This work was supported by National Institute on Drug Abuse (NIDA) grant R01 DA24040 (DCC), University of Colorado Innovative Seed Grant (DCC), and NIDA training grant T32 DA017637 (MVB).

1. Virus Storage and Preparation
<ol>
	<li>The use of replication-incompetent AAV vectors for delivering opsin genes to rodent brain is approved for Biosafety Level 1 (BSL-1) and requires proper protection and handling procedures as outlined in the U.S. Government publication, Biosafety in Microbiology and Biomedical Laboratories, available at the Centers for Disease Control's website at (<a href="http://www.cdc.gov/biosafety/publications/bmbl5/index.htm" target="_blank">http://www.cdc.gov/biosafety/publications/bmbl5/index.htm</a>).</li>
	<li>In order to avoid repeated freeze-thaw cycles, pipette virus from manufacturer's vial into smaller working aliquots in a Class II Biosafety Cabinet and store in a -80 &deg;C freezer.</li>
	<li>Prior to stereotactic injection, allow an aliquot to thaw on ice and then briefly spin down with a bench top centrifuge.</li>
	<li>Slowly backfill a Hamilton glass syringe and needle with a small amount of silicone or mineral oil. Make sure there are no visible air bubbles in the syringe barrel. Insert the syringe into a microinjector pump and attach the pump directly to the vertical stereotaxic arm (i.e. stereotaxic manipulator).</li>
	<li>Lower the stereotaxic arm until the tip of the needle touches the bottom of the virus aliquot tube. Use the controller for the microinjector pump to withdraw the desired volume of virus (1.5 &mu;l for a 1 &mu;l injection).</li>
	<li>All material and surfaces that come into contact with virus should be decontaminated with a disinfectant such as chlorine bleach (10%).</li>
</ol>
2. Surgery and Virus Injection
<ol>
	<li>Anesthetize animal with a ketamine (rat; 125 mg/kg, i.p.) - xylazine (rat; 10 mg/kg, i.p.) cocktail. To gauge effectiveness of anesthesia, check for a reflex response to a toe pinch and give supplemental doses of ketamine if needed. Using standard aseptic surgical methods, place animal into a stereotaxic apparatus (David Kopf Instruments).</li>
	<li>Make a midline incision through the skin on top of the animal skull with small surgical scissors or scalpel. Gently separate connective tissue and clean the top of the skull with a small bone scraper.</li>
	<li>Verify that the head of the animal is level such that the z coordinates for bregma and lambda are equal. Determine the stereotaxic coordinates for the target brain area from a brain atlas such as The Rat Brain in Stereotaxic Coordinates (George Paxinos and Charles Watson, Academic Press, 2007) or The Mouse Brain in Stereotaxic Coordinates (Keith B.J. Franklin and George Paxinos, Academic Press, 2007). Mark the intended site of injection with a surgical pen.</li>
	<li>Carefully thin the skull over the target area using a hand-held drill and stop when the drill bit reaches the bottom of the skull. Using a pair of extra fine forceps, remove the thinned bone in order to expose the dura.</li>
	<li>Position the syringe needle over the target area and lower the needle until it touches the dura and use this point to calculate the z coordinate. Very slowly lower the injection needle into the brain until the proper z position is reached. In this protocol, the prelimbic region of the medial prefrontal cortex (2.7 mm anterior to bregma, &plusmn; 0.5 mm lateral to the midline, and -2.2 mm from dura) or the dorsal subiculum (-6.0 mm anterior to bregma, &plusmn; 3.0 mm lateral to the midline, and -2.0 mm from dura) is targeted in an adult Sprague Dawley male rat (approx. 275 g).</li>
	<li>Inject virus at a rate of 0.1 &mu;l/min. After the injection is completed wait 10 min before withdrawing the needle to avoid backflow of the virus to the brain surface.</li>
	<li>Use a sewing suture with attached needle to seal the skin and apply antibiotics to the wound.</li>
	<li>The time course of ChR2 and NpHR functional expression from AAV vectors will vary depending on the experimental design and viral titer. For this experiment, in vivo recordings were performed 18-21 days post injection.</li>
</ol>
3. Light Delivery and In vivo Recording of ChR2 or NpHR-expressing Neurons
<ol>
	<li>Attach a tungsten electrode (~1-1.5 M&Omega;, MicroProbes) to a glass capillary tube (Fisher Scientific) using super glue.</li>
	<li>Use a fiber stripping tool (Thorlabs) to expose the bare end of a multimode optical fiber (200 &mu;m diameter core, Thorlabs) and clean fiber tip with ethanol. Very lightly score fiber tip with a wedge shaped diamond knife (Thorlabs) and carefully remove the excess fiber (e.g. with fine forceps). The fiber should cleave easily at the score.</li>
	<li>Connect the FC end of the fiber to the PC connector on the output port of a 200 mW single diode laser (<a href="http://www.lumiphy.com" target="_blank">www.Lumiphy.com</a>) with the desired wavelength. Make sure that appropriate protective eyewear is worn at all times when working with lasers.</li>
	<li>Measure the laser intensity at the tip of the optic fiber using an optical power meter (<a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a> ). For in vivo recordings, light intensity as little as 20-100 mW/mm2 can reliably evoke ChR2- or NpHR-mediated activation or silencing, respectively, of neuronal activity.</li>
	<li>Insert the optical fiber into the capillary tube. Position the fiber tip approximately 500 &mu;m above the tip of tungsten electrode and tie a thin suture thread twice at a few points near the tip so that the electrode and the fiber are straight. Make sure that the distance between the tip of electrode and the nearest knot is long enough for insertion to the target region.</li>
	<li>Prepare the animal as in steps 2.1 and 2.2 above. Use the initial craniotomy as a guide to make a new clean small window in the skull for positioning the optrode (<a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a> ). Make a small incision on the dura using a fine needle.</li>
	<li>Carefully lower the optrode through the skull window and into the brain to the transduced region using a micromanipulator (Narishige). Then advance the optrode slowly in 10-50 &mu;m steps until the emergence of a light-responsive cell.</li>
	<li>A custom software user interface (NeuroLux Pro <a href="http://www.lumiphy.com/" target="_blank">www.Lumiphy.com</a>) written in LabVIEW (National Instruments) is used to control the single diode laser and to visualize and record ongoing neural activity. Recorded signals are captured with an ExAmp-20K (Kation Scientific) amplifier band-pass filtered (0.3-8 kHz), and a National Instruments analog to digital board (NI USB-6009). The NeuroLux Pro user interface allows the choice of analog inputs (choose two channels for the neuronal and TTL signal) and digital output. The user may define the sampling rate (20 kHz), the baseline period (pre-light), and post-light period. The user may define the TTL laser stimulation pulse width, frequency and stimulation period (if frequency is 20 Hz and stimulation period is 500 msec, 10 laser pulses with defined pulse width are delivered). The user also can choose continuous light delivery (e.g., Figure 1). For repeated recordings, the user also may define the number of pulse trains and the interval between trains. Data can be acquired with or without recording to disk.</li>
	<li>Following recording, animals are anesthetized with the ketamine/xylazine cocktail and transcardially perfused with physiological saline and paraformaldehyde (4%) in order to verify optrode placement and opsin expression.</li>
</ol>

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P., 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=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446 (7136), 633-639 (2007).</li><li>Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279-2284 (2005).</li><li>Adamantidis, A. R., Zhang, F., Aravanis, A. 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The authors declare that they have no competing financial interests.

A wide array of techniques are available for genetically-targeting microbial opsin genes to discrete brain regions in rodents. Viral gene delivery provides a relatively inexpensive and quick approach for mediating ChR2 and NpHR expression with cell-type specificity. AAV vector systems are a common choice for use in optogenetic experiments due to high production titers that remain stable during storage, its lack of pathogenicity, and its ability to produce long-term gene expression 10. Many of the opsin constructs with cell-type specific promoters are commercially available in a variety of AAV serotypes from vector core facilities such as the University of Pennsylvania (<a href="http://www.med.upenn.edu/gtp/vectorcore" target="_blank">http://www.med.upenn.edu/gtp/vectorcore</a>) or the University of North Carolina at Chapel Hill (<a href="http://genetherapy.unc.edu" target="_blank">http://genetherapy.unc.edu</a>). One drawback to AAV technology is the limited packaging capacity (~4.7 kb), which places a restriction on the transgene cassette size that can be used for cell-specific targeting. As an alternative, lentiviral vectors, which have a larger packaging capacity, are able to accommodate opsin targeting under the control of larger promoter sequences.
The use of an optrode allows for reliable detection of electrophysiological signals in combination with temporally-precise light delivery. As described above, however, sufficient care is needed in its construction. Since the cleaved optical fiber is fragile, tying the suture thread too tightly may cause the fiber to break. Even though the optrode can be used for multiple recordings, the electrode and optical fiber should be cleaned (or manually cleaved in the case of the optical fiber bare end) prior to insertion because the impedance of the electrode and quality of the delivered light will degrade with repeated use.
During electrophysiological recordings it is important that the optrode be advanced slowly. The optrode used here has approximately 350 &mu;m thickness (tungsten electrode: 125 &mu;m; core of optic fiber: 200 &mu;m) and lowering it fast could lead to movement of brain tissue. Light-induced artifacts also might be considered with particular recording and light delivery set-ups 11-12. Although we found no light-induced artifact in our experimental condition, recordings outside the transduced brain area can potentially be used as a control for light artifacts. Another consideration during the recording process is the required light intensity for observing changes in ChR2- or NpHR-expressing neurons, especially for long duration recordings. Strong laser intensity and long laser illumination could result in the tissue damage and/or a decreased response to subsequent delivered light 12-13. For behavioral experimentation the use of a control viral vector is required for eliminating any effect due to heat delivered from the fiber. For many of the commercially available optogenetic constructs there are complementary control constructs in which the opsin gene sequence has been removed.
It should be noted that engineered ChR2 variants with improved properties and kinetics have been developed along with other silencing opsins that may be better suited for certain experimental questions 14-15. For instance, a new ChR2 variant established via targeted mutagenesis, termed "ChETA", can drive high-fidelity neuronal spiking at frequencies (up to at least 200 Hz) above that of the original ChR2 14.
The combination of in vivo light delivery and simultaneous recording of neuronal responses is a critical step in establishing causal relationships between patterned activity in genetically-targeted cell populations and corresponding time-locked behavioral events. The procedures and hardware outlined here provide a straightforward approach for implementing optogenetics for in vivo head-fixed recordings in rodents.

The ability to activate or silence a specific cell type within a neural circuit in a temporally precise fashion is critical for understanding how neural circuits process different types of information underlying emotion and cognition. Experimental control over intact neural activity has employed loss-/gain-of-function tools (e.g., electrical stimulation, pharmacological modulation, lesion) that do not provide the required selectively for controlling specific populations of neurons, either on a temporal or spatial scale. Directly addressing these technological challenges, the development and application of genetically-encodable light-sensitive tools has enabled neuroscientists to control the electrical activity of select cell types during well-defined behavioral events. Many of these light-sensitive proteins are of microbial origin, with the light-gated cation channel, channelrhodopsin-2 (ChR2) 1, and the light-driven chloride pump, halorhodopsin (NpHR) 2,3, in widespread use.
A major advantage of optogenetics is the ability to genetically-target specific cell populations in heterogeneous brain regions and the technique has been successfully applied to a number of model organisms (invertebrates to nonhuman primates) 4-6. Many optogenetic transgenic animals have been generated and are commercially available 7,8, however establishing transgenic lines can be labor intensive and cost prohibitive. Here we describe a protocol for virally-mediated delivery of ChR2 and NpHR genes under the CaMKII&alpha; promoter in rat prelimbic cortex and dorsal subiculum using a recombinant adeno-associated virus (AAV) vector. Within the forebrain, CaMKII&alpha; expression is exclusive to glutamatergic pyramidal neurons 9. AAV is commonly used for basic research due to its relative ease of production and lack of pathogenicity, as well as the strong and persistent transgene expression that has been achieved with these vectors 10. Additionally we outline the steps and hardware for simultaneous light delivery and recording in anesthetized head-fixed rats.

<table><tbody><tr><td>Syringe </td><td>Hamilton</td><td>7653-01</td><td>10 &amp;mu;l</td></tr><tr><td>Removable Needle</td><td>Hamilton</td><td>7803-03</td><td>31 gauge, beveled tip</td></tr><tr><td>Microinjector Pump</td><td>World Precision Instruments</td><td>UMP3</td><td></td></tr><tr><td>Pump Controller</td><td>World Precision Instruments</td><td>SYS-MICRO4</td><td></td></tr><tr><td>Silicone Oil</td><td>Alfa Aesar</td><td>A12728</td><td></td></tr><tr><td>Laser Protective Eyeware</td><td>Kentek</td><td>KMT-4501</td><td></td></tr><tr><td>Multimode Optical Fiber</td><td>Thorlabs</td><td>BFL37-200</td><td>200 &amp;mu;m diameter core, 0.37 NA</td></tr><tr><td>Fiber Stripping Tool</td><td>Thorlabs</td><td>T12S21</td><td>for 200 &amp;mu;m diameter core multimode fiber</td></tr><tr><td>Optical Power Meter</td><td>Lumiphy LLC</td><td>&lt;a href=&quot;http://www.lumiphy.com/&quot; target=&quot;_blank&quot;&gt;www.lumiphy.com&lt;/a&gt;</td><td></td></tr><tr><td>Photodetector</td><td>Newport</td><td>818-SL/DB</td><td></td></tr><tr><td>Blue Laser (445-473 nm, 100-200 mW)</td><td>Lumiphy LLC</td><td>&lt;a href=&quot;http://www.lumiphy.com&quot; target=&quot;_blank&quot;&gt;www.lumiphy.com&lt;/a&gt;</td><td>coupled to a 200 &amp;mu;m multimode fiber with FC/PC adapter </td></tr><tr><td>Green Laser (532 nm, 100-200 mW)</td><td>Lumiphy LLC</td><td>&lt;a href=&quot;http://www.lumiphy.com&quot; target=&quot;_blank&quot;&gt;www.lumiphy.com&lt;/a&gt;</td><td>coupled to a 200 &amp;mu;m multimode fiber with FC/PC adapter </td></tr><tr><td>Tungsten/Fiber Optrode</td><td>Lumiphy LLC</td><td>&lt;a href=&quot;http://www.lumiphy.com/&quot; target=&quot;_blank&quot;&gt;www.lumiphy.com&lt;/a&gt;</td><td>Lumitrode</td></tr><tr><td>Glass Capillary Tube</td><td>Fisher Scientific</td><td>22-362-566</td><td></td></tr><tr><td>Hydraulic Micromanipulator</td><td>Narishige</td><td>MO-22</td><td></td></tr><tr><td>Amplifier</td><td>Kation Scientific</td><td>ExAmp-20K</td><td></td></tr><tr><td>Data Acquistion Device</td><td>National Instruments</td><td>NI USB-6009</td><td></td></tr><tr><td>Rodent Head Restraint for Recording</td><td>Lumiphy LLC</td><td>&lt;a href=&quot;http://www.lumiphy.com/&quot; target=&quot;_blank&quot;&gt;www.lumiphy.com&lt;/a&gt;</td><td></td></tr><tr><td>Small Animal Stereotaxic Unit</td><td>David Kopf Instruments</td><td>Model 963</td><td></td></tr></tbody></table>

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.

Figure 1 depicts a screenshot of the customized software (NeuroLux Pro) developed in LabVIEW environment (National Instruments) used for simultaneous recording of neuronal activity and controlling light pulse parameters (i.e. pulse frequency, pulse width, stimulation period). The software program sends a command signal to a digital acquisition device that generates TTL pulses for control over the single diode laser. Additionally the software program displays and stores the recorded neuronal activity and delivered TTL signals. These signals are digitized through the acquisition device at a defined sample rate (e.g., 20 kHz). Recorded signals are saved as a two-dimensional double-precision array in a binary file.
Figure 2 shows the custom optrode consisting of a tungsten electrode attached to an optical fiber. An optical fiber is fixed to the electrode using thin suture thread at three points. If needed, super glue can be used to adhere them. However, avoid permanently fixing the optical fiber to the electrode because although the electrode may be reused several times, light transmission through the fiber will degrade with repeated use and should be cleaved and cleaned or replaced with every insertion into the brain.
Left panels of Figure 3 show fluorescent images of enhanced yellow fluorescent protein, indicating actual ChR2 and NpHR expression. These photographs show representative NpHR expression in rat dorsal subiculum (Figure 3A) and ChR2 expression in prelimbic cortex (Figure 3B). Viral expression was restricted mostly to the dorsal subiculum and prelimbic cortex (e.g. some fluorescence was observed in dentate gyrus (Figure 3A, left)). The tracks of the electrode (thin track, arrow head) and optical fiber (thick track, arrow) were also observed.
Left panel of Figure 4 shows that ChR2 induced temporally precise spiking in rat prelimbic cortex. Figure 4A is an example trace of ChR2-triggered action potentials in response to 20 Hz delivery of 10 msec blue light pulses to rat prelimbic cortex. Raster plot (Figure 4B) shows ChR2-induced activation in six representative neurons. All recorded neurons showed light-evoked spiking with perfect fidelity. In contrast to ChR2, NpHR rapidly and reversibly silenced spontaneous activity in vivo in rat prelimbic cortex (right panel of Figure 4). Figure 4C is an example trace showing that continuous 532 nm illumination (10 s) of the prelimbic cortex expressing NpHR under the CaMKll&alpha; promoter eliminates spontaneous single-unit activity in vivo. Note that the complete silencing of prelimbic pyramidal cell activity is time-locked to the 10 sec continuous light delivery. Lower raster plot (Figure 4D) shows NpHR-induced silencing in six representative neurons. Both in vivo recordings are typical of those obtained from adult Sprague-Dawley rats in which AAV-mediated delivery of microbial opsin genes occurred 18-21 days prior.
Figure 5 shows the result of repeated ChR2 stimulation of a rat prelimbic pyramidal neuron. Blue light pulse (10 msec) was delivered at 20 Hz for 5 sec (100 pulses total). Voltage traces of the light-evoked spiking were repeatedly acquired every 2 min during a 2 hr recording session (61 repetitions total). Even when using this repetitive stimulation protocol, ChR2 induced stable and robust spiking in response to the light delivery.
Figures 6 and 7 show the results of NpHR-induced photoinhibition and ChR2-driven photoactivation of rat subicular neuron activity. As is the case in prelimbic cortex, NpHR and ChR2 were able to mediate the light-induced time-locked inhibition and activation of the opsin-transduced subicular neurons with high reproducibility.
<img alt="Figure 1" src="/files/ftp_upload/50291/50291fig1.jpg" /> 
Figure 1. Screenshot of the NeuroLux Pro software interface for simultaneous light delivery and electrophysiological recording. Pictured trace shows NpHR induced silencing of spontaneous activity of rat prelimbic pyramidal cell in response to 10 sec of continuous 532 nm light delivery. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50291/50291fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50291/50291fig2.jpg" /> 
Figure 2. High power image of custom optrode. An optical fiber is inserted into glass capillary tube attached to a tungsten electrode and fixed to the electrode using suture thread. The center-to-center distance between the electrode tip and the fiber tip is approximately 500 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/50291/50291fig3.jpg" /> 
Figure 3. Viral expression and optrode placement. (Left) Photographs show representative NpHR expression in dorsal subiculum (A) and ChR2 expression in prelimbic cortex (B). (Right) Schematic that represents the location where each photograph was taken. The arrowhead and arrow in each photograph indicate the location of the tungsten electrode and optical fiber, respectively. SUB: subiculum, DG: dentate gyrus, PL: prelimbic cortex. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50291/50291fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/50291/50291fig4.jpg" /> 
Figure 4. In vivo electrophysiological recordings from ChR2 or NpHR-transduced rat prelimbic cortex. (A) Example trace of ChR2-triggered action potentials in response to 20 Hz delivery of blue (473 nm) light pulses (10 msec, blue bar). (B) Raster plot showing ChR2-induced spiking in six representative neurons. Each unit activity is plotted as a dot. (C) Example trace of NpHR-induced suppression of spontaneous activity during continuous green (532 nm) light illumination (10 sec, green bar). (D) Raster plot showing NpHR-induced silencing in six representative neurons. Each unit activity is plotted as a dot. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50291/50291fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/50291/50291fig5.jpg" /> 
Figure 5. Repetitive 20 Hz ChR2-driven spiking of a rat prelimbic pyramidal neuron in vivo. (A) Voltage traces of the light-evoked spiking acquired at the time points 0, 30, 60, 90, 120 min after the beginning of the recordings. (B) Raster plot showing all 61 repetitions (2 min inter-trial interval, 120 min total) of the light-induced activation. Each unit activity is plotted as a dot. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50291/50291fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 6" src="/files/ftp_upload/50291/50291fig6.jpg" /> 
Figure 6. In vivo electrophysiological recordings from NpHR-transduced rat dorsal subiculum. (A) Example trace showing that 10 sec continuous 532 nm illumination (green bar) of the dorsal subiculum expressing NpHR eliminates spontaneous activity. (B) Average waveforms of the two recorded units from the above trace. The amplitude threshold was used for identifying two distinct neurons. (C) Raster plot showing five repetitions of NpHR-induced silencing of these units. Each unit activity is plotted as a dot.
<img alt="Figure 7" src="/files/ftp_upload/50291/50291fig7.jpg" /> 
Figure 7. Repetitive 20 Hz CR2-driven spiking of a rat dorsal subicular neuron in vivo. (A) Voltage traces of the light-evoked spiking acquired at the time points 0, 30, 60, 90, 120 min after the beginning of the recordings. Inset: typical bursting activity of this cell. (B) Raster plot showing all 61 repetitions (2 min inter-trial interval, 120 min total) of the light-induced activation. Each unit activity is plotted as a dot. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50291/50291fig7large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo at JoVE.com

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.

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

19.0K Views.  University of Colorado Boulder. 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.

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

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

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 ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

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.

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

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 ...

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. ...

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

Fine motor skills are essential in everyday life and can be compromised in several nervous system disorders. The acquisition and performance of these tasks require sensory-motor integration and involve precise control of bilateral brain circuits. Implementing unimanual behavioral paradigms in animal models will improve the understanding of the contribution of brain structures, like the striatum, to complex motor behavior as it allows manipulation and recording of neural activity of specific nuclei in control conditions and disease during the performance of the task. 
Since its creation, optogenetics has been a dominant tool for interrogating the brain by enabling selective and targeted activation or inhibition of neuronal populations. The combination of optogenetics with behavioral assays sheds light on the underlying mechanisms of specific brain functions. Wireless head-mounted systems with miniaturized light-emitting diodes (LEDs) allow remote optogenetic control in an entirely free-moving animal. This avoids the limitations of a wired system being less restrictive for animals' behavior without compromising light emission efficiency. The current protocol combines a wireless optogenetics approach with high-speed videography in a unimanual dexterity task to dissect the contribution of specific neuronal populations to fine motor behavior.

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

The present protocol describes how to use wireless optogenetics combined with high-speed videography in a single pellet reach-to-grasp task to characterize the neural circuits involved in the performance of skilled motor behavior in freely moving mice.

Fine motor skills are essential in everyday life and can be compromised in several nervous system disorders. The acquisition and performance of these ...

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

Summary

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Chapters in this video

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

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

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

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

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior