Name: simultaneous electrophysiological recording and calcium imaging of suprachiasmatic nucleus neurons
Uploaded: 2013-12-08T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons at JoVE.com

The work was funded by a grant from the National Institute of General Medical Sciences (GM096972).

1. Preparation of Hypothalamic Brain Slices
<ol>
	<li>Obtain, in advance, Institutional Animal Care and Use Committee (IACUC) approval for any procedure involving animals. The animal procedures described here are consistent with the AVMA Guidelines for the Euthanasia of Animals 2013 and were approved in advance by the Oregon Health &#38; Science University IACUC.</li>
	<li>Prepare 500 ml of the slicing buffer without the MgCl2 and CaCl2 (Table 1). Bubble th...

<ol>
	<li>Bading, H., Ginty, D. D., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+gene+expression+in+hippocampal+neurons+by+distinct+calcium+signaling+pathways.">Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.</a> Science. 260, 181-186 (1993).</li><li>Ghosh, A., Ginty, D. D., Bading, H., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+regulation+of+gene+expression+in+neuronal+cells.">Calcium regulation of gene expression in neuronal cells.</a> J. Neurobiol. 25, 294-303 (1994).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high+resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260, 3440-3450 (1985).</li><li>Higley, M. J., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+dendrites+and+spines:+practical+and+functional+considerations.">Calcium signaling in dendrites and spines: practical and functional considerations.</a> Neuron. 59, 902-913 (2008).</li><li>Colwell, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+calcium+levels+in+cells+in+the+suprachiasmatic+nucleus.">Circadian modulation of calcium levels in cells in the suprachiasmatic nucleus.</a> Eur. J. Neurosci. 12, 571-576 (2000).</li><li>Ikeda, M., Yoshioka, T., Allen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+circadian+changes+in+Ca2++mobilization+mediated+by+GABAA+and+NMDA+receptors+in+the+suprachiasmatic+nucleus.">Developmental and circadian changes in Ca2+ mobilization mediated by GABAA and NMDA receptors in the suprachiasmatic nucleus.</a> Eur. J. Neurosci. 17, 58-70 (2003).</li><li>Irwin, R. P., Allen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+signaling+induces+divergent+neuronal+Ca2++responses+in+the+suprachiasmatic+nucleus+network.">GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network.</a> Eur. J. Neurosci. 30, 1462-1475 (2009).</li><li>Berson, D. M., Dunn, F. A., Takao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+by+retinal+ganglion+cells+that+set+the+circadian+clock.">Phototransduction by retinal ganglion cells that set the circadian clock.</a> Science. 295, 1070-1073 (2002).</li><li>Hattar, S., Liao, H. W., Takao, M., Berson, D. M., Yau, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanopsin-containing+retinal+ganglion+cells:+architecture,+projections,+and+intrinsic+photosensitivity.">Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity.</a> Science. 295, 1065-1070 (2002).</li><li>Hartwick, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Evoked+Calcium+Responses+of+Isolated+Melanopsin-Expressing+Retinal+Ganglion+Cells.">Light-Evoked Calcium Responses of Isolated Melanopsin-Expressing Retinal Ganglion Cells.</a> J. Neurosci. 27, 13468-13480 (2007).</li><li>Castel, M., Belenky, M., Cohen, S., Ottersen, O. P., Storm-Mathisen, 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-like+immunoreactivity+in+retinal+terminals+of+the+mouse+suprachiasmatic+nucleus.">Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus.</a> Eur. J. Neurosci. 5, 368-381 (1993).</li><li>Card, J. P., Moore, R. Y., Reppert, S. M., Klein, D. C., Moore, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+visual+circuits+influencing+the+circadian+activity+of+the+suprachiasmatic+nucleus.">The organization of visual circuits influencing the circadian activity of the suprachiasmatic nucleus.</a> In Suprachiasmatic Nucleus - The Mind's Clock. , 51-76 (1991).</li><li>Shigeyoshi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+resetting+of+a+mammalian+circadian+clock+is+associated+with+rapid+induction+of+the+mPer1+transcript.">Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript.</a> Cell. 91, 1043-1053 (1997).</li><li>Albrecht, U., Sun, Z. S., Eichele, G., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+response+of+two+putative+mammalian+circadian+regulators,+mper1+and+mper2,+to+light.">A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light.</a> Cell. 91, 1055-1064 (1997).</li><li>Yan, L., Silver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+induction+and+localization+of+mPer1+and+mPer2+during+advancing+and+delaying+phase+shifts.">Differential induction and localization of mPer1 and mPer2 during advancing and delaying phase shifts.</a> Eur. J. Neurosci. 16, 1531-1540 (2002).</li><li>Lowrey, P. L., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+the+mammalian+circadian+system:+Photic+entrainment,+circadian+pacemaker+mechanisms,+and+posttranslational+regulation.">Genetics of the mammalian circadian system: Photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation.</a> Annu. Rev. Genet. 34, 533-562 (2000).</li><li>Ding, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+biological+clock:+Mediation+of+nocturnal+circadian+shifts+by+glutamate+and+NO.">Resetting the biological clock: Mediation of nocturnal circadian shifts by glutamate and NO.</a> Science. 266, 1713-1717 (1994).</li><li>Ding, J. M., Fairman, L. E., Hurst, W. J., Kuriashkina, L. R., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+biological+clock:+mediation+of+nocturnal+CREB+phosphorylation+via+light,+glutamate,+and+nitric+oxide.">Resetting the biological clock: mediation of nocturnal CREB phosphorylation via light, glutamate, and nitric oxide.</a> J. Neurosci. 17, 667-675 (1997).</li><li>Ding, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+ryanodine+receptor+mediates+light-induced+phase+delays+of+the+circadian+clock.">A neuronal ryanodine receptor mediates light-induced phase delays of the circadian clock.</a> Nature. 394, 381-384 (1998).</li><li>Bollmann, J. H., Helmchen, F., Borst, J. G., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+Ca2++influx+mediated+by+three+different+pathways+during+synaptic+transmission+at+a+calyx-type+synapse.">Postsynaptic Ca2+ influx mediated by three different pathways during synaptic transmission at a calyx-type synapse.</a> J. Neurosci. 18, 10409-10419 (1998).</li><li>Irwin, R. P., Allen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Response+to+Retinohypothalamic+Tract+Synaptic+Transmission+in+Suprachiasmatic+Nucleus+Neurons.">Calcium Response to Retinohypothalamic Tract Synaptic Transmission in Suprachiasmatic Nucleus Neurons.</a> J. Neurosci. 27, 11748-11757 (2007).</li><li>Smetters, D., Majewska, A., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+action+potentials+in+neuronal+populations+with+calcium+imaging.">Detecting action potentials in neuronal populations with calcium imaging.</a> Methods. 18, 215-221 (1999).</li><li>Spruston, N., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforated+patch-clamp+analysis+of+the+passive+membrane+properties+of+three+classes+of+hippocampal+neurons.">Perforated patch-clamp analysis of the passive membrane properties of three classes of hippocampal neurons.</a> J. Neurophysiol. 67, 508-528 (1992).</li><li>Horn, R., Marty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscarinic+activation+of+ionic+currents+measured+by+a+new+whole-cell+recording+method.">Muscarinic activation of ionic currents measured by a new whole-cell recording method.</a> J. Gen. Physiol. 92, 145-159 (1988).</li><li>Marty, A., Neher, E., Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight-seal+whole-cell+recording.">Tight-seal whole-cell recording.</a> Single-channel recording. 2, 31-52 (1995).</li><li>Sarantopoulos, C., McCallum, J. B., Kwok, W. M., Hogan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-escin+diminishes+voltage-gated+calcium+current+rundown+in+perforated+patch-clamp+recordings+from+rat+primary+afferent+neurons.">Beta-escin diminishes voltage-gated calcium current rundown in perforated patch-clamp recordings from rat primary afferent neurons.</a> J. Neurosci. Methods. 139, 61-68 (2004).</li><li>Hatae, J., Fujishiro, N., Kawata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+properties+of+fluorescence+dye+fura-2+with+various+divalent+cations.">Spectroscopic properties of fluorescence dye fura-2 with various divalent cations.</a> Jap. J. Physiol. 46, 423-429 (1996).</li><li>Poenie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+intracellular+Fura-2+fluorescence+by+viscosity:+a+simple+correction.">Alteration of intracellular Fura-2 fluorescence by viscosity: a simple correction.</a> Cell Calcium. 11, 85-91 (1990).</li></ol>

The methods described above provide a powerful tool to simultaneously record the link between neuron membrane electrical activity and the intracellular calcium concentration. The method has a number of strengths in that it combines two very well characterized methods - whole cell patch clamp recording and measurement of intracellular calcium using fluorescent dyes. Our approach is similar to those described by other investigators6,23.
A number of items must be taken into ...

Changes in gene expression are known to occur in neurons as a consequence of synaptic signaling. Signaling by the excitatory neurotransmitter glutamate can depolarize the neuronal membrane potential eventually leading to gene transcription and translation1,2. Activation of ionotropic receptors by glutamate allows extracellular calcium ions to enter the cell, which is thought to play a critical role as a second messenger in activating gene transcription. Evaluating the relationship between membrane electrical activity, such as action potential firing frequency, and changes of intracellular calcium concentration requires the combination of two methods - whole cell patch clamping and quantitative imaging of fluorescent calcium probes3-5, allowing the relationship to be studied in individual neurons. The single cell recording technique allows the recording of the activity of individual neurons in identifiable portions of the brain. The whole cell recording technique allows the membrane voltage or current to be controlled allowing for experimental manipulation of specific ion channel currents. Using micropipettes filled with fluorescent calcium probes also ensures that the neuron is well filled with calcium probe. This technique has a clear advantage when working with brain slice preparations from adult slice preparations, since these neurons are particularly difficult to load using the more common cell permeant probes and reduces potential background fluorescence issues6-8.
Light is the principal way mammals adjust their circadian clock, which is located in the hypothalamic suprachiasmatic nucleus (SCN). Light information transduced in the retina is transmitted 9-11 via the retinohypothalamic tract (RHT) where glutamate is released in the SCN12,13. Glutamate opens NMDA and AMPA ionotropic receptors located on SCN neurons producing an influx of calcium and sodium, and initiating an intracellular signaling cascade that ultimately leads to altering the expression of a family of clock genes14-17 and shifts in phase of the circadian clock18-20. However, calcium can enter neurons either directly through ionotropic glutamate receptors or through membrane depolarization and activation of voltage-dependent calcium channels (VDCC)21. We therefore developed an experimental protocol to investigate the relationship between the intracellular calcium concentration and membrane electrical activity in SCN neurons, such as occurs with action potential firing and from synaptic input22.

<table border="0" cellpadding="0" cellspacing="0" width="492">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">NaCl</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">S271-3</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">KCl</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">P217-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">NaH2PO4&#8226;H2O</td>
			<td style="width:123px;">Sigma Chemical Co.</td>
			<td style="width:123px;">S-9638</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">M33-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">C79-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">D-glucose</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">D16-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">NaHCO3</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">S233-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">Sucrose</td>
			<td style="width:123px;">Fisher Scientific Co.</td>
			<td style="width:123px;">S5-500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:123px;">Potassium D-gluconate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">G4500</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">HEPES</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">H4034</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="66">
			<td height="86" rowspan="2" style="height:87px;width:123px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">A8937</td>
			<td rowspan="2" style="width:123px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;"></td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:123px;">Guanosine 5&#8242;-triphosphate tris salt</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">G9002</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:123px;">Agarose (ultrapure)</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:123px;">15510-027</td>
			<td style="width:123px;">Gel pored into sterile Petri dish 6 mm thick layer</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">KOH</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">P-6310</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">NaOH</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">S-5881</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Bis-fura-2, hexapotassium salt</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">B6810</td>
			<td style="width:123px;">Cell impermeant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Name of Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td style="width:123px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">Microtome</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">VT1000S</td>
			<td style="width:123px;">Tissue slicing</td>
		</tr>
		<tr height="21">
			<td height="50" rowspan="2" style="height:51px;width:123px;">Cyanoacrylic glue (Roti-Coll1)</td>
			<td rowspan="2" style="width:123px;">Carl Roth GmbH+Co</td>
			<td rowspan="2" style="width:123px;">Art-Nr. 0258.1</td>
			<td rowspan="2" style="width:123px;"></td>
		</tr>
		<tr height="29">
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:123px;">Microelectrode puller</td>
			<td style="width:123px;">Narshige International USA</td>
			<td style="width:123px;">PP-83</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:123px;">Microelectrode Capillary Tubes</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">1B150F-4</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:123px;">Microfil 34 g</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">MF34G-5</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">Syringe filter</td>
			<td style="width:123px;">Corning</td>
			<td style="width:123px;">#431212</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">Microscope</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">DM LFS</td>
			<td style="width:123px;">With 4X, 40X UV and 63X UV objectives, and epifluorescence</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">CCD Camera</td>
			<td style="width:123px;">Hamamatsu</td>
			<td style="width:123px;">ORCA ER</td>
			<td style="width:123px;">12 bit CCD</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">Fura-2 Filter Cube</td>
			<td style="width:123px;">Chroma</td>
			<td style="width:123px;">71500A</td>
			<td style="width:123px;">Set with UG11 filter</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:123px;">Polychrome IV</td>
			<td style="width:123px;">Till Photonics GmBH</td>
			<td style="width:123px;"></td>
			<td style="width:123px;">Monochronometer</td>
		</tr>
		<tr height="38">
			<td height="55" rowspan="2" style="height:56px;width:123px;">Ultraviolet blocking safety glasses</td>
			<td rowspan="2" style="width:123px;">Ultra-Violet Products</td>
			<td rowspan="2" style="width:123px;"></td>
			<td rowspan="2" style="width:123px;"></td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:123px;">EPC-9 amplifier</td>
			<td style="width:123px;">HEKA Eletronik</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:123px;">Metafluor</td>
			<td style="width:123px;">Molecular Devices</td>
			<td style="width:123px;"></td>
			<td style="width:123px;">Imaging Software</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Patchmaster</td>
			<td style="width:123px;">HEKA Eletronik</td>
			<td style="width:123px;"></td>
			<td style="width:123px;">Data Acquisition Software</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:123px;">Igor version 6</td>
			<td style="width:123px;">Wavemetrics</td>
			<td style="width:123px;"></td>
			<td style="width:123px;">Electrophysiology &#38; Ca2+ Data Analysis</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:123px;">VAPRO 5520</td>
			<td style="width:123px;">Westcor</td>
			<td style="width:123px;"></td>
			<td style="width:123px;">Vapor pressure osmometer</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and calcium imaging of suprachiasmatic nucleus neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Using hypothalamic brain slices, we simultaneously recorded changes in intracellular calcium in whole cell mode under both voltage and current clamp conditions. The microelectrode shown in Figure 1A is lowered into position under high magnification (40X&#160;or 63X&#160;UV objectives) with a small amount of applied positive pressure. After touching the neuron, a gigaohm seal is formed with gentle suction. In voltage-clamp mode after setting the cell membrane potential to -60 mV, additional suction breaks...

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons at JoVE.com

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Research

JoVE Journal

Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...