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J Sleep Res 2005 14 91 101 The ontogeny of mammalian sleep a response to Frank and Heller 2003 MARK S BLUMBERG KARL A3 KARLSSON ADELE M H SEELKE and ETHAN J MOHNS Program in Behavioral and Cognitive Neuroscience Department of Psychology The University of Iowa Iowa City IA USA Accepted in revised form 14 September 2004 received 10 May 2004 SUMMARY In a recent review Frank and Heller 2003 provided support for their presleep theory of sleep development According to this theory rapid eye movement REM and nonrapid eye movement NonREM sleep in rats emerge from a common dissociated state only when the neocortical EEG differentiates at 12 days of age P12 Among the assumptions and inferences associated with this theory is that sleep before EEG differentiation is only sleeplike and can only be characterized using behavioral measures that the neural mechanisms governing presleep are distinct from those governing REM and NonREM sleep and that the presleep theory is the only theory that can account for developmental periods when REM and NonREM sleep components appear to overlap Evidence from our laboratory and others however refutes or casts doubt on these and other assertions For example infant sleep in rats is not sleeplike in that it satis es nearly every criterion used to characterize sleep across species In addition beginning as early as P2 in rats myoclonic twitching occurs only against a background of muscle atonia indicating that infant sleep is not dissociated and that electrographic measures are available for sleep characterization Finally improved techniques are leading to new insights concerning the neural substrates of sleep during early infancy Thus while many important develop mental questions remain the presleep theory at least in its present form does not accurately re ect the phenomenology of infant sleep KEYWORDS INTRODUCTION Beginning with the revitalization of interest in the embryonic origins of behavior in the 19605 sleep in early life has been viewed as a diffuse collection ofphasic and cyclic motor events that gradually coalesce with other sleep components to form the complex differentiated forms of sleep that are most easily recognized in adults Corner 1977 1985 Based on numerous studies of fetuses and infants in a variety of mammalian species it is widely believed that the earliest form of sleep is properly characterized as active sleep that is an immature form of REM sleep Jouvet Mounier et 11 1970 Parmelee et 11 1967 Roffwarg et 11 1966 Ruckebusch et 11 1977 Shimizu and Himwich 1968 Szeto and Hinman 1985 Correspondence Mark S Blumberg Department of Psychology The University of Iowa Iowa City IA 5242 USA Tel 319 335 2424 fax 319 335 0191 e mail mark blumberguiowaedu 2005 European Sleep Research Society active sleep atonia myoclonic twitching rat rem sleep slowwave sleep Accordingly it is thought that quiet sleep an immature form of slow wave sleep SWS emerges or becomes more prom inent as REM sleep s predominance diminishes during onto geny In a recent review Frank and Heller 2003 present evidence and argument to support an alternative theory of sleep development which they call the presleep theory According to their theory infant presleep is comprised of spontaneous dissociated activity that can be characterized as neither REM nor non REM sleep Accordingly any resemblance between the components of presleep and the components of mature forms of sleep is misleading Moreover they argue that the transformation of presleep into REM and non REM sleep does not occur until the neocortical EEG exhibits state dependent differentiated activity To their credit Frank and Heller explicitly delineate the assumptions and inferences that they believe differentiate the presleep theory from other perspectives Speci cally they 91 92 M S Blumberg et a1 argue a that sleep prior to EEG differentiation ie presleep is only sleep like b that only behavioral measures are available for characterizing sleep in rats during early infancy c that the spontaneous motor activity that characterizes presleep may outwardly resemble REM sleep but is in fact fundamentally distinct from this EEG de ned sleep state p 31 d that the central executive mechanisms that govern adult sleep are distinct from the mechanisms that function during presleep and e that evidence of overlapping REM and NREM sleep components during development demands a reconceptualization of sleep along the lines of their presleep theory We applaud Frank and Heller for helping to reinvigorate interest in sleep development With them we believe that this area of sleep research has been neglected for far too long and that we have much to learn about sleep and its neural substrates by studying rats and other species that give birth to altricial young With them we bemoan the maddeningly imprecise range of criteria p 30 that are used to de ne sleep states in infants and that make steady progress in this area so di icult And with them we believe that investigators interes ted in the origins of sleep should begin their experiments as early in development as possible and not restrict them to a single time point p 30 Despite these common goals and attitudes however recent ndings from our laboratory lead us to doubt each of the assumptions and inferences of the presleep theory outlined above In this response we review the basis for this doubt and in the process describe the conceptual perspective that underlies our approach SLEEP NOT PRESLEEP Infant sleep presents a challenge to sleep researchers because it differs from adult sleep on a number of important dimensions Perhaps most critically infant sleep is di cult to categorize because some sleep components are absent or intermittently expressed early in ontogeny For example the neocortical EEG does not exhibit state dependent differenti ation including slow wave activity until 115 120 days postconception in sheep Clewlow et 11 1983 Szeto and Hinman 1985 50 days postconception in guinea pigs Umans et 11 1985 approximately 32 weeks postconception in preterm human infants Dreyfus Brisac 1975 and until 12 days of age P12 in rats Frank and Heller 1997 Gramsbergen 1976 Mirmiran and Corner 1982 When attempting to describe and quantify sleep at ages before EEG differentiation or when measurements of EEG are not possible or are considered unreliable eg in human fetuses and preterm infants investigators have relied on other measures of state including body movements respiration heart rate and muscle tone Gramsbergen et 11 1970 Nijhuis et 11 1984 Parmelee et 11 1967 Perhaps inevit ably disagreement and confusion have emerged as different investigators have relied on different measures and adopted different criteria for categorizing sleep at ages prior to EEG differentiation Dreyfus Brisac 1970 Prechtl 1974 Interestingly a similar problem of categorization has been confronted by those investigating sleep in invertebrates such as the fruit y that do not possess a neocortex Hendricks et 11 2000 Shaw et 11 2000 Categorizing sleep in such non traditional species is relevant to the present discussion because as already mentioned neonatal rats do not exhibit state dependent neocortical EEG activity Accordingly if the neocortical EEG is considered the sine qua non of sleep then we are confronted with the odd juxtaposition whereby sleep in fruit ies is gaining acceptability even as sleep in infant rats is being relegated to the category of presleep We will return to the signi cance of the EEG for infant sleep later First however it is important to address the fundamen tal question of whether sleep during the pre EEG period is more properly categorized as sleep like as Frank and Heller suggest We address this question below by determining whether sleep in infant rats conforms to standard criteria used by other researchers to assess the existence of sleep in a variety of vertebrate and invertebrate species Campbell and Tobler 1984 Hendricks et 11 2000 Sleep is characterized by an absence of voluntary movements Behaviorally an infant rat housed in a thermoneutral humidi ed environment exhibits behavioral activation that entails high amplitude movements of the limbs such as stretching locomoting yawning and kicking Blumberg and Stolba 1996 Gramsbergen et 11 1970 such movements are often designated as voluntary coordinated or purposeful although each of these terms has limitations and are typically considered to indicate periods of wakefulness After brief bursts of awake activity a period of quiet ensues followed by the onset of myoclonic twitching of the limbs and tail Such bursts of twitching are typically considered to indicate periods of active Sleep Periods of twitching are almost always followed by the abrupt onset of high ampli tude awake behaviors thus completing the cycle Experi enced observers can reliably distinguish twitches from wake related movements especially when pups are observed in a supine position so that the limbs are unloaded and their movements are easily visualized Robinson et 11 2000 Clearly twitching does not fall into the category of voluntary movements Therefore infant rats satisfy this criterion Sleep is spontaneous occurring with a circadian rhythm Spontaneous rhythms occur in the absence of an external trigger that is when exogenous conditions remain constant Sleep in infant rats satis es this criterion in that the provisioning of a thermoneutral humidi ed environment permits ultradian cycling between sleep and wakefulness Gramsbergen et 11 1970 Jouvet Mounier et 11 1970 Kar lsson et 11 2004 There is currently little information concerning the onset of circadian sleep wake rhythms in infant rats 2005 European Sleep Research Society J Sleep Res 14 91 101 Sleep is reversible During periods when infant rats are twitching sensory stimulation is su icient to produce arousal Seelke and Blumberg 2004 thus distinguishing this state from coma and other irreversible pathological states Sleep is characterized by a speciesspeci c posture andor resting place that minimizes sensory stimulation In the wild infant rats are reared in species typical nests or burrows in which the combined in uences of the shelter mother and littermates ensures a warm humid environment that is conducive to sleep Sensory and or arousal thresholds increase during sleep It was recently shown that P8 rats exhibit an increased olfactory threshold during periods of myoclonic twitching relative to periods of wakefulness Seelke and Blumberg 2004 In addition in a recent sleep deprivation study using P5 rats see below it was shown that arousal threshold increases as sleep pressure intensi es Blumberg et al in press Sleep is regulated by a homeostatic mechanism Very few p d 39 mrli hm b n conducted in infant rats Feng et al 2001Frank et al 1998 Mirmiran et al 1981 1983 and in none of these previous studies has the effect of short term sleep deprivation been examined before P12 We conducted such an experiment at P5 using electric shock applied to the ank during periods of sleep Blumberg et al in press Over the course ofa 30 min deprivation period it was necessary to increase the intensity of the shock to maintain arousal an indication of increased sleep pressure Surprisingly during the rst 5 min of recovery sleep we also found a signi cant rebound in myoclonic twitching although there was no rebound in sleep duration This study indicates that some aspects of sleep are regulated homeostatically in early infancy in rats Sleep exhibits staterelated changes in neural function including those leading to decreased sensory input to the CNS Few studies have been conducted to examine state dependent neural activity in infant rats Nonetheless in two studies Corner and Bour 1984 Tamasy et al 1980 it was demon strated that neurons in the pontine and mesencephalic reticular formation exhibited state dependent activity at P8 and earlier More recently hippocampal theta and gamma rhythms were found to exhibit state dependency at P2 5 Karlsson and Blumberg 2003 Lahtinen et al 2001 We have also found neurons within the ventromedial medulla that re selectively during sleep at P8 Karlsson and Blumberg in press To our knowledge however there have been no studies that address the issue of neuronal mediation of decreased sensory input during sleep in infants as has been shown in adults Soja et al 2005 European Sleep Research Society J Sleep Res 14 91 101 The ontogeny of mammalian Sleep 93 2001 Thus although there has been a urry of recent progress we agree with Frank and Heller that more work needs to be done characterizing neuronal activity during sleep in the perinatal period p 30 The sleep state should be identi able as a stable species characteristic Sleep in infant rats clearly satis es this criterion in that it exhibits stable and predictable characteristics across litters and across time Although these eight criteria were devised to help charac terize sleep in the adults of diverse species the extent to which infant rats satisfy them is notable It seems then that Frank and Heller s designation of sleep at these early ages as presleep does not accurately re ect the phenomenology of infant sleep ADDING NUCHAL ATONIA AS AN ELECTROGRAPHIC CRITERION OF ACTIVE SLEEP Frank and Heller view the neocortical EEG as a central element in their theoretical approach For example they write that the emergence of REM and NREM sleep from presleep occurs approximately at the time of EEG differentiation in both altricial and precocial species pp 29 30 In a tautological rendition of this idea they write that most studies report that states similar to EEG determined sleep and NREM sleep seem to emerge from spontaneous fetal activity approximately at the time of EEG differentiation p 29 Elsewhere they state Although mechanisms governing EEG differentiation do not I I new cuiiy dAIVC Ofut ucl p the appearance of the EEG is a consistent hallmark of organized sleep behavior in these species and that the concordance of sleep parameters into recognizable sleep states invariably occurs near the time of EEG differentiation p 29 These and other similar comments leave little doubt that Frank and Heller view the EEG as an essential component for assessing sleep in infants as indeed it has been for many other investigators Our question however is whether this single component plays an inordinate role in their conceptualization committing an error akin to in their words restricting the de nition of REM sleep to the presence ofa single behavior p 30 Sleep researchers have long cautioned against the overgen eralization of sleep scoring methods established in one species or age to other species and ages For example in their manual for scoring sleep Rechtschaffen and Kales 1968 were clear in stating that it is well known that human infants show combinations of polygraphic features which defy classi cation by the criteria proposed in this manual A strict adherence to the proposed system would not yield an adequate description of infant sleep p 1 Thus at those ages where the EEG does not provide useful information we must rely on other measures for characterizing sleep 94 M S Blumberg et al In contrast Frank and Heller seem troubled that precursor sleep states are identi ed based solely on their behavioral similarities to EEG determined sleep pp 25 26 It is widely acknowledged however that the neocortical EEG is not causal to sleep but rather is a non causal correlate of sleep Although Frank and Heller come close to making this point mechanisms governing EEG differentiation do not necessarily drive the organization of other sleep phenomena p 29 Siegel 1999 is more clear Both active sleep in the neonate and REM sleep in the adult can be de ned by purely behavioral criteria We must remember that the EEG derives its value because of its correlation with behavioral measures of sleep If animals are responsive and locomoting we say they are awake even if their EEG is high in voltage a condition that can be created by certain brain lesions and by administration of the muscarinic receptor blocker atropine p 89 Conversely patients exhibiting a condition called alpha coma are behavi orally non responsive despite exhibiting a wake like EEG Jones 2000 To emphasize the reliance on behavioral as opposed to electrographic characterizations of sleep in infants at ages where the EEG is not a reliable indicator of sleep Frank and Heller introduce a novel nomenclature bAS and bQS for behavioral active sleep and behavioral quiet sleep respect ively We contend however that this nomenclature is not warranted in light of recent studies showing that infant rats as young as P2 a cycle rapidly between periods of high nuchal muscle tone and atonia and b exhibit myoclonic twitching only against a background of atonia Karlsson and Blumberg 2002 Karlsson et al 2004 In our view this early concor dance between twitching and atonia is not a coincidence but rather indicates a state that is closely related to REM sleep as others have concluded on the basis of less de nitive evidence Siegel 1999 We can now revisit the sleep wake cycle of infant rats already described above but now add information derived from the measurement of the nuchal EMG Karlsson and Blumberg 2002 During high amplitude awake behaviors nuchal tone is high and remains high for several seconds after the movements cease then pups remain behaviorally quiet as nuchal tone decreases this decrease in tone is often abrupt nally after a brief period in which pups exhibit behavioral quiescence against a background of muscle atonia myoclonic twitching begins and continues until one observes the simul taneous expression of high amplitude awake behaviors and the abrupt increase in nuchal muscle tone thus completing the cycle SPONTANEOUS MOTOR ACTIVITY AND CENTRAL EXECUTIVE MECHANISMS Spontaneous motor activity in the form ofmyoclonic twitching plays a central role in Frank and Heller s presleep hypothesis it is viewed as dissociated motor activity that is merely a form of spontaneous fetal activity that continues to be expressed ex utero in altricial species p 30 The notion that myoclonic twitching represents the postnatal expression of fetal motor activity was championed by Corner 1977 and it is clear that the two forms of behavior prenatal and postnatal are closely related Robinson et al 2000 Regardless the nding that twitching is tightly coupled with nuchal atonia at P2 as discussed above belies the notion that twitching in newborn rats is dissociated from other indicators of sleep Frank and Heller consider myoclonic twitching during presleep in infant rats to be the product of spinal mechanisms alone Although our own work supports the notion that spinal mechanisms contribute to spontaneous movements in fetuses Robinson et al 2000 and neonates Blumberg and Lucas 1994 Frank and Heller go further to claim that the normal cycling of high and low periods of spontaneous motility is not controlled by executive sleep centers p 30 by which they apparently mean brain mechanisms implicated in adult REM sleep In an earlier paper Frank et al 1997 they stated this idea even more clearly Brainstem midbrain nuclei important in mediating REM sleep expression do not mediate the expression of AS or AS myoclonia p 64 While we continue to actively explore the neural substrates of infant sleep there is already compelling evidence that supraspinal mechanisms are involved including mechanisms typically associated with adult sleep First our nding of a tight link between twitching and nuchal atonia argues for coordination of these two sleep components within the brain This inference gains perhaps its strongest support from evidence that as early as P7 activation of the ventromedial medulla produces nuchal atonia Karlsson and Blumberg in press just as it does in adults during REM sleep Hajnik et al 2000 It follows then that coordination of nuchal atonia and twitching must involve mechanisms within the brain including at least one neural mechanism that appears functionally identical to that involved in REM sleep in adults Secondly numerous additional ndings support the notion of central coordination of sleep states during the rst postnatal week For example sleep related expression of hippocampal theta Karlsson and Blumberg 2003 and eye muscle activity AMH Seelke and MS Blumberg unpublished observa tions sleep related modulation of olfactory threshold Seelke and Blumberg 2004 and homeostatic regulation of sleep Blumberg et al in press all imply more complex central organization of sleep than Frank and Heller s conceptualiza tion allows Thirdly we have reported substantial decreases in twitching by P8 rats after transections that are caudal but not rostral to the mesopontine region Kreider and Blumberg 2000 Frank and Heller question these ndings writing that they would have been more compelling had younger rats been examined as EEGs begin differentiating very early p 28 in the albino strain of rats used by Kreider and Blumberg The basis for Frank and Heller s suggestion that albino rats exhibit EEG differentiation approximately four days earlier ie at P8 than hooded rats ie at P12 is a methods paper that reports no comparison of strains and no measures of sleep Snead and Stephens 1983 Where such comparisons are available 2005 European Sleep Research Society J Sleep Res 14 91 101 however the evidence indicates that albino rats exhibit state related EEG differentiation only 1 day earlier than hooded rats Gramsbergen 1976 Additional ndings from our laboratory support the view that mesopontine and even hypothalamic mechanisms contribute to sleep regulation during early infancy For example we have found that P2 rats cycle rapidly ie approximately every 10 s between periods of high muscle tone and atonia and that these cycles elongate signi cantly during the rst postnatal week Karlsson et 11 2004 We have also observed in P2 rats that transections caudal to the mesopontine area result in animals that exhibit neither atonia nor myoclonic twitching KJE Karlsson and MS Blumberg unpublished observations as the transections are moved rostral to the mesopontine region atonia and twitching are restored In P8s transections that lie between the mesopontine area and the rostral hypothalamus produce rapid cycling that is characteristic of P2s without disrupting the coupling between nuchal atonia and myoclonic twitching suggesting that rostral hypothalamic structures perhaps those within the ventrolateral preoptic area Saper et 11 2001 play an increasing role in sleep regulation over the rst postnatal week Frank and Heller also examine neuropharmacological differences between infants and adults to support their claim of distinct neurophysiological mechanisms For example they note that the cholinergic system well known to be an important modulator of REM sleep in adults is extremely immature p 28 in infants at an age when active sleep predominates Frank and Heller support this claim in part by citing evidence concerning neurotransmitter and receptor levels in infants For example they cite research in infant mice showing that brainstem and cortical acetylcholine levels are at 10 of adult levels Evidence from rats however tells a somewhat different story Speci cally in infant rats during the rst postnatal week acetylcholine levels are 40 of adult whole brain values cholinergic markers appear sooner in the pons and medulla and then decrease over the next 2 weeks before increasing to adult values around the sixth postnatal week Johnston and Silverstein 1998 Semba 1992 More over although levels of acetylcholine and muscarinic receptor densities are reduced in fetuses and neonates there is a compensatory increase in the responsiveness of muscarinic receptors to cholinergic stimulation Heacock et 11 1987 Johnston and Silverstein 1998 In other words the choliner gic system of infants may not be functionally immature Even more signi cant for the present discussion however is that infusions of the cholinergic agonist carbachol into the pontine reticular formation of adult rats do not evoke the powerful and reliable REM sleep promoting responses that they do in cats Boissard et 11 2002 This striking species difference does not mean that the cholinergic system plays no role in the activation of REM sleep and its components in rats indeed carbachol infusions into the nucleus subcoeruleus of rats activates P waves Datta et 11 1998 But such species differences do highlight the danger of supposing an essential linkage between a complex behavioral process documented 2005 European Sleep Research Society J Sleep Res 14 91 101 The ontogeny of mammalian Sleep 95 across many species and any single neural mechanism docu mented in one or only a few species And if this caution is valid for comparisons between species it should also be valid for comparisons within species at different ages For the eld of infant sleep research to move forward we need detailed developmental information concerning the sleep related functioning of speci c nuclei and the role of speci c neurotransmitters Thus in addition to acetylcholine and the monoamines which Frank and Heller discuss there are many other neurotransmitters whose roles in adult but not infant sleep have been established including glutamate orexin adenosine and GABA Arrigoni et 11 2001 Boissard et 11 2002 2003 Datta 2002 Kiyashchenko et 11 2001 Nitz and Siegel 1997 Of particular importance for our understanding of developmental changes in sleep may be the transition in GABA s effects from excitatory to inhibitory during early development Ben Ari 2002 Ultimately then our goal should be to understand the developing contributions of these and other transmitter systems to infant sleep regulation not merely to document differences between infants and adults THE SIGNIFICANCE OF OVERLAPPING REM AND NREM SLEEP COMPONENTS Our reading of Frank and Heller s papers on sleep develop ment suggests to us that their reconceptualization of sleep was inspired by a single observation Speci cally that with the onset of a differentiated EEG at P12 the age at which their observations began they observed episodes where cortical slow waves were accompanied by myoclonic twitches Frank and Heller 1997 Such periods of half activated REM sleep Jouvet Mounier et 11 1970 were interpreted as a blended state comprising both NREM and REM sleep components ie slow waves and myoclonic twitches respectively Although this overlap sometimes occurs at boundaries between states Frank and Heller contend that the overlap was more evenly distributed across periods of sleep p 26 Noting that these periods of overlap diminish as NREM sleep develops Frank and Heller conclude that they represent instances of adult like NREM sleep emerging from bAS p 26 The observation of slow waves or spindles as has been reported in kittens Jouvet Mounier et 11 1970 during periods of twitching requires explanation Before we take these observations at face value however consider the following we have occasionally observed myoclonic twitches in 1week old rats that appeared to occur against a back ground of high nuchal muscle tone only to nd on closer inspection that the nuchal muscle became brie y atonic at the moment when the twitch was observed Because Frank and Heller 1997 used 10 s epochs to evaluate their sleep data and because they do not report their method of evaluating twitching and whether they distinguished twitching from other movements such as startles the contention that slow waves and myoclonic twitching overlap at P12 deserves closer scrutiny 96 M S Blumberg et a1 But even if an overlap between some sleep components is a reliable nding such a nding does not invalidate the milestones in sleep development that have already been reached In this regard it is signi cant that the process of sleep development is orderly and cumulative in that previously integrated components remain integrated as new components are added Thus when the differentiated EEG comes on line at P12 the temporal disintegration of the previously achieved concordance between twitching and atonia is not observed We repeat The possible overlap ofsleep components at one age does not negate the processes ofsleep development that have already occurred CONCLUDING COMMENTS Frank and Heller have performed an important service by highlighting the need for a theory of sleep development that accounts for the available data and that makes explicit predictions At this point in time however we believe that those features of the presleep theory that distinguish it from the precursor theory however construed are not supported by the available evidence Regardless our view is that the challenge of understanding sleep development is to explain each of the individual components of active sleep in develop mental time and investigate the processes by which these multiple components coalesce cohere and self organize dur ing ontogeny p 4 Blumberg and Lucas 1996 Accordingly any theory of sleep development must account for both the addition and integration of sleep components as well as changes in sleep persistence during ontogeny see Dreyfus Brisac 1970 and Corner 1985 for similar perspectives Critical gaps remain in our understanding of infant sleep For example it remains unclear whether infant sleep is best considered a single state comprising tonic ie atonia and phasic eg twitching components or two states akin to active and quiet sleep In making this determination we will want to avoid mere semantic distinctions and focus instead on the organization of multiple components and their neural sub strates Longitudinal assessments may prove extremely valu able for establishing the developmental relations between infant and adult sleep indeed a recent longitudinal study in two strains of rats has provided intriguing evidence for a correspondence between active and REM sleep and between quiet and SWS Dugovic and Turek 2001 An obvious additional gap in our knowledge concerns the neural circuitry underlying sleep in newborns and how it changes over the course of development This is a daunting task that encompasses changes in the neural mechanisms that activate and integrate sleep components and that alter the temporal regulation of sleep Although we nd little support for Frank and Heller s contention that infant sleep is governed by distinct neurophysiologic mechanisms there is little doubt that the neural and neuropharmacological substrates of sleep undergo signi cant changes during ontogeny Rather than envisioning these substrates as distinct however it seems more likely that component circuits are elaborated and integrated over time similar to the process by which twitches are spinally generated in fetuses and embryos and are gradually brought under the control of more rostral structures during ontogeny in chicks Corner 1973 Provine 1973 as well as rats Blumberg and Lucas 1994 Kreider and Blumberg 2000 Robinson et al 2000 We should be wary however of the notion that this process is merely one of rostralization indeed the fact that rostral hypothalamic structures are already regulating the expression of sleep in rats during the rst postnatal week Karlsson et al 2004 suggests that neural sleep circuits develop concurrently throughout their rostro caudal extent We end by noting that the eld of animal learning made its greatest strides as investigators turned to simple animal models of learning in invertebrates eg Aplysia Kandel and Schwartz 1982 and well de ned model systems in adult mammals eg eye blink conditioning Gormezano etal 1983 Thompson 1986 Similarly sleep researchers are considering the potential bene ts of using simple animal models including invertebrates Hendricks et al 2000 We believe that the infants of altricial species such as rats also offer uniquely valuable opportunities for making rapid pro gress in our 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1985 153 467 471 Unresolved issues in sleep ontogeny a response to Blumberg et al MARCOS G FRANK1 andH CRAIG HELLER2 1Department of Neuroscience University of Pennsylvania School of Medicine Philadelphia PA and 2Department of Biological Sciences Stanford University Stanford CA USA Accepted in revised form 14 September 2004 received 10 May 2004 KEYWORDS In 2003 we presented a review of sleep ontogeny in the hope of stimulating new research in this area Frank and Heller 2003 Based on our own work and that of others we proposed that both REM and NREM sleep develop from a form of spontaneous fetal activity presleep that outwardly resembles REM sleep While our manuscript was in review Blumberg et al published ndings in support of the traditional view that REM sleep is present at birth in altricial species like the rat Their response to our review is thoughtful and their studies are important because they address basic questions about neonatal sleep However the rather strong conclusions in their response are premature It is di cult to assess their views fully because many of the studies on which they are based are unpublished see Table 1 We will deal with their peer reviewed work respond to speci c interpretations of our studies and discuss three main issues raised in their response the relationship between presleep and sleep the use of behavior and electro physiology in neonatal state assignments and subcortical evidence for REM sleep in the pre EEG prior to EEG differentiation period Correspondence Marcos Frank Department of Neuroscience Uni versity ofPennsylvania School of Medicine 215 Stemmler Hall 35th amp Hamilton Walk Philadelphia PA 19104 6074 USA Tel 215 746 0388 OHice 215 746 0389 Lab fax 215 573 9050 email mgfmailmedupennedu atonia ontogeny PRESLEEP AND SLEEP In a classic straw man argument Blumberg et al present a distorted version of our ideas which they then attack in a rather lengthy discussion We have never stated that presleep is not a type of sleep Nor have we claimed that presleep bears no relation to later developing REM and NREM sleep Our argument is that presleep is not a homolog of REM sleep and instead represents a common precursor to REM and NREM sleep By analogy embryonic limb buds are still limbs but they are not hands ngers or feet As discussed in our review the precise relationship between presleep and adult sleep states is unclear but it is quite possible that events in presleep are incorporated into later appearing REM and NREM sleep To what extent this primordial state satis es general criteria for sleep is an interesting question but this is largely irrelevant to our main points EEG TWITCHES AND SLEEP An old debate in the eld ofneonatal sleep research concerns the choice of criteria in assigning vigilance states Our position on this matter as discussed in our review is straightforward EEG differentiation is a consistent hallmark of the appearance of states that satisfy multiple behavioral and neurophysiological criteria for sleep This suggests that organized sleep states appear 2005 European Sleep Research Society J Sleep Res 14 91 101 Table 1 Critical ndings cited by Blumberg et al in their response to our review Claim Status at time of our letter Karlsson and Blumberg 2002 Karlsson and Blumberg 2003 Myoclonia is coupled to atonia P2 rat pups exhibit hippocarnpal theta in A Behavioral sleep deprivation Unpublished increases arousal threshold Behavioral sleep deprivation Unpublished increases myoc onia State speci c ring of ventromedial Unpublished me u a Coupling of hippocampal theta to Unpublished REMs and homeostatic regulation Transections caudal to mesopontine Unpublished area abolish atonia and myoclonia around this time This seems reasonable because most scientists agree that mammalian sleep is a brain phenomenon and eld potentials like the cortical EEG are measurements of brain activity Could there be states homologous to EEG de ned REM and NREM sleep before the appearance of differentiated cortical EEGs Yes but this has not been conclusively demon strated Until this happens we nd it useful to classify states as either behaviorally determined behavioral active sleep bAS or behavioral quiet sleep bQS or electrographically determined REM and NREMslow wave sleep In our original study of neonatal rats we used three separate parameters to determine states video records of behavior EMG and EEG recordings The behavioral criteria we used to determine AS and Q8 were similar to those used by Jouvet Mounier Frank and Heller 1997 Jouvet Mounier et al 1970 In contrast Blumberg et al predominantly use a single measurement motor activity to determine vigilance states Based on the analysis of motor activity they claim to nd evidence of REM sleep a uni ed state characterized by myoclonia coupled to atonia before the appearance of differentiated EEGs Karlsson and Blumberg 2002 We do not nd their data persuasive In the latter study atonia is measured with nuchal EMG recordings Strictly speaking such measurements do not reveal REM sleep atonia per se only gross changes in motor activity Karlsson and Blumberg 2002 Sleeping reptiles also have atonia based on EMG recordings but there is little evidence that reptiles have REM sleep Frank 1999 To prove the presence of REM sleep atonia during AS they must show that REM sleep inhibitory mechanisms are active in newborn rats Presleep and REM sleep 99 Chase and Morales 2000 Fenik et al 2004 This is highly unlikely because GABA and glycine are excitatary not inhibitory in the CNS in the rst postnatal week Leinekugel et al 1999 Singer and Berger 2000 Indeed the absence of glycinergic inhibition in newborn animals may explain the unusual nding that re exes normally suppressed during REM sleep are enhanCed during pre EEG prior to EEG differenti ation AS Chase 1971 In short the signi cance of this study has been overstated The uni ed state they report is simply a period of hypotonia coupled with spontaneous twitches a condition noted decades ago and just as easily explained as a form of spontaneous fetal activity A surprising oversight by Blumberg et al is that they have never shown that cortical EEGs are undifferentiated in their neonatal rats As shown in Table 2 there is some variability regarding the age when differentiated EEGs are rst reported As noted by Gramsbergen 1976 the variability in the timing of EEG development may be due to differences in rat strains For example EEG patterns similar to human trace alternant are detected during sleep in the Lister strain around P9 P10 and transform into SWS EEG patterns by P11 Gramsbergen 1976 We have observed a similar developmental pattern in the Long Evans strain Other studies report differentiated EEGs at much younger ages including ages assumed by Blumberg to be pre EEG The Snead and Stephens 1983 study is informative because it shows that differentiated EEGs in alert rat pups are reported very early in the strain used by Blumberg Sleep speci c EEGs were not examined but in other studies sleep EEGs usually appear near the time wake EEGs are detected Aristakesyan and Vataev 1993 Grams bergen 1976 Jouvet Mounier et al 1970 In light of these ndings Blumberg et al should verify that their rats are pre EEG at the ages they claim Although Blumberg et al do not measure cortical EEGs in their studies they suggest that our observation of EEG slow waves coupled with low EMG activity and REMs and twitches is because of rapid cycling of micro REM sleep and NREM sleep episodes This was not the case The epochs in question were continuous periods of delta waves with no intervening periods of EEG attening as might be expected if there were smaller cycles of fast low amplitude waves interposed between slower waves Spectral analysis of the EEG in these epochs con rmed that these epochs were not mixtures of synchronized and desynchronized EEG states Frank and Heller 1997 Table 2 Onto genetic appearance of state Speci c EEGS vanes by Straw EEG type EEGs reported in Age at dz erentiation Wistar CTX AS QS and wake P5 7 Aristakesyan and Vataev 1993 Wistar CTX Not reported Dux et a 2 Sp a e D wl CTX HPP Wake P1 2 Snead and Stephens 1983 Lister black amp white CTX AS QS and wake P9 Gramsbergen 1976 J ouvet Mounier 1976 CTX AS QS and wake P6 P8 AS wake P11QS Postnatal age at birth has been set to 0 P0 for all studies uvet Mounier did not report the strain use CTX cerebral cortex HPP hippocampus 2005 European Sleep Research Society J Sleep Res 14 91 101 100 M G Frank and H C Heller SUBCORTICAL SIGNS OF REM SLEEP As discussed in our original review the evidence for subcor tical signs of REM sleep prior to cortical EEG differentiation is equivocal Many studies do not nd evidence of REM sleep and those cited by Blumberg et al which were included in our review are open to interpretation For example Corner and Bour 1984 recorded from the FTG area in Wistar rat pups an area now known to be more important in movement than REM sleep Siegel 2000 In light of what we now know about FTG neurons heightened ring in the FTG during AS in pre EEG rats is more likely related to heightened twitchiness and not the activity of REM sleep mechanisms A very interesting nding from this study is that cells more active in AS than wake appear abruptly in the second postnatal week Corner and Bour 1984 which is precisely the time when differen tiated EEGs are commonly detected in the Wistar strain The Lahtinen et al 2002 study used very small numbers of rats so the results should be cautiously interpreted The Tamasy et al 1980 study currently provides the best evidence for state speci c activity in the pre EEG period In this study increases in medial reticular formation and basal forebrain multi unit activity were reported during AS in newborn rats However the identity of these neurons is not known nor was the correspondence between behavioral measurements and multi unit activity quantitatively assessed The situation has not improved much in the last few years Karlsson and Blumberg 2003 report hippocampal theta in P2 rats but these ndings have not been con rmed in another study Leinekugel et al 2002 It is also unclear if the theta recorded by Karlsson and Blumberg is identical to theta normally generated in REM sleep First they nd that 786 of theta bouts in newborn rats occur during a state that sounds like quiet Sleep or quiet woke still periods with low medium motor tone without myoclonia and only 214 during AS as de ned by low motor tone and sporadic twitching Karlsson and Blumberg 2003 Secondly the theta frequencies they report are much faster than REM sleep theta in older rat pups with differentiated EEGs We and others have shown that at ages when the determination of state is not in doubt theta in REM sleep centers around 45 50 Hz and shifts towards faster frequencies during later development Bronzino et al 1987 Cavoy and Delacour 1981 Frank and Heller 1997 Karlsson and Blum berg on the contrary report theta centered at 80 Hz in P2 rats Are they proposing that REM sleep theta is rst fast then slow and then fast again with later development Considering that the majority of theta bouts do not occur in a state characterized by atonia and myoclonia is it possible that they are recording something else entirely There are different types of hippocam pal theta even a form triggered by rotating restrained rats onto their backs a situation remarkably similar to the restrained supine recording position used by Karlsson and Blumberg Buzsaki 2002 Gavrilov et al 1995 Karlsson and Blumberg 2003 Moreover it is unlikely that REM sleep theta could be present at the ages they claim The inhibitory neurotransmitter GABA which is critical in the generation of REM sleep theta is excitatory in newborn rats Buzsaki 2002 Leinekugel et al Their discussion of cholinergic maturation is misleading Reports of heightened muscarinic receptor sensitivity in the neonatal rat were primarily based on assays that use the neuronal uptake of labeled substrates such as 3H inositol Immature neurons tend to be more permeable to these substances Consequently cholinergic receptor mediated metabolism of the labeled substrate appears higher in imma ture neurons than in mature neurons When this is corrected for no such super sensitivity in muscarinic receptors is detected Lee et al 1990 More recent studies of cholinergic nuclei important in REM sleep also cast doubt on assertions made by Blumberg et al As discussed in our original review levels of the synthetic enzyme for acetylcholine ChAT in the rat LDT are extremely low in the rst postnatal week Ninomiya et al 2001 Neurons in the rat PPN are also quite immature in terms of their bursting properties and their inhibition by 5 HT agonists until the third fourth postnatal week Kobayashi et al 2003 Blumberg et al then suggest that as carbacholinfusions do not enhance REM sleep in adult rats this somehow diminishes the importance of cholinergic maturation in REM sleep ontogeny The carbachol issue is a red herring and adds little to the discussion As acknowledged by Blumberg et al acetylcholine is a critical component ofmammalian REM sleep Even if it were true that carbachol does not increase REM sleep amounts in adult rats this would not invalidate previous ndings showing the importance of acetylcholine in rat REM sleep In fact carbachol has been shown to increase REM sleep amounts in adult rats Kubin 2001 Kumar and Raju 2001 Wetzel et al 2003 The variable effects of carbachol in rats as is true in cats is likely due to methodological differences between studies eg location volume ofinjection diffusion from site Kubin 2001 The relevant facts are that the cholinergic system is crucial for rat REM sleep and its immaturity in newborn rats poses a major problem for these investigators CONCLUDING REMARKS In conclusion it is worthwhile to remind the reader why we championed the presleep theory Blumberg et al are correct to point out that our interest in this idea was partly motivated by our nding of states that looked like NREM sleep based on the EEG but were behaviorally indistinguishable from REM sleep We were also motivated by the fact that upon close inspection of the available literature we found little evidence to support the traditional view that neonatal behavioral sleep is homol ogous to EEG de ned states In our opinion this was a view founded on assumptions as much it was on empirical ndings The studies by Blumberg et al are promising but in our opinion Blumberg et al have not demonstrated an orderly and cumulative addition of REM sleep components to a pre existing REM sleep state Whatever the ultimate outcome of these different ideas about sleep ontogeny there is no disagreement over the 2005 European Sleep Research Society J Sleep Res 14 91 101 importance of studying sleep during infancy SIDS is the most obvious stimulant for further research but there is much more to be learned by examining sleep ontogenesis 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