In the developing hippocampus, GABA exerts depolarizing and excitatory actions and contributes to the generation of neuronal network driven giant depolarizing potentials (GDPs). Here, we studied spike time coding at immature GABAergic synapses and its impact on synchronization of the neuronal network during GDPs in the neonatal (postnatal days P2-6) rat hippocampal slices. Using extracellular recordings, we found that the delays of action potentials (APs) evoked by synaptic activation of GABA(A) receptors are long (mean, 65 ms) and variable (within a time window of 10-200 ms). During patch-clamp recordings, depolarizing GABAergic responses were mainly subthreshold and their amplification by persistent sodium conductance was required to trigger APs. AP delays at GABAergic synapses shortened and their variability reduced with an increase in intracellular chloride concentration during whole-cell recordings. Negative shift of the GABA reversal potential (E(GABA)) with low concentrations of bumetanide, or potentiation of GABA(A) receptors with diazepam reduced GDPs amplitude, desynchronized neuronal firing during GDPs and slowed down GDPs propagation. Partial blockade of GABA(A) receptors with bicuculline increased neuronal synchronization and accelerated GDPs propagation. We propose that spike timing at depolarizing GABA synapses is determined by intracellular chloride concentration. At physiological levels of intracellular chloride GABAergic depolarization does not reach the action potential threshold and amplification of GABAergic responses by non-inactivating sodium conductance is required for postsynaptic AP initiation. Slow and variable excitation at GABAergic synapse determines the level of neuronal synchrony and the rate of GDPs propagation in the developing hippocampus.

The developing entorhinal-hippocampal system is embedded within a large-scale bottom-up network, where spontaneous myoclonic movements, presumably via somatosensory feedback, trigger hippocampal early sharp waves (eSPWs). The hypothesis, that somatosensory feedback links myoclonic movements with eSPWs, implies that direct somatosensory stimulation should also be capable of evoking eSPWs. In this study, we examined hippocampal responses to electrical stimulation of the somatosensory periphery in urethane-anesthetized, immobilized neonatal rat pups using silicone probe recordings. We found that somatosensory stimulation in ~33% of the trials evoked local field potential (LFP) and multiple unit activity (MUA) responses identical to spontaneous eSPWs. The somatosensory-evoked eSPWs were delayed from the stimulus, on average, by 188 ms. Both spontaneous and somatosensory-evoked eSPWs (i) had similar amplitude of ~0.5 mV and half-duration of ~40 ms, (ii) had similar current-source density (CSD) profiles, with current sinks in CA1 strata radiatum, lacunosum-moleculare and DG molecular layer and (iii) were associated with MUA increase in CA1 and DG. Our results indicate that eSPWs can be triggered by direct somatosensory stimulations and support the hypothesis that sensory feedback from movements is involved in the association of eSPWs with myoclonic movements in neonatal rats.

No abstract available

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.

The excitatory action of gamma-aminobutyric acid (GABA) is considered to be a hallmark of the developing nervous system. However, in immature brain slices, excitatory GABA actions may be secondary to neuronal injury during slice preparation. Here, we explored GABA actions in the rodent intact hippocampal preparations and at different depths of hippocampal slices during the early post-natal period [post-natal days (P) 1-7]. We found that in the intact hippocampus at P1-3: (i) GABA exerts depolarizing action as seen in cell-attached single GABA(A) channel recordings; (ii) GABA(A) receptor (GABA(A)-R) agonist isoguvacine and synaptic activation of the GABA(A)-Rs increase the frequency of multiple unit activity and the frequency of the network-driven giant depolarizing potentials (GDPs); and that (iii) Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) antagonist bumetanide suppresses GDPs and the excitatory actions of isoguvacine. In the hippocampal slices at P2-5, isoguvacine and synaptic activation of GABA(A)-Rs-evoked excitatory responses at all slice depths, including surface and core. Thus, GABA exerts excitatory actions in the intact hippocampus (P1-3) and at all depths of hippocampal slices (P2-5). Therefore, the excitatory actions of GABA in hippocampal slices during the first post-natal days are not due to neuronal injury during slice preparation, and the trauma-related excitatory GABA actions at the slice surface are a fundamentally different phenomenon observed during the second post-natal week.

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.

Correlated activity in the entorhinal-hippocampal neuronal networks, supported by oscillatory and intermittent population activity patterns is critical for learning and memory. However, when and how correlated activity emerges in these networks during development remains largely unknown. Here, we found that during the first postnatal week in non-anaesthetized head-restrained rats, activity in the superficial layers of the medial entorhinal cortex (MEC) and hippocampus was highly correlated, with intermittent population bursts in the MEC followed by early sharp waves (eSPWs) in the hippocampus. Neurons in the superficial MEC layers fired before neurons in the dentate gyrus, CA3 and CA1. eSPW current-source density profiles indicated that perforant/temporoammonic entorhinal inputs and intrinsic hippocampal connections are co-activated during entorhinal-hippocampal activity bursts. Finally, a majority of the entorhinal-hippocampal bursts were triggered by spontaneous myoclonic body movements, characteristic of the neonatal period. Thus, during the neonatal period, activity in the entorhinal cortex (EC) and hippocampus is highly synchronous, with the EC leading hippocampal activation. We propose that such correlated activity is embedded into a large-scale bottom-up circuit that processes somatosensory feedback resulting from neonatal movements, and that it is likely to instruct the development of connections between neocortex and hippocampus.

During development, sensory systems switch from an immature to an adult mode of function along with the emergence of the active cortical states. Here, we used patch-clamp recordings from neocortical slices in vitro to characterize the developmental changes in the basic electrophysiological properties of excitatory L4 neurons and their connectivity before and after the developmental switch, which occurs in the rat barrel cortex in vivo at postnatal day P8. Prior to the switch, L4 neurons had higher resting membrane potentials, higher input resistance, lower membrane capacity, as well as action potentials (APs) with smaller amplitudes, longer durations and higher AP thresholds compared to the neurons after the switch. A sustained firing pattern also emerged around the switch. Dual patch-clamp recordings from L4 neurons revealed that recurrent connections between L4 excitatory cells do not exist before and develop rapidly across the switch. In contrast, electrical coupling between these neurons waned around the switch. We suggest that maturation of electrophysiological features, particularly acquisition of a sustained firing pattern, and a transition from the immature electrical to mature chemical synaptic coupling between excitatory L4 neurons, contributes to the developmental switch in the cortical mode of function.

Reactive oxygen species (ROS) are implicated in aging, but the neurobiological mechanisms of ROS action are not fully understood. Using electrophysiological techniques and biochemical assays, we studied the age-dependent effect of hydrogen peroxide (H2O2) on acetylcholine release in rat diaphragm neuromuscular junctions. H2O2 significantly inhibited both spontaneous (measured as frequency of miniature end-plate potentials) and evoked (amplitude of end-plate potentials) transmitter release in adult rats. The inhibitory effect of H2O2 was much stronger in old rats, whereas in newborns tested during the first postnatal week, H2O2 did not affect spontaneous release from nerve endings and potentiated end-plate potentials. Proteinkinase C activation or intracellular Ca2+ elevation restored redox sensitivity of miniature end-plate potentials in newborns. The resistance of neonates to H2O2 inhibition was associated with higher catalase and glutathione peroxidase activities in skeletal muscle. In contrast, the activities of these enzymes were downregulated in old rats. Our data indicate that the vulnerability of transmitter release to oxidative damage strongly correlates with aging and might be used as an early indicator of senescence.

In the neonatal rodent hippocampus, the first and predominant pattern of correlated neuronal network activity is early sharp waves (eSPWs). Whether and how eSPWs are organized bilaterally remains unknown. Here, using simultaneous silicone probe recordings from the left and right hippocampus in neonatal rats in vivo we found that eSPWs are highly synchronized bilaterally with nearly zero time lag between the two sides. The amplitudes of eSPWs in the left and right hippocampi were also highly correlated. eSPWs also supported bilateral synchronization of multiple unit activity (MUA). We suggest that bilateral correlated activity supported by synchronized eSPWs participates in the formation of bilateral connections in the hippocampal system.

Rats are born deaf and start hearing at the end of the second postnatal week, when the ear canals open and low-intensity sounds start to evoke responses in the auditory cortex. Here, using μECoG electrode arrays and intracortical silicon probe recordings, we found that bone-conducted (BC) sounds evoked biphasic responses in the auditory cortex starting from postnatal day (P) 8. The initial phase of these responses, generated by thalamocortical input, was followed by intracortical propagation within supragranular layers. BC-evoked responses co-localized with the responses evoked by electrical stimulation of the cochlea and the deepest layers of the inferior colliculus prior to onset of low-threshold hearing (P13), as well as with the responses evoked by high-frequency (30 kHz) low-intensity (70 dB) air-conducted sounds after that. Thus, BC signals reach high-frequency processing regions of the auditory cortex well before the onset of low-threshold hearing, reflecting early integrity of the auditory system.