Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Abstract

Source: Bosch, D., et al. Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices. J. Vis. Exp. (2016).

This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

Protocol

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

1. Visualization and Stimulation of Presynaptic Fibers

1. Prepare patch microscope for optogenetic activation of fibers and cells:
   1. Center the mounted light emitting diode (LED) onto the light delivery pathway.
   2. Measure the LED light intensity at the back focal plane and at the output of each objective with a power meter choosing the appropriate wavelength of 470 nm.
   3. Calculate the light intensity in mW/mm² and create a calibration curve (LED intensity....

Results

Figure 1. Stereotactic Injections, Preparation of Acute Brain Slices, and Visualization of Presynaptic Fibers. (A, B) Stereotactic virus injection. A) Picture of anesthetized mouse placed in a stereotactic frame with skull exposed and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Artificial cerebrospinal fluid (ACSF)	for composition see references #16 and #23
Internal patch solutions	for composition see references #16 and #23
MagnesiumSulfate Heptahydrate	Roth, Germany	P027.1	prepare 2M stock solution in purified water
Stereoscope, SZX2-RFA16	Olympus, Japan
Xcite fluorescent lamp (XI120Q-1492)	Lumen Dynamics Group, Canada	2012-12699
Patch microscope, BX51WI	Olympus, Japan
Multiclamp 700B patch amplifier	Molecular Devices, USA
Digitdata 1440A	Molecular Devices, USA
PClamp software, Version 10	Molecular Devices, USA		used to control data acquisition and stimulation
Bath temperature controler, TC05	Luigs & Neumann, Germany	200-100 500 0145
Three axis micromanipulator Mini 25	Luigs & Neumann, Germany	210-100 000 0010
Micromanipulator controller SM7	Luigs & Neumann, Germany	200-100 900 7311
Glass capillaries for patch pipettes	World Precision Instruments, Germany	GB150F-8P
Cellulose nitrate filterpaper for interface chamber	Satorius Stedim Biotech, Germany	13006--50----ACN
LED unit, CoolLED pE	CoolLED, UK	244-1400	CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation
CoolLED 100 Dual Adapt	CoolLED, UK	pE-ADAPTOR-50E
LED unit, USL 70/470	Rapp Optoelectronic	L70-000
Dual port adapter	Rapp Optoelectronic	inquire with company
Filter set red (excitation)	AHF, Germany	F49-560	Filters can be bought as set F46-008
(beamsplitter)	AHF, Germany	F48-585
(emission)	AHF, Germany	F47-630
Filter set green (excitation)	AHF, Germany	F39-472	Alternatives: filterset F36-149 or F46-002 (with bandpass emission)
(beamsplitter)	AHF, Germany	F43-495W
(emission)	AHF, Germany	F76-490
LaserCheck, handheld power meter	Coherent, USA	1098293
IgorPro Software, Version 6	Wavemetrics, USA		for electrophysiology data analysis, other alternative software packages can also be used
Neuromatic suite of macros for IgorPro	http://www.neuromatic.thinkrandom.com