Name: Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex (Video) | JoVE Journal
Uploaded: 2022-02-25T13:00:00.000+00:00
Duration: 11 min 31 s
Description: Learn optogenetic interrogation of long-range synaptic transmission from medial prefrontal cortex to lateral entorhinal cortex using acute rodent brain slices. This method enables precise electrophysiological characterization of synaptic physiology and plasticity.

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5 minutes

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.

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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

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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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.

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

Research

JoVE Journal

Neuroscience

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.

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

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges.  An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

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

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that 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.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

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.

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

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

Here, we present two protocols for high-density micro-electrocorticography (µEcoG) recording in rats and mice, including surgical, implantation, and recording methods. µECoG recordings are performed in combination with either laminar polytrode recording in the rat auditory cortex or with optogenetic manipulation of neural activity in the mouse somatosensory cortex.

Multiscale Investigations of Cortical Processing by Integrating Laminar Polytrodes and Optogenetics with Micro Electrocorticography in Rodents

This protocol describes a two-phase surgical method to create a large, resealable, dura-sparing cranial window in mice. This technique enables chronic, multi-modal electrophysiological recordings from distributed, deep-brain networks, such as the Default Mode Network, over several weeks.

Surgical Protocol for a Large, Resealable Cranial Window Enabling Longitudinal, Multi-Modal Electrophysiology Recordings of Mouse Default Mode Network

Learn optogenetic interrogation of long-range synaptic transmission from medial prefrontal cortex to lateral entorhinal cortex using acute rodent brain slices. This method enables precise electrophysiological characterization of synaptic physiology and plasticity.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex (Video) | JoVE Journal

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

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