Perchance to dream? Primordial motor activity patterns in vertebrates from fish to mammals: their prenatal origin, postnatal persistence during sleep, and pathological reemergence during REM sleep behavior disorder.

An overview is presented of the literature dealing with sleep-like motility and concomitant neuronal activity patterns throughout the life cycle in vertebrates, ectothermic as well as endothermic. Spontaneous, periodically modulated, neurogenic bursts of non-purposive movements are a universal feature of larval and prenatal behavior, which in endothermic animals (i.e. birds and mammals) continue to occur periodically throughout life. Since the entire body musculature is involved in ever-shifting combinations, it is proposed that these spontaneously active periods be designated as 'rapid-BODY-movement' (RBM) sleep. The term 'rapid-EYE-movement (REM) sleep', characterized by attenuated muscle contractions and reduced tonus, can then be reserved for sleep at later stages of development. Mature stages of development in which sustained muscle atonia is combined with 'paradoxical arousal' of cortical neuronal firing patterns indisputably represent the evolutionarily most recent aspect of REM sleep, but more research with ectothermic vertebrates, such as fish, amphibians and reptiles, is needed before it can be concluded (as many prematurely have) that RBM is absent in these species. Evidence suggests a link between RBM sleep in early development and the clinical condition known as 'REM sleep behavior disorder (RBD)', which is characterized by the resurgence of periodic bouts of quasi-fetal motility that closely resemble RBM sleep. Early developmental neuromotor risk factors for RBD in humans also point to a relationship between RBM sleep and RBD.

Call it sleep -- what animals without backbones can tell us about the phylogeny of intrinsically generated neuromotor rhythms during early development.

A comprehensive overview is presented of the literature dealing with the development of sleep-like motility and neuronal activity patterns in non-vertebrate animals. it has been established that spontaneous, periodically modulated, neurogenic bursts of movement appear to be a universal feature of prenatal behavior. New empirical data are presented showing that such' seismic sleep' or 'rapid-body-movement' bursts in cuttlefish persist for some time after birth. Extensive ontogenetic research in both vertebrates and non-vertebrates is thus essential before current hypotheses about the phylogeny of motorically active sleep-like states can be taken seriously.

From neural plate to cortical arousal-a neuronal network theory of sleep derived from in vitro "model" systems for primordial patterns of spontaneous bioelectric activity in the vertebrate central nervous system.

In the early 1960s intrinsically generated widespread neuronal discharges were discovered to be the basis for the earliest motor behavior throughout the animal kingdom. The pattern generating system is in fact programmed into the developing nervous system, in a regionally specific manner, already at the early neural plate stage. Such rhythmically modulated phasic bursts were next discovered to be a general feature of developing neural networks and, largely on the basis of experimental interventions in cultured neural tissues, to contribute significantly to their morpho-physiological maturation. In particular, the level of spontaneous synchronized bursting is homeostatically regulated, and has the effect of constraining the development of excessive network excitability. After birth or hatching, this "slow-wave" activity pattern becomes sporadically suppressed in favor of sensory oriented "waking" behaviors better adapted to dealing with environmental contingencies. It nevertheless reappears periodically as "sleep" at several species-specific points in the diurnal/nocturnal cycle. Although this "default" behavior pattern evolves with development, its essential features are preserved throughout the life cycle, and are based upon a few simple mechanisms which can be both experimentally demonstrated and simulated by computer modeling. In contrast, a late onto- and phylogenetic aspect of sleep, viz., the intermittent "paradoxical" activation of the forebrain so as to mimic waking activity, is much less well understood as regards its contribution to brain development. Some recent findings dealing with this question by means of cholinergically induced "aroused" firing patterns in developing neocortical cell cultures, followed by quantitative electrophysiological assays of immediate and longterm sequelae, will be discussed in connection with their putative implications for sleep ontogeny.

Long-term relationships between cholinergic tone, synchronous bursting and synaptic remodeling.

Cholinergic neuromodulation plays key roles in the regulation of neuronal excitability, network activity, arousal, and behavior. On longer time scales, cholinergic systems play essential roles in cortical development, maturation, and plasticity. Presumably, these processes are associated with substantial synaptic remodeling, yet to date, long-term relationships between cholinergic tone and synaptic remodeling remain largely unknown. Here we used automated microscopy combined with multielectrode array recordings to study long-term relationships between cholinergic tone, excitatory synapse remodeling, and network activity characteristics in networks of cortical neurons grown on multielectrode array substrates. Experimental elevations of cholinergic tone led to the abrupt suppression of episodic synchronous bursting activity (but not of general activity), followed by a gradual growth of excitatory synapses over hours. Subsequent blockage of cholinergic receptors led to an immediate restoration of synchronous bursting and the gradual reversal of synaptic growth. Neither synaptic growth nor downsizing was governed by multiplicative scaling rules. Instead, these occurred in a subset of synapses, irrespective of initial synaptic size. Synaptic growth seemed to depend on intrinsic network activity, but not on the degree to which bursting was suppressed. Intriguingly, sustained elevations of cholinergic tone were associated with a gradual recovery of synchronous bursting but not with a reversal of synaptic growth. These findings show that cholinergic tone can strongly affect synaptic remodeling and synchronous bursting activity, but do not support a strict coupling between the two. Finally, the reemergence of synchronous bursting in the presence of elevated cholinergic tone indicates that the capacity of cholinergic neuromodulation to indefinitely suppress synchronous bursting might be inherently limited.

The sleep-like nature of early mammalian behavioral rhythms: Theoretical comment on Todd et al. (2010).

Early sleep patterns lack several of the major defining physiological criteria used to identify sleep states in adult animals, but many typical aspects of mature sleep can nevertheless be demonstrated at surprisingly early stages of development. In Todd, Gibson, Shaw, & Blumberg (2010), the ability to compensate for enforced sleep deprivation is found to be present already shortly after birth in laboratory rats, an altricial mammalian species. Whereas the brainstem is capable of resisting enforced wakefulness by an increasing "pressure" to fall asleep, "catch-up" replacement of the lost sleep by means of longer subsequent sleep durations requires forebrain participation. This investigation represents an initial foray into the theoretically important area of the ontogeny of homeostatic regulatory mechanisms for behaviorally crucial neurophysiological processes.

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: differential effects on intrinsically generated bioelectric discharges in a living 'model' system for slow-wave sleep activity.

Within the context of an updated thorough review of the literature concerning activity-dependent cerebro-cortical development, a survey is made of recent experiments which utilize spontaneous spike-trains as the dependent variable in rodent neocortex cultures when synaptic transmission is interfered with during early ontogeny. Emphasis is placed on the complexity of homeostatic adaptations to reduced as well as intensified firing. Two kinds of adaptation are distinguished: (i) rapid recovery (within several hours) towards baseline levels despite sustained blockade of excitatory synaptic transmission, and (ii) the generation of essentially normal firing patterns in cultures assayed in control medium following development in the presence of excitatory receptor blockers. The former category of homeostatic responses is strongly dependent on the type of preparation, with isolated organotypic explants showing greatly limited plasticity in comparison with co-cultures of matching contralateral pieces of cortical tissue. In such co-cultures, compensatory excitatory drive manifests itself even when all three known types of ionotropic glutamate receptors are chronically blocked, and is then mediated by (muscarinic) cholinergic mechanisms which normally do not contribute measurably to spontaneous activity. The rapid return of high levels of spontaneous firing during sustained selective glutamatergic receptor blockade appears to protect neuronal cultures treated in this way from becoming hyperexcitable. In particular, quasi-epileptiform paroxysmal bursting upon return to control medium, such as appears in preparations where bioelectric activity has been totally suppressed during network formation, fails to appear in chronically receptor blocked cultures. On the contrary, desensitization of blocked glutamate receptors, as a physiological compensation for the up-regulation of non-blocked receptors, could be demonstrated for both the AMPA and the NMDA glutamate receptor sub-types. This wide range of homeostatic responses underscores the importance of spontaneous neuronal discharges for setting and maintaining an optimal balance between excitatory and inhibitory mechanisms in developing neocortical networks.

Spontaneous neuronal burst discharges as dependent and independent variables in the maturation of cerebral cortex tissue cultured in vitro: a review of activity-dependent studies in live 'model' systems for the development of intrinsically generated bioelectric slow-wave sleep patterns.

A survey is presented of recent experiments which utilize spontaneous neuronal spike trains as dependent and/or independent variables in developing cerebral cortex cultures when synaptic transmission is interfered with for varying periods of time. Special attention is given to current difficulties in selecting suitable preparations for carrying out biologically relevant developmental studies, and in applying spike-train analysis methods with sufficient resolution to detect activity-dependent age and treatment effects. A hierarchy of synchronized nested burst discharges which approximate early slow-wave sleep patterns in the intact organism is established as a stable basis for isolated cortex function. The complexity of reported long- and short-term homeostatic responses to experimental interference with synaptic transmission is reviewed, and the crucial role played by intrinsically generated bioelectric activity in the maturation of cortical networks is emphasized.

Homeostatically regulated spontaneous neuronal discharges protect developing cerebral cortex networks from becoming hyperactive following prolonged blockade of excitatory synaptic receptors.

In order to further examine the role of spontaneous action potential (SAP) discharges in neocortical development, amino-acid-mediated synaptic transmission was selectively blocked in an improved organotypic neocortex culture preparation. Contralateral occipital cortex slices from neonatal rats were co-cultured for several weeks in a ventricle-to-ventricle orientation known to greatly enhance cyto-morphological and electrophysiological maturation. Such preparations are highly resistant to attempts to suppress neuronal firing by blocking ionotropic glutamate receptors: not only can kainate receptors partly substitute for NMDA- and AMPA-mediated neurotransmission when these receptors are pharmacologically blocked, but (muscarinic) cholinergic receptors also begin to drive SAP activity when the kainate receptors, too, are chronically blocked. Only tetrodotoxin proved able to eliminate SAPs altogether in these co-cultures, while GABAergic receptor blockade (using bicucculine) led to persistent epileptiform discharges. Treatment effects were assayed upon transfer to control medium by means of a quantitative analysis of spontaneously occurring polyneuronal spike trains. Total suppression of action potentials for several weeks (by tetrodotoxin treatment) led, as in earlier experiments, to strongly intensified burst firing upon transfer to control medium. Chronic glutamate receptor blocked cultures, on the other hand, showed only minor deviations from control firing levels and patterns when assayed in normal medium. Protection against the development of hyperactivity despite partial blockade of synaptic transmission was roughly proportional to the degree to which spontaneous firing during the treatment period approximated normal SAP levels. This homeostatic response to chronically reduced excitatory drive thus differs from earlier results using isolated organotypic cortex cultures, in which the restoration of SAP activity failed to prevent the development of network hyperactivity. Chronic bicucculine treatment, in contrast, had little or no homeostatic effect on SAP firing patterns; on the contrary, opposite to earlier findings using isolated occipital cortex explants, paroxysmal discharges persisted even after transfer to control medium.

Spontaneous neuronal discharge patterns in developing organotypic mega-co-cultures of neonatal rat cerebral cortex.

Sagittal slices of neonatal rat neocortex, extending from the prefrontal to the occipital area, were cultured separately or in pairs, oriented in such a way that axons projecting from the ventricular surface of each explant could innervate the other one. Functional connections were made between as well as within the explants, and spontaneous field potentials and associated action potentials occurred in variable bursts, and with varying degrees of synchrony. Spike-train analysis revealed that the activity patterns seen in these 'mega' co-cultures closely mimic 'tracé alternant' patterns, consisting of trains of burst discharges recurring several times per minute, which are characteristic for the immature intact cerebral cortex during slow-wave sleep. The prefrontal region was consistently less active than the occipital area but the two were qualitatively similar with respect to their patterns of neuronal firing. Isolated mega-cultures, on the other hand, despite their large size, exhibited only intermittent brief bursts that closely resembled those observed both in occipital cortex tissue fragments and in dissociated cell cultures. The mega-co-culture preparation thus appears to give the best currently available approximation to intrinsic cerebral discharge patterns in vivo.

Dynamics and plasticity in developing neuronal networks in vitro.

When dissociated cortical tissue is brought into culture, neurons readily grow out by forming axonal and dendritic arborizations and synaptic connections. These developing neuronal networks in vitro display spontaneous firing activity from about the end of the first week in vitro. When cultured on multielectrode arrays firing activity can be recorded from many neurons simultaneously over long periods of time. These experimental approaches provide valuable data for studying firing dynamics in neuronal networks in relation to an ongoing development of neurons and synaptic connectivity in the network. This chapter summarizes recent findings on the characteristics and developmental changes in the spontaneous firing dynamics. These changes include long-lasting transient periods of increased firing at individual sites on a time scale of days to weeks, and an age-specific repetitive pattern of synchronous network firing (network bursts) on a time scale of seconds. Especially the spatio-temporal organization of firing within network bursts showed great stability over many hours. In addition, a progressive day-to-day evolution was observed, with an initial broadening of the burst firing rate profile during the 3rd week in vitro (WIV) and a pattern of abrupt onset and precise spike timing from the 5th WIV onwards. These developmental changes are discussed in the light of structural changes in the network and activity-dependent plasticity mechanisms. Preliminary findings are presented on the pattern of spike sequences within network burst, as well as the effect of external stimulation on the spatio-temporal organization within network bursts.

Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro.

Paired organotypic explants from rat occipital cortex were cultured for up to three weeks in the presence of selective blockers of amino acid receptor blockers, during which period spontaneous action potential generation was monitored electrophysiologically. In contrast to isolated explants (Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E.and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185), which upregulated their initially depressed spontaneous bursting activity only under conditions of N-methyl D-aspartate (NMDA) receptor blockade, cross-innervated co-cultures showed a large degree of functional recovery even when combined NMDA and AMPA receptor blockade was carried out. This compensatory activity could be eliminated by acute addition of a selective kainate receptor blocker to the medium. When kainate along with AMPA and NMDA receptor mediated activity was chronically suppressed, however, considerable functional recovery--in the form of recurrent burst discharges--took place gradually over a period of three weeks in vitro. These spontaneous bursts disappeared rapidly upon treatment with the muscarinic receptor blocker, atropine, but continuous low-level firing emerged at the same time. Similar "tonic" background activity was induced in control cultures as well, but without any noticeable reduction in burst discharges. Co-cultured neocortex explants, in which cyto-morphological maturation proceeds to a far greater degree than in isolated explants (Baker, R.E.and van Pelt, J. (1997) Co-cultured but not isolated cortical explants display normal dendritic development: a longterm quantitative study. Dev. Brain Res., 98: 21-27) are evidently capable of an astonishing degree of functional compensation for loss of excitatory synaptic drive during development. It could be shown, furthermore, that such homeostatic responses are not mediated largely by a weakening of inhibitory mechanisms in the absence of spontaneous firing. Chronic inhibitory synaptic blockade, on the other hand, led to intensified bursting activity which gradually normalized over a 3-week culture period. The cellular basis for this reversal of the disinhibited state, as well as for the residual neuronal firing even after cholinergic mechanisms have been largely eliminated, is at present unknown. The degree to which immature cortical networks attempt to compensate for altered levels of physiological activity, as documented in the present report, is another indication of how important such activity can be for normal development (see Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E. and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks-an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185).. At the same time, the large variations in overall firing levels and "macro-scale" temporal patterns from culture to culture within a given series, despite all attempts at identical preparation of the explants, can only mean that the "set-points" for such regulation are themselves subject to unknown ontogenetic factors which, apparently, are nonuniformly distributed even within a restricted region of the neocortex. On the other hand, it was striking to note that, regardless of age or treatment, an unexpected degree of consistency in temporal patterning existed at "mini-" and "micro-" time-scales (viz., EEG delta and beta frequency ranges, respectively) even when network bursting tendencies became greatly reduced in favor of tonic firing.

Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.

Extracellular action potentials were recorded from developing dissociated rat neocortical networks continuously for up to 49 days in vitro using planar multielectrode arrays. Spontaneous neuronal activity emerged toward the end of the first week in vitro and from then on exhibited periods of elevated firing rates, lasting for a few days up to weeks, which were largely uncorrelated among different recording sites. On a time scale of seconds to minutes, network activity typically displayed an ongoing repetition of distinctive firing patterns, including short episodes of synchronous firing at many sites (network bursts). Network bursts were highly variable in their individual spatio-temporal firing patterns but showed a remarkably stable underlying probabilistic structure (obtained by summing consecutive bursts) on a time scale of hours. On still longer time scales, network bursts evolved gradually, with a significant broadening (to about 2 s) in the third week in vitro, followed by a drastic shortening after about one month in vitro. Bursts at this age were characterized by highly synchronized onsets reaching peak firing levels within less than ca. 60 ms. This pattern persisted for the rest of the culture period. Throughout the recording period, active sites showed highly persistent temporal relationships within network bursts. These longitudinal recordings of network firing have, thus, brought to light a reproducible pattern of complex changes in spontaneous firing dynamics of bursts during the development of isolated cortical neurons into synaptically interconnected networks.