Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Summary

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Abstract

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

Introduction

Neuronal activity in mammals is coordinated by network oscillations, which assist information transfer within and between brain regions^1,2,3,4. Brain rhythms include oscillations ranging from very slow (< 0.8 Hz) up to ultrafast (> 200 Hz) frequencies. A large body of evidence supports involvement of network oscillations in diverse brain functions, including cognition^5,6,7,8,9

Protocol

PV-Cre knock-in male mice⁵⁹, 10-25 weeks old, were used. Mice were housed under standard conditions in the animal facility and kept on a 12 h light/dark cycle. All procedures were performed in accordance with national and international guidelines, and were approved by the local health authorities (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen).

1. Viral Injection

1. During the whole procedure, follow biological safety guidelines⁶⁰. Wear a lab coat, a surgical mask, a hairnet, and two pairs of gloves.
2. Cut tubing with a sterile scalpel or sci....

Results

Targeting of ChR2 to GABAergic cells in the MS as described in section 1 is illustrated in Figure 2A. Optogenetic stimulation of axons of MS GABAergic cells in the dorsal hippocampus via an optic fiber which is implanted above the CA1 area entrains theta oscillations at the frequency of the stimulus in the ipsilateral (Figure 2B) as well as contralateral hemisphere (Figure 2C). Theta oscillations cou.......

Discussion

Here we presented a widely accessible methodology to entrain and elicit hippocampal theta oscillations in the behaving animal. This approach can be useful for studies of theta rhythm's functions in information processing and behavior. Critical aspects of this method include: (1) choice of the opsin and targeting of ChR2 to axons of MS cells in the hippocampus, (2) robust optical and electrical features of implanted optic fiber-wire array assemblies to ensure continuous stimulation and LFP recording in behaving mice, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PV-Cre mice	The Jackson Laboratory	B6;129P2-Pvalbtm1(cre)Arbr/J
Name	Company	Catalog Number	Comments
Surgery
Stereotaxis	David Kopf Instruments, Tujunga, CA, USA	Model 963	Ultra Precise Small Animal Stereotaxic Instrument
Drill bits, 0.8 mm	Bijoutil, Allschwil, Switzerland	49080HM
0.01-1 ml syringe	Braun, Melsungen, Germany	9161406V
Sterican cannulas	Braun	26 G, 0.45x25 mm BL/LB
Fine and sharp scissors	Fine Science Tools Inc., Vancouver, Canada	14060-09
Forceps	Fine Science Tools Inc.	11210-10	Dumont AA - Epoxy Coated Forceps
Blunt stainless steel scissors	Fine Science Tools Inc.	14018-14
Soldering station	Weller Tools GmbH, Besigheim, Germany	WSD 81
Erythromycin	Rotexmedica GmbH, Trittau, Germany	PZN: 10823932	1g Powder for Solution for Infusion
Name	Company	Catalog Number	Comments
Optogenetics
Hamilton pump	PHD Ultra, Harvard Apparatus, Holliston, MA, USA	model 703008	PHD Ultra Syringe Pump with push/pull mechanism
Hamilton 5 µL Syringe, 26 gauge	PHD Ultra, Harvard Apparatus	Model 75 RN SYR
Hamilton 5 µL Plunger	PHD Ultra, Harvard Apparatus	Model 75 RN SYR
Tubing	Fisher Scientific, Pittsburgh, USA	PE 20	Inner diameter 0.38 mm (.015"), Outer diameter 1.09 mm (.043")
Sterican cannulas	Braun, Melsungen, Germany	27 G, 25x0.40 mm, blunt
Precision drill/grinder	Proxxon, Wecker, Luxemburg	fbs 240/e
Cutting disks	Proxxon	NO 28812
Cre dependent channelrhodopsin	Penn Vector Core, Philadelphia, PA, USA	AV-1-18917P	Contruct name: AAV2/1.CAGGS.flex.ChR2.tdTomato, titer: 1.42x1013 vg/ml
Cam kinase dependent halorhodopsin	Penn Vector Core	AV-1-26971P	Construct name: eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH, titer: 2.08_1012 vg/ml
Multimode optic fiber	ThorLabs, Dachau, Germany	FG105LCA	0.22 NA, Low-OH, Ø105 µm Core, 400 - 2400 nm
Ceramic stick ferrule	Precision Fiber Products, Milpitas, CA, USA	CFLC126	Ceramic LC MM Ferrule, ID 126um
Polishing paper	Thorlabs	LF3D	6" x 6" Diamond Lapping (Polishing) Sheet
Power meter	Thorlabs	PM100D	Compact Power and Energy Meter Console, Digital 4" LCD
Multimode fiber optic coupler	Thorlabs	FCMM50-50A-FC	1x2 MM Coupler, 50:50 Split Ratio, 50 µm GI Fibers, FC/PC
Fiberoptic patch cord	Thorlabs	FG105LCA CUSTOM-MUC	custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule
Sleeve	Precision Fiber Products, Milpitas, CA, USA	ADAL1	Ceramic Split Mating Sleeve for Ø1.25 mm (LC/PC) Ferrules
473 nm DPSS laser	Laserglow Technologies, Toronto, ON, Canada	R471005FX	LRS-0473 Series
593 nm DPSS laser	Laserglow Technologies	R591005FX	LRS-0594 Series
MC_Stimulus II	Multichannel Systems, Reutlingen, Germany	STG 4004
Impedance conditioning module	Neural microTargeting worldwide, Bowdoin, USA	ICM
Name	Company	Catalog Number	Comments
Electrophysiology
Tungsten wires	California Fine Wire Company, Grover Beach, CA, USA	CFW0010954	40 µm, 99.95%
Capillary tubing	Optronics	1068150020	ID: 100.4 µm
Omnetics nanoconnector	Omnetics Connector Corporation, Minneapolis, USA	A79038-001
Screws	Bilaney, Düsseldorf, Germany	00-96x1/16	stainless-steel
Silicone probe	NeuroNexus Technologies, Ann Arbor, MI, USA	B32
Headstage	Neuralynx, Bozeman, Montana USA	HS-8	miniature headstage unity gain preamplifiers
Silver conductive paint	Conrad electronics, Germany	530042
Liquid flux	Felder GMBH Löttechnik, Oberhausen, Germany	Lötöl ST	DIN EN 29454.1, 3.2.2.A (F-SW 11)
LED	Neuralynx	HS-LED-Red-omni-10V
Name	Company	Catalog Number	Comments
Software
MATLAB	Mathworks, Natick, MA, USA
MC_Stimulus software	Multichannel, Systems
Neurophysiological Data Manager			NDManager, http://neurosuite.sourceforge.net
Klusters			http://neurosuite.sourceforge.net, Hazan et al., 2006
Software of the recording system	Neuralynx	Cheetah	https://neuralynx.com/software/cheetah
Multi-channel data analysis software	Cambridge Electronic Design Limited, Cambridge, GB	Spike2