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. 2023 Jan 23;12:e76863. doi: 10.7554/eLife.76863

Figure 7. Decreasing extracellular Cl− and overexpression of ncc69 causes nociceptor and behavioral sensitization.

(A) Extracellular application of low Cl− saline to fileted electrophysiology preps causes spontaneous bursting activity. At room temperature, neurons are largely silent but may show occasional, low-frequency spiking (likely associated with mechanosensation from saline flow). (B) The presence of low Cl− saline causes spontaneous firing and sensitizes class III (CIII) neurons to cooling. Room temp: saline (n=12); low Cl− (n=12); wash (n=8); p=0.003, BF10=1.89. 20°C: saline (n=12); low Cl− (n=12); wash (n=8); p=0.003, BF10=66.38. 15°C: saline (n=12); low Cl− (n=12); wash (n=7); p=0.011, BF10=14.51. 10°C: saline (n=12); low Cl− (n=11); wash (n=7); p=0.25, BF10=0.75. (C) Top: Heatmap representation of cold-evoked CIII activity, with each line representing an individual sample prep. Middle: Representative traces of cold-evoked neural activity over graph of temperature ramp. Overexpression of ncc69 results in spontaneous nociceptor activity and increased cold sensitivity. Bottom: Representation of average spike frequency from population binned by 10 s. Red and blue bars show the proportion of bursting vs tonic spiking activity. (D) Overexpression of ncc69 causes spontaneous neural activity and sensitizes neurons to cooling. Room temp: GAL4 control (n=13); ncc69 RNAi (n=12; p=0.99; BF10=0.22); UAS-ncc69 (n=12; p<0.001; BF10=7.86). 20°C: GAL4 control (n=13); ncc69 RNAi (n=12; p=0.96; BF10=0.40); UAS-ncc69 (n=11; p=0.020; BF10=2.23). 15°C: GAL4 control (n=13); ncc69 RNAi (n=12; p=0.90; BF10=0.26); UAS-ncc69 (n=11; p=0.062; BF10=1.17). 10°C: GAL4 control (n=13); ncc69 RNAi (n=12; p=0.77; BF10=0.60); UAS-ncc69 (n=11; p=0.0042; BF10=25.18). (E) Mean peak magnitude in larval contraction in response to noxious cold (10°C). w1118 (n=28); GAL4 control (n=30; p=1; BF10=0.27); wwkMi/Mi (n=30; p=1; BF10=0.48); wwk RNAi (n=30; p=1; BF10=0.52); subduedMi/Mi (n=30; p=1; BF10=0.27); subdued RNAi (n=30; p=1; BF10=0.27); UAS-ncc69 (n=30; p=0.037; BF10=199.79); kcc RNAi (n=30; p=1; BF10=0.29). (F) % of animals which strongly contract (CT) in response to noxious cold (10°C). CIII-specific (GAL419-12) overexpression of ncc69 results in an increased percentage of strong CT in response to less-noxious cold. w1118 (n=28); GAL4 control (n=30; p=1; BF10=0.46); wwkMi/Mi (n=30; p=1; BF10=0.69); wwk RNAi (n=30; p=1; BF10=0.46); subduedMi/Mi (n=30; p=1; BF10=0.69); subdued RNAi (n=30; p=1; BF10=0.46); UAS-ncc69 (n=30; p=0.026; BF10=28.79); kcc RNAi (n=30; p=1; BF10=1.07).

Figure 7.

Figure 7—figure supplement 1. Cold nociception defects of subdued, white walker, ncc69, and kcc at less-noxious cold temperature.

Figure 7—figure supplement 1.

(A) Heatmap representation of cold-evoked contraction (CT) in genetic control (w1118 or GAL419-12), knockdown, and overexpression conditions, where FP is flaccid paralysis. N=239, n=30 for each condition except w1118, where n=28. (B) % of animals from SLC12 manipulation experiments performing a particular CT response, where NR is no response (<10% reduction in length), weak is ≥10% reduction in length, moderate is ≥20% reduction in length, and strong is ≥30% reduction in length; bars show proportions in % ± standard error of the proportion (SEP). (C) % ± SEP of animals which ceased any contractile behavior whatsoever (returned to normal length) within the experimental time frame.