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

Summary

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.

Abstract

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'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.

Introduction

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 dogmas^2,3. 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 ....

Protocol

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 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 wate....

Results

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.

Discussion

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’s health. Here, we described two non-invasive strategies for CNO de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Sigma life science	#A2153-100G	Lyophilized powder ≥96% (agarose gel electrophoresis)
C57BL/6J mice	The Jackson laboratory	#000664	male mice, 3 months old
Capillaries	Drummond Scientific Company	#3-000-203-G/X	Outer diameter: 1.14 inch
Clozapine-N-oxide	Sigma	#C0832	5 mg
Forane	Baxter	#NDC 10019-360-60	Isoflurane, USP
Microinjector III	Drummond Scientific Company	#3-000-207	Nanoject III - Programmable Nanoliter Injector
Mounting media	Invitrogen	#P36930	Prolong Gold antifade reagent
Paraformaldehyde	Electron Microscopy Sciences	#15710	16% aqueous solution (methanol free), 10 mL
Primary c-Fos Antibody	Cell signaling technology	#2250S	c-Fos (9F6) Rabbit mAb (100µL)
rAAV5/hSyn-hm3D-mCherry	UNC Vector Core		Titer: ~3 x 1012 vg/mL
rAAV5/hSyn-mCherry	UNC Vector Core		Titer: ~3x10e12 vg/mL
Secondary Antibody	Invitrogen	#A21206	Alexa Fluor TM 488 Donkey anti-rabbit IgG(H+L), 2mg/ml
Triton X-100	americanbio.com	#AB02025-00100