Extracellular Recording from Coronal, 400µm Hippocampal Slices to Investigate Synaptic Transmission

Abstract

(from miRNA-132/212 Gene-Deletion Aggravates the Effect of Oxygen-Glucose Deprivation on Synaptic Functions in the Female Mouse Hippocampus, Bormann et al, 2021)

Download Full Paper

Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.

Method

Hippocampal Slices Preparation

Acutely dissected hippocampal slices obtained from miRNA 132/212−/− mice and WT littermates (n = 6–7 animals per experimental group) were prepared as previously described [22,51,52,53], with slight modifications. Briefly, mice were sacrificed by swift cervical dislocation and subsequent sharp-blade decapitation. Utilizing one midline incision from the foramen magnum to the frontal suture and two basolateral incisions from the foramen magnum outwards, the parietal bones were mobilized and removed. The brains were removed gently with a spatula and immediately submerged in an ice-cold aCSF solution of the following composition (in mM): 125 NaCl, 2.5 KCl, 20 NaHCO3, 2.5 CaCl2, 1 MgCl2, 25 D-glucose and 1 NaH2PO4 (pH 7.35–7.40). Coronal hippocampal slices (400 μm nominal thickness) were cut while submerged in ice-cold, carbogenated aCSF (95% O2/5% CO2), using a vibrating microtome (Vibratome 7000smz-2, Campden Instruments Ltd.; PO Box 8148, Loughborough, Leics.; LE12 7TJ. U.K.) at a frequency of 90 Hz, an amplitude of 0.75 mm and speed of 0.12 mm/s. Cutting was followed by separation of hemispheres and immediate transfer to a custom-build recovery chamber filled with carbogenated aCSF at 32 °C. Slices were allowed to recover for a minimum of 1.5 h before electrophysiological recording.