eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging
<ol>
	<li>Rinse cultures (5 - 7 days in vitro) quickly with normal HBS, then load cultures with 5 &#181;M Fluo-4 Ca2+ indicator (AM ester) and 0.02% Pluronic F-127 in HBS (2 ml) for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Wash out the Fluo-4 solution with HBS (3 times/5 min). Return the cultures to the incubator (37 &#176;C / 5% CO2) for at least 30 min.</li>
	<li>Maintain cultures (5 - 7 days in vitro) in an imaging chamber and mount the chamber on a spinning disk confocal microscope. Continuously perfuse (1 ml/min) with HBS cocktail at RT.</li>
	<li>Collect Fluo-4 fluorescence images of axonal projections from the vHipp micro-slices with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and a CCD camera.</li>
	<li>Collect baseline Fluo-4 fluorescence images (every 10 sec for 2 min) as a pre-nicotine control. Apply nicotine (1 &#181;M) by rapid perfusion (2 ml/min) for 1 min, then wash the nicotine with an HBS cocktail. Keep capturing time-lapse images before, during, and after nicotine application every 10 sec for 30 min.</li>
	<li>Save all frames of the raw fluo-4 fluorescence images and export them as a series of TIFF images for further analysis.</li>
	<li>Collect the integrated intensity of fluo-4 fluorescence along vHipp axons before and after nicotine application.</li>
	<li>Normalize integrated fluorescence intensity with the following equation: &#916;F/F0 =&#160; (F-F0)/F0, where F0 is the background-corrected pre-nicotine integrated fluorescence intensity, and F is the integrated fluorescence intensity along vHipp axons at each time point after nicotine application. Analyze and plot normalized integrated fluorescence intensity.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HBS cocktail for live imaging pH=7.3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
	</tbody>
</table>

imaging nicotine-induced calcium signaling along ventral hippocampal axons in synaptic co-cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

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

	Rinse ...

Take genetically modified co-cultures of mouse ventral hippocampus slices and nucleus accumbens neurons on a coverslip.
The hippocampal neurons project pre-synaptic axon terminals overexpressing nicotinic acetylcholine receptors, forming synapses with the post-synaptic nucleus accumbens neurons.
Neurons are loaded with a calcium indicator dye for intracellular calcium measurements.
Transfer the coverslip to a confocal microscope chamber perfused with buffer containing calcium ions.
Capture fluorescent images of the hippocampal axonal projections as a control.
Perfuse nicotine, which binds to the nicotinic acetylcholine receptors on hippocampal neurons, triggering calcium influx.
The calcium ions bind to the intracellular dye, increasing fluorescence.
Hippocampal axons release excitatory neurotransmitters, initiating synaptic transmission in nucleus accumbens neurons.
Wash with buffer to remove nicotine.
Over time, nicotinic acetylcholine receptor activation causes calcium release from intracellular stores, leading to increased dye fluorescence and indicating sustained calcium signaling along the ventral hippocampal axons.

imaging-nicotine-induced-calcium-signaling-along-ventral-hippocampal

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

35 Views. Fonte: Zhong, C., et al., Imaging dal vivo della segnalazione del calcio indotta dalla nicotina e dal rilascio di neurotrasmettitori lungo gli assoni dell'ippocampo ventrale. J. Vis. Exp. (2015) Questo video dimostra come l'attivazione mediata dalla nicotina dei recettori nicotinici dell'acetilcolina nell'ippocampo ventrale del topo si traduca in una segnalazione sostenuta del calcio lungo gli assoni. 

Video: Imaging della segnalazione del calcio indotta dalla nicotina lungo gli assoni dell'ippocampo ventrale in co-colture sinaptiche - Concept

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

L&#39;incubazione prolungata di acuta neuronale tessuto per Elettrofisiologia e Calcio imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Ricerca

JoVE Journal

Neuroscienze

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Imaging in vivo del calcio delle risposte del neurone ganglio genicolato di topo agli stimoli del gusto

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Trascrizione

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures