Name: non-invasive strategies for chronic manipulation of dreadd-controlled neuronal activity
Uploaded: 2019-08-25T15:00:00
Description: Watch this Scientific Journal Video about Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity at JoVE.com

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 t...

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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 &#8805;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 in.</td>
		</tr>
		<tr>
			<td>Clozapine-N-oxide</td>
			<td>Sigma</td>
			<td>#C0832</td>
			<td>5mg</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&#181;l)</td>
		</tr>
		<tr>
			<td>rAAV5/hSyn-hm3D-mCherry</td>
			<td>UNC Vector Core</td>
			<td></td>
			<td>Titer: ~3x10e12 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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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 ...

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