This work was supported by the life insurance company "Novalis-Taitbout", Ecole des Neurosciences de Paris (ENP), the Agence Nationale de la Recherche "ANR-09-NEUR-004" in the frame of "ERA-NET NEURON" of FP7 program by the European Commission, "Foundation pour la Recherche Medicale", the Letten Foundation and the Pasteur Foundation.

1. Stereotaxic injections
<ol>
	<li>The virus used in these experiments is a replication-deficient lentiviral vector based on the HIV virus to express Channelrhodopsine2-YFP (yellow fluorescent protein) fusion construct driven by a synapsin promoter6. The plasmid was generously provided by K. Deisseroth's research group (<a href="http://www.stanford.edu/group/dlab/optogenetics/index.html">http://www.stanford.edu/group/dlab/optogenetics/index.html</a>). Lentiviruses can be obtained from commercial sources or produced in the lab according to established protocols7. Average vector titers were in the order of 1010 transduction units/ml. Viral aliquots are stored at -80&deg;C and should be thawed just before pipette loading. Multiple freeze/thaw cycles should be avoided. Never exposure the virus solution directly to dry ice.</li>
	<li>Prepare glass pipettes using borosilicate glass capillaries on standard pipette puller (Sutter P-97 pipette puller). The pulling setting should be properly calibrated to yield a long and rigid pipette. Next, cut the tip of the pipette using a pair of scissors to obtain a tip of 30-40 &mu;m in diameter. Store pulled pipettes in a dust free box.</li>
	<li>Setup the Nanoject II microinjector with the manufacturer's instructions for delivery of 50 nL. Prepare the injection pipette by back-filling with mineral oil and insert it in to the microinjector plunger. Attach the microinjector onto a stereotaxic frame. Partially empty oil from the glass pipette before filling with the virus solution</li>
	<li>With a micropipette, transfer 2 &mu;l of virus solution onto a sterile piece of Parafilm. Gently lower the glass pipette into the drop of virus solution and fill the pipette with 1-2 &mu;l. Make sure that there are no air bubbles in the pipette.</li>
	<li>Anesthetize a mouse with 100 mg/kg Ketamine and 10 mg/kg Xylazine, diluted in sterile saline. Remove the hair from the scalp of the animal using rodent clippers, razor, scissors, or chemical depilitory agent. Disinfect the scalp with three applications of alternating 70% ethanol and betadine. All surgical instruments should also be autoclaved and then disinfected with 70% ethanol. Place the animal in the stereotaxic frame with ear bars and nose bar, assuring that the head is secure. Apply eye ointment to prevent drying of the corneas.</li>
	<li>With a scalpel, cut the scalp from between the eyes to between the ears. Pull the skin aside to expose the skull and anchor the skin with a pair of clamps. Clean the surface of the skull. The stereotaxic apparatus is zeroed with the tip of the glass pipette at bregma. Then the tip is positioned at the injection site (Antero-Posterior, 3.30; Medio-Lateral, &plusmn;0.82, for both right and left hemispheres, respectively). The position of the nose bar has to be adjusted according to the calculation of the stereotaxic coordinates. In our experiment, the nose bar is adjusted until bregma and the injection site are aligned to the same height.</li>
	<li>Identify the injection site. Using a high-speed surgical drill, carefully drill holes into the skull. With a bent syringe needle hooked at the tip, remove the remnants of thin bone to expose the dura mater. Check that the hole is centered at the correct position. Take care not to rupture blood vessels on the surface of the brain while drilling. If blood vessels are ruptured, gently press onto the surface of the brain with a small cotton bud or tissue for several seconds until blood flow stops.</li>
	<li>Lower the pipette and zero the Dorso-Ventral height when the tip of the pipette touches the surface of the brain. Then, lower to the correct Dorso-Ventral depth (Dorso-Ventral, 2.9 from brain surface) and wait 30 seconds for pressure equilibrium. Inject 4 times 50 nl of virus for a total of 200 nl. Wait 30 seconds between each injection.</li>
	<li>One minute after the last injection, slowly withdraw the pipette. Repeat points 1.6-1.8 for the other hemisphere. Remove the animal from stereotaxic apparatus, clean the incision and stitch the cut skin using surgical threads. Allow the animal to recover on warming pad before returning to cage. Additionally, animals can be given a non-steroidal anti-inflammatory analgesic (e.g., Carprofen 4mg/kg) immediately after and 3 days following surgery.</li>
</ol>
2. Chronic LED implantation for in vivo optogenetic stimulation in the olfactory bulb
<ol>
	<li>Animals injected with lentiviral particles bilaterally are next chronically implanted with a miniature blue LED (Osram, LED CMS 4.6W blue). Solder the LED pins to a female miniature electrical connector so that the connector is positioned on the opposite side of the LED. Check that there is no electrical short cut and identify the positive and negative pin on the connector. Test the LED by connecting it to a current controller for LED though a proper cable.</li>
	<li>To measure and/or set the absolute light power of the LED, mount the LED on a micromanipulator and position the LED in direct contact with the photodetector of a power meter of a peak light intensity at 470 nm. Drill a pinhole of 3 mm diameter in an opaque PVC board. Affix this pinhole to the power meter and build a standard curve of optical power versus input current to calculate the power per mm2.</li>
	<li>To measure the propagation of light through the brain, cut blocks of fresh brain tissue of 300, 500, 1000, 1500 and 2500 &mu;m thickness in 0-4&deg;C ACSF using a vibratome14. Place a piece of skull with a craniotomy identical to the one performed in live mice under the LED. First, measure the light power without tissue centered on LED. Then, place each block of tissue between the LED and the photodetector of the power meter, and measure the power. Build a curve of tissue thickness versus relative transmission fraction, calculated as the ratio between the power measured through tissue and without tissue.</li>
	<li>Anesthetize a mouse with 100 mg/kg Ketamine and 10 mg/kg Xylazine, diluted in sterile saline. Clean and disinfect the LED with 70% ethanol. Remove the hair from the scalp of the animal and disinfect the scalp with three applications of alternating 70% ethanol and betadine. Then place the animal in the stereotaxic frame. Apply eye ointment to prevent drying of the corneas. Attach the connector to a stereotaxic holder. The position of the nose bar should be adjusted until the surface of the skull above the olfactory bulb is perfectly horizontal and parallel to the surface of the miniature LED.</li>
	<li>With a scalpel, cut the scalp from 3 mm anterior to the eyes to 3 mm posterior to the ears. Pull the skin aside and anchor it with a pair of clamps. Scrape away the membranes at the surface of the skull using a razor blade. Clean and dry the skull. Apply a thin cover of cyanoacrylate adhesive to allow subsequent adhesion of dental acrylic.</li>
	<li>Using a high-speed surgical drill, perform a rectangular craniotomy (1 mm caudal and 3 mm lateral; centered on 5.1 mm caudal, 0 mm lateral from bregma) over the olfactory bulbs. To do this, carefully thin the skull until only a thin layer of bone remains. Carefully remove the remaining bone with the help of a bent syringe needle. Special care should be taken to avoid puncturing the dura matter and rupturing the major blood vessels that lie between the two hemispheres and in the caudal border of the olfactory bulbs. Blood accumulation and coagulation at the brain surface precludes optimal light penetration into the tissue. Rinse the craniotomy and the surface of the brain with sterile saline and then dry the skull.</li>
	<li>Attach the miniature LED to the connector. Lower the miniature LED on the top of the olfactory bulbs. The main axis of the miniature LED should be parallel to the rostral-caudal axis of the mouse, Clean with sterile saline and dry the skull. Then secure the LED to the skull with a first layer of acrylic (i.e., polycarboxylate cement), followed by a second layer of dental acrylic. The addition of two small screws may be required to fix the LED. The screws should be inserted on the skull (posterior to the LED position) and covered with both acrylics. While the acrylic is still liquid, pull back and glue the free edges of the scalp on the surface of the dental acrylic. Take care not to apply any acrylic on the connector. After the acrylic has hardened, remove the animal from stereotaxic apparatus and let it recover on a warming pad before returning to cage. Animals are left isolated in their cage to recover from the surgery for at least seven days. Additionally, animals can be given a non-steroidal anti-inflammatory analgesic (e.g., Carprofen 4mg/kg) immediately after and 3 days following surgery. LEDs should be tested before implantation and after termination the experiments.</li>
	<li>A light electrical cable is used to tether the animal to a current controller for LED coupled to a pulse generator (Master-8) to control the timing and intensity of the LED. The thin cable allows for complete freedom of movement. Take care to respect the polarity of the connector. Inverting the polarity may break the LED.</li>
</ol>
3. Representative Results:
ChR2-YFP is expressed in transduced neurons approximately 48 hours after injection, and remains stable for several months7. Figure 1 is a representative picture of a sagittal section of a paraformaldehyde-fixed animal 15 days post-injection. Note the dense labeling and the size of the injection site in the RMS. A large number of newborn cells have migrated along the RMS to colonize the different layers of the olfactory bulb (Fig. 1). The fusion protein is expressed in all neuron compartments, including the soma, dendrites, and spines.
Miniature LED implantation allows large scale and inexpensive optical stimulation of a broad region of interest. The animal is tethered to the LED controller with a light electrical cable that allows a complete freedom of movement. By characterizing the light diffusion in the brain tissue and the threshold of ChR2 activation, we estimate that optical activation could be triggered at a depth of ~2mm below the surface of the LED (Fig. 2A). To monitor the functional activation of ChR2-expressing neurons in vivo, the expression of c-fos, a marker of neuronal activity, can be analyzed after repetitive light stimulation (Fig. 2B). As an alternative, in vitro electrophysiological recordings of ChR2-expressing neurons coupled to focal light stimulation can be performed14. Finally, two-photon targeted loose-patch recordings have been used to study the functional activation of ChR2-expressing cortical neurons by similar miniature LED in vivo9.
<img alt="Figure 1" src="/files/ftp_upload/3380/3380fig1.jpg" /> 
Figure 1. ChR2-YFP expression from lentiviral injection into the rostral migratory stream. Representative confocal images of a saggital sections of the olfactory bulb of a mouse 15 days after injections of a lentivirus in the rostral RMS. Note the presence of scattered YFP newborn neurons migrating from the end of the RMS to the different layers of the OB. Most of newborn cells (95%) are granule cells, which soma lie in the Granule Cell Layer (GCL) and dendrites arborize in the External Plexiform Layer (EPL). A small population of newborn neurons migrate up to the most superficial layer of the OB, in the Glomerular Layer (GL). Scale bar, 100&mu;m. Inset, high magnification of a newborn granule cell, showing the membrane expression of ChR2-YFP in the dendrites as well as in spines. Scale bar, 20&mu;m.
<img alt="Figure 2" src="/files/ftp_upload/3380/3380fig2.jpg" /> 
Figure 2. Diagram of implanted LED on the top of the olfactory bulb and light-evoked c-fos expression in ChR2-YFP expressing new granule cells. 
A, Schematic drawing of the implanted LED above the olfactory bulbs. 
B, Representative confocal images showing double-labelled ChR2-YFP+ c-fos+ granule cells one hour after light stimulation of the olfactory bulb. C-fos expression is revealed using a rabbit anti-c-fos primary antibody (Calbiochem, 1:5000) and A568-conjugated anti-rabbit secondary antibody (Invitrogen, 1:1000). Scale bar, 10&mu;m.

<ol>
	<li>Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+and+functional+plasticity+in+neuronal+circuits.">Adult neurogenesis and functional plasticity in neuronal circuits.</a> Nat. Rev. Neurosci. 7, 179-193 (2006).</li><li>Alonso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+astrocytes+from+the+rostral+migratory+stream+into+neurons:+a+role+for+the+olfactory+sensory+organ.">Turning astrocytes from the rostral migratory stream into neurons: a role for the olfactory sensory organ.</a> J. Neurosci. 28, 11089-11102 (2008).</li><li>Mouret, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+survival+of+newly+generated+neurons:+when+time+matters.">Learning and survival of newly generated neurons: when time matters.</a> J. Neurosci. 28, 11511-11516 (2008).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How,+when,+and+where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">How, when, and where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> J. Neurosci. 30, 17023-17034 (2010).</li><li>Haubensak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+an+amygdala+microcircuit+that+gates+conditioned+fear.">Genetic dissection of an amygdala microcircuit that gates conditioned fear.</a> Nature. 468, 270-276 (2010).</li><li>Zhang, F. <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, 633-639 (2007).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+Production.">Lentivirus Production.</a> J. Vis. Exp. (32), e1499-e1499 (2009).</li><li>Cardin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+optogenetic+stimulation+and+recording+of+neurons+in+vivo+using+cell-type-specific+expression+of+Channelrhodopsin-2.">Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2.</a> Nature protocols. 5, 247-254 (2010).</li><li>Huber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+optical+microstimulation+in+barrel+cortex+drives+learned+behaviour+in+freely+moving+mice.">Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice.</a> Nature. 451, 61-64 (2010).</li><li>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=In+vivo+light-induced+activation+of+neural+circuitry+in+transgenic+mice+expressing+channelrhodopsin-2.">In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.</a> Neuron. 54, 205-218 .</li><li>Albeanu, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+arrays+as+cost+effective+and+efficient+light+sources+for+widefield+microscopy.">LED arrays as cost effective and efficient light sources for widefield microscopy.</a> PLoS ONE. 3, 2146-2146 (2008).</li><li>Slotnick, B., Bodyak, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+Discrimination+and+Odor+Quality+Perception+in+Rats+with+Disruption+of+Connections+between+the+Olfactory+Epithelium+and+Olfactory+Bulbs.">Odor Discrimination and Odor Quality Perception in Rats with Disruption of Connections between the Olfactory Epithelium and Olfactory Bulbs.</a> J. Neurosci. 22, 4205-4216 (2002).</li><li>Gradinaru, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+approaches+for+diversifying+and+extending+optogenetics.">Molecular and cellular approaches for diversifying and extending optogenetics.</a> Cell. 141, 154-165 (2010).</li><li>Valley, M., Wagner, S., Gallarda, B. W., Lledo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Affordable+LED+Arrays+for+Photo-Stimulation+of+Neurons.">Using Affordable LED Arrays for Photo-Stimulation of Neurons.</a> J. Vis. Exp. (57), e3379-e3379 (2011).</li></ol>

No conflicts of interest declared.

Optogenetic tools have recently increased our control over selective neuronal populations, including adult born neurons in the olfactory system. Here we describe how to perform a stereotaxic injection of a lentiviral vector expressing ChR2-YFP in a specific population of adult-born neurons. By carefully monitoring the period post-injection, we are able to analyze and manipulate the activity of a cohort of adult-born neurons with a relative homogeneous age.
Because of their low diffusion spread, their high transduction efficiency and their relative good tropism for neuroblasts of the RMS, lentiviral vectors should be favored compared to others viral vectors such as retroviruses or adeno-associated viruses (AAV). Because they selectively infect dividing cells, such as stem cells or progenitors, retrovirus vectors must be injected into the subventricular zone and transduce a smaller population of newborn neurons. Compared to lentiviral vectors, AAV vectors provide a much higher spread of infection. However, AAV transduction of neuroblasts in the RMS should be avoided because of the transduction of surrounding structures such as the accessory olfactory nucleus, a region that projects axons back into the olfactory bulb. Lastly, use of implanted LEDs can be applied to other superficial structures of the brain such as cortical regions in which ChR2 expression is driven in specific populations of neurons through viral vectors or via a transgenic mouse line10.
With LED implantation, we are able to apply chronic optical stimulation to study the functional role of olfactory bulb adult neurogenesis in awake behaving animals. This simple light source provides a fast temporal control of light thanks to the intrinsically rapid on/off speed of LEDs. In addition to being inexpensive, a miniature LED light source produces sufficient power (~100mW) for ChR2 activation in a large volume of brain tissue11, even bilaterally in the case of olfactory bulbs. This is particularly important in this brain region where significant lesions may be without effect on odor quality perception due to a high degree of redundancy in the inputs from the olfactory epithelium12. Thanks to its power and its spectral bandwidth (25nm), these miniature LEDs may be used for light-evoked excitation using ChR2 as well as others versions of channelrhodopsins such as ChETA or CatCh. Miniature yellow (597nm) and red (624 or 637nm) LEDs are available to be used for red-shifted rhodopsins such as halorhodopsin and VChR113.
Miniature LEDs also show limitations. To avoid heating the brain, a compromise must be found between pulse duration, pulse frequency, and light intensity. For instance, short light pulses of 5 milliseconds are recommended at high frequencies (&gt;20Hz) and light intensity of 100 mW. Above this duration, a significant elevation of the temperature (~1-4&deg;C) in the surrounding tissue can be measured. This parameter should be taken into account for longer light pulses (&gt;5ms) or continuous light stimulation, notably in the case of light-evoked hyperpolarization using halorhodopsin. In addition, the current supplying the LED provides strong electrical artifact, which may preclude in vivo electrophysiological recordings when the LED is in direct contact with the tissue. Laser-coupled optic fibers constitute a possible alternative, allowing a smaller and more restricted optical stimulation. However, the rigidity of optic fibers may interfere with certain behaviors. 
After characterizing of the light penetration and the functional stimulation of neurons by light, in vivo optical stimulation can be triggered in combination with behavioral experiments. Special care should be taken regarding the timing of the stimulation. Synchronization of the photostimulation with a specific behavior or task in a behavioral apparatus, notably during operant conditioning, could be decisive for establishing a causal link between cell activation and behavior.

<table><tbody><tr><td>Ketamine</td><td>Merial</td><td>Imalg&amp;#232;ne 1000</td><td></td></tr><tr><td>Xylazine</td><td>Bayer AG</td><td>Rompum 0.2%</td><td></td></tr><tr><td>Pipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Glass Capillaries (3.5&quot;)</td><td>Drummond Scientific</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Nanoject II Microinjector</td><td>Drummond Scientific</td><td>3-000-204</td><td></td></tr><tr><td>High speed surgical drill</td><td>Fine Science Tools</td><td>18000-17</td><td></td></tr><tr><td>Micro Drill Steel Burrs 0.7mm tip diameter</td><td>Fine Science Tools</td><td>19008-07</td><td></td></tr><tr><td>LED CMS blue 4.6 W</td><td>OSRAM</td><td>LBW5SN</td><td></td></tr><tr><td>Female electrical connector (2.54 mm)</td><td>Fisher Scientific</td><td>BL5.36Z</td><td></td></tr><tr><td>Current controller for LED (max: 1A)</td><td>A1W Electronik</td><td>HKO-KL50-1000</td><td></td></tr><tr><td>Pulse generator</td><td>AMPI</td><td>Master-8</td><td></td></tr><tr><td>Optical power meter</td><td>Thorlabs Inc.</td><td>PM100</td><td></td></tr><tr><td>Cyanoacrylate adhesive</td><td>Loctite</td><td>454</td><td></td></tr><tr><td>Polycarboxylate cement (first layer cement)</td><td>Septodont</td><td>Selfast (#0068Q)</td><td></td></tr><tr><td>Methacrylate cement (second layer cement)</td><td>GC International</td><td>Unifast Trad &amp;#8211; Liquid (#339292) Unifast Trad &amp;#8211; Powder (#339114)</td><td></td></tr><tr><td>Carprofen</td><td>Pfizer Pharma GmbH</td><td>Rimadyl</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation at JoVE.com

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.

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 ...

selective-viral-transduction-adult-born-olfactory-neurons-for-chronic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.4K Views.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). 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.

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

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

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 ...

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 ...

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 ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

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 ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

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 ...

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

Summary

Explore More Videos

Chapters in this video

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Using Affordable LED Arrays for Photo-Stimulation of Neurons

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

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

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