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.

Anoxic 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 out-side 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.

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.

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

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.

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

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.