Submillisecond Optical Reporting of Membrane Potential in Situ Using a Neuronal Tracer Dye

Overview

Journal J Neurosci

Specialty Neurology

Date 2009 Jul 24

PMID 19625510

Citations 50

Authors

Jonathan Bradley

Ray Luo

Thomas S Otis

David A DiGregorio

Affiliations

Soon will be listed here.

Abstract

A major goal in neuroscience is the development of optical reporters of membrane potential that are easy to use, have limited phototoxicity, and achieve the speed and sensitivity necessary for detection of individual action potentials in single neurons. Here we present a novel, two-component optical approach that attains these goals. By combining DiO, a fluorescent neuronal tracer dye, with dipicrylamine (DPA), a molecule whose membrane partitioning is voltage-sensitive, optical signals related to changes in membrane potential based on FRET (Förster resonance energy transfer) are reported. Using DiO/DPA in HEK-293 cells with diffraction-limited laser spot illumination, depolarization-induced fluorescence changes of 56% per 100 mV (tau approximately 0.1 ms) were obtained, while in neuronal cultures and brain slices, action potentials (APs) generated a Delta F/F per 100 mV of >25%. The high sensitivity provided by DiO/DPA enabled the detection of subthreshold activity and high-frequency APs in single trials from somatic, axonal, or dendritic membrane compartments. Recognizing that DPA can depress excitability, we assayed the amplitude and duration of single APs, burst properties, and spontaneous firing in neurons of primary cultures and brain slices and found that they are undetectably altered by up to 2 microm DPA and only slightly perturbed by 5 microm DPA. These findings substantiate a simple, noninvasive method that relies on a neuronal tracer dye for monitoring electrical signal flow, and offers unique flexibility for the study of signaling within intact neuronal circuits.

Citing Articles

Fxr1 Deletion from Cortical Parvalbumin Interneurons Modifies Their Excitatory Synaptic Responses.

Scheuer K, Jansson A, Shen M, Zhao X, Jackson M eNeuro. 2025; 12(1).

PMID: 39753370 PMC: 11735682. DOI: 10.1523/ENEURO.0363-24.2024.

Inter and intralaminar excitation of parvalbumin interneurons in mouse barrel cortex.

Scheuer K, Jansson A, Zhao X, Jackson M PLoS One. 2024; 19(6):e0289901.

PMID: 38870124 PMC: 11175493. DOI: 10.1371/journal.pone.0289901.

Velocity of conduction between columns and layers in barrel cortex reported by parvalbumin interneurons.

Scheuer K, Judge J, Zhao X, Jackson M Cereb Cortex. 2023; 33(17):9917-9926.

PMID: 37415260 PMC: 10656945. DOI: 10.1093/cercor/bhad254.

Inter and Intralaminar Excitation of Parvalbumin Interneurons in Mouse Barrel Cortex.

Scheuer K, Jansson A, Zhao X, Jackson M bioRxiv. 2023; .

PMID: 37398428 PMC: 10312540. DOI: 10.1101/2023.06.02.543448.

Imaging Voltage Globally and in Isofrequency Lamina in Slices of Mouse Ventral Cochlear Nucleus.

Ma Y, Shu W, Lin L, Cao X, Oertel D, Smith P eNeuro. 2023; 10(3).

PMID: 36792362 PMC: 9997695. DOI: 10.1523/ENEURO.0465-22.2023.

References

Dombeck D, Sacconi L, Blanchard-Desce M, Webb W . Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. J Neurophysiol. 2005; 94(5):3628-36. DOI: 10.1152/jn.00416.2005. View

Baker B, Lee H, Pieribone V, Cohen L, Isacoff E, Knopfel T . Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells. J Neurosci Methods. 2006; 161(1):32-8. DOI: 10.1016/j.jneumeth.2006.10.005. View

Honig M, Hume R . Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J Cell Biol. 1986; 103(1):171-87. PMC: 2113786. DOI: 10.1083/jcb.103.1.171. View

Cohen L, Salzberg B, Davila H, Ross W, Landowne D, Waggoner A . Changes in axon fluorescence during activity: molecular probes of membrane potential. J Membr Biol. 1974; 19(1):1-36. DOI: 10.1007/BF01869968. View

Morgan J, Wong R . Ballistic labeling with fluorescent dyes and indicators. Curr Protoc Neurosci. 2008; Chapter 2:Unit 2.11. DOI: 10.1002/0471142301.ns0211s43. View

Zhou W, Yan P, Wuskell J, Loew L, Antic S . Dynamics of action potential backpropagation in basal dendrites of prefrontal cortical pyramidal neurons. Eur J Neurosci. 2008; 27(4):923-36. PMC: 2715167. DOI: 10.1111/j.1460-9568.2008.06075.x. View

Petersen C, Grinvald A, Sakmann B . Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage-sensitive dye imaging combined with whole-cell voltage recordings and neuron reconstructions. J Neurosci. 2003; 23(4):1298-309. PMC: 6742278. View

Stuart G, Hausser M . Initiation and spread of sodium action potentials in cerebellar Purkinje cells. Neuron. 1994; 13(3):703-12. DOI: 10.1016/0896-6273(94)90037-x. View

Lu C, Kabakov A, Markin V, Mager S, Frazier G, Hilgemann D . Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp. Proc Natl Acad Sci U S A. 1995; 92(24):11220-4. PMC: 40603. DOI: 10.1073/pnas.92.24.11220. View

10.

Grinvald A, Hildesheim R . VSDI: a new era in functional imaging of cortical dynamics. Nat Rev Neurosci. 2004; 5(11):874-85. DOI: 10.1038/nrn1536. View

11.

Wu C, Russell R, Nguyen R, Karten H . Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit. Neuroscience. 2003; 117(4):831-45. DOI: 10.1016/s0306-4522(02)00833-3. View

12.

Dombeck D, Blanchard-Desce M, Webb W . Optical recording of action potentials with second-harmonic generation microscopy. J Neurosci. 2004; 24(4):999-1003. PMC: 6729824. DOI: 10.1523/JNEUROSCI.4840-03.2004. View

13.

Parsons T, Salzberg B, Obaid A, Kleinfeld D . Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons. J Neurophysiol. 1991; 66(1):316-33. DOI: 10.1152/jn.1991.66.1.316. View

14.

Reddy G, Kelleher K, Fink R, Saggau P . Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat Neurosci. 2008; 11(6):713-20. PMC: 2747788. DOI: 10.1038/nn.2116. View

15.

Blunck R, Chanda B, Bezanilla F . Nano to micro -- fluorescence measurements of electric fields in molecules and genetically specified neurons. J Membr Biol. 2006; 208(2):91-102. DOI: 10.1007/s00232-005-0822-z. View

16.

Palmer L, Stuart G . Site of action potential initiation in layer 5 pyramidal neurons. J Neurosci. 2006; 26(6):1854-63. PMC: 6793621. DOI: 10.1523/JNEUROSCI.4812-05.2006. View

17.

Oberhauser A, Fernandez J . Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion. Biophys J. 1995; 69(2):451-9. PMC: 1236270. DOI: 10.1016/S0006-3495(95)79918-0. View

18.

Cohen L, Salzberg B, Grinvald A . Optical methods for monitoring neuron activity. Annu Rev Neurosci. 1978; 1:171-82. DOI: 10.1146/annurev.ne.01.030178.001131. View

19.

Djurisic M, Antic S, Chen W, Zecevic D . Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones. J Neurosci. 2004; 24(30):6703-14. PMC: 6729725. DOI: 10.1523/JNEUROSCI.0307-04.2004. View

20.

Otsu Y, Bormuth V, Wong J, Mathieu B, Dugue G, Feltz A . Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope. J Neurosci Methods. 2008; 173(2):259-70. DOI: 10.1016/j.jneumeth.2008.06.015. View