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 the solution with 95% O2 and 5% CO2 at room temperature then add the MgCl2 and CaCl2 while continuing to aerate. Adjust the osmolarity to 300 mOsm with sucrose. The osmolarity is measured with a vapor pressure osmometer.</li>
	<li>Prepare 1 L of the recording media without the MgCl2 and CaCl2 (Table 2). Bubble the solution with 95% O2 and 5% CO2 then add the MgCl2 and CaCl2 while continuing to aerate. Adjust the pH to 7.2-7.4 with NaOH (10 N) and the osmolarity to 300 mOsm with sucrose. The addition of HEPES buffer is optional and was used to further stabilize pH.</li>
	<li>The slicing and recording solutions may be stored in the refrigerator for several days prior to use.</li>
	<li>Prepare 3% agarose and pour into a sterile Petri dish (6 mm deep) and refrigerate.</li>
	<li>Cut three cubes of agarose about 6 mm x 6 mm x 6 mm.</li>
	<li>Deeply anesthetize a four to eight week old Sprague-Dawley rat with isoflurane. Test for the depth of anesthesia. The depth of anesthesia is deemed appropriate when there is no withdrawal reflex of the hind limb following pinching of the footpad. Once appropriately anesthetized, the rat is euthanized by rapid decapitation.&#160;</li>
	<li>Rapidly remove the brain being careful to cut the optic nerves first, and place in ice-cold slicing solution saturated with 5% CO2 and 95% O2 and allow to cool for 2-3 min.</li>
	<li>Trim the brain. Place the brain using forceps on a moistened (with slicing solution) circular filter paper set on a glass Petri dish resting on ice.</li>
	<li>Cut the brain coronally to remove the remaining cerebellum and brain stem, then the rostral cortex and the sides, to leave a block of brain containing the hypothalamus carefully preserving the side containing SCN, optic nerves, and optic chiasm.</li>
	<li>Spread a thin layer of cyanoacrylic glue large enough to attach the brain and agarose blocks in an ice-cold but dry microtome-slicing chamber.</li>
	<li>Transfer the ice-cold brain block caudal side down onto the glue with the side containing the SCN positioned for cutting first with the optic nerves forming a &#34;V&#34; pointing up.</li>
	<li>Place the agarose blocks on the other 3 sides. Fill the chamber with ice-cold slicing buffer continuously bubbled with 5% CO2 and 95% O2. The cyanoacrylic glue will solidify with moisture holding the brain and agarose blocks in place.</li>
	<li>Cut coronal hypothalamic slices (220-250 &#181;m thick) containing the SCN with the vibrating blade microtome. Typically a slice is chosen where the optic chiasm is about 2-4 mm across, appearing like a white band with SCN just dorsal appearing as two small &#8220;indentations&#8221; in the optic chiasm on either side of the 3rd ventricle (Figure 1A).</li>
	<li>Place the slices in the recording chamber (36 &#176;C) mounted on the stage of a Leica DMLFS microscope. The chamber is filled with continuously flowing recording solution (2 ml/min) aerated with 95% O2 and 5% CO2 and preheated with an inline heater.</li>
	<li>Perform whole-cell patch clamp and calcium imaging recordings in SCN neurons 0.5-8 hr after slice preparation.</li>
</ol>
2. Patch Clamp Recording
<ol>
	<li>Prepare the electrode solution (intracellular) solution in half the total final volume of water.</li>
	<li>Adjust the solution to pH 7.3 with KOH (1 N).</li>
	<li>Add the remaining water. The final osmolarity of the solution should be in the range of 280-290 mOsm.</li>
	<li>Pass the electrode solution through a 0.2 &#181;m filter, aliquot (0.5 ml) into microfuge tubes, and store at -20 &#176;C.</li>
	<li>Prepare the calcium probe bis-fura-2 hexapotassium salt stock solution (10 mM) by mixing 1 mg bis-fura-2 hexapotassium salt with 99.29 &#181;l of 0.2 &#181;m filtered water.</li>
	<li>Aliquot into separate tubes (2-5 &#181;l each) and freeze (-20 &#176;C).</li>
	<li>Thaw an aliquot of internal solution and the Ca2+ probe shortly before use. Add 2 &#181;l of the bis-fura-2 stock solution to 400 &#181;l of the internal solution for a final 50 &#181;M calcium probe concentration. Briefly triturate to mix the solutions. The calcium probe concentration (10-250 &#181;M) varies depending on experimental design and imaging system. Note that bis-fura-2 is comprised of two linked fura-2 molecules.</li>
	<li>Pull whole-cell patch electrodes, in two stages to an outside tip diameter of approximately 1 &#181;m and resistances of 7-10 M&#937; when filled with the intracellular solution.</li>
	<li>Backfill the electrode with a few &#181;l of the internal solution containing the calcium probe filtered with a 4 mm syringe filter (0.2 &#181;m) for low volume samples via a Microfil tube.</li>
	<li>Apply a small amount of positive pressure to the electrode as it is guided into the recording chamber under red light illumination while being viewed on a video monitor.&#160;</li>
	<li>Place the microelectrode over the SCN using a low power objective (4X). The SCN is identified as the two translucent areas on either side of the third ventricle and dorsal to the optic chiasm (Figure 1A).</li>
	<li>Switch the microscope to a high power magnification (40X or 63X). Subregions of the SCN and individual SCN neurons can be targeted for recording at the higher magnification.</li>
	<li>Apply gentle positive pressure to the microelectrode as it is advanced onto the surface of a neuron.</li>
	<li>Apply gentle negative pressure to the microelectrode to form a seal with resistances of 3-10 G&#937;.</li>
	<li>Adjust the membrane voltage to -60 mV with the amplifier controlled with acquisition software.</li>
	<li>Apply additional negative pressure to the electrode to rupture the cell membrane to whole cell mode.</li>
	<li>Upon entering whole cell mode the SCN neuron soma and dendrites fill with fluorescent probe.</li>
	<li>Membrane voltage is measured in current-clamp mode or current in voltage-clamp mode while the calcium probe image data is being monitored and stored (described below). View acquired images along with a graph of 340 nm/380 nm ratio fluorescence data during the experiment.</li>
</ol>
3. Measurement of Intracellular Ca2+
<ol>
	<li>Quantitative Ca2+ measurements are obtained by recording a pair of images by rapidly exciting the tissue with UV light at 340 nm and 380 nm via a monochronometer with a 10 nm bandwidth and passed through a UG11 optical filter to restrict harmonic wavelengths above 400 nm, a 400 nm DCLP dichroic and emitted light through a 510&#177;40 nm emission filter.</li>
	<li>The images are acquired using a cooled CCD camera with acquisition time and binning adjusted to minimize photobleaching and maximize recording speed.</li>
	<li>Quantitative imaging software is used to acquire and display the images.</li>
	<li>Select regions of interest from the initial image to be converted to relative fluorescence intensity unit data (i.e.&#160;soma, dendrite,&#160;etc.). Also select a region for measurement of the background fluorescence (Figure 1B).</li>
	<li>Raw images and quantified optical data are continually saved during the experiment. After the experiment the saved images are generally reanalyzed when more time can be taken to select more accurately the regions of interest to be quantified.</li>
	<li>The data can be presented as the estimated intracellular calcium concentration (Est[Ca2+]i). The ratio (R) of emitted light following excitation and background subtraction at 340 nm divided by 380 nm along with the maximum and minimum fluorescence at 340 nm and 380 nm is used to calculate the calcium concentration4. Background subtraction and other calculations are performed using data analysis software.</li>
	<li>An in&#160;vitro determination of maximum and minimum bis-fura-2 fluorescence can be performed using 10 mM CaCl2 for Ca2+ bound and 10 mM EGTA for Ca2+ free conditions. The Ca2+ disassociation constant, Kd of the probe is taken to be 370 nM for bis-fura-2 (Invitrogen).</li>
</ol>

<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 authors declare that they have no competing financial interests.

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 consideration when using the method. First is the use of the whole cell patch configuration, which leads to the dialyzation of the cellular contents with the intracellular microelectrode solution. The dialyzation can lead to the rundown of voltage-gated ion channels and the loss of second messenger mediated signals24-26. Second, cell injury or damage to the gigaohm seal can result in increasing levels of calcium that may be associated with decreasing cellular input resistance24. Third, the intercellular solution containing the probe should be filtered just before filling the microelectrode to avoid plugging the microelectrode tip. By minimizing these factors, intracellular calcium and membrane electrical activity may be simultaneously recorded in patch clamped neurons for more than 30 min. We have had some success using the beta-Escin perforated patch method27 and bis-fura-2 to slow dialyzation and increase recording time. Fourth, calcium and a chelator EGTA or BAPTA are common constituents of intercellular solutions used to stabilize the intracellular calcium concentration. However, we have not included these under our recording conditions and dialyzing the neuron may alter calcium homeostasis and lower the basal calcium levels. We also did not add magnesium to the internal solution since it can also bind to fura-2. However, since fura-2 binds Mg2+ with a much lower affinity than calcium (Kd 5.6 mM)28 this may be optional. Fifth, the affinity of bis-fura-2 for calcium can be altered by a number of intracellular factors within a cell including pH and cell viscosity4,29. Finally, bis-fura-2 can buffer the intracellular calcium concentration and this should be considered when deciding on the probe concentration in the internal solution5. Thus the constituents of the internal solution may be modified based on neuronal type and conditions, experimental questions being addressed,&#160;and the equipment available for the study.
It is important to note that quantitative calcium imaging using bis-fura-2 typically uses the ultraviolet light wavelengths at 340 nm and 380 nm. Protective equipment must be used to avoid injury to eyes (UV eye protection) and skin (lab coats, etc.). In addition, UV producing bulbs may produce ozone requiring adequate ventilation especially important since such equipment is often located in small rooms where ambient light can be controlled.
The affinity of the calcium probe for calcium is an important consideration. If a high affinity probe is chosen then large calcium currents may saturate the probe leading to an under estimation of the magnitude of the calcium signal5. Whereas a low affinity probe may be more useful in regions where high calcium concentrations occur such as in distal dendritic processes and spines. Similarly, low affinity probes may miss changes at low calcium concentrations. In general, somatic changes of calcium can be measured using a quantitative ratiometric calcium probe such as bis-fura-2 or fura-2.
Data may be presented as a simple ratio quantified from a region of interest from a pair of images following background (BG) subtraction at each wavelength. While not as quantitative, ratiometric probes may also be excited at a single wavelength as one would use other single excitation wavelength probes for experiments requiring very fast recording time frames or if a monochronometer or other wavelength changing (e.g. filter wheel) device is not available.
We chose to use an in&#160;vitro calibration to obtain the parameters needed to estimate the intracellular calcium concentration along with a published Kd. However, more detailed techniques are available for in&#160;situ calibrations and to measure the Kd of the probe under specific experimental conditions. We used the excitation wavelengths of 340 nm and 380 nm for bis-fura-2 and estimated the intracellular Ca2+ concentration using the formula: Est[Ca2+]i = (R-Rmin)/(Rmax-R) x (Ff380/Fb380) x Kd, where F is fluorescence intensity at 380 nm of the probe in it&#39;s free (f) and bound (b) states, R is the emission ratio following excitation at 340 nm divided by the emission following excitation at 380 nm (i.e.&#160;F340/F380), Rmin is the minimum (Ff340/Ff380) and Rmax the maximum (Fb340/Fb380) ratio obtained in the free and bound states respectively, and Kd is the Ca2+ disassociation constant of the indicator4. Note that both Kd and (Ff380/Fb380) are scalar terms while Rmin and Rmax can affect the magnitude of the change in calcium. Care must be taken when estimating the concentration values for calcium. There are a number of unknowns that may be altered by the intracellular chemical environment that may affect the values in the equation.
Table 1. Slicing solution.
<table border="1">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td>Final Conc (mM)</td>
			<td>FW (g/M)</td>
			<td>Final (g/L)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>120</td>
			<td>58.44</td>
			<td>7.013</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
			<td>74.55</td>
			<td>0.186</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>5</td>
			<td>203.31</td>
			<td>1.017</td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>0.5</td>
			<td>147.02</td>
			<td>0.074</td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;H2O</td>
			<td>1.2</td>
			<td>137.99</td>
			<td>0.166</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>84.01</td>
			<td>2.184</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>10</td>
			<td>180.16</td>
			<td>1.802</td>
		</tr>
	</tbody>
</table>
Table 2. Recording solution.
<table border="1">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td>Final Conc (mM)</td>
			<td>FW (g/M)</td>
			<td>Final (g/L)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>120</td>
			<td>58.44</td>
			<td>7.013</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
			<td>74.55</td>
			<td>0.186</td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>1.2</td>
			<td>203.31</td>
			<td>0.244</td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>2.4</td>
			<td>147.02</td>
			<td>0.353</td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;H2O</td>
			<td>1.2</td>
			<td>137.99</td>
			<td>0.166</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>84.01</td>
			<td>2.184</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>10</td>
			<td>180.16</td>
			<td>1.802</td>
		</tr>
		<tr>
			<td>HEPES (optional)</td>
			<td>10</td>
			<td>238.3</td>
			<td>2.383</td>
		</tr>
	</tbody>
</table>
Table 3. Intracellular (microelectrode) solution.
<table border="1">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td>Final Conc (mM)</td>
			<td>FW (g/M)</td>
			<td>Final (mg/25 ml)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5</td>
			<td>74.55</td>
			<td>9.32</td>
		</tr>
		<tr>
			<td>Potassium D-gluconate</td>
			<td>140</td>
			<td>234.2</td>
			<td>819.70</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>238.3</td>
			<td>59.58</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate dipotassium salt</td>
			<td>4</td>
			<td>583.4</td>
			<td>58.34</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate tris salt</td>
			<td>0.4</td>
			<td>523.2</td>
			<td>5.23</td>
		</tr>
	</tbody>
</table>
Table of Specific Reagents and Equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific Co.</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific Co.</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;H2O</td>
			<td>Sigma Chemical Co.</td>
			<td>S-9638</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>Fisher Scientific Co.</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>Fisher Scientific Co.</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Fisher Scientific Co.</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific Co.</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific Co.</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium D-gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate tris salt</td>
			<td>Sigma-Aldrich</td>
			<td>G9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose (ultrapure)</td>
			<td>Life Technologies</td>
			<td>15510-027</td>
			<td>Gel pored into sterile Petri dish 6 mm thick layer</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>P-6310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S-5881</td>
			<td></td>
		</tr>
		<tr>
			<td>bis-fura-2, hexapotassium salt</td>
			<td>Invitrogen</td>
			<td>B6810</td>
			<td>cell impermeant</td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Alomone Labs</td>
			<td>T-550</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>P-1675</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td>Tissue slicing</td>
		</tr>
		<tr>
			<td>Cyanoacrylic glue (Roti-Coll1)</td>
			<td>Carl Roth GmbH+Co</td>
			<td>Art-Nr. 0258.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Narshige International USA</td>
			<td>PP-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Capillary Tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil 34g</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Corning</td>
			<td>#431212</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM LFS</td>
			<td>With 4X, 40 UV and 63X UV objectives, and epifluorescence</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA ER</td>
			<td>12 bit CCD</td>
		</tr>
		<tr>
			<td>Fura-2 Filter Cube</td>
			<td>Chroma</td>
			<td>71500A</td>
			<td>Set with UG11 filter</td>
		</tr>
		<tr>
			<td>Polychrome IV</td>
			<td>Till Photonics GmBH</td>
			<td></td>
			<td>Monochronometer</td>
		</tr>
		<tr>
			<td>Ultraviolet blocking safety glasses</td>
			<td>Ultra-Violet Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EPC-9 amplifier</td>
			<td>HEKA Eletronik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metafluor</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Imaging Software</td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>HEKA Eletronik</td>
			<td></td>
			<td>Data Acquisition Software</td>
		</tr>
		<tr>
			<td>Igor version 6</td>
			<td>Wavemetrics</td>
			<td></td>
			<td>Electrophysiology &#38; Ca2+ Data Analysis</td>
		</tr>
		<tr>
			<td>VAPRO 5520</td>
			<td>Westcor</td>
			<td></td>
			<td>Vapor pressure osmometer</td>
		</tr>
	</tbody>
</table>

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 the membrane creating the whole cell configuration. The neuron will rapidly fill with probe when viewed on the monitor. Dendritic processes in a similar focal plane as the soma can be visualized (Figure 1A inset). Alternatively, the focus could be adjusted to measure calcium changes in distal dendrites. Recording in voltage clamp mode at -60 mV we simulated action potential firing using brief (2 msec) steps to +20 mV at 10 Hz, which is in the physiological range for the daytime action potential firing rate of many SCN neurons (Figures 1B and&#160;1C). Different regions of interest (ROI) of the neuron including the proximal dendrite were quantified and the background at each wavelength was subtracted to determine the ratio of emitted fluorescence from excitation at 340 nm and 380 nm of light (Figure 1B, inset). The bis-fura-2 filled microelectrode can be seen but is mainly out of the focal plane. Pseudocolor applied to the image with software can sometimes facilitate visualization. Differences in the rate of intracellular calcium influx can be observed between different regions of the neuron and the magnitude of the response increased with depolarizing current steps. Combining location data from images taken with 63X&#160;and 4X&#160;objectives (Figure 1A) we can assess the anatomical location within the SCN from each neuron recorded.
Similarly, recording in current-clamp mode the intracellular calcium response to changes in membrane potential can be measured. A small change of current near the action potential firing threshold can induce action potential firing and a corresponding elevation of calcium (Figure 2). Recorded in the soma, the calcium concentration increase lags behind the changes in membrane voltage but decays after cessation of action potential firing to a new plateau. Using these methods we have previously shown that synaptically-evoked excitatory postsynaptic potentials (EPSPs) do not significantly alter somatic intracellular calcium without subsequently induced action potential firing22, suggesting that postsynaptic action potential firing is required for calcium to affect subsequent transcriptional and translation events which may represent a way these neurons differentiate synaptic input signaling at different times of the day and night.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/50794/50794fig1.jpg" width="400px" /> 
Figure 1. Calcium imaging of a voltage clamped SCN neuron. A: Bright field image of a coronal slice of hypothalamus containing the SCN and optic chiasm (OC). A glass microelectrode filled with a bis-fura-2 containing internal solution is sealed onto a single neuron. Inset: Recorded neuron with bis-fura-2 fluorescence (380 nm excitation) images in grayscale and pseudocolor. B: Calcium responses to a series of 50 voltage steps recorded from a voltage-clamped SCN neuron. Voltage-gated sodium currents were inhibited with TTX (0.5 &#181;M). Inset: Screen capture image of somatic and dendritic regions of interest corresponding to the calcium responses shown in the graph. C: Calcium responses to a variable number of voltage steps. The right recording is the same data shown in B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50794/50794fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50794/50794fig2.jpg" width="500px" /> 
Figure 2. Simultaneous recording of membrane voltage and intracellular calcium in an SCN neuron. The current-clamp experiment was performed in the presence of picrotoxin (50 &#181;M). A small depolarization of the membrane potential triggers several bouts of action potential firing with a corresponding rise and subsequent fall of intracellular calcium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50794/50794fig2highres.jpg" target="_blank">Click here to view larger image</a>.

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 ...

simultaneous-electrophysiological-recording-calcium-imaging

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.9K Views.  Oregon Health & Science University. 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.

Video: Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

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 anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

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

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

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

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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 seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

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

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

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

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

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 recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

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

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 researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

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 system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

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

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...