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 Dye Loading
<ol>
	<li>Select calcium dyes based on the preference of the individual researcher. Here we use Fluo-8AM or Fluo-4AM. However, this method can be applied to other dyes as well: dissolve calcium dyes in DMSO to a 1 mM solution and 1% block copolymers (e.g., pluronic acid-127) to a final volume of 50 &#181;L and sonicate for 10 min.</li>
	<li>Add solution to aCSF to a final concentration of 10 &#181;M, 0.01% block copolymers for brain slices.
	<ol>
		<li>For adult animals, (&#62;12 weeks), pipette the dyes (50 &#181;L) directly onto the brain slices, and maintain for 75 min to allow better penetration of the dye into the deep layers.</li>
		<li>To ensure adequate oxygenation of the submerged slice during dye incubation, prepare a glass loading chamber with a closed lid (circular jar of diameter 2.5 cm, 3.5 cm height) with calcium dyes diluted in 2.5 mL of aCSF. Oxygenate continuously with 95% O2/5% CO2. Do not bubble.</li>
	</ol>
	</li>
	<li>Following dye loading, wash the tissue with aCSF and transfer it to the incubation system, slowly reducing the temperature to 15 - 16 &#176;C until it is ready for experimental use.</li>
</ol>
2. Electrophysiological Recordings and Imaging
<ol>
	<li>Place tissue in a submerged recording chamber under a microscope and perfuse with oxygenated aCSF at a flow rate of 4 - 5 mL/min either at RT (~22 &#176;C) or physiological temperature (~35 &#176;C). Hold the tissue in place by using a custom-made &#39;&#39;harp,&#39;&#39; made of nylon or gold threads stretched and glued across a U-shaped piece of gold or platinum wire.</li>
	<li>For electrophysiology:
	<ol>
		<li>Prepare recording pipettes from 1.5 mm (1.19 mm ID) borosilicate glass using a micropipette puller to achieve a final resistance of 5-6 M&#937;.</li>
		<li>Fill the pipette with 3 - 4 &#181;L of internal solution and visualize cells under infrared Differential Interference Contrast (IR-DIC) using a CCD camera. The intracellular solution should be carefully tailored to each experiment to achieve experimental outcomes.</li>
		<li>Position the pipette while maintaining positive pressure applied through a suction port on the pipette holder on the membrane of a cell using a micromanipulator. No prior membrane scraping is required since the ILM has been removed from the retina. Once the pipette is on the cell, apply gentle negative pressure to the pipette to achieve a gigaohm seal. Then, rupture the cell membrane with a brief amount of negative pressure.</li>
		<li>Make whole-cell current- or voltage-clamp recordings using standard techniques as appropriate.</li>
	</ol>
	</li>
	<li>Calcium-imaging:
	<ol>
		<li>For a single excitation wavelength of Fluo-4, filter excitation light through a 460 - 490 nm bandpass filter and emitted light through a 515 - 550 nm bandpass filter. Use a high-speed digital camera for the fastest and most sensitive recording. Acquire images as required.</li>
		<li>Measure alterations in fluorescence as a function of time either at a single wavelength (Fluo-4).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich VETEC</td>
			<td>V800372</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S2554</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 Low UV absorbance*</td>
			<td>Life technologies</td>
			<td>P-6867</td>
			<td>20% solution in DMSO</td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Life Technologies</td>
			<td>F23917</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-8 AM</td>
			<td>Abcam</td>
			<td>Ab142773</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Olympus</td>
			<td>XLUMPlanFLN 20x/1.00w</td>
			<td>20X</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Olympus</td>
			<td>LUMPlanFLN 60x/1.00w</td>
			<td>60X</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Andor</td>
			<td>Ixon +</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch clamp amplifier</td>
			<td>Molecular Devices</td>
			<td>MultiClamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td>Axon Digidata&#174; System</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Sutter Instrument Co</td>
			<td>BF150-86-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter Instrument Co</td>
			<td>P-97</td>
			<td>Flaming/Brown type micropipette puller</td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-40LP</td>
			<td>Low Profile Open Bath Chambers</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Molecular Devices</td>
			<td>pClamp 10.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor iQ Live Cell Imaging Software</td>
			<td>Andor</td>
			<td>Andor iQ3</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>TE Technology</td>
			<td>TC-36-25 RS232</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring neural activity in a mouse brain slice after prolonged incubation

Source: Cameron, M. A., et al., <a href="https://app.jove.com/v/55396">Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.</a> J. Vis. Exp. (120), (2017)
This video demonstrates a technique for measuring neuronal activity in mouse brain slices using calcium imaging and electrophysiological recordings. By perfusing the tissue with potassium ions and recording intracellular calcium changes through high-speed digital imaging, this method reveals real-time neuronal responses.

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

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

	...

Secure a mouse brain slice in a recording chamber, perfused with oxygenated aCSF after prolonged incubation.
The cells in the slice are loaded with a calcium indicator, facilitating intracellular calcium measurements.
Take a recording pipette comprising an electrode in an electrolyte&#160; solution.
Identify a target neuron. Position the recording pipette with positive pressure on the cell membrane, creating a dimple.
Apply negative pressure to create a tight seal.
Further, apply negative pressure pulses, rupturing the membrane and establishing a whole-cell configuration .
Perfuse aCSF with a high potassium concentration, which depolarizes the neuron&#39;s membrane.
Depolarization opens sodium channels, triggering an action potential, which in turn activates voltage-gated calcium channels and allows calcium influx.
Calcium ions bind to the indicator, enhancing fluorescence, which can be visualized under suitable illumination.
The increase in intracellular fluorescence, caused by calcium influx following potassium application, indicates neuronal viability after prolonged incubation.

measuring-neural-activity-mouse-brain-slice-after-prolonged

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

36 Aufrufe. Quelle: Cameron, M. A., et al., Verlängerte Inkubation von akutem neuronalem Gewebe für Elektrophysiologie und Kalzium-Bildgebung. J. Vis. Exp. (120), (2017) Dieses Video demonstriert eine Technik zur Messung der neuronalen Aktivität in Gehirnschnitten von Mäusen mit Hilfe von Kalzium-Bildgebung und elektrophysiologischen Aufzeichnungen. Durch die Perfusion des Gewebes mit Kaliumionen und die Aufzeichnung intrazellulärer Kalziumveränderungen durch digitale Hochgeschwindigkeitsbildgebung z...

Serie: Messung der neuronalen Aktivität in einem Maushirnschnitt nach längerer Inkubation - Konzept

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Forschung

JoVE Journal

Entwicklungsbiologie

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Ratiometrischen Kalzium Imaging einzelner Neuronen im Verhalten Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Neurowissenschaften

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.

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Reaktionen auf Geschmacksreize

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

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

Transkript

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation