Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Summary

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Abstract

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Introduction

Precise tools for visualization and concurrent activation of specific connections between brain areas and specific types of neurons are becoming more important in understanding the functional connectivity underlying healthy brain function and disease states. Ideally, this entails physiological investigation of precise synaptic properties with which identified neurons communicate. This is particularly true for connections between brain areas that cannot be preserved in a single acute brain slice. In the past, this has been largely achieved in separate experiments. On the one hand, neural tracers injected in vivo have been employed combined with subsequent light or electron microscopic analysis of pre- and postsynaptic partners. On the other hand, when fiber tracts from the region of origin are preserved and accessible in the slice preparation, electrical stimulation has been used to assess synaptic communication mechanisms with cells in the target region.

With the advent of optogenetics, the targeted expression of light-gated cation-channels, such as Channelrhodopsins (ChRs) fused to fluorescent proteins, now enables activation of neurons and their axonal trajectories while allowing for their visualization and post-hoc anatomical analysis ^1-4. Because ChR-expressing axons can be stimulated even when severed from parent somata ⁵, it is possible in brain slices to: 1) assess inputs from brain regions that were not accessible with conventional electrical stimulation, because fiber tracts are not separable or the specific trajectory is not known; 2) unequivocally identify the region of origin for specific inputs that were postulated but incompletely understood; and 3) investigate the functional connectivity between defined cell types, both locally and in long-range projections. Because of a number of advantages, this optogenetic mapping of circuits in brain slices has become widely used in the last years, and a variety of viral vectors for expression of fluorescently-tagged ChRs are readily available from commercial suppliers. Some key advantages of optogenetic activation over conventional electrical stimulation are no damage to the tissue due to placement of stimulation electrodes, specificity of fiber stimulation because electrical stimulation may also recruit fibers of passage or other nearby cells, and an equally rapid and temporally precise stimulation. In addition, stereotactic injection of viral vectors can easily be targeted to specific brain areas ⁶ and conditional or cell-type specific expression can be achieved using Cre-dependent expression and/or specific promoters ⁷. Here, this technique is applied for mapping of long-range and local circuits in the fear system.

The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories ^8,9. Apart from the amygdala, the medial prefrontal cortex (mPFC) and hippocampus (HC), structures that are reciprocally connected to the amygdala, are implicated in aspects of acquisition, consolidation and retrieval of fear and extinction memories ^10,11. Activity in subdivisions of the mPFC appears to play a double role in controlling both high and low fear states ^12,13. This could in part be mediated by direct connections from mPFC to the amygdala that would control amygdala activity and output. Therefore, in the last years, several studies started in ex vivo slice experiments to investigate synaptic interactions between mPFC afferents and specific target cells in the amygdala ^14-17.

During fear learning, sensory information about conditioned and unconditioned stimuli reaches the amygdala via projections from specific thalamic and cortical regions. Plasticity of these inputs to neurons in the lateral part (LA) of the basolateral amygdala (BLA) is an important mechanism underlying fear conditioning ^9,18. Increasing evidence suggests that parallel plastic processes in the amygdala involve inhibitory elements to control fear memory ¹⁹. A group of clustered inhibitory neurons are the GABAergic medial paracapsular intercalated cells (mpITCs), but their precise connectivity and function is incompletely understood ^20-22. Here, optogenetic circuit mapping is used to assess afferent and efferent connectivity of these cells and their impact on target neurons in the amygdala, demonstrating that mpITCs receive direct sensory input from thalamic and cortical relay stations ²³. Specific expression of ChR in mpITCs or BLA neurons allows mapping of local interactions, revealing that mpITCs inhibit, but are also mutually activated by, BLA principal neurons, placing them in novel feed-forward and feedback inhibitory circuits that effectively control BLA activity ²³.

Protocol

Ethics statement: All experimental procedures were in accordance with the EU directive on use of animals in research and were approved by the local Animal Care and Use Committee (Regierungspräsidium Tuebingen, state of Baden-Württemberg, Germany) responsible for the University of Tübingen. 

1. Stereotactic Injection Procedure

1. Prepare sterile tools (scissors, scalpel, clamps, drill, needles, suture material) using a sterilizer. Arrange sterile tools and other required solutions and surgery supplies such as sterile cotton swaps, disinfectant, sterile phosphate buffered saline (PBS, pH 7.4), and H₂O₂ on a sterile surgical drape.
2. Pull glass micropipettes for injections (Figure 1A, inset) using a 3 mm wide box filament on a horizontal microelectrode puller according to the manufacturer's instructions.
   Note: For deep injections, the electrode taper should be sufficiently long to reach the ventral most coordinates (approx. 5 mm; for coordinates, see 1.10).
3. Premix 1 µl of virus solution and 0.2 µl of 0.1% fast green solution in sterile PBS (for better visibility of solution in the glass pipette). Fill glass pipettes with mixture of virus solution and fast green using a microliter pipette with a fine tip (Figure 1A, inset).
   Note: Work with recombinant Adeno-associated viral vectors (rAAV) is considered biosafety level 1 (BSL 1) work and needs to be performed in a BSL 1 laboratory. rAAVs used in this study (Figure 1C) were: 1) mPFC: rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9) ¹⁶; 2) MGm/PIN: rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9) ²³; 3) mpITC/BLA: rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1) ²³
4. Anesthetize a mouse using a small animal anesthesia machine (Isoflurane: 3% in oxygen for induction).
   Note: Mice used in this study were 4 - 7 weeks old and of the following strains and genotypes: C57Bl6/J wild type (experiment in Figures 2A and 3A-D); GAD67-GFP ²⁴ (experiment in Figures 2B and 3E-H); Tac2-Cre ²⁵ (experiment in Figures 2C and 3I-J).
5. Shave head between ears and eyes. Apply disinfectant (providone-iodine based) to shaved head using cotton swabs.
6. Apply an eye ointment to prevent drying of eyes during anesthesia. Subcutaneously inject mouse with analgesic (meloxicam-based, 0.1 ml of 5 mg/ml solution).
7. Place mouse in stereotactic frame (Figure 1A) and maintain anesthesia via a gas anesthesia mask (Isoflurane: 2% in oxygen for maintenance). Check anesthesia depth using limb withdrawal reflex before continuing.
   Note: Maintain sterile conditions as well as possible during the entire surgical procedure; wear disposable facemask, surgical gown, and gloves.
8. Make skin incision on top of the head using scissors. Gently pull skin to the side using blunt forceps, fix with clamps to expose skull surface, and clean skull with H₂O₂.
9. Mark injection sites on skull using a fine tip permanent marker and drill holes for both hemispheres (Figure 1B). Coordinates for injections in this study are (from Bregma (mm)): mPFC: anterior 1.9, lateral ± 0.3, ventral 2.1; MGm/PIN: posterior 3.0, lateral ± 1.8, ventral 3.8; mpITC/BLA: posterior 1.45, lateral ± 3.35, and ventral 4.75.
10. Mount filled glass pipette onto stereotactic frame connected to a pressure injection device and bring the pipette to Bregma position.
11. Break off the tip of the glass pipette using fine straight-tip forceps. Do this gently to prevent aerosol generation of virus solution. Make sure the pipette tip is open by applying a few pressure pulses and observing extrusion of drops of virus solution.
12. Go to the desired injection coordinates and inject half of the pipette content (~0.5 µl) using the following settings on the pressure injection device: Pressure: 20 psi, average pulse length: 30 ms, average number of pulses: 50.
13. Leave pipette in place for ~1 min before slowly (1 mm/min) retracting it.
    Note: Make sure pipette is not blocked before repeating procedure with same pipette on other hemisphere. In case of blocked tip, clean or break off tip and zero pipette position at Bregma again.
14. Clean skull with PBS (pH 7.4), remove clamps, gently pull the skin together, and suture the incision with individual button suture (3 - 4 knots). Apply disinfectant (providone-iodine based) around the wound.
15. Stop the anesthesia and do not leave mouse unattended until it is fully awake. Keep single housed or return to company of other animals only when fully recovered. Postoperatively, continue to monitor the health status, and administer analgesic if necessary. Follow procedures according to rules put forward by the local Animal Care and Use Committee.

2. Preparation of Acute Slices

1. Prepare ACSF, cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks.
   Note: 1 L of artificial cerebro-spinal fluid (ACSF) is required per experiment, and is prepared by dissolving chemicals in double-distilled H₂O as previously published ^16,23. Cutting solution is prepared by supplementing 200 ml of ACSF with 0.87 ml of 2 M MgSO₄ stock solution.
2. Oxygenate ACSF and cutting solution throughout the experiment with 95% O₂ and 5% CO_2.
3. Deeply anesthetize mouse using a small animal anesthesia machine using isoflurane (3% in oxygen). Check anesthesia depth using limb withdrawal reflex before continuing.
4. Decapitate mouse using large scissors and immediately chill head in ice-cold cutting solution.
5. Open skull by a single midline incision from caudal to rostral and gently push pieces of skull to the sides using forceps. Rapidly remove brain by gently lifting it out of the skull using a small rounded spatula. Cut off cerebellum with scalpel. Place brain in ice-cold cutting solution.
6. For mPFC injection sites: cut off anterior part of the brain (containing mPFC) using a scalpel and put in ice-cold cutting solution until slicing.
7. Remove excess cutting solution with a filter paper and glue the posterior part of the brain onto the vibratome stage.
8. For tilted amygdala slices, glue the brain on an agar block (4%) cut at a 35° angle (Figure 1D, middle). For coronal amygdala slices, MGm/PIN injection sites and mPFC injection sites, glue the brain directly on stage (Figure 1D, left and right). Place an additional agar block behind brain for stability while slicing.
9. Place stage in cutting chamber with ice-cold oxygenated cutting solution that is maintained at 4 °C using a cooling unit. Prepare acute slices of the amygdala (320 µm) using a sapphire blade. Place slices in an interface chamber supplied with oxygenated ACSF at RT.
10. After preparation of acute amygdala slices, place the interface chamber in a waterbath at 36 °C to recover slices for 35 - 45 min. Subsequently, return the interface chamber to RT. For recording from these slices, proceed with step 3.2.
11. During commencement of step 2.10, cut slices of the injection sites as described before for acute amygdala slices (steps 2.8 - 2.9). Recovery of slices in the waterbath is not required here. Optional: To quickly estimate injection site location, observe slices on a stereoscope equipped with a fluorescent lamp and appropriate filter sets.
12. Fix slices containing injection sites for post-hoc analysis by sandwiching them between two filter papers and submerging them in 4% paraformaldehyde (PFA) in PBS solution O/N. For analysis of injection sites proceed with step 4.1.

3. 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 ASCF 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 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 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 3.4) in the microscope light pathway as desired for the next recorded cell and/or in the presence of specific drugs.
7. After recording, fix slices for post-hoc analysis by sandwiching them between two filter papers and submerging them in 4% PFA O/N.
8. Analyze electrophysiological data.
   Note: Use appropriate software to visualize recorded sweep data for each individual stimulation offline. When analyzing effects of drugs, use peak detection routines to obtain time course of drug effect on synaptic current amplitudes for all individual sweeps. Determine when the drug effect reaches steady state. To prepare representative average responses for figures, use software to average ≥10 individual sweeps per experimental condition (c.f. Figure 3B-D, F-H, J right panel).
   Note: several alternative software packages can be used for data analysis.

4. Post-hoc Analysis of Injection Sites

1. Wash slices of injection sites (from step 2.12) and recording sites (if re-imaging is desired, from step 3.7) three times in PBS. This is done by repeatedly replacing solution with fresh PBS on a rotating shaker three times for 10 min.
2. Prepare 2% agar-agar solution in PBS, and let it cool down to ~65 °C. Place slices flat on the bottom of a small 30 mm diameter petri dish, embed slices in agar-agar and let it cool down until solid.
3. Glue an agar block with embedded slice or slices to the stage of a vibratome and place stage in cutting chamber with PBS.
4. Resection embedded slices to 70 µm thickness. Optional: After resectioning, slices can be stained, i.e., with Neurotrace to reveal cytoarchitecture (Figure 3A, C), and/or by immunofluorescence staining for YFP or mCherry to boost the signal from the ChR fusion protein.
5. Mount slices on slides and coverslip with mounting media. Image injection sites and, if desired, also image slices containing fibers in the projection areas using a fluorescent microscope or a confocal laser-scanning microscope (Figure 2A-C).

Results

This section shows the workflow of an ex vivo optogenetic approach and representative results from different experimental strategies to investigate the physiological properties of sensory and modulatory long-range projections to BLA and mpITC neurons as well as properties of local connectivity between mpITC and BLA.

After stereotactic injection of the selected viral vector at the desired coordinates into the mouse brain...

Discussion

This protocol describes a method for ex vivo optogenetic investigation of neural circuits and local connectivity that can be easily implemented on most, if not all, upright slice patch-clamp recording setups by equipping them with a ~470 nm LED at the epifluorescence light port. A major advantage of optogenetic stimulation of axonal projections in slices is that it allows for specific activation and investigation of properties of connections that were not accessible with conventional electrical stimulation,...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Surgery
Stereotactic frame	Stoelting, USA	51670	can be replaced by other stereotactic frame for mice
Steretoxic frame mouse adaptor	Stoelting, USA	51625	can be replaced by other stereotactic frame for mice
Gas anesthesia mask for mice	Stoelting, USA	50264	no longer available, replaced by item no. 51609M
Pressure injection device, Toohey Spritzer	Toohey Company, USA	T25-2-900	other pressure injection devices (e.g. Picospritzer) can be used
Kwik Fill glass capillaries	World Precision Instruments, Germany	1B150F-4
Anesthesia machine, IsoFlo	Eickemeyer, Germany	213261
DC Temperature Controler and heating pad	FHC, USA	40-90-8D
Horizontal Micropipette Puller Model P-1000	Sutter Instruments, USA	P-1000
Surgical tool sterilizer, Sterilizator 75	Melag, Germany	08754200
rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9)	Penn Vector Core, USA	AV-9-26973P
rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9)	Penn Vector Core, USA	AV-9-20938M
rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1)	Penn Vector Core, USA	AV-1-20298P
fast green	Roth, Germany	0301.1
Isoflurane Anesthetic, Isofuran CP (1ml/ml)	CP Pharma, Germany
Antiseptic, Betadine (providone-iodine)	Purdure Products, USA	BSOL32	can be replaced by other disinfectant
Analgesic, Metacam Solution (5mg/ml meloxicam)	Boehringer Ingelheim, Germany		can be replaced by other analgesics
Bepanthen eye ointment	Bayer, Germany	0191	can be replaced by other eye ointment
Drill NM3000 (SNKG1341 and SNIH1681)	Nouvag, Switzerland
Sutranox Suture Needle	Fine Science Tools, Germany	12050-01
Braided Silk Suture	Fine Science Tools, Germany	18020-60
Recordings, light stimulation, and analysis
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
Slicer, Microm HM650V	Fisher Scientific, Germany	920120
Cooling unit for tissue slicer, CU65	Fisher Scientific, Germany	770180
Sapphire blade	Delaware Diamond Knives		custom order, inquire with company
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	CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation
LED unit, USL 70/470	Rapp Optoelectronic	L70-000	CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation
Dual port adapter	Rapp Optoelectronic	inquire with company	CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation
Filter set red (excitation)	AHF, Germany	F49-560	Filters can be bought as set F46-008
(beamsplitter)	AHF, Germany	F48-585	Filters can be bought as set F46-008
(emission)	AHF, Germany	F47-630	Filters can be bought as set F46-008
Filter set green (excitation)	AHF, Germany	F39-472	Alternatives: filterset F36-149 or F46-002 (with bandpass emission)
(beamsplitter)	AHF, Germany	F43-495W	Alternatives: filterset F36-149 or F46-002 (with bandpass emission)
(emission)	AHF, Germany	F76-490	Alternatives: filterset F36-149 or F46-002 (with bandpass emission)
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		for electrophysiology data analysis, other alternative software packages can also be used
Post hoc analysis of injections and projections
Paraformaldehyde powder (PFA)	Roth, Germany	0335.2
Neurotrace 435/455 blue fluorescent Nissl stain	Invitrogen	N-21479
agar-agar for embedding and resectioning	Roth, Germany	5210.3
30 x 10 mm petri dishes for embedding	SPL Life Sciences		alternatives can be used
Slides, Super Frost	R. Langenbrinck, Germany	61303802	alternatives can be used
cover slips	R. Langenbrinck, Germany	3000302	alternatives can be used
Vecta Shield mounting medium	Vector Laboratories, USA	H-1000	alternative mounting media can be used
cellulose nitrate filter for flattening slices for fixation	Satorius Stedim Biotech, Germany	11406--25------N
Confocal Laser Scanning Microscope LSM 710	Zeiss, Germany