Extracellular Recordings from 450µm Hippocampal Slices cut with 5100mz Vibrotome

Abstract

(from: Increased hippocampal excitability in miR-324-null mice, Hayman et al, 2021)

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MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3′UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

Methods

Hippocampus Slice Preparation

Female miR-324 KO and WT mice (aged 5 months) were anaesthetised by inhalation of 100% isoflurane, before intramuscular injections with ketamine (≥ 100 mg/kg) and xylazine (≥ 10 mg/kg). After the pedal withdrawal reflex had ceased, the mice underwent a transcardial perfusion with 30 ml of sucrose artificial cerebrospinal fluid (aCSF), via injection into the left ventricle. The sucrose aCSF was composed of the following: 204.5 mM sucrose, 3.0 mM KCl, 1.25 mM NaH2PO4, 2.0 mM MgSO4, 2.0 mM CaCl2·2H2O, 10.0 mM of D-glucose and 24.0 mM NaHCO3 (all from Sigma-Aldrich). The brain was excised and 450 µm horizontal hippocampal sections were cut using a vibratome (Model 5100 mz, Campden Instruments). The overlying cortex was trimmed from around the hippocampus, before being placed in a holding chamber at room temperature at an interface between carbogen gas and aCSF (with sucrose replaced by 126 mM NaCl) for 1 h. In order to avoid any confounding issues due to dorsal–ventral (DV) differences within the hippocampus we used only slices from the central portion of the hippocampus (between − 3.24 mm and − 5.04 mm along the DV axis relative to the bregma).