Patch Clamp Techniques Used for Studying Synaptic Transmission in Slices of Mammalian Brain

Abstract

SUMMARYProcedures are described for recording postsynaptic currents from neurones in slices of rat brain using patch clamp techniques. The method involves cutting brain slices (120-300 ,m thick) with a vibrating microtome followed by localization of cell somata, which can be clearly seen with Nomarski differential interference contrast optics in the light microscope. Tissue covering the identified cell is then removed mechanically and standard patch clamp techniques are applied. Using these methods, spontaneously occurring and stimulus-evoked inhibitory postsynaptic currents (IPSCs) were recorded from neurones in rat hippocampus at greatly improved resolution. In the presence of tetrodotoxin, to block presynaptic action potentials, spontaneous IPSCs seldom exceeded 25 pA. Evoked IPSCs elicited by constant electrical stimulation of a presynaptic neurone were larger and fluctuated in their amplitudes. Singlechannel currents, activated by the putative inhibitory transmitter y-aminobutyric acid (GABA), had a size of about 1 pA. The number of postsynaptic channels activated by a packet of inhibitory transmitter is probably not more than thirty, nearly two orders of magnitude smaller than previously reported estimates for CNS synapses. This might reflect matching of synaptic efficacy to the high input resistance of hippocampal neurones and could be a requirement for fine tuning of inhibition.

Gene Profiles and Electrophysiology of Doublecortin-Expressing Cells in the Subventricular Zone after Ischemic Stroke

“…Brain slices were prepared according to published protocols (Sakmann et al , 1989) that were modified. Briefly, rats or mice were anaesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneally) and perfused with ice-cold artificial cerebral spinal fluid (ACSF) composed of the following (in mmol/L): 125 NaCl, 3 KCl, 2.5 CaCl 2 , 1.5 MgCl 2 , 25 NaHCO 3 , and 10 Dextrose at pH 7.35 to 7.45 when equilibrated with 95% O 2 and 5% CO 2 for 3 mins.…”

In vitro neuronal depolarization and increased synaptic activity induced by infrared neural stimulation

“…The membrane current channel was first DC-filtered to eliminate slow changes in holding current, differentiated to facilitate detection of overlapping synaptic events, and then window-discriminated to detect downward deflections corresponding to post-synaptic currents [21]. Peristimulus time histograms of laser-evoked synaptic events were constructed from sequential laser stimuli using 100 ms bin width in Spike 2.…”

Metabolic Therapy for Temporal Lobe Epilepsy in a Dish: Investigating Mechanisms of Ketogenic Diet using Electrophysiological Recordings in Hippocampal Slices

“…In vitro electrophysiological recording is done using single-cell intracellular sharp electrodes (Abe and Ogata, 1981) or patch-clamp electrodes (Kawamura et al, 2004), or extracellular field recording with single electrodes (Masino and Dunwiddie, 1999) or electrode arrays (Knowles et al, 1987) using acute slices of hippocampus. Compared with in vivo hippocampal recordings, the advantages of hippocampal slices are several-fold: (1) Ease of use and tissue access: acute brain slices must be maintained by perfusion with oxygenated artificial cerebrospinal fluid (Sakmann et al, 1989). Continuous perfusion allows for changing the extracellular fluid, making it easy to apply and wash out agonists and/or antagonists of various proteins such as ion channels, receptors and transporters.…”