The authors wish to thank J. Vazquez for illustrations, D. Kaplan and C. Preda for reagents and helpful guidance, and the labs of B. Sabatini and C. Harvey for in vivo imaging. Microscopy was made possible by M. Ocana and the Neurobiology Imaging Center, supported in part by the Neural Imaging Center as part of a National Institute of Neurological Disorders and Stroke (NINDS) P30 Core Center grant (NS072030). This work was supported by the GVR Khodadad Family foundation, the Nancy Lurie Marks foundation, and by NIH grants, NINDS R21NS093498, U01NS108177 and NINDS R35NS097284 to W.G.R, and by an NIH postdoctoral fellowship F32NS101889 to C.H.C.

All experiments involving animals were performed in accordance with protocols approved by the Harvard Standing Committee on Animal Care following guidelines described in the US NIH Guide for the Care and Use of Laboratory Animals. Adult C57BL/6 mice of either sex (6-15 weeks of age) were used for all experiments.
1. Obtain Aqueous Silk Fibroin
<ol>
	<li>Prepare or purchase aqueous silk fibroin (5-7.5% w/v).</li>
</ol>
2. Mix Aqueous Silk with AAV Expression Vec...

<ol>
	<li>Klapoetke, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature Methods. 11 (3), 338-346 (2014).</li><li>Tervo, D. 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=A+Designer+AAV+Variant+Permits+Efficient+Retrograde+Access+to+Projection+Neurons.">A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons.</a> Neuron. 92 (2), 372-382 (2016).</li><li>Jackman, S. L., 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=Silk+Fibroin+Films+Facilitate+Single-Step+Targeted+Expression+of+Optogenetic+Proteins.">Silk Fibroin Films Facilitate Single-Step Targeted Expression of Optogenetic Proteins.</a> Cell Reports. 22 (12), 3351-3361 (2018).</li><li>Ung, K., Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-optic+implantation+for+chronic+optogenetic+stimulation+of+brain+tissue.">Fiber-optic implantation for chronic optogenetic stimulation of brain tissue.</a> Journal of Visualized Experiments. (68), e50004 (2012).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">Intracranial injection of adeno-associated viral vectors.</a> Journal of Visualized Experiments. (45), (2010).</li><li>Ghosh, K. K., 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=Miniaturized+integration+of+a+fluorescence+microscope.">Miniaturized integration of a fluorescence microscope.</a> Nature Methods. 8 (10), 871-878 (2011).</li><li>Cai, D. J., 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=A+shared+neural+ensemble+links+distinct+contextual+memories+encoded+close+in+time.">A shared neural ensemble links distinct contextual memories encoded close in time.</a> Nature. 534 (7605), 115-118 (2016).</li><li>Goldey, G. J., 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=Removable+cranial+windows+for+long-term+imaging+in+awake+mice.">Removable cranial windows for long-term imaging in awake mice.</a> Nature Protocols. 9 (11), 2515-2538 (2014).</li><li>Sparta, D. R., 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=Construction+of+implantable+optical+fibers+for+long-term+optogenetic+manipulation+of+neural+circuits.">Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits.</a> Nature Protocols. 7 (1), 12-23 (2011).</li><li>Resendez, S. L., 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=Visualization+of+cortical,+subcortical+and+deep+brain+neural+circuit+dynamics+during+naturalistic+mammalian+behavior+with+head-mounted+microscopes+and+chronically+implanted+lenses.">Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.</a> Nature Protocols. 11 (3), 566-597 (2016).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> Journal of Visualized Experiments. (3), 194 (2007).</li><li>Cao, Y., Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+silk+biomaterials.">Biodegradation of silk biomaterials.</a> International Journal of Molecular Sciences. 10 (4), 1514-1524 (2009).</li><li>Jackman, S. L., Beneduce, B. M., Drew, I. R., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+high-frequency+optical+control+of+synaptic+transmission.">Achieving high-frequency optical control of synaptic transmission.</a> Journal of Neuroscience. 34 (22), 7704-7714 (2014).</li><li>Ortinski, P. I., 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nature Neuroscience. 13 (5), 584-591 (2010).</li><li>Hines, D. J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+controlled+release+from+silk+fibroin+films.">Mechanisms of controlled release from silk fibroin films.</a> Biomacromolecules. 12 (3), 804-812 (2011).</li><li>Hu, X., 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=Regulation+of+silk+material+structure+by+temperature-controlled+water+vapor+annealing.">Regulation of silk material structure by temperature-controlled water vapor annealing.</a> Biomacromolecules. 12 (5), 1686-1696 (2011).</li><li>Rockwood, D. N., 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=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6 (10), 1612-1631 (2011).</li><li>Yucel, T., Cebe, P., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vortex-induced+injectable+silk+fibroin+hydrogels.">Vortex-induced injectable silk fibroin hydrogels.</a> Biophysical Journal. 97 (7), 2044-2050 (2009).</li><li>Wang, X., Kluge, J. A., Leisk, G. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonication-induced+gelation+of+silk+fibroin+for+cell+encapsulation.">Sonication-induced gelation of silk fibroin for cell encapsulation.</a> Biomaterials. 29 (8), 1054-1064 (2008).</li><li>Lee, J., Park, S. H., Seo, I. H., Lee, K. J., Ryu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+repeatable+fabrication+of+high+A/R+silk+fibroin+microneedles+using+thermally-drawn+micromolds.">Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds.</a> European Journal of Biopharmaceutics. 94, 11-19 (2015).</li></ol>

The use of silk/AAV to target the expression of optogentic proteins overcomes limitations of approaches that are currently in use. Although many studies successfully use AAV injections to express optogenetic proteins, it is challenging to align expression to the tip of optical fibers, to regions around the length of tapered fibers, and to the viewing region of a GRIN lens. Because of misalignment between optical components and optogenetic expression, stereotaxic injections can be unreliable, and many experiments fail. Th...

The past decade has produced an explosion of engineered light-sensitive proteins for monitoring and manipulating neural activity1. Viruses offer unparalleled flexibility for expressing these optogenetic tools in the brain. Compared to transgenic animals, viruses are far easier to produce, transport, and store, allowing fast implementation of the newest optogenetic tools. Expression can be targeted genetically to distinct neuronal populations, and viruses designed for the retrograde transport can even be used to target expression based on neuronal connectivity2.
Viruses are usually introduced with stereotaxic injections, which can be time-consuming and challenging. Precisely targeting small regions can be difficult, while driving expression over broad areas often requires many injections. Moreover, when an optical device is subsequently implanted into the brain to deliver light in vivo, the implant must be properly aligned with the viral injection. Here, we describe an easily-implemented method for delivering viral vectors to the tissue around an implanted device using silk fibroin films3. Silk fibroin is commercially available, well-tolerated by neural tissues, and can be used to produce materials with varied properties. Silk films can be applied to implants using common laboratory equipment like microinjection pipettes or hand pipettes. Silk/AAV films eliminate the requirement for two surgical procedures and ensure that virus-mediated expression is properly aligned to the optical implant. The resulting expression is constrained to the tip of fibers, and results in less unwanted expression along the fiber track than stereotaxic injections.
In addition to producing targeted expression at the tip of small fibers, silk/AAV films can be used to drive widespread (&#62;3 mm diameter) cortical expression beneath cranial windows. In vivo 2-photon imaging of fluorescent activity sensors has become an indispensable tool for evaluating the role of neuronal activity in driving sensory and cognitive processing. However, to drive uniform expression over the broad cortical areas, experimenters often perform multiple injections. These injections can be extremely time-consuming and can lead to inconsistent expression across the field of view. In contrast, silk/AAV-coated cranial windows are extremely easy to manufacture, greatly reduce the time required for surgeries, and most remarkably drive expression hundreds of microns below the cortical surface.

<table>
	<tbody>
		<tr>
			<td>Aqueous silk fibroin</td>
			<td>Sigma</td>
			<td>5154-20ML</td>
			<td>Aqueous Silk Fibroin (5% w/v) for making films</td>
		</tr>
		<tr>
			<td>Microinjector to deposit silk/AAV</td>
			<td>Drummond</td>
			<td>3-000-207</td>
			<td>Nanoject III nanoliter injector</td>
		</tr>
		<tr>
			<td>Manipulator to hold implants</td>
			<td>Narashige</td>
			<td>MM-33</td>
			<td>Micromanipulator</td>
		</tr>
		<tr>
			<td>Stereoscope to visualize silk deposits</td>
			<td>AmScope</td>
			<td>SM-6TX-FRL</td>
			<td>3.5X-45X Trinocular articulating zoom microscope with ring light</td>
		</tr>
		<tr>
			<td>Vacuum chamber to store implants</td>
			<td>Ablaze</td>
			<td>N/A</td>
			<td>3.5 Quart Vacuum Vac Degassing Chamber</td>
		</tr>
		<tr>
			<td>Optional, implant holder for storage</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To store premade optical fibers, drill a grid of ~4 mm-deep holes with a diameter just larger than the ferrule diameter into a plastic block.</td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>FT200EMT</td>
			<td>&#216;200 &#181;m Core Multimode Optical Fiber for fiber implants</td>
		</tr>
		<tr>
			<td>Ferrules</td>
			<td>Kientec</td>
			<td>FZI-LC-230</td>
			<td>LC Zirconia Ferrule for fiber implants</td>
		</tr>
		<tr>
			<td>Various materials for manufacturing chronic fiber implants</td>
			<td>Various</td>
			<td>N/A</td>
			<td>For detailed procedure, see Ung K, Arenkiel BR. Fiber-optic implantation for chronic optogenetic stimulation of brain tissue. Journal of visualized experiments: JoVE. 2012(68).</td>
		</tr>
		<tr>
			<td>Tapered fiber implants</td>
			<td>Optogenix</td>
			<td>Lambda-B</td>
			<td>Tapered fiber implants</td>
		</tr>
		<tr>
			<td>GRIN lenses</td>
			<td>GoFoton</td>
			<td>CLH-100-WD002-002-SSI-GF3</td>
			<td>GRIN lenses</td>
		</tr>
		<tr>
			<td>Small glass cranial windows</td>
			<td>Warner</td>
			<td>64-0726 (CS-3R-0)</td>
			<td>Small round cover glass, #0 thickness</td>
		</tr>
		<tr>
			<td>Large glass cranial windows</td>
			<td>Warner</td>
			<td>64-0731 (CS-5R-0)</td>
			<td>Small round cover glass, #0 thickness</td>
		</tr>
		<tr>
			<td>Various materials for manufacturing cranial windows</td>
			<td>Various</td>
			<td>N/A</td>
			<td>For detailed procedure, see Goldey GJ et al. Removable cranial windows for long-term imaging in awake mice. Nature protocols. 2014 Nov;9(11):2515.</td>
		</tr>
	</tbody>
</table>

in vivo targeted expression of optogenetic proteins using silk/aav films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

To assess the success of silk/AAV films in driving expression, we perfused animals 2-3 weeks after implantation and prepared brain slices from the region of interest. Fluorescence images of fluorophore-tagged optogenetic proteins (ChR2-YFP) provided a measure of the extent of expression (Figure 1D). Typical optical fibers (230 &#181;m diameter) can readily accommodate 200 nL of silk/AAV. With practice, experimenters can achieve highly reliable expression arou...

Watch this Scientific Journal Video about In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films at JoVE.com

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

This method can help answer key questions in the field of systems neuroscience. Such as how the activity of neuronal ensembles supports sensory and cognitive processing in the brain. The main advantage of this technique is that it facilitates the accurate targeting of optogenetic proteins like Chan rhodopsin and GCaMP to brain regions of interest, without requiring a separate virus injection procedure.Visual demonstration of this method is critical, as the deposition of silk and virus to small optical implants can take some practice in order to achieve reproducible results. First, mix thawed AAV and five to seven point five percent aqueous silk fibroin in a one to one ratio in a 200 microliter PCR tube. Gently pipette the solution in and out several times to thoroughly mix the AAV and fibroin.To deposit the silk/AAV, load an injection pipette with the amount of solution required for the number of fiber implants being made, plus approximately 30 percent extra to accommodate losses due to pipette clogging. Next, mount a ferrule holder into a stereotaxic apparatus equipped with a microinjector. Place the ferrule holder above the microinjector and apply the silk/AAV mixture from below.Maneuver the injection pipette until it is touching or nearly touching the center of the optical fiber surface, and eject 10 to 20 nanoliters of silk/AAV solution. Then, withdraw the pipette. Next, observe the bolus of silk/AAV on the flat surface which appears as a liquid dome that dries to a flat film within approximately one minute.Repeat the previous steps until the desired amount of silk/AAV is deposited. When preparing multiple implants, apply the silk/AAV solution to one implant and then move on to coat other implants, before returning to the first. After one hour of drying, place the entire ferrule holder into a vacuum chamber and vacuum desiccate overnight at approximately 125 torr and four degrees Celsius.On the following day, evaluate the shape and position of the resulting silk film under a high-power microscope. Ensure that films are confined to the tip of the optical fiber surface, relatively thin, and symmetrical. For coating tapered optical fibers, position the silk/AAV injection pipette against the side of the optical fiber at the beginning of the taper, making sure that the pipette is touching the optical fiber.Eject 20 nanoliters of silk/AAV to start the coating process, ensuring that the droplet adheres to the optical fiber and remains at the interface of the fiber and pipette. Gently wick the droplet towards the end of the fiber tip as the silk/AAV dries, and keep the injection pipette in contact with the drying droplet to avoid clogging the pipette tip. When the first bolus has almost completely dried, eject another 20 nanoliters and continue wicking the droplet along the taper.Repeat the previous step by ejecting small amounts of silk/AAV, and gradually drying the solution up the side of the taper. After drying, evaluate the shape and position of the resulting silk film under a high-power microscope. For coating GRIN lens implants, deposit silk/AAV in a single injection on a GRIN lens implant.After drying, evaluate the shape and position of the resulting silk film under a high-power microscope to ensure that the film covers the surface of the lens. For coating glass cranial windows, hand-pipette a five-microliter droplet of silk/AAV onto the surface of a three millimeter round cover slip so that the droplet spreads out to cover the entire glass surface. After drying, store the silk/AAV-coated optical fibers in a cooled vacuum desiccator at 125 torr and four degrees Celsius prior to implanting the devices.Fluorescence images of fluorophore-tagged optogenetic proteins provided a measure of the extent of expression of the silk/AAV films, and typical optical fibers can readily accommodate 200 nanoliters of silk/AAV. With practice, experimenters can achieve highly reliable expression around the tip of implanted fibers. Two possible issues with coated cranial windows are insufficient expression, and silk films that fail to dissolve, and obscure the field of view.To increase expression, a durectomy can be performed prior to implanting the window, and/or increasing the amount of virus in the film. The best expression was achieved using a one to four mixture of silk and stock-titer AAV, respectively. The amount of silk in coated windows is 10 to 100 times more than on fiber implants, and the film is less embedded in tissue, and thus may not be exposed to the same levels of proteolytic activity that can dissolve silk films.However, the presence of some silk is essential to achieving your expression beneath windows, likely because a film made of virus alone is washed away by interstitial fluid during surgery. While attempting this procedure, it's important to prepare and store silk-coated implants in the manner we describe. Improper storage may change the properties of silk films, or decrease the efficacy of viral expression vectors.This technique can be of great utility for researchers in the field of systems neuroscience to explore how neural activity drives sensory and cognitive processing in the brains of mammals such as mice, rats, or even nonhuman primates. Don't forget that working with viral expression vectors can be hazardous and personal protective equipment, such as gloves, goggles, and lab coats, should always be worn while working with Adeno-associated virus.

in-vivo-targeted-expression-optogenetic-proteins-using-silkaav

Research

JoVE Journal

Neuroscience

8.4K ビュー.  Oregon Health and Science University. この方法は、システム神経科学の分野における重要な質問に答えるのに役立ちます。ニューロンアンサンブルの活動が脳内の感覚と認知処理をどのようにサポートしているかなど。この技術の主な利点は、別のウイルス注射手順を必要とせずに、関心のある脳領域にチャンロドプシンやGCaMPのような光遺伝学的タンパク質の正確な標的化を容易にすることです。こ?...

ビデオ: In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

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

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

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

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

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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

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

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

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

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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

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

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.

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

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

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

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

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

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...