Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Abstract

Source: Richevaux, L., et al. In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices. J. Vis. Exp. (2019)

The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Solutions for acute slice recordings

1. Prepare stock solutions of 10x artificial cerebrospinal fluid (aCSF) solution (124 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, 1 mM NaH₂PO₄, and 11 mM D-glucose) in pure deionized water prior to electrophysiology experiments. Store the solution at 4 °C in 1 L bottle without CaCl₂ and MgCl₂.
2. Prepare the potassium-gluconate-based pipette solution to contain 135 mM K-gluconate, 1.2 mM KCl, 10 ....

Results

Figure 1: Presubicular layer III neuron: intrinsic properties, response to light stimulation of thalamic afferents, and post-hoc revelation of cell morphology. (A) Firing pattern and membrane potential variations of layer III neuron for hy.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KCl	Sigma	P4504	final 1.2 mM
Magnesium chloride	Sigma	63069	final 2 mM
MultiClamp 700B	Axon Instruments
pClamp acquisition software	Axon Instruments
Peristaltic pump	Gilson	Minipuls 3	14-16 on the display for 2-3 ml/min
Potassium gluconate (K-gluconate)	Sigma	G4500	Final 135 mM
Borosilicate Capillaries	Havard Apparatus	GC150-10	1.5 mm outer, 0.86 inner diameter
Brown Flaming electrode puller	Sutter Instruments	P-87
EGTA	Sigma	E4368	Final 0.2 mM
470 nm LED	Cairn Research	P1105/470/LED DC/59022m	Use with matched excitation filter 470/40x and emission filter for GFP
AAV5.Syn.Chronos-GFP.WPRE.bGH	Penn Vector Core	AV-5-PV3446	Lot V6026R, qTiter GC/ml 4.912e12, ddTiter GC/ml 2.456e13
CCD Camera	Andor	DL-604M
Confocal Microscope	Zeiss	LSM710	20X
Digidata 1440A	Axon Instruments
Digital handheld optical meter	ThorLabs	PM100D	Parametered on 475 nm
HEPES	Sigma	H3375	Final 10 mM
LED Power Supply	Cairn Research	OptoLED Light Source