Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique

Abstract

Source: Stefani, S. P., et al. Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro. J. Vis. Exp. (2019).

This video demonstrates a protocol involving the application of subthreshold noise currents to medial vestibular nucleus neurons using the whole-cell patch-clamp technique to study the impact of the noise currents on neuronal sensitivity to electrical stimuli.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Animals

NOTE: Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the Medical Foundation Building Animal Facility at the University of Sydney.

1. Maintain the mice on a standard 12 h light/dark cycle with environmental enrichment.
2. Use male and female C57BL/6 mice (3–5 weeks old) for all experiments.

2. Preparation of Solutions

1. Prepare 1 L of....

Results

Figure 1: Objective determination of 12 pA threshold. Firing rates for less than 12 pA (3 and 6 pA) and more than 12 pA (18 and 24 pA) were pooled and averaged. These averages were then analyzed using an ANOVA and statistical significance between sham and &#.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CaCl2	Scharlau	CA01951000	Used for ACSF and sACSF
D-(+)-Glucose	Sigma	G8270	Used for ACSF and sACSF
EGTA	Sigma	E0396-25G	Used for K-based intracellular solution
HEPES	Sigma	H3375-25G	Used for K-based intracellular solution
KCl	Chem-supply	PA054-500G	Used for ACSF, sACSF and intracellular solution
K-gluconate	Sigma	P1847-100G	Used for K-based intracellular solution
Mg-ATP	Sigma	A9187-500MG	Used for K-based intracellular solution
MgCl	Chem-supply	MA00360500	Used for ACSF and sACSF
Na3-GTP	Sigma	G8877-100MG	Used for K-based intracellular solution
NaCl	Chem-supply	SO02270500	Use for ACSF and intracellular solution
NaH2PO4.2H2O	Ajax	AJA471-500G	Used for ACSF and sACSF
NaHCO3	Sigma	S5761-1KG	Used for ACSF and sACSF
Sucrose	Chem-supply	SA030-500G	Used for sACSF
Isoflurane	Henry Schein	1169567762	Used for anaesthetising mice
EQUIPMENT
Borosilicate glass capillaries	Warner instruments	GC150T-7.5	1.5mm OD, 1.16mm ID, 7.5cm length
Data acquisition software	Axograph		Used for electrophysiology and analysis
Friedmen-Pearson Rongeurs	World precision instruments	14089	Used for dissection
Micropipette puller	Narishige	PP-830	Used for micropipette
Multiclamp amplifier	Axon instruments	700B	Used for electrophysiology
pH meter	Sper scientific	860033	Used for internal solution
Standard pattern scissors	FST	14028-10	Used for dissection
Sutter micromanipulator	Sutter	MP-225/M	Used for electrophysiology
Upright microscope	Olympus	BX51WI	Used for electrophysiology
Vibratome	Leica	VT1200	Used for slicing brain tissue