<0.03). The striking finding that juveniles had significantly heavier brains than did adults may be consistent with findings of increased brain weights in young animals reared in enriched environments (Bennett 22.31., 196k). This interpretation, however, does not satisfactorily explain the fact that the isolated juveniles, presumably those experiencing the least varied environment, had the second heaviest brains. In view of this difficulty in inter- pretation and the very small variance in brain weights, it seems wiser to reserve judgment until more animals can be studied. The possible age effect must be further investigated with more precise measuring techniques involving a variety of neuroanatomical structures such as those studied by Bennett and his associates (196h). Table 8 summarizes the means for brain weight divided by the final measure of body weight. Again, the treatment and age x 1*7 Table 7 Mean brain weight in grams as a function of condition and age group. Experimental g Yoked Sleep Deprived, Isolated Juvenile Adult ;Juvenile Adult Juvenile Adult Juvenile Adult Q'i ( 0.559 £0.h83 f 0.512 ? 0.521 0.525 ? 0.h96 0.557 ;0608 i - , I 3 SD? 0.000 0.031 0.031 0.000 0.000 0.000 0.0uu 0.000 all .7 . h8 Table 8 Mean brain weight to body weight ratio as a function of condition and.age group. Experimental .H_ ' Juvenile Adult {x 0.0377 0.03000 I . . SD; 0.000 {0.000 '- Yoked Juvenile Adult 0.0358 OOOOh h9 . 1 1 0.0316: 0.0357 I 0.000 i 0.00h Sleep Deprived Juvenile‘Adult 0.030h 0.000 i 0.005 Isolated Juvenile Adult _ 0.0337 0.0322 0.000 treatment effects were not Significant (all.§e<:l.00, all dfs = l/h, all ps)’0.68). The age effect was Significant (§.= 6.63, df = l/h, p}:0.02). This result, however is clearly related to the age differences in brain weight and the lack of age differences in final body weight. Therefore, these ratios add little additional information, but further underscore the necessity for more elaborate future research. Circadian Activity Measures. Figure 2 illustrates mean activity per half-hour for juvenile and adult animals as a func— tion of condition. Activity means, expressed in frequenty of movement, were computed for each 5-day interval. Contrasts dis- closed no treatment effect (all gs < 1.00, all dfs = l/II, all ps> 0.25). With respect to age x treatment interactions, the I yg. S and the Y XE: I + S contrasts revealed no significant interactions (all {a (1.00, all dfs = l/h, all ps7 0.25). However, the E IE.- I + S + Y age x treatment contrasts disclosed significant differences. Table 9 presents mean activity for the E and I + S + Y groups as a function of age and 5-day interval (pa = 11.07, 6.71 and 11.92; all dfs = 1/h; ps<0.03, 0.062, and 0.03 for the three 5-day intervals respectively). The age effect was significant for the first two 5-day intervals (Es = 9.78 and 10.98, dfs = 1/I+, and ps0.133). SO .aao>apowmmou mmsoaw copmfloma use .vm>auo0o ammam .aoapooo 60x0» .HmpsoaHaOQX0 psomoamoa :H: can .rm: .rw: .rm: one .zososdoam usefio>oa mo mayo» ca ommmmaoxm ma hpfl>wpo< .aohao pudendum H a escapades moswa Hmowpao> .mam>a0psw admin m 90 none a0>o povsasoo one: memo: .oowuwcooo mo oprossm a ma mamsHsm paste was oawom>5n pom usonnmaerwoo hpw>apom new: .m madman _ m m m m H m m H m m a m m (To 888 III namesake. I ON mu om mm ow AllA llOV 51 Activity means (standard deviations in parentheses) expressed in frequency of movement per half-hour for the E :3. Table 9 I + S + Y contrasts, as a function of age group and 5-day interval. S-Day Interval Juveniles ' Adults Juveniles Adults Juveniles ‘ Adults 5.90 (51975) (3.886) 11. 03 (11 388) H (3 677) 17. 51 (11 593) _ s 53 (2 267) -_. —. ...— ._.— .. -.a ..-—_...— I + S + Y 1. 02 (o 951) 16. 81 (12.198) 1. 37 ”(1. 268) 22. 78 (16.6h6) §_w(6. 689) 52 ” 21. 36 ""— (16.158) 11.07 6.71 11.92 .3 (age x treatment) df l/h l/h l/h 0.03 0.06 0.03 There seemed to be no systematic differences among conditions or age groups in the time of activity onset. All animals began activity shortly after light offset during the first and third 5-day intervals. During continuous darkness (second 5-day interval), all animals tended to begin activity successively earlier by 10 to 15 minute increments across successive 2h hour intervals. The finding is consistent with other circadian activity research on dark-active animals (Marler and Hamilton, 1967). In this ten- dency, treatment or age differences were not apparent. A further age difference was apparent during examination of data for indi- vidual subjects. For the 11 juveniles who survived the entire procedure, five were most active during continuous darkness the remaining six were most active during the third 5-day interval. For adults, nine of 12 were most active during continuous dark- ness and three most active during the third 5-day interval. There seemed to be no relation between interval of greatest activity and treatment condition. These age differences may be due to a true age x light schedule interaction. On the other hand, they may be artifacts of either diminishing differences in body weights or of the juveniles reaching sexual maturity between the second and third 5-day intervals. Changing differences in body weight may have differentially influenced the likelihood of movements being recorded between the second and third 5-day5. 53 The onset of sexual maturity, and hence oestrous, in juvenile. females may have directly increased female activity and indirect- ly increased.male activity. In view of the high sensitivity of the activity recording devices the weight-gain interpretation seems least likely. The sexual maturity interpretation is plausible, yet Layne (1968) concluded from a review of the ontogenetic literature on Peromyscus that P. m. bairdi; reach sexual maturity between 33 and ho days of age. This would place the onset of sexual maturity in juveniles well before activity recording even began. Nevertheless, more data are certainly necessary before any conclusions may be drawn on the age x light schedule interaction implied by this individual subject analysis. Table 10 summarizes the data on number of Significant periods computed from a Fourier analysis of movement frequency over each 5-day interval. Such significant period lengths ranged from a maximum of 29h.95 hours (projected) to a minimum of 5.05 hours. There seemed to be no systematic pattern among conditions, age groups, or lighting schedules either in period length or likeli- hood of a particular length to emerge repeatedly over successive 5-day intervals. This was true with only one exception; virtually each animal showed a significant period in the range of 23.50 to 2h.50 hours in length. Such periods emerged even during the 2h-hour 5-day interval having constant darkness. Such a finding 5h 0mm.o oma.m >m.oa OO.® mm mm mwm.a sea.a sm.m 00.6 Hm ma Pm:.m HOP.H a6.aa mm.m i. s eased assesses nossaonH m:m.o omN.H Fm.® mm.OH mm mm oma.m ma>.m 00.0H oo.mH paw we wam.o >~.m mm.m em.» a» a: pa56< mafioo>on po>wamon mmoam mm 00m.m OmH.N mm.® O0.0H mm mm omH.N o>®.N OO.HH No.0H an m: Hma.: mm:.m ~m.m mm.m up a: sasc< oaaso>ss some» 0mm.: mam.m oo.aa No.6 mm mm omm.m Hma.a >0.HH >®.HH am a: msm.m m-.m 00.6 mm.m as as easn< assesses Hmpooaaaomxm .Hs>aopoH hmcum dos .asoam 0mm sooapfiesoo mo soapoosm a no upofiaoa posOHMstfim mo assess one: OH memH Ed Ed Ed mm m m ow< mm m. m om< mm m. H mmd Hm>aopoH. been suggests that in the present study neither age or treatment factors disrupted the tendency for at least one activity period to depend on lighting schedule. Table 11 summarizes rhythmicity index data as a function of condition, age group, and 5-day interval. No contrast disclosed significant treatment or age x treatment effects (all Fa<:2.02, all dfs = l/h, all ps>.0.23). For the age effect, analyses indi- cated that across treatments juveniles had smaller rhythmicity indices. Tests for the first and third 5-day intervals disclosed significant age differences in rhythmicity (F = 8.55, 1.96, 39.1I0, all dfs .-. 1/h, p< 0.0h3, 0.2M, 0.003 for the three 5-day intervals respectively). These data suggest that neither treatment nor the age x treatment interaction affected regularity of activity patterns, and that animals in the juvenile group appeared to be more regular than their adult counterparts. Activity data indicated juveniles were also less active. Thus, this age effect may be simply an artifact of a tendency for the index itself to correlate with mean activity. A correlation between scores for activity and rhythmicity for all animals disclosed a Pearson product-moment r = 0.70. Implications. With respect to overall deve10pment, the body weight data suggest that prolonged LVF deprivation had no Significant effect. However, in view of the activity differences, 56 (.1 -"Ill-.I‘g.‘ ' mam.m mam.m mm.oa mo.m mm mm oso.s ssm.m as.aa mm.m aw ms .1. I'i. .- .' 1| me.m mam.m :m.oa HP.m as a: peace oaasu>ss oopsaomH 0mm.s oom.a mm.w mm.m mm mm wmm.m msa.a om.ma as.m am we Nom.w mwm.o mw.ma mo.m m . n as as pase< maaso>ou e0>Hamoo mooaw .soapmpSQEOo pm amorous .msoaw 0mm *O_D‘-VQO‘--p- ...-_..w--coa _..-_...-h———.-. a (I ...}.Illqlv... .1!!! I‘a‘li‘lwln 1'3! «I: 3...!!! (Ii-1.. ..sii sm mma.m ms6.s wa.s mo.m mm mm omH.s Hmo.a aH.0H Hm.m am we mem.s mwm.o mm.aa rs sa.m as sase< uaaso>se sumo» vn.d‘.‘cll..lcl.\lvl a! {3.1. 19...?! Ii'QE'IIIi! bit)! I, II to _ 1 m M mam.m mms.m mo.m mm.aa mm mm ms.m mma.s mm.aa ms.oa am we mma.m aas.m mm.m mm.s ss as easc<_.uaaso>sw ornamented asofipfieooo ho ooapoodm s as amuse apaoasnphsa one: HH OHQNH t.- » dill. c\ (I I‘ll, :‘II Illl“... . . . _ mm H a. m 1 $5.93 ow¢ mm M m Amhmuv mmd mm 1 M H , Amadov om¢ U Hs>aopaH . mama that such deprivation seemed to induce subsequently in P, p, Epipdi, it seems unlikely that anatomical differences will not be found with.more exacting techniques. Brain weight also seems too crude a measure to reveal treat- ment or age x treatment effects. The age effect Observed in brain weights is only suggestive of possible developmental differences in brain plasticity such as those noted by Bennett and his associates (196A): For further research at the CNS level, such techniques as brain section weighings, histological examination of cell-size and density, neurochemical analysis, and single cell activity recordings may be very useful. For example, it would be interesting to compare cell activity during various stages of arousal, brain weights, histological results, and acetylcholinesterase content in P, m, pgipdi for an age x treatment factorial design similar to that of the present re- search. Considering the Observed differences in activity, areas in the brain stem reticular formation should surely be included among the portions of brain selected for such analyses. These activity data in combination with the possibility that LVF provides non-visual input to the visual cortex (pointed out in Chapter I) suggest that the primary and secondary visual cortices may also be profitable areas for extended analysis. The data on circadian activity generally indicate that with measures employed here, age and age x treatment interactions can 58 be detected only in quantity of daily activity. Differences in the regularity of such activity were not convincingly disclosed with the exceptions of the age effect on the first and third rhythmicity indices. But even these differences are suSpect because of the high correlation noted between activity scores and the index itself. Thus, there seems to be a clear need for a measure of rhythmicity which is less dependent on quantity of activity. The significant age x treatment interaction observed in activity does not mesh well with any theory of LVF function. This difficulty arises because the data actually indicate a double, or mirror-image interaction. When treatment began on the 20th day of life, LVF-deprived animals were subsequently more active than animals in the three control groups, but when treatment began on the 53rd day, LVF-deprived animals were less active than controls. The Roffward, Muzio, and Dement hypothesis can explain the effect on juvenile animals in terms of CNS input regulation acting to counteract losses in endogenous input by increasing exogenous input. However, the mirror image effect Observed for adults is not so simply handled. According to the supplemental stimulation hypothesis of Roffwarg and his associates, LVF is a less important source of input for adults than for juveniles. Therefore, such a hypothesis would predict activity differences in the same 59 direction as those of the juvenile group, but decidedly less pronounced. This was clearly not the case. An alternative interpretation combines the supplemental sti- mulation hypothesis with findings of large increases in LVF propor- tions after LVF deprivation (LVF rebound). With such a combination, the age x treatment interaction could be explained solely in terms of differences in input regulation strategies. The LVF-deprived juveniles should have lost more input than LVF-deprived adults. In counteracting such losses, LVF rebound.was insufficient and required supplemental input via increases in exogenous activity. For LVF-deprived adults, LVF rebound was sufficient provided sleep time was adequate to allow required LVF. Adults could then get by with just slight increases in their sleep time. Such in- creases could account for the reduced activity observed in LVF- deprived adults. However, this "sufficient measure" interpretation involves almost pure speculation. Furthermore, both the former and latter interpretations fail to deal with the fact that 21 days post-treatment seems long enough for any input deficit correction to take place. Yet the data do not show any merging trends in activity among treatments or among age groups. On the contrary, activity differences seem to increase over the 21 days. Such results suggest that age x treatment effects are non-static and long term (if not permanent). Perhaps some of these questions could 6O be answered.with continuous polygraphic monitoring of arousal states in post-treatment animals. Difficulties also arise in trying to integrate the present data with other findings on the effects of LVF-deprivation. Other than the present findings for juveniles, there seems to be no systematic LVF-deprivation data for young animals. For adult subjects various measures have been used. Dement (1965) has noted in LVF-deprived cats increased sexual activity and a general inability to attend to cues in a Y-maze learning task. Dawson and associates (1965) have Observed in LVF-deprived rats threshold decreases in excitability of the auditory system and in electroconvulsive seizures. These findings would seem in direct opposition to the present findings of decreased activity in LVF-deprived P. m. m. One possible explanation of this apparent discrepancy is that LVF-deprived adult animals may be p932 inactive 22d hyper- responsive. Another Observation by Dement (1965) lends some support to such an interpretation. He noted that some LVF- deprived cats, presumably some of the same animals who could not learn his Y-maze, tended to seek dark corners when they were undisturbed. It may be that, had circadian activity been measured in Dement's LVF-deprived cats, less than normal activity would have been found. In qualitative Observations in the present research, it was noted that the post-treatment E animals tended to be most violent in avoiding capture for cage transfer and 61 most responsive when someone entered the laboratory. However, these speculations can only be tested with more extensive research. Thus the entire body of Study IV data can only be regarded as exploratory. The findings raise more questions than they answer. The use of locomotor activity as a dependent variable clearly indicated that LVF-deprivation during early life affects P, m, pgipdi differently than does such deprivation during adult- hood. Furthermore, such differences appear to increase over time. However, the remaining dependent variables proved too crude to further examine the phenomenon's biological underpinnings. Substantially more work will be necessary to uncover such under- pinnings. In summary, Study I demonstrated that adult 2. g, bairdi have both HVS and LVF. Study II demonstrated that LVF could be blocked in P, p, Epigdi'by restriction to a small pedestal over a shock grid. Study III compared the proportion of LVF in adult and juvenile E. p. pair—db and disclosed that mice over 53 days of age had 25.0% less LVF than did 20-day-old mice. 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