This work is supported by Wellcome grant 206401/Z/17/Z. We would like to thank Zafar Bashir for his expert mentorship and Dr. Clair Booth for technical assistance and comments on the manuscript.

<ol>
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The authors have nothing to disclose.

The protocol presented here describes a method to explore highly specific long-range synaptic projections using a combination of stereotaxic surgery to deliver AAVs encoding optogenetic constructs, and electrophysiology in acute brain slices (Figure 1). Together these techniques offer tools to characterize the physiology and plasticity of brain circuitry with high precision in long-range and anatomically diffuse pathways that were previously inaccessible using traditional, non-specific, elec...

Understanding the physiology of synapses and how they undergo plastic changes is fundamental for understanding how brain networks function in the healthy brain1, and how they malfunction in brain disorders. The use of acute ex vivo brain slices allows for the recording of the electrical activity of synapses from single neurons with a high signal-to-noise ratio using whole-cell patch-clamp recordings. Control of membrane potential and straightforward pharmacological manipulation allows isolation of receptor subtypes. These recordings can be made with exquisite specificity to identify the post-synaptic neuron, including laminar and sub-regional position2, cellular morphology3, presence of molecular markers4, its afferent projections5, or even if it was recently active6.
Achieving specificity of pre-synaptic inputs is, however, somewhat more challenging. The conventional method has used stimulation electrodes to excite the axons which run in a particular lamina. An example of this is in the hippocampus where local stimulation in the stratum radiatum activates synapses that project from the CA3 to the CA1 subfield7. In this instance, presynaptic specificity is achieved as CA3 input represents the sole excitatory input located within stratum radiatum which projects to CA1 pyramidal cells8. This high degree of input specificity achievable with conventional electrical presynaptic activation of CA3-CA1 axons is, however, an exception which is reflected in the intense study that this synapse has been subject to. In other brain regions, axons from multiple afferent pathways co-exist in the same lamina, for example, in layer 1 of neocortex9, thus rendering input-specific presynaptic stimulation impossible with conventional stimulating electrodes. This is problematic as different synaptic inputs may have divergent physiological properties; therefore, their co-stimulation may lead to mischaracterization of synaptic physiology.
The advent of optogenetics, the genetic encoding of photosensitive membrane proteins (opsins) such as channelrhodopsin-2 (ChR2), has allowed a vast expansion of possibilities for studying isolated synaptic projections between brain regions10,11. Here we describe a generalizable and low-cost solution to studying long-range synaptic physiology and plasticity. The optogenetic constructs are delivered in a highly specific manner using viral vectors allowing for extremely precise control of the pre-synaptic region of interest. Efferent projections will express the light-activated channel allowing for activation of these fibers in a target region. Thus, long-range, anatomically diffuse pathways that cannot be independently activated by traditional, non-specific, electrical stimulation can be studied.
We describe, as an example pathway, transduction of medial prefrontal cortex (mPFC) with adeno-associated viruses (AAVs) encoding excitatory cation-channel opsins. We then describe the preparation of acute slices from lateral entorhinal cortex (LEC), patch-clamp recordings from layer 5 LEC pyramidal neurons, and light-evoked activation of glutamatergic mPFC-LEC projections (Figure 1). We also describe the histological assessment of the injection site to confirm the location of the pre-synaptic region of interest and identification of post-synaptic cell morphology.

<table><tbody><tr><td>0.2 mL tube</td><td>Fisher Scientific Ltd</td><td>12134102</td><td></td></tr><tr><td>10 &amp;micro;L pipette</td><td>Gilson</td><td>FD10001</td><td></td></tr><tr><td>24 well plate</td><td>SARSTEDT</td><td>83.3922</td><td></td></tr><tr><td>3 way luer valve</td><td>Cole-Parmer</td><td>WZ-30600-02</td><td></td></tr><tr><td>3,3&amp;prime;-Diaminobenzidine (DAB) substrate</td><td>Vector Laboratories</td><td>SK-4105</td><td></td></tr><tr><td>40x objective</td><td>Olympus</td><td>LUMPLFLN40XW</td><td></td></tr><tr><td>4-aminopyridine</td><td>Hello Bio</td><td>HB1073</td><td></td></tr><tr><td>4x objective</td><td>Olympus</td><td>PLN4X/0.1</td><td></td></tr><tr><td>AAV9-CaMKiia-hChR2(E123T/T159C)-mCherry</td><td>Addgene</td><td>35512</td><td>Viral titre: 3.3x10&lt;sup&gt;13&lt;/sup&gt; GC/ml</td></tr><tr><td>Achromatic lens</td><td>Edmund Optics</td><td>49363</td><td>Focusses visual spectrum and near-IR</td></tr><tr><td>Benchtop microcentrifuge</td><td>Benchmark Scientific</td><td>C1005*</td><td></td></tr><tr><td>Biocytin</td><td>Sigma-Aldrich</td><td>B4261</td><td></td></tr><tr><td>Borosillicate glass capillary</td><td>Warner Instruments</td><td>G150F-6</td><td></td></tr><tr><td>Burr</td><td>Fine science tools</td><td>19008-07</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Camera - Qimaging Retiga Electro</td><td>Photometrics</td><td>01-ELECTRO-M-14-C</td><td></td></tr><tr><td>Carbachol</td><td>Tocris</td><td>2810</td><td></td></tr><tr><td>Chlorhexidine surgical scrub</td><td>Vetasept</td><td>XHG008</td><td></td></tr><tr><td>Clippers</td><td>Andis</td><td>22445</td><td>AGC Super 2-Speed Detachable Blade Clipper</td></tr><tr><td>Collimation condenser lens</td><td>ThorLabs</td><td>ACL2520-A</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific Ltd</td><td>10011913</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 S</td><td></td></tr><tr><td>CsMeSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1426</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Rapid Electronics Ltd</td><td>84-4557</td><td></td></tr><tr><td>Data acquisition device</td><td>National Instruments</td><td>USB-6341 BNC</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Dichroic mirror 500 nm long-pass</td><td>Edmund Optics</td><td>69899</td><td></td></tr><tr><td>Dichroic mirror 600 nm long-pass</td><td>Edmund Optics</td><td>69901</td><td></td></tr><tr><td>Dichroic mirror cube</td><td>ThorLabs</td><td>CM1-DCH/M</td><td></td></tr><tr><td>EGTA</td><td>Millpore</td><td>324626</td><td></td></tr><tr><td>Electrode holder with side port</td><td>HEKA</td><td>895150</td><td></td></tr><tr><td>Emission filter</td><td>Chroma</td><td>59022m</td><td></td></tr><tr><td>Excitation filter</td><td>Chroma</td><td>ET570/20x</td><td></td></tr><tr><td>Eye gel</td><td>Dechra</td><td>Lubrithal</td><td></td></tr><tr><td>Fine paint brush</td><td>Scientific Laboratory Supplies</td><td>BRU2052</td><td></td></tr><tr><td>Guillotine</td><td>World Precision Instruments</td><td>DCAP</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>H1009</td><td>30% (w/w)</td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>988-3245</td><td></td></tr><tr><td>Isopentane</td><td>Sigma-Aldrich</td><td>M32631</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>k-gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td></td></tr><tr><td>Kinematic fluorescence filter cube</td><td>ThorLabs</td><td>DFM1T1</td><td></td></tr><tr><td>LED driver</td><td>ThorLabs</td><td>LEDD1B</td><td></td></tr><tr><td>Lidocaine ointment</td><td>Teva</td><td>80007150</td><td></td></tr><tr><td>MgATP</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>MgCl</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Micro drill</td><td>Harvard Apparatus</td><td>75-1887</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P-87</td><td></td></tr><tr><td>Microinjection syringe</td><td>Hamilton</td><td>7634-01/00</td><td></td></tr><tr><td>Microinjection syringe needle</td><td>Hamilton</td><td>7803-05</td><td>Custom specification: gauge 33, length 15mm, point style 4 - 12&amp;deg;</td></tr><tr><td>Microinjection syringe pump</td><td>World Precision Instruments</td><td>UMP3T-1</td><td></td></tr><tr><td>Mounted blue LED</td><td>ThorLabs</td><td>M470L5</td><td></td></tr><tr><td>Mounted green LED</td><td>ThorLabs</td><td>M565L3</td><td></td></tr><tr><td>Na2HPO4.7H2O</td><td>Sigma-Aldrich</td><td>S9390</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NaGTP</td><td>Sigma-Aldrich</td><td>G8877</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4.&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>NIR LED</td><td>OSRAM</td><td>SFH4550</td><td>Used for refracted IR imaging of slice, differential interference contrast (DIC) optics is another commonly used method</td></tr><tr><td>OCT medium</td><td>VWR International</td><td>RAYLLAMB/OCT</td><td>Optimal cutting temperature medium</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>700A</td><td></td></tr><tr><td>Peristaltic pump</td><td>World Precision Instruments</td><td>Ministar</td><td></td></tr><tr><td>Poly-L-lysine coated microscope slides</td><td>Fisher Scientific Ltd</td><td>23-769-310</td><td></td></tr><tr><td>Recording chamber</td><td>Warner Instruments</td><td>RC-26G</td><td></td></tr><tr><td>Scalpel blade</td><td>Swann Morton</td><td>#24</td><td></td></tr><tr><td>Slice anchor</td><td>Warner Instruments</td><td>SHD-26-GH/15</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Kopf</td><td>Model 902</td><td></td></tr><tr><td>Stereotaxic holder for micro drill</td><td>Harvard Apparatus</td><td>75-1874</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Surgical Microscope</td><td>Carl Zeiss</td><td>OPMI 1 FR pro</td><td></td></tr><tr><td>Suture</td><td>Ethicon</td><td>W577H</td><td></td></tr><tr><td>Syringe filter for intracellular recording solution</td><td>Thermo Scientific Nalgene</td><td>171-0020</td><td></td></tr><tr><td>Tetrodotoxin citrate</td><td>Hello Bio</td><td>HB1035</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific Ltd</td><td>10458842</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Upright fluorescence microscope</td><td>Leica</td><td>DM6 B</td><td></td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200-10</td><td></td></tr><tr><td>VECTASTAIN ABC-HRP kit</td><td>Vector Laboratories</td><td>PK-4000</td><td></td></tr><tr><td>Vibratome</td><td>Campden Instruments</td><td>7000smz-2</td><td></td></tr><tr><td>WinLTP</td><td>https://www.winltp.com/</td><td>Version 2.32</td><td>Data acquisition software</td></tr><tr><td>&lt;strong&gt;Solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>aCSF</td><td></td><td></td><td></td></tr><tr><td>sucrose cutting solution</td><td></td><td></td><td></td></tr><tr><td>PFA</td><td></td><td></td><td></td></tr><tr><td>Intracellular?</td><td></td><td></td><td></td></tr></tbody></table>

ex vivo optogenetic interrogation of long-range synaptic transmission and plasticity from medial prefrontal cortex to lateral entorhinal cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

In this protocol, we describe how to study long-range synaptic physiology and plasticity using viral delivery of optogenetic constructs. The protocol can be very easily adapted to studying almost any long-range connection in the brain. As an example, we describe the injection of AAVs encoding an opsin into rat mPFC, the preparation of acute slices from LEC, patch-clamp recordings from layer 5 LEC pyramidal neurons, and light-evoked activation of mPFC terminals in LEC (Figure 1).
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Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

ex-vivo-optogenetic-interrogation-long-range-synaptic-transmission

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Neuroscience

2.2K Views.  University of Bristol. Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Video: Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

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 ...

Behavior

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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.

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 ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce ...

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://app.jove.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

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Electrophysiology Techniques

Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Source: Kinnavane, L., et al. <a href="https://app.jove.com/v/63077">Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for studying synaptic efficiency in the lateral entorhinal cortex (LEC) by optogenetically stimulating medial prefrontal cortex (mPFC) axons and recording the responses of pyramidal neurons in the LEC. This method combines patch-clamp recordings with synaptic activation by light stimulation to study synaptic plasticity.

Stymulacja optogenetyczna i elektrofizjologiczny zapis zdarzeń synaptycznych w wycinkach mózgu szczura

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

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

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Rozdziały w tym wideo

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

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

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Stymulacja optogenetyczna i elektrofizjologiczny zapis zdarzeń synaptycznych w wycinkach mózgu szczura