This work was supported by NIH/NIDCD grant R03 DC 011361, SVM CORP grant LAV3202, NSF-LA EPSCoR grant PFUND86, and a publication grant from Leica Microsystems (CCL) and NIH/NIDCD grant R01 DC 008794 (SMS).

1. Surgery and Injections
<ol>
	<li>Anesthetize mice using an intraperitoneal injection (22-25 G needle recommended) of ketamine (100-120 mg/kg) and xylazine (7.5-16 mg/kg).
	<ol>
		<li>Assess sedation using strong toe pinches until no responses are elicited. For all animal procedures described, modify to follow local IACUC guidelines as necessary.</li>
	</ol>
	</li>
	<li>Place sedated mice in a stereotaxic device by securing the head and ears in their respective holders.
	<ol>
		<li>Lubricate the eyes prior to placing the animal in the stereotaxic device with a sterile ophthalmic ointment.</li>
	</ol>
	</li>
	<li>Shave the scalp with a razor and sterilize the area with Betadine swabs.</li>
	<li>Using a scalpel, cut a midline incision and reflect the skin and underlying muscles above the area to be injected.</li>
	<li>Rotate a scalpel blade as a trephine to perform a craniotomy above the region to be exposed. Alternatively, use a small drill bit attached to a Dremel to perform the craniotomy.
	<ol>
		<li>To perform the craniotomy, place scalpel blade or drill bit on surface of skull and slowly rotate until enough bone has been excavate to reveal the pia mater underneath.</li>
		<li>Once the initial excavation is made, enlarge the craniotomy using fine forceps to carefully remove bone from the edges.</li>
	</ol>
	</li>
	<li>Fill a 1 &#956;l syringe with the viral solution to be injected (UNC Vector Core).
	<ol>
		<li>To enhance transfection efficacy, prepare viral solution with 4 &#956;g/ml polybrene.</li>
	</ol>
	</li>
	<li>Lower the needle into the area to be injected and pressure infuse 200 nl of solution containing virus with a syringe pump.
	<ol>
		<li>Let syringe sit for 10 min before withdrawing the needle.</li>
	</ol>
	</li>
	<li>Suture the skin with nonabsorbable monofilament Nylon and cover the incision with antibiotic ointment.
	<ol>
		<li>Sutures should be removed after 7-10 days.</li>
	</ol>
	</li>
	<li>Inject buprenorphine (0.05-0.1 mg/kg) for post-operative analgesia.</li>
	<li>Allow animals to post-operatively recover for 14-21 days to allow for adequate expression of channel rhodopsin.</li>
</ol>
2. Slice Preparation
<ol>
	<li>Following the recovery period, deeply anesthetize the animal with isoflurane anesthesia, as verified by absence of withdrawal reflex when the hind foot and/or toes are firmly pinched.</li>
	<li>Dissect the brain by decapitation followed by midline and frontal cuts to the skull.</li>
	<li>Peel apart the skull from the midline to expose the brain.</li>
	<li>Carefully remove the brain using a fine scoop spatula to separate the cranial nerves from the base of the brain.</li>
	<li>Quickly place dissected brain in small 100 ml beaker containing chilled, ice-cold artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 25 glucose).</li>
	<li>Remove brain from ACSF and place on a microscope slide covered with filter paper prewetted with ACSF.</li>
	<li>Make a blocking cut of the brain in the desired orientation using a clean razor blade.</li>
	<li>Affix the blocked surface of the brain to cutting stage using instant adhesive glue.</li>
	<li>Transfer stage to a vibratome cutting chamber (kept in freezer until ready for cutting).</li>
	<li>Pour ice cold ACSF into cutting chamber.</li>
	<li>Slice brain sections at 400-500 &#181;m using a vibrating tissue slicer.</li>
	<li>Transfer brain slices to a holding chamber containing oxygenated ACSF, and incubate at 30 &#176;C for at least 1 hr before experiment.</li>
</ol>
3. Physiological Recording
<ol>
	<li>After the incubation period, transfer a slice to a recording chamber on a modified microscope stage.</li>
	<li>Secure the slice in the bath during the experiment using a few threads of Nylon filaments attached to a platinum wire slice holder.</li>
	<li>Perfuse the slice with ACSF constantly.</li>
	<li>Pull recording pipettes to a tip resistance of 3-6 M&#937;&#160;when filled with intracellular solution containing the following (in mM): 127 K-gluconate, 3 KCl, 1 MgCl2, 0.07 CaCl2, 10 HEPES, 2 Na2-ATP, 0.3 Na-GTP, 0.1 EGTA. The intracellular solution should have pH 7.3, adjusted with KOH or gluconic acid and the osmolality of 280-290 mOsm.</li>
	<li>Using a standard visualized whole-cell recording setup, place recording pipette near neuron to be recorded while applying positive pressure22,23.</li>
	<li>Release positive pressure while applying suction to form Gigaohm seal.</li>
	<li>Apply brief suction to break membrane and form whole-cell configuration recording.</li>
	<li>Record the responses of neurons in either voltage or current clamp.</li>
</ol>
4. Laser-scanning Photostimulation
<ol>
	<li>Start data acquisition software and hardware, described in previous reports23-29. 
	Note: We have utilized both Tidelwave25 and, more recently, Ephus26 acquisition programs, written in Matlab and developed by the laboratory of Karel Svboda at Janelia Farms30. For a full description of laser-scanning photostimulation in Ephus, see Suter et al.26&#160;and the step-by-step guide30. 
	Note: The setup described in this article is derived from that described by Shepherd (2012)23. The laser beam is guided via an open beam&#160;system of mirrors to the top port of the microscope through the objective lens onto the slice (Figure&#160;2).</li>
	<li>Divert a small portion of the laser onto a photodiode with a microscope coverslip placed in the laser path and use the current output from this photodiode to monitor the laser intensity during experiments.
	<ol>
		<li>Calibrate the photodiode current output recorded in the software to the measured laser power at the back focal plane of the objective using a power meter during equipment setup23.</li>
	</ol>
	</li>
	<li>Control the laser power by rotating a variable neutral density wheel. The laser power used varied between 25-70 mW (at the back focal plane) in our experiments.</li>
	<li>Set the length and timing of the laser stimulating by modifying the pulse parameters for the laser and shutter. Laser stimulation is typically 2 msec long.</li>
	<li>Capture image of the region to be stimulated using the &#39;Grab Video&#39; function.</li>
	<li>Select the stimulation grid according to desired dimensions. We typically use a 16 x 16 stimulation array with 80 &#956;m spacing between adjacent rows and columns.</li>
	<li>Overlay the mapping grid with the acquired image and adjust the x, y, and degree offset to the desired location and orientation.</li>
	<li>Activate the &#39;Map&#39; function to start stimulation and acquisition of data.</li>
	<li>Following acquisition of photostimulation evoked responses, carry out analysis of mapped data using either the offline map analysis program in Tidelwave25 or the &#39;MapAnalysis&#39; program in Ephus26.</li>
</ol>
5. Fixed Slice Imaging
<ol>
	<li>After recordings, transfer brain slice to 4% paraformaldehyde (PFA) in 0.01 M PBS (pH7.4) overnight at 4 &#186;C.</li>
	<li>Transfer the slice in 30% sucrose in 4% PFA/0.01 M PBS overnight at 4 &#186;C.</li>
	<li>Cut 50 &#181;m sections using a cryostat and collect in 0.01 M PBS.</li>
	<li>Rinse sections in 0.01 M PBS twice at room temperature.</li>
	<li>Mount sections and coverslip in an appropriate antifade medium.</li>
	<li>Transfer sections to the confocal microscope.</li>
	<li>Focus into the ROI using white light.</li>
	<li>Choose the wavelength, imaging size, and repetition for image acquisition. For YFP, we use 514 nm (Ex)/527 nm (Em).</li>
</ol>

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J., Mogri, M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+in+neural+systems.">Optogenetics in neural systems.</a> Neuron. 71, 9-34 (2011).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 13940-13945 (2003).</li><li>Zemelman, B. V., Lee, G. 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The authors declare they have no competing interests.
The author (CCL) received a publication grant from Leica Microsystems, which produces an instrument used in this filming of this protocol.

The described protocol details an approach for the in vitro assessment of functional topography and physiology of neuronal circuits, which is not limited to the investigation of forebrain circuits, i.e. those of the thalamus and cortex. Several viral constructs for optogenetic transfection have been made available to the scientific community and are readily obtainable from the UNC Vector Core. For a description of viral constructs readily available, see the UNC Vector Core31, the optogenetics resource page at the Deisseroth Lab at Stanford University32 and the Boyden synthetic neurobiology lab at MIT33. Variants of these opsins have been developed by these labs and enable wide-ranging modes for controlling neuronal excitability1,2. Obtaining these vectors from the UNC Vector Core requires a Material Transfer Agreement from the originating labs. In this protocol, we note that the addition of polybrene enhances transfection efficacy. In addition, another variable influencing expression is the post-surgical recovery period, which we note that a period of at least 14 days is sufficient to obtain robust expression in the forebrain pathways of mice.
For the targeted transfection of specific neuronal groups, several Cre-transgenic mouse lines have been developed, which can be used in combination with &#39;floxed&#39; reversed open reading frame viral constructs that have been developed11. Several strains pertinent to neuroscience research are available through the Mutant Mouse Regional Resource Centers34. Atlases of Cre-recombinase expression patterns in these transgenic lines are offered through the GENSAT database35. In addition, Jackson Labs offers several Cre-transgenic strains, as well as others that constitutively express opsins in specific neuronal groups. A critical step to utilizing these mouse lines is to factor in the time necessary for obtaining and establishing a breeding colony, which may vary based on the particular strain of interest.
For photostimulation, we have utilized and adapted the setup developed by the Shepherd Lab at Northwestern University, described in a recent protocol29. Although optimized for glutamate uncaging, this setup is amenable also for photostimulation of channelrhodopsin without modification. This setup enables precise spatial control and positioning of the loci of photostimulation, which enables an assessment of the functional topography of channelrhodopsin innervation of recorded neurons. Although spatial control is enhanced with this method of photostimulation, it is more cost prohibitive than methods utilizing LED based field illumination20. In addition, the nonuniformity of brain tissue is a limitation of optogenetics, or any techniques involving manipulation of light. In practice though, it is rarely a factor since results are interpreted mostly in a qualitative manner. In case more quantitative comparison between brain regions is needed, one can always use maximum light intensity to achieve &#34;saturated&#34; responses.
In addition, we have used both Tidelwave and Ephus software programs written in Matlab for controlling the laser-scanning photostimulation and data acquisition25,26. However, it is recommended that those setting up a similar system employ the newer Ephus software26, which has support available through the Janelia Farms development site30. A key step is choosing which of these software packages to use, since their hardware requirements differ slightly. In addition, the map analysis programs are tailored to their respective data acquisition programs.
Several of these steps can be modified to suit the practitioner&#39;s particular setup. Injections of viral solutions can be performed with delivery mechanisms besides a syringe, e.g. capillary glass pipettes pulled to tip diameters of ~40 &#956;m also work well. For surgeries, a nonstereotaxic headholder can be substituted if such coordinates are not required for guiding injections. In addition, ACSF and intracellular solution compositions may be altered depending on the particular experimental requirements, e.g. substituting a cesium or high chloride intracellular solution to isolate inhibitory currents. Also, an alternative to imaging of expression patterns in fixed slices may also be done in the live slice on a suitable imaging rig with a bath perfusion system.
In summary, this protocol will enable the practitioner to characterize the functional topography and synaptic physiology of projections in vitro, which may not be possible using standard methods, e.g. characterizing long-range projections that are unable to be preserved in a slice. The transfection protocol described can be applied to in vivo&#160;optogenetic experiments1-3,12,14, since the delivery mechanism is the same. Finally, although this protocol describes transfection of mice, many of the viral vectors available can be used to theoretically transfect and express these opsins in a wide range of mammalian species, thus enabling a myriad of comparative and species-specific studies1-3.

The elaborate circuitry of the mammalian forebrain poses an experimental challenge to dissecting the functional roles of its intertwined components. Untangling these relationships has been aided recently by the development of optogenetic methods for specifically exciting and inhibiting neuronal populations and their projections1-3. These approaches make use of the genetically controlled expression of membrane channels responsive to light4-6. Of particular note, channelrhodopsin, a light-gated ion channel, depolarizes neuronal membranes following optical stimulation, leading to excitation of the expressing neuron and its projections4,7. Conversely, halorhodopin, a light-gated chloride pump, hyperpolarizes the membrane, thus inhibiting the targeted projection6,8,9. Several variants of these opsins have been engineered, multiple vectors produced for transfection and expression in neuronal populations of interest, and transgenic mouse lines engineered that constitutively express these opsins or that enable cre-lox mediated control of their expression1,2,10,11.
The utility of these optogenetic methods and materials has revolutionized modern experimental neuroscience, enabling the dissection of the role of specific neural circuits in the physiology and behavior of the organism12-15. In particular, these methods can be powerfully applied to the functional dissection of brain circuits using in vitro slice preparations16-20. In this context, one of the main advantages of this method is the ability to characterize the physiology of long range projections that are difficult, if not impossible, to preserve in a thick slice, such as the commissural connections between cerebral cortical hemispheres16. Furthermore, this technology enables manipulations not possible with other methods, such as the selective inhibition of projections with fine temporal precision using halorhodopsin6,8,9. Moreover, the cell-type specific expression of these opsins allows isolated components in a neural circuit to be selectively stimulated or inhibited1,2,11,15,21. As such, these novel optogenetic tools enable the unprecedented analysis of the neuronal contributions to physiology and behavior.
Here, we describe a protocol for expressing channelrhodopsin in the mouse brain using adeno-associated viral (AAV) vectors1,2. Specific transfection of targeted neuronal populations can be accomplished using this protocol, with the appropriate choice of promoter constructs or transgenic mice that express Cre-recombinase in the targeted neurons of interest and a &#39;floxed&#39; viral construct1,11,15,21 (Figure&#160;1). As examples here, we examine the corticocortical projections between the primary visual cortex (V1) and the secondary visual cortex (V2) and we also investigate the layer 6 corticothalamic projection from the primary somatorsensory cortex (S1) to the ventroposterior medial nucleus (VPm). We combine this specific expression with laser-scanning photostimulation&#160;in vitro&#160;to assess synaptic physiology and functional topography. The setup presented here enables the precise spatial positioning and control of the loci of photostimulation. Finally, we describe the visualization of expression patterns using confocal microscopy.

<table><tbody><tr><td>Polybrene</td><td>Santa Cruz Biotech</td><td>SC134220</td><td></td></tr><tr><td>Paraformaldehyde 32% solution</td><td>EMS</td><td>15714</td><td></td></tr><tr><td>VECTASHIELD</td><td>Vector Labs</td><td>H-1400</td><td></td></tr><tr><td>AAV viral constructs</td><td>UNC Vector Core</td><td>NA</td><td></td></tr><tr><td>Mouse stereotactic headholder</td><td>Stoelting</td><td>51730</td><td></td></tr><tr><td>Nanocool injector</td><td>Harvard Apparatus</td><td>703040</td><td></td></tr><tr><td>UV laser: 355 nm, Nd:YVO4; 1 W;</td><td>DPSS Lasers, Inc</td><td>3500</td><td></td></tr><tr><td>Galvonometers</td><td>Cambridge Scanning</td><td>6210</td><td></td></tr><tr><td>mirrors</td><td>Cambridge Scanning</td><td>6m2003S-A</td><td></td></tr><tr><td>mount</td><td>Cambridge Scanning</td><td>6102103L</td><td></td></tr><tr><td>cable</td><td>Cambridge Scanning</td><td>6010-19-120</td><td></td></tr><tr><td>driver/controller</td><td>Cambridge Scanning</td><td>67121-1</td><td></td></tr><tr><td>Shutter - ZM coated (UV-compatible)</td><td>Uniblitz</td><td>L23-ZM2</td><td></td></tr><tr><td>Shutter Controller</td><td>Uniblitz</td><td>VMM-D1</td><td></td></tr><tr><td>Current Amplifier</td><td>Stanford Research Systems</td><td>SR570</td><td></td></tr><tr><td>6713 board</td><td>National Instruments</td><td>777741-01</td><td></td></tr><tr><td>2110 break-out box</td><td>National Instruments</td><td>777643-01</td><td></td></tr><tr><td>PCI-MIO-16E-4</td><td>National Instruments</td><td>777383-01</td><td></td></tr><tr><td>BNC-2090</td><td>National Instruments</td><td>777270-01</td><td></td></tr><tr><td>Cables</td><td>National Instruments</td><td>184749-01</td><td></td></tr><tr><td>MATLAB</td><td>Mathworks</td><td></td><td></td></tr><tr><td>TCS SP2</td><td>Leica Microsystems</td><td></td><td></td></tr></tbody></table>

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.

Laser-scanning photostimulation of projections between the primary visual area (V1) and the secondary visual area (V2) in the mouse was accomplished following injections of pAAV-CaMKIIa-hChR2(H134R)-EYFP into V1 of a Balb/C mouse (Figure 3). The CaMKIIa promoter in this viral construct drives specific expression of channelrhodopsin-EYFP in excitatory glutamatergic neurons1,15. After preparation of slices for physiological recording, expression of channelrhodopsin-EYFP fibers, i.e. epifluorescence of the conjugated EYFP, was observed traversing from V1 to V2 (Figure 3A). In addition, the corticothalamic fibers in the lateral geniculate nucleus (LGN) were also observed (Figure 3A). Whole-cell patch clamp recordings from a neuron in V2 revealed EPSCs elicited following laser-scanning photostimulation of channelrhodopsin-EYFP expressing fibers in the lower layers of V2 (Figure 3B).
In another example, laser-scanning photostimulation of the corticothalamic projections from layer 6 of the primary somatosensory cortex (S1) to the ventroposterior nucleus (VP) was accomplished using this protocol. Here, we used a Cre-Lox approach to specifically target channelrhodopsin expression in the layer 6 neurons and corticothalamic projections of interest. Explicitly, we injected pAAV-Ef1a-DIO-hChR2(H134R)-EYFP-WPRE-pA into S1 of B6.Cg-Tg(Ntsr1-cre)GN220Gsat/Mmcd mice (Figures 1 and&#160;4). This transgenic mouse strain expresses Cre-recombinase specifically in layer 6 corticothalamic neurons21, and therefore results in ChR-YFP expression only in those neurons following transfection by the &#39;floxed&#39; AAV construct. After preparation of slices, channelrhodopsin-EYFP fibers were observed in the layer 6 neurons and fibers projecting to layer 4 and VP (Figures 4A and 4B). Physiological recording from a VPm neuron using whole-cell patch clamp revealed that EPSCs could be elicited following laser-scanning photostimulation near the recorded neuron (Figures 4C and 4D).
The morphology of labeled fibers was assessed more readily following physiological recordings. Here, we resectioned (50 &#956;m) slice preparations with channelrhodopsin-EYFP expression in layer 6 corticothalamic neurons (Figure 5). This revealed a fine pattern of labeled fibers traversing from layer 6 to layer 4, where they terminated and arborized extensively (Figure 5A). In addition, corticothalamic fibers were observed entering the white matter traversing towards the thalamus (Figures 5A and 5B).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50915/50915fig1.jpg" width="500px" /> 
Figure 1. Schematic illustration of targeted expression of channelrhodopsin via a Cre-Lox approach. Transgenic mice expressing Cre-recombinase in the neuronal populations of interest are injected with a &#39;floxed&#39; virus encoding channelrhodopsin fused to a fluorescent protein, such as YFP. Following transfection, only neurons with Cre-recombinase will express channelrhodopsin-EYFP. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50915/50915fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50915/50915fig2.jpg" width="500px" /> 
Figure 2. Schematic illustration of photostimulation setup. (A) Diagram of the laser-scanning photostimulation setup. The laser beam is guided successively through an open beam&#160;system to a shutter and a neutral density (N.D.) filter. A coverslip reflects part of the beam to a photodiode connected to an amplifier for laser power measurements. Mirror galvanometers are used to position the laser beam through a scan lens, which guides the beam through a side port on the top of the microscope. The beam is then reflected down through the objective lens to the slice preparation using a dichroic mirror. For further details see Lam and Sherman23&#160;and Shepherd29. (B) Example of the photostimulation grid pattern for the representative experiment shown in Figure 3. The 16 x 16 stimulation grid is overlaid on a photomicrograph of the slice preparation. A red star indicates the location of a neuron recorded using whole-cell patch clamp. The laser-beam position is guided to each of the 256 positions in the stimulation grid by computerized positioning of the mirror galvanometers. The laser positions of the 1st 5 stimulation sites are depicted in the figure. The responses from the recorded neuron to photostimulation at each of the sites is depicted in Figure 3. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50915/50915fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50915/50915fig3.jpg" width="500px" /> 
Figure 3. Laser-scanning photostimulation of corticocortical projections from primary visual cortex (V1) to secondary visual cortex (V2) in the mouse. (A) Epifluorescent image of the slice preparation showing channelrhodopsin-EYFP fibers from V1 to V2 and in the lateral geniculate nucleus (LGN). Specific expression of channelrhodopsin-EYFP in excitatory glutamatergic neurons is driven by a viral construct containing the CaMKIIa promoter. (B) Excitatory postsynaptic currents (EPSCs) elicited from a neuron in V2 following laser-scanning photostimulation. Red star indicates recording site. Scale bars: (A) 500 &#181;m, (B) 200 pA, 100 msec. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50915/50915fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50915/50915fig4.jpg" width="500px" /> 
Figure 4. Laser-scanning photostimulation of corticothalamic projections from primary somatosensory cortex (S1) to the ventroposterior nucleus (VP) in the mouse. (A) Epifluorescent image of the slice preparation showing channelrhodopsin-EYFP fibers in S1. (B) Epifluorescent image of labeled-fibers in VP. (C) Excitatory postsynaptic currents (EPSCs) elicited from a VP neuron following laser-scanning photostimulation. (D) Plot of mean EPSCs. Red star indicates recording site. Boxed traces in (C) correspond with boxed values in (D). Scale bars: (A) 250 &#181;m, (B) 250 &#181;m, (C) 100 pA, 100 msec. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50915/50915fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50915/50915fig5.jpg" width="500px" /> 
Figure 5.&#160;Imaging of layer 6 neurons expressing channelrhodopsin-EYFP in the primary somatosensory cortex (S1) of the mouse. (A-B) Labeled neurons in layer 6 and fibers projecting to layer 4 and the white matter. Scale bars: (A) 250 &#181;m, (B) 250 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50915/50915fig5highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits at JoVE.com

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

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.

Laser-scanning Fotostimolazione di circuiti prosencefalo Optogenetically mirati

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

laser-scanning-photostimulation-optogenetically-targeted-forebrain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.2K Views.  Louisiana State University. We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations in vitro.

Video: Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Mappatura dei circuiti neuronali inibitori da fotostimolazione scansione laser

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Ricerca

Neuroscienze

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.

Fibra ottica Impianto per la Stimolazione Optogenetic cronica del tessuto cerebrale

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

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Un metodo per High Fidelity optogenetic controllo dei singoli Piramidale neuroni<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetic Perturbazione di attività neurale con illuminazione laser in semi-intatte<em&gt; Drosophila</em&gt; Larve in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of 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.

Manipolazione optogenetica dell'attività neuronale per modulare il comportamento nei topi in movimento libero

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

Comportamento

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.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

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

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

Optogenetic risonanza magnetica funzionale

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetica trascinamenti di teta Hippocampal oscillazioni nel comportarsi topi

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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.

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.

In Vivo Intracerebral Stereotaxic Iniezioni per la stimolazione optogenetica degli ingressi a lungo raggio nelle fette di cervello del topo

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

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Laser-scanning Fotostimolazione di circuiti prosencefalo Optogenetically mirati

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