Abstract
Since the 1970s, a multitude of fluorescence-based methods for observing changes in neuronal membrane voltage have been developed, progressively improving in terms of signal sensitivity, speed, and targeting. While significant development has been seen in the past decade in engineering novel variants of genetically encoded voltage indicators (GEVI), synthetic dyes can, in many cases, be more straightforward and flexible tools to be used. Here, we describe in detail an improved dye-labeling method where the synthetic, pure electrochromic voltage-sensitive dye ANNINE-6plus is introduced into the brain region of interest via stereotactical injection in a mouse. Specifically, we have optimized the labeling protocol for effective voltage imaging of subthreshold activity in inferior olive. The homogenous labeling and excellent signal-to-noise ratio allow this method to reveal olivary subthreshold oscillations in high spatiotemporal resolution. In addition to the procedures related to dye introduction and optical imaging, we give a detailed description of the process of acute slice preparation from the mouse inferior olive.
Key words
- Inferior olive
- Voltage imaging
- ANNINE-6plus
- One-photon
- In vitro
- Hot slicing
- Stereotactic microinjection
This is a preview of subscription content, access via your institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Hodgkin AL, Huxley AF (1939) Action potentials recorded from inside a nerve fibre. Nature 44:710–711
Peterka PS, Takahashi H, Yuste R (2011) Imaging voltage in neurons. Neuron 69(1):9–21. https://doi.org/10.1016/j.neuron.2010.12.010
Cohen LB et al (1974) Changes in axon fluorescence during activity: molecular probes of membrane potential. J Membr Biol 19(1):1–36. https://doi.org/10.1007/BF01869968
Loew LM et al (1992) A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. J Membr Biol 130(1):1–10. https://doi.org/10.1007/BF00233734
Hübener G, Lambacher A, Fromherz P (2003) Anellated hemicyanine dyes with large symmetrical solvatochromism of absorption and fluorescence. J Phys Chem B 107:7896–7902. https://doi.org/10.1021/jp0345809
Fromherz P et al (2008) ANNINE-6plus, a voltage-sensitive dye with good solubility, strong membrane binding and high sensitivity. Eur Biophys J 37(4):509–514. https://doi.org/10.1007/s00249-007-0210-y
Kuhn B, Roome CJ (2019) Primer to voltage imaging with ANNINE dyes and two-photon microscopy. Front Cell Neurosci 13:321. https://doi.org/10.3389/fncel.2019.00321
Knöpfel T (2012) Genetically encoded optical indicators for the analysis of neuronal circuits. Nat Rev Neurosci 13(10):687–700. https://doi.org/10.1038/nrn3293
Roome CJ, Kuhn B (2019) Voltage imaging with ANNINE dyes and two-photon microscopy. NeuroMethods 13:321. https://doi.org/10.1007/978-1-4939-9702-2\13
Nilsson J, Panizza M, Hallett M (1993) Principles of digital sampling of a physiologic signal. In: Electroencephalography and clinical neurophysiology/ evoked potentials. Wiley, New York, pp 348–358
Kuhn B, Denk W, Bruno RM (2008) In vivo two-photon voltage- sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness. Proc Natl Acad Sci U S A 105(21):7588–7593. https://doi.org/10.1073/pnas.0802462105
Dalphin N et al (2020) Voltage imaging of cortical oscillations in layer 1 with two-photon microscopy. eNeuro 7(3):ENEURO.0274-19.2020. https://doi.org/10.1523/ENEURO.0274-19.2020
De Zeeuw CI et al (1998) Microcircuitry and function of the inferior olive. Trends Neurosci 21(9):391–400. https://doi.org/10.1016/s0166-2236(98)01310-1
Llinás R, Baker R, Sotelo C (1974) Electrotonic coupling between neurons in cat inferior olive. J Neurophysiol 37(3):560–571. https://doi.org/10.1152/jn.1974.37.3.560
Lampl I, Y. Yarom Y. (1993) Subthreshold oscillations of the membrane potential: A functional synchronizing and timing device. J Physiol 70(5):2181–2186. https://doi.org/10.1152/jn.1993.70.5.2181
De Zeeuw CI et al (1990) Intracellular labeling of neurons in the medial accessory olive of the cat: II ultrastructure of dendritic spines and their gabaergic innervation. J Comp Neurol 300(4):478–494. https://doi.org/10.1002/cne.903000404
Leznik E, Makarenko V, Llinás R. (2002) Electrotonically mediated oscillatory patterns in neuronal Ensembles: An in vitro voltage-dependent dye-imaging study in the inferior olive. J Neurosci 22(7):2804–2815. https://doi.org/10.1523/jneurosci.22-07-02804.2002
Devor A, Yarom Y (2002) Coherence of subthreshold activity in coupled inferior Olivary neurons. Ann N Y Acad Sci 978:508. https://doi.org/10.1111/j.1749-6632.2002.tb07594.x
Leznik E, Llinás R. (2005) Role of gap junctions in synchronized neuronal oscillations in the inferior olive. J Neurophysiol 94(4):2447–2456. https://doi.org/10.1152/jn.00353.2005
Dorgans K et al (2019) Short-term plasticity at cerebellar granule cell to molecular layer interneuron synapses expands information processing. Elife 8:e41586. https://doi.org/10.7554/eLife.41586
Candelas M et al (2019) Cav3.2 T-type calcium channels shape electrical firing in mouse lamina II neurons. Sci Rep 9(1):3112. https://doi.org/10.1038/s41598-019-39703-3
Eguchi K et al (2020) Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions. Front Cell Neurosci 14:63. https://doi.org/10.3389/fncel.2020.00063
Ankri L, Yarom Y, Uusisaari MY (2014) Slice it hot: acute adult brain slicing in physiological temperature. J Vis Exp 92:e52068. https://doi.org/10.3791/52068
Huang S, Uusisaari MY (2013) Elevated temperature during slicing enhances acute slice preparation quality. Front Cell Neurosci 7:48. https://doi.org/10.3389/fncel.2013.00048
Dorgans K, Kuhn B, Uusisaari MY (2020) Imaging subthreshold voltage oscillation with cellular resolution in the inferior olive in vitro. Front Cell Neurosci 14:440. https://doi.org/10.3389/fncel.2020.607843
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dorgans, K., Kuhn, B., Uusisaari, M.Y. (2022). In Vitro Voltage Imaging of Subthreshold Activity in Inferior Olive Neurons with ANNINE-6plus. In: Sillitoe, R.V. (eds) Measuring Cerebellar Function. Neuromethods, vol 177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2026-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2026-7_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2025-0
Online ISBN: 978-1-0716-2026-7
eBook Packages: Springer Protocols