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...

<ol>
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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160; 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&#160; 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&#38;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 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 ...

The current protocol contributes to the study of the brain mechanisms underlying skilled motor behavior. Wireless optogenetics permits the manipulation of specific neurons while subjects perform a task in free movement. In combination with high-speed videography, it is possible to have a detailed analysis of fine motor behavior.The method could help to understand motor problems in neurodevelopmental and neurodegenerative disorders. These techniques can be used to study brain function in other behavioral paradigms as well. Diana Rodriguez-Munoz, an undergrad student from my lab, will be assisting with experiment.For the surgical procedures, start by preparing an LED cannula of the desired length according to the dorso-ventral coordinates of the structure-of-interest. To do so, cut the glass fiber to a length longer than the final desired size and grind the fiber tip to the target length with rough sandpaper. Then, polish the fiber tip with fine sandpaper.For microinjections, fill a pipette with the mineral oil and place the pipette in the microinjector. Ensure the microinjector works correctly by injecting some mineral oil. Next, prepare the anesthetized mouse for the surgery by applying an ophthalmic ointment and removing hair from the scalp with a trimmer and hair removal cream.Then, wipe the scalp with cotton swabs having 8%povidone iodine and 70%ethanol alternated three times each. After sanitization, place the mouse in the stereotaxic apparatus and secure the head, ensuring that the skull is leveled in the medial-lateral and anterior-posterior axes. Use a scalpel to make a one-centimeter incision through the scalp at the level of the eyes along the sagittal axis.Then, retract the skin to expose the skull and clean the periosteum with cotton swabs. Clean the cranium surface with saline solution and cotton swabs and resolve any bleeding at the surface using sterile absorbent eye spears. Then, 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 the area thoroughly with a clean cotton swab. With a glass pipette of 15-micron final tip diameter, locate bregma and lambda to check that the skull is leveled in the anterior-posterior axis. Then, paint a reference point in the scalp above the selected coordinates with a marker.In the reference point, perform a one-millimeter diameter craniotomy applying gentle pressure to the skull with a rotary tool at a low to medium speed with a small, round dental drill bit. Once done, move the capillary toward the selected anterior-posterior, or AP, and medial-lateral, or ML, coordinates. Load the capillary with 300 to 400 nanoliters of the CreE-dependent adeno-associated virus, or AAV, such as AAV1, D-flox channelrhodopsin mCherry to express channelrhodopsin in the region-of-interest.An AAV can be used as a control to express the reporter protein. After ensuring that the tip is not clogged, introduce the glass pipette in the brain at the dorso-ventral minus 3.35 millimeter coordinates in the dorsal-lateral striatum and inject 200 nanoliters of the virus using an automatic injector at a rate of 23 nanoliters per second. Wait for 10 minutes post-injection before withdrawing the glass pipette slowly to avoid spillage.Then, clean and dry any residues with cotton swabs. Next, attach the glass LED cannula to the stereotaxic arm and calibrate the coordinates using bregma as a reference. Then, insert the cannula slowly to avoid tissue damage and place it 100 micrometers above the injection site.Once the LED cannula is in place, add a drop of 100 microliters of tissue adhesive at the edge of the craniotomy. Use a sterile brush to apply a freshly prepared 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. When the cement dries completely, close the skin around the implant using tissue adhesive.Then, place the mouse in a recovery cage over a heating pad at 33 degrees Celsius while monitoring for the signs of discomfort or pain. Record the behavior of the animal with a regular camera and capture 30 to 60 frames per second from the front of the chamber. A mirror can be placed under the training chamber at a 45-degree angle to monitor the animal's posture.When mice start reaching the pellet, turn the LED cannula manually with the remote controller to have a continuous stimulation for the time the behavior is performed for no longer than two seconds. The stimulation device triggers an LED of 470 nanometers with intensity at the tip of one milliwatt per millimeter square. The fine motor behavior of the animal under optogenetic manipulations was studied with the reach-to-grasp task.The mice learned to execute the task in a couple of days and achieved more than 55%accuracy, reaching a plateau after five days of training. The trajectory of movements was tracked with high-speed videography, making it possible to analyze kinematics. The quantifiable assessment of parameters such as distance traveled, velocity, acceleration, endpoint, and trajectory was performed.In the missed trials, the mice started the grasping movement further away from the pellet than the hit trials. Additionally, errors were significantly associated with the mouse posture, shown by differences in the body angle during hit and missed trials. The contralateral activation of D1 dopamine expressing spiny projection neurons, or SPNs, reduced the grasping success compared to the control conditions.During an optogenetic stimulation, the paw trajectory increased in the travel distance, leading to the incapability to target the pellet and an increase in initial error type one. Principle component analysis, or PCA, showed that all trials trajectories during contralateral D1 SPNs activation separated in a cluster with almost no overlap with the control cluster, indicating a low similarity. Activation of the DSPNs in the ipsilateral side led to an increase in trajectory dispersion shown by PCA analysis.Hence, the ipsilateral D1 SPNs activation modified the reaching trajectory without changing the behavioral outcome. When performing this protocol, be careful to place the cannula correctly so it doesn't detach during training sessions. This method can be used in other behavioral paradigms to study motor behavior, sensory processing, learning, and memory.Wireless optogenetics will allow to study naturalistic behavior and its neurobiological underpinnings in truly free-moving subjects.

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

Research

JoVE Journal

Neuroscience

3.1K visualizações.  National University of Mexico. O protocolo atual contribui para o estudo dos mecanismos cerebrais subjacentes ao comportamento motor qualificado. A optogenética sem fio permite a manipulação de neurônios específicos enquanto os sujeitos realizam uma tarefa em livre circulação. Em combinação com a videografia de alta velocidade, é possível ter uma análise detalhada do comportamento motor fino.O método poderia ajudar a entender problemas motores em distúrbios neurodesenvolvimentantes e neurodegenerativos...