Simultaneous Imaging and Current Clamp Recordings from Hippocampal Slices during Simulated Ischemia
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Following sudden failure of the Na+/K+ATPase pump, the acute neuronal swelling and dendritic damage that occurs within minutes of stroke onset are consequences of anoxic depolarization (AD). The AD front in our protocol is imaged as an increase in tissue light transmittance (LT) propagating across gray matter of the hippocampal slice preparation. Under current clamp in the single neuron, AD is recorded as a sharp depolarization within 6 min of O2/glucose deprivation (OGD). Simultaneous imaging and current clamp recordings show that the increased LT front and sudden depolarization are coincident. AD onset in CA1 hippocampal neurons is delayed in slices pretreated with 10 μM dibucaine (dib), a local anaesthetic understood to block voltage-gated sodium channels, and 10 to 100 µM carbetapentane (CP), a sigma1 receptor agonist. We examined if changes to single cell excitability could explain how dibucaine and CP work to inhibit AD. Pretreatment of slices with dibucaine for 30 min had no effect upon resting membrane potential, or whole cell input resistance (n=11). However dib pretreatment consistently raised spike threshold, decreased AP frequency and increased the fast afterhyperpolarization (fAHP). Orthodromic and antidromic APs were also eliminated within 15 min. Intracellular dibucaine application in addition to similar effects upon intrinsic electophysiological properties reduced the peak potential of the fast AD while extending the time until the persistent depolarization of AD reached zero. In contrast, 30 -100 μM CP had no effect upon orthodromic or antidromic responses, probably because unlike dibucaine it did not markedly raise spike threshold. Also unlike dibucaine, the fAHP was eliminated while the slow AHP was accentuated, resulting in a lowering of the AP frequency during steady depolarization. Both drugs appear to inhibit AD onset by reducing cortical excitability at the level of the single pyramidal neuron, but through strikingly different mechanisms. Our simultaneous imaging and single cell recording under current clamp allowed for further examination of potential cell recovery after AD in CA1 neurons and astrocytes, as well as confirmation of AD generation in the CA3 region. Indirect evidence for a more robust AD generation and propagation was evident in the transverse slices of dorsal hippocampal CA3 region compared to coronal slices. AD in astrocytes was lower in amplitude and more prolonged, as well as often displaying recovery to near resting potential. This supported our previous imaging experiments showing that astrocytes quickly recover their volume post-AD.