J Comp Physiol A (2014) 200:53–76 DOI 10.1007/s00359-013-0861-3
Original Paper
Mechanisms underlying odorant‑induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus Tizeta Tadesse · Charles D. Derby · Manfred Schmidt
Received: 18 June 2013 / Revised: 3 October 2013 / Accepted: 4 October 2013 / Published online: 1 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca2+ imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca2+ signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations in the somata of olfactory receptor neurons. Both Ca2+ signals are primarily mediated by an influx of extracellular Ca2+ through voltage-activated Ca2+ channels that can be blocked by CoCl2 and the L-type Ca2+ channel blocker verapamil. Intracellular Ca2+ stores contribute little to odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations. The odorant-induced Ca2+ transients as well as the spontaneous Ca2+ oscillations depend on action potentials mediated by Na+ channels that are largely TTXinsensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of T. Tadesse · C. D. Derby · M. Schmidt (*) Neuroscience Institute and Department of Biology, Georgia State University, P.O. Box 5030, Atlanta, GA 30302‑5030, USA e-mail: [email protected]; [email protected] Present Address: T. Tadesse Department of Neurology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
Ca2+ signals represent experimentally accessible proxies of spiking. Keywords Ca imaging · Olfaction · Oscillation · Arthropod · Crustacean Abbreviations ΔF/F Relative change in fluorescence intensity NMDG N-methyl-d-glucamine OGB Oregon Green 488 BAPTA-1 AM ORN Olfactory receptor neuron SERCA Sarco/endoplasmic reticulum Ca2+ ATPase pump TM TetraMarine extract TTX Tetrodotoxin
Introduction Olfactory receptor neurons (ORNs) across numerous vertebrate and invertebrate species show robust odorant-induced changes in intracellular Ca2+ that can be readily assessed in different cellular compartments by Ca2+ imaging. Ca2+ imaging was first used to study responsiveness in catfish ORNs to odorants (Restrepo et al. 1990), and has since been used to show that vertebrate ORNs have rapid increases in intracellular Ca2+ in the cilia that are associated with olfactory transduction and are mediated by cAMP-gated channels (Leinders-Zufall et al. 1997, 1998). The rise in intraciliary Ca2+ amplifies the depolarizing receptor potential through activation of Cl− channels, and it mediates odor adaptation (Menini 1999; Matthews and Reisert 2003). Odorant-induced increases in intracellular Ca2+ occur in the somata of ORNs in a wide range of species (fish: Restrepo et al. 1990/squid: Piper and Lucero 1999/salamander: Zufall et al. 2000/rat: Restrepo et al. 1993b;
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Tareilus et al. 1995/humans: Restrepo et al. 1993a/insect: Nakagawa-Inoue et al. 1998; Pelz et al. 2006) including the spiny lobster, Panulirus argus (Schmidt et al. 2010; Tadesse et al. 2011; Ukhanov et al. 2011; Bobkov et al. 2012). In vertebrate and insect ORNs, the somatic increase in intracellular Ca2+ is based on an influx of extracellular Ca2+ (Restrepo and Teeter 1990; Restrepo et al. 1990, 1993a; Tareilus et al. 1995; Nakagawa-Inoue et al. 1998) mediated by voltage-gated Ca2+ channels (Restrepo and Teeter 1990). In squid and salamander ORNs, release of Ca2+ from intracellular stores contributes to the odorantinduced increase in somatic Ca2+ (Piper and Lucero 1999; Zufall et al. 2000). It is generally believed that the odorantinduced somatic influx of extracellular Ca2+ through voltage-gated Ca2+ channels is associated with spiking activity elicited in the soma by the odorant-induced depolarizing receptor potential (Schild and Restrepo 1998; Narusuye et al. 2003). This assumption, however, has not been validated experimentally, and it has been shown in rat ORNs that odorant-induced Ca2+ transients can be directly propagated from the cilia to the soma through low voltage-gated, T-type Ca2+ channels (Gautam et al. 2007). Recording odorant-induced Ca2+ signals from ORN somata by Ca2+ imaging has become a popular technique to study odorant responsiveness of ORNs in diverse animal species (Rawson et al. 1997; Bozza and Kauer 1998; Ma and Shepherd 2000; Elsaesser et al. 2005; Pelz et al. 2006; Vielma et al. 2008; Gliem et al. 2009; Nara et al. 2011; Bazaes and Schmachtenberg 2012; Hassenklöver et al. 2012). The main advantage of this technique compared to electrophysiological methods that target single cells such as patch clamping is that it allows recording from many ORNs simultaneously and, therefore, is a highly efficient way to characterize responses of large populations of ORNs and to detect rare ORNs responding to select odorants (Nara et al. 2011). One basic technical requirement for Ca2+ imaging is that an indicator dye or protein that changes fluorescence upon binding of free Ca2+ must be introduced into the ORNs, and a variety of methods have been used, including bath loading, back filling, and expression of Ca2+ sensitive proteins. The second basic requirement is that loaded ORNs have to be visually accessible to be imaged by fluorescence microscopy, and in most cases this is achieved by dissociating ORNs or by cutting tissue slices from the epithelium. Obviously, these procedures perturb the tissue context in which ORNs function and may introduce changes in ORN properties. Until now, in vivo Ca2+ imaging of ORN responses from intact animals has only been achieved in Drosophila by expression of a Ca2+ sensitive protein in antennal ORNs and imaging Ca2+ signals through the transparent cuticle (Pelz et al. 2006; Galizia et al. 2010). Three further criteria must be met to make Ca2+ imaging from ORN somata a useful tool for studying olfactory
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response properties of ORNs. The somatic odorant-induced Ca2+ signals must arise from odorant-induced events in the ORN dendrites, must be stable long enough for application of multiple odorants, and must reflect spiking activity so that they can serve as proxy for the chemosensory information conveyed to the CNS. Meeting the first criterion is particularly important in aquatic species, such as fish and crustaceans (including P. argus), because for them natural odorants are small water-soluble molecules (e.g., amino acids, nucleotides, amines), many of which also function as neurotransmitters (Carr et al. 1989; Carr 1990; Shaham 2010). Thus, stimulus application to dissociated cells or tissue slices via bath perfusion as is regularly used in aquatic species (Vielma et al. 2008; Gliem et al. 2009; Bazaes and Schmachtenberg 2012; Hassenklöver et al. 2012) can be problematic. The goal of this study was to determine if a recently developed antennule slice preparation allowing imaging of odorant-induced Ca2+ signals in ORN somata of the spiny lobster P. argus (Schmidt et al. 2010; Tadesse et al. 2011) fulfills the three criteria detailed above. Towards this end, we had three aims. First, we tested if a newly developed pressure-activated stimulation pipette allows focal application of chemical stimuli to the ORN dendrites. Second, we performed control experiments to determine the stability of odorant-induced Ca2+ signals in the somata over realistic experimental times and numbers of stimulations. Third, we performed a series of pharmacological experiments to determine if the Ca2+ signals in the ORN somata—odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations—reflect spiking activity.
Materials and methods Animals Young adult male and female Caribbean spiny lobsters (P. argus) of 50–75 mm carapace length were collected near the Florida Keys Marine Laboratory and shipped to Georgia State University where they were held in 400-l aquaria with re-circulated, filtered, and aerated artificial seawater (Instant Ocean™, Aquarium Systems Inc., Mentor, OH, USA) at 20–25 °C. Animals were maintained in a 12 h: 12 h light: dark cycle and fed shrimp or squid three times per week. Chemicals All chemicals are of >99 % purity and were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted.
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Tissue preparation The olfactory organ of spiny lobsters, P. argus, and other decapod crustaceans consists of olfactory sensilla, called aesthetascs, located on the lateral flagella of the paired first antennae, or antennules (see Fig. 1b, c) (Schmidt and Mellon 2011). The lateral flagella are subdivided into series of annuli and in P. argus ~100 annuli bear ~20–30 aesthetascs each. Each aesthetasc is innervated by ~300 ORNs whose somata form a dense cluster beneath the cuticle. Distally, each soma gives rise to a dendrite that crosses the cuticle and develops numerous ciliary branches within a slender cuticular seta whose thin cuticle is permeable for water-soluble odorants (Grünert and Ache 1988; Derby et al. 1997). Antennular lateral flagella were collected from intermolt spiny lobsters and immersed in Panulirus saline (459 mM NaCl, 13.4 mM KCl, 13.6 mM CaCl2·2H2O, 3 mM MgCl2·6H2O, 14.1 mM Na2SO4, 9.8 mM HEPES, pH 7.4, 977 mOsmol). Guard setae (stout sensilla accompanying aesthetascs) were removed and two- or three-annulus pieces were cut from the mature zone (the area of the lateral flagellum containing functionally mature aesthetascs: Steullet et al. 2000). These pieces, which we call ‘antennule slices’ (Fig. 1b), were enzymatically digested for 5–7 min in a solution of 2 ml Panulirus saline, 500 μl of 2.5 mg/ ml papain, and 8 mg of l-cysteine. Antennule slices were rinsed in Panulirus saline for 15–20 min on a shaker followed by desheathing of ORN clusters using forceps. Pilot data were collected using the acetoxymethyl (AM) ester Ca2+ indicator dyes Fura-2 AM, Fluo-4 AM, Fluo-3 AM, Fluo-5F AM, and Oregon Green 488 BAPTA-1 AM (OGB) (Molecular Probes, Invitrogen, Carlsbad, CA, USA). All showed odorant-induced Ca2+ transients; however, OGB was used in subsequent studies because it loaded the maximum number of ORNs and showed a robust response. 50 μg of OGB was dissolved in 45–50 μL Pluronic® F127 with DMSO (Molecular Probes, Invitrogen) and diluted to a final concentration of 50–60 μM (according to Stosiek et al. 2003; Grewe et al. 2010), in Panulirus saline in an Eppendorf tube. Antennule slices were transferred to the Eppendorf tube and shaken vigorously in the dark for 1 h at room temperature. Loaded antennule slices were rinsed in Panulirus saline for 20 min and then transferred into another dish containing Panulirus saline until imaging. Calcium imaging Antennule slices were secured in an experimental flowthrough chamber consisting of a polycarbonate ring (inner diameter 12 mm, outer diameter 24 mm, thickness 5 mm) glued on a 60 mm plastic Petri dish with a layer of Sylgard® (Dow Corning, Midland, MI, USA) on the bottom (Fig. 1a). The chamber was mounted on the stage of
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an upright epifluorescence microscope (Zeiss Axioplan 2, Jena, Germany) equipped with a 40× water immersion objective with long working distance (Fig. 1a). Antennule slices were continuously perfused with Panulirus saline at a rate of 3.0–3.6 ml/min for the duration of the experiment. Excitation light (488 nm for OGB) was provided by a monochromator (Polychrome V, Till Photonics, Munich, Germany), and fluorescent images (688 × 520 pixels and 2 × 2 binning) were captured with a fast CCD camera (Imago QE, Till Photonics). A customized experimental protocol (Vision software, Till Photonics) synchronized image acquisition with an external processor (Imaging System Controller, Till Photonics), allowing control of the stimulus onset and offset via solenoids. For Ca2+ signal quantification, a region of interest was outlined for each selected OBG-loaded ORN soma, and the average pixel value of mean fluorescence intensity was calculated using Vision software. These raw data were transferred to Excel (Microsoft, Redmond, WA, USA) and from there to PSIPlot 8.5 (Poly Software International, Pearl River, NY, USA), Spike2 (Cambridge Electronic Design, Cambridge, UK), and Matlab (Version 8.0.0.783—R2012b; Math Works, Natick, MA, USA) for further data analysis (see below). Chemical stimulus preparation and delivery We used an extract of the fish food TetraMarine (TM; Tetra Werke, Melle, Germany) prepared in Panulirus saline as described by Michel et al. (1991), as chemical stimulus. Briefly, 2 g of TM was mixed in 60 ml Panulirus saline for ~3 h, centrifuged at 1,400×g for 20 min, filtered, aliquoted, and stored at −20 °C until used for each experiment. For an experiment, an aliquot of TM was thawed and diluted 1:100 with Panulirus saline; Panulirus saline was used as a negative control. We constructed a multi-barrel stimulation pipette from fused silica tubing (Polymicro Technologies, Phoenix, AZ, USA) that consisted of a center suction channel (250 μm inner diameter) surrounded by 6 pressurized stimulus channels (100 μm inner diameter) (Fig. 1a, inset). We used a Picospritzer III (Parker, Pine Brook, NJ, USA) and two sets of solenoid valves (NResearch, West Caldwell, NJ, USA) to control the delivery of the chemical stimulus. One set of valves (3-way valves) controlled pressurization of one of 6 reservoirs (5-ml syringes) containing chemical stimuli, and the other set (pinch valves) controlled the delivery of the selected stimulus to one stimulus channel of the stimulation pipette. The suction channel of the stimulation pipette was continuously open except when a stimulus event was triggered. The triggering of an event by the imaging system controller opened the two solenoid valves of a selected stimulus channel for 1 s and simultaneously
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closed the suction channel resulting in the ejection of the selected stimulus. After 1 s, the stimulus solenoid valves closed and the suction valve opened resulting in the rapid termination of stimulus ejection. The stimulation pipette was mounted onto a micromanipulator and its tip was brought close to the antennule slice secured onto the bottom of the flow-through chamber. Application of Panulirus saline with 0.01 % Fast Green or 0.001 % fluorescein
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with the stimulation pipette to antennule slices allowed visualization of the stimulus distribution and quantification of its time course by capturing images with the Ca2+ imaging system. These control experiments showed that the stimulus reached the aesthetasc setae but not their somata (Fig. 1d) and that a stimulus elicited by a 1-s long solenoid activation had a rise-time of ~1 s and a total duration of ~4 s (Fig. 1d).
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◂ Fig. 1 Experimental approach of Ca2+ imaging of spiny lobster
ORNs. a Schematic drawing of the experimental setup (by Lynn Milstead, Whitney Laboratory, University of Florida); stimulation pipette (SP); inset schematic drawing of the multi-channel stimulation pipette in cross section. The stimulation pipette has six pressurized channels containing chemical stimuli and a thicker suction channel all made of fused silica tubes bonded together with epoxy glue. b Transmitted light microscopic image of antennule slice preparation consisting of two aesthetasc-bearing annuli secured on a layer of Sylgard® used for Ca2+ imaging. c Schematic drawing of a single aesthetasc of P. argus (modified from Grünert and Ache 1988). The aesthetasc is innervated by ~300 bipolar olfactory receptor neurons (ORNs) whose somata form a cluster below the cuticle (C). Each soma gives rise to a dendrite projecting into the cuticular seta where it branches into many ciliated outer segments and to an axon projecting to the brain. d Time course of stimulus measured by delivering a 1-s pulse of Panulirus saline containing 0.001 % fluorescein by the stimulation pipette (SP) and quantifying the change in mean fluorescence intensity (ΔF/F) at the aesthetasc setae (red) and the ORN somata (blue). Note that the total duration of the stimulus at the setae, although at varying concentration, is ~4 s. Inset Visualization of stimulus distribution by application of saline containing Fast Green to the antennule slice with the stimulation pipette (SP). Note that stimulus reaches the aesthetasc setae but not the ORN somata. e Time course of a fluid exchange in the experimental chamber measured by switching from Panulirus saline to Panulirus saline containing 0.0001 % fluorescein and quantifying ΔF/F at the antennule slice. Note that the fluid exchange starts ~10 s and is complete ~60 s after switching (indicated by dashed lines). f Time line of the experimental protocol (no-stimulation trial = NoStim)
Experiments As a prerequisite for the interpretation of pharmacological experiments, we measured the stability of baseline fluorescence and amplitude of odorant-induced Ca2+ transients of ORN somata in antennule slice preparations under experimental conditions over time. For this, two antennule slices were stimulated every 4 min with 1 % TM for a total of 40 min, exceeding the typical duration of the experiments (~30 min). For each stimulation, 500 images with an exposure time of 4 ms were acquired at 150 ms/ frame (for a total recording time of 75 s), and the stimulus was applied at 15 s. This data acquisition scheme was used in most experiments except those testing the effect of low extracellular Ca2+ and 1 μM thapsigargin; in some of these experiments, 800 images were acquired at 100 ms/ frame (for a total recording time of 80 s). We also measured the duration of fluid exchange in the experimental chamber by switching from Panulirus saline to Panulirus saline containing 0.0001 % fluorescein and back while capturing images every 500 ms with the Ca2+ imaging system. These experiments showed that the fluid exchange in the experimental chamber is complete in less than 60 s (Fig. 1e). Based on the results of the stability and chamberexchange measurements, we developed the experimental protocol (Fig. 1f). Starting at 0 min, the antennule slice was stimulated by 1 % TM in Panulirus saline every 4 min
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and in every 2nd inter-stimulus interval spontaneous activity was recorded in no-stimulation trials. At 6 min, the bath perfusion was switched from Panulirus saline to Panulirus saline containing low Ca2+ or a drug and at 14 min the perfusion was switched back to Panulirus saline. Data analysis was usually based on the 4, 12, and 24 or 28 min 1 % TM stimulation trials and the 2, 10, and 26 min no-stimulation trials reflecting, respectively, the start condition, the treatment condition, and the recovery condition. We conducted four experiments, each comprising one or more treatments. The first experiment addressed the contribution of extracellular Ca2+, the second experiment focused on the role of voltage-activated Ca2+ channels, the third experiment addressed the contribution of intracellular Ca2+ stores, and the fourth experiment focused on the role of action potentials in generating the Ca2+ signals in ORN somata. Treatment 1 was low extracellular Ca2+, for which low Ca2+ Panulirus saline containing 459 mM NaCl, 13.4 mM KCl, 0.1 mM CaCl2·2H2O, 3 mM MgCl2·6H2O, 14.1 mM Na2SO4, and 9.8 mM HEPES (pH 7.4) was prepared before the experiments and stored at room temperature. Treatment 2 was CoCl2, for which 10 or 20 mM CoCl2 was prepared by dissolving the appropriate amount of CoCl2·6H20 in Panulirus saline on the day of the experiment. Treatment 3 was verapamil, for which 1 mM verapamil was prepared by dissolving the appropriate amount of verapamil HCl (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Panulirus saline on the day of the experiment. Treatment 4 was thapsigargin, for which 1 μM thapsigargin was prepared by diluting 10 mM thapsigargin (Tocris Bioscience, Ellisville, MO, USA) stock in DMSO that was stored at −20 °C with Panulirus saline on the day of the experiment (as it is known that thapsigargin is a slowacting irreversible blocker, treatment testing started 15– 18 min after switching the bath perfusion to 1 μM thapsigargin in Panulirus saline and no post-treatment testing was performed). Treatment 5 was caffeine, for which 2 mM caffeine (Tocris Bioscience) was prepared by dissolving the appropriate amount in Panulirus saline on the day of the experiment. Treatment 6 was tetrodotoxin (TTX), for which 1, 5, or 8 μM TTX was prepared by diluting 10 mM TTX citrate (Tocris Bioscience; Abcam Inc., Cambridge, MA, USA) stock that was stored at −20 °C with Panulirus saline on the day of the experiment. Treatment 7 was NMDG (N-methyl-d-glucamine) saline in which Na+ in Panulirus saline was replaced by 459 mM NMDG (VWR International, Radnor, PA, USA). NMDG saline was prepared before the experiments, and the pH was adjusted to 7.4 by addition of 6 N HCl. Treatment 8 was tetracaine, for which 0.3 or 1 mM tetracaine was prepared by dissolving the appropriate amount of tetracaine HCl in Panulirus saline on the day of the experiment. Treatment 9 was lidocaine, for which 1 or 5 mM lidocaine was prepared by
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J Comp Physiol A (2014) 200:53–76
◂ Fig. 2 Characteristics of Ca2+ signals in ORN somata (a–f). Typi-
cal odorant-induced transients in a cluster of ORN somata. Pseudocolored fluorescence images of OGB loaded ORNs before stimulation (a) and at the peak of the response following stimulation with 1 % TM (b). Dotted lines mark four ORNs (1–4) whose Ca2+ signals are shown in c–f. c–f Time courses of Ca2+ responses in ORNs 1–4 highlighted in a and b. The four ORNs show robust, long-lasting increases in intracellular Ca2+ following stimulation (time of stimulus indicated by vertical dashed line at 15 s; times when images in a and b were taken indicated by arrows in c). Note that the odorant-induced Ca2+ transients of the 4 ORNs are similar in rise-time, but differ substantially in amplitude (~0.15–0.30 ΔF/F), duration, time constant of decay, and occurrence of Ca2+ oscillations (visible in c, e). d Parameters used to quantify the Ca2+ transients (baseline, peak, amplitude) are illustrated in blue. g–i Stability of baseline fluorescence and odorant-induced transients recorded from 20 ORNs (from 2 antennule slices) every 4 min over 40 min. g Box plot (median, 10, 25, 75, 90 percentiles) of baseline fluorescence normalized to the baseline fluorescence of the first trial (0 min). Note that the baseline fluorescence declines steadily but remains >80 % throughout the duration of the experiments. h Recordings of odorant-induced transients of one ORN stimulated with 1 % TM at 0 min (black), 20 min (blue), and 40 min (magenta). Note that amplitude and time course of odorant-induced transients fluctuate slightly between trials but without a distinct trend over experimental time. i Box plot (median, 10, 25, 75, 90 percentiles) of amplitudes of odorant-induced transients normalized to the baseline fluorescence of the first trial (0 min). Note that the median amplitudes of odorant-induced transients vary from ~90 to 110 % between trials but without a distinct trend over experimental time. j–l Recordings of spontaneous Ca2+ oscillations. j Three ORNs (ORN1– ORN3) from different preparations. k Three ORNs (ORN1–ORN3) of one ORN cluster. l One ORN recorded at different time points (0, 20, 36 min) of an experiment. Note that all Ca2+ oscillations have a characteristic time course (fast rise and slower decay) but that they differ between ORNs and also over time within an ORN in amplitude and frequency of occurrence
dissolving the appropriate amount of lidocaine HCl (VWR) in Panulirus saline on the day of the experiment. Data analysis and preparation of illustrations To generate illustrations of time series of mean fluorescence intensity measurements and to quantify the amplitude of odorant-activated Ca2+ transients and the frequency and amplitude of Ca2+ oscillations, raw data were converted to relative fluorescence changes expressed as ΔF/F in PSI-Plot 8.5 or Matlab. The baseline fluorescence F was calculated as the arithmetic mean of the first 100 data points (Fig. 2d). The amplitude of odorant-activated Ca2+ transients was determined as the most positive value of ΔF/F in a time series (Fig. 2d). Visual inspection of the traces ensured that the amplitude measurements were not assigned to rarely occurring glitches. Only ORNs whose odorant-activated Ca2+ transient had an amplitude of ≥0.05 ΔF/F at the initial stimulation with 1 % TM were included in further analyses. Spontaneous Ca2+ oscillations were quantified with Spike2 software. A spontaneous event was defined as having two troughs and one peak exceeding a preset threshold (2
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standard deviations different from F). The amplitude of each peak was calculated as peak/average of pre- and post-troughs. Since spontaneous oscillations showed some variability over experimental time, only ORNs in which spontaneous oscillations were present upon return to Panulirus saline (Recovery) were included in further analyses (since treatment with thapsigargin did not include recovery measurements, all ORNs showing spontaneous oscillations during pre-treatment in Panulirus saline were included in the analysis). For each ORN, the amplitude of the odorant-activated Ca2+ transient and the amplitude and frequency of spontaneous oscillations were normalized to the respective values during the pre-treatment phase (set to 100 %). PSI-Plot 8.5 was used to generate box plots (median, 10, 25, 75, 90 percentiles) of selected data and representative time series of Ca2+ signals. Final figures were composed with Illustrator CS3 (Adobe Systems Incorporated, San Jose, CA, USA). Statistical analyses (Friedman’s two-way analysis of variance by ranks–ANOVA/post hoc analysis with Wilcoxon signed ranks test) were conducted using Statistica (StatSoft, Inc., Tulsa, OK, USA).
Results Calcium imaging of spiny lobster ORNs To study physiological properties of spiny lobster ORNs, we established Ca2+ imaging of ORN somata in an in vitro ‘antennule slice’ (Fig. 1a, b) developed in parallel with that of Ukhanov et al. (2011). This antennule slice, consisting of two or three aesthetasc-bearing annuli, maintains the structural integrity of the aesthetascs and thus allows analysis of physiological properties of ORNs in situ. Antennule slices loaded with OGB typically showed numerous loaded ORN somata, most of which could clearly be delineated (Fig. 2a, b). When the aesthetasc setae were stimulated with 1 % TM, many of the loaded ORN somata (67 % of 214 analyzed ORN somata from 6 antennule slices) showed a robust and transient increase in fluorescence intensity (Fig. 2c–f). We chose TM as standard chemical stimulus due to its ability to cause excitation and a transient increase in somatic Ca2+ in many spiny lobster ORNs (Michel et al. 1991; Ukhanov et al. 2011). In control experiments (data not shown), we stimulated ORNs with 1 % TM and Panulirus saline (negative control) and found that Panulirus saline did not cause an increase in fluorescence intensity >0.05 ΔF/F (the criterion to identify odorant-induced responses) in any of them. We typically used antennule slices for only one experiment lasting ~30 min, but ORN somata showed robust odorant-induced responses for up to 1 h (Fig. 2h, i). Overall, this demonstrates the functionality of the Ca2+ imaging set-up for spiny lobster ORNs.
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Odorant‑induced Ca2+ transients and spontaneous Ca2+ oscillations in ORNs Stimulation of aesthetasc setae with 1 % TM caused a stimulus-related transient increase in mean fluorescence intensity in many ORN somata, which we call ‘odorant-induced Ca2+ transient’ (Fig. 2a–f). The odorant-induced Ca2+ transients varied considerably between ORNs in maximum amplitude and time course of fluorescence increase and decay (Fig. 2c–f), but in a given ORN they were stable over repeated stimulations. The amplitude of odorant-induced Ca2+ transients ranged from 0.05 to 0.35 ΔF/F (0.05 ΔF/F being the minimum cutoff criterion) with amplitudes between 0.10 and 0.20 ΔF/F being most common. Upon stimulus presentation, the fluorescence typically started to increase with very short delay (
Original Paper
Mechanisms underlying odorant‑induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus Tizeta Tadesse · Charles D. Derby · Manfred Schmidt
Received: 18 June 2013 / Revised: 3 October 2013 / Accepted: 4 October 2013 / Published online: 1 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca2+ imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca2+ signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations in the somata of olfactory receptor neurons. Both Ca2+ signals are primarily mediated by an influx of extracellular Ca2+ through voltage-activated Ca2+ channels that can be blocked by CoCl2 and the L-type Ca2+ channel blocker verapamil. Intracellular Ca2+ stores contribute little to odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations. The odorant-induced Ca2+ transients as well as the spontaneous Ca2+ oscillations depend on action potentials mediated by Na+ channels that are largely TTXinsensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of T. Tadesse · C. D. Derby · M. Schmidt (*) Neuroscience Institute and Department of Biology, Georgia State University, P.O. Box 5030, Atlanta, GA 30302‑5030, USA e-mail: [email protected]; [email protected] Present Address: T. Tadesse Department of Neurology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
Ca2+ signals represent experimentally accessible proxies of spiking. Keywords Ca imaging · Olfaction · Oscillation · Arthropod · Crustacean Abbreviations ΔF/F Relative change in fluorescence intensity NMDG N-methyl-d-glucamine OGB Oregon Green 488 BAPTA-1 AM ORN Olfactory receptor neuron SERCA Sarco/endoplasmic reticulum Ca2+ ATPase pump TM TetraMarine extract TTX Tetrodotoxin
Introduction Olfactory receptor neurons (ORNs) across numerous vertebrate and invertebrate species show robust odorant-induced changes in intracellular Ca2+ that can be readily assessed in different cellular compartments by Ca2+ imaging. Ca2+ imaging was first used to study responsiveness in catfish ORNs to odorants (Restrepo et al. 1990), and has since been used to show that vertebrate ORNs have rapid increases in intracellular Ca2+ in the cilia that are associated with olfactory transduction and are mediated by cAMP-gated channels (Leinders-Zufall et al. 1997, 1998). The rise in intraciliary Ca2+ amplifies the depolarizing receptor potential through activation of Cl− channels, and it mediates odor adaptation (Menini 1999; Matthews and Reisert 2003). Odorant-induced increases in intracellular Ca2+ occur in the somata of ORNs in a wide range of species (fish: Restrepo et al. 1990/squid: Piper and Lucero 1999/salamander: Zufall et al. 2000/rat: Restrepo et al. 1993b;
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Tareilus et al. 1995/humans: Restrepo et al. 1993a/insect: Nakagawa-Inoue et al. 1998; Pelz et al. 2006) including the spiny lobster, Panulirus argus (Schmidt et al. 2010; Tadesse et al. 2011; Ukhanov et al. 2011; Bobkov et al. 2012). In vertebrate and insect ORNs, the somatic increase in intracellular Ca2+ is based on an influx of extracellular Ca2+ (Restrepo and Teeter 1990; Restrepo et al. 1990, 1993a; Tareilus et al. 1995; Nakagawa-Inoue et al. 1998) mediated by voltage-gated Ca2+ channels (Restrepo and Teeter 1990). In squid and salamander ORNs, release of Ca2+ from intracellular stores contributes to the odorantinduced increase in somatic Ca2+ (Piper and Lucero 1999; Zufall et al. 2000). It is generally believed that the odorantinduced somatic influx of extracellular Ca2+ through voltage-gated Ca2+ channels is associated with spiking activity elicited in the soma by the odorant-induced depolarizing receptor potential (Schild and Restrepo 1998; Narusuye et al. 2003). This assumption, however, has not been validated experimentally, and it has been shown in rat ORNs that odorant-induced Ca2+ transients can be directly propagated from the cilia to the soma through low voltage-gated, T-type Ca2+ channels (Gautam et al. 2007). Recording odorant-induced Ca2+ signals from ORN somata by Ca2+ imaging has become a popular technique to study odorant responsiveness of ORNs in diverse animal species (Rawson et al. 1997; Bozza and Kauer 1998; Ma and Shepherd 2000; Elsaesser et al. 2005; Pelz et al. 2006; Vielma et al. 2008; Gliem et al. 2009; Nara et al. 2011; Bazaes and Schmachtenberg 2012; Hassenklöver et al. 2012). The main advantage of this technique compared to electrophysiological methods that target single cells such as patch clamping is that it allows recording from many ORNs simultaneously and, therefore, is a highly efficient way to characterize responses of large populations of ORNs and to detect rare ORNs responding to select odorants (Nara et al. 2011). One basic technical requirement for Ca2+ imaging is that an indicator dye or protein that changes fluorescence upon binding of free Ca2+ must be introduced into the ORNs, and a variety of methods have been used, including bath loading, back filling, and expression of Ca2+ sensitive proteins. The second basic requirement is that loaded ORNs have to be visually accessible to be imaged by fluorescence microscopy, and in most cases this is achieved by dissociating ORNs or by cutting tissue slices from the epithelium. Obviously, these procedures perturb the tissue context in which ORNs function and may introduce changes in ORN properties. Until now, in vivo Ca2+ imaging of ORN responses from intact animals has only been achieved in Drosophila by expression of a Ca2+ sensitive protein in antennal ORNs and imaging Ca2+ signals through the transparent cuticle (Pelz et al. 2006; Galizia et al. 2010). Three further criteria must be met to make Ca2+ imaging from ORN somata a useful tool for studying olfactory
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response properties of ORNs. The somatic odorant-induced Ca2+ signals must arise from odorant-induced events in the ORN dendrites, must be stable long enough for application of multiple odorants, and must reflect spiking activity so that they can serve as proxy for the chemosensory information conveyed to the CNS. Meeting the first criterion is particularly important in aquatic species, such as fish and crustaceans (including P. argus), because for them natural odorants are small water-soluble molecules (e.g., amino acids, nucleotides, amines), many of which also function as neurotransmitters (Carr et al. 1989; Carr 1990; Shaham 2010). Thus, stimulus application to dissociated cells or tissue slices via bath perfusion as is regularly used in aquatic species (Vielma et al. 2008; Gliem et al. 2009; Bazaes and Schmachtenberg 2012; Hassenklöver et al. 2012) can be problematic. The goal of this study was to determine if a recently developed antennule slice preparation allowing imaging of odorant-induced Ca2+ signals in ORN somata of the spiny lobster P. argus (Schmidt et al. 2010; Tadesse et al. 2011) fulfills the three criteria detailed above. Towards this end, we had three aims. First, we tested if a newly developed pressure-activated stimulation pipette allows focal application of chemical stimuli to the ORN dendrites. Second, we performed control experiments to determine the stability of odorant-induced Ca2+ signals in the somata over realistic experimental times and numbers of stimulations. Third, we performed a series of pharmacological experiments to determine if the Ca2+ signals in the ORN somata—odorant-induced Ca2+ transients and spontaneous Ca2+ oscillations—reflect spiking activity.
Materials and methods Animals Young adult male and female Caribbean spiny lobsters (P. argus) of 50–75 mm carapace length were collected near the Florida Keys Marine Laboratory and shipped to Georgia State University where they were held in 400-l aquaria with re-circulated, filtered, and aerated artificial seawater (Instant Ocean™, Aquarium Systems Inc., Mentor, OH, USA) at 20–25 °C. Animals were maintained in a 12 h: 12 h light: dark cycle and fed shrimp or squid three times per week. Chemicals All chemicals are of >99 % purity and were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted.
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Tissue preparation The olfactory organ of spiny lobsters, P. argus, and other decapod crustaceans consists of olfactory sensilla, called aesthetascs, located on the lateral flagella of the paired first antennae, or antennules (see Fig. 1b, c) (Schmidt and Mellon 2011). The lateral flagella are subdivided into series of annuli and in P. argus ~100 annuli bear ~20–30 aesthetascs each. Each aesthetasc is innervated by ~300 ORNs whose somata form a dense cluster beneath the cuticle. Distally, each soma gives rise to a dendrite that crosses the cuticle and develops numerous ciliary branches within a slender cuticular seta whose thin cuticle is permeable for water-soluble odorants (Grünert and Ache 1988; Derby et al. 1997). Antennular lateral flagella were collected from intermolt spiny lobsters and immersed in Panulirus saline (459 mM NaCl, 13.4 mM KCl, 13.6 mM CaCl2·2H2O, 3 mM MgCl2·6H2O, 14.1 mM Na2SO4, 9.8 mM HEPES, pH 7.4, 977 mOsmol). Guard setae (stout sensilla accompanying aesthetascs) were removed and two- or three-annulus pieces were cut from the mature zone (the area of the lateral flagellum containing functionally mature aesthetascs: Steullet et al. 2000). These pieces, which we call ‘antennule slices’ (Fig. 1b), were enzymatically digested for 5–7 min in a solution of 2 ml Panulirus saline, 500 μl of 2.5 mg/ ml papain, and 8 mg of l-cysteine. Antennule slices were rinsed in Panulirus saline for 15–20 min on a shaker followed by desheathing of ORN clusters using forceps. Pilot data were collected using the acetoxymethyl (AM) ester Ca2+ indicator dyes Fura-2 AM, Fluo-4 AM, Fluo-3 AM, Fluo-5F AM, and Oregon Green 488 BAPTA-1 AM (OGB) (Molecular Probes, Invitrogen, Carlsbad, CA, USA). All showed odorant-induced Ca2+ transients; however, OGB was used in subsequent studies because it loaded the maximum number of ORNs and showed a robust response. 50 μg of OGB was dissolved in 45–50 μL Pluronic® F127 with DMSO (Molecular Probes, Invitrogen) and diluted to a final concentration of 50–60 μM (according to Stosiek et al. 2003; Grewe et al. 2010), in Panulirus saline in an Eppendorf tube. Antennule slices were transferred to the Eppendorf tube and shaken vigorously in the dark for 1 h at room temperature. Loaded antennule slices were rinsed in Panulirus saline for 20 min and then transferred into another dish containing Panulirus saline until imaging. Calcium imaging Antennule slices were secured in an experimental flowthrough chamber consisting of a polycarbonate ring (inner diameter 12 mm, outer diameter 24 mm, thickness 5 mm) glued on a 60 mm plastic Petri dish with a layer of Sylgard® (Dow Corning, Midland, MI, USA) on the bottom (Fig. 1a). The chamber was mounted on the stage of
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an upright epifluorescence microscope (Zeiss Axioplan 2, Jena, Germany) equipped with a 40× water immersion objective with long working distance (Fig. 1a). Antennule slices were continuously perfused with Panulirus saline at a rate of 3.0–3.6 ml/min for the duration of the experiment. Excitation light (488 nm for OGB) was provided by a monochromator (Polychrome V, Till Photonics, Munich, Germany), and fluorescent images (688 × 520 pixels and 2 × 2 binning) were captured with a fast CCD camera (Imago QE, Till Photonics). A customized experimental protocol (Vision software, Till Photonics) synchronized image acquisition with an external processor (Imaging System Controller, Till Photonics), allowing control of the stimulus onset and offset via solenoids. For Ca2+ signal quantification, a region of interest was outlined for each selected OBG-loaded ORN soma, and the average pixel value of mean fluorescence intensity was calculated using Vision software. These raw data were transferred to Excel (Microsoft, Redmond, WA, USA) and from there to PSIPlot 8.5 (Poly Software International, Pearl River, NY, USA), Spike2 (Cambridge Electronic Design, Cambridge, UK), and Matlab (Version 8.0.0.783—R2012b; Math Works, Natick, MA, USA) for further data analysis (see below). Chemical stimulus preparation and delivery We used an extract of the fish food TetraMarine (TM; Tetra Werke, Melle, Germany) prepared in Panulirus saline as described by Michel et al. (1991), as chemical stimulus. Briefly, 2 g of TM was mixed in 60 ml Panulirus saline for ~3 h, centrifuged at 1,400×g for 20 min, filtered, aliquoted, and stored at −20 °C until used for each experiment. For an experiment, an aliquot of TM was thawed and diluted 1:100 with Panulirus saline; Panulirus saline was used as a negative control. We constructed a multi-barrel stimulation pipette from fused silica tubing (Polymicro Technologies, Phoenix, AZ, USA) that consisted of a center suction channel (250 μm inner diameter) surrounded by 6 pressurized stimulus channels (100 μm inner diameter) (Fig. 1a, inset). We used a Picospritzer III (Parker, Pine Brook, NJ, USA) and two sets of solenoid valves (NResearch, West Caldwell, NJ, USA) to control the delivery of the chemical stimulus. One set of valves (3-way valves) controlled pressurization of one of 6 reservoirs (5-ml syringes) containing chemical stimuli, and the other set (pinch valves) controlled the delivery of the selected stimulus to one stimulus channel of the stimulation pipette. The suction channel of the stimulation pipette was continuously open except when a stimulus event was triggered. The triggering of an event by the imaging system controller opened the two solenoid valves of a selected stimulus channel for 1 s and simultaneously
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closed the suction channel resulting in the ejection of the selected stimulus. After 1 s, the stimulus solenoid valves closed and the suction valve opened resulting in the rapid termination of stimulus ejection. The stimulation pipette was mounted onto a micromanipulator and its tip was brought close to the antennule slice secured onto the bottom of the flow-through chamber. Application of Panulirus saline with 0.01 % Fast Green or 0.001 % fluorescein
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with the stimulation pipette to antennule slices allowed visualization of the stimulus distribution and quantification of its time course by capturing images with the Ca2+ imaging system. These control experiments showed that the stimulus reached the aesthetasc setae but not their somata (Fig. 1d) and that a stimulus elicited by a 1-s long solenoid activation had a rise-time of ~1 s and a total duration of ~4 s (Fig. 1d).
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◂ Fig. 1 Experimental approach of Ca2+ imaging of spiny lobster
ORNs. a Schematic drawing of the experimental setup (by Lynn Milstead, Whitney Laboratory, University of Florida); stimulation pipette (SP); inset schematic drawing of the multi-channel stimulation pipette in cross section. The stimulation pipette has six pressurized channels containing chemical stimuli and a thicker suction channel all made of fused silica tubes bonded together with epoxy glue. b Transmitted light microscopic image of antennule slice preparation consisting of two aesthetasc-bearing annuli secured on a layer of Sylgard® used for Ca2+ imaging. c Schematic drawing of a single aesthetasc of P. argus (modified from Grünert and Ache 1988). The aesthetasc is innervated by ~300 bipolar olfactory receptor neurons (ORNs) whose somata form a cluster below the cuticle (C). Each soma gives rise to a dendrite projecting into the cuticular seta where it branches into many ciliated outer segments and to an axon projecting to the brain. d Time course of stimulus measured by delivering a 1-s pulse of Panulirus saline containing 0.001 % fluorescein by the stimulation pipette (SP) and quantifying the change in mean fluorescence intensity (ΔF/F) at the aesthetasc setae (red) and the ORN somata (blue). Note that the total duration of the stimulus at the setae, although at varying concentration, is ~4 s. Inset Visualization of stimulus distribution by application of saline containing Fast Green to the antennule slice with the stimulation pipette (SP). Note that stimulus reaches the aesthetasc setae but not the ORN somata. e Time course of a fluid exchange in the experimental chamber measured by switching from Panulirus saline to Panulirus saline containing 0.0001 % fluorescein and quantifying ΔF/F at the antennule slice. Note that the fluid exchange starts ~10 s and is complete ~60 s after switching (indicated by dashed lines). f Time line of the experimental protocol (no-stimulation trial = NoStim)
Experiments As a prerequisite for the interpretation of pharmacological experiments, we measured the stability of baseline fluorescence and amplitude of odorant-induced Ca2+ transients of ORN somata in antennule slice preparations under experimental conditions over time. For this, two antennule slices were stimulated every 4 min with 1 % TM for a total of 40 min, exceeding the typical duration of the experiments (~30 min). For each stimulation, 500 images with an exposure time of 4 ms were acquired at 150 ms/ frame (for a total recording time of 75 s), and the stimulus was applied at 15 s. This data acquisition scheme was used in most experiments except those testing the effect of low extracellular Ca2+ and 1 μM thapsigargin; in some of these experiments, 800 images were acquired at 100 ms/ frame (for a total recording time of 80 s). We also measured the duration of fluid exchange in the experimental chamber by switching from Panulirus saline to Panulirus saline containing 0.0001 % fluorescein and back while capturing images every 500 ms with the Ca2+ imaging system. These experiments showed that the fluid exchange in the experimental chamber is complete in less than 60 s (Fig. 1e). Based on the results of the stability and chamberexchange measurements, we developed the experimental protocol (Fig. 1f). Starting at 0 min, the antennule slice was stimulated by 1 % TM in Panulirus saline every 4 min
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and in every 2nd inter-stimulus interval spontaneous activity was recorded in no-stimulation trials. At 6 min, the bath perfusion was switched from Panulirus saline to Panulirus saline containing low Ca2+ or a drug and at 14 min the perfusion was switched back to Panulirus saline. Data analysis was usually based on the 4, 12, and 24 or 28 min 1 % TM stimulation trials and the 2, 10, and 26 min no-stimulation trials reflecting, respectively, the start condition, the treatment condition, and the recovery condition. We conducted four experiments, each comprising one or more treatments. The first experiment addressed the contribution of extracellular Ca2+, the second experiment focused on the role of voltage-activated Ca2+ channels, the third experiment addressed the contribution of intracellular Ca2+ stores, and the fourth experiment focused on the role of action potentials in generating the Ca2+ signals in ORN somata. Treatment 1 was low extracellular Ca2+, for which low Ca2+ Panulirus saline containing 459 mM NaCl, 13.4 mM KCl, 0.1 mM CaCl2·2H2O, 3 mM MgCl2·6H2O, 14.1 mM Na2SO4, and 9.8 mM HEPES (pH 7.4) was prepared before the experiments and stored at room temperature. Treatment 2 was CoCl2, for which 10 or 20 mM CoCl2 was prepared by dissolving the appropriate amount of CoCl2·6H20 in Panulirus saline on the day of the experiment. Treatment 3 was verapamil, for which 1 mM verapamil was prepared by dissolving the appropriate amount of verapamil HCl (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Panulirus saline on the day of the experiment. Treatment 4 was thapsigargin, for which 1 μM thapsigargin was prepared by diluting 10 mM thapsigargin (Tocris Bioscience, Ellisville, MO, USA) stock in DMSO that was stored at −20 °C with Panulirus saline on the day of the experiment (as it is known that thapsigargin is a slowacting irreversible blocker, treatment testing started 15– 18 min after switching the bath perfusion to 1 μM thapsigargin in Panulirus saline and no post-treatment testing was performed). Treatment 5 was caffeine, for which 2 mM caffeine (Tocris Bioscience) was prepared by dissolving the appropriate amount in Panulirus saline on the day of the experiment. Treatment 6 was tetrodotoxin (TTX), for which 1, 5, or 8 μM TTX was prepared by diluting 10 mM TTX citrate (Tocris Bioscience; Abcam Inc., Cambridge, MA, USA) stock that was stored at −20 °C with Panulirus saline on the day of the experiment. Treatment 7 was NMDG (N-methyl-d-glucamine) saline in which Na+ in Panulirus saline was replaced by 459 mM NMDG (VWR International, Radnor, PA, USA). NMDG saline was prepared before the experiments, and the pH was adjusted to 7.4 by addition of 6 N HCl. Treatment 8 was tetracaine, for which 0.3 or 1 mM tetracaine was prepared by dissolving the appropriate amount of tetracaine HCl in Panulirus saline on the day of the experiment. Treatment 9 was lidocaine, for which 1 or 5 mM lidocaine was prepared by
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◂ Fig. 2 Characteristics of Ca2+ signals in ORN somata (a–f). Typi-
cal odorant-induced transients in a cluster of ORN somata. Pseudocolored fluorescence images of OGB loaded ORNs before stimulation (a) and at the peak of the response following stimulation with 1 % TM (b). Dotted lines mark four ORNs (1–4) whose Ca2+ signals are shown in c–f. c–f Time courses of Ca2+ responses in ORNs 1–4 highlighted in a and b. The four ORNs show robust, long-lasting increases in intracellular Ca2+ following stimulation (time of stimulus indicated by vertical dashed line at 15 s; times when images in a and b were taken indicated by arrows in c). Note that the odorant-induced Ca2+ transients of the 4 ORNs are similar in rise-time, but differ substantially in amplitude (~0.15–0.30 ΔF/F), duration, time constant of decay, and occurrence of Ca2+ oscillations (visible in c, e). d Parameters used to quantify the Ca2+ transients (baseline, peak, amplitude) are illustrated in blue. g–i Stability of baseline fluorescence and odorant-induced transients recorded from 20 ORNs (from 2 antennule slices) every 4 min over 40 min. g Box plot (median, 10, 25, 75, 90 percentiles) of baseline fluorescence normalized to the baseline fluorescence of the first trial (0 min). Note that the baseline fluorescence declines steadily but remains >80 % throughout the duration of the experiments. h Recordings of odorant-induced transients of one ORN stimulated with 1 % TM at 0 min (black), 20 min (blue), and 40 min (magenta). Note that amplitude and time course of odorant-induced transients fluctuate slightly between trials but without a distinct trend over experimental time. i Box plot (median, 10, 25, 75, 90 percentiles) of amplitudes of odorant-induced transients normalized to the baseline fluorescence of the first trial (0 min). Note that the median amplitudes of odorant-induced transients vary from ~90 to 110 % between trials but without a distinct trend over experimental time. j–l Recordings of spontaneous Ca2+ oscillations. j Three ORNs (ORN1– ORN3) from different preparations. k Three ORNs (ORN1–ORN3) of one ORN cluster. l One ORN recorded at different time points (0, 20, 36 min) of an experiment. Note that all Ca2+ oscillations have a characteristic time course (fast rise and slower decay) but that they differ between ORNs and also over time within an ORN in amplitude and frequency of occurrence
dissolving the appropriate amount of lidocaine HCl (VWR) in Panulirus saline on the day of the experiment. Data analysis and preparation of illustrations To generate illustrations of time series of mean fluorescence intensity measurements and to quantify the amplitude of odorant-activated Ca2+ transients and the frequency and amplitude of Ca2+ oscillations, raw data were converted to relative fluorescence changes expressed as ΔF/F in PSI-Plot 8.5 or Matlab. The baseline fluorescence F was calculated as the arithmetic mean of the first 100 data points (Fig. 2d). The amplitude of odorant-activated Ca2+ transients was determined as the most positive value of ΔF/F in a time series (Fig. 2d). Visual inspection of the traces ensured that the amplitude measurements were not assigned to rarely occurring glitches. Only ORNs whose odorant-activated Ca2+ transient had an amplitude of ≥0.05 ΔF/F at the initial stimulation with 1 % TM were included in further analyses. Spontaneous Ca2+ oscillations were quantified with Spike2 software. A spontaneous event was defined as having two troughs and one peak exceeding a preset threshold (2
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standard deviations different from F). The amplitude of each peak was calculated as peak/average of pre- and post-troughs. Since spontaneous oscillations showed some variability over experimental time, only ORNs in which spontaneous oscillations were present upon return to Panulirus saline (Recovery) were included in further analyses (since treatment with thapsigargin did not include recovery measurements, all ORNs showing spontaneous oscillations during pre-treatment in Panulirus saline were included in the analysis). For each ORN, the amplitude of the odorant-activated Ca2+ transient and the amplitude and frequency of spontaneous oscillations were normalized to the respective values during the pre-treatment phase (set to 100 %). PSI-Plot 8.5 was used to generate box plots (median, 10, 25, 75, 90 percentiles) of selected data and representative time series of Ca2+ signals. Final figures were composed with Illustrator CS3 (Adobe Systems Incorporated, San Jose, CA, USA). Statistical analyses (Friedman’s two-way analysis of variance by ranks–ANOVA/post hoc analysis with Wilcoxon signed ranks test) were conducted using Statistica (StatSoft, Inc., Tulsa, OK, USA).
Results Calcium imaging of spiny lobster ORNs To study physiological properties of spiny lobster ORNs, we established Ca2+ imaging of ORN somata in an in vitro ‘antennule slice’ (Fig. 1a, b) developed in parallel with that of Ukhanov et al. (2011). This antennule slice, consisting of two or three aesthetasc-bearing annuli, maintains the structural integrity of the aesthetascs and thus allows analysis of physiological properties of ORNs in situ. Antennule slices loaded with OGB typically showed numerous loaded ORN somata, most of which could clearly be delineated (Fig. 2a, b). When the aesthetasc setae were stimulated with 1 % TM, many of the loaded ORN somata (67 % of 214 analyzed ORN somata from 6 antennule slices) showed a robust and transient increase in fluorescence intensity (Fig. 2c–f). We chose TM as standard chemical stimulus due to its ability to cause excitation and a transient increase in somatic Ca2+ in many spiny lobster ORNs (Michel et al. 1991; Ukhanov et al. 2011). In control experiments (data not shown), we stimulated ORNs with 1 % TM and Panulirus saline (negative control) and found that Panulirus saline did not cause an increase in fluorescence intensity >0.05 ΔF/F (the criterion to identify odorant-induced responses) in any of them. We typically used antennule slices for only one experiment lasting ~30 min, but ORN somata showed robust odorant-induced responses for up to 1 h (Fig. 2h, i). Overall, this demonstrates the functionality of the Ca2+ imaging set-up for spiny lobster ORNs.
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Odorant‑induced Ca2+ transients and spontaneous Ca2+ oscillations in ORNs Stimulation of aesthetasc setae with 1 % TM caused a stimulus-related transient increase in mean fluorescence intensity in many ORN somata, which we call ‘odorant-induced Ca2+ transient’ (Fig. 2a–f). The odorant-induced Ca2+ transients varied considerably between ORNs in maximum amplitude and time course of fluorescence increase and decay (Fig. 2c–f), but in a given ORN they were stable over repeated stimulations. The amplitude of odorant-induced Ca2+ transients ranged from 0.05 to 0.35 ΔF/F (0.05 ΔF/F being the minimum cutoff criterion) with amplitudes between 0.10 and 0.20 ΔF/F being most common. Upon stimulus presentation, the fluorescence typically started to increase with very short delay (
