Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Protocolo

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 (%) versus light output (mW/mm²)) for each objective for values measured for 470 nm wavelength.
2. Retrieve an acute amygdala slice from the interface chamber and place in the slice chamber mounted onto the upright microscope equipped with a fluorescent lamp. Take care to position the slice such that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse slice with fresh, oxygenated artificial cerebrospinal fluid (ACSF) at a rate of 1 - 2 ml/min at a temperature of ~31 °C.
3. Observe presynaptic fibers in the slice using the fluorescent lamp in combination with appropriate filter sets for the specific fluorescent protein expressed. Use 5x objective to obtain an overview (Figure 1E), and 60x objective for assessment of fiber density within the target area.
   Note: For GFP and YFP, use Filter set "green" (Excitation 472/20, Beamsplitter 495, Emission 490 LP) for mCherry use filter set "red" (Excitation 560/40, Beamsplitter 585, Emission 630/70) as specified in the materials/equipment table.
4. Open or restrict the aperture in the microscope light pathway as desired for the experiment (Figure 2D).
5. To obtain a patch recording, fill a patch pipette with internal solution and mount it in electrode holder. Apply positive pressure to the patch pipette and slowly lower it first into the bath solution and then under visual control into the slice using the micromanipulator.
   1. Approach the neuron of interest with the patch pipette from the side and top. Release positive pressure when the pipette is on the surface of the cell (dimple visible on cell surface) and obtain a "gigaseal" by applying negative pressure.
   2. Apply further suction to rupture the membrane patch to obtain whole cell recording. Subsequently, stimulate labeled fibers with the connected LED using the appropriate wavelength for activating channelrhodopsin (ChR) (470 nm) while recording electrical responses from the cell.
   3. For synaptic stimulation start with a low LED intensity and increase until the desired synaptic current amplitude is reached. Trigger the LED by configuring digital outputs in the data acquisition software to control the timing and pulse length (examples in Figure 3).
      Note: other software and/or TTL-generating devices can be used to trigger LED.
6. Repeat stimulation with opened or restricted aperture (step 1.4) in the microscope light pathway as desired for the next recorded cell and/or in the presence of specific drugs.

Resultados

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 the injection pipette. Inset: Zoom in picture of injection pipette filled with virus solution mixed with fast green. Scale bar: 3 mm. (B

Materiales

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