optogenetic stimulation and electrophysiological recording of synaptic events in rat brain slices

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

For target cell identification, place the slice into the recording chamber, and immobilize it using a slice anchor. Under low magnification, using infrared illumination, navigate to the LEC layer 5. Then, change to the high magnification water-immersion objective, and identify the pyramidal neurons.
Mark the position of the cell on the monitor with tape. Next, to form a whole-cell patch clamp, fabricate a borosilicate glass micropipette, and fill it with intracellular recording solution. Place the filled micropipette in the electrode holder, and apply positive pressure by blowing hard into a mouthpiece connected to the electrode holder side port.
Raise the microscope objective such that a meniscus forms, and insert the electrode into the meniscus until it can be seen on the microscope. Open the seal test window, and determine whether the pipette resistance is three to five megaohms. Then, touch the identified cell with a pipette tip resulting in an indentation in the cell membrane.
Next, apply negative pressure by suction at the mouthpiece, increasing pipette resistance. Continue applying negative pressure gradually until the cell membrane ruptures, resulting in whole-cell capacitance transients. To enter the current-clamp configuration, use a mounted LED directed into the microscope light path with filter cubes and appropriate optics, and apply light pulses to the slice via the 40x objective. Deliver trains of multiple light pulses at 5 Hertz, 10 Hertz, and 20 Hertz to investigate the presynaptic release properties.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology and optogenetic stimulation
<ol>
	<li>Identification of target cell.
	<ol>
		<li>Place the slice into a submerged recording chamber at 34 &#176;C perfused with artificial cerebrospinal fluid (aCSF) at a rate of 2 mL/min by a peristaltic pump. Immobilize the slice using a slice anchor.</li>
		<li>Under the low magnification (4x) objective of a widefield microscope using oblique infrared illumination, navigate to lateral entorhinal cortex (LEC) layer 5. Measure the distance from the pial surface to the required layer. 
		NOTE: Oblique infrared illumination was achieved by positioning a near-infra-red LED approximately 3 mm below the recording chamber coverslip at an angle of ~55&#176; to the plane of the coverslip (Figure 1B). Differential interference contrast is an alternative and commonly used imaging technique for slice electrophysiology.</li>
		<li>Change to a high-magnification water immersion objective (40x) and identify pyramidal neurons. Mark the position of the cell on the computer monitor with tape. 
		NOTE: The pyramidal neurons have a roughly triangular morphology with prominent apical dendrites projecting toward the pial surface of the slice (Figure 2A). Cell health can be assessed by the absence of a condensed, visible nucleus and inspection of the plasma membrane which should appear smooth. It is unlikely that clear fluorescent labeling of axons by the opsin-fluorophore fusion protein will be visible when using a wide-field fluorescence microscope. To visualize axonal projections, perform immunohistochemistry post-hoc using a primary antibody against the opsin&#39;s fluorophore and amplify with a secondary antibody conjugated to a fluorophore of the same or similar wavelength.</li>
	</ol>
	</li>
	<li>Formation of whole-cell patch clamp.&#8203;
	<ol>
		<li>Fabricate a borosilicate glass micropipette using a pipette puller and fill it with filtered intracellular recording solution (120 mM k-gluconate, 40 mM HEPES (N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid), 10 mM KCl (potassium chloride), 2 mM NaCl (sodium chloride), 2 mM MgATP (magnesium adenosine triphosphate), 1 mM MgCl (magnesium chloride), 0.3 mM NaGTP (sodium guanosine triphosphate), 0.2 mM EGTA (Ethylene glycol tetraacetic acid), and 0.25% biocytin made in UPW (ultrapure water), pH 7.25, 285-300 mOsm). Place the filled micropipette in the electrode holder on the patch-clamp amplifier headstage, ensuring the electrode wire is in contact with the intracellular solution.</li>
		<li>Apply positive pressure by mouth by blowing hard into a mouthpiece (such as a 1 mL syringe with the plunger removed) connected by tubing to the electrode holder side port and maintain pressure by closing an in-line three-way valve. Raise the microscope objective such that a meniscus forms and insert the electrode into the meniscus until it can be seen on the microscope.</li>
		<li>Open the Seal Test window in WinLTP16 (or other acquisition software/oscilloscope), and with the amplifier in the voltage-clamp mode, apply a 5 mV square pulse to determine whether the pipette resistance is 3-6 M&#937;.</li>
		<li>Approach and touch the identified cell with the pipette tip; this should result in an indentation in the cell membrane (Figure 2A; right panel) and a small increase in pipette resistance (0.1 M&#937;).</li>
		<li>Release positive pressure and apply negative pressure by applying moderate suction at the mouthpiece; this should result in a vast increase in pipette resistance(&#62;1000 M&#937;). Pressure can now be left neutral. Apply negative pressure in a gradually increasing ramp until cell membrane ruptures resulting in whole-cell capacitance transients.</li>
	</ol>
	</li>
	<li>Record optogenetically evoked synaptic events.
	<ol>
		<li>Enter current-clamp configuration. 
		&#8203;NOTE: In most instances, long-range synaptic transmission is glutamatergic, therefore recording at membrane potentials close to the chloride reversal potential will best isolate AMPA receptor (AMPAR)-mediated transmission and minimize measurement of any feed-forward inhibition (FFI) evoked. Chloride reversal is dependent on the composition of intracellular recording solution and aCSF and can be calculated using the Goldman-Hodgkin-Katz equation; for the above solutions, this was -61.3 mV. Layer 5 LEC pyramidal neurons had an average resting membrane potential of -62 mV and, where necessary, were maintained at this potential by injection of constant current. Alternatively, cells can be voltage-clamped to the desired potential. To record long-range inhibitory projections16 or to record FFI, voltage-clamp at cation reversal potential to isolate GABAergic chloride conductance. When voltage-clamping neurons at membrane potentials above action potential threshold, a cesium-based intracellular solution containing voltage-gated sodium channel blockers is used to improve voltage-clamp and prevent initiation of action potentials (130 mM CsMeSO4 (cesium methanesulfonate), 10 mM HEPES, 8 mM NaCl, 5 mM QX 314 chloride, 4 mM MgATP, 0.5 mM EGTA, 0.3 mM NaGTP, 0.25% biocytin made in UPW, pH 7.25, 285-300 mOsm).</li>
		<li>Using data acquisition software, send transistor-transistor logic (TTL) signals to an LED (light emitting diode) driver to activate a mounted 470 nm LED. The mounted LED is directed into the microscope light path using filter cubes and appropriate optics (Figure 1B) to apply light pulses to the slice via the 40x objective to evoke optogenetic excitatory post-synaptic potentials (oEPSPs). 
		NOTE: Light pulses can be applied perisomatically/over the dendrites, which will result in the activation of opsins in the axons and presynaptic bouton, or the investigator can move the objective to axons away from the recorded cell to avoid over-bouton stimulation. Maximal oEPSP amplitude depends on the strength of the synaptic projection, the efficacy of viral injections, and opsin used. oEPSPs can be titrated to the desired amplitude by varying light intensity and/or duration18; varying the duration of light pulses (typical duration between 0.2-5 ms at maximal LED power output, which results in 4.4 mW/mm light density19) gives more consistent oEPSP amplitudes than altering the power output of the LED.</li>
		<li>Investigate presynaptic release properties (change in voltage) by delivering trains of multiple light pulses with differing inter-stimulus intervals (Figure 2E); care should be taken while interpreting optogenetically evoked transmission.</li>
		<li>Investigate long-term plasticity either by repetitively evoking oEPSPs19 or application of ligands. Monitor oEPSP amplitude for 5-10 min to ensure stability before induction of plasticity, and then monitor until a stable amplitude is reached (typically 30-40 min). 
		NOTE: Most current opsins are not capable of reliably evoking multiple action potentials at high frequencies, e.g., 100 stimuli delivered at 100 Hz, as is typically used to induce LTP.</li>
		<li>To confirm oEPSPs are monosynaptic, perform over-bouton activation of the transduced pathway by positioning the objective over the dendritic arbor and stimulating in the presence of 0.5 &#181;M tetrodotoxin and 100 &#181;M aminopyridine. Application of tetrodotoxin will abolish transmission if the responses are action potential-dependent, and subsequent inclusion of aminopyridine will partially restore transmission if the oEPSPs are generated monosynaptically19,20.</li>
		<li>To allow biocytin to fill the neuron, wait for at least 15 min after entering the whole-cell configuration. In voltage-clamp, monitor membrane capacitance and input resistance.</li>
		<li>Slowly withdraw the pipette along the approach angle away from the soma of the cell, observing the slow disappearance of capacitance transients and membrane current, indicating the re-sealing of the cell membrane and formation of an outside-out patch at the pipette tip. Note the orientation of the slice and the location of the cell(s) within the slice. Put the slice into PFA (paraformaldehyde) in a 24-well plate, incubate overnight at 4 &#176;C, and then transfer to 0.1 M PB. 
		&#8203;NOTE: Slices can be stored for up to a week. If longer storage is required, change the PB (phosphate buffer) regularly or use PB containing sodium azide (0.02%-0.2% of sodium azide).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 way luer valve</td>
			<td>Cole-Parmer</td>
			<td>WZ-30600-02</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>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>CaCl2</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>CsMeSO4</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>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>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</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&#176;</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>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>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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>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>Slice anchor</td>
			<td>Warner Instruments</td>
			<td>SHD-26-GH/15</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>Syringe filter for intracellular recording solution</td>
			<td>Thermo Scientific Nalgene</td>
			<td>171-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Leica</td>
			<td>DM6 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution</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>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22874/22874_Figure1.jpg" /> 
Figure 1: Slice collection chamber and optical configuration for visualized whole-cell recordings, optogenetic excitation, and identification of tdTomato-positive neurons. (A) The slice collection chamber14 is custom-made from a microcentrifuge tube rack glued onto a sheet of nylon mesh and placed into a beaker in which it is submerged in aCSF (B) LEDs for excita...

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

Begin with a rat brain slice containing the target neuron region connected with a light-sensitive axon from a different brain region.
Place the slice into the submerged recording chamber and secure it with a slice anchor.
Under a microscope, locate the desired layer and identify the target pyramidal neuron.
Raise the microscope objective to create a meniscus with the slice surface.
Lower a glass micropipette filled with intracellular recording solution into this meniscus.
Touch the identified pyramidal cell with the pipette tip to create an indent in the membrane.
Apply suction to form a seal.
Increase the suction until the membrane ruptures, establishing a whole-cell configuration.
Using a mounted light source, apply light pulses to the slice.
These light pulses trigger an action potential in the light-sensitive axon, releasing neurotransmitters at the synaptic cleft.
Record the pyramidal neuron&#39;s responses to these releases to analyze synaptic efficiency.

24 Görüntüleme. Sıçan Beyin Dilimlerinde Sinaptik Olayların Optogenetik Stimülasyonu ve Elektrofizyolojik Kaydı

Video: Sıçan Beyin Dilimlerinde Sinaptik Olayların Optogenetik Stimülasyonu ve Elektrofizyolojik Kaydı - Deney

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.

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

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

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Nörobilim

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.

Fare Beyin Dilimlerinde Uzun Menzilli Girdilerin Optogenetik Stimülasyonu için Vivo İntraserebral Stereotaksik Enjeksiyonlarda

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

Bilişsel Araştırma ve İlaç Analizi için Fare Beyninde LFP Kaydı

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

Yazar Spotlight: Farelerde Çift LFP Kayıt Protokolü Kullanarak Beyin Sinyalleri Üzerindeki İlaç Etkilerinin İncelenmesi

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

Fare Hipokampusu ve Prefrontal korteksinde ikili hücre dışı kayıtlar

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

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

TRANSKRIPT