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>

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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 de...

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.
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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 ...

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13.3K Views.  National Institute of Mental Health. 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’ stress.

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

Simultaneous EEG Monitoring During Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that delivers weak electric currents through the scalp. This constant electric current induces shifts in neuronal membrane excitability, resulting in secondary changes in cortical activity. Although tDCS has most of its neuromodulatory effects on the underlying cortex, tDCS effects can also be observed in distant neural networks. Therefore, concomitant EEG monitoring of the effects of tDCS can provide valuable information on the mechanisms of tDCS. In addition, EEG findings can be an important surrogate marker for the effects of tDCS and thus can be used to optimize its parameters. This combined EEG-tDCS system can also be used for preventive treatment of neurological conditions characterized by abnormal peaks of cortical excitability, such as seizures. Such a system would be the basis of a non-invasive closed-loop device. In this article, we present a novel device that is capable of utilizing tDCS and EEG simultaneously. For that, we describe in a step-by-step fashion the main procedures of the application of this device using schematic figures, tables and video demonstrations. Additionally, we provide a literature review on clinical uses of tDCS and its cortical effects measured by EEG techniques.

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time.

Transcranial  direct current stimulation (tDCS) is a technique that delivers weak electric  currents through the scalp. This constant electric current ...

Behavior

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 ...

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 ...

Non-Invasive Electrical Brain Stimulation Montages for Modulation of Human Motor Function

Non-invasive electrical brain stimulation (NEBS) is used to modulate brain function and behavior, both for research and clinical purposes. In particular, NEBS can be applied transcranially either as direct current stimulation (tDCS) or alternating current stimulation (tACS). These stimulation types exert time-, dose- and in the case of tDCS polarity-specific effects on motor function and skill learning in healthy subjects. Lately, tDCS has been used to augment the therapy of motor disabilities in patients with stroke or movement disorders. This article provides a step-by-step protocol for targeting the primary motor cortex with tDCS and transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range. The setup of two different stimulation montages is explained. In both montages the emitting electrode (the anode for tDCS) is placed on the primary motor cortex of interest. For unilateral motor cortex stimulation the receiving electrode is placed on the contralateral forehead while for bilateral motor cortex stimulation the receiving electrode is placed on the opposite primary motor cortex. The advantages and disadvantages of each montage for the modulation of cortical excitability and motor function including learning are discussed, as well as safety, tolerability and blinding aspects.

Non-invasive electrical brain stimulation can modulate cortical function and behavior, both for research and clinical purposes. This protocol describes different brain stimulation approaches for modulation of the human motor system.

Non-invasive electrical brain stimulation (NEBS) is used to modulate brain function and behavior, both for research and clinical purposes. In particular, ...

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 ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

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 ...

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

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.

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce ...

Neuronavigated tDCS for Enhanced Electrode Placement Accuracy in fMRI Studies

Neuronavigated Focalized Transcranial Direct Current Stimulation Administered During Functional Magnetic Resonance Imaging

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that allows the modulation of the excitability and plasticity of the human brain. Focalized tDCS setups use specific electrode arrangements to constrain the current flow to circumscribed brain regions. However, the effectiveness of focalized tDCS can be compromised by electrode positioning errors on the scalp, resulting in significant reductions of the current dose reaching the target brain regions for tDCS. Electrode placement guided by neuronavigation based on the individual&#39;s head and brain anatomy derived from structural magnetic resonance imaging (MRI) data may be suited to improve positioning accuracy.
This protocol describes the method of neuronavigated electrode placement for a focalized tDCS setup, which is suitable for concurrent administration during functional MRI (fMRI). We also quantify the accuracy of electrode placement and investigate electrode drift in a concurrent tDCS-fMRI experiment. Critical steps involve the optimization of electrode positions based on current modeling that considers the individual&#39;s head and brain anatomy, the implementation of neuronavigated electrode placement on the scalp, and the administration of optimized and focal tDCS during fMRI.
The regional precision of electrode placement is quantified using the Euclidean norm (L2&#160;Norm) to determine deviations of the actual from the intended electrode positions during a concurrent tDCS-fMRI study. Any potential displacement of electrodes (drift) during the experiment is investigated by comparing actual electrode positions before and after the fMRI acquisition. In addition, we directly compare the placement accuracy of neuronavigated tDCS to that achieved by a scalp-based targeting approach (a 10-20 Electroencephalography (EEG) system). These analyses demonstrate superior placement accuracy for neuronavigation compared to scalp-based electrode placement and negligible electrode drift across a 20 min scanning period.

This protocol describes the method of neuronavigated electrode placement for focal, transcranial direct current stimulation (tDCS) administered during functional magnetic resonance imaging (fMRI).

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that allows the modulation of the excitability and plasticity ...