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, ...

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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Surgery&lt;/u&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Optogenetics&lt;/u&gt;</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 &amp;micro;L Syringe, 26 gauge</td><td>PHD Ultra, Harvard Apparatus</td><td>Model 75 RN SYR</td><td></td></tr><tr><td>Hamilton 5 &amp;micro;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&quot;), Outer diameter 1.09 mm (.043&quot;)</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, &amp;Oslash;105 &amp;micro;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&quot; x 6&quot; 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&quot; 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 &amp;micro;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 &amp;Oslash;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Electrophysiology&lt;/u&gt;</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 &amp;micro;m, 99.95%</td></tr><tr><td>Capillary tubing</td><td>Optronics</td><td>1068150020</td><td>ID: 100.4 &amp;micro;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&amp;uuml;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&amp;ouml;ttechnik, Oberhausen, Germany</td><td>L&amp;ouml;t&amp;ouml;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</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...

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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 ...

optogenetic-entrainment-hippocampal-theta-oscillations-behaving

Research

JoVE Journal

Neuroscience

11.7K Views.  Heinrich Heine University Düsseldorf. We beschrijven het gebruik van optogenetics en elektrofysiologische opnames voor selectieve manipulaties van hippocampal theta oscillaties (5-10 Hz) in gedragend muizen. De werkzaamheid van de entrainment ritme wordt gecontroleerd met behulp van lokale veld potentieel. Een combinatie van opto- en Farmacogenetische inhibitie adressen de efferent uitlezing van hippocampal synchronisatie.

Video: Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

Behavior

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

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 ...

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 ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://app.jove.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp (2018)
This video demonstrates a protocol to modulate theta oscillations in a genetically engineered mouse model with light-sensitive ion channels in its GABAergic medial septum neurons. By delivering light pulses to the hippocampus, the protocol aims to synchronize neuronal activity and generate theta oscillations, which are recorded as local field potentials.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
PV-Cre ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Implanting an Electrode Array and Optic Fiber into the Mouse Hippocampus

Source: Bender, F., et. al., <a href="https://app.jove.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp. (2018)
This video demonstrates the stereotactic implantation of a wire electrode array and an optic fiber in the hippocampus of a mouse model.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Optogenetic Control of Hippocampal Network Dynamics

Source: Manseau, F., et al. <a href="https://app.jove.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017)
This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

	Optogenetic control in the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

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