Cortical Auditory-Evoked Responses in Preterm Neonates: Revisited by Spectral and Temporal Analyses.

Characteristic preterm EEG patterns of "Delta-brushes" (DBs) have been reported in the temporal cortex following auditory stimuli, but their spatio-temporal dynamics remains elusive. Using 32-electrode EEG recordings and co-registration of electrodes' position to 3D-MRI of age-matched neonates, we explored the cortical auditory-evoked responses (AERs) after 'click' stimuli in 30 healthy neonates aged 30-38 post-menstrual weeks (PMW). (1) We visually identified auditory-evoked DBs within AERs in all the babies between 30 and 33 PMW and a decreasing response rate afterwards. (2) The AERs showed an increase in EEG power from delta to gamma frequency bands over the middle and posterior temporal regions with higher values in quiet sleep and on the right. (3) Time-frequency and averaging analyses showed that the delta component of DBs, which negatively peaked around 550 and 750 ms over the middle and posterior temporal regions, respectively, was superimposed with fast (alpha-gamma) oscillations and corresponded to the late part of the cortical auditory-evoked potential (CAEP), a feature missed when using classical CAEP processing. As evoked DBs rate and AERs delta to alpha frequency power decreased until full term, auditory-evoked DBs are thus associated with the prenatal development of auditory processing and may suggest an early emerging hemispheric specialization.

Ketamine-Midazolam Anesthesia Induces Total Inhibition of Cortical Activity in the Brain of Newborn Rats.

The effects of general anesthetics ketamine and midazolam, the drugs that cause neuroapoptosis at the early stages of CNS development, on electrical activity of the somatosensory cortex in newborn rats were studied using extracellular recording of local field potentials and action potentials of cortical neurons. Combined administration of ketamine (40 mg/kg) and midazolam (9 mg/kg) induced surgical coma and almost completely suppressed early oscillatory patterns and neuronal firing. These effects persisted over 3 h after injection of the anesthetics. We concluded that general anesthesia induced by combined administration of ketamine and midazolam profoundly suppressed cortical activity in newborn rats, which can trigger neuroapoptosis in the developing brain.

Hunting for origins of migraine pain: cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers.

Trigeminal nerves in meninges are implicated in generation of nociceptive firing underlying migraine pain. However, the neurochemical mechanisms of nociceptive firing in meningeal trigeminal nerves are little understood. In this study, using suction electrode recordings from peripheral branches of the trigeminal nerve in isolated rat meninges, we analyzed spontaneous and capsaicin-induced orthodromic spiking activity. In control, biphasic single spikes with variable amplitude and shapes were observed. Application of the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin to meninges dramatically increased firing whereas the amplitudes and shapes of spikes remained essentially unchanged. This effect was antagonized by the specific TRPV1 antagonist capsazepine. Using the clustering approach, several groups of uniform spikes (clusters) were identified. The clustering approach combined with capsaicin application allowed us to detect and to distinguish "responder" (65%) from "non-responder" clusters (35%). Notably, responders fired spikes at frequencies exceeding 10 Hz, high enough to provide postsynaptic temporal summation of excitation at brainstem and spinal cord level. Almost all spikes were suppressed by tetrodotoxin (TTX) suggesting an involvement of the TTX-sensitive sodium channels in nociceptive signaling at the peripheral branches of trigeminal neurons. Our analysis also identified transient (desensitizing) and long-lasting (slowly desensitizing) responses to the continuous application of capsaicin. Thus, the persistent activation of nociceptors in capsaicin-sensitive nerve fibers shown here may be involved in trigeminal pain signaling and plasticity along with the release of migraine-related neuropeptides from TRPV1 positive neurons. Furthermore, cluster analysis could be widely used to characterize the temporal and neurochemical profiles of other pain transducers likely implicated in migraine.

[Secondary epileptogenesis in the immature brain: a role of GABA]. / Vtorichnyi épileptogenez v nezrelom mozge: rol' GAMK.

Clinical studies show that the probability of recurrent epileptiform discharges and formation of an epileptic focus (epileptogenesis) in young children is much higher than in adults. Repetitive epileptiform discharges and their potential contribution to the mechanisms of the development of the epileptic focus - an important object of clinical and scientific research. This review is based on the data from animal studies, and summarizes the current understanding of the mechanisms underlying the increased excitability of the immature brain, the formation of a secondary epileptogenic focus, and the functional changes of neurons due to deleterious effects of repetitive epileptiform discharges on the excitation and inhibition in the immature neuronal networks. The review discusses the relevance of experimental data in light of the general mechanisms of epileptogenesis in infants and identifies the gaps in current scientific knowledge, including the relationship between the data obtained in animal studies and processes underlying human acquired epilepsy.

Gamma oscillations in the somatosensory cortex of newborn rats.

Here we addressed a question of whether gamma oscillations previously described in the whisker-related barrel cortex are a universal pattern of activity in the somatosensory cortex of newborn rats. Intracortical recording of local field potentials and action potentials in neurons using multisite silicon electrodes in 2-7-day-old rats showed that mechanical stimulation of single fingers or specific areas on the plantar or back side of the foot evoked early gamma oscillations followed by spindle-burst oscillations in the corresponding regions of the somatosensory cortex. Early gamma oscillations had maximum amplitude in layer IV of the somatosensory cortex and effectively synchronized action potentials in layer IV neurons. It was concluded that early gamma oscillations evoked by activation of the topographic sensory input are a universal activity pattern of the entire somatosensory cortex of newborn rats.

Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system.

Indirect evidence suggests the increased production of reactive oxygen species (ROS) in migraine pathophysiology. In the current study we measured lipid peroxidation product in the rat cortex, trigeminal ganglia and meninges after the induction of cortical spreading depression (CSD), a phenomenon known to be associated with migraine aura, and tested nociceptive firing triggered by ROS in trigeminal nerves ex vivo. Application of KCl to dura mater in anesthetized rats induced several waves of CSD recorded by an extracellular electrode in the cortex. Following CSD, samples of cortex (affected regions were identified with blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI)), meninges from left and right hemispheres and trigeminal ganglia were taken for biochemical analysis. We found that CSD increased the level of the lipid peroxidation product malondialdehyde (MDA) in the ipsilateral cerebral cortex and meninges, but also in both ipsi- and contralateral trigeminal ganglia. In order to test the pro-nociceptive action of ROS, we applied the mild oxidant hydrogen peroxide to isolated rat hemiskull preparations including preserved trigeminal innervations. Application of hydrogen peroxide to meninges transiently enhanced electrical spiking activity of trigeminal nerves showing a pro-nociceptive action of ROS. In the presence of hydrogen peroxide trigeminal nerves still responded to capsaicin by burst of spiking activity indicating integrity of neuronal structures. The action of hydrogen peroxide was mediated by TRPA1 receptors as it was abolished by the specific TRPA1 antagonist TCS-5861528. Using dorsal root ganglion sensory neurons as test system we found that hydrogen peroxide promoted the release of the migraine mediator calcitonin gene-related peptide (CGRP), which we previously identified as a trigger of delayed sensitization of trigeminal neurons. Our data suggest that, after CSD, oxidative stress spreads downstream within the trigeminal nociceptive system and could be involved in the coupling of CSD with the activation of trigeminovascular system in migraine pathology.

Early gamma oscillations.

Gamma oscillations have long been considered to emerge late in development. However, recent studies have revealed that gamma oscillations are transiently expressed in the rat barrel cortex during the first postnatal week, a "critical" period of sensory-dependent barrel map formation. The mechanisms underlying the generation and physiological roles of early gamma oscillations (EGOs) in the development of thalamocortical circuits will be discussed in this review. In contrast to adult gamma oscillations, synchronized through gamma-rhythmic perisomatic inhibition, EGOs are primarily driven through feedforward gamma-rhythmic excitatory input from the thalamus. The recruitment of cortical interneurons to EGOs and the emergence of feedforward inhibition are observed by the end of the first postnatal week. EGOs facilitate the precise synchronization of topographically aligned thalamic and cortical neurons. The multiple replay of sensory input during EGOs supports long-term potentiation at thalamocortical synapses. We suggest that this early form of gamma oscillations, which is mechanistically different from adult gamma oscillations, guides barrel map formation during the critical developmental period.

[Excitatory action of GABA in ontogenesis].

GABA (gamma-aminobutyric acid) is the main inhibitory mediator in central nervous system. However, at early stages of ontogenesis, GABA has an excitatory effect on immature neurons. This review surveys modern concepts of the mechanisms of GABAergic excitation and physiological role of excitatory GABA in generation of patterns of network activity in developing brain.

Early development of neuronal activity in the primate hippocampus in utero.

Morphological studies suggest that the primate hippocampus develops extensively before birth, but little is known about its functional development. Patch-clamp recordings of hippocampal neurons and reconstruction of biocytin-filled pyramidal cells were performed in slices of macaque cynomolgus fetuses delivered by cesarean section. We found that during the second half of gestation, axons and dendrites of pyramidal cells grow intensively by hundreds of micrometers per day to attain a high level of maturity near term. Synaptic currents appear around midgestation and are correlated with the level of morphological differentiation of pyramidal cells: the first synapses are GABAergic, and their emergence correlates with the growth of apical dendrite into stratum radiatum. A later occurrence of glutamatergic synaptic currents correlates with a further differentiation of the axodendritic tree and the appearance of spines. Relying on the number of dendritic spines, we estimated that hundreds of new glutamatergic synapses are established every day on a pyramidal neuron during the last third of gestation. Most of the synaptic activity is synchronized in spontaneous slow ( approximately 0.1 Hz) network oscillations reminiscent of the giant depolarizing potentials in neonatal rodents. Epileptiform discharges can be evoked by the GABA(A) receptor antagonist bicuculline by the last third of gestation, and postsynaptic GABA(B) receptors contribute to the termination of epileptiform discharges. Comparing the results obtained in primates and rodents, we conclude that the template of early hippocampal network development is conserved across the mammalian evolution but that it is shifted toward fetal life in primate.

Neuronal mechanisms of the anoxia-induced network oscillations in the rat hippocampus in vitro.

1. A spindle of fast network oscillations precedes the ischaemia-induced rapid depolarisation in the rat hippocampus in vivo. However, this oscillatory pattern could not be reproduced in slices and the underlying mechanisms remain poorly understood. We have found that anoxia-induced network oscillations (ANOs, 20-40 Hz, lasting for 1-2 min) can be reproduced in the intact hippocampi of postnatal day P7-10 rats in vitro, and we have examined the underlying mechanisms using whole-cell and extracellular field potential recordings in a CA3 pyramidal layer. 2. ANOs were generated at the beginning of the anoxic depolarisation, when pyramidal cells depolarised to subthreshold values. Maximal power of the ANOs was attained when pyramidal cells depolarised to -56 mV; depolarisation above -47 mV resulted in a depolarisation block of pyramidal cells and a waning of ANOs. 3. A multiple unit activity in extracellular field recordings was phase locked to the negative and ascending phases of ANOs. Pyramidal cells recorded in current-clamp mode generated action potentials with an average probability of about 0.05 per cycle. The AMPA receptor-mediated EPSCs and the GABA receptor-mediated IPSCs in CA3 pyramidal cells were also phase locked with ANOs. 4. ANOs were prevented by tetrodotoxin and glutamate receptor antagonists CNQX and APV, and were slowed down by the allosteric GABA(A) receptor modulator diazepam. In the presence of the GABA(A) receptor antagonist bicuculline, ANOs were transformed to epileptiform discharges. 5. In the presence of the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the anoxia induced an epileptiform activity and no ANOs were observed. 6. In normoxic conditions, a rise of extracellular potassium to 10 mM induced an epileptiform activity. Increasing extracellular potassium in conjunction with a bath application of the adenosine A1 receptor agonist cyclopentyladenosine induced oscillations similar to ANOs. 7. Multisite recordings along the septo-temporal hippocampal axis revealed that ANOs and anoxic depolarisation originate in the temporal part, and propagate towards the septal pole at a speed of 1.9 mm x min(-1). 8. ANOs were observed starting from P7, i.e. at a developmental stage when the effects of GABA change from depolarisation to hyperpolarisation. 9. These results suggest that the synchronisation of anoxia-induced oscillations relies on synaptic mechanisms; that the inhibition by GABA and adenosine sets the tune for a generation of oscillations and prevents an epileptiform activity; and that a synchronous GABAergic inhibition is instrumental in a phase locking neuronal activity similarly to other types of oscillatory activities in the gamma frequency range.

Seizures accelerate anoxia-induced neuronal death in the neonatal rat hippocampus.

Seizures occurring in infants with hypoxia are frequently associated with an ominous prognosis. There is, however, no direct evidence that seizures are involved in the pathogenesis of hypoxia-induced neuronal damage. Here, we report that seizures significantly aggravate the hypoxic state by accelerating rapid anoxic depolarization (AD) and associated neuronal death in preparations of the intact hippocampus of neonatal rats in vitro. Under control conditions, prolonged episodes of anoxia/aglycemia induced rapid suppression of synaptic activity followed sequentially by brief bursts of epileptiform activity and then by rapid AD. AD was associated with irreversible neuronal damage manifested by irreversible loss of the membrane potential, synaptic responses, and neuronal degeneration. Aggravation of electrographic seizure activity during anoxic episodes by the adenosine A1 receptor antagonists DPCPX and caffeine or the gamma-aminobutyric acid-A receptor antagonist bicuculline or pretreatment with 4-aminopyridine accelerated AD and associated neuronal death by up to twofold, whereas blockade of seizure activity by the glutamate receptor antagonists or tetrodotoxin significantly delayed the onset of AD. This report provides direct evidence for the need to prevent seizures during neonatal brain hypoxia.

Dual role of GABA in the neonatal rat hippocampus.

The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0-5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1-2 microM) and the allosteric modulator diazepam (2 microM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 microM) and picrotoxin (100 microM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual - both excitatory and inhibitory - action on the network activity.

Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro.

In vivo studies suggest that ontogenesis of limbic seizures is determined by the development of the limbic circuit. We have now used the newly-developed in vitro intact interconnected neonatal rat limbic structures preparation to determine the developmental profile of kainate-induced epileptiform activity in the hippocampus and its propagation to other limbic structures. We report gradual alterations in the effects of kainate during the first postnatal week on an almost daily basis; from no epileptiform activity at birth, through interictal seizures around postnatal day (P) 2 and ictal seizures by the end of the first week. The developmental profile of kainate-induced hippocampal seizures is paralleled by the expression of postsynaptic kainate receptor-mediated currents in CA3 pyramidal cells. Intralimbic propagation of the hippocampal seizures is also age-dependent: whereas seizures readily propagate to the septum and to the contralateral hippocampus via the commissures on P2, propagation to the entorhinal cortex only takes place from P4 onwards. Finally, repeated brief applications of kainate to the hippocampus induce recurrent spontaneous glutamatergic ictal and interictal discharges which persist for several hours after the kainate is washed away and which replace the physiological pattern of network activity. Paroxysmal activities are thus generated by kainate in the hippocampus at an early developmental stage and are initially restricted to this structure. Before the end of the first week of postnatal life, kainate generates the epileptiform activities that may perturb activity-dependent mechanisms that modulate neuronal development. Although at this stage neurons are relatively resistant to the pathological effects of kainate, the epileptiform activities that it generates will perturb activity-dependent mechanisms that modulate neuronal development.

GABA is the principal fast-acting excitatory transmitter in the neonatal brain.

gamma-aminobutyric acid (GABA) is the principal neurotransmitter of inhibition in the adult mammalian brain. However, at early stages of development, including the embryonic period and first week of postnatal life, GABA plays the role of main neurotransmitter of excitation. The paradoxical excitatory effect of GABA is caused by an inverted chloride gradient and, therefore, a depolarizing direction of GABA type A (GABAA) receptor mediated responses. In addition, another type of GABAergic inhibition mediated by postsynaptic GABA type B (GABAB) receptors is not functional at early stage of life. In the neonatal rat hippocampus, GABA, acting via GABAA receptors, activates voltage-gated sodium and calcium channels and potentiates the activity of N-methyl-D-aspartate (NMDA) receptors by reducing their voltage-dependent Mg2+ block. The temporal window when GABA exerts excitatory actions coincides with a particular pattern of activity of hippocampal neuronal network that is characterized by periodical giant depolarizing potentials (GDPs) reminiscent of interictal-like epileptiform discharges. Recent studies have shown that GDPs result from the synchronous discharge of GABAergic interneurons and principal glutamatergic pyramidal cells, and they are mediated by the synergistic excitatory actions of GABAA and glutamate receptors. GDPs provide synchronous intracellular Ca2+ oscillations and may, therefore, be implicated in hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network.

Epileptogenic action of caffeine during anoxia in the neonatal rat hippocampus.

Excessive maternal caffeine consumption can lead to fetal and neonatal pathology, but the underlying mechanisms have not been determined. Here, we report that low doses of caffeine generate seizures when applied in conjunction with brief anoxic episodes in the hippocampus of neonatal rats in vitro. In control conditions, brief (4-6 minutes) anoxic episodes reversibly depressed evoked synaptic responses and blocked the physiological pattern of network activity. In the presence of caffeine (50 microM), similar anoxic episodes generated ictal (29%) or interictal (33%) epileptiform activities often followed during reoxygenation by recurrent spontaneous seizure activity that persisted for several hours. These effects are likely mediated by a blockade of adenosine receptors by caffeine because (1) in control conditions, caffeine antagonized the inhibitory effect of selective A1 receptor agonist N6-cyclopentyladenosine on excitatory synaptic responses, and (2) epileptogenic effects of caffeine were reproduced by selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and theophylline. Our findings suggest that endogenous adenosine released during anoxia acting via A1 receptors prevents seizures in the neonatal hippocampus and that the antagonism of these receptors by caffeine leads to epileptogenesis. This study suggests concerns about the safety of caffeine in the fetus and newborn.

Three-independent-compartment chamber to study in vitro commissural synapses.

We describe a novel chamber in which the two intact neonatal rat hippocampi and the commissural fibers are placed in three independent compartments separated by latex membranes and perfused selectively with different solutions. A set of control tests showed that the compartments are well isolated: 1) methylene blue or eosin applied to one compartment did not diffuse to other compartments when verified via the microscope, and spectrophotometry revealed that <1/10.000th of the dye diffuses to other compartments; 2) tetrodotoxin (1 microM) applied to the commissural compartment blocked the synaptic responses evoked contralaterally without affecting those evoked on the ipsilateral side. This chamber enables a wide range of experiments that cannot be performed in conventional chambers, e.g., to study the maturation and plasticity of the commissural connections, bilateral synchronization of the rhythmic activities in the limbic system, commissural propagation of the epileptiform activities, etc.

Giant depolarizing potentials: the septal pole of the hippocampus paces the activity of the developing intact septohippocampal complex in vitro.

In neonatal hippocampal slices, recurrent spontaneous giant depolarizing potentials (GDPs) provide neuronal synchronized firing and Ca2+ oscillations. To investigate the possible role of GDPs in the synchronization of neuronal activity in intact neonatal limbic structures, we used multiple simultaneous electrophysiological recordings in the recently described preparation of intact neonatal septohippocampal complex in vitro. Combined whole-cell (in single or pairs of cells) and extracellular field recordings (one to five simultaneous recording sites) from the CA3 hippocampal region and various parts of the septum indicated that spontaneous GDPs, which can be initiated anywhere along the longitudinal hippocampal axis, are most often initiated in the septal poles of hippocampus and propagate to medial septum and temporal poles of both hippocampi simultaneously. GDPs were abolished in the medial septum but not in the hippocampus after surgical separation of both structures, suggesting hippocampal origin of GDPs. The preferential septotemporal orientation of GDP propagation observed in the intact hippocampus was associated with a corresponding gradient of GDP frequency in isolated portions of hippocampus. Accordingly, most GDPs propagated in the septotemporal direction in both septal and temporal hippocampal isolated halves, and whereas GDP frequency remained similar in the septal part of hippocampus after its surgical isolation, it progressively decreased in more temporally isolated portions of the hippocampus. Because GDPs provide most of the synaptic drive of neonatal neurons, they may modulate the development of neuronal connections in the immature limbic system.

[GABA--the basic mediator of excitation in the early stages of hippocampal development]. / GAMK--osnovnoi mediator vozbuzhdeniia na rannikh étapakh razvitiia gippokampa.

GABA is the principal neurotransmitter of inhibition in the adult mammalian brain. However, at early stages of development, including embryonic period and first week of postnatal life, GABA plays the role of main neurotransmitter of excitation. The paradoxical excitatory effect of GABA is due to an inversed chloride gradient and therefore a depolarizing direction of GABA-A receptor mediated responses. In addition, another type of GABAergic inhibition mediated by postsynaptic GABA-B receptors is not functional at early stage of life. In the neonatal rat hippocampus, GABA, acting via GABA-A receptors, activates voltage gated sodium and calcium channels and potentiates the activity of NMDA receptors by reducing their voltage dependent Mg2+ block. The temporal window when GABA exerts excitatory actions coincides with a particular pattern of activity of hippocampal neuronal network that is characterized by periodical giant depolarizing potentials (GDPs) reminiscent of interictal-like epileptiform discharges. Recent studies have shown that GDPs result from the synchronous discharge of GABAergic interneurons and principal glutamatergic pyramidal cells and are mediated by the synergistic excitatory actions of GABA-A and glutamate receptors. GDPs provide synchronous intracellular Ca2+ oscillations and may therefore be implicated in hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network.

GABAA, NMDA and AMPA receptors: a developmentally regulated 'ménage à trois'.

The main ionotropic receptors (GABAA, NMDA and AMPA) display a sequential participation in neuronal excitation in the neonatal hippocampus. GABA, the principal inhibitory transmitter in the adult CNS, acts as an excitatory transmitter in early postnatal stage. Glutamatergic synaptic transmission is first purely NMDA-receptor based and lacks functional AMPA receptors. Therefore, initially glutamatergic synapses are 'silent' at resting membrane potential, NMDA channels being blocked by Mg2+. However, when GABA and glutamatergic synapses are coactivated during the physiological patterns of activity, GABAA receptors can facilitate the activation of NMDA receptors, playing the role conferred to AMPA receptors later on in development. Determining the mechanisms underlying the development of this 'ménage à trois' will shed light not only on the wide range of trophic roles of glutamate and GABA in the developing brain, but also on the significance of the transition from neonatal to adult forms of plasticity.

A novel in vitro preparation: the intact hippocampal formation.

The intact hippocampal formation (IHF) of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation. Field or patch-clamp recordings, intracellular Ca2+ measurements, and 3-D reconstruction of biocytin-filled neurons can be performed routinely. The generation and propagation of network-driven activities can be studied within the IHF or between connected intact structures such as the septum and the hippocampus or two hippocampi, and the use of a dual chamber enables the application of drugs separately to each structure. This preparation will be useful to study intact neuronal networks in the developing hippocampus in vitro.