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

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

Transfer the cultures one at a time to the imaging chamber. Collect the baseline fluorophore fluorescent images of axonal projections from the ventral hippocampal micro-slices as pre-nicotine control.
Next, apply one micromolar of nicotine by rapid perfusion at 2 milliliters per minute for one minute, and then, wash the nicotine away with HBS cocktail. Keep capturing the time-lapse images before, during, and after nicotine application every 10 seconds for 30 minutes.

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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>

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.

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.

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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.

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

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