Name: Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation (Video) | JoVE Journal
Uploaded: 2011-12-28T12:00:00.000+00:00
Duration: 12 min
Description: Learn optogenetic stimulation of adult-born olfactory neurons using lentiviral Channelrhodopsin2 delivery and chronic LED photostimulation in mice. This method enables precise control of neuronal activation for behavioral studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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°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 μm 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 μl 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 μl. 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, ±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 μm thickness in 0-4°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="Fluorescence microscopy; neural structure visualization; RMS; GCL, EPL, GL regions; biological analysis." src="/files/ftp_upload/3380/3380fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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, 100μm. 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, 20μm.
<img alt="Optogenetic experiment diagram with LED setup on brain; fluorescence microscopy results shown." src="/files/ftp_upload/3380/3380fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
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, 10μm.

<ol><li>Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+neurogenesis+and+functional+plasticity+in+neuronal+circuits%5BTitle%5D)+AND+%22Nat.+Rev.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Turning+astrocytes+from+the+rostral+migratory+stream+into+neurons%3A+a+role+for+the+olfactory+sensory+organ%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Learning+and+survival+of+newly+generated+neurons%3A+when+time+matters%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((How%2C+when%2C+and+where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Genetic+dissection+of+an+amygdala+microcircuit+that+gates+conditioned+fear%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Multimodal+fast+optical+interrogation+of+neural+circuitry%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Lentivirus+Production%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Targeted+optogenetic+stimulation+and+recording+of+neurons+in+vivo+using+cell-type-specific+expression+of+Channelrhodopsin-2%5BTitle%5D)+AND+%22Nature+protocols%22%5BJournal%5D)">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/pubmed?term=((Sparse+optical+microstimulation+in+barrel+cortex+drives+learned+behaviour+in+freely+moving+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((In+vivo+light-induced+activation+of+neural+circuitry+in+transgenic+mice+expressing+channelrhodopsin-2%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.</a> Neuron. 54, 205-218 Forthcoming.</li>
<li>Albeanu, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LED+arrays+as+cost+effective+and+efficient+light+sources+for+widefield+microscopy%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Odor+Discrimination+and+Odor+Quality+Perception+in+Rats+with+Disruption+of+Connections+between+the+Olfactory+Epithelium+and+Olfactory+Bulbs%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Molecular+and+cellular+approaches+for+diversifying+and+extending+optogenetics%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Using+Affordable+LED+Arrays+for+Photo-Stimulation+of+Neurons%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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...

<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

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.

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 ...

The goal of this procedure is to use remote control to activate olfactory bulb neurons and observe how their activation affects behavior. This is accomplished by introducing a trends gene of channel Redin two, coupled to YFP into target neurons of adults with stereotaxic injections of a lentiviral vector. Then an LED is calibrated and prepared for implantation producing a general idea of the amount of light transmitted into the brain.The final step of the procedure is the implantation of the LED over the olfactory bulb, ensuring its proper positioning and attachment. Ultimately, neuronal activation is confirmed through CFOs, immuno staining, and the results of behavioral testing. The main advantage of this technique over existing methods like standard electrical stimulation or pharmacological perturbations, is that optogenetic stimulation of labeled neurons is highly specific regarding both type and number of neurons activated.The implication of this technique stand forward the therapy or diagnosis of neurodegenerative disease because of the precise temporal and spatial control offer. This method can provide insight into the role of adult bone neuron in the olfactory bone network. It can be applied to other superficial regions such as the cortical or cerebral lung neurons.Begin by retrieving prepared vector titers from the negative 80 degrees Celsius freezer. Keep them frozen before they use and discard any leftover titers. Cut the tip of pooled boro silica glass pipettes using a pair of scissors.The goal is to make 30 to 40 micron diameter tips. Store the prepared pipettes in a dust-free box. Next, set up a micro injector to inject 50 nanoliters back filler prepared pipette with mineral oil and insert it into the micro injector plunger, which should be attached to the stereotaxic frame.Once the viral titer has thawed, partially empty oil from the glass pipette, and then refill it with the titer. Transfer two microliters of virus onto a sterile piece of para film. Gently lower the glass pipette into the drop and fill the pipette with one to two microliters of virus.Check that there are no air bubbles in the pipette, and then proceed with preparing the animal after anesthetizing a mouse using ketamine and xylazine diluted in sterile saline. Make sure the mouse is non-responsive to pain stimulus, such as a toe pinch before proceeding. Now, remove the hair from the scalp of the mouse and disinfect the scalp.Place the animal in a stereotaxic frame and use ear barss and a nose bar to secure its head 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.Then clean the surface of the skull using sterile saline solution. Zero the stereotaxic apparatus with the tip of the glass pipette at breg ma. Then position the tip at the injection site.Adjust the position of the nose bar according to the calculation of the stereotaxic coordinates. Using these coordinates, the bgma and the injection site are aligned to the same height. Using a high speed surgical drill, carefully drill a small hole into the skull at each injection site.Take care not to rup your blood vessels on the surface of the brain while drilling. Now with a bent syringe needle hooked at the tip, remove the remnants of thin bone to expose the URA mater. Check that the hole is centered at the correct position.Lower the pipette and zero the dorso ventral height. When the tip of the pipette touches the surface of the brain, then lower the pipette to the correct dorsa ventral depth and wait 30 seconds for pressure equilibrium. Now inject 50 nanoliters of virus four times for a total of 200 nanoliters of virus waiting 30 seconds between injections.One minute after the last injection, slowly withdraw the pipette and remove the mouse from the apparatus. Clean the incision site with Betadine solution and stitch the skin. Using surgical thread, allow the mouse to recover on a warming pad before finally returning it to its cage before implanting the miniature blue LED.For optogenetic stimulation, it must be prepared and tested. Sold are the LED pins to a female electrical connector so that the connector is positioned on the opposite side of the LED. Check that there is no electrical short circuit, and identify the positive and negative pin on the connector.Lastly, test the LED by connecting it to an LED controller to measure or set the absolute light power of the LED mount, the LED on a micro manipulator and prepare the light meter. Drill a three millimeter hole in an opaque PVC board and affix this hole to the power meter. Now position the LED in direct contact with the photo detector of the power meter and take measurements at the peak LED light intensity, which is usually four 70 nanometers.Use the power meter to build a standard curve of optical power versus input current. Then use the to calculate the power per square millimeter to measure the propagation of light through the brain. Obtain vibram cut blocks of fresh brain tissue of 300, 500, 1000, one thousand five hundred and two thousand five hundred micron thicknesses using a piece of skull with a craniotomy identical to the one performed in the live mice.Place it between the LED and the detector. Now, measure the light power without tissue. Then place each block of tissue between the LED and the photo detector of the power meter and measure the change in power.Build a curve of tissue thicknesses versus relative transmission fraction. Transmission fraction is the ratio of the power measured through the tissue compared to without the tissue. Use this data to calibrate the LED and then proceed with the implantation surgery.Prepare the LED and connector by disinfecting them with 70%ethanol and attaching the connector to a stereotaxic holder. After placing an anesthetized mouse in the stereotaxic frame, use a nose bar to align the surface of the skull perfectly horizontal and parallel to the surface of the miniature LED. In this case, the region of the skull corresponding with the olfactory bulb is aligned.Now with a scalpel, cut the scalp from three millimeters anterior to the eyes to three millimeters 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.Prepare to perform a rectangular craniotomy over the off effect bulbs. First, use a high-speed drill to carefully thin the skull until only a thin layer of bone remains. Second, carefully remove the remaining bone with the help of a bent syringe needle.Carefully avoid puncturing the dura mater. Now rinse the surgical area with sterile saline and then dry the skull. After cleaning and drying the exposed skull, apply a thin base layer of cyanoacrylate adhesive to the skull for later adhesion of dental acrylic.The addition of two small screws may be required to fix the LED. The screws should be inserted into the skull posterior to the LED and covered with both acrylics. After attaching the LED connector to the holder, lower the miniature LED above the olfactory bulbs with its main axis parallel to the rostral cordal axis.Then secure the LED to the skull with two layers of dental acrylic. While the acrylic is still liquid, pull back the free edges of the scalp and glue them to two layers of dental acrylic. Be careful not to apply any acrylic to the connector after the acrylic has hardened.Remove the animal from the frame and let it wake up on a warming pad. Once returned to its cage, full recovery takes at least seven days later. A thin light electrical cable is used to tether the animal to an LED controller, allowing ample ambulatory freedom, respect the polarity of the wire as inverting.The polarity may break the LED. After the experiment, remove the LED to verify that it was functional CH opsin. Two YFP was expressed in transduced neurons approximately 48 hours after injection and remained stable for several months.A sagittal section of a paraldehyde fixed animal 15 days post injection showed dense labeling and the size of the injection site. In the RMS, A large number of newborn cells migrated along the RMS to colonize the different layers of the olfactory bulb. The fusion protein was expressed in all neuron compartments, including the soma dendrites and spines.Miniature LED implantation allowed large scale and inexpensive optical stimulation of a broad region of interest by characterizing the light diffusion in the brain tissue and the threshold of channel redin two activation. Optical activation was estimated to be triggered at a depth of about two millimeters below the surface of the LED to monitor the functional activation of the channel adoption, two expressing neurons in vivo. The expression of CFOs.A marker of neuronal activity was analyzed after repetitive light stimulation. One master. This technique can be done in one hour to inject the virus, and after at least three weeks, two more hours to implant LAD.Calibration of LAD is important and take additional time Following this procedure. Other methods, such as in vivo electrosurgical recording can be performed to answer additional question like about the relationship between activating one type of cell and the response in another After its development. This technique paved the way for researchers in the field of systems neuroscience to explore precise correlations between newborn neuron activation and olfactory perception in the mouse olfactory system.After watching this video, you should have a good understanding of how to inject a viral vector using STEREOTAXIC coordinates, how to prepare and calibrate an LED for implantation, how to implant the LED and how to use oxygen and stimulation to conduct behavioral experiments. Also, please remember that using viral vectors can be extremely hazardous and proper precautions, such as proper training and disposal of hazardous materials should be taken.

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

Research

JoVE Journal

Neuroscience

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

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

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

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb.  Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

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.

Using Affordable LED Arrays for Photo-Stimulation of Neurons

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

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

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.

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

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.

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

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

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

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Learn optogenetic stimulation of adult-born olfactory neurons using lentiviral Channelrhodopsin2 delivery and chronic LED photostimulation in mice. This method enables precise control of neuronal activation for behavioral studies.

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

Explore More Videos