Name: live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons
Uploaded: 2015-06-24T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 2012) and studies were approved by Institutional Animal Care and Use for Research Committees at Stony Brook University (#1618 and #1792).
1. vHipp-nAcc Synaptic Co-cultures
<ol>
	<li>Sacrifice mice (postnatal day 0 - 3, from wild-type (WT) or &#945;7 nAChRs transgenic mouse line) with CO2. Decapitate each...

<ol>
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The co-culture preparation described re-capitulates ventral hippocampal&#8211;accumbens circuits in vitro. This preparation permits relatively straightforward and reliable examination of the spatial and temporal profiles by which activation of pre-synaptic nAChRs elicit enhanced glutamatergic transmission5, 21.
Co-cultures are defined as the growth of different specific cell types in one dish which can provide physiological conditions in vitro to demonstrate in...

Cholinergic modulation of circuit excitability contributes to fundamental aspects of cognition, and altered cholinergic modulation is a feature of neurodegenerative and neuropsychiatric disorders including Alzheimer&#8217;s disease, Parkinson&#8217;s disease, schizophrenia and addiction1-4. An established mechanism of cholinergic facilitation of synaptic transmission in the CNS is via direct activation of nAChRs localized at pre-synaptic sites. Activation of these pre-synaptic receptors leads to increased intracellular Ca2+ ([Ca2+]i) in pre-synaptic terminals &#8211; both directly, due to the relatively high calcium conductance of certain nAChR subtypes, and indirectly, via intracellular signaling cascades5, thereby enhancing neurotransmitter release. In fact, the activation of pre-synaptic nAChRs has been linked with changes in release of a wide variety of neurotransmitters including glutamate, GABA, ACh, and dopamine6-10. Although this process has been studied indirectly using electrophysiological methods at various synapses, optical reporters of [Ca2+]i and synaptic vesicle recycling allow more direct and temporally precise measurement of pre-synaptic phenomena.
Pre-synaptic localization of nAChRs has been demonstrated convincingly with direct immuno-gold labeling of nAChRs at the electron microscopic (EM) level11,12. Several other techniques have also been used to address the nAChR localization indirectly, including detecting locations of nAChRs subunit- fluorescent protein chimeras in cultured neurons13,14, electrophysiological recording of nAChR currents in synaptic terminals15,16, monitoring nicotine induced changes in [Ca2+]i in synaptic nerve terminals by live cell imaging17, and indirect monitoring of neurotransmitter release at the synaptic terminal by live cell imaging techniques with fluorescent indicators, including exocytosis of synaptic vesicles viewed by styryl amphipathic FM dyes (FM1-43 and FM4-64) and/or synapto-pHluorin and by specific fluorescent neurotransmitter reporters, such as CNiFERs for ACh and iGluSnFr for glutamate18-20. Overall, these current approaches for identifying pre-synaptic localization of nAChRs are complicated, and require special systems and techniques to allow reliable identification and physiological monitoring of pre-synaptic activity.
Here we describe protocols and equipment for an in vitro co-culture system of a ventral hippocampal (vHipp) &#8211; nucleus accumbens (nAcc) circuit that provides direct access to identify and analyze both pre- and post-synaptic components of synaptic transmission. We show examples of pre-synaptic localization of nAChRs and the live cell imaging of nAChR mediated Ca2+ signaling and neurotransmitter release along vHipp axons. A natural (and straightforward) extension of the protocol presented here is the preparation of pre- and post-synaptic contacts comprised of neurons from different genotypes. In this manner the contribution of a particular gene product to the pre- and/or post-synaptic mechanisms of modulation can be assessed directly.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</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>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

The preparation employed consists of gene chimeric co-cultures of vHipp&#8211;nAcc circuits in vitro. Projections emanating from vHipp microslices, as pre-synaptic axonal input, can make synaptic contacts with post-synaptic targets, the dispersed neurons from nAcc. Nicotine induced a sustained (&#8805; 30 min) facilitation of glutamatergic transmission from nAcc neurons innervated by vHipp axons21 and prolonged calcium signaling along vHipp axons5 via pre-synaptic &#945;7*nAChRs.
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Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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