Piotr Bregestovski

Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices.

1. Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by activation of GABAA receptors (synaptic stimulation or application of the GABAA agonist isoguvacine) were studied on pyramidal cells and interneurons from hippocampal slices of rats from two age groups (postnatal days (P) 2‐5 and P12‐13) using the fluorescent dye fluo‐3 and a confocal laser scanning microscope. Cells were loaded with the dye either intracellularly, using patch pipettes containing fluo‐3 in the internal solution, or extracellularly, using pressure pulses applied to an extracellular pipette containing the permeant dye fluo‐3 AM. 2. Interneurons and pyramidal cells from P2‐5 slices loaded with fluo‐3 AM responded by an increase in [Ca2+]i to isoguvacine and to glutamate, in contrast to cells from P12‐13 slices which responded to glutamate but not to isoguvacine. 3. The isoguvacine‐induced rise in [Ca2+]i was reversibly blocked by bath application of the GABAA receptor antagonist bicuculline (20 microM), suggesting the specific involvement of GABAA receptors. The sodium channel blocker tetrodotoxin (TTX, 1 microM in the bath) did not prevent the isoguvacine‐induced rise in [Ca2+]i. 4. The isoguvacine‐induced rise in [Ca2+]i was reversibly blocked by bath application of the calcium channel blocker D600 (50 microM) suggesting the involvement of voltage‐dependent Ca2+ channels. 5. Electrical stimulation of afferent fibres induced a transient increase in [Ca2+]i in neonatal pyramidal cells and interneurons (P5) loaded non‐invasively with fluo‐3 AM. This elevation of [Ca2+]i was reversibly blocked by bicuculline (20 microM) but not by APV (50 microM) and CNQX (10 microM). 6. During simultaneous electrophysiological recording in the current‐clamp mode and [Ca2+]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibres, in the presence of APV (50 microM) and CNQX (10 microM), caused synaptic depolarization accompanied by a few action potentials and a transient increase in [Ca2+]i. In voltage clamp (‐70 mV) however, there was no increase in [Ca2+]i following synaptic stimulation, showing that it is depolarization dependent. 7. Using a non‐invasive method of [Ca2+]i monitoring, we demonstrate here that in neonatal (P2‐5) hippocampus, GABA is an excitatory neurotransmitter which can cause an elevation of [Ca2+]i in interneurons and pyramidal cells via activation of voltage‐dependent Ca2+ channels. This action may underlie the trophic role of GABA in hippocampal development.

Genetically encoded chloride indicator with improved sensitivity.

Chloride (Cl) is the most abundant physiological anion. Abnormalities in Cl regulation are instrumental in the development of several important diseases including motor disorders and epilepsy. Because of difficulties in the spectroscopic measurement of Cl in live tissues there is little knowledge available regarding the mechanisms of regulation of intracellular Cl concentration. Several years ago, a CFP-YFP based ratiometric Cl indicator (Clomeleon) was introduced [Kuner, T., Augustine, G.J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000; 27: 447-59]. This construct with relatively low sensitivity to Cl (K(app) approximately 160 mM) allows ratiometric monitoring of Cl using fluorescence emission ratio. Here, we propose a new CFP-YFP-based construct (Cl-sensor) with relatively high sensitivity to Cl (K(app) approximately 30 mM) due to triple YFP mutant. The construct also exhibits good pH sensitivity with pK(alpha) ranging from 7.1 to 8.0 pH units at different Cl concentrations. Using Cl-sensor we determined non-invasively the distribution of [Cl](i) in cultured CHO cells, in neurons of primary hippocampal cultures and in photoreceptors of rat retina. This genetically encoded indicator offers a means for monitoring Cl and pH under different physiological conditions and high-throughput screening of pharmacological agents.

Energy substrate availability as a determinant of neuronal resting potential, GABA signaling and spontaneous network activity in the neonatal cortex in vitro

J. Neurochem. (2009) 112, 900–912.

Calcium-dependent inactivation of heteromeric NMDA receptor-channels expressed in human embryonic kidney cells.

1. Whole‐cell current through heteromeric NR1‐NR2A and NR1‐NR2B subunit combinations of NMDA channels transiently expressed in human embryonic kidney cells (HEK 293) were studied using the patch‐clamp technique. 2. With 4 mM Mg‐ATP in the internal pipette solution, the responses of cells expressing NR1‐NR2A channels to glutamate application gradually decreased, reaching 50% of control during the first 20 min of recording. This process was accompanied by acceleration of desensitization. 3. Conditioning (5‐15 s) applications of glutamate (100 microM) induced a transient inactivation of NR1‐NR2A and NR1‐NR2B channels (20‐40%) with a slow time course of recovery (tau r = 10‐60 s). Both the degree of inactivation and the time constant of recovery increased with the duration of conditioning applications of glutamate, and with an elevation of Ca2+ in the external solution. 4. These results show that both NR1‐NR2A and NR1‐NR2B recombinant NMDA receptor‐channels expressed in HEK 293 cells can be transiently inhibited by Ca2+ ions in a similar way to that described for hippocampal neurones.

Excitatory GABA: How a Correct Observation May Turn Out to be an Experimental Artifact.

The concept of the excitatory action of GABA during early development is based on data obtained mainly in brain slice recordings. However, in vivo measurements as well as observations made in intact hippocampal preparations indicate that GABA is in fact inhibitory in rodents at early neonatal stages. The apparent excitatory action of GABA seems to stem from cellular injury due to the slicing procedure, which leads to accumulation of intracellular Cl− in injured neurons. This procedural artifact was shown to be attenuated through various manipulations such as addition of energy substrates more relevant to the in vivo situation. These observations question the very concept of excitatory GABA in immature neuronal networks.

Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival

Non‐technical summary  ‘To be, or not to be’– thousands of neurons are facing this Shakespearean question in the brains of patients suffering from epilepsy or the consequences of a brain traumatism or stroke. The destiny of neurons in damaged brain depends on tiny equilibrium between pro‐survival and pro‐death signalling. Numerous studies have shown that the activity of the neuronal potassium chloride co‐transporter KCC2 strongly decreases during a pathology. However, it remained unclear whether the change of the KCC2 function protects neurons or contributes to neuronal death. Here, using cultures of hippocampal neurons, we show that experimental silencing of endogenous KCC2 using an RNA interference approach or a dominant negative mutant reduces neuronal resistance to toxic insults. In contrast, the artificial gain of KCC2 function in the same neurons protects them from death. This finding highlights KCC2 as a molecule that plays a critical role in the destiny of neurons under toxic conditions and opens new avenues for the development of neuroprotective therapy.

Neuronal activity in vitro and the in vivo reality: the role of energy homeostasis

The energy demands of the brain are exceptionally high compared with any other organ of the body. A complex control system maintains brain energy homeostasis, mobilizing appropriate energy substrates to satisfy the energy requirements. It is a common belief that many fundamental neuronal properties, including those governing excitability, are dependent on the energy supply. However, surprisingly little is known about how the specific factors underlying neuronal activity are affected by energy status. Most of these parameters have been studied in acute brain slices, in which the homeostatic system is absent and neurons in the artificial extracellular milieu are arbitrarily supplied with energy substrates. In this paper, we discuss the relationships between availability of energy substrates and neuronal excitability, and suggest that for in vitro studies, it is crucial to optimize the composition of the energy pool in the extracellular milieu.

CALCIUM-INDUCED INACTIVATION OF NMDA RECEPTOR-CHANNELS EVOLVES INDEPENDENTLY OF RUN-DOWN IN CULTURED RAT BRAIN NEURONES

1. Calcium‐induced transient inactivation of NMDA receptor (NMDAR) channels was studied in cultured rat hippocampal and cerebellar granule neurones using patch‐clamp techniques and confocal scanning microscopy. 2. During whole‐cell recordings, in the presence of 2 mM external Ca2+, conditioning (2‐20 s) pulses of NMDA (20‐100 microM) caused a transient decrease in NMDA responses. Recovery developed in two phases with time constants of 0.6 and 40 s. The slow phase of the recovery could be prevented either by strong intracellular Ca2+ ([Ca2+]i) buffering with 30 mM BAPTA or by using Ca(2+)‐free extracellular solution. 3. Simultaneous measurement of currents and Ca(2+)‐dependent fluorescence revealed a close correlation between the time constants of [Ca2+]i decay and the slow component of NMDA‐activated test current recovery. 4. During prolonged recordings, the transient inactivation was not related to irreversible NMDA‐activated current run‐down. After 25 min of recording with ATP‐free intracellular solution, NMDA‐activated currents in hippocampal neurones irreversibly decreased by 49 +/‐ 5% while inactivation decreased by 8% (n = 9). Calyculin A and FK‐506 (phosphatase inhibitors) significantly delayed run‐down but did not modulate the transient inactivation. 5. In cerebellar granule cells that did not show run‐down (4 mM MgATP in the pipette) the percentage of transient inactivation strongly decreased during 25 min of recording (from 28 +/‐ 6 to 7 +/‐ 5%, n = 15). 6. In cell‐attached recordings (5 microM NMDA in the pipette), elevation of [Ca2+]i (application of 100 microM NMDA to the soma) caused a reversible reduction of single NMDAR channel open probability (NPo) due to a decrease in the frequency of channel opening. 7. In inside‐out patches, application of Ca2+ to the cytoplasmic side of the membrane caused a rapid and reversible decrease in NPo (13 out of 29 patches). In the absence of run‐down, the ability of Ca2+ to transiently inhibit NMDAR channel activity disappeared after 3‐5 min of recording. 8. These results indicate that Ca(2+)‐induced transient inactivation of NMDAR currents develops independently from the run‐down and suggest that a diffusible Ca2+ ‐dependent factor mediates NMDAR channel inactivation.

Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity

This review briefly discusses the main approaches for monitoring chloride (Cl−), the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i) is a challenging task owing to two main difficulties: (i) the low transmembrane ratio for Cl−, approximately 10:1; and (ii) the small driving force for Cl−, as the Cl− reversal potential (ECl) is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP) exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM), it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM) has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR) channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening pharmacological agents, analysis of Cl− homeostasis and functions of Cl−-selective channels under different physiological and pathological conditions.

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre‐ and postsynaptically regulated, although mechanisms are poorly understood. Using patch‐clamp recording and Ca2+ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca2+ (Cai) in glycinergic synapse modulation. Ca2+ influx through voltage‐gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired‐pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212‐2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca2+‐dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Cai buffering (20 mm BAPTA). Our findings demonstrate two independent mechanisms by which Ca2+ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR‐mediated responses. This dual Ca2+‐induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.