DOI: 10.5061/dryad.fxpnvx161
title: Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception

description: Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a
 neurotensin analogue, has been demonstrated to be safe and potentially
 analgesic in humans. However, the mechanism of action for CGX analgesia is
 unknown. We hypothesized that spinal application of CGX produces
 antinociception through activation of the presynaptic neurotensin receptor
 (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception
 in rodent models of inflammatory and neuropathic pain. Intrathecal
 administration of CGX, dose dependently, inhibited thermal and mechanical
 hypersensitivities in rodents of both sexes. Pharmacological and clustered
 regularly interspaced short palindromic repeats/Cas9 editing of NTSR2
 reversed CGX-induced antinociception without affecting morphine analgesia.
 Electrophysiological and gene editing approaches demonstrated that CGX
 inhibition was dependent on the R-type voltagegated calcium channel
 (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression
 of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic
 fractionation and slice electrophysiology recordings confirmed a
 predominantly presynaptic effect. Together, these data reveal a nonopioid
 pathway engaged by a human-tested drug to produce antinociception.
description: # Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium
 channel (Cav2.3) pathway to mediate spinal antinociception Dataset DOI:
 [10.5061/dryad.fxpnvx161](10.5061/dryad.fxpnvx161) ## Description of the
 data and file structure There are 6 figures in this paper and the data in
 the excel sheet is organized by figure.  figure 1: in the excel sheet
 under figure 1 is the raw numerical outputs for the von frey animal
 behavior. and area under the curve. the data is listed under the
 corresponding panel **Panel B and C**:  **Row Labels:** 
 `Animals: ` numerically represented, but placed by male and female groups
 each with experimental treatment variables ( Saline, CGX 0.02nmol, CGX
 0.1nmol, CGX 0.5nmol) `BL` = Baseline measurement (before surgery).
 `PostSx` = 24 hours post-surgery, immediately before treatment injection.
 `0.5`, `1`, `2`, `4`, `5` = Hours after treatment injection. And for the
 units: **Units:** All values in Panel B (von Frey test) are reported as
 [e.g., withdrawal threshold in grams].  All values in Panel C (Hargreaves
 test) are reported as [e.g., paw withdrawal latency in seconds]. Panel
 D: Area under the curve analysis for mechanical withdrawal thresholds is
 presented in panel (B) Panel E: Area under the curve analysis for thermal
 withdrawal latencies id presented in panel (C). The AUC was calculated
 from the time of injection (time 0, which corresponds to the
 "PostSx" baseline) through the 5-hour time point for each
 individual animal. A higher AUC value indicates a greater overall pain
 relief (antinociceptive effect) over the 5-hour observation period.
 **Panel G and H**: **Row Labels:**  `Animals: ` numerically represented,
 but placed by male and female groups each with experimental treatment
 variables ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol) `BL` =
 Baseline measurement (before surgery). `PostSx` = 2 weeks post-surgery,
 immediately before treatment injection. `0.5`, `1`, `2`, `4`, `5` = Hours
 after treatment injection. And for the units: **Units:** All values in
 Panel G (von Frey test) are reported as [e.g., withdrawal threshold in
 grams].  All values in Panel H (Hargreaves test) are reported as [e.g.,
 paw withdrawal latency in seconds]. Panel I: Area under the curve analysis
 for mechanical withdrawal thresholds is presented in panel (G) Panel
 J: Area under the curve analysis for thermal withdrawal latencies id
 presented in panel (H). The AUC was calculated from the time of injection
 (time 0, which corresponds to the "PostSx" baseline) through the
 5-hour time point for each individual animal. A higher AUC value indicates
 a greater overall pain relief (antinociceptive effect) over the 5-hour
 observation period. **figure 2**:  **Panel B:** **Row Labels:** 
 `Animals: ` numerically represented, but placed by group ( Saline, CGX
 0.02nmol, CGX 0.1nmol, CGX 0.5nmol) `Baseline `= Baseline measurement
 (before surgery). `BL PostSx` = 24 hours post-surgery, immediately before
 treatment injection. `0.5`, `1`, `2`, `4`, `5` = Hours after treatment
 injection. **Panel E:** `Animals: ` numerically represented, but placed by
 group ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol) `Baseline `=
 Baseline measurement (before surgery). `BL PostSx` = 2 weeks post-surgery,
 immediately before treatment injection. `0.5`, `1`, `2`, `4`, `5` = Hours
 after treatment injection. And for the units: **Units:** All values in
 Panel B and E (von Frey test) are reported as [e.g., withdrawal threshold
 in grams].  **Panel C and F**: Area under the curve analysis for
 mechanical withdrawal thresholds is presented in panel (B and E) The AUC
 was calculated from the time of injection (time 0, which corresponds to
 the "PostSx" baseline) through the 5-hour time point for each
 individual animal. A higher AUC value indicates a greater overall pain
 relief (antinociceptive effect) over the 5-hour observation period.
 **figure 3:** **Panel B**: quantification western blot images NTSR1 and
 NTSR2 expression calculated by percent. groups (Non Injected, Ctrl gRNA,
 NTSR2 gRNA) **Panel C:**  `Animals: ` numerically represented, but placed
 by group (Morphine-ctrl gRNA-ipsilateral, Morphine-ctrl
 gRNA, contralateral, 0.15 nM Cont-G-ctrl
 gRNA-ipsilateral, 0.15 nM Cont-G-ctrl gRNA, contralateral, Morphine-nstr2
 gRNA-ipsilateral, Morphine-nstr2 gRNA, contralateral, 0.15 nM Cont-G-nstr2
 gRNA-ipsilateral, 0.15 nM Cont-G-nstr2 gRNA, contralateral) `BL `=
 Baseline measurement (before surgery). `BL PostSx` = 2 weeks post-surgery,
 immediately before treatment injection. `1`, `2`, `4`= Hours after
 treatment injection. And for the units: **Units:** All values in Panel C
 (von Frey test) are reported as [e.g., withdrawal threshold in grams]. 
 **Panel D**: Mechanical withdrawal thresholds before and 1 hour after
 morphine or CGX injections, in the same conditions as panel (C). 2 groups
 ( Ctrl gRNA, ntsr2 gRNA) treatments under groups (Mor ipsi BL Mor ipsi 1h
 CGX ipsi BL CGX ipsi 1h). **figure 4:**  **Panel B:** Current–voltage
 relationships for total calcium currents. Peak currents were normalized to
 cell capacitance. units are Mb pot = membrane potential in (mV)
 (millivolts). groups are ( saline, CGX (10nM)) **Panel C:** peak current
 density (pA/pF) measured at +10 mV for CGX-treated neurons. groups
 (saline, CGX 10nM). units in  (mV) (millivolts) **Panel
 E:** Current–voltage relationships for R-type calcium currents are shown
 in. Peak currents were normalized to cell capacitance.units are Mb pot =
 membrane potential in (mV) (millivolts). groups are ( saline, CGX (10nM))
 **Panel F:** peak current density (pA/pF) measured at +10 mV for
 CGX-treated neurons. groups (saline, CGX 10nM). units in  (mV)
 (millivolts) **figure 5**:  **Panel B**: quantification western blot
 images Cav2.3 and Cav2.2 expression calculated by percent. groups
 (Ctrl gRNA, Cav2.3 gRNA ) **Panel C:**  `Animals: ` numerically
 represented, but placed by group (Morphine-ctrl
 gRNA-ipsilateral, Morphine-ctrl gRNA, contralateral, 0.15 nM Cont-G-ctrl
 gRNA-ipsilateral, 0.15 nM Cont-G-ctrl gRNA, contralateral, Morphine-nstr2
 gRNA-ipsilateral, Morphine-nstr2 gRNA, contralateral, 0.15 nM Cont-G-nstr2
 gRNA-ipsilateral, 0.15 nM Cont-G-nstr2 gRNA, contralateral) `BL `=
 Baseline measurement (before surgery). `BL PostSx` = 2 weeks post-surgery,
 immediately before treatment injection. `1`, `2`, `4`= Hours after
 treatment injection. And for the units: **Units:** All values in Panel C
 (von Frey test) are reported as [e.g., withdrawal threshold in grams]. 
 **Panel D**: Mechanical withdrawal thresholds before and 1 hour after
 morphine or CGX injections, in the same conditions as panel (C). 2 groups
 ( empty vector, Cav2.3 CRISPR) treatments under groups (Mor ipsi BL Mor
 ipsi 1h CGX ipsi BL CGX ipsi 1h). **Figure 6:** **Panel B**:
 Quantification of cells expressing ntsr2, cacna1e, or both mRNA . numbers
 represented by percent cells positive in y axis and group in x axis.
 groups (Ntsr2 cells, Cacna1e cells, Co-exp) **Panel
 C: **Relative expression of NSTR2 and Cav2.3 mRNA
 depending on cells expressing one of the mRNAs. . relative expression
 percent in y axis, number represent %. groups
 (Cav2.3 pos cell expressing Ntsr2, Ntsr2 pos cell expressing Cav2.3)
 **Panel D**: DRG cell surface, depending on NTR2 mRNA expression, CaV2.3
 mRNA expression, or both. x axis represents values in cell surface
 units=uM squared. groups (CoExp, CaV2.3, NTR2, Total) **Panel F**:Values
 represent mean amplitude and frequency of sEPSC after treatments. data
 shown in values of amplitude and frequency (hertz). variables (Ctrl,
 Contulakin-G, 10 nM). **Panel G:** KCl-depolarization evoked CGRP release
 from spinal afferents in mice. Isolated mouse lumbar spinal cords were
 washed (90 minutes), 2 baselines were collected (10 minutes fraction each,
 BL1 and BL2), and CGX (1-1000 nM) was pretreated (Tx) and cotreated with
 KCl (90 mM). Supernatant was analyzed for immunoreactive CGRP (iCGRP)
 using ELISA. data represented in CGRP release from pg/ml/mg of spinal
 cord. groups ( Saline, CGX 1nM, CGX 10nM, CGX 1000nM). **Panel I and
 J:** data represents the quantification of Western blot data for Cav2.3 by
 synaptic region. represented as expression a.u. (arbitrary units). groups
 ( Postsyn, Presyn). **Panel J**: A bar graph with scatterplot represents
 the quantification of Western blot data for NTSR2 by synaptic
 region. represented as expression a.u. groups ( Postsyn, Presyn). ##
 Code/software primary software used to analyze and collect data: microsoft
 excel graphpad prism ImageJ