We thank Bertrand Mathon, M&#233;rie Nassar, Li-Wen Huang, and Jean Simonnet for their help in the development of previous versions of the stereotaxic injection protocol and Marin Manuel and Patrice Jegouzo for technical help. This work was supported by the French Ministry for Education and Research (L. R., L. S.), Centre National des Etudes Spatiales (M. B.), and Agence Nationale de la Recherche Grant&#160;<span lang="FR" style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">ANR-18-CE92-0051-01&#160;(D. F.).

All procedures were performed in accordance with the European Community Council Directive (2010/63/EU) and approved by the ethics committee of Paris Descartes University. The experimenter must obtain authorization for the procedure to comply with local regulations.
1. Planning of the experiment
<ol>
	<li>Define the brain area to be targeted. Determine the stereotaxic coordinates of the injection site with the help of a mouse brain atlas15. For the right antero-dorsal thalamic nucleus (ADN), the coordinates are: -0.82 posterior, 0.75 lateral, -3.2 depth (mm) relative to bregma. Coordinates may need to be adjusted for animals of different age, sex, or strain.</li>
	<li>Confirm and document the exactitude of the coordinates by injecting a fluorescent tracer (150 to 300 nL) observable with an epifluorescence microscope in a pilot experiment (Figure 1A,B).</li>
	<li>Define the type of virus to be injected. Store the virus in 6 &#181;L aliquots at -80 &#176;C as recommended by the producer. Bring 1 aliquot placed on ice to the surgery room, for injection of one to six animals on a given day. Biosafety regulations for the use of AAV may depend on the country or institution, and the use of a PSM 2 hood may be required. 
	NOTE: Here, we use a AAV2/5 serotype expressing Chronos, a fast channelrhodospin-2 variant, fused to green fluorescent protein under the control of the Synapsin promoter: AAV5.Syn.Chronos-GFP.WPRE.bGH.</li>
</ol>
2. Stereotaxic surgery
<ol>
	<li>Install a stereotaxic frame equipped with a pump holder on a stable standard laboratory bench. Adjust stereoscope so as to clearly see the zone where the animal&#8217;s head will be placed. Use a LED light source for illumination. Rotate the stereoscope away to access the pump holder, which is not needed for the first steps of the surgery.</li>
	<li>Install a 10 &#181;L Hamilton syringe equipped with a 33 G beveled metal needle in the pump holder. Test the ejection system with water.</li>
	<li>Anesthetize a 4- to 5-week old C57BL6 mouse with an intraperitoneal injection of a mix of ketamine hydrochloride and xylazine (100 mg/kg and 10 mg/kg, respectively). Prepare a mix of 1 mL of ketamine and 0.5 mL of xylazine in 8.5 mL of 0.9% NaCl. This will result in 10 mg/mL ketamine and 1 mg/mL xylazine in the mix. Of this mix, inject intraperitoneally 10 &#181;L per gram of the animal&#8217;s body weight. Duration of anesthesia is about 1 h.</li>
	<li>Verify that the animal is well-anesthetized with a toe pinch. Then, pull out the tongue to facilitate breathing. Shave the cranial hair.</li>
	<li>Inject 20 &#181;L of lidocaine hydrochloride (4 mg/mL; 2 mg/kg) under the skin of the head for local anesthesia and wait 5 min for the effect to begin. To avoid eye damage due to dryness, cover the eyes with topical ophthalmic ointment.</li>
	<li>To expose the skull, create a straight cut in the scalp with small surgery scissors. Place the animal in a stereotaxic frame, inserting the ear bars slightly rostral to the actual ear to rest on the bone and pull down the skin, which should create good access to the skull. Tighten into place. Install the nose piece.</li>
	<li>Maintain the body of the animal horizontally at the level of the head using a height-adjusted support. Place a heating pad under the mouse to keep it at physiological temperature.</li>
	<li>Clean the skull by applying 0.9% NaCl with a cotton swab to remove soft tissue from the bone. Use the stereoscope for the rest of the surgery.</li>
	<li>Adjust the skull so that the bregma-lambda axis is level, moving up or down the nose and teeth piece. This necessitates iterative measures of bregma and lamba, as both will change following adjustment of the nose level.</li>
	<li>Find the location of the injection site on the skull. Adjust the injection needle above the injection site according to posterior and medial coordinates and mark the skull with a disposable needle. Move the injection needle upward by 4 cm.</li>
	<li>Use a 0.5 mm burr with a drill to realize a 1 mm diameter craniotomy on the mark, at one-half of maximum speed. Swab eventual bleeding with a paper tissue.</li>
	<li>Empty the water contained in the Hamilton syringe for storage by completely ejecting it with the pump. Only the needle will still be filled with water.&#160;The needle is washed between each use with pure deionized water. Sterilization is not required.</li>
	<li>Take the&#160;aliquot of virus that is to be used for this day. Make sure that the viral solution is not frozen anymore but has remained cooled (close to 0 &#176;C, on ice). Only briefly remove from the ice to obtain 700 nL with a micropipette for small volumes. Deposit the drop on a 5 cm x 5 cm piece of paraffin film. Avoid creating bubbles. Put the remaining viral solution back on ice. 
	NOTE: The drop volume should be greater than the desired injection volume (700 nL for 200 nL injected). This will give a safety margin in case some of the liquid is lost during the transfer and allows performing a small test ejection (step 2.16) before proceeding.</li>
	<li>Place the paraffin film on top of the craniotomy. Plunge the needle in the drop of viral solution without changing the antero-posterior and lateral position.</li>
	<li>Use the &#34;withdraw&#34;&#160;function of the pump to fill the syringe with about 500 nL of viral solution disposed on the paraffin film. &#160;Do this under visual control (stereoscope), watch the drop disappear, and make sure not to aspirate air.</li>
	<li>Make sure the syringe has been filled correctly. Verify the functioning of the ejection system by driving down the plunger to test eject a small drop of liquid of 50 nL under visual control. Wipe the drop.</li>
	<li>Insert the needle into the brain to the chosen depth, by turning the knob controlling the dorso-ventral axis of the stereotaxic apparatus clockwise. Push the &#34;run&#34;&#160;button (speed 15 nL/min per volume of 150 nL injected). A small volume (50-300 nL, depending on the virus used) is slowly ejected over 10 min with an automatic pump.</li>
	<li>Wait 10 min after the injection to avoid leaking from the injection site. Then, slowly remove the needle over 3-5 min by turning the knob controlling the dorso-ventral axis counterclockwise. &#160;</li>
	<li>Rotate the vertical part of the stereotaxic frame with the syringe away from the animal. Immediately wash the needle in clean distilled water by filling-emptying it several times, in order to avoid clogging. Store the syringe filled with water.</li>
	<li>Remove the mouse from the stereotaxic frame. Suture the skin with 4-0 polyamide suture filament. Make three or four stiches, tied with 2-1-1 standard surgical knots.</li>
	<li>Place the mouse in a heated cage until it completely wakes up from anesthesia, and provide water and soaked food in a Petri dish placed on the ground. If the heat source is below the cage, use a spacer grid to avoid overheating. &#160; 
	NOTE: According to local guidelines, a single dose of ketoprofen (2-5 mg/kg, subcutaneously) or buprenorphine (0.05-0.1 mg/kg, subcutaneously) may be applied to prevent pain.</li>
	<li>When the animal is fully awake, return it to its home cage and monitor its well-being, particularly on the day following injection. Check for signs of pain. If any behavioral modification is observed, the animal is weighed to monitor its body weight.</li>
	<li>Depending on the virus used, the time for full expression may vary. Here, we allow 3 weeks for expression of AAV5.Syn.Chronos-GFP.</li>
</ol>
3. Solutions for acute slice recordings and fixation
<ol>
	<li>Prepare stock solutions of 10x concentrated cutting solution (125 mM NaCl, 25 mM sucrose, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, and 2.5 mM D-glucose) and artificial cerebrospinal fluid (ACSF) solution (124 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, and 11 mM D-glucose) in pure deionized water prior to electrophysiology experiments. Store these solutions at 4 &#176;C in 1 L bottles without CaCl2 and MgCl2.</li>
	<li>On the day of the experiment, dilute the stock solutions of cutting solution and ACSF 10x to a final volume of 0.5 L each. Agitate with a magnetic stirrer and oxygenize by bubbling with 95%/5% O2/CO2. Add divalent ions to obtain final concentrations of 0.1 mM CaCl2 and 7 mM MgCl2 for the cutting solution, and 2 mM CaCl2 and 2 mM MgCl2 for ACSF.</li>
	<li>Prepare the potassium-gluconate based pipette solution to contain: 135 mM K-gluconate, 1.2 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 2 mM MgCl2, 4 mM MgATP, 0.4 mM Tris-GTP, 10 mM Na2-phosphocreatine, and 2.7&#8211;7.1 mM biocytin for post-hoc cell morphology revelation. Adjust the solution&#8217;s pH to 7.3 and osmolarity to 290 mOsm. Store 1 mL aliquots at -20 &#176;C.</li>
	<li>Prepare 0.1 M PBS by diluting BupH PBS dry-blend powder pouches in 500 mL of distilled water, resulting in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2.</li>
	<li>To prepare 1 L of 4% PFA solution, dilute 111 mL of 36% liquid PFA and 90 mL of 10x PBS solution in distilled water.</li>
	<li>Prepare 30% sucrose solution containing 150 g of sucrose in 500 mL of 0.1 M PBS.</li>
</ol>
4. Preparation of brain slices
<ol>
	<li>Prepare the bench space with absorbent bench paper before perfusion.</li>
	<li>Install a drip about 1 m above the bench for gravity-fed perfusion. Attach a 24 G butterfly needle.</li>
	<li>Surround the cutting chamber of the vibratome with ice and store it in a freezer.</li>
	<li>Anesthetize the mouse with intraperitoneal injection of the same ketamine-xylazine mixture used for surgery. Assess the stage of the anesthesia by pinching the toe with forceps. When fully asleep, inject 100 &#181;L of heparin (5000 U.I./mL) intraperitoneally.</li>
	<li>Fix the animal with adhesive tape on the absorbent paper, lying on its back. Open the thoracic cage by cutting the ribs on the left and right sides with small scissors, from the diaphragm upwards. Maintain the thoracic cage open with the help of adhesive tape.</li>
	<li>Clamp the descending aorta with a hemostat and perfuse via the left ventricle of the heart with 4 &#176;C cooled and oxygenated (95%/5% O2/CO2), cutting the solution through the 24 G butterfly needle. After 5 s, open the right atrium with small scissors.</li>
	<li>After 5 min of perfusion, when the organs are bloodless, stop the perfusion. Decapitate the animal with big scissors and immerge the head into 4 &#176;C cooled and oxygenated cutting solution in a Petri dish.</li>
	<li>To extract the brain, cut the skin from neck to nose, then section the last vertebrae from the skull with scissors. Manually retract the skin and use small scissors to open the skull, cutting it along the midline, from caudal to rostral, upward to between the eyes.</li>
	<li>Carefully remove the parietal bone and caudal part of frontal bone with curved or bone forceps. Extract the brain with a small rounded spatula by inserting the instrument between the brain and the cranial floor, sectioning the olfactory bulb, optic nerve and other cranial nerves, and cerebellum.</li>
	<li>Gently submerge the brain in ice-cold cutting solution (4 &#176;C) in a beaker. Position the brain on filter paper to gently dry the cortical surface. Glue the brain cortex-down to the specimen holder of a vibratome, with the caudal side facing the blade, in order to cut horizontal brain slices.</li>
	<li>Fill the cutting chamber with ice-cold oxygenated cutting solution so the brain is fully immerged. Make a cut on the left hemisphere (contralateral to the injected side) to avoid potential left-right ambiguity on slices. 
	CAUTION: Always oxygenate the solution and protect slices from light exposure.</li>
	<li>Cut 300 &#181;m thick slices with the vibratome, at a speed of 0.07 mm/s at 1 mm amplitude. At this stage, it is recommended to briefly check the Chronos-GFP expression in the thalamus using a fluorescent flashlight (440-460 nm) and corresponding filter glasses (500 nm long pass).</li>
	<li>Isolate the hippocampal region with a scalpel and transfer it to a chamber positioned in a beaker filled with bath-warmed (34 &#176;C), oxygenated (95%/5% O2/CO2) ACSF.</li>
	<li>After 15 min, take the chamber out of the heated water bath and let the slices rest at room temperature, still oxygenated for at least 45 min until use.</li>
</ol>
5. Whole-cell patch-clamp recording
<ol>
	<li>Gently transfer a brain slice containing the hippocampal complex with a custom-made glass transfer pipette to the recording chamber mounted on an upright microscope. A transfer pipette is made of a shortened Pasteur pipette (inner diameter 6.5 mm) attached to a rubber pipette bulb. Continuously perfuse the recording chamber (3 mL) with 34 &#176;C (warmed) ACSF bubbled with 95%/5% O2/CO2. Set the speed of the peristaltic pump to 2-3 mL/min.</li>
	<li>Briefly examine Chronos-GFP expression in axon terminals in the region of interest with blue LED illumination (470 nm) and observe with a 4x objective. GFP fluorescence is visualized through an appropriate emission filter, with a CCD camera image displayed on a computer screen.</li>
	<li>Place a slice anchor made from a U-shaped platinum wire with tightly spaced nylon strings (&#34;harp&#34;) on the slice to maintain it.</li>
	<li>Change to a 63x immersion objective and adjust the focus. Check for axons expressing Chronos-GFP and choose a pyramidal neuron for patch recording.</li>
	<li>Move the objective upward.</li>
	<li>Pull pipettes using a Brown-Flaming electrode puller from borosilicate glass. The puller is set to produce pipettes with approximately 1 &#956;m in tip diameter. Fill the pipettes with K-gluconate-based internal solution.</li>
	<li>Mount the pipette in the pipette holder on the head-stage. Lower the pipette in the chamber and find the tip under the objective. Pipette resistance should be between 3&#8211;8 M&#937;. Apply a light positive pressure with a syringe so as to see a cone of solution outflow out of the pipette and progressively lower the pipette and objective to the surface of the slice.</li>
	<li>Patch the cell in voltage-clamp configuration: approach the identified neuron and delicately press the pipette tip onto the soma. The positive pressure should produce a dimple on the membrane surface. Release the pressure to create a giga-ohm seal (&#62;1 G&#937; resistance). Once sealed, set the holding voltage to -65 mV. Break the membrane with a sharp pulse of negative pressure: this is achieved by applying strong suction to a tube connected to the pipette holder.</li>
	<li>Record in whole-cell current clamp mode the responses of the neuron to hyperpolarizing and depolarizing current steps (Figure 2A). 
	NOTE: This protocol will be used to determine active and passive intrinsic properties of the cell. Custom-written MATLAB routines are used for off-line analysis10,16.</li>
	<li>Record in current- or voltage-clamp postsynaptic responses to whole-field 475 nm LED stimulation of afferent fibers expressing Chronos. Stimulate with trains of 10 stimulations of 2 ms durations at 20 Hz (Figure 2B,C). Light intensity may vary from 0.1&#8211;2 mW. 
	NOTE: Light intensity was measured with a digital handheld optical power console equipped with a photodiode sensor, positioned under the objective. Response latencies of 2&#8211;4 ms are characteristic for a monosynaptic connection.</li>
	<li>To investigate the nature of the synaptic transmission between the long-range afferents and the recorded neuron, different pharmacological agents may be used. To pharmacologically distinguish direct, monosynaptic responses from indirect responses via network activation, add 1 &#181;M TTX and 100 &#181;M 4-AP to the ACSF. 
	NOTE: Bath application of glutamate receptor blockers allows to determine the nature of the neurotransmitter that is released and the identity of postsynaptic receptors. For example, AMPA type glutamate receptors will be blocked by NBQX (10 &#181;M) and NMDA receptors by APV (100 &#181;M). Depending on the aim of the study, protocols may be conceived to investigate voltage dependence of synaptic responses or response dynamics over time.</li>
	<li>Wash with original ACSF solution to patch another cell, or transfer the slice containing a biocytin-filled neuron in a small vial filled with 4% PFA.</li>
	<li>After overnight fixation in 4% PFA, wash the slice in 0.1 M PBS (2x for 5 min, 1x for 20 min).</li>
	<li>Store in 30% sucrose at 4 &#176;C.</li>
</ol>
6. Biocytin revelation
<ol>
	<li>Transfer the fixed slices containing biocytin-filled neurons onto a glass blade in a drop of 30% sucrose and perform three cycles of freezing-thawing: place the blade onto dry ice disposed in a styrofoam box for 1 min until drops of sucrose are completely frozen, then press the blade against the hand palm to thaw.</li>
	<li>Wash the slice 3x in 0.1 M PBS (2x for 5 min, 1x for 1 h and 50 min), gently agitated. Do not exceed 2 h for the last washing.</li>
	<li>Pre-incubate the slice at RT for 2 h in agitated buffer solution containing 2% milk powder (0.4 g in 20 mL) to saturate non-specific sites and 0.5% Triton X100 (0.1 mL in 20 mL) to permeabilize the membranes in 0.1 M PBS.</li>
	<li>Incubate overnight at 4 &#176;C in a solution containing 2% milk powder, 1% Triton X100, streptavidin-Cy5 conjugate (1/500), and DAPI (1/1000) in 0.1 M PBS, gently agitated.</li>
	<li>Wash the slice three times in 0.1 M PBS (2x for 5 min, 1x for 2 h). The last wash can last longer, up to 4 h, to reduce background staining.</li>
	<li>Before mounting the slice, use an epifluorescence microscope at 10x magnification configured to observe Cy5 fluorescent markers in order to identify the side of the slice containing the marked cell in a chamber filled with PBS.</li>
	<li>Transfer the slice onto a blade, cell-side up, dry it with a paper tissue, and mount it using high-resistance mounting medium.</li>
	<li>Use an epifluorescence microscope at 10x magnification in Cy5 and DAPI configuration to examine the cell body location, and in GFP configuration to observe the marked afferents, or a high-resolution confocal microscope at 20x for detailed somatic, axonal, and dendritic morphology (Figure 2D,E). Filter settings are detailed in the Table of Materials.</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature Methods. 11 (3), 338-346 (2014).</li><li>Hass, C. A., Glickfeld, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fidelity+optical+excitation+of+cortico-cortical+projections+at+physiological+frequencies.">High-fidelity optical excitation of cortico-cortical projections at physiological frequencies.</a> Journal of Neurophysiology. 116 (5), 2056-2066 (2016).</li></ol>

The authors declare no competing financial interests.

In vivo viral injection to express light-sensitive opsins in a defined brain area is a choice method for the optogenetic analysis of long-range functional connectivity10,11,17,18. Stereotaxic injections offer the possibility to precisely target a specific area of the brain. The coexpression of an opsin with a fluorescent reporter conveniently allows evaluation of the successful expression and confirmation of the precise injection site. The use of AAV serotype 2/5 typically restricts expression to the targeted brain region. In this way, a restricted population of neurons is transfected, expressing light-sensitive ion channels in their cell bodies and axon terminals. In subsequent ex vivo slice experiments, it is possible to stimulate these axon terminals with light pulses directly in their target area, while reading out successful synaptic transmission via patch-clamp recording of a post-synaptically connected neuron. The above protocol is robust and convenient, and some additional notes may help performance of successful experiments.
Different types of anesthesia may be used. Described here is the intraperitoneal injection of a ketamine-xylazine combination as an easy-to-use, short-term anesthesia with convenient analgesia19. The depth and duration of anesthesia may vary to some extent. In some cases, it may be necessary to inject another half-dose of ketamine-xylazine during the protocol. Isoflurane anesthesia can be a good alternative to induce more quickly and better control the depth of anesthesia. Coordinates of injection sites may be determined with the help of a mouse brain atlas. In practice, coordinates need to be tested and adjusted, if necessary.
Clean working conditions are also key. It is recommended to use disposable protective gear, including gloves, a mob cap, and a lab coat. When positioning the animal in the stereotaxic frame, special attention should be paid to the comfort of the animal, which will greatly improve efficiency of the anesthesia. The body of the animal should be aligned with the head and neck. The most critical step in positioning the animal and before craniotomy is adjustment of the bregma-lambda axis. Especially when targeting deep brain structures, even a small deviation will generate errors when lowering the injection needle into the brain. In some cases, one may deliberately choose and calculate an oblique needle trajectory.
The injection volume is a determinant factor for obtaining precisely localized opsin expression. A small volume is ideal to privilege a tightly restricted transfection zone. Higher volumes may be useful to cover the full extent of a large target area. If a large area needs to be covered, such as the septum18, it may be helpful to place several small injections with a range of neighboring coordinates. The interval until the ex vivo electrophysiological recording is also critical. A minimum time for full expression is necessary. While 3 weeks seem to be an optimal delay for these experiments11, the necessary delay may vary depending on the virus, its serotype, and the distance to the postsynaptic brain region.
The approach described here is even more powerful when combined with injections in transgenic animals. Previous work has exploited different mouse lines for subtypes of GABAergic neurons, in order to specifically target either PV- or SST-expressing interneurons for patch-clamp recordings20. Simultaneous double recording of neighboring PV and pyramidal neurons or SST and pyramidal neurons then allows comparison of strengths of long-range inputs between two neuron types11. This yields results that are standardized with respect to one neuron type. This standardization is particularly important in cases where the expression levels of opsins vary between different animals or different slices.
Slice health is essential for high-quality patch-clamp recordings. Constant oxygenation of the slices is crucial, and a slow cutting speed significantly improves slice surface quality. A slice thickness of 300 &#181;m preserves, to some extent, the microcircuit integrity in horizontal presubicular sections, including pyramidal neurons with their cell bodies, dendritic and local axonal ramifications, and local synaptic connections. The type of light-gated channels chosen to induce activation of afferent fibers will greatly influence the stimulation parameters (duration, light intensity). Chronos is a blue light-sensitive channelrhodopsin, and a broad range of illumination wavelengths can be used for activation (peak sensitivity around 500 nm, even with minimal light intensity of 0.05 mW/mm2, also activated at 405 nm, and up to 530 nm21). Furthermore, Chronos has fast kinetics properties in comparison to classical ChR2, which enables high frequency stimulations and reliable activation of long-range projections22. In combination with the expression of Chrimson, a red-shifted opsin variant, the independent optical excitation of distinct neural populations becomes feasible.

Defining connectivity between brain regions is necessary to understand neural circuits. Classical anatomical tracing methods allow establishing interregional connectivity, and lesion studies help to understand the hierarchical organization of information flow. For example, brain circuits for spatial orientation and head direction signaling involve the directional flow of information from the thalamus to the presubiculum. This has been demonstrated by lesion studies of antero-dorsal thalamic nuclei (ADN) that degrade the head direction signal in the downstream dorsal presubiculum, as well as the parahippocampal grid cell signal1,2.
The functional connectivity between brain areas is more difficult to establish at a cellular and subcellular level. In the hippocampus, a highly organized anatomy allows to investigate pathway-specific synaptic connections using electrical simulation in the slice preparation. Stimulation electrodes placed in stratum radiatum of CA1 can be used to specifically stimulate Schaffer collateral input from CA33. Stimulating electrodes placed in stratum lacunosum moleculare of CA1 will activate the perforant path input to CA14,5. Electrical stimulation activates neurotransmitter release from axon terminals; however, it activates neurons with somata near the stimulation site as well as axons of passage. It is therefore of limited use for studying afferents from defined brain regions when fibers of different regions of origin intermingle in the target structure, as is typically the case in the neocortex.
Neurons may also be stimulated with light. Optical methods include the photoactivation of caged glutamate, which can be combined with one- or two-photon laser scanning. Multiple closely spaced sites may be stimulated sequentially, with no mechanical damage to the tissue6. This has been successfully used to map synaptic receptors as well as activate individual neurons7. While glutamate uncaging can be used for local circuit analysis, it does not allow for specific activation of long-range inputs.
A method of choice for the investigation of long-range connectivity in neuronal circuits is the use of virus-mediated channelrhodopsin expression. Using in vivo stereotaxic injections as described here, the expression of light-gated ion channels can be targeted and spatially restricted to a desired brain region. In this way, channelrhodopsins are effective for mapping excitatory or inhibitory connectivity from one region to its target. Transfected axons terminals may be stimulated with light in a brain slice preparation, and patch-clamp recordings as a read-out allow examination of the functions and strengths of specific circuit components in the brain8. The optogenetic approach combined with stereotaxic injection of a virus offers unprecedented specificity and genetic control9. Stimulating with light additionally allows for both high temporal and spatial precision10,11.
The presubiculum is a six-layered cortical structure at the transition of the hippocampus and the para-hippocampal formation12,13. It receives important synaptic input from the ADN11 but also from several other cortical and subcortical regions14. Thus, the selective stimulation of thalamic axons terminals within a presubicular slice is not possible with electrical stimulation nor glutamate uncaging. Described in this protocol are methods to determine functional connectivity between brain regions (ADN and presubiculum) using precise stereotaxic injections of viral vectors expressing light-gated channels. Also described is the photostimulation of axons terminals of projecting neurons in their target region, coupled with whole-cell patch-clamp recordings of post-synaptic neurons in the brain slice preparation.

<table border="0" cellpadding="0" cellspacing="0" style="width:927px;" width="927">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.5 mm bur&#160;</td>
			<td style="width:183px;">Harvard Apparatus</td>
			<td style="width:136px;">724962</td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10 &#181;L Hamilton syringe</td>
			<td style="width:183px;">Hamilton</td>
			<td style="width:136px;">1701 RN - 7653-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X PBS solution</td>
			<td style="width:183px;">Thermofisher Scientific</td>
			<td style="width:136px;">AM9624</td>
			<td>&#160;text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">36% PFA</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:136px;">F8775</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">470 nm LED&#160;</td>
			<td style="width:183px;">Cairn Research</td>
			<td style="width:136px;">P1105/470/LED&#160; DC/59022m</td>
			<td style="width:365px;">use with matched excitation filter 470/40x&#160; and emission filter for GFP&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">AAV5.Syn.Chronos-GFP.WPRE.bGH</td>
			<td>Penn Vector Core</td>
			<td>AV-5-PV3446</td>
			<td>lot V6026R, qTiter GC/ml 4.912e12, ddTiter GC/ml 2.456e13&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">All chemicals</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bath temperature controler</td>
			<td style="width:183px;">Luigs &#38; Neumann</td>
			<td style="width:136px;">SM7</td>
			<td>Set at 34&#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">beveled metal needle</td>
			<td style="width:183px;">Hamilton</td>
			<td style="width:136px;">7803-05</td>
			<td>33 gauge, 13mm, point style 4-20&#176;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Big scissors</td>
			<td style="width:183px;">Dahle Allround</td>
			<td style="width:136px;">50038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Biocytin</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">B4261</td>
			<td>final 1-3 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Borosilicate Capillaries</td>
			<td style="width:183px;">Havard Apparatus</td>
			<td style="width:136px;">GC150-10</td>
			<td>1.5 mm outer, 0.86 inner diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Brown Flaming electrode puller</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:136px;">P-87</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">BupH Phosphate Buffered Saline pack</td>
			<td style="width:183px;">Thermofisher Scientific</td>
			<td style="width:136px;">28372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">butterfly needle for perfusion</td>
			<td style="width:183px;">Braun&#160;</td>
			<td style="width:136px;">Venofix A</td>
			<td>24G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CCD Camera</td>
			<td style="width:183px;">Andor&#160;</td>
			<td style="width:136px;">DL-604M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Confocal Microscope</td>
			<td style="width:183px;">Zeiss</td>
			<td style="width:136px;">LSM710</td>
			<td>20X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">curved forceps</td>
			<td style="width:183px;">FST&#160;</td>
			<td style="width:136px;">11011-17</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">CY5 configuration (confocal)</td>
			<td style="width:183px;"></td>
			<td style="width:136px;"></td>
			<td style="width:365px;">Helium-Neon 633nm (5,0 mW) laser; Mirror: MBS 488/561/633&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">CY5 configuration (epifluo)</td>
			<td style="width:183px;">Nikon/Chroma</td>
			<td style="width:136px;"></td>
			<td style="width:365px;">Fluorescent light (Intensilight); Excitation filter: BP645/30; Dichroic mirror: 89100 BS ; Emission filter: BP705/72</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">D9542</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">DAPI configuration (epifluo)</td>
			<td style="width:183px;">Nikon/Chroma</td>
			<td style="width:136px;"></td>
			<td style="width:365px;">Fluorescent light (Intensilight); Cube: Semrock Set DAPI-5060C-000-ZERO (Excitation: BP 377/50; Mirror: BS 409; Emission: BP 447/60)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Digidata 1440A</td>
			<td style="width:183px;">Axon Instruments</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Digital handheld optical meter</td>
			<td style="width:183px;">ThorLabs</td>
			<td style="width:136px;">PM100D</td>
			<td>Parametered on 475 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Double egde stainless steel razor blades</td>
			<td style="width:183px;">Electron Microscopy Sciences</td>
			<td style="width:136px;">72000</td>
			<td>Use half of the blade in the slicer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dual Fluorescent Protein Flashlight</td>
			<td style="width:183px;">Nightsea</td>
			<td style="width:136px;">DFP-1</td>
			<td style="width:365px;">excitation, 440-460 nm; emission filter on glasses, 500 nm longpass.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:243px;">EGTA</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">E4368</td>
			<td>final 0,2 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:243px;">Epifluorescence Microscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:136px;">Eclipse TE-2000E</td>
			<td>10 or 20X</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:243px;">Filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fluoro-Ruby 10%</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:136px;">AG335</td>
			<td>disolve 10 mg in 100 &#181;l of distilled water ; inject 150 to 300 nl</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">GFP configuration (epifluo)</td>
			<td style="width:183px;">Nikon/Chroma</td>
			<td style="width:136px;"></td>
			<td style="width:365px;">Fluorescent light (Intensilight); Cube: Filter Set Nikon B-2E/C FITC (Excitation: BP 465-495; Mirror: BS 505; Emission: BP 515-555)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Heatingplate</td>
			<td style="width:183px;">Physitemp</td>
			<td style="width:136px;">HP4M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Heparin choay 5000 U.I./ml</td>
			<td style="width:183px;">Sanofi</td>
			<td style="width:136px;"></td>
			<td>5 ml vial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEPES</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">H3375</td>
			<td>final 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">High speed rotary micromotor kit</td>
			<td style="width:183px;">Foredom</td>
			<td style="width:136px;">K.1070</td>
			<td>maximum drill speed 38,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Internal solution compounds :</td>
			<td style="width:183px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Isolated Pulse Stimulator</td>
			<td style="width:183px;">A-M Systems</td>
			<td style="width:136px;">2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">KCl</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">P4504</td>
			<td>final 1,2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ketamine 1000</td>
			<td style="width:183px;">Virbac</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ketofen 10%</td>
			<td style="width:183px;">Merial</td>
			<td style="width:136px;"></td>
			<td>100 mg/ml : dilute 1 &#181;l in 1ml total (0,1%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Laocaine (lidocaine)</td>
			<td style="width:183px;">MSD</td>
			<td style="width:136px;"></td>
			<td>16,22 mg/ml : dilute 1 ml in 4 ml total (around 4%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LED hi power spot for surgery</td>
			<td>Photonic (via Phymep)</td>
			<td style="width:136px;">10044</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:243px;">LED Power Supply</td>
			<td style="width:183px;">Cairn Research</td>
			<td style="width:136px;">OptoLED Light Source</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Manipulators</td>
			<td style="width:183px;">Luigs &#38; Neumann</td>
			<td style="width:136px;">SM-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mg-ATP 2H20</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">A9187</td>
			<td>final 4 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MgCl2</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">63069</td>
			<td>final 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Micro temperature controler</td>
			<td>Physitemp</td>
			<td style="width:136px;">MTC-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Milk powder</td>
			<td style="width:183px;">Carnation</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MultiClamp 700B</td>
			<td style="width:183px;">Axon Instruments</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Na Phosphocreatine</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">P7936</td>
			<td>final 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Na3-GTP 2H20</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">G9002</td>
			<td>final 0.4 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:243px;">needle holder/hemostat</td>
			<td style="width:183px;">FST</td>
			<td style="width:136px;">13005-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">pClamp acquisition software</td>
			<td style="width:183px;">Axon Instruments</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Peristaltic pump</td>
			<td style="width:183px;">Gilson</td>
			<td style="width:136px;">Minipuls 3</td>
			<td>14-16 on the display for 2-3 ml/min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium gluconate (K-gluconate)</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:136px;">G4500</td>
			<td>Final 135 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">ProLong Gold antifade mounting medium</td>
			<td style="width:183px;">Thermofisher Scientific</td>
			<td style="width:136px;">P36390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Rompun 2% (xylazine)</td>
			<td style="width:183px;">Bayer</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">small scissors</td>
			<td style="width:183px;">FST</td>
			<td style="width:136px;">14060-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium chloride 0.9%&#160;</td>
			<td style="width:183px;">Virbac</td>
			<td style="width:136px;"></td>
			<td>dilute 8.5 mL in 10 ml total</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope VISISCOPE SZT</td>
			<td style="width:183px;">VWR</td>
			<td style="width:136px;">630-1584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotaxic frame with digital display</td>
			<td style="width:183px;">Kopf</td>
			<td style="width:136px;">Model 940</td>
			<td>Small animal stereotaxic instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Streptavidin-Cy3 conjugate</td>
			<td style="width:183px;">Life technologies&#160;</td>
			<td style="width:136px;">434315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Streptavidin-Cy5 conjugate</td>
			<td style="width:183px;">Thermofisher Scientific</td>
			<td style="width:136px;">S32357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Superglue3 Loctite</td>
			<td style="width:183px;">Dutscher</td>
			<td style="width:136px;">999227</td>
			<td>1g tube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Suture filament Ethilon II 4-0 polyamid</td>
			<td style="width:183px;">Ethicon</td>
			<td style="width:136px;">F3210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Syringe pump</td>
			<td style="width:183px;">kdScientific</td>
			<td>Legato 130 - 788130</td>
			<td>Use Infuse and Withdraw modes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tissue slicer</td>
			<td style="width:183px;">Leica</td>
			<td style="width:136px;">VT1200S</td>
			<td>speed 0.07, amplitude 1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">tubing</td>
			<td style="width:183px;">Gilson</td>
			<td style="width:136px;">F117942, F117946</td>
			<td>Yellow/Black, Purple/Black</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">upright microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:136px;">BX51W1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Versi-dry bench absorbant paper</td>
			<td style="width:183px;">Nalgene</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo intracerebral stereotaxic injections for optogenetic stimulation of long-range inputs in mouse brain slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

The procedure presented here was used to express a blue light-sensitive channelrhodopsin (Chronos) fused to GFP in the antero-dorsal nucleus of the thalamus (ADN), by stereotaxic injection of anterograde adeno-associated virus. The stereotaxic coordinates were determined according to a mouse brain atlas and tested by injecting 200 nL of fluorescent tracer fluoro-ruby. The animal was sacrificed 10 min after the injection, and the brain was extracted and fixated overnight. Coronal brain sections were prepared to examine the injection site, which was correctly placed in and limited to ADN (Figure 1A,B). &#160;
In order to express Chronos-GFP in neurons of ADN, we injected 300 nL of AAV5.Syn.Chronos-GFP.WPRE.bGH. Three weeks after the injection, acute horizontal brain slices were prepared. Figure 1C shows a brain slice containing the thalamic injection site in the right hemisphere, with GFP expression in green. Upon inspection with an epi-fluorescence microscope equipped with a 4x objective, GFP labeled thalamic axons were observed in the presubiculum (Figure 1C,D). It was noted that thalamic axons densely innervated the superficial layers I and III of the presubiculum (Figure 1D).
The activity of presubicular neurons in layer III was recorded in the whole-cell patch-clamp configuration. Hyperpolarizing and depolarizing current steps were applied while recording the membrane potential variations (Figure 2A). Data was stored on a computer for later offline analysis of active and passive membrane properties. Presubicular layer III principal cells typically possessed a negative resting potential close to -63 mV and required depolarizing current injections to drive the membrane potential to firing threshold. A full description of their intrinsic properties has been published11.
Stimulating ADN axon terminals expressing Chronos-GFP elicited excitatory post-synaptic potentials (EPSPs) in presubicular layer III principal cells in current clamp mode (Figure 2B). Depending on light intensity, the EPSPs could reach action potential threshold. Postsynaptic responses were also observed in voltage-clamp mode as excitatory post-synaptic currents (EPSCs) were elicited (Figure 2C). Onset latencies of EPSCs evoked by light stimulations were short (median, 1.4 ms10), indicating a direct synaptic contact between thalamic axons and layer III presubicular neurons. Persisting EPSCs in TTX-4AP condition confirmed this monosynaptic activation. It is noteworthy that these cells responded reliably to the light stimulations of afferent axons with a regular firing pattern.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59534/59534fig1.jpg" /> 
Figure 1: Stereotaxic injection in the anterodorsal thalamic nucleus (ADN). (A) Schematic representation of the injection. (B) Injection site confirmation with fluoro-ruby in a coronal section. Inset indicates antero-dorsal level and distance from bregma. (C) Horizontal slice following AAV-Chronos-GFP injection in the thalamus. The axonal projections to the ipsilateral presubiculum should be noted. An incision on the left side of the slice (indicated by a black triangle) marks the contralateral hemisphere. (B, C) Scale bar 1 mm. (D) Magnified view of inset in (C) with ADN projections to the presubicular superficial layers. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59534/59534fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59534/59534fig2.jpg" /> 
Figure 2: Presubicular layer III neuron: intrinsic properties, response to light stimulation of thalamic afferents, and post-hoc revelation of cell morphology. (A) Firing pattern and membrane potential variations of layer III neuron for hyperpolarizing and depolarizing current steps. (B, C) Responses of layer III neuron to 2 ms light stimulations (blue bars) of thalamic axons recorded in (B) current-clamp and (C) voltage-clamp modes. (D, E) Layer III pyramidal neuron (white, indicated by filled yellow triangle) surrounded by thalamic axons expressing Chronos-GFP (green) in presubicular superficial layers with DAPI staining (blue) in horizontal slice imaged with an epifluorescence microscope (D, scale bar = 100 &#181;m) and confocal microscope at a high magnification (E, scale bar = 50 &#181;m). The cell in (A) is indicated with filled yellow triangles. A second, partially filled neuron is present in this slice indicated with empty yellow triangles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59534/59534fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices at JoVE.com

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

in-vivo-intracerebral-stereotaxic-injections-for-optogenetic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.3K Views.  CNRS (Integrative Neuroscience and Cognition Center, UMR 8002). This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Video: In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

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.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;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.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

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Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains