Analyzing the Glutamate Release and Clearance at a Single Neuron Synapse in a Mouse Brain Slice

Abstract

Source: Dvorzhak, A. et al., Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice. J. Vis. Exp. (2020)

This video demonstrates evaluating glutamate release and clearance at single corticostriatal synapses in a mouse brain slice. Electrical stimulation facilitates glutamate release at the synapse. The released glutamate alters sensor protein's fluorescence, which decays over time, indicating release and clearance efficiency.

Protocol

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

NOTE: Recordings from Q175 wild-type (WT) and heterozygotes (HETs) can be performed at any age and sex. Here we studied males and females at an age of 51 to 76 weeks.

1. Injection of the Glutamate Sensor iGlu_u for Expression in Corticostriatal Axons

1. Use the pipette puller (one step mode) to prepare borosilicate glass pipettes for the injection of the virus. After pulling, break the pipette manually to obtain a tip diameter of 30–5....

Results

Figure 1: Identification of iGlu_u-positive varicosities. (A) Fluorescence image obtained with a 510 nm high pass filter ("yellow image"). (B) Same view field acquired with a 600 nm high pas.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo microsope	WPI	PZMIII	Precision Stereo Zoom Binocular Microscope
Stereotaxic frame	Stoelting	51500D	Digital Lab New Standard stereotaxic frame
High speed drill equipment	Stoelting	514439V	Foredom K1070 cromoter Kit
Injection system	Stoelting	53311	Quintessential Stereotaxic Injector (QSI)
Hamilton syringe 5 µl	Hamilton	87930	75RN Syr (26s/51/2)
Laser positioning system	Rapp OptoElectronic	UGA-40	UGA-40
Blue laser for iGluu excitation	Rapp OptoElectronic	DL-473-020-S	473 nm laser
Dichroic mirror for 473 nm	Rapp OptoElectronic	ROE TB-355-405-473	Dichroic
1P upright microscope	Carl Zeiss	000000-1066-600	Axioskop 2 FS Plus
Objective 63x/1.0	Carl Zeiss	421480-9900	W Plan-Apochromat
4x objective	Carl Zeiss	44-00-20	Achroplan 4x/0,10
Dichroic mirror for iGluu	Omega optical	XF2030
Emission filter for iGluu	Omega optical	XF3086
Dichroic mirror	Omega optical	QMAX_DI580LP
Emission filter for autofluorescence subtr.	Omega optical	QMAX EM600-650
sCMOS camera	Andor	ZYLA4.2PCL10	ZYLA 4.2MP Plus
Acqusition software	Andor	4.30.30034.0	Solis
AD/DA converter	HEKA Elektronik	895035	InstruTECH LIH8+8
Aquisition software	HEKA Elektronik	895153	TIDA5.25
Electrode positioning system	Sutter Instrument	MPC-200	Micromanipulator
Electrical stimulator	Charite workshops	STIM-26
Slicer	Leica	VT1200 S	Vibrotome
Brown/Flaming-type puller	Sutter Instr	SU-P1000	P-1000
Glass tubes for injection pipettes	WPI	1B100F3
Glass tubes forstimulation pipettes	WPI	R100-F3
Tetrodotoxin	Abcam	ab120054	TTX
iGluu plasmid	Addgene	106122	pCI-syn-iGluu
Q175 mice	Jackson Lab	27410	Z-Q175-KI