This work was supported by the intramural research program at the National Institute of Mental Health (ZIA MH002964-02). We would like to thank the support of the NIMH IRP Rodent Behavioral Core (ZIC MH002952).

All animals were handled in accordance with guidelines of the Animal Care and Use Committees of the National Institute of Mental Health (NIMH). All efforts were made to minimize the pain and the number of animals used.
1. Adeno-associated virus injections in the hippocampus
NOTE: Wild type male mice of mixed background (B6/129 F1 hybrid, 3 months old) were for stereotaxically injected with&#160;an AAV encoding the M3 muscarinic receptor (hM3Dq) into the hippocampus. During the entire experiment, mice were single-housed, under a regular 12 h light: 12 h dark (T24) cycle, with access to food and water ad libitum.
<ol>
	<li>Before performing stereotaxic surgeries, clean and sterilize the stereotaxic frame and all needed instruments. 
	NOTE: Surgical drapes could be used to maintain a sterile field and reduce mouse&#39;s heat loss.</li>
	<li>Deeply anesthetize the mouse using isoflurane. To do this, first adjust the oxygen flow meter to approximately 1.5 L/ min, and then adjust the isoflurane vaporizer to approximately 3-5% for induction and approximately 1-3% for maintenance.
	<ol>
		<li>To ensure that the animal is fully unconscious, pinch the mouse&#8217;s paw; the animal is properly anesthetized when the flinching response to pinch is absent.</li>
	</ol>
	</li>
	<li>Place the mouse on a heating pad to maintain the stability of the mouse&#8217;s body temperature.</li>
	<li>Shave the top of the head and fix the head of the mouse to the stereotaxic frame. Then, apply ocular protective lubricant on the eyes, clean the surface by scrubbing with povidone-iodine and 70% ethanol, and expose the skull using a sterile scalpel.</li>
	<li>Calibrate the frame to bregma point, then drill at a medial-lateral coordinate of 2.9 mm and an anterior-posterior coordinate -2.7 mm to target the hippocampus. 
	NOTE: If other brain target needs to be injected, determine the desired coordinates for injection using the Paxinos and Franklin mouse atlas16.</li>
	<li>Once the brain is exposed, unilaterally inject 90 nL of the AAV at the dorsal-ventral depth of -3.0 mm in the hippocampus using a microinjector and pulled microcapillary pipettes (Figure 1A). 
	NOTE: See Table of Materials for the titer of AAV used in this experiment. For other brain areas, adjust the AAV volume of injection as needed.</li>
	<li>At the end of the surgical procedure, close the incision with nylon sutures and apply topical antibiotics to the wound site.</li>
	<li>Administer analgesics (buprenorphine, 0.1 mg/kg) systemically immediately following surgery, and 4-6 hours after.</li>
	<li>Beginning 4 weeks post-injection, subject mice to any of the paradigms described in the following section to chronically control neurons expressing the designer excitatory receptor.</li>
</ol>
2. Repetitive CNO delivery using eye-drops
<ol>
	<li>Acclimate the animals to handling by scruffing each mouse 3 min daily for 3-4 days prior to the administration of eye-drops.</li>
	<li>Dissolve Clozapine-N-oxide (CNO, 5 mg) in 1 mL of sterile 0.9% saline solution (stock solution: 5 mg CNO/ mL). Keep the solution refrigerated at 4 &#176;C.</li>
	<li>Weigh each mouse before starting the experiment to determine the amount of CNO to be delivered. Use 1-3 &#956;L drop (per eye) to achieve 1.0 mg CNO/ kg body weight. 
	NOTE: As an example, a 20 g mouse should receive bilateral (2&#160;&#956;L each) eye-drops.</li>
	<li>Deliver the eye-drops during the inactive (light) phase of mice, 2 h before lights turn off (zeitgeber time (ZT) 10). In cases where CNO needs to be delivered during the active (dark) phase of mice, ensure the presence of dim red light for proper animal handling. 
	NOTE: Precautions should be taken to avoid disrupting the circadian (and light/dark) cycle of experimental animals.
	<ol>
		<li>Using a P10 micropipette, load the required amount (1-3 &#956;L) of CNO solution to achieve 1.0 mg CNO/ kg. 
		NOTE: Use a new and sterile pipette tip for each eye-drop. In this set of experiments, bilateral eye-drops of CNO were performed; however, if a lower CNO concentration is required, unilateral eye-drops could be also applied.</li>
		<li>Immobilize the mouse via scruff.</li>
		<li>Slowly expel the solution until a stable droplet forms on the pipette tip.</li>
		<li>Carefully bring the droplet close to the cornea of the mouse&#8217;s eye until the solution is delivered. The pipette tip should never contact the mouse&#8217;s eye.</li>
		<li>Release the mouse, placing it back in its home cage.</li>
	</ol>
	</li>
	<li>Repeat this procedure every day for 5 days. 
	NOTE: This duration can be adjusted as per the experimental requirements.</li>
	<li>For control experiments, use AAV/DREADD-injected mice subjected to sham treatment (eye-drops containing only saline solution), and mice injected with an empty vector (e.g., AAV/mCherry) exposed to the described CNO eye-drops protocol.</li>
</ol>
3. Chronic CNO treatment delivered through drinking water
<ol>
	<li>Make small bottles using 50 mL (plastic) conical tubes and rubber stopper spouts; cover with aluminum foil to avoid any light-mediated effects on CNO stability.</li>
	<li>Three days before starting with the CNO treatment, replace regular water bottles with small bottles, containing 10 mL of regular water, to allow mice to acclimate to them. Secure the bottles to the cages using tape.</li>
	<li>Measure the daily water consumption for each mouse.</li>
	<li>Weigh each mouse before starting the experiment.</li>
	<li>Dissolve Clozapine-N-oxide (CNO, 5 mg) in 1 mL of 0.9% sterile saline solution. Refrigerate the stock solution at 4 &#176;C.</li>
	<li>Use the body weight and the average amount of water consumed to define the concentration of CNO solution to achieve 1.0 mg CNO/ kg (body weight). 
	NOTE: Adult male mice (~20 g body weight) consume ~5 mL of water per day (Figure 2A). Therefore, to achieve a CNO concentration of 1 mg CNO/ kg, 6.4 &#956;L of CNO stock solution should be added to a final volume of 8 mL of water (final concentration: 4 &#956;g CNO/ mL). Thus, the dose of CNO for a 20 g animal that drinks 5 mL water per day results in 1 mg CNO/ kg.</li>
	<li>Determine the optimal CNO dose that displays the maximum effectiveness with minimal CNO concentration by testing a range of concentrations. Perform a dose response analysis to determine the optimal CNO dose for the drinking water method. 
	NOTE: The following CNO doses were tested for this experiment: 1.0 mg/ mL, 0.5 mg/ mL, 0.25 mg/ mL, 0.1 mg/ mL, and saline. 1.0 mg CNO/ kg was first tested, based on i.p. and eye-drops protocols.</li>
	<li>On day 1, fill the bottle with 8 mL of regular water and add the required amount of CNO. 
	NOTE: This amount of water is enough for 24 h of ad libitum water access for an adult male mouse. In case other rodent species are used, first measure the amount of water consumed daily to determine the volume needed.</li>
	<li>Monitor the health of the animals throughout the protocol to ensure that there are no adverse side effects caused by water + CNO consumption.</li>
	<li>After 24 h, replace the bottles with fresh water + CNO solution. Record the volume consumed during the previous day.</li>
	<li>Dispose of the water + CNO solution that was not consumed in waste containers. Discard plastic bottles and replace the rubber stoppers every day, after sanitizing them according to the animal facility guidelines. 
	NOTE: Do not mix aqueous wastes with organic solvents. Contact the Chemical Disposal Service for instructions for storage and pick-up.</li>
	<li>Replace the bottles at the same time every day for 5 days. 
	NOTE: This duration can be adjusted as per the experimental requirements.</li>
	<li>Include control groups, as described in step 2.6.</li>
</ol>
4. Restricted CNO treatment using mice&#8217;s preference for sucrose
<ol>
	<li>3 days before starting with the CNO treatment, place a small bottle containing 10 mL of water + 1% sucrose on the cage, preferably away from the original water bottle. 
	NOTE: Use the same small bottles described in Step 3.1.</li>
	<li>Expose animals to water + 1% sucrose during the last portion of their active phase (ZT 18 &#8211; 24). After this exposure, remove the bottle with water + sucrose from the cage. 
	NOTE: Different time windows of CNO delivery could be used. Additionally, mice could be placed under an inverted light/dark cycle, where the onset of light occurs in the evening hours to facilitate the CNO delivery.</li>
	<li>Measure the daily water + 1% sucrose consumption for each mouse.</li>
	<li>Weigh each mouse before starting the experiment.</li>
	<li>Use the body weight and the average amount of water + 1% sucrose consumed to determine the dose of CNO solution to achieve 1.0 mg CNO/ kg (body weight). 
	NOTE: The optimal CNO dose that displays the maximum effectiveness with minimal CNO concentration should be tested, as explained in step 3.6.</li>
	<li>On day 1, fill bottles with 5 mL of water + 1% sucrose + CNO (1 mg CNO/ Kg) and place them on the cage (always at the same location) during the determined time window.</li>
	<li>At the end of the restricted time window, remove the bottles and measure the amount of water + sucrose + CNO consumed. 
	NOTE: Materials and solutions are sanitized or discarded as previously described in step 3.11.</li>
	<li>Repeat this procedure every day for 5 days. 
	NOTE: This duration can be adjusted as per the experimental requirements.</li>
	<li>Include a control group, as described in step 2.6.</li>
</ol>
5. Data analysis
<ol>
	<li>Perfuse mice intracardially with 4% paraformaldehyde (PFA) either 2 or 6 h after receiving the last repetitive (5th day) CNO eye-drop. When CNO is delivered through drinking water, replace CNO + water with water at the end of the mouse&#8217;s active phase, then perfuse the mouse after either 2 or 6 h post-CNO access. 
	NOTE: If light exposure could affect the c-Fos induction in the area of interest, keep mice in constant darkness during the last day of the experiments, and before the perfusion.</li>
	<li>Carefully dissect the brain out and submerge in 4% PFA solution for 9-12h.</li>
	<li>After PFA fixation, cryoprotect the brain tissue using a 30% sucrose solution (wait until the brain sinks), then section the brain using a cryostat.</li>
	<li>Transfer the coronal brain sections (35 &#956;m) into a solution containing 1x PBS, 10% bovine serum albumin, and 0.3% Triton X-100 for 1 h at room temperature.</li>
	<li>Incubate the brain sections with an anti-c-Fos (1:2500) antibody solution at 4 &#176;C overnight with constant agitation.</li>
	<li>After 3 washes of 5 min each with a solution containing 1x PBS and 0.3% Triton X-100, incubate the samples with an Alexa-conjugated secondary antibody (1:500) solution for 1 h at room temperature away from light and with constant agitation.</li>
	<li>Obtain digital images using a confocal microscope. Assemble and process captured images with a photo editing and analysis software (e.g., Adobe Photoshop).</li>
	<li>For data analysis, outline and measure the AAV-infected area (mCherry(+) cells) using ImageJ software, and quantify the number of c-Fos(+) cells within this region to obtain the number of activated cells per area. Combine the results obtained from 3 separate sections per animal.</li>
</ol>

<ol>
	<li>Park, H. G., Carmel, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Manipulation+of+Neural+Circuits.">Selective Manipulation of Neural Circuits.</a> Neurotherapeutics. 13 (2), 311-324 (2016).</li><li>Muir, J., Lopez, J., Bagot, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+the+depressed+brain:+optogenetic+and+chemogenetic+circuit+interrogation+in+animal+models+of+depression.">Wiring the depressed brain: optogenetic and chemogenetic circuit interrogation in animal models of depression.</a> Neuropsychopharmacology. 1, (2018).</li><li>Wiegert, J. S., Mahn, M., Prigge, M., Printz, Y., Yizhar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Review Silencing Neurons: Tools, Applications, and Experimental Constraints. , (2017).</li><li>Urban, D. J., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+(Designer+Receptors+Exclusively+Activated+by+Designer+Drugs):+Chemogenetic+Tools+with+Therapeutic+Utility.">DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic Tools with Therapeutic Utility.</a> Annual Review of Pharmacology and Toxicology. 55 (1), 399-417 (2015).</li><li>Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+for+Neuroscientists.">DREADDs for Neuroscientists.</a> Neuron. 89 (4), 683-694 (2016).</li><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G+protein-coupled+receptors+potently+activated+by+an+inert+ligand.">Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.</a> Proceedings of the National Academy of Sciences. 104 (12), 5163-5168 (2007).</li><li>Donato, F., Jacobsen, R. I., Moser, M. -. B., Moser, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stellate+cells+drive+maturation+of+the+entorhinal-hippocampal+circuit.">Stellate cells drive maturation of the entorhinal-hippocampal circuit.</a> Science. 355 (6330), (2017).</li><li>Mahler, S. V., 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=Designer+receptors+show+role+for+ventral+pallidum+input+to+ventral+tegmental+area+in+cocaine+seeking.">Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking.</a> Nature Neuroscience. 17 (4), 577-585 (2014).</li><li>Lichtenberg, N. 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=Basolateral+Amygdala+to+Orbitofrontal+Cortex+Projections+Enable+Cue-Triggered+Reward+Expectations.">Basolateral Amygdala to Orbitofrontal Cortex Projections Enable Cue-Triggered Reward Expectations.</a> The Journal of Neuroscience. 37 (35), 8374-8384 (2017).</li><li>Schotman, P., Reith, M. E. A., Gispen, W. H. <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+stressful+procedures+as+ether+anesthesia+and+intracranial+injections+on+amino+acid+incorporation+into+brain+protein.">Effects of stressful procedures as ether anesthesia and intracranial injections on amino acid incorporation into brain protein.</a> Brain Research Bulletin. , (1977).</li><li>Frumberg, D. B., Fernando, M. S., Lee, D. E., Biegon, A., Schiffer, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+behavioral+deficits+following+a+routine+surgical+procedure+in+rats.">Metabolic and behavioral deficits following a routine surgical procedure in rats.</a> Brain Research. , (2007).</li><li>Keenan, W. T., Fernandez, D. C., Shumway, L. J., Zhao, H., Hattar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye-Drops+for+Activation+of+DREADDs.">Eye-Drops for Activation of DREADDs.</a> Frontiers in Neural Circuits. 11, 93 (2017).</li><li>LeGates, T. A., Altimus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+circadian+and+acute+light+responses+in+mice+using+wheel+running+activity.">Measuring circadian and acute light responses in mice using wheel running activity.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Bainier, C., Mateo, M., Felder-Schmittbuhl, M. -. P., Mendoza, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+of+hedonic+drinking+behavior+in+mice.">Circadian rhythms of hedonic drinking behavior in mice.</a> Neuroscience. 349, 229-238 (2017).</li><li>Fernandez, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Affects+Mood+and+Learning+through+Distinct+Retina-Brain+Pathways.">Light Affects Mood and Learning through Distinct Retina-Brain Pathways.</a> Cell. 175 (1), 71-84 (2018).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Paxinos and Franklin&#8217;s The mouse brain in stereotaxic coordinates. , (2019).</li><li>Urban, D. J., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+(designer+receptors+exclusively+activated+by+designer+drugs):+chemogenetic+tools+with+therapeutic+utility.">DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility.</a> Annual Review of Pharmacology and Toxicology. 55, 399-417 (2015).</li><li>Urban, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+The+Behavioral+Program+and+Neuronal+Network+Encoded+by+Dorsal+Raphe+Serotonergic+Neurons.">Elucidation of The Behavioral Program and Neuronal Network Encoded by Dorsal Raphe Serotonergic Neurons.</a> Neuropsychopharmacology official publication of the American College of Neuropsychopharmacology. 41 (5), 1404-1415 (2016).</li><li>Jain, S., Ruiz De Azua, I., Lu, H., White, M. F., Guettier, J. -. M., Wess, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+activation+of+a+designer+G+q-coupled+receptor+improves+&#946;+cell+function.">Chronic activation of a designer G q-coupled receptor improves &#946; cell function.</a> The Journal of Clinical Investigation. 123, (2013).</li><li>MacLaren, D. A. A., 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=Clozapine+N-Oxide+Administration+Produces+Behavioral+Effects+in+Long-Evans+Rats:+Implications+for+Designing+DREADD+Experiments.">Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.</a> eNeuro. 3 (5), (2016).</li><li>Gomez, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemogenetics+revealed:+DREADD+occupancy+and+activation+via+converted+clozapine.">Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.</a> Science. 357 (6350), 503-507 (2017).</li></ol>

The authors have nothing to disclose.

DREADDs have emerged as a popular and effective approach to remotely manipulate neuronal activity17. The design of alternative strategies for CNO delivery will broadly increase the spectrum of options available for specific experimental settings. In addition, non-invasive strategies for the delivery of CNO minimize any potential misinterpretation of results by reducing adverse side effects that can directly impact the animal&#8217;s health. Here, we described two non-invasive strategies for CNO delivery that confer a robust activation of DREADDs (hM3Dq) and offer a wide spectrum of possibilities. Further, we believe that the protocols described here might also be useful for different DREADD variants for neuronal manipulation, including genetically engineered muscarinic or opioid receptors.
CNO delivery using repetitive eye-drops represents a painless alternative to repetitive intraperitoneal CNO injections while preserving the power to precisely control dosage and timing of CNO delivery. Therefore, we recommend using this protocol when repetitive DREADD activation is required. Eye-drops are also the least expensive option for CNO delivery, particularly compared with the protocol using CNO added to the drinking water. CNO delivered through drinking water, on the other hand, confers a chronic and sustained activation of DREADDS, avoiding any mouse handling. It is important to mention that this protocol lacks precise control over the timing of CNO delivery. A third alternative, time-restricted access to a sucrose solution containing CNO, combines advantages of both protocols previously discussed. This strategy is at the same time non-invasive, repetitive and easy to perform. Additionally, it offers a better control of the timing of CNO delivery compared with the 24 h access to water with CNO. A caveat of this approach is that it can only be used during the active phase of animals. We recommend using both strategies involving CNO in drinking water in combination with infrared cameras or a lick-o-meter system to obtain precise temporal information about CNO consumption and, therefore, DREADD activation.
Long-lasting effects conferred by CNO delivered through drinking water were previously reported. We have successfully applied a chronic CNO (5 &#956;g/ mL) treatment during 14 consecutive days15 to evaluate the behavioral consequences of tonic activation of a thalamo-cortical circuit involved in mood control. Alternatively, CNO provided in the drinking water at a concentration of 40 &#8201;mg/L has been used to chronically modulate the activity of serotonergic neurons of the dorsal raphe nucleus18, whereas the function of pancreatic &#946;-cells was controlled using CNO at a concentration of 0.25 mg/mL water19. Combined, these results suggest that different CNO concentrations can be tuned to effectively control DREADDs. Here, we found that different doses of CNO added to drinking water elicited similar c-Fos activation, suggesting that a dose-response analysis should be performed to define the lowest and effective CNO dose required. Recent studies have shown that CNO is not entirely pharmacologically inert20; in addition, it was also demonstrated that the in vivo activation of DREADDs is mediated by the CNO metabolite clozapine, which has several endogenous targets21. Therefore, the authors suggest using subthreshold doses of clozapine, instead of high CNO doses. Although we have not evaluated the effectiveness of clozapine in the methods described, we found that CNO concentration could be reduced without significantly reducing neuronal activation, and therefore, minimizing side effects caused by the CNO-to-clozapine conversion.
In summary, the strategies presented here represent potential schemes for CNO delivery that can be easily adapted to a variety of experimental designs. They were conceived as non-invasive strategies that may be useful for repetitive or chronic CNO-mediated activation of DREADD-controlled neurons, reducing the impact of CNO delivery on animal behavior.

Technical advances in the field of neuroscience have allowed scientists to precisely identify and control the activity of particular neuronal populations1. This has contributed to better understand the basis of neuronal circuits and their impact on animal behavior, as well as, revising established dogmas2,3. Among these novel tools, optogenetic and chemogenetic strategies have had a profound impact not only on the quality of discoveries but also on the way experiments are conceived and designed4. In the present manuscript, we focus on chemogenetic strategies for controlling the activation of neurons via engineered receptor-ligand strategies. Designer receptors exclusively activated by designer drugs (DREADDs) represent one of the most popular chemogenetic tools for the remote control of neuronal activity, as reviewed by Roth 20165. DREADDs utilize modified muscarinic acetylcholine receptors that are specifically activated by an inert ligand, clozapine-N-oxide (CNO)6.
Most studies use CNO administered by intraperitoneal (i.p.) injections, which effectively controls the dosage and timing of engineered receptors activation in an acute fashion. However, when repetitive or chronic DREADD activation is required, the use of multiple i.p. injections become unfeasible. To address this issue, different strategies for the chronic CNO delivery have been reported, including implanted minipumps7 and intracranial cannulas8,9. To different extents, all these strategies cause the animals stress and pain10, and require a surgical intervention that could also have a direct impact on the behavioral responses to be tested11. Here, we describe three non-invasive strategies for the chronic CNO delivery.
For this purpose, mice were stereotaxically injected in the hippocampus with an adeno-associated virus (AAV) encoding an engineered version of the excitatory M3 muscarinic receptor (hM3Dq) that when activated by the ligand CNO leads to the burst-like firing of neurons6. It was previously shown that a single eye-drop containing CNO can effectively elicit a robust activation of DREADD-expressing neurons12. Here we describe a modified method for the repetitive delivery of eye drops. To achieve chronic and sustained control of the designer receptors, we next describe a non-invasive strategy to deliver CNO to mice through the drinking water. Finally, we describe an alternative paradigm for delivering CNO in drinking water during a restricted time window. Mice locomotor activity, as well as drinking behavior and the consumption of sweet caloric solutions, are mostly restricted to the dark portion of the light/dark cycle13,14. Therefore, we adopted a protocol based on the mouse&#8217;s preference for sucrose. By measuring the induction of the immediate-early gene c-Fos in AAV-infected cells, as a readout for neuronal activation12,15, we found that these CNO delivery strategies robustly activate DREADD-controlled neurons over extended durations.

<table><tbody><tr><td>BSA</td><td>Sigma life science</td><td>#A2153-100G</td><td>Lyophilized powder &amp;ge;96% (agarose gel electrophoresis)</td></tr><tr><td>C57BL/6J mice</td><td>The Jackson laboratory</td><td>#000664</td><td>male mice, 3 months old</td></tr><tr><td>Capillaries</td><td>Drummond Scientific Company</td><td>#3-000-203-G/X</td><td>Outer diameter: 1.14 inch</td></tr><tr><td>Clozapine-N-oxide</td><td>Sigma</td><td>#C0832</td><td>5 mg</td></tr><tr><td>Forane</td><td>Baxter</td><td>#NDC 10019-360-60</td><td>Isoflurane, USP</td></tr><tr><td>Microinjector III</td><td>Drummond Scientific Company</td><td>#3-000-207</td><td>Nanoject III - Programmable Nanoliter Injector</td></tr><tr><td>Mounting media</td><td>Invitrogen</td><td>#P36930</td><td>Prolong Gold antifade reagent</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>#15710</td><td>16% aqueous solution (methanol free), 10 mL</td></tr><tr><td>Primary c-Fos Antibody</td><td>Cell signaling technology</td><td>#2250S</td><td>c-Fos (9F6) Rabbit mAb (100&amp;micro;L)</td></tr><tr><td>rAAV5/hSyn-hm3D-mCherry</td><td>UNC Vector Core</td><td></td><td>Titer: ~3 x 10&lt;sup&gt;12&lt;/sup&gt; vg/mL</td></tr><tr><td>rAAV5/hSyn-mCherry</td><td>UNC Vector Core</td><td></td><td>Titer: ~3x10e12 vg/mL</td></tr><tr><td>Secondary Antibody</td><td>Invitrogen</td><td>#A21206</td><td>Alexa Fluor TM 488 Donkey anti-rabbit IgG(H+L), 2mg/ml</td></tr><tr><td>Triton X-100</td><td>americanbio.com</td><td>#AB02025-00100</td><td></td></tr></tbody></table>

non-invasive strategies for chronic manipulation of dreadd-controlled neuronal activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal&#39;s drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

We observed that repetitive CNO delivery using eye-drops elicited a robust induction of c-Fos expression in most infected neurons (Figure 1C), showing that the effectiveness of CNO delivery is sustained during the repetitive exposure. Furthermore, a significant induction of c-Fos was observed in samples collected 2 h after CNO treatment, compared to samples obtained 6 h after CNO exposure (Figures 1D-E), demonstrating that changes induced by CNO are time-dependent.
We then measured the effectiveness of the chronic CNO treatment delivered through drinking water. We observed that the daily consumption of water + CNO was not significantly different compared with the total volume of regular water consumed (Figure 2A). Similarly, the amount of water + 1% sucrose consumed during the night (6 h time window) was not affected by the addition of CNO (Figure 2B). Further, no differences in the daily consumption (5 days) of both water + CNO (Figure 2C) and water + sucrose + CNO (Figure 2D) were found throughout the experiment for all the animals.
Similar to what we found using CNO eye-drops, robust induction of c-Fos was observed after 2 h but not 6 h upon CNO access (Figures 2E-F).
Finally, we measured the dose response of CNO added to drinking water. To do this, mice were exposed to the following CNO doses: 1.0 mg/ mL, 0.5 mg/ mL, 0.25 mg/ mL, 0.1 mg/ mL, saline. In all cases, animals were perfused 2 h after CNO exposure. We found that there is a clear threshold of effectiveness for CNO, where a low CNO dose (0.1 mg/ mL) does not elicit c-Fos activation compared to saline control, whereas higher doses (0.25 mg/ mL, 0.5 mg/mL and 1.0 mg/ mL) induced robust and similar c-Fos induction (Figure 2G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59439/59439fig1.jpg" /> 
Figure 1: Repetitive CNO delivery using eye-drops. (A) AAV/hM3Dq-mCherry was stereotaxically injected in the hippocampus of adult (3 months old) male mice. (B) Four weeks post-injection, CNO was administered using eye-drops once daily for 5 consecutive days. A dose of 1.0 mg CNO/ kg was used. (C) Finally, mice were sacrificed, and brain tissue was tested for c-Fos (green) immunoreactivity in the AAV-infected area (mCherry-positive cells, red). A representative coronal section of the injection site and the CNO-mediated c-Fos activation is shown. (D) The number of c-Fos positive cells in the AAV-infected area was measured in mice that were perfused 2 or 6 h after the last CNO administration. Data are mean &#177; SEM. ***p &#60; 0.001; by Student&#8217;s t-test (n = 2-3 mice). (E) Representative images for the two groups are shown. Scale bar: 100 &#956;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59439/59439fig1large.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/59439/59439fig2.jpg" /> 
Figure 2: Chronic CNO treatment delivered through drinking water. (A) No differences in the total liquid consumption were observed between control (water) or treated (water + CNO, dose: 1.0 mg CNO/ kg) animals. Data are mean &#177; SEM (n = 13-14 mice). (B) Similarly, no significant differences were observed in the volume of water + 1 % sucrose consumed (during a 6 h time window), after adding CNO (1.0 mg/ Kg). Data are mean &#177; SEM (n = 5 mice). (C) Daily consumption of water + CNO (1.0 mg/ kg) for individual mice is shown. No differences in the daily consumption were observed. Data are mean &#177; SEM (n = 5 mice). (D) Daily liquid consumption (during a 6 h time window) of 1% sucrose + CNO (1.0 mg/ kg) for individual mice is shown. No differences in the daily consumption were observed. Data are mean &#177; SEM (n = 3 mice). (E) 2 or 6 h after the last CNO administration, mice were sacrificed, and the number of c-Fos positive cells was quantified in the AAV-infected area. Data are mean &#177; SEM. ***p &#60; 0.001; by Student&#8217;s t-test (n = 5 mice). (F) Brain coronal sections were tested for c-Fos (green) immunoreactivity in the AAV-infected (mCherry-positive cells, red) region. Representative images are shown. (G) Four CNO doses were administered (0.1, 0.25, 0.5, and 1.0 mg CNO/ kg), and the c-Fos induction was measured. Data are mean &#177; SEM. ***p &#60; 0.001; by ANOVA, followed by Tukey&#8217;s test (n = 2 mice). Scale bar: 100 &#956;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59439/59439fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity at JoVE.com

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals&#8217; stress.

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

non-invasive-strategies-for-chronic-manipulation-dreadd-controlled

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.4K Views.  National Institute of Mental Health. DREADD's zijn de meest populaire chemogenetische aanpak voor afstandsbediening van neuronale activiteit. Hier bieden we nieuwe alternatieven voor repetitieve of chronische DREADD controle, gericht op minder invasieve CNO leveringsmethoden. We beschrijven twee strategieën voor de langdurige CNO-levering bij muizen door herhaalde oogdruppels, of chronisch door het drinkwater van het dier.De hier beschreven protocollen zijn voorbeelden van chronische en niet-invasieve opties voor CNO-lev...

Video: Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

Behavior

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

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

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

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

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

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