Name: in vivo wireless optogenetic control of skilled motor behavior
Uploaded: 2021-11-22T14:30:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about In Vivo Wireless Optogenetic Control of Skilled Motor Behavior at JoVE.com

This work was supported by the UNAM-PAPIIT project IA203520. We thank the IFC animal facility for their help with mouse colonies maintenance and the computational unit for IT support, especially to Francisco Perez-Eugenio.

The procedures involving animal use were conducted following local and national guidelines and approved by the corresponding Institutional Animal Care and Use Committee (Institute of Cellular Physiology IACUC protocol VLH151-19). Drd1-Cre transgenic male mice27, 35-40 days postnatal with C57BL/6 background were used in the current protocol. Mice were kept under the following conditions: temperature 22&#177;1 &#176;C; humidity 55%; light schedule 12/12 h with lights off at 7 p.m. and were weaned at postnatal day 21. Weaned pups were housed in same-sex groups of 2-5.&#160;Animals were housed in static housing with micro-barrier tops. Bedding consisted of sterile aspen shavings. Rodent pellets&#160;and RO-purified water were provided ad libitum, except when noted.
1. Surgical procedures
<ol>
	<li>Prepare an LED cannula at the desired length according to the dorsoventral coordinates of the structure of interest (ideally 0.5 mm longer to account for the thickness of the skull, for the dorsolateral striatum 3.5 mm) (Figure 1).
	<ol>
		<li>Cut the glass fiber to a length longer than the final desired size, grind the fiber tip to the target length with rough sandpaper, and finally, polish the fiber tip with fine sandpaper. 
		NOTE: LED cannula is a glass optical fiber of 250 &#181;m diameter attached to an infrared receiver (see Table of Materials).</li>
	</ol>
	</li>
	<li>Pull glass pipettes (1.14 mm outer diameter, 0.53 mm inner diameter, and 3.5 in length) for the nano-injector with a horizontal puller (see Table of Materials) and store them for later. Program the puller in one loop to get a 15-20 &#181;m tip diameter with a long gradual slope taper (4-5 mm).</li>
	<li>Prepare the surgery area by thoroughly disinfecting the stereotaxic apparatus, hood, micro-injector (see Table of Materials), and surrounding surfaces with 70% ethanol. 
	NOTE: A mouse stereotaxic apparatus is essential to inject Adeno Associated Virus (AAVs) precisely and place the LED cannula in the region of interest.</li>
	<li>Wear the appropriate personal protective equipment for the procedure, including a clean lab coat or disposable surgical gown, sterile gloves, face mask, and disposable head cap.</li>
	<li>Place the necessary equipment close to the surgery area, such as sterile surgical tools, cotton tips, solutions, micropipette, pipette tips, capillaries, micro-fill with mineral oil, and marker.</li>
	<li>Fill a pipette for microinjections with mineral oil and place it in the micro-injector. Make sure that the micro-injector is working correctly by ejecting some mineral oil. 
	NOTE: All instruments used during the surgery should be autoclaved and sterile. Aseptic technique should be used.</li>
	<li>Anesthetize animals with gaseous isoflurane 4-5% to induce anesthesia and 1.2% throughout the surgery with 0.5-1 L/min pure oxygen. The surgery begins only after the animal has reached a point of deep anesthesia, assessed by the absence of paw withdrawal after a slight pinch.
	<ol>
		<li>Continuously monitor the breathing rate and temperature of the animal. Maintain the body temperature by a heating pad set at 34 &#176;C.</li>
	</ol>
	</li>
	<li>Apply an ophthalmic ointment. Remove hair from the scalp with a trimer and hair removal cream. Wipe the scalp with cotton swabs having 8% povidone-iodine (see Table of Materials) and 70% ethanol alternated three times each.</li>
	<li>Place the mouse in the stereotaxic apparatus and secure the head, ensuring that the skull is leveled in the mediolateral and anterior-posterior axes.</li>
	<li>Make a 1 cm incision with a scalpel through the scalp at the level of the eyes along the sagittal axis. Retract the skin to expose the skull and clean the periosteum with cotton swabs.</li>
	<li>Clean the cranium surface with saline solution and sterile cotton swabs. Resolve any bleeding at the surface using sterile absorbent eye spears (see Table of Materials) or similar sterile absorbent material.</li>
	<li>Apply a drop of 2.5% hydrogen peroxide with a cotton swab and let it act for a few seconds to make the skull sutures visible and have a better reference. After a few seconds, clean thoroughly with a clean cotton swab.</li>
	<li>With the glass pipette (15 &#181;m final tip diameter), locate bregma and lambda to check that the skull is leveled in the anterior-posterior axis. 
	NOTE: It is recommended to have a stereoscopic microscope or USB microscope to see the tip of the glass pipette. In case it is needed, adjust the height of the mouth holder to level the skull.</li>
	<li>Move the capillary toward the selected anterior-posterior (AP) and medial-lateral (ML) coordinates (dorsolateral striatum AP 1.2 mm, ML 2.28 mm). Paint a reference point in the scalp above the selected coordinates with a sterile marker.</li>
	<li>In the reference point, perform an ~1 mm diameter craniotomy applying gentle pressure to the skull with a sterile rotary tool or dental drill at a low to medium speed with a small round dental drill bit (see Table of Materials).</li>
	<li>Load the capillary with 300-400 nL of Cre-dependent adeno-associated virus (AAV) such as AAV1-dflox-hChR-2-mCherry to express Channelrhodopsin or an AAV to express only the reporter protein (e.g., mCherry) as a control in the region of interest (see Table of Materials). Check that the tip is not clogged, then introduce the glass pipette in the brain at the desired dorso-ventral (DV) coordinates (dorsolateral striatum DV -3.35 mm).
	<ol>
		<li>Inject 200 nL using an automatic injector at a rate of 23 nL/s. Wait for 10 min after finishing the injection, withdraw the glass pipette slowly to avoid spillage. 
		NOTE: It is possible to use a 30 G needle to inject with the appropriate micro-injector.</li>
	</ol>
	</li>
	<li>Clean and dry any residues with cotton swabs.</li>
	<li>Attach the sterile&#160;glass LED cannula to the stereotaxic arm and calibrate the coordinates using bregma as a reference. Insert the cannula very slowly (300 &#181;m/min) to avoid tissue damage and place it 100 &#181;m above the injection site.</li>
	<li>Once the LED cannula is in place, add a drop (100 &#181;L) of tissue adhesive at the edge of the craniotomy.</li>
	<li>Prepare dental cement mixture (see Table of Materials) following the manufacturer&#39;s instructions to fix the fiber to the skull. 
	NOTE: Briefly, use a chilled porcelain dish to have more working time before cement sets. Add 2 scoops of resin clear powder to the porcelain dish, add 4 drops of quick base and 1 drop of catalyst, then mix well. The powder/liquid ratio can be adjusted if a thinner or thicker viscosity is needed.</li>
	<li>Using a sterile brush, apply the dental cement mixture around the cannula connector little by little, building layers until the skull is covered and the connector is securely attached to the skull, leaving the pins completely free. Avoid getting dental cement on the skin of the mouse.</li>
	<li>Allow drying completely.</li>
	<li>Close the skin around the implant using tissue adhesive (see Table of Materials).</li>
	<li>Place the mouse in a recovery cage over a heating pad at 33&#176; C. Monitor for the presence of one or more of the following signs of pain/discomfort: 1) Hunched up, lack or reduction of motor activity, 2) Failure to groom reflected in a unkept dirty coat, 3) Excessive licking or scratching, redness in the incision site, 4) aggressive behavior, 5) anorexia or dehydration, and 6) Lack of nest formation. 
	NOTE: Keep the mouse individually caged during all the procedures to avoid implant detaching. In case of detachment of the cannula, perform euthanasia by injecting 150 mg/kg of sodium pentobarbital followed by decapitation after deep anesthesia is reached.</li>
	<li>Inject subcutaneously (SC) meloxicam 1 mg/kg once daily for three days post-surgery to provide analgesia.</li>
	<li>Wait at least 7 days for complete recovery and 14 days for opsin expression before further procedures. 
	NOTE: Perform a postoperative follow-up every 12 hours for three days, then check animals every day until the day of euthanasia at the end of the experiment.</li>
</ol>

2. Reach-to-grasp training
<ol>
	<li>On day 7 post-surgery, start the food deprivation protocol28. Weigh mice for three consecutive days to determine their average ad libitum body weight. Then, schedule food restrictions so that the animals receive enough nutrients to maintain approximately 90% and not less than 85% of body weight. 
	NOTE: This is achieved by providing 2.5-3 g of food daily. Monitor animals&#39; weight daily and score for overall well-being observing animals&#39; behavior and appearance, for example, coat and eyes appearance. Use the body condition scoring system from Reference29.</li>
	<li>During the pre-training, training, and testing periods, provide each mouse with 20 pellets (20 mg of dustless chocolate-flavored pellets) daily (see Table of Materials) (eaten during the task or after) besides the standard food pellets.</li>
	<li>Three days before habituation, scatter 0.4 g/animal/day 20 mg of dustless chocolate-flavored pellets in their home cages, so mice get acquainted with the pellets that serve as a reward during the reach-to-grasp task.</li>
	<li>Habituate mice by placing them 10 min in the testing chamber one day before pre-training with pellets scattered on the chamber floor (Figure 1A).</li>
	<li>Allow food daily after training and testing. Keep a similar schedule every day.</li>
	<li>On the first day of pre-training, place the mice in the reach-to-grasp chamber and observe from the front. Place the pellets in front of the chamber close to the opening so that they start consuming the pellets. At this stage, mice are allowed to grab the pellets in any form.</li>
	<li>On day two of pre-training, place the pellets further and further from the opening until getting them to the indentation (1 cm from the opening) so mice can shape their reach-to-grasp movement (Figure 1C).</li>
	<li>Train mice to run to the rear of the cage and return to the cage opening to receive the next food pellet as a strategy to individualize trials. 
	NOTE: This can be achieved by waiting until the mouse is in the rear of the cage before placing a pellet in the indentation for each trial.</li>
	<li>Place pellets to be grasped by either their right or left paw. 
	NOTE: Mice start using preferentially one paw to grasp, which will be used the following days of training and testing.</li>
	<li>Train animals for 6 days in daily sessions lasting 20 trials or until a maximum of 10 min elapse. From day 2 of training, put the mock receiver (dimensions 12 x 18 x 7 mm, 1 g, see Table of Materials), so mice get habituated to the weight while performing the task (Figure 1B). Each day score the number of hit&#160;and missed trials.</li>
	<li>Record behavior with a regular camera and capture 30-60 frames/s from the front of the chamber. Additionally, one can place a mirror under the training chamber at a 45&#176; angle to monitor the animals&#39; posture (Figure 1D,E).</li>
	<li>For post-hoc kinematic analysis (Figure 2), mount a high-speed camera (see Table of Materials) at an angle of 45&#176; to record from the side of the cage. If a 3D analysis is required, place a second high-speed camera to record at a 35&#176; angle from the front of the chamber; both cameras should be placed in the right or left side of the cage depending on animals&#39; sidedness and should capture at the same frame rate and be synchronized7 (Figure 3D,E).</li>
	<li>Set the high-speed cameras to 100 frames/s with a resolution of 376 x 252 pixels or more if possible. Place white Styrofoam walls behind the sides and back of the chamber to reduce background and increase contrast (Figure 1E).</li>
	<li>On test day, replace the mock unit with an infrared receiver for wireless optogenetic stimulation (Figure 1B,C).</li>
	<li>When mice start reaching, turn the LED cannula manually with the remote controller to have a continuous stimulation for the time the behavior is performed and for no longer than 2 s. Programming an automatic stimulation paradigm is preferable. The stimulation device triggers an LED of 470 nm (blue light) with intensity at the tip of 1.0 mW/mm2.</li>
	<li>Collect the videos for further examination, including scoring and kinematic analysis.</li>
</ol>
3. Post-hoc histological confirmation
<ol>
	<li>Upon completion of an experiment, confirm viral expression and LED cannula placement. Anesthetize the animal with a cocktail of ketamine 100 mg/kg and xylazine 10 mg/kg. Once the mouse presents signs of deep anesthesia (step 1.7), perfuse with ice-cold phosphate-buffered saline (PBS) followed by 4% PFA.</li>
	<li>Remove implanted cannula carefully by firmly grasping the connector with forceps and pulling up gently.</li>
	<li>Extract and post-fix the brain for 24 h in 4% PFA23.</li>
	<li>Perform 3-10 min washes with PBS.</li>
	<li>Cut the brain in 50 &#181;m sections using a microtome (see Table of Materials).</li>
	<li>Mount the sections in slides with hard-set mounting media with DAPI to stain nuclei and cover slides.</li>
	<li>After drying, observe the sections under the confocal microscope and verify the implanted cannula location and expression of Ch2R fused with any fluorescent protein.</li>
</ol>

<ol>
	<li>Balbinot, 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=Post-stroke+kinematic+analysis+in+rats+reveals+similar+reaching+abnormalities+as+humans.">Post-stroke kinematic analysis in rats reveals similar reaching abnormalities as humans.</a> Scientific Report. 8 (1), 8738 (2018).</li><li>Klein, A., Sacrey, L. A., Whishaw, I. Q., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+rodent+skilled+reaching+as+a+translational+model+for+investigating+brain+damage+and+disease.">The use of rodent skilled reaching as a translational model for investigating brain damage and disease.</a> Neuroscience &amp; Biobehavioral Reviews. 36 (3), 1030-1042 (2012).</li><li>MacLellan, C. L., Gyawali, S., Colbourne, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+impairments+follow+intrastriatal+hemorrhagic+stroke+in+rats.">Skilled reaching impairments follow intrastriatal hemorrhagic stroke in rats.</a> Behavioural Brain Research. 175 (1), 82-89 (2006).</li><li>Evenden, J. L., Robbins, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unilateral+6-hydroxydopamine+lesions+of+the+caudate-putamen+on+skilled+forepaw+use+in+the+rat.">Effects of unilateral 6-hydroxydopamine lesions of the caudate-putamen on skilled forepaw use in the rat.</a> Behavioural Brain Research. 14 (1), 61-68 (1984).</li><li>Rodgers, R. A., Travers, B. G., Mason, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimanual+reach+to+grasp+movements+in+youth+with+and+without+autism+spectrum+disorder.">Bimanual reach to grasp movements in youth with and without autism spectrum disorder.</a> Frontiers in Psychology. 9, 2720 (2019).</li><li>Sacrey, L. A. -. O., Zwaigenbaum, L., Bryson, S., Brian, J., Smith, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reach-to-grasp+movement+in+infants+later+diagnosed+with+autism+spectrum+disorder:+a+high-risk+sibling+cohort+study.">The reach-to-grasp movement in infants later diagnosed with autism spectrum disorder: a high-risk sibling cohort study.</a> Journal of Neurodevelopmental Disorders. 10 (1), 41 (2018).</li><li>Azim, E., Jiang, J., Alstermark, B., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+relies+on+a+V2a+propriospinal+internal+copy+circuit.">Skilled reaching relies on a V2a propriospinal internal copy circuit.</a> Nature. 508 (7496), 357-363 (2014).</li><li>Marques, J. M., Olsson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+juvenile+mice+in+a+reach-to-grasp+task.">Performance of juvenile mice in a reach-to-grasp task.</a> Journal of Neuroscience Methods. 193 (1), 82-85 (2010).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+Impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: Impairments in movement and posture and compensatory adjustments.</a> The Journal of Neuroscience. 14 (11), 7148-7158 (1994).</li><li>Vaidya, M., Kording, K., Saleh, M., Takahashi, K., Hatsopoulos, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+coordination+during+reach-to-grasp.">Neural coordination during reach-to-grasp.</a> Journal of Neurophysiology. 114 (3), 1827-1836 (2015).</li><li>Wang, 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=Deconstruction+of+corticospinal+circuits+for+goal-directed+motor+skills.">Deconstruction of corticospinal circuits for goal-directed motor skills.</a> Cell. 171 (2), 440-455 (2017).</li><li>Xu, T., 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=Rapid+formation+and+selective+stabilization+of+synapses+for+enduring+motor+memories.">Rapid formation and selective stabilization of synapses for enduring motor memories.</a> Nature. 462 (7275), 915-919 (2009).</li><li>Ian, Q. W., Sergio, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+skilled+forelimb+reaching+in+the+rat:+A+proximally+driven+movement+with+a+single+distal+rotatory+component.">The structure of skilled forelimb reaching in the rat: A proximally driven movement with a single distal rotatory component.</a> Behavioural Brain Research. 41 (1), 49-59 (1990).</li><li>Proske, U., Gandevia, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprioceptive+senses:+their+roles+in+signaling+body+shape,+body+position+and+movement,+and+muscle+force.">The proprioceptive senses: their roles in signaling body shape, body position and movement, and muscle force.</a> Physiological Reviews. 92 (4), 1651-1697 (2012).</li><li>Yttri, E. A., Dudman, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opponent+and+bidirectional+control+of+movement+velocity+in+the+basal+ganglia.">Opponent and bidirectional control of movement velocity in the basal ganglia.</a> Nature. 533 (7603), 402-406 (2016).</li><li>Donchin, O., Gribova, A., Steinberg, O., Bergman, H., Vaadia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+motor+cortex+is+involved+in+bimanual+coordination.">Primary motor cortex is involved in bimanual coordination.</a> Nature. 395 (6699), 274-278 (1998).</li><li>Brus-Ramer, M., Carmel, J. B., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortex+bilateral+motor+representation+depends+on+subcortical+and+interhemispheric+interactions.">Motor cortex bilateral motor representation depends on subcortical and interhemispheric interactions.</a> The Journal of Neuroscience. 29 (19), 6196-6206 (2009).</li><li>d'Avella, A., Saltiel, P., Bizzi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinations+of+muscle+synergies+in+the+construction+of+a+natural+motor+behavior.">Combinations of muscle synergies in the construction of a natural motor behavior.</a> Nature Neuroscience. 6 (3), 300-308 (2003).</li><li>Fattori, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+orientation+during+reach-to-grasp+movements+modulates+neuronal+activity+in+the+medial+posterior+parietal+area+V6A.">Hand orientation during reach-to-grasp movements modulates neuronal activity in the medial posterior parietal area V6A.</a> The Journal of Neuroscience. 29 (6), 1928-1936 (2009).</li><li>vanden Berg, F. E., Swinnen, S. P., Wenderoth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+the+motor+cortex+ipsilateral+to+the+moving+body+side+depends+on+spatio-temporal+task+complexity+and+hemispheric+specialization.">Excitability of the motor cortex ipsilateral to the moving body side depends on spatio-temporal task complexity and hemispheric specialization.</a> PLoS One. 6 (3), 17742 (2011).</li><li>vanden Berg, F. E., Swinnen, S. P., Wenderoth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+primary+motor+cortex+in+controlling+movements+executed+with+the+ipsilateral+hand+differs+between+left-+and+right-handers.">Involvement of the primary motor cortex in controlling movements executed with the ipsilateral hand differs between left- and right-handers.</a> Journal of Cognitive Neuroscience. 23 (11), 3456-3469 (2011).</li><li>Verstynen, T., Diedrichsen, J., Albert, N., Aparicio, P., Ivry, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipsilateral+motor+cortex+activity+during+unimanual+hand+movements+relates+to+task+complexity.">Ipsilateral motor cortex activity during unimanual hand movements relates to task complexity.</a> Journal of Neurophysiology. 93 (3), 1209-1222 (2005).</li><li>Lopez-Huerta, V. 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=Striatal+bilateral+control+of+skilled+forelimb+movement.">Striatal bilateral control of skilled forelimb movement.</a> Cell Reports. 34 (3), 108651 (2021).</li><li>Lopez-Huerta, V. 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=The+neostriatum:+two+entities,+one+structure.">The neostriatum: two entities, one structure.</a> Brain Structure and Function. 221 (3), 1737-1749 (2016).</li><li>Becker, M. I., Calame, D. J., Wrobel, J., Person, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+control+of+reach+accuracy+in+mice.">Online control of reach accuracy in mice.</a> Journal of Neurophysiology. 124 (6), 1637-1655 (2020).</li><li>Jaidar, O., 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=Synchronized+activation+of+striatal+direct+and+indirect+pathways+underlies+the+behavior+in+unilateral+dopamine-depleted+mice.">Synchronized activation of striatal direct and indirect pathways underlies the behavior in unilateral dopamine-depleted mice.</a> European Journal of Neuroscience. 49 (11), 1512-1528 (2019).</li><li>Gong, S., 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=Targeting+Cre+recombinase+to+specific+neuron+populations+with+bacterial+artificial+chromosome+constructs.">Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs.</a> The Journal of Neuroscience. 27 (37), 9817-9823 (2007).</li><li>Rowland, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+or+fluid+restriction+in+common+laboratory+animals:+balancing+welfare+considerations+with+scientific+inquiry.">Food or fluid restriction in common laboratory animals: balancing welfare considerations with scientific inquiry.</a> Comparative Medicine. 57 (2), 149-160 (2007).</li><li>Ullman-Culler&#233;, M. H., Foltz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+condition+scoring:+a+rapid+and+accurate+method+for+assessing+health+status+in+mice.">Body condition scoring: a rapid and accurate method for assessing health status in mice.</a> Laboratory Animal Science. 49 (3), 319-323 (1999).</li><li>Chen, C. C., Gilmore, A., Zuo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+motor+skill+learning+by+single-pellet+reaching+tasks+in+mice.">Study motor skill learning by single-pellet reaching tasks in mice.</a> Journal of Visualized Experiments. (85), e51238 (2014).</li><li>Fink, A. 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=Presynaptic+inhibition+of+spinal+sensory+feedback+ensures+smooth+movement.">Presynaptic inhibition of spinal sensory feedback ensures smooth movement.</a> Nature. 509 (7498), 43-48 (2014).</li><li>Li, Q., 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=Refinement+of+learned+skilled+movement+representation+in+motor+cortex+deep+output+layer.">Refinement of learned skilled movement representation in motor cortex deep output layer.</a> Nature Communication. 8, 15834 (2017).</li><li>Overduin, S. A., d'Avella, A., Carmena, J. M., Bizzi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstimulation+activates+a+handful+of+muscle+synergies.">Microstimulation activates a handful of muscle synergies.</a> Neuron. 76 (6), 1071-1077 (2012).</li><li>Miyazaki, T., 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=Large+Timescale+interrogation+of+neuronal+function+by+fiberless+optogenetics+using+lanthanide+micro-particles.">Large Timescale interrogation of neuronal function by fiberless optogenetics using lanthanide micro-particles.</a> Cell Reports. 26 (4), 1033-1043 (2019).</li><li>Yang, Y., 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=Wireless+multilateral+devices+for+optogenetic+studies+of+individual+and+social+behaviors.">Wireless multilateral devices for optogenetic studies of individual and social behaviors.</a> Nature Neuroscience. 24 (7), 1035-1045 (2021).</li><li>Kampasi, 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=Fiberless+multicolor+neural+optoelectrode+for+in+vivo+circuit+analysis.">Fiberless multicolor neural optoelectrode for in vivo circuit analysis.</a> Scientific Reports. 6, 30961 (2016).</li><li>Allen, B. D., Singer, A. C., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+designing+interpretable+optogenetic+behavior+experiments.">Principles of designing interpretable optogenetic behavior experiments.</a> Learning &amp; Memory. 22 (4), 232-238 (2015).</li><li>Packer, A. M., 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> Nature Methods. 9, 1202-1205 (2012).</li></ol>

The authors declare no disclosures.

The use of optogenetic manipulation of neuronal populations in well-defined behavioral paradigms is advancing our knowledge about the mechanisms underlying motor control7,23. Wireless methods are especially suitable for tasks that require tests on multiple animals or free movement34,35. Nevertheless, as techniques and devices are refined, it should be the go-to option for any behavioral task combined with optogenetics34,36.
The current method has many advantages because miniaturized LEDs provide a reliable light source with high intensity, and implants can be used in studies requiring stimulation over several days. Nevertheless, insertion of an optic fiber for opsin stimulation may mechanically damage brain tissue, cause infections and occasionally inflammation at the location of the canula37. Long-lasting high-frequency optogenetic stimulation has been demonstrated to produce heat and could cause phototoxity37. It is possible to reduce phototoxicity by using red-shifted effector opsins that are activated with red or even near-infrared light, which reduces the generation of heat38.
Also, since the pins to connect the receiver remain outside of the skull, sometimes mice can cause displacement or detachment of the cannula if dental cement is not applied correctly; this often leads to damage of brain tissue and decreases the number of subjects to be taken into account for further analysis. Recent developments have introduced fiberless optogenetics, which uses particles that can emit visible light through up-conversion luminescence in response to near-infrared light penetrating deep in the brain tissue36. Fiberless devices bring the opportunity to stimulate optogenetically over longer time frames in freely behaving animals with unnoticeable implants35. This allows for unrestrained motion even in water mazes, to have multiple animals housed together (to avoid the impact of social isolation), and to study animals in more naturalistic settings35,36.
Even with all the advantages that fiberless optogenetics offers, it still faces biocompatibility and heat generation challenges. The efficiency of photon conversion also limits it. Finally, further improvement is required for high emission efficiency34,36.
The combination of this paradigm with high-speed videography allows for kinematic analysis under different experimental conditions. This offers sensitive detection of even subtle effects over distinct components of behavior and motor control. As more analytic tools develop, it is possible to have online kinematic analysis and an in-depth characterization of motor behavior in different contexts. A thorough quantification of mouse reaching movement kinematics has been recently published by Becker et al.25.
The possibility of selectively manipulating neuronal populations in freely moving animals with minimally invasive techniques allows one to dissect the contribution of specific neuronal types in precise behavioral tasks23. The reach-to-grasp task is a translatable paradigm for motor behavior13,19. It is known that conserved brain structures participate in the different phases of acquisition, learning, and performance of the task7,12,23. Revealing the neural circuits that underlie this behavior will increase the understanding of motor control. Several studies highlight the importance of bi-hemispheric control over unimanual tasks, especially when high dexterity is needed20,21,22. The kinematic analysis combined with optogenetic manipulations allows for the investigation of the different mechanisms of this complex behavior. It could help to analyze the contribution of sensory-motor feedback in normal conditions and disease models.

Motor skilled behavior is present during most movements performed by us, and it is known to be affected in several brain disorders1,2,3,4,5,6. Implementing tasks that allow studying the development, learning, and performance of skilled movements is crucial to understanding the motor function&#39;s neurobiological underpinnings, especially in models of brain injury, neurodegenerative and neurodevelopmental disorders2,7,8,9,10,11,12,13. Reaching for and retrieving objects is done routinely in everyday life actions, and it is one of the first motor skills acquired during early development and then refined through the years5,6. It comprises a complex behavior that requires sensory-motor processes such as the perception of the object&#39;s features, movement planning, action selection, movement execution, body coordination, and speed modulation7,14,15,16. Thus, unimanual high dexterity tasks require the participation of many brain structures of both hemispheres16,17,18,19,20,21,22. In mice, the single pellet reach-to-grasp task is characterized for several phases that can be controlled and analyzed separately7,13,23. This feature allows to study the contribution of specific neuronal subpopulations at different stages of acquisition and behavior performance and provides a platform for detailed studies of motor systems13,23,24. The movement occurs in a couple of seconds; thus, high-speed videography should be used for kinematic analysis in distinct stages of the skilled motor trajectory7,25. Several parameters can be extracted from the videos, including body posture, trajectory, velocity, and type of errors25. Kinematic analysis can be used to detect subtle changes during wireless optogenetic manipulation7,23.
Using miniaturized light-emitting diodes (LEDs) to deliver light via a wireless head-mounted system makes it possible to have remote optogenetic control while the animal performs the task. The wireless optogenetic controller accepts single-pulse or continuous trigger commands from a stimulator and sends infrared (IR) signals to a receiver connected to the miniaturized LED23,26. The current protocol combines this wireless optogenetics approach with high-speed videography of a dexterity task to dissect the role of specific neuronal populations during the performance of fine motor behavior23. Since it is a unimanual task, it allows for assessing the participation of structures in both hemispheres. Traditionally, the brain controls the body movement in a highly asymmetric manner; however, high dexterity tasks require careful coordination and control from many brain structures, including ipsilateral nuclei and differential contribution of neuronal subpopulations within nuclei10,20,21,22,23. This protocol shows that subcortical structures from both hemispheres control the trajectory of the forelimb23. This paradigm can be suitable to study other brain regions and models of brain disease.

<table><tbody><tr><td>Anaesthesia machine</td><td>RWD</td><td>R583S</td><td>Isoflurane vaporizer</td></tr><tr><td>Anesket</td><td>PiSA</td><td></td><td>Ketamine</td></tr><tr><td>Breadboard</td><td>Thorlabs</td><td>MB3090/M</td><td>Solid aluminum optical breadboard</td></tr><tr><td>Camera lense</td><td>Canon</td><td></td><td>50mmf/ 1.4 manual focus lenses (c-mount)</td></tr><tr><td>Camera system</td><td>BrainVision</td><td>MiCAM02</td><td>Camera controller and synchronizer</td></tr><tr><td>Cotton swabs</td><td></td><td></td><td></td></tr><tr><td>CS solution</td><td>PiSA</td><td></td><td>Sodium chloride solution 9%</td></tr><tr><td>Customized training chamber</td><td>In house</td><td></td><td></td></tr><tr><td>Drill bit #105</td><td>Dremel</td><td>2 615 010 5AE</td><td>Engraving cutter</td></tr><tr><td>Dustless precission chocolate pellets</td><td>Bio-Serv</td><td>F05301</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>J.T.&amp;nbsp; Baker</td><td>9000-02</td><td>Ethanol</td></tr><tr><td>Eyespears</td><td>Ultracell</td><td>40400-8</td><td>Eyespears of absorbent PVA material</td></tr><tr><td>Fluriso</td><td>VetOne</td><td>V1 502017-250</td><td>Isoflurane</td></tr><tr><td>Glass capillaries</td><td>Drumond Scientific</td><td>3-000-203-G/X</td><td>Pipettes for NanoJect II</td></tr><tr><td>Hidrogen peroxide</td><td>Farmacom</td><td></td><td>Antiseptic</td></tr><tr><td>High-speed camera</td><td>BrainVision</td><td>MiCAM02-CMOS</td><td>Monochrome high-speed cameras</td></tr><tr><td>Infrared emmiter</td><td>Teleopto</td><td></td><td></td></tr><tr><td>Insulin syringe</td><td></td><td></td><td></td></tr><tr><td>LED cannula</td><td>Teleopto</td><td>TelC-c-l-d</td><td>LED cannula 250um 487nm light</td></tr><tr><td>Micropipette 10 uL</td><td>Eppendorf</td><td>Z740436</td><td></td></tr><tr><td>Micro-pipette puller</td><td>Sutter</td><td>P-87</td><td>Horizontal puller</td></tr><tr><td>Microscope LSM780</td><td>Zeiss</td><td></td><td>Confocal microscope</td></tr><tr><td>Microtome</td><td></td><td></td><td></td></tr><tr><td>Mock receiver</td><td>Teleopto</td><td></td><td></td></tr><tr><td>NanoJect II</td><td>Drumond Scientific</td><td>3-000-204</td><td>Micro injector</td></tr><tr><td>Oxygen tank</td><td>Infra</td><td>na</td><td></td></tr><tr><td>pAAV-EF1a-double.floxed-hChR2(H134R)-mCherry-WPRE- HGHpA</td><td>Addgene</td><td>20297</td><td>Viral vector for ChR-2 expression</td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td></td></tr><tr><td>Phosphate saline buffer</td><td>Sigma</td><td>P-4417</td><td>Phosphate saline buffer tablets</td></tr><tr><td>Pipette tips 10 uL</td><td>ThermoFisher</td><td>AM12635</td><td>0.5-10 uL&amp;nbsp; volume</td></tr><tr><td>Pisabental</td><td>PiSA</td><td></td><td>Sodium pentobarbital</td></tr><tr><td>Plexiglass</td><td>commercial</td><td></td><td>Acrylic sheet</td></tr><tr><td>Povidone iodine</td><td>Farmacom</td><td></td><td>Antiseptic</td></tr><tr><td>Procin</td><td>PiSA</td><td></td><td>Xylacine</td></tr><tr><td>Puralube</td><td>Perrigo pharma</td><td>1228112</td><td>Eye lubricant 15% mineral oil/85% petrolatum</td></tr><tr><td>Rotary tool</td><td>Kmoon</td><td>Mini grinder</td><td>Standard</td></tr><tr><td>Scalpel</td><td></td><td></td><td></td></tr><tr><td>Scalpel blade</td><td></td><td></td><td></td></tr><tr><td>Stereotaxic apparatus</td><td>Stoelting</td><td>51730D</td><td>Digital apparatus</td></tr><tr><td>Super-Bond C&amp;amp;B</td><td>Sun Medical</td><td></td><td>Dental cement</td></tr><tr><td>Surgical dispossable cap</td><td></td><td></td><td></td></tr><tr><td>Teleopto remote controller</td><td>Teleopto</td><td></td><td></td></tr><tr><td>Tg Drd1-Cre mouse line</td><td>Gensat</td><td>036916-UCD</td><td>Transgene insertion FK150Gsat</td></tr><tr><td>Tissue adhesive</td><td>3M Vetbond</td><td>1469SB</td><td></td></tr><tr><td>TPI Vibratome 1000 plus</td><td>Peico</td><td></td><td>Microtome</td></tr><tr><td>Vectashield mounting media with DAPI</td><td>Vector laboratories</td><td>H-1200</td><td>Mounting media</td></tr><tr><td>Wireless receiver</td><td>Teleopto</td><td>TELER-1-P</td><td></td></tr></tbody></table>

in vivo wireless optogenetic control of skilled motor behavior

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

The reach-to-grasp task is a paradigm widely used to study shaping, learning, performance, and kinematics of fine skill movement under different experimental manipulations. Mice learn to execute the task in a couple of days and achieve more than 55% accuracy reaching a plateau after 5 days of training (Figure 2A,B). Similar to what has been previously reported, a percentage of animals do not perform the task appropriately (29.62%), and those should be excluded from further analysis30. These include a subset of non-learner mice (6/54 mice, 11.1%) that from the beginning of training miss the target aiming too far from the pellet or perform the grasping movement before they are in the correct position over the pellet. During the first training days, another group performed the task with high accuracy but started performing poorly by aiming too far from the pellet by day 3-4 (10/54 mice, 18.51%). Within this group, some mice start training using a preferred paw but change their preference after a few days; this has been previously discussed by Chen et al., 201430.
The reach-to-grasp movement is highly stereotypical from trial to trial and within animals (Figure 2). The use of high-speed videography allows tracking the trajectory of movements making it possible to analyze kinematics at different phases in the control condition and during optogenetic stimulation (Figure 1E and Figure 2C). This approximation results in a quantifiable assessment of parameters such as distance traveled, velocity, acceleration, end-point, and trajectory (Figure 2 C-E). It is possible to analyze both multi-reach trials, where the mouse reaches multiple times before retrieving the pellet, and single-trial events, where the mouse retrieves the pellet in a single reaching movement. The trial is finished when animals push the pellet away or reinitiate the trial by going to the rear of the cage. A quantitative comparison of trajectories under different experimental conditions is achieved with principal component analysis (PCA) followed by k-means clustering (Figure 3J-K)23,25.
During most training sessions, mice sometimes fail to grasp the pellet (missed trials). Some manipulations change the number of missed trials and hence the accuracy of the task. Then, it is essential to analyze differences between hit and missed trials. In our hands, missed trials result from changes in three distinct phases of the movement: (1) the paw modifies its trajectory before it crosses the chamber opening (initial error), (2) the paw modifies its trajectory after the paw crosses the opening (final error), and, (3) failure to collect the pellet (grasp error) (Figure 2 I,J)13. A general characteristic of missed trials is that mice start the grasping movement further away from the pellet (end-point) compared to hit trials (Figure 2G). Additionally, misses associated with the mouse posture are measured as significant differences in body angle between hit and missed trials (Figure 2H).
Depending on the structure or neuronal population targeted with optogenetics, one can expect differential effects over behavior7,19,23,31,32,33. The current protocol describes the impact of activating spiny projection neurons (SPNs) in the striatum in contralateral or ipsilateral hemispheres about the preferred paw used by the mouse during the reaching movement (Figure 3). Contralateral activation of D1 dopamine expressing SPNs, which give origin to the basal ganglia direct pathway, reduced grasping success to 64.9&#177;8.8% compared to control conditions (Figure&#160;3B). Kinematic analysis reveals that during optogenetic stimulation, the paw trajectory described an oscillatory pattern, shown by an increase in the traveled distance to 218.4&#177;19.2% of control, leading to the incapability to target the pellet and an increase in initial error type I (Figure 3F). PCA analysis shows that all trials&#39; trajectories during contralateral D1 SPNs activation separated in a cluster with almost no overlap with a control cluster, indicating a low similarity (Figure 3J-K).
On the other hand, activation of dSPNs in the ipsilateral side lead to an increase in trajectory dispersion shown by PCA analysis (Figure 3K) without affecting reaching success (120.7&#177;23.6%, n = 4), total distance traveled (136.3&#177;35.5%), or maximum velocity during the reaching phase (117.3&#177;10.3%) (Figure 3C), indicating that ipsilateral D1 SPNs activation modified in some degree the reaching trajectory without changing the behavioral outcome (Figure 3G-I). Kinematic analysis indicates subtle changes in movement control by ipsilateral manipulation. Finally, body posture analysis shows a shift in body angle during contralateral D1SPNs activation (Figure 3L). It is highlighted that even this simple task has many components that permit proper movement execution to attain a goal.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63082/63082fig01.jpg" /> 
Figure 1: Experimental setup. (A) Schematics of the behavioral chamber. A chamber made with acrylic sheet having the following dimensions in cm: 18.5 (h) x 8.5 (w) x 20 (d) with a front window 1 (w) x 5 (h) and a small shelf 8.5 (w) x 4 (d). (B) Photograph of LED cannula (left) and wireless receiver (right). (C) Side view of a mouse with the implanted LED cannula connected to the receiver while performing the reach-to-grasp task (the white arrowhead shows the receiver, the asterisk shows the dental cement holding the cannula, and the empty arrowhead shows the pellet). (D) Sketch of the experimental setup. Two high-speed cameras record the reach in two dimensions, while a third collected a panoramic view of the task, including the mouse&#39;s location from the mirror under the chamber. Animals were free to choose their preferred paw, and stimulation sides always refer to the side of the preferred paw. (E) The exact position of the cameras and representative image of each camera during a trial. This figure has been adapted from Reference23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63082/63082fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63082/63082fig02.jpg" /> 
Figure 2: Kinematic analysis of reaching behavior. (A) Timeline of the experiment from day-0 (d0) to day-25 (d25). (B)&#160;Performance during the reach-to-grasp task over time measured as pellet retrieval accuracy (total number of successful grasps/total number of trials x 100). (C) Example of trajectory tracking from a high-speed video. (D)&#160;Individual trajectories of the paw during the hit and missed trials. (E) Total distance traveled by the paw during the hit and missed trials. (F) Acceleration of the paw through the trajectory in the hit and missed trials plotted as distance vs. velocity. (G) Summary of end-points distance in hits = 3.16 mm, misses&#160;6.08 mm (Mann-Whitney-Wilcoxon testmissed vs. hit U = 4184, p&#60;0.0001, n = 28 mice). (H) Differences in body angle in the two kinds of trials, misses = 8.4&#177;5.3&#176;, hits 6.7&#177;4&#176; (Mann-Whitney-Wilcoxon test U = 6437, P = 0.0243, n = 28 mice). (I) Schematics of the three types of errors. (J) Proportions of the three kinds of errors made by the mice in control conditions. This figure has been adapted from Reference23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63082/63082fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63082/63082fig03.jpg" /> 
Figure 3: Optogenetic activation of contralateral and ipsilateral D1 SPNs during reach-to-grasp behavior. (A) Schematics of the stimulation paradigm. (B) Success rate compared to non-stimulation trials. (C) Change in traveled distance compared to control conditions. (D), (G) Two-dimensional plots of paths made by the paw with and without optogenetic stimulation. (E), (H) Total distance traveled by the paw during reaching movements. (F), (I) Summary of the distributions of the different kinds of errors. D1 contralateral: Initial error or type I (control = 18.2&#177;11.6 %, stimulation = 79.9&#177;8.2 % Fisher&#39;s exact test, p&#60;0.0001). (J) Example of PCA analysis of the trajectories in control conditions compared to contralateral activation of D1SPNs. The shaded area represents the cluster of each condition, and the star is the cluster centroid. (K) Summary of the overlap between clusters of the different experimental conditions. (L) Changes in body angle. This figure has been modified from Reference23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63082/63082fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Wireless Optogenetic Control of Skilled Motor Behavior at JoVE.com

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

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

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

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

in-vivo-wireless-optogenetic-control-of-skilled-motor-behavior

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.2K Views.  National University of Mexico. Het huidige protocol draagt bij aan de studie van de hersenmechanismen die ten grondslag liggen aan geschoold motorisch gedrag. Draadloze optogenetica maakt de manipulatie van specifieke neuronen mogelijk terwijl proefpersonen een taak uitvoeren in vrije beweging. In combinatie met high-speed videografie is het mogelijk om een gedetailleerde analyse van fijn motorisch gedrag te hebben.De methode kan helpen om motorische problemen bij neurologische en neurodegeneratieve aandoeningen te ...

Video: In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Behavior

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

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

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

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

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

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

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

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

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

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

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

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

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

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

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

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

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

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 ...

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.

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.

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

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

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior

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