Name: neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations
Uploaded: 2011-06-17T12:00:00
Duration: 4 min 50 s
Description: Watch this Scientific Journal Video about Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations at JoVE.com

We would like to thank Donald Hutson for contributing his technical expertise and great skill during the design and fabrication of the stage-top incubator. We are also grateful to Ayda Dashtaei for her excellent technical assistance. All work was supported by Neurosciences Research Foundation.

<ol>
	<li>Ligon, L. A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+mitochondria+in+the+axons+and+dendrites+of+cultured+hippocampal+neurons.">Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.</a> J Comp Neurol. 427, 340-350 (2000).</li><li>Miller, K. E., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+coherent+low+velocity+axonal+transport+of+mitochondria.">Direct evidence for coherent low velocity axonal transport of mitochondria.</a> J Cell Biol. 173, 373-381 (2006).</li><li>Macaskill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miro1+is+a+calcium+sensor+for+glutamate+receptor-dependent+localization+of+mitochondria+at+synapses.">Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.</a> Neuron. 61, 541-555 (2009).</li><li>Morris, R. L., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+mitochondria+along+microtubules+and+F-actin+in+living+vertebrate+neurons.">Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.</a> J Cell Biol. 131, 1315-1326 (1995).</li><li>Rintoul, G. L., Filiano, A. J., Brocard, J. B., Kress, G. J., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+decreases+mitochondrial+size+and+movement+in+primary+forebrain+neurons.">Glutamate decreases mitochondrial size and movement in primary forebrain neurons.</a> J Neurosci. 23, 7881-7888 (2003).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in neuroscience. , (1999).</li><li>Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for neural cell culture. , (2001).</li><li>Chen, S., Owens, G. C., Crossin, K. L., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+stimulates+mitochondrial+transport+in+hippocampal+neurons.">Serotonin stimulates mitochondrial transport in hippocampal neurons.</a> Mol Cell Neurosci. 36, 472-483 (2007).</li><li>Chen, S., Owens, G. C., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+inhibits+mitochondrial+motility+in+hippocampal+neurons.">Dopamine inhibits mitochondrial motility in hippocampal neurons.</a> PLoS One. 3, e2804-e2804 (2008).</li><li>Chen, S., Owens, G. C., Makarenkova, H., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+regulates+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 regulates mitochondrial transport in hippocampal neurons.</a> PLoS One. , e10848-e10848 (2010).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol Ther. 1, 31-38 (2000).</li></ol>

No conflicts of interest declared.

Employing lentivirus-mediated expression of a fluorescent protein targeted to mitochondria in infected cultured neurons and an inexpensive lab-built stage-top incubator that allows imaging of live cells for extended durations, we have been able to investigate the link between mitochondrial movement and neuromodulatory signals, such as serotonin (5-HT), dopamine (DA), and acetylcholine (ACh). Our studies have helped to elucidate a signaling pathway that, for the first time, links mitochondrial trafficking to changes in the activity of neurons-modulated by neurotransmitters such as 5-HT and DA--that are at the heart of neural function. We find that the use of targeted fluorescent proteins permits the observation of labeled mitochondria in living cultured neurons over extended periods that may be more physiologically relevant than the much shorter durations that are possible using vital dyes. Furthermore, the intensity of the fluorescent protein signal allows us to keep exposure times short during image acquisition, minimizing the possibility of photobleaching or phototoxicity. Finally, a simple and inexpensive stage-top incubator that maintains ambient temperature, humidity, and CO2 levels, while minimizing the evaporation of media, allows us to follow mitochondrial movement in living neurons over hours or even days. Researchers who wish to fabricate a stage-top incubator for the long-term observations of mitochondria in live neurons need not follow the precise details of our design, provided that the properties of the materials used (e.g., gas permeable membrane to avoid evaporation of media) and principles applied (e.g., temperature and humidity control, buffering of pH, maintenance of osmolarity) are generally consistent with what is described in this protocol.

<table border="1"><tbody>
	<tr>
 	<th>Quantity</th>
 <th>Description (and Location in Figure 1) </th>
 </tr>
 <tr>
 	<td>1</td>
 <td>Tissue culture incubator (H)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Heat-resistant plastic enclosure (delrin or comparable) (A)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Plastic frame for 35mm GBM lid (for affixing membrane to petri dish) (M)</td>
 </tr>
 <tr>
 	<td>1-2</td>
 <td>linear ft. Clear PFTE (Teflon) membrane material</td>
 </tr>
 <tr>
 	<td>4</td>
 <td>Brass thumb screws</td>
 </tr>
 <tr>
 	<td>2</td>
 <td>Small 10kOhm heatsink resistors (used as heating elements inside stage-top incubator enclosure)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Transformer (supply of 9V current to resistors)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Terrarium temperature controller and probe (thermostatic regulation of power to heatsink resistors via transformer)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>2000ml Erlenmeyer flask (muffler for vibration suppression in hose before stage-top incubator) (K)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>1/8" sorbothane sheet (gasket material for base of stage-top incubator)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Airtight ABS (or comparable) plastic box (housing for aquarium pump) (J)</td>
 </tr>
 <tr>
 	<td>1</td>
 <td>Aquarium pump (I)</td>
 </tr>
	<tr>
 	<td>1</td>
 <td>Small computer cooling fan</td>
 </tr>
	<tr>
 	<td>1</td>
 <td>Plastic enclosure for cooling fan (L)</td>
 </tr>
	<tr>
 	<td>1</td>
 <td>9V transformer for computer cooling fan</td>
 </tr>
	<tr>
 	<td>20-30ft</td>
 <td>Nalgene or silicone hose (connections between tissue culture and stage-top incubators; moisture traps) (F,G,N)</td>
 </tr>
	<tr>
 	<td>2</td>
 <td>Plastic stopcocks (to open and close air flow before and after stage-top incubator) (O)</td>
 </tr>
	<tr>
 	<td>4</td>
 <td>Barbed brass hose connectors (for hose connections to/from stage-top incubator and aquarium pump enclosure)</td>
 </tr>
 <tr>
 	<td>2</td>
 <td>Brass quick couplers (for hose connections to/from stage-top incubator)</td>
 </tr>
 <tr>
 	<td>2</td>
 <td>Brass quick couplers (for hose connections to/from stage-top incubator)</td>
 </tr>
</tbody></table>
 Table 1. Stage-top incubator parts:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma-Aldrich</td>
 <td>P7280-5MG</td>
 </tr>
 <tr>
 <td>laminin</td>
 <td>Roche Applied Science</td>
 <td>11243217001</td>
 </tr>
 <tr>
 <td>35 mm glass-bottom dishes</td>
 <td>MatTek</td>
 <td>P35GC-0-14-C</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Life Technologies</td>
 <td>10567</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Life Technologies</td>
 <td>17504-044</td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Life Technologies</td>
 <td>35050</td>
 </tr>
 <tr>
 <td>Lipid-rich BSA</td>
 <td>Life Technologies</td>
 <td>11020-021</td>
 </tr>
 <tr>
 <td>L-Asparagine</td>
 <td>Sigma-Aldrich</td>
 <td>P0380-100G</td>
 </tr>
 <tr>
 <td>L-Proline</td>
 <td>Sigma-Aldrich</td>
 <td>A8381-100G</td>
 </tr>
 <tr>
 <td>Vitamin B-12</td>
 <td>Sigma-Aldrich</td>
 <td>V2876-100MG</td>
 </tr>
 <tr>
 <td>5-HT</td>
 <td>Sigma-Aldrich</td>
 <td>H9523-25MG</td>
 </tr>
 <tr>
 <td>8-OH-DPAT</td>
 <td>Sigma-Aldrich</td>
 <td>H8520-25MG</td>
 </tr>
 <tr>
 <td>Dopamine</td>
 <td>Sigma-Aldrich</td>
 <td>H8502-5G</td>
 </tr>
 <tr>
 <td>Bromocriptine</td>
 <td>Sigma-Aldrich</td>
 <td>B2134-25MG</td>
 </tr>
 <tr>
 <td>SKF38393</td>
 <td>Sigma-Aldrich</td>
 <td>D047-100MG</td>
 </tr>
 </tbody>
</table>

neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

Watch this Scientific Journal Video about Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations at JoVE.com

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

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