Combining Voltage and Calcium Imaging from Neuronal Dendrites
- 251 Downloads
The ability to monitor membrane potential (V m) and calcium (Ca2+) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V m and Ca2+ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca2+ indicators. In all cases V m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca2+ optical signals depends on the indicator Ca2+ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 μM Bis-Fura-2, V m optical signals cannot be calibrated and the physiological Ca2+ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V m and Ca2+ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V m optical signals can be calibrated in terms of mV and Ca2+ optical signals quantitatively reveal the physiological changes in free Ca2+. Using these two examples, the method is explained in detail.
KeywordsVoltage-sensitive dyes Calcium-sensitive dyes Imaging Neuronal dendrites
This work was supported by the University of Basel and the NIH Grant RO1NS42739. We are grateful to Leslie Loew and Joe Wuskel for kindly providing voltage-sensitive dyes, and to Helene Pierre for technical help.
- Berger T, Borgdorff A, Crochet S, Neubauer FB, Lefort S, Fauvet B, Ferezou I, Carleton A, Luscher HR, Petersen CC (2007) Combined voltage and calcium epifluorescence imaging in vitro and in vivo reveals subthreshold and suprathreshold dynamics of mouse barrel cortex. J Neurophysiol 97:3751–3762PubMedCrossRefGoogle Scholar
- Borst JG, Helmchen F, Sakmann B (1995) Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J Physiol (Lond) 489:825–840Google Scholar
- Fierro L, Llano I (1996) High endogenous calcium buffering in Purkinje cells from rat cerebellar slices. J Physiol (Lond) 496:617–625Google Scholar
- Fisher JA, Barchi JR, Welle CG, Kim GH, Kosterin P, Obaid AL, Yodh AG, Contreras D, Salzberg BM (2008) Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. J Neurophysiol 99:1545–1553Google Scholar
- Kremer SG, Zeng W, Skorecki KL (1992) Simultaneous fluorescence measurement of calcium and membrane potential responses to endothelin. Am J Physiol 263:1302–1309Google Scholar
- Llinas R, Sugimori M (1980) Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. J Physiol (Lond) 305:197–213Google Scholar
- Martinez-Zaguilan R, Martinez GM, Lattanzio F, Gillies RJ (1991) Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2. Am J Physiol 260:297–307Google Scholar