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. 2012;7(8):e42602.
doi: 10.1371/journal.pone.0042602. Epub 2012 Aug 3.

Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons

Yu-Jin Won  1 , Fumihito Ono, Stephen R Ikeda
Affiliations

Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons

Yu-Jin Won et al. PLoS One. 2012.

Abstract

Background: Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/principal findings: We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+) currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I(Na) were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na) was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na) components. Voltage-gated Ca(2+) currents (I(Ca)) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I(Ca) in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/significance: Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. EGFP expression driven by the Isl2b promoter facilitates observation of dorsal root ganglia development concurrent with degeneration of Rohon-Beard (R-B) neurons.
A, At 2 days post-fertilization (dpf), EGFP was intensely expressed in R-B and DRG neurons. B-a–c, Higher magnification confocal images of the spinal cord in isl2b:EGFP at 30, 40 and 50 dpf. A few EGFP-labeled cells remained over the dorsal longitudinal fasciculus (DFL, white triangles) that progressively decreased until only ∼1–3 cells/field remained (B-b and c, green arrow heads). All images are maximum intensity projections of z-stacks acquired in the lateral plane using confocal microscopy. The contrast of the images was adjusted to emphasize EGFP-labeled sensory neurons. Scale bar in A represents 0.5 mm and the other scale bars represent 50 µm.
Figure 2
Figure 2. Isolation of single DRG neurons from isl2b:EGFP transgenic zebrafish.
A, Dissected central trunk region including the spinal cord and notochord from juvenile isl2b:EGFP fish (left panel). Enlarged dashed region shows EGFP-labeled DRG present in every trunk segment from the primary preparation (A-a and b). Note that the trunk segment had been exposed to enzymes at this point. B, Phase-contrast (B-a) and fluorescent (B-b) photomicrographs of acutely dissociated DRG neurons (circled) from the primary preparation. Scale bar in A (top) represent 0.1 mm and scale bar in B (bottom) represent 50 µm. C, Dot plots representing cell diameter of single dissociated DRG (filled circles) and R-B (open circles) neurons. Error bars represent standard deviation and statistical significant was determined using an unpaired t-test with Welch's correction.
Figure 3
Figure 3. Immunohistochemistry of DRG neurons in juvenile isl2b:EGFP fish sections.
A, Confocal image displaying EGFP-labeled DRG neurons and DFL in transverse section. Note that both dorsal and ventrolateral positioned DRG (arrows) cell bodies were observed. B, Higher magnification images of DRG ganglion reveal a variety of cell body sizes (B-a). Counterstaining with DAPI (B-b, magenta) revealed that some EGFP-negative or dim cell bodies were also present in DRG sections (asterisks). C, EGFP-positive and -negative DRG neurons from juvenile isl2b:EGFP fish. A few DRG neurons stained with anti-HuC/HuD neuronal protein antibody (magenta) did not express EGFP. D, Isolectin B4 (IB4) staining. Most DRG cell bodies and some spinal neurons were labeled with IB4 (magenta). Dashed rectangular region was enlarged for displaying labeled DRG neurons (D-a). Solid line represents the dorsal boundary of the spinal cord. Inset cartoons represent orientation of images. D, dorsal; V, ventral; SC, spinal cord; DFL, dorsal longitudinal fasciculus. All scale bars represent 10 µm.
Figure 4
Figure 4. Distinct subpopulations of DRG neurons based on Na+ current (INa) inactivation kinetics.
A, Representative traces ensemble for Rapidly/Slowly-inactivating INa acquired using whole-cell patch-clamp. Holding potential: −80 mV; test potentials are indicated for each trace. Superimposed Rapidly/Slowly-inactivating INa were evoked by a series of voltages steps to potentials ranging between −70 and +40 mV from a holding potential of −80 mV (bottom). For illustrative purposes, current traces from −50 to +30 mV are shown. B, Curve fitting examples and time constants (tau) for the decay phases from Rapidly/Slowly-inactivating INa traces. Single exponential functions are represented by gray lines. Holding potential: −80 mV; test potentials are indicated for each trace. C, Dot plots represent current decay/inactivation time constant fitted to a single exponential process at the indicated potential. D, Cumulative probability distribution of inactivation ταυ values obtained at the indicated conditioning potentials.
Figure 5
Figure 5. Characterization of R-and S-INa recorded in zebrafish DRG neurons.
A, The current density versus test potential (I–V) relationship for R- and S-INa. B, Dot plots represent the membrane capacitance of DRG neurons displaying R- (open circles) and S-INa (filled circles). Vertical bars represent the median. *** P<0.001, Mann-Whitney U-test. C, Concentration-response curves for block of the R- and S-INa by tetrodotoxin (TTX). Solid lines represent nonlinear regression least-square fits of experimental points to a Hill equation (see under Material and Methods). D, The voltage-dependence of the Na+ conductance (GNa, left panel) and steady-state inactivation (right panel) of R- and S-INa. Conductance was calculated from GNa = INa/(Vm−Vrev), in which INa is the peak current, Vm is the potential of the test pulse, and Vrev is the reversal potential for INa. The solid line represents a nonlinear regression fit to the Boltzmann function: 1/(1+exp[−(V−V1/2)/k]), where V is the step membrane potential, V1/2 is the half-activation potential, and k is a slope factor. Voltage-dependence of inactivation was determined using a 200 ms conditioning pulse followed by a test pulse to −10 mV. Test pulse currents were normalized to the maximal value. Solid line is a fit to the Boltzmann equation (k is negative for inactivation curve). Error bars on each symbol represent the mean ± s.e.m. The number of neurons tested is shown in parentheses.
Figure 6
Figure 6. RT-PCR analysis of mRNA encoding Na+ channels from zebrafish DRG.
A, Upper and lower electrophoresis images show RT-PCR products generated from specific primer sets coding for Na+ channel isoforms (zscn1Laa/ab, zscn4aa/ab, zscn8aa/ab and zscn5Laa/ab) from zebrafish whole brain and muscles, respectively. B, Phase-contrast (upper) and fluorescent (bottom) images of an example dissociated a DRG cluster as was used for RT-PCR. Scale bar represent 50 µm. C, RT-PCR products generated from a DRG cluster. PCR products of each lane represent they were generated from same RT reaction tube. Zscn primer sets were used in the same order as arranged in Fig. 6A. PCR was also performed with β-actin as well as EGFP primer sets to establish successful DRG neurons isolation. The resultant PCR products were separated and visualized on 1.5% agarose gel. L: DNA ladder, m: culture media, Filled and opened arrows represent 1 Kbp and 0.5 Kbp size markers, respectively.
Figure 7
Figure 7. Ca2+ currents (ICa) from single dissociated DRG neurons.
A, Two representative traces of ICa acquired from separated neurons using whole-cell patch-clamp. ICa were evoked by a 160 ms ramp from −80 to +80 mV (top of traces) from a holding potential of −80 mV. A tiny “hump” (arrow) in the ramp current, indicative of low-voltage-activated ICa was observed in some neurons (right). B, Dot plots representing current amplitude (left panel), membrane capacitances (middle panel) and current density (pA/pF, right panel) of DRG neurons with (closed circles) and without (filled circles) low-voltage-activated components. Statistical significance was determined using an unpaired t-test with Welch's correction for unequal variance (P>0.05, middle panel). Error bars represent s.e.m.
Figure 8
Figure 8. Ca2+ current-voltage (I–V) relationships from zebrafish DRG neurons.
A–B, Superimposed ICa traces (A) and means normalized (to maximal amplitude) I–V relationships (B). Current traces were evoked by a series of voltages steps to potentials ranging between −80 to +60 mV from a holding potential of −80 mV. For illustrative purposes, only traces from −50 mV to −30 mV are shown. Error bars on each symbol represent the mean ± s.e.m. The number of neurons tested is shown in parentheses.
Figure 9
Figure 9. Pharmacological dissection of HVA-ICa in zebrafish DRG neurons.
A, Left, Time courses of ICa amplitude during serial application of nifedipine (10 µM), ω-agatoxin IVA (0.5 µM), ω-conotoxin GVIA (3 µM), SNX-482 (300 nM) and CdCl2 (100 µM). ICa was evoked every 10 s by 70 ms test pulses to 0 mV from a holding potential of −80 mV. The horizontal bars indicate the duration of drug application. Right, superimposed current traces obtained at different time points during drug application (labeled as a–f). B, Bar graph representing the mean ICa inhibition (%) produced by application of the indicated antagonists or toxins. Error bars represent s.e.m. The number of neurons tested is indicated in parentheses. C, Effect of non-dihydropyridine Ca2+ channel agonist FPL 64176 (FPL) on ICa in DRG neurons. FPL (1 µM) was applied to rat superior cervical ganglion (SCG) neurons as a positive control (left panel). Note that FPL applied to zebrafish DRG ICa display neither an increase in macroscopic inward currents nor greatly prolonged trajectory of the tail currents (right panel).
Figure 10
Figure 10. Modulation of HVA-ICa by neurotransmitters in zebrafish DRG neurons.
A–B, Time courses of ICa (left) and superimposed current traces (right) evoked with the double-pulse voltage protocol (inset on the traces in A) during application of 5-HT (10 µM) and DAMGO (1 µM), respectively. The ICa amplitude generated by the pre-pulse (filled circles) and post-pulse (open circles) are plotted. Facilitation ratio (FR) was calculated as the ratio of the post-pulse to pre-pulse ICa amplitude (open squares). The horizontal bars indicate the duration of drugs application. C, Dot plots represent inhibition of prepulse ICa (%) produced by application of norepinephrine (NE, 10 µM), GABA (100 µM), 5-HT, l-glutamic acid hydrochloride (l-Glu, 100 µM), oxotremorine M (OxoM, 10 µM) and DAMGO. D, Summary of the mean FR (Post/Pre) before (basal, open bars) or after (filled bars) application of agonists. Data are presented as mean ± sem. * P<0.05, ** P<0.001 by paired t-test.
Figure 11
Figure 11. Heterogeneous expression pattern of anti-GABAb receptor antibody staining on DRG neurons.
A, Labeling with an anti-GABAb R1 antibody. Enlarged images (A-a, left and right) show only a specific subset of DRG neurons were labeled (asterisks). B, Labeling with an antibody against 5-HT. 5-HT antibody binding was mostly in spinal cord tracts rather than DRG neurons somata (enlarged box B-a). Inset cartoons represent orientation of images. D, dorsal; V, ventral; SC, Spinal Cord; NC, Notochord. Scale bars represent 20 µm.
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