measuring neural activity in a mouse brain slice after prolonged incubation

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

For recording, place tissue in a submerged recording chamber under a microscope, and perfuse it with oxygenated aCSF at a flow rate of 4 to 5 milliliters per minute. Hold the tissue in by using a custom-made harp. Next, prepare some recording pipettes using a micropipette puller to achieve a final resistance of 5 to 6 mega ohms.
Fill the pipette with 3 to 4 microliters of internal solution, and then place the solution on ice. Then, visualize the cells using a CCD camera under IR-DIC. Position the pipette on the cell membrane using a micromanipulator. Maintain positive pressure through a suction port on the pipette holder. 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 brief negative pressure. Subsequently, start whole-cell current or voltage clamp recording. For a single excitation wavelength of flow 4, filter the excitation light through a 460- to 490-nanometer bandpass filter and emitted light through a 515- to 550-nanometer bandpass filter.

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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>

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.

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.

12 Views. Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

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