Dexmedetomidine as a Short-Use Analgesia for the Immature Nervous System.

Pain management in neonates continues to be a challenge. Diverse therapies are available that cause loss of pain sensitivity. However, because of side effects, the search for better options remains open. Dexmedetomidine is a promising drug; it has shown high efficacy with a good safety profile in sedation and analgesia in the immature nervous system. Though dexmedetomidine is already in use for pain control in neonates (including premature neonates) and infants as an adjunct to other anesthetics, the question remains whether it affects the neuronal activity patterning that is critical for development of the immature nervous system. In this study, using the neonatal rat as a model, the pharmacodynamic effects of dexmedetomidine on the nervous and cardiorespiratory systems were studied. Our results showed that dexmedetomidine has pronounced analgesic effects in the neonatal rat pups, and also weakly modified both the immature network patterns of cortical and hippocampal activity and the physiology of sleep cycles. Though the respiration and heart rates were slightly reduced after dexmedetomidine administration, it might be considered as the preferential independent short-term therapy for pain management in the immature and developing brain.

Early suppression of excitability in subcortical band heterotopia modifies epileptogenesis in rats.

Malformations of cortical development represent a major cause of epilepsy in childhood. However, the pathological substrate and dynamic changes leading to the development and progression of epilepsy remain unclear. Here, we characterized an etiology-relevant rat model of subcortical band heterotopia (SBH), a diffuse type of cortical malformation associated with drug-resistant seizures in humans. We used longitudinal electrographic recordings to monitor the age-dependent evolution of epileptiform discharges during the course of epileptogenesis in this model. We found both quantitative and qualitative age-related changes in seizures properties and patterns, accompanying a gradual progression towards a fully developed seizure pattern seen in adulthood. We also dissected the relative contribution of the band heterotopia and the overlying cortex to the development and age-dependent progression of epilepsy using timed and spatially targeted manipulation of neuronal excitability. We found that an early suppression of neuronal excitability in SBH slows down epileptogenesis in juvenile rats, whereas epileptogenesis is paradoxically exacerbated when excitability is suppressed in the overlying cortex. However, in rats with active epilepsy, similar manipulations of excitability have no effect on chronic spontaneous seizures. Together, our data support the notion that complex developmental alterations occurring in both the SBH and the overlying cortex concur to creating pathogenic circuits prone to generate seizures. Our study also suggests that early and targeted interventions could potentially influence the course of these altered developmental trajectories, and favorably modify epileptogenesis in malformations of cortical development.

Diadenosine-Polyphosphate Analogue AppCH2ppA Suppresses Seizures by Enhancing Adenosine Signaling in the Cortex.

Epilepsy is a multifactorial disorder associated with neuronal hyperexcitability that affects more than 1% of the human population. It has long been known that adenosine can reduce seizure generation in animal models of epilepsies. However, in addition to various side effects, the instability of adenosine has precluded its use as an anticonvulsant treatment. Here we report that a stable analogue of diadenosine-tetraphosphate: AppCH2ppA effectively suppresses spontaneous epileptiform activity in vitro and in vivo in a Tuberous Sclerosis Complex (TSC) mouse model (Tsc1+/-), and in postsurgery cortical samples from TSC human patients. These effects are mediated by enhanced adenosine signaling in the cortex post local neuronal adenosine release. The released adenosine induces A1 receptor-dependent activation of potassium channels thereby reducing neuronal excitability, temporal summation, and hypersynchronicity. AppCH2ppA does not cause any disturbances of the main vital autonomous functions of Tsc1+/- mice in vivo. Therefore, we propose this compound to be a potent new candidate for adenosine-related treatment strategies to suppress intractable epilepsies.

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.

Transient microstructural brain anomalies and epileptiform discharges in mice defective for epilepsy and language-related NMDA receptor subunit gene Grin2a.

OBJECTIVE: The epilepsy-aphasia spectrum (EAS) is a heterogeneous group of age-dependent childhood disorders characterized by sleep-activated discharges associated with infrequent seizures and language, cognitive, and behavioral deficits. Defects in the GRIN2A gene, encoding a subunit of glutamate-gated N-methyl-d-aspartate (NMDA) receptors, represent the most important cause of EAS identified so far. Neocortical or thalamic lesions were detected in a subset of severe EAS disorders, and more subtle anomalies were reported in patients with so-called "benign" phenotypes. However, whether brain structural alterations exist in the context of GRIN2A defects is unknown. METHODS: Magnetic resonance diffusion tensor imaging (MR-DTI) was used to perform longitudinal analysis of the brain at 3 developmental timepoints in living mice genetically knocked out (KO) for Grin2a. In addition, electroencephalography (EEG) was recorded using multisite extracellular electrodes to characterize the neocortical activity in vivo. RESULTS: Microstructural alterations were detected in the neocortex, the corpus callosum, the hippocampus, and the thalamus of Grin2a KO mice. Most MR-DTI alterations were detected at a specific developmental stage when mice were aged 30 days, but not at earlier (15 days) or later (2 months) ages. EEG analysis detected epileptiform discharges in Grin2a KO mice in the third postnatal week. SIGNIFICANCE: Grin2a KO mice replicated several anomalies found in patients with EAS disorders. Transient structural alterations detected by MR-DTI recalled the age-dependent course of EAS disorders, which in humans start during childhood and show variable outcome at the onset of adolescence. Together with the epileptiform discharges detected in young Grin2a KO mice, our data suggested the existence of early anomalies in the maturation of the neocortical and thalamocortical systems. Whereas the possible relationship of those anomalies with sleep warrants further investigations, our data suggest that Grin2a KO mice may serve as an animal model to study the neuronal mechanisms of EAS disorders and to design new therapeutic strategies.

Dynamic Changes from Depolarizing to Hyperpolarizing GABAergic Actions during Giant Depolarizing Potentials in the Neonatal Rat Hippocampus.

During development, GABA exerts depolarizing action on immature neurons and, acting in synergy with glutamate, drives giant depolarizing potentials (GDPs) in the hippocampal network. Yet, blockade of the GABA(A) receptors transforms GDPs to epileptiform discharges suggesting dual, both excitatory and inhibitory, actions of GABA in the immature hippocampal network. However, the nature of this dualism in early GABA actions is poorly understood. Here we characterized the dynamics of synaptic currents mediated by GABA(A) and glutamate receptors through an estimation of the changes in their conductance and driving forces in neonatal rat CA3 pyramidal cells during GDPs. We found that depolarizing GABAergic and glutamatergic currents act in synergy at the GDPs' onset. However, during the peak of the population discharge, the inward synaptic current was essentially mediated by glutamate receptors whereas GABA currents transiently switched their direction from depolarizing to hyperpolarizing as a result of neuronal depolarization above the GABA(A) reversal potential. Thus, the action of GABA on CA3 pyramidal cells dynamically changes during GDPs from excitatory at the GDPs' onset to inhibitory at the GDPs' peak. We propose that the dynamic changes in GABA actions occurring during GDPs enable GABAergic interneurons not only to initiate the discharge of pyramidal cells but also to control excitation in the recurrent CA3 network preventing epileptiform synchronization. SIGNIFICANCE STATEMENT: During development GABA exerts a depolarizing action on immature neurons. However, at the network level the effects of GABA are complex involving both excitatory and inhibitory actions. Here we show that GABA actions critically depend on the network state. Although GABA depolarizes neurons at rest and at the onset of population bursts, it transiently becomes hyperpolarizing at the peak of the population bursts. These dynamic changes in GABA actions enable GABAergic interneurons not only to initiate the network discharge but also to control excitation to prevent epileptiform synchronization.

Imprecise Whisker Map in the Neonatal Rat Barrel Cortex.

The somatosensory barrel cortex in rodents contains a topographic map of the facial whiskers where each cortical barrel is tuned to a corresponding whisker. However, exactly when this correspondence is established during development and how precise the functional topography of the whisker protomap is at birth, before the anatomical formation of barrels, are questions that remain unresolved. Here, using extracellular and whole-cell recordings from the barrel cortex of 0- to 7-day-old (P0-7; P0 = day of birth) rat pups in vivo, we report a low level of tuning to the principal whisker at P0-1, with multiple adjacent whiskers evoking large multi- and single-unit responses and excitatory postsynaptic currents in cortical neurons. Additionally, we found broad and largely overlapping projection fields (PFs) for neighboring whiskers in the barrel cortex at P0-1. Starting from P2-3, a segregated whisker map emerged, characterized by preferential single whisker tuning and segregated whisker PFs. These results indicate that the functional whisker protomap in the somatosensory cortex is imprecise at birth, that for 2-3 days after birth, whiskers compete for the cortical target territories, and that formation of a segregated functional whisker map coincides with emergence of the anatomical barrel map.

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.

Simple and Efficient 3D-Printed Superfusion Chamber for Electrophysiological and Neuroimaging Recordings In Vivo.

In vitro and in vivo experimentation in the central nervous system are effective approaches to study its functioning. Manipulations in vitro are characterized by easy experimental control and stable experimental conditions. However, transferring these advantages to in vivo research remains technically and ethically challenging, preventing many research teams from acquiring critical recordings in their animal models. In order to transfer the benefits of in vitro experimentation to in vivo experimentation, we developed a suite of 3D-printed tools (a superfusion chamber with an independent brain presser and animal stand). Using the immature rat barrel cortex as a model, we show that our set of tools (further "superfusion preparation") provides stable conditions for electrophysiological and neuroimaging recordings in the neonatal rat neocortex in vivo Highly correlated intracellular and extracellular activity was recorded during spontaneous and evoked cortical activity, supporting the possibility of simultaneous long-lasting electrophysiological recordings from a single cortical column in vivo The optical intrinsic signal of evoked cortical responses was also recorded from the skull-free neocortex, suggesting the effective combination of the superfusion preparation with neuroimaging approaches. Modulation of immature activity by epicortical application of pharmacological agents via superfusion equally supports the use of the superfused cortex preparation in pharmacological screening. In addition to high efficiency (in affordability, reliability, and ease of use in vivo), the 3D-printed set of tools developed should reduce animal use, supporting the 3Rs principle (Replacement, Reduction, and Refinement) of ethical use of animals.

The rapid developmental rise of somatic inhibition disengages hippocampal dynamics from self-motion.

Early electrophysiological brain oscillations recorded in preterm babies and newborn rodents are initially mostly driven by bottom-up sensorimotor activity and only later can detach from external inputs. This is a hallmark of most developing brain areas, including the hippocampus, which, in the adult brain, functions in integrating external inputs onto internal dynamics. Such developmental disengagement from external inputs is likely a fundamental step for the proper development of cognitive internal models. Despite its importance, the developmental timeline and circuit basis for this disengagement remain unknown. To address this issue, we have investigated the daily evolution of CA1 dynamics and underlying circuits during the first two postnatal weeks of mouse development using two-photon calcium imaging in non-anesthetized pups. We show that the first postnatal week ends with an abrupt shift in the representation of self-motion in CA1. Indeed, most CA1 pyramidal cells switch from activated to inhibited by self-generated movements at the end of the first postnatal week, whereas the majority of GABAergic neurons remain positively modulated throughout this period. This rapid switch occurs within 2 days and follows the rapid anatomical and functional surge of local somatic GABAergic innervation. The observed change in dynamics is consistent with a two-population model undergoing a strengthening of inhibition. We propose that this abrupt developmental transition inaugurates the emergence of internal hippocampal dynamics.

Effects of urethane and isoflurane on the sensory evoked response and local blood flow in the early postnatal rat somatosensory cortex.

Functional studies in the central nervous system are often conducted using anesthesia. While the dose-dependent effects of anesthesia on neuronal activity have been extensively characterized in adults, little is known about the effects of anesthesia on cortical activity and cerebral blood flow in the immature central nervous system. Substitution of electrophysiological recordings with the less-invasive technique of optical intrinsic signal imaging (OIS) in vivo allowed simultaneous recordings of sensory-evoked functional response and local blood flow changes in the neonatal rat barrel cortex. Using OIS we characterize the effects of two widely used anesthetics-urethane and isoflurane. We found that both anesthetics suppressed the sensory-evoked optical intrinsic signal in a dose-dependent manner. Dependence of the cortical response suppression matched the exponential decay model. At experimental levels of anesthesia, urethane affected the evoked cortical response less than isoflurane, which is in agreement with the results of electrophysiological recordings demonstrated by other authors. Changes in oxygenation and local blood flow also showed negative correlation with both anesthetics. The high similarity in immature patterns of activity recorded in different regions of the developing cortex suggested similar principles of development regardless of the cortical region. Therefore the indicated results should be taken into account during functional explorations in the entire developing cortex. Our results also point to urethane as the anesthetic of choice in non-survival experimental recordings in the developing brain as it produces less prominent impairment of cortical neuronal activity in neonatal animals.

Inhibitory actions of the gamma-aminobutyric acid in pediatric Sturge-Weber syndrome.

OBJECTIVE: The mechanisms of epileptogenesis in Sturge-Weber syndrome (SWS) are unknown. We explored the properties of neurons from human pediatric SWS cortex in vitro and tested in particular whether gamma-aminobutyric acid (GABA) excites neurons in SWS cortex, as has been suggested for various types of epilepsies. METHODS: Patch-clamp and field potential recordings and dynamic biphoton imaging were used to analyze cortical tissue samples obtained from four 6- to 14-month-old pediatric SWS patients during surgery. RESULTS: Neurons in SWS cortex were characterized by a relatively depolarized resting membrane potential, as was estimated from cell-attached recordings of N-methyl-D-aspartate channels. Many cells spontaneously fired action potentials at a rate proportional to the level of neuronal depolarization. The reversal potential for GABA-activated currents, assessed by cell-attached single channel recordings, was close to the resting membrane potential. All spontaneously firing neurons recorded in cell-attached mode or imaged with biphoton microscopy were inhibited by GABA. Spontaneous epileptiform activity in the form of recurrent population bursts was suppressed by glutamate receptor antagonists, the GABA(A) receptor agonist isoguvacine, and the positive allosteric GABA(A) modulator diazepam. Blockade of GABA(A) receptors aggravated spontaneous epileptiform activity. The NKCC1 antagonist bumetanide had little effect on epileptiform activity. INTERPRETATION: SWS cortical neurons have a relatively depolarized resting membrane potential and spontaneously fire action potentials that may contribute to increased network excitability. In contrast to previous data depicting excitatory and proconvulsive actions of GABA in certain pediatric and adult epilepsies, GABA plays mainly an inhibitory and anticonvulsive role in SWS pediatric cortex.

NMDA receptors pattern early activity in the developing barrel cortex in vivo.

N-methyl-D-aspartate (NMDA) type of glutamate receptors play an important role in activity-dependent plasticity in the developing cortex. However, the physiological patterns of cortical activity that activate NMDA receptors in vivo remain largely unknown. We performed full-band recordings from the barrel cortex of neonatal rats in vivo and found that the dominant pattern of the early activity, network driven spindle bursts, are associated with large amplitude NMDA receptor-dependent delta waves. The major sink of delta waves was in the dense cortical plate, which coincided with the sinks of sensory-evoked responses as well as fast spindle-burst oscillations. Pharmacological analysis revealed major contributions from NMDA and alpha-aminopropionate (AMPA) type of glutamate receptors in the generation of delta waves, whereas fast oscillations primarily involved only AMPA receptors. Our results suggest that the 2 component spindle burst is generated by rhythmic, presumably thalamocortical, synaptic input which entrains an AMPA receptor-mediated fast oscillation and who's summation generates an NMDA and AMPA receptor mediated delta wave. The massive summation of thalamocortical activity during the spindle bursts thus provides a long time window for co-incident activation of cortical neurons by the thalamocortical cells which may contribute to the formation of thalamocortical synapses in the barrel cortex during the critical period of developmental plasticity.

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.

Horizontal Synchronization of Neuronal Activity in the Barrel Cortex of the Neonatal Rat by Spindle-Burst Oscillations.

During development, activity in the somatosensory cortex is characterized by intermittent oscillatory bursts at gamma (early gamma-oscillations, EGOs) and alpha-beta (spindle-bursts, SBs) frequencies. Here, we explored the topography of EGOs and SBs in the neighbor barrels of the whisker-related barrel cortex of neonatal rats (P4-7) during responses evoked by simultaneous activation of multiple whiskers as it occurs during natural conditions. We found that brief simultaneous deflection of all whiskers evoked complex neuronal responses comprised of EGOs and SBs. In contrast to EGOs, that specifically synchronized neuronal activity in each individual barrel, SBs efficiently synchronized activity between neighboring barrels. After plucking a single whisker, synchronous stimulation of spared whiskers evoked EGO-lacking responses in the whisker-deprived barrel, even though the remaining neuronal activity was synchronized by SBs in neighboring barrels. Thus, EGOs specifically support topographic synchronization of neuronal activity within barrels, whereas SBs support horizontal synchronization between neighboring barrels during stimulation of multiple whiskers. We suggest that these two co-existing activity patterns coordinate activity-dependent formation of topographic maps and support the emergence of integrative functions in the primary somatosensory cortex during the critical period of somatosensory maps development.

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.

Postsynaptic GABA(B) Receptors Contribute to the Termination of Giant Depolarizing Potentials in CA3 Neonatal Rat Hippocampus.

During development, hippocampal CA3 network generates recurrent population bursts, so-called Giant Depolarizing Potentials (GDPs). GDPs are characterized by synchronous depolarization and firing of CA3 pyramidal cells followed by afterhyperpolarization (GDP-AHP). Here, we explored the properties of GDP-AHP in CA3 pyramidal cells using gramicidin perforated patch clamp recordings from neonatal rat hippocampal slices. We found that GDP-AHP occurs independently of whether CA3 pyramidal cells fire action potentials (APs) or remain silent during GDPs. However, the amplitude of GDP-AHP increased with the number of APs the cells fired during GDPs. The reversal potential of the GDP-AHP was close to the potassium equilibrium potential. During voltage-clamp recordings, current-voltage relationships of the postsynaptic currents activated during GDP-AHP were characterized by reversal near the potassium equilibrium potential and inward rectification, similar to the responses evoked by the GABA(B) receptor agonists. Finally, the GABA(B) receptor antagonist CGP55845 strongly reduced GDP-AHP and prolonged GDPs, eventually transforming them to the interictal and ictal-like discharges. Together, our findings suggest that the GDP-AHP involves two mechanisms: (i) postsynaptic GABA(B) receptor activated potassium currents, which are activated independently on whether the cell fires or not during GDPs; and (ii) activity-dependent, likely calcium activated potassium currents, whose contribution to the GDP-AHP is dependent on the amount of firing during GDPs. We propose that these two complementary inhibitory postsynaptic mechanisms cooperate in the termination of GDP.

Ontogeny of kainate-induced gamma oscillations in the rat CA3 hippocampus in vitro.

GABAergic inhibition, which is instrumental in the generation of hippocampal gamma oscillations, undergoes significant changes during development. However, the development of hippocampal gamma oscillations remains largely unknown. Here, we explored the developmental features of kainate-induced oscillations (KA-Os) in CA3 region of rat hippocampal slices. Up to postnatal day P5, the bath application of kainate failed to evoke any detectable oscillations. KA-Os emerged by the end of the first postnatal week; these were initially weak, slow (20-25 Hz, beta range) and were poorly synchronized with CA3 units and synaptic currents. Local field potential (LFP) power, synchronization of units and frequency of KA-Os increased during the second postnatal week to attain gamma (30-40 Hz) frequency by P15-21. Both beta and gamma KA-Os are characterized by alternating sinks and sources in the pyramidal cell layer, likely generated by summation of the action potential-associated currents and GABAergic synaptic currents, respectively. Blockade of GABA(A) receptors with gabazine completely suppressed KA-Os at all ages indicating that GABAergic mechanisms are instrumental in their generation. Bumetanide, a NKCC1 chloride co-transporter antagonist which renders GABAergic responses inhibitory in the immature hippocampal neurons, failed to induce KA-Os at P2-4 indicating that the absence of KA-Os in neonates is not due to depolarizing actions of GABA. The linear developmental profile, electrographic features and pharmacological properties indicate that CA3 hippocampal beta and gamma KA-Os are fundamentally similar in their generative mechanisms and their delayed onset and developmental changes likely reflect the development of perisomatic GABAergic inhibition.

Isoflurane suppresses early cortical activity.

OBJECTIVE: Isoflurane and other volatile anesthetics are widely used in children to induce deep and reversible coma, but they may also exert neurotoxic actions. The effects of volatile anesthetics on the immature brain activity remain elusive, however. METHODS: The effects of isoflurane on spontaneous and sensory-evoked activity were explored using intracortical extracellular field potential and multiple unit recordings in the rat barrel cortex from birth to adulthood. RESULTS: During the first postnatal week, isoflurane suppressed cortical activity in a concentration-dependent manner. At surgical anesthesia levels (1.5-2%), isoflurane completely suppressed the electroencephalogram and silenced cortical neurons. Although sensory potentials evoked by the principal whisker deflection persisted, sensory-evoked early gamma and spindle-burst oscillations were completely suppressed by isoflurane. Isoflurane-induced burst-suppression pattern emerged during the second postnatal week and matured through the first postnatal month. Bursts in adolescent and adult rats were characterized by activation of entire cortical columns with a leading firing of infragranular neurons, and were triggered by principal and adjacent whiskers stimulation, and by auditory and visual stimuli, indicating an involvement of horizontal connections in their generation and horizontal spread. INTERPRETATION: The effects of isoflurane on cortical activity shift from total suppression of activity to burst-suppression pattern at the end of the first postnatal week. Developmental emergence of bursts likely involves a development of the intracortical short-and long-range connections. We hypothesize that complete suppression of cortical activity under isoflurane anesthesia during the first postnatal week may explain neuronal apoptosis stimulated by volatile anesthetics in the neonatal rats.

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.