Optogenetic Control of Neural Circuits in the Mongolian Gerbil

Stefan Keplinger,Lars Kunz,Stylianos Michalakis,Benedikt Grothe,Martin Biel,Barbara Beiderbeck

doi:10.3389/fncel.2018.00111

Abstract

The Mongolian gerbil (Meriones unguiculatus) is widely used as a model organism for the human auditory system. Its hearing range is very similar to ours and it uses the same mechanisms for sound localization. The auditory circuits underlying these functions have been characterized. However, important mechanistic details are still under debate. To elucidate these issues, precise and reversible optogenetic manipulation of neuronal activity in this complex circuitry is required. However, genetic and genomic resources for the Mongolian gerbil are poorly developed. Here, we demonstrate a reliable gene delivery system using an AAV8(Y337F)-pseudotyped recombinant adeno-associated virus (AAV) 2-based vector in which the pan-neural human synapsin (hSyn) promoter drives neuron-specific expression of CatCH (Ca2+-permeable channelrhodopsin) or NpHR3.0 (Natronomonas pharaonis halorhodopsin). After stereotactic injection into the gerbil’s auditory brainstem (medial nucleus of the trapezoid body, dorsal nucleus of the lateral lemniscus) and midbrain [inferior colliculus (IC)], we characterized CatCH- and/or NpHR3.0-transduced neurons in acute brain slices by means of whole-cell patch-clamp recordings. As the response properties of optogenetic tools strongly depend on neuronal biophysics, this parameterization is crucial for their in vivo application. In a proof-of-principle experiment in anesthetized gerbils, we observed strong suppression of sound-evoked neural responses in the dorsal nucleus of the lateral lemniscus (DNLL) and IC upon light activation of NpHR3.0. The successful validation of gene delivery and optogenetic tools in the Mongolian gerbil paves the way for future studies of the auditory circuits in this model system.

Highlights

After the indicated expression periods, animals were sacrificed by administering a lethal dose of pentobarbital (IC: 7, 14, 21, and 28 dpi; dorsal nucleus of the lateral lemniscus (DNLL): 28 dpi; medial nucleus of the trapezoid body (MNTB): 21 dpi)
Additional expression of EYFP was detected in the ECIC (n = 3/12) upon injection into the lateral part of the central inferior colliculus (IC) (CIC), and along the injection tract when virus particles were injected into the MNTB (n = 1/3) or into the DNLL (n = 1/3)
A glial scar caused by bolus injection of 250 nl of AAV8(Y733F).human synapsin (hSyn).EYFP into the IC displayed extensive labeling of the S100B marker specific for astrocytes at 14 dpi, but no EYFP expression was detected in these cells (Supplementary Figure S1)

Summary

Introduction

The discovery of channelrhodopsins and light-activated Cl− pumps and their application as highly versatile optogenetic tools for controlling light-stimulated excitation and inhibition of neurons have made many exciting neurobiological discoveries possible over the past decade (Boyden et al, 2005; Nagel et al, 2005; Deisseroth et al, 2006; Zhang et al, 2010; Fenno et al, 2011; Abbreviations: AAV, adeno-associated virus; AP, action potential; CatCH, Ca2+-permeable channelrhodopsin; ChR2, channelrhodopsin 2; D-AP5, D-(-)-2-amino-5-phosphonopentanoic acid; DNLL, dorsal nucleus of the lateral lemniscus; DNQX, 6,7-dinitroquinoxaline-2,3-dione; hSyn, human synapsin; IC, inferior colliculus; LSO, lateral superior olive; MAP2, microtubule-associated protein 2; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; NpHR3.0, Natronomonas pharaonis halorhodopsin 3.0; P, post-natal day; PBS, phosphate-buffered saline; SNR, signal to noise ratio; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.Optogenetics in Mongolian Gerbil BrainYizhar et al, 2011a). Since the application of optogenetics requires gene transfer, most in vivo studies of neural networks have been performed in the mouse, for which methods of gene transfer and genome manipulation are well established. For model organisms such as the Mongolian gerbil (Meriones unguiculatus), the use of optogenetic tools is far less well developed. The Mongolian gerbil is the model of choice for human hearing research because – unlike the case in mice and rats – its audiogram includes most of the human low-frequency hearing range (Ryan, 1976) and age-related hearing loss occurs in both species (Mills et al, 1990). Annotation of the gerbil genome is only on its way (Zorio et al, 2018). and techniques for the generation of transgenic or knock-out strains are difficult because breeding is time consuming and reproductive performance is poor compared to mice (Ågren, 1984; Salo and French, 1989)

Results

Discussion

Conclusion