Frequency-Dependent Cannabinoid Receptor-Independent Modulation of Glycine Receptors by Endocannabinoid 2-AG

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1–1 μM), 2-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4–10 Hz) application of short (2 ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10–20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner

Dynamics of the Hypoxia—Induced Tissue Edema in the Rat Barrel Cortex in vitro

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

ISCHEMIC RESPONSES IN THE RAT BARREL CORTEX IN VITRO AT DIFFERENT POSTNATAL AGES

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Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo

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

International audienceDuring 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

Anoxic spreading depolarization in the neonatal rat cortex in vitro

Anoxic spreading depolarization (aSD) is a hallmark of ischemic injury in the cerebral cortex. In adults, aSD is associated with rapid and nearly complete neuronal depolarization and loss of neuronal functions. While ischemia also evokes aSD in the immature cortex, developmental aspects of neuronal behavior during aSD remain largely unknown. Here, using oxygen-glucose deprivation (OGD) ischemia model in slices of the postnatal rat somatosensory cortex, we found that immature neurons displayed much more complex behaviors: they initially moderately depolarized during aSD, then transiently repolarised (for up to tens of minutes), and only then passed to terminal depolarization. The ability to fire action potentials was maintained in neurons mildly depolarized during aSD without reaching the level of depolarization block, and these functions were regained in the majority of immature neurons during post-aSD transient repolarization. The amplitude of depolarization and the probability of depolarization block during aSD increased, whereas transient post-SD repolarization levels and duration, and associated recovery in neuronal firing decreased with age. By the end of the first postnatal month, aSD acquired an adult-like phenotype, where depolarization during aSD merged with terminal depolarization and the phase of transient recovery was lost. Thus, changes in neuronal function during aSD undergo remarkable developmental changes that may contribute to lower susceptibility of the immature neurons to ischemia

Lactate Effectively Covers Energy Demands during Neuronal Network Activity in Neonatal Hippocampal Slices

Although numerous experimental data indicate that lactate is efficiently used for energy by the mature brain, the direct measurements of energy metabolism parameters during neuronal network activity in early postnatal development have not been performed. Therefore, the role of lactate in the energy metabolism of neurons at this age remains unclear. In this study, we monitored field potentials and contents of oxygen and NAD(P)H in correlation with oxidative metabolism during intense network activity in the CA1 hippocampal region of neonatal brain slices. We show that in the presence of glucose, lactate is effectively utilized as an energy substrate, causing an augmentation of oxidative metabolism. Moreover, in the absence of glucose lactate is fully capable of maintaining synaptic function. Therefore, during network activity in neonatal slices, lactate can be an efficient energy substrate capable of sustaining and enhancing aerobic energy metabolism

Reappraisal of anoxic spreading depolarization as a terminal event during oxygen–glucose deprivation in brain slices in vitro

International audienceAnoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen-glucose deprivation (OGD) in submerged cortical slices in vitro. However, mechanical artifacts caused by aSD-triggered edema may introduce error in the assessment of neuronal viability. Here, using continuous patch-clamp recordings from submerged rat cortical slices, we first confirmed that vast majority of L4 neurons permanently lost their membrane potential during OGD-induced aSD. In some recordings, spontaneous transition from whole-cell to outside out configuration occurred during or after aSD, and only a small fraction of neurons survived aSD with reperfusion started shortly after aSD. Secondly, to minimize artifacts caused by OGD-induced edema, cells were short-term patched following OGD episodes of various duration. Nearly half of L4 cells maintained membrane potential and showed the ability to spike-fire if reperfusion started less than 10 min after aSD. The probability of finding live neurons progressively decreased at longer reperfusion delays at a rate of about 2% per minute. We also found that neurons in L2/3 show nearly threefold higher resistance to OGD than neurons in L4. Our results suggest that in the OGD ischemia model, aSD is not a terminal event, and that the "commitment point" of irreversible damage occurs at variable delays, in the range of tens of minutes, after OGD-induced aSD in submerged cortical slices. Duration of ischemia is the key factor determining neuronal survival, neurological, and behavioral outcome. During global brain ischemia caused by cardiac arrest, irreversible loss of brain function and legal death are conventionally thought to occur within 5-10 min of cardiac arrest 1. Paradoxically, however, neuronal function can be restored even after much longer periods of global ischemia under several experimental settings including extracorporeal reperfusion with special cytoprotective reperfusate up to 4 h after cardiac arrest in pigs 2 or maintenance of rat brain slices that were prepared 1-6 h after cardiac arrest 3 , or after 30 min middle cerebral artery occlusion 4 , in ACSF. These observations raised the hypothesis that neurons may survive much longer episodes of ischemia than traditionally thought, thus opening questions for further research on the possibility of expanding the time window for resuscitation. Development of brain injury during ischemia proceeds through the initial phase of compensation, during which brain activity ceases but neurons maintain their membrane potential, and recovery of cardiovascular function restores brain activity without major damage to the brain 1,5,6. Passage to the decompensation phase is associated with a wave of collective and nearly complete neuronal depolarization, so-called anoxic spreading depolarization (aSD) occurring at ~ 5 min after the onset of ischemia 5,7,8. Generation of aSD is caused by depletion of intracellular reserves of energy metabolites, loss of function of the energy-dependent active ion transporters and rupture in transmembrane ionic gradients 5,7,8. aSD is a highly energy-demanding event which severely aggravates metabolic status 9. Considerable evidence indicates that aSD is the key ischemic event that triggers cascades of intracellular reactions leading to acute and delayed neuronal death 5,7,8. Similarly, periinfarct depolarizations (PIDs) severely compromise the metabolic state in the penumbra and cause expansion of the ischemic core during the development of the focal ischemic injury 10. The oxygen-glucose deprivation (OGD) model of an ischemia-like condition has been extensively used to explore acute changes in neuronal function during metabolic insult in brain slices in vitro. In this model, metabolite deprivation causes a series of events largely recapitulating the development of ischemic injury OPE

Genetically encoded chloride indicator with improved sensitivity.

International audienceChloride (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