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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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

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

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

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

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Preferential Initiation and Spread of Anoxic Depolarization in Layer 4 of Rat Barrel Cortex

International audienceAnoxic 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

Coupling Cortical Neurons through Electronic Memristive Synapse

International audienceof electrophysiological spike-sorting [4] and optogenetic [5] approaches enables an efficient readout and control over activity of single or groups of neurons leading to a development of prosthetic devices. For example, fMRI-guided electrophysiological recordings of single motor cortex neurons involved in specific motor tasks combined with muscle electrostimulation enabled to produce prosthesis with a remarkable alleviation of the neurological deficits in hemiplegic patients with a traumatic lesion of synaptic connections between the corticospinal neurons and motor neurons in spinal cord. [6] Restoration of synaptic connections as in the case of traumatic injury above as well as in other patholo-gies associated with a synaptic loss of function and various synaptopathies could be also solved through an introduction of electronic synapses to connect neurons directly, given that these artificial synapses recapitulate the main feature of natural synapses including their plasticity. Moreover, development of electronic synapses with unprecedented, due to biological restraints, features during evolution could result in creation of cyborgs with unprecedented capacities. We used patch-clamp recordings from nonconnected pairs of cortical layer 5 pyramidal neurons in rat brain slices (Figure 1a). Action potentials (APs) evoked by suprathreshold depolarizing current injection in either neuron failed to evoke any response in another cell in the pair (Figure 1c), indicating that these cells were not connected by natural synapses in either direction. These neurons were then connected through an electronic circuit with an OMD, playing the role of a synapse analog (Figure 1b). The structure of the OMD included a conducting polymer-polyaniline (PANI), with a solid electrolyte-lithium salt doped polyethylene oxide (PEO), and its memristive features were based on the high difference in PANI conductivity in the oxidized and reduced forms. [7,8] The ratio of PANI conductivity in oxidized and reduced forms is in the order of 10 8. [8] However, the need to have a medium for these reactions reduces the maximum reported ratio for the entire device till about 10 5. [9] After setting the OMD resistance initially at high values by negative voltage loading (Figure 1d, bottom), APs in a "presynaptic" Cell 1 (Figure 1d, plot 2) were induced by a suprathreshold depolarizing steps (Figure 1d, plot 1). However, these APs in Cell 1 evoked only a subthreshold depolarizing response in the "postsynaptic" Cell 2 (Figure 1d, plot 4, the first Functional coupling live neurons through artificial synapses is the primary requirement for their implementation as prosthetic devices or in building hybrid networks. Here, the first evidence of unidirectional, activity dependent, coupling of two live neurons in brain slices via organic memristive devices (OMD) is demonstrated. ODM is a polymeric electrochemical element, which has two terminals for the connection in electrical circuits and which displays hysteresis and rectifying features. OMD coupling is characterized by nonlinear relationships determined by the instantaneous values of OMD resistance that can be controlled by the neuronal activity, and the excitation threshold in the postsynaptic neuron. OMD coupling also has the spike-timing features similar to that of the natural excitatory synapses. Also, OMD-synapses support synchronized delta-oscillations in the two-neuron network. It is proposed that OMD-synapses may enable realization of prosthetic synapses and building hybrid neuronal networks endowed with a capacity of learning, memory, and computation. A synapse is a biological structure, which connects two neu-rons enabling specific and unidirectional information flow (excitation or inhibition) from one neuron to another. Synaptic connections are the key elements of the neuronal networks and their plasticity underlies learning and memory. Recent progress in building artificial neuronal networks is largely based on the elements mimicking features of natural synapses in silico or in electrico. [1-3] Hybrid networks, in which brain-computer systems read and control the activity of live cells also require interphase devices with cellular resolution. Us