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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Ex-vivo endogenous monoamine release from brain slices or punches
NOTE: The device used for this section consists of a 48-well plate and a tissue holder made of six microcentrifuge filter units without the inset filters connected to a carbogen line (Figure 1). To make the holder, use a sturdy plastic rod (e.g., from a cell scraper) and super glue the microcentrifuge filter units without the inset filters to it. Let it dry for 1-2 days. Time required for the endogenous monoamine release experiment and concentrations of amphetamine, fluoxetine, and cocaine are based on the current literature and previous protocols.
<ol>
	<li>Tissue activation
	<ol>
		<li>Transfer the brain tissue to each well of the efflux chamber and allow recovery for 30-50 min at 37 &#176;C on a slide warmer in 0.5-1 mL of oxygenated efflux buffer, with constant, gentle bubbling (Figure 1B1).</li>
		<li>During this incubation, dilute the drugs to the desired concentration for the experiment. All the drugs must be dissolved in efflux buffer, and concentrations are based off the current literature.</li>
	</ol>
	</li>
	<li>First incubation
	<ol>
		<li>Move the tissue holder with brain tissue to wells containing 500 &#956;L of oxygenated efflux buffer and incubate for 20 min at 37 &#176;C. Ensure that minimal to no buffer is transported over by tapping the holder on the edge of the well until no excess buffer is in the holder.</li>
		<li>In experiments with pharmacological agents such as monoamine transporter inhibitors, incubate the tissue samples with the drugs diluted in oxygenated efflux buffer (e.g., 10 &#956;M Fluoxetine, 40 &#956;M cocaine; see Figure 1B2). The final volume in each well will be 500 &#956;L.</li>
	</ol>
	</li>
	<li>Second incubation
	<ol>
		<li>Move the holder with the tissue to a new set of wells containing 500 &#956;L total efflux buffer with or without the desired concentration of each drug. Ensure there is no excess buffer left over. Each well represents an n= 1 for experimental conditions. Each experimental condition is performed in triplicate.</li>
		<li>One well includes an oxygenated efflux buffer, the next 10-30 &#956;M AMPH, and the final well includes 10-30 &#956;M AMPH plus monoamine transporter inhibitors. Each drug is dissolved in oxygenated efflux buffer.</li>
		<li>Incubate the tissue for 20 min at 37 &#176;C with 500 &#956;L of the drug condition. 
		NOTE: Additional wells may include an oxygenated high K+ efflux buffer with or without the monoamine transporter inhibitors. Dissolve each drug in the oxygenated efflux buffer (500 &#956;L).</li>
		<li>During this second incubation of 20 min, collect the solution from the wells from the first incubation in step 1.2.1 and transfer to microcentrifuge tubes containing 50 &#956;L of 1 N perchloric acid or phosphoric acid (dependent on the type of HPLC, final concentration 0.1 N). The final volume of the sample will be 550 &#956;L. Maintain microcentrifuge tubes on ice and label the tubes #1.</li>
		<li>After the second incubation of 20 min, move the tissue holder with brain sections or punches to an empty well and maintain the plate on ice. Transfer the supernatant to microcentrifuge tubes containing 50 &#956;L of 1 N perchloric acid or phosphoric acid. The final volume of the sample will be 550 &#956;L. Maintain microcentrifuge tubes on ice and label the tubes #2.</li>
		<li>Add 1 mL of ice-cold dissection buffer to each well containing tissue. Collect all of the tissue using small tweezers, and transfer to clean microcentrifuge tubes.</li>
		<li>Maintain tubes with brain tissue at -80 &#176;C. Discard the 1 mL of dissection buffer (Figure 1B4).</li>
		<li>Filter solutions obtained from each incubation using microcentrifuge filter tubes (0.22 &#956;m) at 2,500 x g for 2min. Use the filtrate to determine monoamine content using HPLC with electrochemical detection (Figure 1B5).</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>48-well plate</td><td>NA</td><td>NA</td><td>Assay</td></tr><tr><td>Calcium Chloride Anhydrous</td><td>Sigma Aldrich</td><td>C1016</td><td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>1002608421</td><td>Dissection Buffer</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma Aldrich</td><td>A5960</td><td>Dissection Buffer</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>7487-88-9</td><td>KH Buffer</td></tr><tr><td>Microcentrifuge Filter Units UltraFree</td><td>Millipore</td><td>C7554</td><td>Assay - 6 to fit in 48-well plate</td></tr><tr><td>Pargyline Clorohydrate</td><td>Sigma Aldrich</td><td>P8013</td><td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>12636</td><td>KH Buffer</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma</td><td>1001655559</td><td>KH Buffer</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td>Dissection Buffer</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td>Dissection Buffer</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S3014</td><td>KH Buffer</td></tr></tbody></table>

a plate-based assay to measure monoamine release from brain slices on test drug exposure

Source: Pino, J. A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62127">A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices.</a> J. Vis. Exp. (2021)
In this video, we demonstrate an assay that investigates monoamine release mechanisms from the neurons of metabolically active ex vivo rat brain slices, when treated with test drugs. The assay demonstrates that neurostimulant drugs induce monoamine release via the cellular monoamine transporters, whereas high KCl concentration induces synaptic vesicle exocytosis-mediated vesicular monoamine release.

<img alt="Figure 1" src="/files/ftp_upload/21279/21279_Figure1.jpg" /> 
Figure 1: Experimental setup for efflux experiment. (A) The efflux chamber consists of a 48-well tissue culture plate and a tissue holder tray connected to the carbogen line. (B) A diagram showing the experimental design for the endogenous monoamine efflux experiment in which the tissue activation (B1), pre-incubation with/without monoamine transporter inhibitors (B2), efflux experiment (B3), and the final sample processing are presented (B4-B5). (C) The left-hand panel depicts an experimenter loading the perfusate into the HPLC in preparation for auto-injection. The right-hand panel shows a representative chromatogram denoting the monoamine standard peaks. The area under the curve (AUC) is measured for each monoamine standard and brain samples. After calibration, the AUC measured for each brain sample is converted to nM concentration.

A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure

Source: Pino, J. A. et al. A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices. J. Vis. Exp. (2021)
In this ...

Monoamine neurotransmitters, endogenous chemical messengers, relay signals across the synapse, the point of contact between neurons. Tightly regulated monoamine neurotransmitter system is essential for normal neuronal function.
To screen the effect of a test drug on monoamine release ex vivo, obtain rat brain tissue slices containing the region of interest in a chilled oxygenated buffer, maintaining tissue viability.
Transfer the tissue slices to wells of a multi-well plate, with porous filter inserts connected to a gaseous mixture supply and comprising oxygenated artificial cerebrospinal fluid, aCSF, supplemented with monoamine oxidase inhibitors. Incubate.
The inhibitors diffuse into the cells and inactivate monoamine oxidase enzymes, preventing degradation of monoamines and increasing their concentration available for storage and release.
Transfer the tissue-containing inserts to wells comprising oxygenated aCSF, and the test drug. Incubate. The drug may stimulate the monoamine neurotransmitter system, increasing the monoamine concentration in the extraneuronal space, leading to their release from the brain tissue into solution.
The released monoamines pass through the porous inserts into the wells, separating from the tissue.
Post-incubation, remove the tissue slice-containing inserts. Collect the monoamine solution in a tube comprising a solvent compatible with high-performance liquid chromatography, HPLC. Transfer the tube contents into pre-assembled filter units. Centrifuge, removing major contaminants.
Load the obtained filtrate into the HPLC column and perform a chromatography run to determine monoamine concentration, indicative of the test drug effect on monoamine release.

a-plate-based-assay-to-measure-monoamine-release-from-brain-slices-on

Nanyang Technological University

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94 次观看. 来源:Pino， J. A. et al. 一种基于板的测定法，用于测量急性脑切片中内源性单胺释放。J. Vis. Exp. （2021 年） 在本视频中，我们演示了一种分析方法，该分析研究了用测试药物处理时代谢活跃的离体大鼠脑切片神经元的单胺释放机制。该测定表明，神经刺激药物通过细胞单胺转运蛋白诱导单胺释放，而高 KCl 浓度诱导突触囊泡胞吐作用介导的囊泡单胺释放。 

视频: 一种基于板的测定法，用于测量受试药物暴露时脑切片中单胺的释放 - 概念

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

科研

JoVE Journal

神经科学

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure

成績單

A Plate-Based Assay to Measure Monoamine Release from Brain Slices on Test Drug Exposure