Dynamic changes from depolarizing to hyperpolarizing GABAergic actions during giant depolarizing potentials in the neonatal rat hippocampus

© 2015 the authors. 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

Intracellular blockade of GABA<inf>A</inf> receptors in the rat hippocampal neurons

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABAA receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl- and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AM PA receptors were not affected by the dialysis, whereas the NM DA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg2+, ATP-free solution either fully blocked giant depolarizing potentials (G DPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during G DPs and shifted the G DP reversal potential to more positive values (n = 5). The dialysis-resistant component of G DPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NM DA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of G DPs at negative voltages were very similar to those of AM PA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities

Dynamics of the hypoxia—induced tissue edema in the rat barrel cortex in vitro

© 2018 Juzekaeva, Gainutdinov, Mukhtarov and Khazipov. Cerebral edema is a major, life threatening complication of ischemic brain damage. Previous studies using brain slices have revealed that cellular swelling and a concomitant increase in tissue transparency starts within minutes of the onset of metabolic insult in association with collective anoxic spreading depolarization (aSD). However, the dynamics of tissue swelling in brain slices under ischemia-like conditions remain elusive. Here, we explored the dynamics of brain tissue swelling induced by oxygen-glucose deprivation (OGD) in submerged rat barrel cortex slices. Video monitoring of the vertical and horizontal position of fluorescent dye-filled neurons and contrast slice surface imaging revealed elevation of the slice surface and a horizontal displacement of the cortical tissue during OGD. The OGD-induced tissue movement was also associated with an expansion of the slice borders. Tissue swelling started several minutes after aSD and continued during reperfusion with normal solution. Thirty minutes after aSD, slice borders had expanded by ~130 μm and the slice surface had moved up to attain a height of ~70 μm above control levels, which corresponded to a volume increase of ~30%. Hyperosmotic sucrose solution partially reduced the OGD-induced slice swelling. Thus, OGD-induced cortical slice tissue swelling in brain slices in vitro recapitulates many features of ischemic cerebral edema in vivo, its onset is tightly linked to aSD and it develops at a relatively slow pace after aSD. We propose that this model of cerebral edema in vitro could be useful for the exploration of the pathophysiological mechanisms underlying ischemic cerebral edema and in the search for an efficient treatment to this devastating condition

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl- and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg 2+, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities. © 2014 Pleiades Publishing, Ltd

Postsynaptic GABA(B) receptors contribute to the termination of Giant Depolarizing Potentials in CA3 neonatal rat hippocampus

© 2017 Khalilov, Minlebaev, Mukhtarov, Juzekaeva and Khazipov. 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

Microchannel avalanche photodiode with wide linearity range

Design and physical operation principles of new microchannel avalanche photodiode (MC APD) with gain up to 10^5 and linearity range improved an order of magnitude compared to known similar devices. A distinctive feature of the new device is a directly biased p-n junction under each pixel which plays role of an individual quenching resistor. This allows increasing pixel density up to 40000 per mm^2 and making entire device area sensitive.Comment: Submitted to Journal of Technical Physic

Preferential initiation and spread of anoxic depolarization in layer 4 of rat barrel cortex

© 2017 Juzekaeva, Nasretdinov, Gainutdinov, Sintsov, Mukhtarov and Khazipov. Anoxic depolarization (AD) is a hallmark of ischemic brain damage. AD is associated with a spreading wave of neuronal depolarization and an increase in light transmittance. However, initiation and spread of AD across the layers of the somatosensory cortex, which is one of the most frequently affected brain regions in ischemic stroke, remains largely unknown. Here, we explored the initiation and propagation of AD in slices of the rat barrel cortex using extracellular local field potential (LFP) recordings and optical intrinsic signal (OIS) recordings. We found that ischemia-like conditions induced by oxygen-glucose deprivation (OGD) evoked AD, which manifested as a large negative LFP shift and an increase in light transmittance. AD typically initiated in one or more barrels and further spread across the entire slice with a preferential propagation through L4. Elevated extracellular potassium concentration accelerated the AD onset without affecting proneness of L4 to AD. In live slices, barrels were most heavily labeled by the metabolic level marker 2,3,5-triphenyltetrazolium chloride, suggesting that the highest metabolic demand is in L4 when compared to the other layers. Thus, L4 is the layer of the barrel cortex most prone to AD, which may be due to the highest metabolic demand and cell density in this layer

An optogenetic approach for investigation of excitatory and inhibitory network GABA actions in mice expressing channelrhodopsin-2 in GABAergic neurons

© 2016 the authors.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

Developmental changes in electrophysiological properties and a transition from electrical to chemical coupling between excitatory layer 4 neurons in the rat barrel cortex

© 2016 Valiullina, Akhmetshina, Nasretdinov, Mukhtarov, Valeeva, Khazipov and Rozov.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

Complex formation, chemical exchange, species structure, and stereoselective effects in the copper(II)- L/DL-histidine systems

The formation of copper(ii) complexes with l- and dl-histidine (HisH) has been studied by means of pH-potentiometry and spectrophotometry over a wide range of pH (2-14), ligand-to-metal ratio (1:1-15:1), and temperature (15-55°C) in aqueous solutions with 1.0 mol dm -3 KNO 3 as background. Formation constants and spectral characteristics of 13 complex types were found. Fine stereoselective effects have been detected with preferential coordination of two ligands with identical configuration in Cu(His)(HisH) + and opposite configuration in Cu(His) 2. The stereoselective effect for Cu(His)(HisH) + is explained by hydrogen bond formation between the carboxyl and imidazolyl groups of neighboring ligands at cis-arrangement of amino groups (3N eq-form). The opposite sign of stereoselective effect for Cu(His) 2 is derived from favourable axial coordination of the imidazole group in meso-form with cis-structure (3N eqN ax-form). A significant tetrahedral distortion was revealed for the first time in the prevalent cis-isomer of the Cu(l-His) 2 4N eq-form. These findings were confirmed by EPR data and DFT computations at the B3LYP/TZVP level. The prevalence of cis-isomers for these complexes has been assigned to the rather strong trans effect of the amino groups. The structures of other detected complexes are briefly discussed on the basis of spectroscopic data. Chemical exchange reactions in the copper(ii)- l/dl-hishidine systems have been investigated by the NMR relaxation of water protons. A unique proton exchange reaction with short-term proton dissociation from the coordinated imidazolyl group catalyzed by hydroxide ion was characterised for the first time. The discovered enantioselective effects in the ligand exchange reactions between Cu(His) 2 and HisH or His - species were attributed to the associative substitution mechanism. © 2012 The Royal Society of Chemistry