Serotonin differentially modulates Ca2+ transients and depolarization in a C. elegans nociceptor

Abstract

Monoamines and neuropeptides modulate neuronal excitability and synaptic strengths, shaping circuit activity to optimize behavioral output. In C. elegans, a pair of bipolar polymodal nociceptors, the ASHs, sense 1-octanol to initiate escape responses. In the present study, 1-octanol stimulated large increases in ASH Ca(2+), mediated by L-type voltage-gated Ca(2+) channels (VGCCs) in the cell soma and L-plus P/Q-type VGCCs in the axon, which were further amplified by Ca(2+) released from intracellular stores. Importantly, 1-octanol-dependent aversive responses were not inhibited by reducing ASH L-VGCC activity genetically or pharmacologically. Serotonin, an enhancer of 1-octanol avoidance, potentiated 1-octanol-dependent ASH depolarization measured electrophysiologically, but surprisingly, decreased the ASH somal Ca(2+) transients. These results suggest that ASH somal Ca(2+) transient amplitudes may not always be predictive of neuronal depolarization and synaptic output. Therefore, although increases in steady-state Ca(2+) can reliably indicate when neurons become active, quantitative relationships between Ca(2+) transient amplitudes and neuronal activity may not be as straightforward as previously anticipated.

Keywords: 1-octanol; 5-HT; ASH; C. elegans; Ca2+ dynamics; Ca2+ imaging; electrophysiology; neuromodulation; nociception.

Fig. 1.

Ca²⁺ channels mediating 1-octanol-evoked Ca²⁺ influx into the ASH soma and axon. A: 1-octanol (oct) stimulates Ca²⁺ transients in the soma and axon of ASHs; nemadipine-A (NemA) preferentially inhibited somal Ca²⁺ signals. B: loss or block of the L-type (EGL-19), but not the P/Q-type (UNC-2) Ca²⁺ channels strongly decreased the somal Ca²⁺ signal. C: axonal Ca²⁺ signals required both L-type and P/Q-type channels. ΔF/F_o, change in fluorescence over original fluorescence intensity. Values are means ± SE; nos. within/above bars indicate n. *P < 0.05 compared with wild type (wt)/untreated. †P < 0.05 compared with unc-2 untreated.

Fig. 2.

Ca²⁺ release from intracellular stores amplifies and shapes Ca²⁺ dynamics in ASH neurons. A and C: time courses of 1-octanol-evoked Ca²⁺ transients in ASH somata (A) and axons (C) in wild-type and inositol tris-phosphate receptor (IP₃R) mutants (left) or ryanodine receptor (RYR) mutants (right; itr-1 and unc-68, respectively). 1-Octanol was applied between 0 and 20 s (gray box). B and D: peak amplitudes of somal (B) and axonal (D) Ca²⁺ transients. E–H: kinetic analysis of somal (E and F) and axonal (G and H) Ca²⁺ transients: signal rise times (E and G), decay slopes (F and H). Black bars in F and H indicate desensitization in the continued presence of 1-octanol; white bars indicate decay rate after 1-octanol removal (rates measured at the intervals denoted by the black and white boxes in A and C, respectively). Values are means ± SE; nos. within bars indicate n. n = 13 wild type, 12 itr-1, and 7 unc-68 (A and F); n = 6 wild type, 8 itr-1, and 6 unc-68 (C and H). *P < 0.05 compared with corresponding wild-type measurement. †P < 0.05 compared with itr-1 desensitization rate.

Fig. 3.

Aversive responses not affected by reduction of Ca²⁺ influx through EGL-19 L-type voltage-gated Ca²⁺ channel (VGCC). ASH-selective RNA interference (RNAi) knockdown of the EGL-19 L-type channel (using the sra-6 promoter) did not alter 1-octanol response times (P > 0.05; A), even though Ca²⁺ signals in the ASH soma in response to 1-octanol exposure were strongly reduced (B). Values are means ± SE; nos. within bars indicate n. *P < 0.05 compared with wild type.

Fig. 4.

5-HT modulation of ASH chemosensory responses. A: aversive behavioral responses to 1-octanol in Psra-6::GCaMP3 worms. Basal responses and 5-HT stimulation were normal, indicating that GCaMP3 expression in ASHs does not interfere with ASH function or 5-HT modulation. B: 5-HT treatment reduced Ca²⁺ signals in ASHs stimulated by 1-octanol (saturated aqueous solution); n = 7 and 13 for untreated and +5-HT, respectively, dashed lines are SEM, gray box indicates 1-octanol exposure. C: 5-HT modulation of ASH Ca²⁺ transients required the 5-HT receptor SER-5 and the Gα_q subunit EGL-30. D: axonal Ca²⁺ signals were not significantly reduced by 5-HT. E: ASH responses to 1 mM dihydrocaffeic acid (DHCA) were also reduced by 5-HT treatment. F: ASH-selective RNAi knockdown of EGL-19 prevented 5-HT potentiation of 1-octanol aversive responses; instead, 5-HT slightly inhibited them. Two different ASH-selective promoters were used, sra-6 and srb-6, as indicated in each bar. Values are means ± SE; nos. within bars indicate n. *P < 0.05, **P < 0.0001 compared with untreated.

Fig. 5.

5-HT potentiates ASH depolarization in response to 1-octanol. A: diagram of recording setup. Left: arrangement of pipettes before exposure, as whole cell recording is being established. At bottom right, the recording pipette and protruding neuronal cell bodies are shown. At top left, flow pipette providing 1-octanol solution and a fluorescent dye to monitor flow (shaded plume) is shown; at bottom left, flow pipette providing a stream of buffer to shield exposed neuron is shown. Exposure is initiated by moving 1-octanol pipette to preset position closer to nose (middle), and terminated by returning 1-octanol pipette to original position (right). B: representative traces of ASH depolarization in response to 1-octanol exposure in control (left) or 5-HT-treated worms (right). C: 5-HT effect on ASH 1-octanol-induced depolarization. ΔVm, change in membrane potential. *P < 0.0005. D: 1-octanol responses of ASH neurons dissected for electrophysiology, but analyzed by Ca²⁺ imaging, showing that 5-HT signaling to reduce Ca²⁺ signals remained intact through the dissection process. *P < 0.05. Values are means ± SE; nos. within/above bars indicate n.

Fig. 6.

Model of a hypothetical Ca²⁺-driven negative feedback loop controlling ASH excitability. The activation of the ASH sensory neuron by exposure to 1-octanol involves an as-yet-unidentified amphidial signal transduction cascade, resulting in a membrane depolarization (ΔV), which spreads down the ASH dendrite to the soma and axon, probably passively, given the apparent isopotentiality of C. elegans neurons (Goodman et al. 1998). The amphidial ASH signal transduction cascade activated by other stimuli includes the ODR-3 G protein and polyunsaturated fatty acid signaling to activate the cationic OSM-9/OCR-2 transient receptor potential channels (Bargmann 2006; Colbert et al. 1997; Kahn-Kirby et al. 2004; Roayaie et al. 1998), and these components are also probably involved in activation by 1-octanol. The stimulus-dependent ASH depolarization activates L-type VGCCs (EGL-19) in dendritic, somal, and axonal membranes, and P/Q-type VGCCs (UNC-2) at synaptic vesicle (SV) release sites in the axon, leading to glutamate release. Ca²⁺ influx through EGL-19 leads to a rise in cytoplasmic Ca²⁺ that is further amplified by the Ca²⁺-dependent release of Ca²⁺ from endogenous stores through both the RYR (UNC-68) and IP₃R (ITR-1). This cytoplasmic Ca²⁺ pool can potentially drive neuronal repolarization by activating Ca²⁺-dependent inhibitory ion channels (Ca²⁺-activated K⁺ channels, anoctamins, or bestrophins; dashed line). Importantly, ASH 5-HT signaling through the SER-5 receptor has the potential to regulate ASH excitability at many levels, including the modulation of ASH Ca²⁺ dynamics. CICR, calcium-induced calcium release; NT, neurotransmitter.