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).

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, (3) application of an optimal amount of light at theta frequencies, (4) post hoc quantification of the entrainment fidelity, and (5) control of optoelectrical artifacts.
The main caveat of the first point is a secure virus injection sparing the medially located venous plexus. Suboptimal surgical execution of this step can decrease the success rate of injections and potentially delay the acquisition of results. Kinetics of an opsin should be considered, ChR2 (activation time: 2 ms, inactivation time: 9 ms65). The second point demands control of the light transmission fidelity and impedance of recording electrodes before implantation, and usually benefits from timely fabrication of additional implants.
The third consideration is common for optogenetics aiming at large scale circuit manipulations, and involves light sources and optical interconnections, which deliver via a 100 &#181;m fiber light power in the brain of 5-15 mW. For each mouse and before each recording, a test recording can be performed to set the light output to the optimal intensity for the experiment. The light output should be high enough to activate a sufficient number of projections to allow reliable entrainment of theta oscillations, but not too high, in order to prevent thermally-evoked responses and tissue damage.
The forth aspect regards the synchronization of light pulses and LFP data, achieved with the highest precision by sampling both data types via the same AD converter. Synchronized time stamps are particularly required for other potential applications aiming at studies of neuronal discharge and LFP. Entrainment efficacy can vary between and within stimulation epochs; this most likely occurs because of rhythmicity interference set by optogenetic stimulation with intrinsic- and/or sensory driven signals, due to multiple generators of the theta rhythm16,66,67,68. Quantification of the instantaneous fidelity of this highly dynamic optogenetic manipulation69 is therefore extremely helpful for causal inference, i.e., for revealing relationship between an effect magnitude (e.g., change in behavior) and the momentary efficacy of the optogenetic control of selected synchronization aspects. Importantly, the multiple oscillation parameters may be influenced by optogenetic stimulation, e.g., entrainment mediates not only frequency locking but also a more regular amplitude of theta oscillations11.
Fifth, the optogenetic stimulation is often associated with optoelectrical artifacts, evoked in metal electrodes by the photoelectrochemical effect. Their degree depends on the electrode material, proximity of the electrode tip to the optic fiber, and light power. In the experiments described here, optoelectrical artifacts can be avoided by increasing the distance between the fiber and the recording electrode, close positioning of which is not essential for theta oscillation monitoring. Optoelectrical artifacts display consistent shape in time and between channels and, therefore, during spike sorting they are typically grouped in a distinct cluster, and do not overlap with recorded neurons70. At the same time, a small fraction of action potentials, waveforms of which are altered by artifacts, are not grouped by sorting algorithms with other spikes fired by a neuron. Laminar LFP profiles obtained using various electrode configurations, including wire arrays (Figure 2B) and linear silicone probes, enable a clear differentiation of artifacts and true LFP patterns, based on the constant phase of the former and physiological laminar phase offsets of the latter. Baseline recordings prior to stimulation enable exploration of characteristic theta features such as typical laminar phase profiles.
Optogenetic control of theta oscillations leads to theta entrainment in all layers of the hippocampal CA1 layer and even to contralateral entrainment. This should be taken to account if an experiment is aimed at a local stimulation (if the effects of a stimulation of a confined subregion or a subpopulation are aimed to be studied). On the other hand, the spatially limited stimulation of hippocampal projections of MS PV+ cells presented here enables a less deterministic manipulation of theta oscillations, i.e., entrainment, than does somatic optogenetic stimulation of MS PV+ cells recently applied in anaesthetized mice71. The latter somatic stimulation consistently results in frequencies of hippocampal oscillation up to 40 Hz and thus represents the case of a highly reliable rhythmic pacing. In contrast, the axonal stimulation presented here is effective preferentially in the theta frequency band (Figure 3C), and therefore is more similar to the entrainment, possibly via the Kuramoto transition72, i.e., working at frequencies close to the ones present at the stimulation onset spontaneous theta oscillations.
While the optogenetic preparation described here enables manipulation of theta oscillation features, somatic optogenetic inhibition of MS inhibitory neurons have been applied for efficient inhibition of theta oscillations during REM sleep73. Integration of the two techniques using optogenetic actuators for opposing control of neuronal excitability (e.g., 12) can potentially enable bidirectional control of the theta rhythm in the same animal for further studies of causal connections between network oscillations and various aspects of behavior.

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 could be more or less efficiently entrained by the optogenetic stimulation (Figure 3A), the efficacy of which was computed for each recording epoch as a relative theta LFP power around the stimulation frequency, i.e., entrainment fidelity (Figure 3B). Entrainment fidelity above 0.3, i.e., higher than in the spontaneous light-off recordings, was observed in approximately 80% of the recording epochs. Optostimulation at non-theta frequencies was less effective (Figure 3C).
Explicit i.e., parametric manipulation of theta oscillations frequency is accompanied by emergent changes of theta regularity: the temporal regularity of amplitude and frequency of the theta oscillations was increased during epochs with high entrainment fidelity. The stability of oscillations can also be regulated parametrically by applying trains of light pulses, periods of which follow Gaussian distributions with different dispersions (Figure 4).
Optogenetic control over the oscillations frequency eliminated the correlation between theta frequency and running speed, in agreement with the frequency control via the MS by ascending afferents during movement (Figure 5A). Optostimulation also induced theta oscillations during immobility (Figure 5B). The preferential firing phases recorded in the CA1 area in putative pyramidal cells and interneurons were unchanged relative to the optogenetically entrained theta oscillation when compared to spontaneous theta (Figure 6).
To study the contribution of the hippocampus to the lateral septum pathway in theta-mediated regulation of locomotion, we optogenetically inhibited this pathway. Halorhodopsin (eNpHR3.0) was bilaterally expressed in hippocampal pyramidal cells (Figure 7A), whereas ChR2 was expressed in MS GABAergic cells as above and theta oscillations were optogenetically entrained (Figure 7B). The theta entrainment reduced variability of running speed but not when the hippocampus to the LS pathway was inhibited (Figure 7C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57349/57349fig1v2.jpg" /> 
Figure 1: Illustration of optic fibers, electrodes and surgery. (A) Illustration of an optic fiber. (B) Illustration of a wire array glued to an optic fiber for the recording of hippocampal LFP during entrainment of hippocampal theta oscillations. (C) For recording of the hippocampal cellular activity, a silicone probe is mounted on a microdrive. (D) Miniature screws are positioned on the skull. Copper wires are presoldered to the ground and reference screw before positioning them above the cerebellum. (E) Cement is applied to cover and connect the screws. The upper blue circle indicates where the craniotomy was performed for the implantation of the silicone probe. Lower blue circle indicates where the craniotomy was performed for the implantation of the optic fiber in the hippocampus. (F) One optic fiber is implanted in a caudal-rostral angle to target the hippocampal CA1 region. A second fiber can be implanted in the medial septum if stimulation of cell somata is desired (optional). (G) The silicone probe is lowered to just above the hippocampal CA1 area. (H) The borders of the microdrive and connector are cemented to the implant and ground, and the reference wires are soldered. (I) Copper mesh is constructed to surround the implant and serve as a Faraday cage. <a href="/files/ftp_upload/57349/57349fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57349/57349fig2.jpg" /> 
Figure 2: Preparation for optogenetic hippocampal theta entrainment. (A) ChR2 was expressed in PV+ medial septal cells in PV-Cre mice (upper scheme). Bright fluorescence in MS (1, 2) confirms successful construct expression in somata. MS fibers project via fornix (f) and fimbria (fi) to the hippocampus (3-6); aca: anterior commissure; anterior part. HDB: nucleus of the horizontal limb of the diagonal band; Or: stratum oriens. The optic fiber for optogenetic stimulation with blue light is implanted above the pyramidal layer of hippocampal area CA1 (lower scheme). Scale bars: 500 &#181;m (images 1, 3, 4) and 50 &#181;m (images 2, 5, 6). (B) Hippocampal LFP during spontaneous theta oscillations (left) and 7 Hz (middle) or 10 Hz (right) optogenetic entrainment. Blue stripes indicate the time windows of light application. Note the phase reset by the light pulse indicated by an arrow. Note gamma envelopes during spontaneous and entrained theta, an indicator of physiological theta rhythm. Phase reversal between stratum oriens (str. or.) and stratum radiatum (str. rad.) is also maintained during entrainment. (C) Entrainment is reliable during ipsilateral (upper plots), as well as contralateral (lower plots) optogenetic stimulation. Schemes illustrate positions of fibers in respect to electrodes positions. Example LFP traces during theta and application of light pulses are shown in the middle. On the right, power spectra of hippocampal LFP during ipsi- and contralateral stimulation color-coded according to stimulation frequency. This figure has been modified from ref. 11. <a href="/files/ftp_upload/57349/57349fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57349/57349fig3.jpg" /> 
Figure 3: Fidelity of optogenetic hippocampal theta entrainment. (A) Example hippocampal LFP traces during low and high entrainment fidelity. (B) Power spectral densities of 10 s epochs during spontaneous theta, with rows ordered according to leading theta frequency (left), and during 7 Hz (middle) and 10 Hz (right) optogenetic stimulation, with rows ordered according to entrainment fidelity. Respective example power spectra (indicated by an arrow) are plotted above. Note reliable entrainment fidelity across epochs. On the right, the cumulative probability of entrainment fidelity for theta frequencies is shown. (C) Entrainment requires theta rhythmic stimulation. Hippocampal network activity can be successfully entrained using frequencies between 6-12 Hz. At lower frequencies (e.g., 2 or 4 Hz) or higher frequencies (e.g., 20 Hz) entrainment is not reliable. This figure has been modified from ref. 11. <a href="/files/ftp_upload/57349/57349fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57349/57349fig4.jpg" /> 
Figure 4: Parametric manipulation of theta oscillations regularity. (A) Stimulation was applied at varying frequencies within the theta range with a mean frequency of 7.8 Hz following a Gaussian distribution. The standard deviation of the inter-pulse intervals was increased across protocols from &#963; = 3.19 to &#963; = 15.09. In total, 11 protocols were generated and applied, each with a total duration of the stimulation epoch of 1 min. Of those, the probability distribution of 5 protocols are shown on the left of the figure. The power spectral densities within a range of 1-14 Hz of the hippocampal LFP during application of the respective protocols are plotted in the middle of the figure. The probabilities of the theta periods during application of the respective protocols are illustrated on the right. (B) The variance of the applied inter-pulse intervals determined the variance of the concurrent theta period (Pearson&#39;s r = 0.94, p = 0.0002). (C) The relationship between the theta amplitude variability and the inter-pulse interval (Pearson&#39;s r = 0.61, p = 0.08). This figure has been modified from ref. 70. <a href="/files/ftp_upload/57349/57349fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57349/57349fig5.jpg" /> 
Figure 5: Optogenetic theta rhythmic entrainment determines hippocampal LFP during behavior. (A) Optogenetic stimulation frequency determined the theta frequency during locomotion. Hence, speed-related afferents do not impact hippocampal theta frequency, and as a consequence, speed is not correlated with theta frequency (blue) as it is during spontaneous theta (black). Data are presented as mean &#177; s.e.m. (B) During quiet wakefulness, the hippocampal theta can be elicited in the absence of movement. Hippocampal LFP traces before and during successful entrainment are shown above, and example speed traces recorded during entrainment are shown below (the red trace corresponds to the hippocampal LFP trace depicted above). Blue stripes mark the time windows of light stimulation pulses. This figure has been modified from ref. 11. <a href="/files/ftp_upload/57349/57349fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57349/57349fig6.jpg" /> 
Figure 6: Hippocampal cellular activity during theta entrainment. (A) Cellular activity was recorded using silicone probes (scheme). Single interneurons and pyramidal cells were isolated and identified according to their respective waveform. Shown here is the average waveform (middle) and auto-correlogram of an example isolated pyramidal cell. (B) Preferred discharge phase of pyramidal cells (Pyr) was not different during spontaneous (in black, n = 29 neurons) and optogenetically entrained (in blue, n = 30) theta (p = 0.79). (C) Shown here is the auto-correlogram (left) and preferential firing phase of a fast-firing interneuron during spontaneous and optogenetically entrained theta. Below the corresponding hippocampal LFP rhythm during spontaneous (left) and entrained (right) theta. (D) Preferred discharge phase of fast-firing interneurons was not different during spontaneous (in black) and optogenetically entrained (in blue, n = 28 neurons) theta (p = 0.97). Average auto-correlogram is shown on the left. (E) Average auto-correlogram str. oriens cells. (F) Preferred discharge phase of str. oriens interneurons was not different during spontaneous (black) and optogenetically entrained (blue, n = 10 neurons) theta (p = 0.56). Histograms of preferred discharge phases are shown on the right. (G) Average firing rates were not affected by theta entrainment in pyramidal cells (p = 0.98), fast-firing interneurons (p = 0.96) or str. oriens interneurons (p = 0.85). This figure has been modified from ref. 11. <a href="/files/ftp_upload/57349/57349fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57349/57349fig7v2.jpg" /> 
Figure 7: Combination of hippocampal theta entrainment and optogenetic inhibition of the hippocampal subcortical output through the LS. (A) eNpHR3.0 (halorhodopsin) was expressed in hippocampal pyramidal cells (upper scheme). Successful expression of the construct was confirmed by bright fluorescence in somata in the hippocampus (upper images) and axons in the LS (lower images). Optic fibers were implanted bilaterally above the LS (lower scheme). Scale bars: 500 &#181;m (images on the left), 50 &#181;m (images on the right). (B) Hippocampal theta is successfully entrained during inhibition of the hippocampus to LS pathway. Here shown are power spectral densities for 9 Hz blue light stimulation during output inhibition. (C) Inhibition of the major hippocampal subcortical output pathway prevents effects of hippocampal theta entrainment on speed. Here shown is decrease in speed variability upon optogenetic entrainment (white bar with blue borders), with is absent upon simultaneous inhibition of the hippocampus to LS pathway (yellow bar with blue borders). Respective average baseline speed is shown on the left. This figure has been modified from ref. 11. <a href="/files/ftp_upload/57349/57349fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.6K Views.  Heinrich Heine University Düsseldorf. 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.

Video: Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...