Impaired vocal communication, sleep-related discharges, and transient alteration of slow-wave sleep in developing mice lacking the GluN2A subunit of N-methyl-d-aspartate receptors.

OBJECTIVE: Glutamate-gated N-methyl-d-aspartate receptors (NMDARs) are instrumental to brain development and functioning. Defects in the GRIN2A gene, encoding the GluN2A subunit of NMDARs, cause slow-wave sleep (SWS)-related disorders of the epilepsy-aphasia spectrum (EAS). The as-yet poorly understood developmental sequence of early EAS-related phenotypes, and the role of GluN2A-containing NMDARs in the development of SWS and associated electroencephalographic (EEG) activity patterns, were investigated in Grin2a knockout (KO) mice. METHODS: Early social communication was investigated by ultrasonic vocalization (USV) recordings; the relationship of electrical activity of the cerebral cortex with SWS was studied using deep local field potential or chronic EEG recordings at various postnatal stages. RESULTS: Grin2a KO pups displayed altered USV and increased occurrence of high-voltage spindles. The pattern of slow-wave activity induced by low-dose isoflurane was altered in Grin2a KO mice in the 3rd postnatal week and at 1 month of age. These alterations included strong suppression of the delta oscillation power and an increase in the occurrence of the spike-wave bursts. The proportion of SWS and the sleep quality were transiently reduced in Grin2a KO mice aged 1 month but recovered by the age of 2 months. Grin2a KO mice also displayed spontaneous spike-wave discharges, which occurred nearly exclusively during SWS, at 1 and 2 months of age. SIGNIFICANCE: The impaired vocal communication, the spike-wave discharges occurring almost exclusively in SWS, and the age-dependent alteration of SWS that were all seen in Grin2a KO mice matched the sleep-related and age-dependent manifestations seen in children with EAS, hence validating the Grin2a KO as a reliable model of EAS disorders. Our data also show that GluN2A-containing NMDARs are involved in slow-wave activity, and that the period of postnatal brain development (postnatal day 30) when several anomalies peaked might be critical for GluN2A-dependent, sleep-related physiological and pathological processes.

An Optogenetic Approach for Investigation of Excitatory and Inhibitory Network GABA Actions in Mice Expressing Channelrhodopsin-2 in GABAergic Neurons.

UNLABELLED: To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo SIGNIFICANCE STATEMENT: We report a novel optogenetic approach for investigating excitatory and inhibitory GABA actions in mice with conditional expression of channelrhodopsin-2 in GABAergic interneurons. This approach shows a developmental excitatory-to-inhibitory switch in the actions of GABA on glutamatergic neurons in neocortical and hippocampal slices from neonatal mouse pups in vitro, but also reveals inhibitory GABA actions in the neonatal mouse neocortex and hippocampus in vivo.

Traumatic alterations in GABA signaling disrupt hippocampal network activity in the developing brain.

Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl(-)](i)) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl(-)](i) correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl(-)](i) demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures.

Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate.

GABA depolarizes immature neurons because of a high [Cl(-)](i) and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite DL-3-hydroxybutyrate (DL-BHB) (4 mM), lactate (4 mM), or pyruvate (5 mM) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4-7), plasma D-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mM, respectively. Then, we show that DL-BHB (4 mM) and pyruvate (200 µM) do not affect (i) the driving force for GABA(A) receptor-mediated currents (DF(GABA)) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA(A) receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high nonphysiological concentrations of pyruvate (5 mM) reduced DF(GABA) and blocked GDPs. Therefore, DL-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high nonphysiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.

Spontaneous activity in developing sensory circuits: Implications for resting state fMRI.

The immature brain spontaneously expresses unique patterns of electrical activity that are believed to contribute to the development of neuronal networks. Certain electrographic features of this activity, particularly modulation on an infraslow time scale, resemble activity patterns observed in the mature brain at 'rest', loosely defined as the absence of an investigator imposed task. However, it is not clear whether the immature activity patterns observed at rest are precursors of the spontaneous neuronal activity that forms resting state networks in the adult. Here, we review recent studies that have explored the generative mechanisms of resting state activity during development in the primary sensory systems of premature human neonates and neonatal rodents. The remarkable hypothesis suggested by this work is that while resting state activity during the pre- and possibly near-term period can bear superficial resemblance to adult activity it is fundamentally different in terms of function and origin. During early development spontaneous thalamocortical activity in primary sensory regions is determined largely by transitory generators in the sensory periphery. This is in contrast to the adult, where spontaneous activity generated within thalamocortex, particularly by cortico-cortical connections, dominates. We therefore suggest a conservative interpretation of developmental mapping studies which are based on indirect measurement of activity (e.g. fMRI), or on the partitioning of EEG frequency using bands derived from adult studies. The generative mechanisms for brain activity at early ages are likely different from those of adults, and may play very different roles; for example in circuit formation as opposed to attention.

"Slow activity transients" in infant rat visual cortex: a spreading synchronous oscillation patterned by retinal waves.

A primary feature of the preterm infant electroencephalogram is the presence of large infra-slow potentials containing rapid oscillations called slow activity transients (SATs). Such activity has not been described in animal models, and their generative mechanisms are unknown. Here we use direct-current and multisite extracellular, as well as whole-cell, recording in vivo to demonstrate the existence of regularly repeating SATs in the visual cortex of infant rats before eye opening. Present only in absence of anesthesia, SATs at postnatal day 10-11 were identifiable as a separate group of long-duration (approximately 10 s) events that consisted of large (>1 mV) negative local-field potentials produced by the summation of multiple bursts of rapid oscillatory activity (15-30 Hz). SATs synchronized the vast majority of neuronal activity (87%) in the visual cortex before eye opening. Enucleation eliminated SATs, and their duration, interevent interval, and sub-burst structure matched those of phase III retinal waves recorded in vitro. Retinal waves, however, lacked rapid oscillations, suggesting that they arise centrally. Multielectrode recordings showed that SATs spread horizontally in cortex and synchronize activity at coactive locales via the rapid oscillations. SATs were clearly different from ongoing cortical activity, which was observable as a separate class of short bursts from postnatal day 9. Together, our data suggest that, in vivo, early cortical activity is primarily determined by peripheral inputs-retinal waves in visual cortex-that provide excitatory input, and by thalamocortical circuitry, which transforms this input to beta oscillations. We propose that the synchronous oscillations of SATs participate in the formation of visual circuitry.

Recurrent neonatal seizures result in long-term increases in neuronal network excitability in the rat neocortex.

Neonatal seizures are associated with a high likelihood of adverse neurological outcomes, including mental retardation, behavioral disorders, and epilepsy. Early seizures typically involve the neocortex, and post-neonatal epilepsy is often of neocortical origin. However, our understanding of the consequences of neonatal seizures for neocortical function is limited. In the present study, we show that neonatal seizures induced by flurothyl result in markedly enhanced susceptibility of the neocortex to seizure-like activity. This change occurs in young rats studied weeks after the last induced seizure and in adult rats studied months after the initial seizures. Neonatal seizures resulted in reductions in the amplitude of spontaneous inhibitory postsynaptic currents and the frequency of miniature inhibitory postsynaptic currents, and significant increases in the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in pyramidal cells of layer 2/3 of the somatosensory cortex. The selective N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate eliminated the differences in amplitude and frequency of sEPSCs and mEPSCs in the control and flurothyl groups, suggesting that NMDA receptors contribute significantly to the enhanced excitability seen in slices from rats that experienced recurrent neonatal seizures. Taken together, our results suggest that recurrent seizures in infancy result in a persistent enhancement of neocortical excitability.

Early motor activity drives spindle bursts in the developing somatosensory cortex.

Sensorimotor coordination emerges early in development. The maturation period is characterized by the establishment of somatotopic cortical maps, the emergence of long-range cortical connections, heightened experience-dependent plasticity and spontaneous uncoordinated skeletal movement. How these various processes cooperate to allow the somatosensory system to form a three-dimensional representation of the body is not known. In the visual system, interactions between spontaneous network patterns and afferent activity have been suggested to be vital for normal development. Although several intrinsic cortical patterns of correlated neuronal activity have been described in developing somatosensory cortex in vitro, the in vivo patterns in the critical developmental period and the influence of physiological sensory inputs on these patterns remain unknown. We report here that in the intact somatosensory cortex of the newborn rat in vivo, spatially confined spindle bursts represent the first and only organized network pattern. The localized spindles are selectively triggered in a somatotopic manner by spontaneous muscle twitches, motor patterns analogous to human fetal movements. We suggest that the interaction between movement-triggered sensory feedback signals and self-organized spindle oscillations shapes the formation of cortical connections required for sensorimotor coordination.

Cholinergic modulation of spindle bursts in the neonatal rat visual cortex in vivo.

Acetylcholine (ACh) is known to shape the adult neocortical activity related to behavioral states and processing of sensory information. However, the impact of cholinergic input on the neonatal neuronal activity remains widely unknown. Early during development, the principal activity pattern in the primary visual (V1) cortex is the intermittent self-organized spindle burst oscillation that can be driven by the retinal waves. Here, we assessed the relationship between this early activity pattern and the cholinergic drive by either blocking or augmenting the cholinergic input and investigating the resultant effects on the activity of the rat visual cortex during the first postnatal week in vivo. Blockade of the muscarinic receptors by intracerebroventricular, intracortical, or supracortical atropine application decreased the occurrence of V1 spindle bursts by 50%, both the retina-independent and the optic nerve-mediated spindle bursts being affected. In contrast, blockade of acetylcholine esterase with physostigmine augmented the occurrence, amplitude, and duration of V1 spindle bursts. Whereas direct stimulation of the cholinergic basal forebrain nuclei increased the occurrence probability of V1 spindle bursts, their chronic immunotoxic lesion using 192 IgG-saporin decreased the occurrence of neonatal V1 oscillatory activity by 87%. Thus, the cholinergic input facilitates the neonatal V1 spindle bursts and may prime the developing cortex to operate specifically on relevant early (retinal waves) and later (visual input) stimuli.

Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.

The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

Early patterns of electrical activity in the developing cerebral cortex of humans and rodents.

During prenatal and early postnatal development, the cerebral cortex exhibits synchronized oscillatory network activity that is believed to be essential for the generation of neuronal cortical circuits. The nature and functional role of these early activity patterns are of central interest in neuroscience. Much of the research is performed in rodents and in vitro, but how closely do these model systems relate to the human fetal brain? In this review, we compare observations in humans with in vivo and in vitro rodent data, focusing on particular oscillatory activity patterns that share many common features: delta brushes, spindle bursts and spindle-like oscillations. There is considerable evidence that the basic functional properties of immature cortical networks are conserved through mammalian evolution, making the neonatal rodent an excellent model for studying early cortical activity and associated plasticity during the developmental period corresponding to the human fetal stage. This review is part of the INMED/TINS special issue "Nature and nurture in brain development and neurological disorders", based on presentations at the annual INMED/TINS symposium (http://inmednet.com/).

Postnatal changes in somatic gamma-aminobutyric acid signalling in the rat hippocampus.

During postnatal development of the rat hippocampus, gamma-aminobutyric acid (GABA) switches its action on CA3 pyramidal cells from excitatory to inhibitory. To characterize the underlying changes in the GABA reversal potential, we used somatic cell-attached recordings of GABA(A) and N-methyl-D-aspartate channels to monitor the GABA driving force and resting membrane potential, respectively. We found that the GABA driving force is strongly depolarizing during the first postnatal week. The strength of this depolarization rapidly declines with age, although GABA remains slightly depolarizing, by a few millivolts, even in adult neurons. Reduction in the depolarizing GABA driving force was due to a progressive negative shift of the reversal potential of GABA currents. Similar postnatal changes in GABA signalling were also observed using the superfused hippocampus preparation in vivo, and in the hippocampal interneurons in vitro. We also found that in adult pyramidal cells, somatic GABA reversal potential is maintained at a slightly depolarizing level by bicarbonate conductance, chloride-extrusion and chloride-loading systems. Thus, the postnatal excitatory-to-inhibitory switch in somatic GABA signalling is associated with a negative shift of the GABA reversal potential but without a hyperpolarizing switch in the polarity of GABA responses. These results also suggest that in adult CA3 pyramidal cells, somatic GABAergic inhibition takes place essentially through shunting rather than hyperpolarization. Apparent hyperpolarizing GABA responses previously reported in the soma of CA3 pyramidal cells are probably due to cell depolarization during intracellular or whole-cell recordings.

Neuronal activity patterns in the developing barrel cortex.

The developing barrel cortex reveals a rich repertoire of neuronal activity patterns, which have been also found in other sensory neocortical areas and in other species including the somatosensory cortex of preterm human infants. The earliest stage is characterized by asynchronous, sparse single-cell firing at low frequencies. During the second stage neurons show correlated firing, which is initially mediated by electrical synapses and subsequently transforms into network bursts depending on chemical synapses. Activity patterns during this second stage are synchronous plateau assemblies, delta waves, spindle bursts and early gamma oscillations (EGOs). In newborn rodents spindle bursts and EGOs occur spontaneously or can be elicited by sensory stimulation and synchronize the activity in a barrel-related columnar network with topographic organization at the day of birth. Interfering with this early activity causes a disturbance in the development of the cortical architecture, indicating that spindle bursts and EGOs influence the formation of cortical columns. Early neuronal activity also controls the rate of programed cell death in the developing barrel cortex, suggesting that spindle bursts and EGOs are physiological activity patterns particularly suited to suppress apoptosis. It remains to be studied in more detail how these different neocortical activity patterns control early developmental processes such as formation of synapses, microcircuits, topographic maps and large-scale networks.

Pharmacodynamics of the Glutamate Receptor Antagonists in the Rat Barrel Cortex.

Epipial application is one of the approaches for drug delivery into the cortex. However, passive diffusion of epipially applied drugs through the cortical depth may be slow, and different drug concentrations may be achieved at different rates across the cortical depth. Here, we explored the pharmacodynamics of the inhibitory effects of epipially applied ionotropic glutamate receptor antagonists CNQX and dAPV on sensory-evoked and spontaneous activity across layers of the cortical barrel column in urethane-anesthetized rats. The inhibitory effects of CNQX and dAPV were observed at concentrations that were an order higher than in slices in vitro, and they slowly developed from the cortical surface to depth after epipial application. The level of the inhibitory effects also followed the surface-to-depth gradient, with full inhibition of sensory evoked potentials (SEPs) in the supragranular layers and L4 and only partial inhibition in L5 and L6. During epipial CNQX and dAPV application, spontaneous activity and the late component of multiple unit activity (MUA) during sensory-evoked responses were suppressed faster than the short-latency MUA component. Despite complete suppression of SEPs in L4, sensory-evoked short-latency multiunit responses in L4 persisted, and they were suppressed by further addition of lidocaine suggesting that spikes in thalamocortical axons contribute ∼20% to early multiunit responses. Epipial CNQX and dAPV also completely suppressed sensory-evoked very fast (∼500 Hz) oscillations and spontaneous slow wave activity in L2/3 and L4. However, delta oscillations persisted in L5/6. Thus, CNQX and dAPV exert inhibitory actions on cortical activity during epipial application at much higher concentrations than in vitro, and the pharmacodynamics of their inhibitory effects is characterized by the surface-to-depth gradients in the rate of development and the level of inhibition of sensory-evoked and spontaneous cortical activity.

Retinal waves trigger spindle bursts in the neonatal rat visual cortex.

During visual system development, the light-insensitive retina spontaneously generates waves of activity, which are transmitted to the lateral geniculate nucleus. The crucial question is whether retinal waves are further transmitted to the cortex and influence the early cortical patterns of activity. Using simultaneous recordings from the rat retina and visual cortex during the first postnatal week in vivo, we found that spontaneous retinal bursts are correlated with spindle bursts (intermittent network bursts associated with spindle-shape field oscillations) in the contralateral visual cortex (V1). V1 spindle bursts could be evoked by electrical stimulation of the optic nerve. Intraocular injection of forskolin, which augments retinal waves, increased the occurrence of V1 spindle bursts. Blocking propagation of retinal activity, or removal of the retina reduced the frequency, but did not completely eliminate the cortical spindle bursts. These results indicate that spontaneous retinal waves are transmitted to the visual cortex and trigger endogenous spindle bursts. We propose that the interaction between retinal waves and spindle bursts contributes to the development of visual pathways to the cortex.

Developmental Changes in Sensory-Evoked Optical Intrinsic Signals in the Rat Barrel Cortex.

Optical Intrinsic Signal imaging (OISi) is a powerful technique for optical brain studies. OIS mainly reflects the hemodynamic response (HR) and metabolism, but it may also involve changes in tissue light scattering (LS) caused by transient cellular swelling in the active tissue. Here, we explored the developmental features of sensory-evoked OIS in the rat barrel cortex during the first 3 months after birth. Multispectral OISi revealed that two temporally distinct components contribute to the neonatal OIS: an early phase of LS followed by a late phase of HR. The contribution of LS to the early response was also evidenced by an increase in light transmission through the active barrel. The early OIS phase correlated in time and amplitude with the sensory-evoked electrophysiological response. Application of the Modified Beer-Lambert Law (MBLL) to the OIS data revealed that HR during the early phase involved only a slight decrease in blood oxygenation without any change in blood volume. In contrast, HR during the late phase manifested an adult-like increase in blood volume and oxygenation. During development, the peak time of the delayed HR progressively shortened with age, nearly reaching the stimulus onset and overlapping with the early LS phase by the fourth postnatal week. Thus, LS contributes to the sensory-evoked OIS in the barrel cortex of rats at all ages, and it dominates the early OIS phase in neonatal rats due to delayed HR. Our results are also consistent with the delayed blood oxygen level dependent (BOLD) signal in human preterm infants.

Seizure-induced damage in the developing human: relevance of experimental models.

A considerable amount of money and effort is spent every year investigating the effects of seizure on the developing rodent brain. A critical question is the relevance of these studies to children. The goal of this chapter is to review the relationship between seizures during early development and cognitive impairment in children and rodents. While the majority of children with epilepsy have normal cognitive development, a small group of children with frequent, recurrent seizures show progressive cognitive impairment. Likewise, in rodent models recurrent seizures during early development are associated with cognitive impairment and histological changes including mossy fiber sprouting and reduced neurogenesis. Status epilepticus is associated with a lower morbidity and mortality rate in children than in adults. Status epilepticus in rodent models is associated with less cell loss and cognitive impairment than in adults. While rodent studies can offer a great deal of insight into mechanisms of seizure-induced brain damage, they also have significant limitations. No animal models have yet been developed that mimic human epileptic syndromes, such as infantile spasms, Lennox-Gastaut syndrome, or the severe myoclonic epilepsies. In addition, rodent studies supply only crude measures of learning and memory. Disturbances of language or higher cortical functions such as visual or auditory processing cannot be tested in animal models.

Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons.

We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A) channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by RuBi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A) receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A) channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A)-Rs mediated signals in small cell compartments.

Auditory stimuli mimicking ambient sounds drive temporal "delta-brushes" in premature infants.

In the premature infant, somatosensory and visual stimuli trigger an immature electroencephalographic (EEG) pattern, "delta-brushes," in the corresponding sensory cortical areas. Whether auditory stimuli evoke delta-brushes in the premature auditory cortex has not been reported. Here, responses to auditory stimuli were studied in 46 premature infants without neurologic risk aged 31 to 38 postmenstrual weeks (PMW) during routine EEG recording. Stimuli consisted of either low-volume technogenic "clicks" near the background noise level of the neonatal care unit, or a human voice at conversational sound level. Stimuli were administrated pseudo-randomly during quiet and active sleep. In another protocol, the cortical response to a composite stimulus ("click" and voice) was manually triggered during EEG hypoactive periods of quiet sleep. Cortical responses were analyzed by event detection, power frequency analysis and stimulus locked averaging. Before 34 PMW, both voice and "click" stimuli evoked cortical responses with similar frequency-power topographic characteristics, namely a temporal negative slow-wave and rapid oscillations similar to spontaneous delta-brushes. Responses to composite stimuli also showed a maximal frequency-power increase in temporal areas before 35 PMW. From 34 PMW the topography of responses in quiet sleep was different for "click" and voice stimuli: responses to "clicks" became diffuse but responses to voice remained limited to temporal areas. After the age of 35 PMW auditory evoked delta-brushes progressively disappeared and were replaced by a low amplitude response in the same location. Our data show that auditory stimuli mimicking ambient sounds efficiently evoke delta-brushes in temporal areas in the premature infant before 35 PMW. Along with findings in other sensory modalities (visual and somatosensory), these findings suggest that sensory driven delta-brushes represent a ubiquitous feature of the human sensory cortex during fetal stages and provide a potential test of functional cortical maturation during fetal development.

Antiepileptic effects of endogenous beta-hydroxybutyrate in suckling infant rats.

Physiological ketosis is a hallmark of metabolism in suckling infants. However, little is known on the impact of physiological ketosis on brain excitability. We addressed this question in suckling rats in vivo. 16-channel extracellular field potential recordings were performed from somatosensory barrel cortex at postnatal days 5-9 non-anaesthetized rat pups. Seizures were induced by the volatile convulsant agent flurothyl. One hour after blockade of physiological ketogenesis using combined administration of beta-oxidation inhibitors mercaptoacetate, insulin and glucose to prevent hypoglycemia, we found no significant change in the flurothyl-induced electrographic seizures. However, build-up of seizures during two repetitive flurothyl applications was strongly aggravated in the animals with blocked ketogenesis. The effect of ketogenesis inhibitors was reversed by exogenous beta-hydroxybutyrate. Diazepam exerted anticonvulsive action both under physiological ketosis and after blockade of ketogenesis, and bumetanide had no significant anticonvulsive effects in both conditions. Thus, physiological ketosis reduces excitability in the immature brain and elimination of physiological ketosis results in elimination of this anticonvulsant effect. Our study raises concern that the changes in diet, and pharmacological manipulations such as glucose infusion, and pathologies such as hyperinsulinism which break natural ketosis, may be a potential risk factor for epileptogenesis in nursing infants.