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EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Visualization and Stimulation of Presynaptic Fibers
<ol>
	<li>Prepare patch microscope for optogenetic activation of fibers and cells:
	<ol>
		<li>Center the mounted light emitting diode (LED) onto the light delivery pathway.</li>
		<li>Measure the LED light intensity at the back focal plane and at the output of each objective with a power meter choosing the appropriate wavelength of 470 nm.</li>
		<li>Calculate the light intensity in mW/mm2 and create a calibration curve (LED intensity (%) versus light output (mW/mm2)) for each objective for values measured for 470 nm wavelength.</li>
	</ol>
	</li>
	<li>Retrieve an acute amygdala slice from the interface chamber and place in the slice chamber mounted onto the upright microscope equipped with a fluorescent lamp. Take care to position the slice such that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse slice with fresh, oxygenated artificial cerebrospinal fluid (ACSF) at a rate of 1 - 2 ml/min at a temperature of ~31 &#176;C.</li>
	<li>Observe presynaptic fibers in the slice using the fluorescent lamp in combination with appropriate filter sets for the specific fluorescent protein expressed. Use 5x objective to obtain an overview (Figure 1E), and 60x objective for assessment of fiber density within the target area. 
	Note: For GFP and YFP, use Filter set &#34;green&#34; (Excitation 472/20, Beamsplitter 495, Emission 490 LP) for mCherry use filter set &#34;red&#34; (Excitation 560/40, Beamsplitter 585, Emission 630/70) as specified in the materials/equipment table.</li>
	<li>Open or restrict the aperture in the microscope light pathway as desired for the experiment (Figure 2D).</li>
	<li>To obtain a patch recording, fill a patch pipette with internal solution and mount it in electrode holder. Apply positive pressure to the patch pipette and slowly lower it first into the bath solution and then under visual control into the slice using the micromanipulator.
	<ol>
		<li>Approach the neuron of interest with the patch pipette from the side and top. Release positive pressure when the pipette is on the surface of the cell (dimple visible on cell surface) and obtain a &#34;gigaseal&#34; by applying negative pressure.</li>
		<li>Apply further suction to rupture the membrane patch to obtain whole cell recording. Subsequently, stimulate labeled fibers with the connected LED using the appropriate wavelength for activating channelrhodopsin (ChR) (470 nm) while recording electrical responses from the cell.</li>
		<li>For synaptic stimulation start with a low LED intensity and increase until the desired synaptic current amplitude is reached. Trigger the LED by configuring digital outputs in the data acquisition software to control the timing and pulse length (examples in Figure 3). 
		Note: other software and/or TTL-generating devices can be used to trigger LED.</li>
	</ol>
	</li>
	<li>Repeat stimulation with opened or restricted aperture (step 1.4) in the microscope light pathway as desired for the next recorded cell and/or in the presence of specific drugs.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artificial cerebrospinal fluid (ACSF)</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Internal patch solutions</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagnesiumSulfate Heptahydrate</td>
			<td>Roth, Germany</td>
			<td>P027.1</td>
			<td>prepare 2M stock solution in purified water</td>
		</tr>
		<tr>
			<td>Stereoscope, SZX2-RFA16</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xcite fluorescent lamp (XI120Q-1492)</td>
			<td>Lumen Dynamics Group, Canada</td>
			<td>2012-12699</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch microscope, BX51WI</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiclamp 700B patch amplifier</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digitdata 1440A</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PClamp software, Version 10</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td>used to control data acquisition and stimulation</td>
		</tr>
		<tr>
			<td>Bath temperature controler, TC05</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>200-100 500 0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Three axis micromanipulator Mini 25</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>210-100 000 0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator controller SM7</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>200-100 900 7311</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries for patch pipettes</td>
			<td>World Precision Instruments, Germany</td>
			<td>GB150F-8P</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose nitrate filterpaper for interface chamber</td>
			<td>Satorius Stedim Biotech, Germany</td>
			<td>13006--50----ACN</td>
			<td></td>
		</tr>
		<tr>
			<td>LED unit, CoolLED pE</td>
			<td>CoolLED, UK</td>
			<td>244-1400</td>
			<td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolLED 100 Dual Adapt</td>
			<td>CoolLED, UK</td>
			<td>pE-ADAPTOR-50E</td>
			<td></td>
		</tr>
		<tr>
			<td>LED unit, USL 70/470</td>
			<td>Rapp Optoelectronic</td>
			<td>L70-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual port adapter</td>
			<td>Rapp Optoelectronic</td>
			<td>inquire with company</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter set red (excitation)</td>
			<td>AHF, Germany</td>
			<td>F49-560</td>
			<td>Filters can be bought as set F46-008</td>
			<td></td>
		</tr>
		<tr>
			<td>(beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F48-585</td>
			<td></td>
		</tr>
		<tr>
			<td>(emission)</td>
			<td>AHF, Germany</td>
			<td>F47-630</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter set green (excitation)</td>
			<td>AHF, Germany</td>
			<td>F39-472</td>
			<td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td>
			<td></td>
		</tr>
		<tr>
			<td>(beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F43-495W</td>
			<td></td>
		</tr>
		<tr>
			<td>(emission)</td>
			<td>AHF, Germany</td>
			<td>F76-490</td>
			<td></td>
		</tr>
		<tr>
			<td>LaserCheck, handheld power meter</td>
			<td>Coherent, USA</td>
			<td>1098293</td>
			<td></td>
		</tr>
		<tr>
			<td>IgorPro Software, Version 6</td>
			<td>Wavemetrics, USA</td>
			<td></td>
			<td>for electrophysiology data analysis, other alternative software packages can also be used</td>
		</tr>
		<tr>
			<td>Neuromatic suite of macros for IgorPro</td>
			<td>http://www.neuromatic.thinkrandom.com</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization and mapping of neuronal pathways in an amygdala brain slice

Source: Bosch, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

<img alt="Figure 1" src="/files/ftp_upload/22856/22856_Figure1.jpg" /> 
Figure 1. Stereotactic Injections, Preparation of Acute Brain Slices, and Visualization of Presynaptic Fibers. (A, B) Stereotactic virus injection. A) Picture of anesthetized mouse placed in a stereotactic frame with skull exposed and the injection pipette. Inset: Zoom in picture of injection pipette filled with virus solution mixed with fast green. Scale bar: 3 mm. (B</s...

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

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

In the mouse brain, the amygdala contains neurons that form synaptic connections with the medial prefrontal cortex, or mPFC neurons.
Take an amygdala brain slice in which the mPFC neurons express a light-sensitive channel rhodopsin fused to a fluorescent protein.
Place the slice in a recording chamber and visualize it under a fluorescence microscope to locate the mPFC neurons.
Switch to a brightfield view and introduce a patch pipette containing an internal solution.
Apply positive pressure to advance the pipette toward an amygdala neuron.
Upon neuronal contact, a dimple forms.
Apply suction to form a tight seal.
Continue suction to rupture the membrane, establishing contact with the cell&#39;s interior.
Illuminate the slice to activate the mPFC neuronal channelrhodopsin, triggering positive ion influx, action potential generation, and neurotransmitter release.
The neurotransmitters bind to postsynaptic amygdala neuron receptors, causing ion influx and current generation.
Record the postsynaptic neuronal current to analyze mPFC-amygdala connectivity.

visualization-mapping-neuronal-pathways-an-amygdala-brain

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

250 조회수. 출처: Bosch, D., et al. Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices. J. Vis. 특급 (2016). 이 비디오는 생쥐의 급성 편도체 뇌 절편을 사용하여 내측 전전두엽 피질(mPFC)과 편도체 뉴런 사이의 시냅스 연결성을 분석하는 절차를 보여줍니다. 이 마우스의 mPFC 뉴런은 형광 단백질에 융합된 채널로돕신을 발현합니다. 채널로돕신은 광 활성화 시 mPFC 뉴런에서 이온 유입 및 활동 전위 생...

비디오: Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice(편도체 뇌 절편에서 신경 경로의 시각화 및 매핑) - 개념

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

생체 발광 광유전학 2.0 : 생체 발광을 활용하여 체외 및 생체 내에서 광감각 단백질을 활성화 합니다.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

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JoVE Journal

생화학

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

초파리 애벌레 동작 Optogenetic 후 각 신경의 자극에 대 한 응답 추적

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

신경과학

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.

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

내레이션 대본

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice