Journal of Neuroscience Methods 164 (2007) 255–260 Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons Ulli H ¨ oger, P¨ aivi H. Torkkeli, Andrew S. French ∗ Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Received 19 March 2007; received in revised form 5 May 2007; accepted 5 May 2007 Abstract In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70 nM and rose to a maximum of about 400 nM at firing rates above 20 action potentials per second. © 2007 Elsevier B.V. All rights reserved. Keywords: Calcium; Ratiometric; Mechanosensory; Sensory neurons; Dye; Fluorescence; Invertebrate; Action potential 1. Introduction Calcium ions play important roles in mechanotransduc- tion by auditory and vestibular hair cells, entering through transduction channels and regulating dynamic sensitivity via several mechanisms (Fettiplace and Ricci, 2003; Hudspeth et al., 2000). Evidence for calcium involvement in vertebrate cuta- neous mechanoreception is indirect, based on morphological labeling of calcium ions, calcium pumps and calcium binding proteins (Pawson et al., 2000; Vega, 1996). Calcium is thought to enter crayfish stretch receptor neurons through the mechani- cally activated ion channels and cause adaptation by acting on calcium-activated potassium channels (Erxleben, 1993). Similar calcium feedback adaptation mechanisms have been proposed in bullfrog vestibular hair cells (Hudspeth and Lewis, 1988) and in cockroach tactile spine neurons (Torkkeli and French, 1995). The compound slit sense organ VS-3 in the patella of the spider, Cupiennius salei, consists of seven to eight slits each innervated by a pair of bipolar mechanosensory neurons ∗ Corresponding author. Tel.: +1 902 494 1302; fax: +1 902 494 2050. E-mail address: andrew.french@dal.ca (A.S. French). (French et al., 2002). VS-3 neurons are accessible to intracellu- lar recording and mechanical stimulation in the periphery, where mechanotransduction occurs (Juusola et al., 1994). VS-3 neu- rons also possess a low-voltage-activated calcium current that is large enough to support calcium-dependent action potentials when other voltage-activated currents are blocked (Sekizawa et al., 2000). However, calcium does not seem to regulate adapta- tion of these neurons (H¨ oger and French, 2005; Sekizawa et al., 2000), and its possible roles in sensory transduction, adaptation or other cellular functions are unknown. In an earlier study using Oregon Green BAPTA-1 as a cal- cium indicator, we showed that calcium is elevated in VS-3 neurons during mechanotransduction via the opening of voltage- activated calcium channels by action potentials (H¨ oger et al., 2005). No evidence could be found for entry through mechani- cally activated channels or release from internal stores. However, we were unable to provide reliable estimates of resting or ele- vated calcium concentrations. We have now developed a ratiometric method of calcium concentration estimation in VS-3 neurons during mechanotrans- duction using Fura Red. Ratiometric measurements allow more accurate calcium estimation by removing dependence on the concentration of dye in the cell, which allows calibration to 0165-0270/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2007.05.006