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 c...

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.4K 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

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Behavior

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 Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Source: Kinnavane, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63077">Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for studying synaptic efficiency in the lateral entorhinal cortex (LEC) by optogenetically stimulating medial prefrontal cortex (mPFC) axons and recording the responses of pyramidal neurons in the LEC. This method combines patch-clamp recordings with synaptic activation by light stimulation to study synaptic plasticity.

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

Stimulation and Modulation Techniques

MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices

Source: Thalhammer, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57223">Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits.</a> J. Vis. Exp. (2018)
This video demonstrates microRNA-mediated inhibition of excitatory synaptic currents in mouse hippocampal brain slices. The CA3 neurons were infected with a recombinant viral vector harboring a construct for expressing a fluorescent reporter, a light-activated membrane channel, and a microRNA that downregulates the expression of voltage-gated calcium channels. Upon placing a slice in a recording chamber and patching a CA1 neuron with a recording pipette, light pulses were used to stimulate the light-activated channel, resulting in an ion influx and generating an action potential. The absence of voltage-gated calcium channels in CA3 neurons inhibits neurotransmitter release, causing inhibition of excitatory postsynaptic currents in the synaptically-connected CA1 neurons.

1. Assessing the Role of Presynaptic Proteins in Intact Neuronal Circuits by Targeted Stimulation of Knocked-down Neurons with Optogenetics
NOTE: The ...

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

Summary

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