We would like to thank Maria Gorbati for expert help with data analysis and Jennifer Kupferman for comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft (DFG; Exc 257 NeuroCure, TK and AP; Priority Program 1665, 1799/1-1(2), Heisenberg Programme, 1799/2-1, AP), the German-Israeli Foundation for Scientific Research and Development (GIF; I-1326-421.13/2015, TK) and the Human Frontier Science Program (HFSP; RGY0076/2012, TK).

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&#160;(Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen).
1. Viral Injection
<ol>
	<li>During the whole procedure, follow biological safety guidelin...

The authors have nothing to disclose.

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&#39;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, ...

Neuronal activity in mammals is coordinated by network oscillations, which assist information transfer within and between brain regions1,2,3,4. Brain rhythms include oscillations ranging from very slow (&#60; 0.8 Hz) up to ultrafast (&#62; 200 Hz) frequencies. A large body of evidence supports involvement of network oscillations in diverse brain functions, including cognition5,6,7,8,9,10, innate behaviors11,12 as well as neuropsychiatric disorders such as Parkinson&#39;s disease and epilepsy13,14,15. Selective and temporally precise methods for experimental manipulation of network oscillations are therefore essential for the development of physiologically plausible models of synchronization and for establishing causal links with behavior.
Network synchronization is mediated by diverse biological substrates and processes, ranging from molecular identity of ion channels and their kinetics to neuromodulation of excitability and network connectivity. The biological design of rhythm generators16 has been revealed for many brain rhythms, distinct aspects of which (e.g., frequency, amplitude) are often brought about by dynamics of distinct cell types and networks. For instance, inhibitory interneurons targeting the somata of principal cells are the most important players across frequency bands and brain regions17,18, including theta19,20, gamma20,21, and ripple (140-200 Hz)22 oscillations. In turn, phase synchronization of distant cells is ensured by robust feed-forward signaling of pyramidal cells, which resets the firing of interneurons. A crucial parameter of oscillations, the size of the synchronized neuronal population, is closely related to the measured LFP oscillation&#39;s amplitude and, at least for fast oscillations, depends on the excitatory drive onto interneurons2. In contrast, slower oscillations, like delta and theta rhythms, are generated by long-range reentrant loops, formed by cortico-thalamic23,24 and hippocampal-medial septal projections25,26,27, respectively. Oscillations in such circuits are brought about by interactions of signal propagation delays, excitable responses, and their frequency preference in participating cells28,29,30,31,32. Inhibitory projections from GABAergic parvalbumin (PV)-positive cells of the medial septum (MS) to interneurons in the hippocampus25,33, parahippocampal regions and entorhinal cortex26 are essential for the generation of theta oscillations in the medial temporal lobe. Thus, physiological mechanisms of network oscillations and neuronal synchronization can be manipulated using optogenetics with a real-time precision.
Cell type-specific optogenetic manipulations have been applied for studies of the hippocampal and cortical oscillations in vitro34,35,36,37,38 and in vivo30,39,40,41,42,43,44,45, including functional investigations of gamma5,12,36,46,47,48,49,50,51,52 and ripple oscillations40,53,54 and sleep spindles55,56. Recently we expressed a Cre-dependent ChR2 virus in the MS, a key region for the generation of the hippocampal theta rhythm, of PV-Cre mice. Using this preparation, features of the hippocampal theta oscillations (frequency and temporal stability) were controlled by optogenetic stimulation of inhibitory projections of the MS in the hippocampus11. Furthermore, theta-frequency optogenetic stimulation of inhibitory septo-hippocampal projections evoked theta rhythm during awake immobility. The optogenetically entrained theta rhythm displayed properties of spontaneous theta oscillations in the mouse at LFP and neuronal activity levels.
Key features of this protocol include: (1) utilization of an inhibitory pathway that is physiologically critical for spontaneous theta oscillations while avoiding unspecific effects on hippocampal excitability; (2) axonal, i.e., projection-specific stimulation to minimize a direct influence on non-hippocampal MS efferents; (3) local theta-rhythmic light stimulation, ensuring a minimal direct interference with theta-rhythmic septo-hippocampal dynamics and a global bilateral entrainment of theta oscillations; (4) parametric control of theta oscillations frequency and regularity; and (5) quantification of entrainment fidelity with high temporal resolution using LFP to enable quantitative causality analysis in behaving animals. Since this preparation essentially capitalizes on a well-known role of the septo-hippocampal disinhibition in theta generation25,30, it enables robust control over several parameters of theta oscillations in behaving mice. Studies where other less investigated pathways and cell types of the septo-hippocampal circuitry were manipulated38,39,47,49,50,51,52,53,54,55,56,57,58 reveal further mechanisms of the theta rhythm.

<table>
	<tbody>
		<tr>
			<td>PV-Cre mice</td>
			<td>The Jackson Laboratory</td>
			<td>B6;129P2-Pvalbtm1(cre)Arbr/J</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxis</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>Model 963</td>
			<td>Ultra Precise Small Animal Stereotaxic Instrument</td>
		</tr>
		<tr>
			<td>Drill bits, 0.8 mm</td>
			<td>Bijoutil, Allschwil, Switzerland</td>
			<td>49080HM</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01-1 ml syringe</td>
			<td>Braun, Melsungen, Germany</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterican cannulas</td>
			<td>Braun</td>
			<td>26 G, 0.45x25 mm BL/LB</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine and sharp scissors</td>
			<td>Fine Science Tools Inc., Vancouver, Canada</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools Inc.</td>
			<td>11210-10</td>
			<td>Dumont AA - Epoxy Coated Forceps</td>
		</tr>
		<tr>
			<td>Blunt stainless steel scissors</td>
			<td>Fine Science Tools Inc.</td>
			<td>14018-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering station</td>
			<td>Weller Tools GmbH, Besigheim, Germany</td>
			<td>WSD 81</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Rotexmedica GmbH, Trittau, Germany</td>
			<td>PZN: 10823932</td>
			<td>1g Powder for Solution for Infusion</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optogenetics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton pump</td>
			<td>PHD Ultra, Harvard Apparatus, Holliston, MA, USA</td>
			<td>model 703008</td>
			<td>PHD Ultra Syringe Pump with push/pull mechanism</td>
		</tr>
		<tr>
			<td>Hamilton 5 &#181;L Syringe, 26 gauge</td>
			<td>PHD Ultra, Harvard Apparatus</td>
			<td>Model 75 RN SYR</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 5 &#181;L Plunger</td>
			<td>PHD Ultra, Harvard Apparatus</td>
			<td>Model 75 RN SYR</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Fisher Scientific, Pittsburgh, USA</td>
			<td>PE 20</td>
			<td>Inner diameter 0.38 mm (.015&#34;), Outer diameter 1.09 mm (.043&#34;)</td>
		</tr>
		<tr>
			<td>Sterican cannulas</td>
			<td>Braun, Melsungen, Germany</td>
			<td>27 G, 25x0.40 mm, blunt</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision drill/grinder</td>
			<td>Proxxon, Wecker, Luxemburg</td>
			<td>fbs 240/e</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting disks</td>
			<td>Proxxon</td>
			<td>NO 28812</td>
			<td></td>
		</tr>
		<tr>
			<td>Cre dependent channelrhodopsin</td>
			<td>Penn Vector Core, Philadelphia, PA, USA</td>
			<td>AV-1-18917P</td>
			<td>Contruct name: AAV2/1.CAGGS.flex.ChR2.tdTomato, titer: 1.42x1013 vg/ml</td>
		</tr>
		<tr>
			<td>Cam kinase dependent halorhodopsin</td>
			<td>Penn Vector Core</td>
			<td>AV-1-26971P</td>
			<td>Construct name: eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH, titer: 2.08_1012 vg/ml</td>
		</tr>
		<tr>
			<td>Multimode optic fiber</td>
			<td>ThorLabs, Dachau, Germany</td>
			<td>FG105LCA</td>
			<td>0.22 NA, Low-OH, &#216;105 &#181;m Core, 400 - 2400 nm</td>
		</tr>
		<tr>
			<td>Ceramic stick ferrule</td>
			<td>Precision Fiber Products, Milpitas, CA, USA</td>
			<td>CFLC126</td>
			<td>Ceramic LC MM Ferrule, ID 126um</td>
		</tr>
		<tr>
			<td>Polishing paper</td>
			<td>Thorlabs</td>
			<td>LF3D</td>
			<td>6&#34; x 6&#34; Diamond Lapping (Polishing) Sheet</td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Compact Power and Energy Meter Console, Digital 4&#34; LCD</td>
		</tr>
		<tr>
			<td>Multimode fiber optic coupler</td>
			<td>Thorlabs</td>
			<td>FCMM50-50A-FC</td>
			<td>1x2 MM Coupler, 50:50 Split Ratio, 50 &#181;m GI Fibers, FC/PC</td>
		</tr>
		<tr>
			<td>Fiberoptic patch cord</td>
			<td>Thorlabs</td>
			<td>FG105LCA CUSTOM-MUC</td>
			<td>custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule</td>
		</tr>
		<tr>
			<td>Sleeve</td>
			<td>Precision Fiber Products, Milpitas, CA, USA</td>
			<td>ADAL1</td>
			<td>Ceramic Split Mating Sleeve for &#216;1.25 mm (LC/PC) Ferrules</td>
		</tr>
		<tr>
			<td>473 nm DPSS laser</td>
			<td>Laserglow Technologies, Toronto, ON, Canada</td>
			<td>R471005FX</td>
			<td>LRS-0473 Series</td>
		</tr>
		<tr>
			<td>593 nm DPSS laser</td>
			<td>Laserglow Technologies</td>
			<td>R591005FX</td>
			<td>LRS-0594 Series</td>
		</tr>
		<tr>
			<td>MC_Stimulus II</td>
			<td>Multichannel Systems, Reutlingen, Germany</td>
			<td>STG 4004</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance conditioning module</td>
			<td>Neural microTargeting worldwide, Bowdoin, USA</td>
			<td>ICM</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Electrophysiology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wires</td>
			<td>California Fine Wire Company, Grover Beach, CA, USA</td>
			<td>CFW0010954</td>
			<td>40 &#181;m, 99.95%</td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Optronics</td>
			<td>1068150020</td>
			<td>ID: 100.4 &#181;m</td>
		</tr>
		<tr>
			<td>Omnetics nanoconnector</td>
			<td>Omnetics Connector Corporation, Minneapolis, USA</td>
			<td>A79038-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>Bilaney, D&#252;sseldorf, Germany</td>
			<td>00-96x1/16</td>
			<td>stainless-steel</td>
		</tr>
		<tr>
			<td>Silicone probe</td>
			<td>NeuroNexus Technologies, Ann Arbor, MI, USA</td>
			<td>B32</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Neuralynx, Bozeman, Montana USA</td>
			<td>HS-8</td>
			<td>miniature headstage unity gain preamplifiers</td>
		</tr>
		<tr>
			<td>Silver conductive paint</td>
			<td>Conrad electronics, Germany</td>
			<td>530042</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid flux</td>
			<td>Felder GMBH L&#246;ttechnik, Oberhausen, Germany</td>
			<td>L&#246;t&#246;l ST</td>
			<td>DIN EN 29454.1, 3.2.2.A (F-SW 11)</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Neuralynx</td>
			<td>HS-LED-Red-omni-10V</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks, Natick, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MC_Stimulus software</td>
			<td>Multichannel, Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurophysiological Data Manager</td>
			<td></td>
			<td></td>
			<td>NDManager, http://neurosuite.sourceforge.net</td>
		</tr>
		<tr>
			<td>Klusters</td>
			<td></td>
			<td></td>
			<td>http://neurosuite.sourceforge.net, Hazan et al., 2006</td>
		</tr>
		<tr>
			<td>Software of the recording system</td>
			<td>Neuralynx</td>
			<td>Cheetah</td>
			<td>https://neuralynx.com/software/cheetah</td>
		</tr>
		<tr>
			<td>Multi-channel data analysis software</td>
			<td>Cambridge Electronic Design Limited, Cambridge, GB</td>
			<td>Spike2</td>
			<td></td>
		</tr>
	</tbody>
</table>

optogenetic entrainment of hippocampal theta oscillations in behaving mice

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.

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...

Watch this Scientific Journal Video about Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice at JoVE.com

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

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.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

This method can help answer key questions in the neurophysiology field, such as causal role of theta oscillation in special navigation and associated behaviors. The main advantage of this technique is that it enables a real-time control over the frequency and regularity of hippocampal theta oscillations in behaving mice. Demonstrating the procedure will be Dr.Franziska Bender, a former grad student in my laboratory.Begin with the fully anesthetized mouse secured in the stereotaxic frame. Drill a hole above the medial septum. Submerge the tip of the injection needle in a droplet of virus on a piece of paraffin film and carefully draw up the virus at about 500 nanoliters per minute while observing the level of the fluid.To prevent air from entering the injection needle, stop withdrawal before the virus is entirely taken up. Clean the needle with a paper tissue. Check that the virus and oil are separated by an air bubble and mark the level of the virus on the tube.Position the needle above the craniotomy and slowly insert it into the brain at the first injection point. Inject 450 nanoliters of the virus at a rate of 100 to 150 nanoliters per minute. Following the injection, wait for 10 minutes.After 10 minutes carefully move the needle up 0.1 millimeters and wait another five minutes. Use a micro stripper to strip 125 micrometers of cladding from a multi-mode optic fiber while the fiber is still attached to the fiber spool. Then, use a diamond knife to cut the fiber to a length of approximately two to three centimeters.Insert the optic fiber into a zirconia ceramic stick ferrule with an internal diameter of 126 microns. Approximately 0.5 to one millimeter of the fiber should protrude from the convex side of the ferrule. Using a needle, apply one drop of epoxy glue to both ends of the ferrule, but not onto the sides of the ferrule.After drying use diamond lapping fiber polishing film with three micron grits to polish the convex side of the ferrule. To prepare the tungsten wire array, first glue several 100 micron silica tube guides to the sticky side of a piece of tape. Then use forceps to thread formvar insulated 45 micron tungsten wires through the guide tubes.Next use a scalpel to strip the insulation from both ends of six enamel insulated fine copper binding wires of approximately five millimeters long. Then strip the insulation from both ends of a grounding wire of approximately two to three centimeters long. Solder each stripped wire to the nanoconnector pins.Connect each bonding wire to one tungsten wire using one drop of silver conductive paint. After drying, apply a small amount of cement to cover the copper wires. Avoid applying cement to the upper part of the nanoconnector.After allowing the cement to dry for at least 30 minutes, use blunt stainless steel scissors to cut the tungsten wires at a five to 20 degree angle. Begin by drilling four holes of 0.8 mm diameter in the clean skull of the anesthetized animal. drill two holes rostral to the coronal suture, and two above the cerebellum.Next, place stainless steel bone screws for grounding and stabilization of the implant. Position the ground screw connected to a copper wire above the cerebellum. Cover the ground screw completely with cement to prevent muscle artifacts during the electrophysiological recordings.Build a cment ring connecting all screws. Perform a craniotomy above the implantation site, then apply approximately one microliter of sterile saline on the surface of the brain tissue. Use the stereotax to slowly lower the wire array into the craniotomy.Then apply approximately five microliters of warm liquid wax above the implantation site to protect the brain tissue. Apply cement around the wire array and cover the skull with cement. Apply one drop of flux to the pre soldered ground wire or the pre soldered wire connected to the ground screw, and fuse the wires using a soldering machine.Lastly, cover the entire ground wire with cement. Check the light output from the patch cord. If the transmission rate of the implanted fiber was 50%ensure that the light output at the tip of the patch cord is 20 milliwatts.Connect the head stage preamplifier to the implanted connector, and connect the fiber optic patch cord to the implanted hippocampal fiber for optogenetic stimulation. Record the baseline behavior for a duration appropriate for the parameters being measured. Open the software to control the stimulus generator.Click file, open, and select the protocol file of choice. Click download and start to initiate the light stimulation. Observe control of the theta rhythm by the light pulses.Hippocampal local field potential during spontaneous theta oscillations is seen here. Note the gamma envelopes, an indicator of physiological theta rhythm. Hippocampal local field potential at seven hertz during optogenetic entrainment is seen here.Blue stripes indicate the time windows of light application. Note the phase reset by the light pulse indicated here by an arrow. Hippocampal local field potential at 10 hertz reflects optogenetic entrainment.Note phase-locked gamma envelopes and the phase reversal between stratham orians and stratum radiatum is also maintained during entrainment. Once mastered, this technique can be done in four hours of experiment time, plus six weeks for viral expression. While attempting this procedure, it's essential to remember to ensure efficient viral transduction.Properly connect optic fiber implant to the patch cord, and obtain good quality of spontaneous local field potential theta oscillations.

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11.6K Views.  Heinrich Heine University Düsseldorf. This method can help answer key questions in the neurophysiology field, such as causal role of theta oscillation in special navigation and associated behaviors. The main advantage of this technique is that it enables a real-time control over the frequency and regularity of hippocampal theta oscillations in behaving mice. Demonstrating the procedure will be Dr.Franziska Bender, a former grad student in my laboratory.Begin with the fully anesthetized mouse secured in the stereotaxic fram...