Trpv1 -dependent Cacna1b gene inactivation reveals cell-specific functions of CaV2.2 channels in vivo

Trpv1-dependent Cacna1b gene inactivation reveals cell-specific functions of CaV2.2 channels in vivo | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Trpv1 -dependent Cacna1b gene inactivation reveals cell-specific functions of Ca V 2.2 channels in vivo View ORCID Profile Remy Y. Meir , View ORCID Profile Martin S. Sisti , View ORCID Profile Arturo Andrade , View ORCID Profile Diane Lipscombe doi: https://doi.org/10.1101/2025.05.09.652131 Remy Y. Meir 1 Department of Neuroscience, Brown University , Providence, RI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Remy Y. Meir Martin S. Sisti 1 Department of Neuroscience, Brown University , Providence, RI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martin S. Sisti Arturo Andrade 1 Department of Neuroscience, Brown University , Providence, RI, USA 2 Carney Institute for Brain Science, Brown University , Providence, RI Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arturo Andrade Diane Lipscombe 1 Department of Neuroscience, Brown University , Providence, RI, USA 2 Carney Institute for Brain Science, Brown University , Providence, RI Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Diane Lipscombe For correspondence: diane_lipscombe{at}brown.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Voltage-gated Ca V 2.2 channels underlie the N-type current, and they regulate calcium entry at many presynaptic nerve endings to control transmitter release. A role for Ca V 2.2 channels has been well-established in the transmission of pain information using pharmacological and global gene inactivated mouse models. However, investigation of the cell-specific actions of Ca V 2.2 channels would benefit from the availability of cell-restricted knockout mouse models and particularly in dissecting behavioral responses that depend on Ca V 2.2 channel activity. Here, we show the importance of Ca V 2.2 channels in Trpv1 -lineage neurons in behavioral responses to sensory stimuli using Cre-dependent inactivation of the Cacna1b gene. Our work shows the cell- type specificity of Ca V 2.2 channels in mediating rapidly developing heat hypersensitivity and the utility of Cre-dependent inactivation of Cacna1b to discern cell-specific Ca V 2.2 channel functions. INTRODUCTION Voltage-gated Ca V 2.2 channels regulate calcium entry at many presynaptic nerve endings to control transmitter release. At some synapses, Ca V 2.2 channels co-regulate synaptic transmission with Ca V 2.1 channels whereas, at other synapses including in the brain, and in sympathetic and sensory neurons, Ca V 2.2 channels are the dominant source of calcium that triggers transmitter release 1 , 2 . Ca V 2.2 channels are well known targets of numerous neuromodulators and drugs that down regulate synaptic efficacy via G protein coupled receptor activation 3 . At sensory nerve endings in skin, Ca V 2.2 channels play a key role in neuroimmune signaling during neuroinflammation 4 , 5 . The availability of highly specific toxins for Ca V 2.2, ω-CgTx-GVIA and ω-CgTx-MVIIA, have helped define functions of Ca V 2.2 channels in certain cells and tissues 6 – 9 . Cacna1b global knockout mouse models have linked Ca V 2.2 channel activity to several behaviors, including the threshold for withdrawal responses to sensory stimuli, overall activity, Rapid Eye Movement (REM) sleep and vigilance 4 , 5 , 10 – 12 . But conditional Cacna1b -/- mouse strains would benefit studies aimed at isolating cell-specific actions of Ca V 2.2 channels and dissecting their contributions to specific neuronal circuits and behaviors. In addition, such models would improve analyses aimed at discerning the contribution of Ca V 2.2 channels to specific behaviors as compared to global Cacna1b -/- mouse models which all exhibit a strong hyperactivity phenotype 10 , 11 , 13 , 14 . Here we use a Cacna1b fl/fl floxed mouse strain for Cre-dependent inactivation of Cacna1b in Trpv1 -lineage neurons. We show the dual function of Ca V 2.2 channels in Trpv1 -expressing, noxious heat-sensing neurons; controlling the efficacy of transmission from peripheral to central sites and triggering heat hypersensitivity in skin associated with neuroinflammation. We also show that Ca V 2.2 channels contribute to paw withdrawal responses to cold, as well as heat stimuli consistent with their importance in triggering transmitter release at presynaptic sites. RESULTS Ca V 2.2 protein levels reduced in DRG but not in brain of conditional knockout mice Cacna1b fl/fl (WT-fl) and Trpv1 Cre/Cre (WT-cre) breeding pairs were used to generate Cacna1b fl/fl / Trpv1 Cre /- (cKO) mice lacking functional Ca V 2.2 channels in Trpv1 -lineage neurons ( Fig. 1A ). cKO mice were viable and fecund and, by these metrics, similar to WT-fl littermate controls ( Table 1 ). Trpv1 is expressed primarily in sensory neurons of dorsal root ganglia (DRG) including in heat and cold sensing subtypes 15 – 17 . We used polyclonal anti-Ca V 2.2 antibodies 10 , 18 , 19 , to compare levels of Ca V 2.2-specific signals in protein preparations from brain and DRG of cKO, Cacna1b -/- (global knockout; gKO) and WT-fl mice ( Fig. 1B ). Polyclonal anti-Ca V 2.2 target an epitope located on the intracellular loop between domains II and III of Ca V 2.2. These polyclonal antibodies recognizes a number of protein bands covering a range of molecular weights in protein preparations of the brain and DRG ( Fig. 1B ) consistent with previously published findings from our group using polyclonal anti-Ca V 2.2 in native tissue 19 , 20 . The use of gKO tissue was therefore essential to verify the identity of Ca V 2.2-specific bands in brain (a single band at ∼280 kDa) and in DRG (two bands at ∼280 kDa and ∼230 kDa). In cKO and WT-fl control brain protein preparations, the intensity of Ca V 2.2-specific bands were similar ( Fig. 1B ), as expected given the very low level of Trpv1 expression in mouse brain 17 , 21 , 22 . By contrast, in DRG which contains relatively high abundance of Trpv1 -lineage DRG neurons 23 , 24 , the intensity of Ca V 2.2-specific bands from cKO were ∼50% of WT-fl controls ( Fig. 1B ). These data are as expected for selective inactivation of Cacna1b in Trpv1 -lineage neurons and they highlight the importance of using gKO tissue to identify Ca V 2.2-specific signals using polyclonal antibodies in native tissue 25 . Download figure Open in new tab Figure 1. Cacna1b gene targeting strategy and Ca V 2.2 protein analysis of brain and dorsal root ganglia. Gene targeting strategy ( A ). CRISPR-Cas9 insertion of loxP sites in introns flanking exons 5 and 6 of Cacna1b (mm10 chr2:24,603,887-24,763,152). B, C , Western analyses of protein preparations from brain ( B : lanes 1-6) and DRG ( C : lanes 1-3) of Cacna1b -/- (gKO; B , lanes 1, 2; C , lane 1), Cacna1b fl/fl / Trpv1 Cre/- (cKO; B , lanes 3, 4; C, lane 2) and Cacna1b fl/fl (WT-fl, B , lanes 5, 6; C , lane 3) mice. Anti-Ca V 2.2 polyclonal antibody (ACC-002, Alomone Labs) identified one prominent Ca V 2.2-specific band in brain at ∼280 kDa in cKO and WT-fl; and two Ca V 2.2-specific bands at ∼280 and ∼240 kDa in cKO and WT-fl DRG. D , Intensity of Ca V 2.2- specific signals in brain and DRG of cKO relative to WT-fl are plotted for 3 experiments. The average band intensity (in arbitrary units) for brain was WT-fl 95.3 ± 5.7 and cKO 101.7 ± 2.3 and were not significantly different from control (WT-fl | cKO: t = 0.9759 p = 0.3668; Student’s t-test). View this table: View inline View popup Download powerpoint Table 1. Cacna1b fl/fl /Trpv1 Cre/- mice are viable and fecund and show no differences respect to WT-fl littermate controls. Whole cell Ca V 2.2 currents absent in Trpv1 -positive DRG neurons of cKO mice To confirm selective loss of Ca V 2.2 protein in Trpv1 -lineage neurons of DRG, we compared whole cell Ca V currents in DRG neurons isolated from cKO, WT-cre and WT-fl mice. Recordings were intentionally biased toward small diameter neurons which contain the highest percentage of Trpv1 -expressing neurons 18 ( Fig. 2A ). The average size of neurons in this analysis, based on measurements of total cell capacitance, was not statistically different among the three genotypes ( Fig. 2A : WT-fl = 15.3 ± 0.88 pF, cKO = 16.8 ± 1.10 pF and WT-cre = 15.2 ± 0.65 pF; WT-fl | cKO | WT-cre: H = 0.105, p = 0.9489; Kruskal-Wallis). Ca V currents were recorded from a total of 107 DRG neurons in response to step depolarizations applied in 5 mV increments, from a holding potential of -80 mV (2 mM Ca 2+ was used as the charge carrier; Fig. 2B ). Total Ca V current densities recorded from WT-cre and WT-fl control DRG neurons were not statistically distinguishable ( Fig. 2C ; WT-cre | WT-fl: t = -0.446, p = 0.6574; Student’s t-test) so we used WT- cre controls for all electrophysiology experiments due to ability to label Trpv1 -lineage neurons. Download figure Open in new tab Figure 2. Whole cell voltage-gated Ca V currents are reduced in Trpv1 -positive neurons from Cacna1b fl/fl /Trpv1 Cre/- cKO mice. Ca V current-voltage relationships recorded from acutely dissociated DRG neurons from Cacna1b f l/fl (WT-fl, light blue; n = 31), Trpv1 Cre/Cre (WT-cre, dark blue; n = 35) and Cacna1b fl/fl /Trpv1 Cre/- (cKO, red; n = 41). A , Whole cell capacitance measurements from DRG neurons from WT-fl, (light blue; 15.3 ± 0.88 pF), WT-cre (dark blue; 15.2 ± 0.65 pF) and cKO (red; 16.8 ± 1.10 pF) mice were not statistically distinguishable ( H = 0.105, p = 0.9489; Kruskal-Wallis). B , A series of Ca V currents from a single neuron elicited by depolarizing steps from a holding potential of -80 mV to voltages between -50 mV and + 70 mV in 5 mV increments. C , D , E , Average Ca V current densities (pA/pF) plotted as a function of the test voltage for all neurons from each of the three genotypes ( C ), Trpv1 -positive (tdT+; D ), and Trpv1 -negative (tdT−; E ) neurons. Insets for each panel show representative Ca V current recordings evoked by steps to 0 mV from -80 mV; scale bars correspond to 200 pA and 10 ms ( C, D, E ). C , Total inward Ca V current of current-voltage relationships generated from recordings from DRG neurons of WT-fl (light blue) and WT-cre (dark blue) mice are not statistically different ( t = -0.446, p = 0.6574; Student’s t-test). Viral delivery of tdTomato in cKO and WT-cre is used to identify Trpv1 -lineage neurons (tdT+) ( D , E ). D , In tdT− neurons, the total Ca V current calculated as the integral of current-voltage relationships is not statistically different between cKO (red, n = 9) and WT-cre (blue, n = 8) (WT-cre | cKO: t = -0.575, p = 0.5735; Student’s t-test). E , In tdT+ neurons, the total Ca V current from current-voltage relationships is statistically different between cKO (red, n = 32) and WT-Cre (blue, n = 27) recordings (WT-cre | cKO: t = 2.911, p = 0.005; Student’s t-test). In cKO/ tdT− neurons a prominent low threshold Ca V 3-type current is evident. Ca V 3 currents are a prominent feature of non- Trpv1 -expressing mechanosensory DRG neurons which have slightly larger cell diameters compared to Trpv1 -lineage neurons 31 (as seen in the capacitance plot ( A ) and see 18 ). Ca V current-voltage relationships show average (solid line) ± SEM (shaded area) values together with the average fitted curves using one ( C, E ) or the sum of two ( D ) Boltzmann-linear line functions. Average parameters calculated from the fitted curves of individual cells. We used adeno-associated viral (AAV) delivery of Cre-dependent tdTomato (tdT) to label Trpv1 - lineage neurons in WT-cre and cKO mice and compared Ca V currents in non- Trpv1 -lineage (non- Cre expressing; tdT−) and Trpv1 -lineage (Cre-expressing; tdT+) neurons ( Figs. 2D , 2E ). In cKO mice, total Ca V current densities in tdT− neurons ( n = 9) were statistically indistinguishable from tdT− neurons of WT-cre controls ( n = 8) ( Fig. 2D ; t = -0.571, p = 0.5735; Student’s t-test). By contrast, in cKO mice, total Ca V current densities in tdT+ neurons ( n = 32) were significantly lower compared to tdT+ neurons of WT-cre controls ( n = 27) ( Fig. 2E ; t = 2.911, p = 0.0051; Student’s t-test). These data show there is selective loss of Ca V currents in tdT+, but not tdT− neurons, of cKO mice and they confirm selective inactivation of Cacna1b in Trpv1 -lineage neurons. There is a low threshold Ca V 3 current in recordings from tdT−, but not in tdT+ neurons of cKO mice ( Fig. 2D ). This current corresponds to Cacna1h -expressing mechanoreceptors which do not express Trpv1 23 , 24 , 26 – 29 and which are slightly larger than Trpv1 -expressing neurons 18 (see Fig. 2A ). ω-Conotoxin GVIA (ω-CgTx-GVIA) is a potent, selective inhibitor of Ca V 2.2 channels. We compared the effects of ω-CgTx-GVIA on Ca V currents in tdT− and tdT+ neurons of WT-cre and cKO DRG. ω-CgTx-GVIA inhibited Ca V currents in tdT− neurons from WT-cre (∼50%) and cKO (∼42%) mice ( Fig. 3A , tdT−/WT-cre, control | ω-CgTx: n = 6, t = -2.573, p = 0.0277; Fig. 3B , cKO, control | ω-CgTx: n = 5, t = -2.761, p = 0.0246; Student’s t-test). ω-CgTx-GVIA also reduced Ca V currents in tdT+/WT-cre neurons (∼50%) ( Fig. 3C , tdT+/WT-cre, control | ω-CgTx: n = 6, t = - 3.584, p = 0.0050; Student’s t-test), whereas, Ca V current densities in tdT+/cKO neurons were only slightly smaller in the presence of ω-CgTx-GVIA ( Fig. 3D ; tdT+/cKO, control | ω-CgTx: n = 6, t = -2.101, p = 0.082; Student’s t-test). The slight reduction in maximum Ca V current densities in cKO neurons in the presence of ω-CgTx-GVIA, as compared to WT-cre controls, likely represents Ca V 1 current run-down during whole cell recording ( Fig. 3C ; also see supplementary data) 30 , 31 . Download figure Open in new tab Figure 3. Whole cell calcium current recordings using ω-CgTx-GVIA confirms selective loss of Ca V 2.2 channels in Trpv1 -lineage neurons of Cacna1b fl/fl /Trpv1 Cre/- mice. Whole cell Ca V currents were recorded in Trpv1 -negative (tdT−, A ) and Trpv1 -positive (tdT+, B ) DRG neurons isolated from Trpv1 Cre/Cre (WT-cre, left ) and Cacna1b fl/fl /Trpv1 Cre/- (cKO, right ) mice. Ca V currents were isolated using a TEA-based extracellular solution with 2 mM calcium as the charge carrier (gray) in the absence (gray) and presence (blue or red) of 2 µM ω-CgTx-GVIA to inhibit Ca V 2.2 channels ( A, B ). Representative current recordings evoked by steps to 0 mV from -80 mV are shown in insets for each average current-voltage plot. Scale bars are 400 pA and 10 ms. A, Ca V currents in tdT negative cells from WT-cre ( left, n = 6) and in cKO ( right , n = 5) mice before (gray) and after (blue/red) ω-CgTx-GVIA (tdT−: Control | ω-CgTx: t = -2.573, p = 0.028; Student’s t-test; tdT+: Control | ω-CgTx: t = -2.761, p = 0.025, Student’s t-test). B , Ca V currents in tdT positive cells from WT-cre mice (n = 7, blue/ left ) were inhibited ∼50% by ω-CgTx-GVIA ( t = -3.584, p = 0.005, Student’s t-test), whereas Ca V currents in tdT positive cells from cKO mice (cKO, n = 7, red/ right ) were slightly, but not statistically smaller in the presence of ω-CgTx-GVIA ( t = -1.893, p = 0.083; Student’s t-test). The reduced currents at the peak of the ω-CgTx-GVIA current-voltage relationship is likely due to rundown of Ca V current which has been well documented particularly for Ca V 1 channels (see Supplemental Fig. 1). Ca V current-voltage relationships show average (solid line) ± SEM (shaded area) values together with the average fitted curves using one ( A, left; B ) or the sum of two ( A, right ) Boltzmann-linear line functions. Average parameters calculated from the fitted curves of individual cells. Selective inactivation of Cacna1b in Trpv1 -lineage neurons is associated with increased neuronal excitability After validating the cKO mouse model, we compared current clamp recordings from tdT+ WT-cre and cKO DRG neurons to assess the contribution of Ca V 2.2 currents to overall excitability of Trpv1 -lineage neurons. Average resting membrane potentials of tdT+/WT-cre ( n = 14) and tdT+/cKO ( n = 10) neurons were similar (WT-cre: -62 ± 2.58 mV; cKO: -63 ± 2.62 mV; U = 32.000, p = 0.4711; Mann-Whitney U), but neurons of cKO mice were more excitable; they fired action potentials in response to smaller currents ( Fig. 4A , 4B; Rheobase: WT-cre = 1.09 ± 0.21 nA, cKO = 0.56 ± 0.12 nA; WT-cre | cKO: t = 2.160, p = 0.0435; Welch’s t-test) as well as more action potentials during the current step compared to WT-cre controls ( Fig. 4A , 4B , 4D ). The trajectory of action potentials in tdT+/WT-cre and tdT+/cKO neurons are compared in the dV/dt plots ( Fig. 4C ); action potentials in tdT+/cKO neurons exhibit steeper rising phases and deeper after hyperpolarizations compared to tdT+/WT-cre neurons ( Fig. 4C ). The threshold voltage at the inflection point of the first action potential generation ( Fig. 4E ; WT-cre = -33.93 ± 2.66 mV, cKO = -31.70 ± 2.61 mV; WT-cre | cKO: t = -0.579, p = 0.5683; Student’s t-test) and the average action potential amplitudes ( Fig. 4F ; WT-cre = 58.52 ± 3.75 mV, cKO = 65.04 ± 2.26 mV; WT-cre | cKO: t = -0.704, p = 0.4886; Student’s t-test) were the same in neurons of cKO and WT-cre mice. Action potential widths were shorter (Fig, 4G) in cKO neurons compared to WT-cre controls (WT-cre = 2.94 ± 0.22 ms, cKO = 2.45 ± 0.31 ms; WT-cre | cKO: U = 109.000, p = 0.0242; Mann-Whitney U). Download figure Open in new tab Figure 4. The absence of Ca V 2.2 channels in Trpv1 -lineage neurons is associated with increased cell excitability. Whole cell recordings from Trpv1 -lineage DRG neurons (tdT+) were used to assess cell excitability in Trpv1 Cre/Cre (WT-cre, dark blue; n = 14) and Cacna1b fl/fl /Trpv1 Cre/- (cKO, red; n = 10) mice. Resting membrane potentials were not significantly different between WT-cre (-62.4 ± 2.6 mV) and cKO (-63.3 ± 2.6 mV) neurons ( U = 32.0, p = 0.471; Mann-Whitney U). A , Examples of voltage recordings from neurons of WT-cre (blue, left ) and cKO (red, right ) mice using sodium-based external solution with 2 mM calcium. Action potentials (APs) were evoked by current injections of 100 ms applied in 0.1 nA increments. In Trpv1 -positive neurons from cKO mice, APs were triggered with smaller current injections ( B ), had slightly larger amplitudes ( A, C, F ) and occurred at higher frequencies ( D ), and had shorter widths ( C, G ) compared to WT-cre controls. Minimum currents required to evoke APs (Rheobase, B ) for WT- cre = 1.09 ± 0.21 nA, cKO = 0.56 ± 0.12 nA (WT-cre | cKO: t = 2.16, p = 0.0435; Welch’s t-test). Small symbols represent values from individual cells together with average ± SEM (solid circle, vertical bars) for each condition. C , Average phase plots show dV/dt trajectory for first APs evoked by max current injection for WT-cre ( blue ) and cKO ( red ) recordings. Data shown are average ± SEM (solid line, shaded region). D , Average AP firing rates as a function of current injection size. Current amplitude significantly affects spike rate ( β = 4.413, p < 0.0001) and there is a significant interaction between mouse strain and current amplitude (WT-cre | cKO: β = -2.456, p < 0.0001; mixed-effects regression model). Values are average ± SEM (circles, shaded region). E, F, G, Features of action potentials evoked by current injections: E , threshold voltage of first APs (WT- cre = -33.93 ± 2.66 mV, cKO = -31.70 ± 2.61 mV; WT-cre | cKO: t = -0.5793, p = 0.5683; Student’s t-test); F , average AP amplitudes for each current injection (WT-cre = 58.52 ± 3.75 mV, cKO = 62.03 ± 2.64 mV; WT-cre | cKO: t = -0.7044, p = 0.4886; Student’s t-test); and G , average AP widths for each current step (WT-cre = 2.94 ± 0.22 ms, cKO = 2.31 ± 0.303 ms; WT-cre | cKO: U = 109.000, p = 0.0242; Mann-Whitney U). Values are individual current injections that elicited an AP (small circles) together with mean (large circles) ± SEM (vertical line); current injection amplitudes are distinguished by the color gradient of the symbol (light to dark). Acute block of Ca V 2.2 channels in Trpv1 -lineage neurons has little effect on neuronal excitability The increased neuronal excitability in Trpv1 -lineage neurons of cKO mice compared to WT controls was unexpected. The faster rising phase of action potentials and increased firing rates are consistent with upregulation of Na V channels which has been reported to occur following prolonged periods of neuronal inhibition 32 , 33 . We therefore tested the effects of acute inhibition of Ca V 2.2 channels in Trpv1 -lineage neurons using ω-CgTx-GVIA ( Fig. 5 , n = 11). Acute block of Ca V 2.2 channels did not alter neuronal excitability ( Fig. 6D ; pre | post: t = -0.389, p = 0.7057; Paired t-test) and only slightly altered the action potential trajectory ( Fig. 5A , 5B). First action potential thresholds ( Fig. 5D ; pre | post: W = 30.000, p = 0.8311; Wilcoxin signed-rank test), average action potential amplitude ( Fig. 5E ; pre | post: t = 0.974, p = 0.3530; Paired t-test), and average action potential width ( Fig. 5F ; pre | post: W = 16.000 , p = 0.1475; Wilcoxin signed-rank test) were not significantly different before and after ω-CgTx-GVIA. The loss of Ca V 2.2 channel function does not result in an increase in neuronal excitability and therefore suggests that the increase in neuronal excitability observed in Trpv1 -lineage neurons from cKO mice may reflect a compensatory response to the loss of Ca V 2.2 channel function and reduced synaptic transmission 10 , 32 , 33 . Download figure Open in new tab Figure 5. Acute pharmacological inhibition of Ca V 2.2 currents in Trpv1 -lineage neurons does not alter cell excitability. Whole cell voltage recordings from Trpv1 Cre/Cre neurons (WT-cre, n = 11) in the absence (gray) and presence (dark blue) of ω-CgTx-GVIA. A , Voltage recordings in the absence (gray, left ) and presence (dark blue, right ) of 2 µM ω-CgTx. Recordings were obtained using sodium-based external solution with 2 mM calcium. Action potentials were evoked by current injections of 100 ms applied in 0.1 nA increments. B , average dV/dt phase plots of first action potentials evoked by max current injections in the absence (gray) and presence of ω-CgTx (dark blue). C , D, E, F , features of action potentials: amplitude of minimum current injected to trigger an action potential ( C , Rheobase; before ω-CgTx = 0.95 ± 0.17 nA and after ω-CgTx = 0.97 ± 0.15 nA; pre | post: t = -0.3886, p = 0.7057; Paired t-test); D, first action potential thresholds (before ω-CgTx = -34.56 ± 3.73 mV; after ω-CgTx = -33.30 ± 3.78 mV; pre | post: W = 30.000, p = 0.8311; Wilcoxin signed-rank test); E , average action potential amplitude for each current injection (before ω-CgTx = 55.15 ± 5.97 mV, after ω-CgTx = 52.84 ± 5.82 mV; pre | post: t = 0.9742, p = 0.3530; Paired t-test); and F , average action potential widths for each current step (before ω-CgTx = 3.10 ± 0.48 ms, after ω-CgTx = 3.13 ± 0.42 ms; pre | post: W = 16.000, p = 0.1475; Wilcoxin signed-rank test). Values are individual cells (small circles) with mean (large circles) ± SEM (vertical lines). Horizontal lines connect individual cell recordings before and after ω-CgTx application. Download figure Open in new tab Figure 6. The hyperactivity behavior typical of global Ca V 2.2 knockout mice is not a feature of conditional KO mice lacking Ca V 2.2 in Trpv1 -lineage neurons. Open field assay of total distance travelled (in 5 mins) for wildtype (WT, green; n = 8) and global Cacna1b -/- knockout (gKO, orange; n = 8); and for Cacna1b fl/fl controls (WT-fl, blue; n = 7) and conditional knockout Cacna1b fl/fl / Trpv1 Cre/- (cKO, red; n = 7) mice were significantly different by strain (WT | gKO | WT- fl | cKO: F = 26.140, p < 0.0001; One-way ANOVA). WT = 4107.7 ± 158.2 cm, gKO = 5672.0 ± 438.9 cm, WT-fl = 3172.3 ± 174.5 cm and cKO = 2431.1 ± 130.1 cm (WT | gKO: p = 0.0009; WT-fl | cKO: p = 0.2619, One-way ANOVA, post hoc Tukey HSD). Values shown are measures from individual mice (small circles) together with mean (large darker circles) ± SEM (vertical lines). cKO and WT-fl control mice have similar overall activity levels cKO and WT-fl mice exhibited similar levels of activity including distance travelled in an open field assay ( Fig 6A ; F = 26.140, p < 0.0001, One-way ANOVA; p = 0.2619 post hoc Tukey HSD). The WT-like behavior of cKO mice was very different from the pronounced hyperactivity of gKO mice 10 , 11 (see supplemental video). In open field, gKO mice travelled 1.4 times the distance of WT controls ( Fig. 6A ; WT: n = 8, gKO: n = 8; WT | gKO: p = 0.0009; One-way ANOVA, post hoc Tukey HSD). Our data show that Cacna1b expression in non- Trpv1 neurons underlies the pronounced hyperactivity phenotype associated with gKO mice 11 . Ca V 2.2 channels in Trpv1 -lineage neurons regulate paw withdrawal response thresholds to heat and cold We measured paw withdrawal thresholds in WT-fl and cKO mice to heat, mechanical, and cold stimuli applied to the plantar surface of hind paws ( Fig. 7A - 7C). Cacna1b is expressed in all sensory neurons 23 , 24 , but Trpv1 has a more restricted expression pattern including in heat and cold responsive, but not mechanosensitive neurons 24 , 34 . In cKO mice, the delay to paw withdrawal following exposure to heat ( Fig. 7A ) and cold ( Fig. 7B ) stimuli were significantly longer compared to WT-fl controls ( Fig. 7A , 7B; Heat: U = 20.000, p = 0.0001; Cold: U = 33.000, p = 0.0444; Mann- Whitney U). Whereas stimulus-response thresholds to mechanical stimuli were not statistically different between cKO and WT-fl mice ( Fig. 7C ; t = 1.311, p = 0.2002; Welch’s t-test). These results illustrate cell-specific deletion of Cacna1b in cKO mice, confirm the involvement of Ca V 2.2 channels in withdrawal responses to heat 4 , 5 , 10 and demonstrate a role for Ca V 2.2 channels in regulating response thresholds to cold ( Fig. 7B ). Download figure Open in new tab Figure 7. Reduced paw withdrawal sensitivity to heat and cold stimuli reduced in mice lacking Ca V 2.2 channels in Trpv1 -lineage neurons compared to wild-type. Paw withdrawal responses to heat ( A , latency), cold ( B , latency) and force ( C , grams) were measured in Cacna1b fl/fl (WT-fl, blue symbols) and Cacna1b fl/fl /Trpv1 Cre/- (cKO, red symbols) mice (WT-fl are littermate controls). Responses to heat ( A ; n = 15) and force ( C ; n = 18) are the baseline responses for both capsaicin and saline injected mice used in Fig. 8 . Small symbols represent averages of 6 measurements from each mouse (3 from each hindpaw) together with mean values (large symbols) ± SEM (vertical bars) for each genotype. Cold responses ( B ) were measured from a separate set of cKO ( n = 11) and WT-fl ( n =12) littermate control mice. Comparison of responses to heat ( A) for WT-fl = 10.69 ± 0.48 s and cKO = 13.85 ± 0.53 s (WT-fl | cKO: U = 20.000, p = 0.0001; Mann-Whitney U); cold ( B ) for WT-fl = 4.92 ± 0.62 s and cKO = 10.18 ± 1.69 s (WT-fl | cKO: U = 33.0000, p = 0.0444; Mann-Whitney U); and force ( C ) for WT-fl = 4.93 ± 0.22 g and cKO = 4.60 ± 0.14 g (WT-fl | cKO: t = 1.2109 , p = 0.2002; Welch’s t-test). Ca V 2.2 channels in Trpv1 -lineage neurons necessary for capsaicin-induced rapid heat, but not mechanical hypersensitivity We previously reported that Ca V 2.2 channels were essential for the development of capsaicin- induced heat, but not mechanical hypersensitivity in skin, using a gKO mouse model and local intradermal injection of ω-CgTx-MVIIA in wild-type mice 4 , 10 . Our data suggested that Ca V 2.2 channels in Trpv1 neurons were critical for capsaicin-induced hypersensitivity, but we could not rule out involvement of non- Trpv1 expressing cells 4 , 10 . We therefore compared withdrawal thresholds in cKO and WT-fl littermate controls to heat (Hargreaves, Fig. 8A ) and mechanical (von Frey, Fig 8B ) stimuli before, 15 min, and 30 min following intradermal administration of 0.1% w/v capsaicin or saline. Download figure Open in new tab Figure 8. Capsaicin-induced heat hypersensitivity absent in mice lacking Ca V 2.2 channels in Trpv1 -lineage neurons. Paw withdrawal responses measured at 0, 15 min and 30 min intervals from wild-type Cacna1b fl/fl (WT-fl, blue; A , B ) and Cacna1b fl/fl /Trpv1 Cre/- (cKO, red; A , B ) mice to radiant heat (latency, A ) and force (threshold, B ) applied to plantar hind paw. Right paws: i.d. 20 μl, 0.1% capsaicin (blue, red) or 20 μl, 0.1% saline (gray). Values shown are mean (solid line) ± SEM (shaded area). A , Saline ( n = 7, gray) and capsaicin ( n = 8, blue). At 15 min, WT-fl mice ( left ): saline = 12.25 ± 1.34 s, capsaicin = 5.13 ± 0.41 s (saline | capsaicin: F = 20.580, p = < 0.0001, Two-way ANOVA; p = 0.0004 post hoc Tukey HSD). cKO mice ( right ): saline = 15.00 ± 1.28 s, capsaicin = 12.50 ± 1.08 s (saline | capsaicin: F = 20.580, p = < 0.0001, Two-way ANOVA; p = 0.3600, post hoc Tukey HSD). B , Saline ( n = 7, gray) and capsaicin ( n = 11, blue). At 15 min, WT-fl mice ( left ): saline = 4.13 ± 0.26 g, capsaicin = 1.75 ± 0.20 g (saline | capsaicin: F = 53.310, p < 0.0001, Two-way ANOVA; p < 0.0001, post hoc Tukey HSD). cKO mice ( right ) saline ( n = 9, gray) and capsaicin ( n = 9, red). Saline = 4.13 ± 0.26 g, capsaicin = 2.89 ± 0.34 g (saline | capsaicin: F = 53.310, p < 0.0001, Two-way ANOVA; p < 0.0001, post hoc Tukey HSD). The capsaicin-induced hypersensitivity response in skin develops within minutes and is robust at 15 mins post capsaicin injection 4 , 10 . At this time point, WT-fl mice exhibited capsaicin-induced hypersensitivity to both heat ( Fig. 8A , middle ; saline: n = 7, capsaicin: n = 8; saline | capsaicin: F = 20.580, p = 0.0001, Two-way ANOVA; p = 0.0004, post hoc Tukey HSD) and mechanical ( Fig. 8B , middle ; saline: n = 7, capsaicin: n = 11; saline | capsaicin: F = 53.310, p < 0.0001, Two-way ANOVA; p = 0.0001, post hoc Tukey HSD) stimuli compared to saline controls. By contrast, cKO mice failed to develop capsaicin-induced hypersensitivity to heat; average paw withdrawal latencies for capsaicin-injected and saline-injected paws were not different ( Fig. 8A , right ; capsaicin: n = 8, saline: n = 7) (saline | capsaicin: p = 0.3600; Two-way ANOVA, post hoc Tukey HSD). Whereas capsaicin-induced mechanical hypersensitivity was robust and significantly different from saline control responses ( Fig. 8B , right ; mechanical stimuli, capsaicin: n = 9, saline: n = 9; saline | capsaicin: p = 0.0001; Two-way ANOVA, post hoc Tukey HSD). These results show that Ca V 2.2 channels expressed in Trpv1 -lineage neurons are the source of depolarization- induced calcium entry and this event is essential for the development of rapid, short-term hypersensitivity to heat. Ca V 2.2 channels in Trpv1 -lineage neurons contribute to the prolonged time course of CFA- induced heat, but not mechanical hypersensitivity We previously reported that Ca V 2.2 channels are essential for heat hypersensitivity associated with chronic inflammation following intraplantar CFA 5 . CFA-induced heat hypersensitivity in response to intradermal CFA in gKO mice was eliminated 5 . Here we use the cKO mouse model to measure paw withdrawal responses to heat (Hargreaves, Fig. 9A ) and mechanical (von Frey, Fig. 9B ) stimuli before and for 7 days following intraplantar administration of CFA to assess the involvement of Ca V 2.2 channels expressed in Trpv1 -lineage neurons. In WT-fl mice ( n = 8), CFA- induced heat (ipsi | contra: day 1, p < 0.0001; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p = 0.0084; day 5, p = 0.0042; day 6, p = 0.008; day 7, p = 0.0007; repeated measures ANOVA, post hoc Tukey HSD) and mechanical hypersensitivity (ipsi | contra: day 1, p = 0.0001; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p < 0.0001; day 5, p = 0.0003; day 6, p = 0.0003; day 7, p = 0.0001; repeated measures ANOVA, post hoc Tukey HSD) was different in ipsilateral, compared to contralateral paws, for all 7 days of the measurement period. In cKO ( n = 8) mice, CFA-induced heat hypersensitivity was different from day 1 to 3, but on days 4-7 the response was curtailed and responses in contralateral and ipsilateral paws were not significantly different (ipsi | contra: day 1, p = 0.0079; day 2, p = 0.002; day 3, p = 0.0002; day 4, p = 0.3286; day 5, p = 0.1545; day 6, p = 1766; day 7, p = 0.7123). By contrast, mechanical hypersensitivity was different in ipsilateral compared to contralateral paws for all 7 days (ipsi | contra: day 1, p = 0.0002; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p = 0.0017; day 5, p = 0.0015; day 6, p = 0.0002; day 7, p = 0.0067; repeated measures ANOVA, post hoc Tukey HSD). Mechanical hypersensitivity following CFA administration remained intact in the absence of Ca V 2.2 channels on Trpv1 -lineage neurons. Download figure Open in new tab Figure 9. Ca V 2.2 channels in Trpv1 -lineage neurons contribute to the late phase of CFA- induced heat hypersensitivity. Paw withdrawal responses to heat (A, latency) and mechanical (B, force) stimuli applied to the plantar surface of mouse hind paws. Contralateral (gray) and ipsilateral (blue and red) paw responses measured daily from wild-type Cacna1b fl/fl (WT-fl, blue/left; A , B ) and Cacna1b fl/fl / Trpv1 Cre/- (cKO, red/right; A , B ) mice before, and for 7 days after ipsilateral paw injected with i.d. 20 μl CFA. Values shown are mean (solid line) ± SEM (shaded area). A , Heat WT-fl ( n = 8, left ) main effect by day (ipsi | contra: F = 3.20, p = 0.0072), injection (ipsi | contra: F = 181.94, p < 0.0001), and day by injection interaction (ipsi | contra: F = 8.44, p < 0.0001; repeated measures ANOVA). For each of day 1-7 days post-CFA (ipsi | contra: day 1, p < 0.0001; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p = 0.0084; day 5, p = 0.0042; day 6, p = 0.008; day 7, p = 0.0007; repeated measures ANOVA, post hoc Tukey HSD). cKO ( n = 8, right ) main effect by day (ipsi | contra: F = 2.30, p = 0.0412), injection (ipsi | contra: F = 121.84, p < 0.0001), and day by injection interaction (ipsi | contra: F = 3.71, p = 0.0027; repeated measures ANOVA). cKO 1-7 days post-CFA (ipsi | contra: day 1, p = 0.0079; day 2, p = 0.002; day 3, p = 0.0002; day 4, p = 0.3286; day 5, p = 0.1545; day 6, p = 1766; day 7, p = 0.7123; repeated measures ANOVA, post hoc Tukey HSD). B , Mechanical responses: WT-f ( n = 7, left ) main effect by day (ipsi | contra: F = 2.33, p = 0.0420), injection (ipsi | contra: F = 133.74, p < 0.0001), and day by injection interaction (ipsi | contra: F = 14.63, p < 0.0001; repeated measures ANOVA). Ipsilateral and contralateral responses compared days 1-7 post-CFA (ipsi | contra: day 1, p = 0.0001; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p < 0.0001; day 5, p = 0.0003; day 6, p = 0.0003; day 7, p = 0.0001; repeated measures ANOVA, post hoc Tukey HSD). cKO ( n = 7, right ) main effect by day (ipsi | contra: F = 3.57, p = 0.0042), injection (ipsi | contra: F = 37.37, p = 0.0009), and day by injection interaction (ipsi | contra: F = 6.04, p = 0.0001; repeated measures ANOVA). cKO ipsilateral and contralateral responses compared days 1-7 post-CFA (ipsi | contra: day 1, p = 0.0002; day 2, p < 0.0001; day 3, p < 0.0001; day 4, p = 0.0017; day 5, p = 0.0015; day 6, p = 0.0002; day 7, p = 0.0067; repeated measures ANOVA, post hoc Tukey HSD). These data, combined with our previous studies of gKO mice which do not exhibit CFA-induced heat hypersensitivity 5 , suggest that Ca V 2.2 channels in both Trpv1 -lineage neurons and Trpv1 - lineage cells combined are required for heat hypersensitivity following chronic neuroinflammation induced by CFA. DISCUSSION Voltage-gated Ca V 2.2 channels control presynaptic calcium entry and neurotransmitter release at many synapses 1 , 2 . Studies using both global knockout of Ca V 2.2 channels and local inhibition through small molecule blockers have also established a critical role for Ca V 2.2 channels in the transmission of pain information 4 , 5 , 10 , 35 , 36 . Furthermore, a peptide inhibitor of Ca V 2.2 channels administered intrathecally is analgesic in patients with otherwise intractable pain 37 , 38 . More recently, our lab reported new data showing a critical and specific role of Ca V 2.2 channels at peripheral sites in skin, in the development of heat but not mechanical hypersensitivity 4 , 5 , 10 . Ca V 2.2 channels are enriched in Trpv1 -expressing, heat-sensitive neurons 24 , so we used a Trpv1 - conditional gene inactivation strategy to: 1) establish the relative contribution of Ca V 2.2 channels in Trpv1- expressing and Trpv1 -non-expressing neurons to behavioral responses associated with neuroinflammatory events, and 2) establish the acute and longer term consequences of eliminating the activity of Ca V 2.2 channels on neuronal excitability. In our studies we also validated the Cre-dependent mouse model for use to explore and define the contributions of the Cacna1b gene and Ca V 2.2 channels in other cell types for in vivo , circuit and cell-based studies. Although not ideal for in vivo studies, because of the pronounced hyperactivity phenotype, global Cacna1b KO mice proved critical as a negative control for Ca V 2.2 protein analysis. Specifically, to identify Ca V 2.2 specific bands in native tissue, and to reveal tissue-specific difference in protein expression. The polyclonal anti-Ca V 2.2 used here detect Ca V 2.2-specific proteins and they have been used by our lab 10 , 18 , 19 but they also binds several non-Ca V 2.2 proteins in native tissue which can only be identified with certainty using an appropriate negative control. The non-Ca V 2.2 bands labelled by polyclonal antibodies raised against Ca V 2.2 have typically not been shown in published data 20 , 39 – 42 but, as we show here and in our previous publications 10 , 18 – 20 , non-Ca V 2.2 signals are a prominent feature in Western analyses of native tissues. By comparing to gKO protein preparations, we also demonstrated tissue-specific differences in the pattern of both non- specific signals (e.g. a non-Ca V 2.2 signal at 125 kDa in brain is absent in DRG; Fig 1B ) as well as Ca V 2.2-specific protein signals (an additional ∼240 kDa Ca V 2.2-specific protein band in DRG but not in brain; Fig. 1B ) 20 , which are easily overlooked without comparing to a gKO negative control. The heterogeneity of neurons in DRG can make it difficult to identify cell-specific differences in the biophysical properties of specific classes of neurons. We therefore combined the use of Cre- reporter mice for gene-specific inactivation of Cacna1b with AAV viral delivery of a Cre-dependent tdTomato (tdT) reporter to separate neurons visually into Cre- and non-Cre-expressing cells (Cre- positive/tdT+ and Cre-negative/ tdT−) for biophysical analyses of individual cells. We used both voltage and current clamp recordings to establish specificity of the cKO model, and we showed that the trajectory of action potentials in Cre-expressing neurons from cKO animals were slightly shorter from WT-cre control neurons ( Fig. 4C , 4G). A subset of nociceptor neurons have a characteristic plateau phase which has been partly attributed to the presence and action of calcium channels 43 – 45 , and our data show that the percentage of cells with longer half widths is reduced in recordings from cKO mice, supporting a role for Ca V 2.2 channels in defining the action potential plateau in Trpv1 -expressing neurons ( Fig. 4G ). We also assessed the impact of Ca V 2.2 channels on the overall excitability of Trpv1 -lineage neurons and unexpectedly found that Trpv1 -lineage neurons from cKO mice exhibited a gain of function phenotype ( Figs. 2 - 4 ). Trpv1 -lineage neurons from cKO mice were more excitable as compared to WT-cre controls based on several criteria including, the amount of current required to fire an AP and the number of APs during a current step ( Fig. 4 ). The biophysical features of Trpv1 -lineage neurons in cKO mice were unexpected based on the behavioral phenotype of cKO mice, which were overall less sensitive to sensory stimuli ( Figs. 7 - 9 ). We conclude that there is a compensatory increase in membrane excitability in cKO neurons associated with the absence of Ca V 2.2 channel activity, most likely because of the reduction in the efficacy of synaptic transmission 10 . Data supporting the presence of a compensatory change in neuronal excitably includes showing that acute inhibition of Ca V 2.2 channel activity in Trpv1 -lineage neurons of WT- cre mice does not alter neuronal excitability and only slightly reduced the action potential half width ( Fig. 5 ). Additionally, analyses of cell excitability and action potential trajectories are consistent with the upregulation of voltage-gated Na+ currents, as seen by a reduced current required to trigger AP generation, no difference in the resting membrane potential, and a more depolarized peak value of action potentials in Trpv1 -lineage neurons of cKO mice ( Fig. 4 ). Compensatory homeostatic mechanisms in neurons have been reported following prolonged changes in synaptic efficacy 32 , 33 , 46 . In tectal neurons, changes in the level of Na V channels underlies the homeostatic mechanisms used to maintain a constant input–output function during development 47 , and the expression of Na V channels and other ion channels changes in models of multiple sclerosis to compensate for reduced signal conductance following demyelination 48 – 50 . In our studies, the increase in neuronal excitability of Trpv1 -neurons measured at the single cell level in cKO mice compared to WT-cre controls did not fully compensate for change in synaptic strength. Trpv1 -cKO mice have reduced responsiveness to heat and cold stimuli, and gKO mice are also less sensitive to mechanical stimuli 4 , 5 , 10 . Furthermore, Cacna1b cKO and gKO mice do not develop heat hypersensitivity in response to capsaicin and have shorter (cKO) or no heat hypersensitivity (gKO) response to CFA suggesting that the loss of Ca V 2.2 channels dominates in defining the neuroinflammatory response in skin 4 , 5 , 10 . Thus, neuronal excitability can change quite substantially in Trpv1 -expressing neurons in response to prolonged alterations in synaptic transmission without impacting overall behavior. Our observations might have relevance to current therapeutic strategies aimed at reducing neuronal excitability (e.g. blocking Na V 1.8 and Na V 1.7) for pain management 51 – 55 . Sensory neurons undergo dynamic and, as we show here, remarkably sustained changes in excitability so while it is possible that targeting ion channels to down regulate neuronal excitability could be effective for short-term analgesia, an inhibitor of transmitter release from peripheral nerve endings and/or presynaptic termini in the spinal cord might prove more effective for longer term, chronic pain management. The use of the cKO approach reported here, allowed us to define cell-specific actions of Ca V 2.2 channels in mediating in heat hypersensitivity using both acute (capsaicin, Fig. 8 ) and long-lasting (CFA, Fig. 9 ) models of neuroinflammation. We reenforced and extended our previous findings using Cacna1b gKO and pharmacological approaches 4 , 5 , 10 to show that Ca V 2.2 channel activity in Trpv1 -lineage neurons is the key initiating signal mediating capsaicin-induced heat hypersensitivity, while having no effect on the development of mechanical hypersensitivity in the same animals. Other signaling molecules independent of Ca V 2.2 channel activity must mediate mechanical hypersensitivity. We know from our previous studies that IL-1a is a key player in capsaicin-induced heat and mechanical hypersensitivity 4 so it is possible that capsaicin acts on TRPV1 receptors on immune cells 56 – 59 to trigger cytokine release with the potential to sensitize mechanosensitive neuron populations. The importance of non- Trpv1 -expressing cells in mediating long-lasting inflammation triggered by intraplantar CFA is shown conclusively in our studies using cKO mice ( Fig. 9 ). Whereas CFA failed to induced heat hypersensitivity in gKO mice 5 , in cKO mice heat hypersensitivity developed but the duration of the response was shortened. CFA-induced mechanical hypersensitivity in cKO mice was unaffected by the absence of Ca V 2.2 channels. It is likely that calcium influx through Ca V 2.2 channels plays a critical role in triggering the release of pro-inflammatory mediators, engaging immune cells and initiating an inflammatory cascades that promote pain and inflammation 4 , 5 , 36 . Inhibiting Ca V 2.2 channels—and thereby disrupting these cascades—may help promote recovery in models of neuroinflammation. Here, we provide conclusive evidence that Ca V 2.2 channels expressed in Trpv1 -lineage neurons are critical for the development of heat hypersensitivity. Ca V 2.2 channels are the critical link between prolonged membrane depolarization of Trpv1 neurons (e.g. by capsaicin) to neuroinflammation that results in a rapid increase in the responsiveness of sensory nerve endings to heat. Our studies also uncover a significant change in the excitability of sensory neurons in response to the loss of Ca V 2.2 channels. Interestingly, this compensatory increase in excitability of Trpv1 -expressing neurons is not manifested at the level of behavior which is dominated by Ca V 2.2-dependent signaling at peripheral and presynaptic sensory nerve endings. Given the dynamic changes in neuronal excitability observed here using the Cacna1b cKO model and the known developmental changes in Trpv1 expression 60 , 61 , it will be important to assess the effects of cell-specific and temporally controlled inactivation of Cacna1b during development. MATERIALS AND METHODS Experimental animals All mice used were bred at Brown University, and all protocols and procedures were approved by the Brown University Institutional Animal Care and Use Committee (IACUC). Mice were housed in a 12 hr light/dark cycle and tested between 10 am and 6 pm during the light cycle. 3 - 6-month- old male and female mice were used in all experiments. Experimenters were blind to animal genotype and solution injected for the experimental and data analysis steps. Cacna1b fl/fl mice were generated by Biocytogen in the C57BL6/J background using the CRISPR/Cas9 Extreme Genome Editing (CRISPR/EGE TM ) platform. To enable Cre-mediated recombination, loxP sites were inserted in intron 4-5 and intron 5-6, both oriented in the 5’ direction. Cre-mediated recombination deletes constitutive exons 5 and 6 resulting in a frame shift and premature stop codon. The sequences of guide RNAs (sgRNA) used in CRISPR/EGT TM and of primers for the targeting vector are in Table 2. The 5.6 kb targeting vector contained 1.5 kb homologous 5’ and 3’ arms. Cacna1b f l/fl were crossed to Cacna1b fl/fl /Trpv1 Cre/- mice to generate F1 progeny (∼50% Trpv1 - lineage specific Cacna1b knockout (cKO) and ∼50% Cacna1b f l/fl littermate wild-type controls. Routine genotyping was performed by qPCR from ear punch biopsies. Probes were designed to detect the 5’ loxP site between exons 5 and 6, the Cacna1b WT allele, and Cacna1b allele with the intended deletion. We vigilantly monitored for the presence of mosaic mice for the Cacna1b deletion in our crosses. Mosaic mice were not used for experiments or breeding. View this table: View inline View popup Download powerpoint The Cacna1b −/− global deletion (gKO) mouse strain ( Cacna1b tm5.1DiLi, MGI) generated in our lab has been described previously 10 . A wild-type strain was bred in parallel from the same genetic background and used for comparison in hyperactivity experiments. Western blot analysis Harvested brain was immediately homogenized on ice in Syn-PER buffer (87793, Thermo Fisher) containing protease inhibitor (Complete, mini; Roche Diagnostics). The homogenate was centrifuged at 1200 xg for 10 min at 4°C, supernatant transferred to a clean tube and centrifuged at 15000 xg for 30 min at 4°C and the pellet resuspended in ice-cold CL-91 buffer (CL-91–01, Logopharm) containing a protease inhibitor (Halt, Thermo Fisher). DRG were pooled from 5 mice, homogenized on ice in RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS pH 7.6) containing protease inhibitor (Complete, mini; Roche Diagnostics). After 3 hr incubation on ice, the homogenate was centrifuged at 10000 xg for 10 min and the supernatant was collected. Homogenate protein concentrations were estimated using BCA Protein Assay and BSA as a standard (23225, Pierce, Thermo Scientific). Protein preparations were separated in tris-acetate 3-8% polyacrylamide gels (EA03752, Nu Page Thermo Fisher). Protein samples were incubated at room temperature for 20 min with Laemmli protein sample buffer (200 mM Tris-HCl, 2% w/v SDS, 10% v/v Glycerol, 100 mM DTT, pH 6.8). 20 µg of protein were loaded per well and the electrophoresis was run at 140 V (constant) using in MES-SDS as a running buffer (50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3, Thermo Fisher). After electrophoresis, proteins were transferred from polyacrylamide gels to 0.2 µm PVDF membranes using a semi dry transference system (Biorad Trans-Blot Turbo) at 1.3 A, 25 V for 10 min with a transfer buffer (Trans-Blot Turbo 5X, 20% v/v ethanol, Bio-Rad). Total protein was estimated using the LI-COR Revert™ 700 Total Protein Stain, and imaging was performed on a Bio-Rad ChemiDoc MP Imaging System. Total protein was visualized at IRDye 680. Transfer membranes were blocked with 5% skimmed milk in PBS-Tween for 1 hr (PBST, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 0.1% v/v Tween-20). Membranes were incubated with polyclonal rabbit anti-Ca V 2.2 (ACC-002, Alomone Labs; 1:200 dilution in PBST, 2% w/v BSA) for 1 hr at room temperature, and detected using an anti-rabbit IgG (whole molecule) peroxidase conjugated antibody (Jackson Inmunoresearch 711-036-152; 1:10000 dilution in PBST, 2 % w/v BSA, 1h, room temp). Membranes were washed with PBST between each step in the protocol. Immunoblot membranes were processed using the SuperSignal West Pico PLUS Chemiluminescent substrate and the image was acquired using a Chemidoc MP (Bio-Rad). Images were analyzed using Image Lab (version 6.1.0 build 7, Bio-rad). Band intensities were calculated and normalized to total protein for each lane. Protein intensities were calculated by normalizing to the control group. Adjustments of image brightness and contrast was applied to the entire gel. Cell isolation and electrophysiology To visually identify Trpv1 -lineage neurons for recording we used a tdTomato reporter AAV viral vector. Cre-dependent tdTomato expression was achieved by intrathecal injection of 10 µL of pAAV-FLEX-tdTomato packaged with AAV-PHP.s (AAV-PHP.s-FLEX-tdTomato; Addgene #: 28306-PHP.S, titer: 2E13 GC/mL) delivered at least 2 weeks prior to DRG isolation. For intrathecal injections, mice were lightly anesthetized with 2% isoflurane. A 26-gauge needle was manually inserted between L5 and L6 vertebrae and solution was injected once a tail flick was evoked (method adapted from Li et al .) 62 . A WPI microinjector (Model UMP3 Serial 191648 M06B; Sarasota, Florida USA) was used to inject virus at a flow rate of 250 nL/s. Mice were monitored for any abnormalities in gait after recovery. Bilateral L3-L5 DRG were isolated 2-3 weeks post-injection from 8-10-week-old isoflurane euthanized mice. DRG were kept on ice in Hank’s Balanced Salt Solution (HBSS, GIBCO) before being transferred to HBSS containing 2 mg/mL collagenase (GIBCO) and 2.5 mg/mL trypsin (Sigma) at 37°C for 30 min. The pellet was lightly disturbed every 10 min. Protease activity was halted with the addition of Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (v/v, FBS, GIBCO) and 1 ng/mL nerve growth factor (NGF, Sigma). Single cells were isolated by trituration using a fire polished Pasteur pipette and resuspended in 500 µL 10% FBS-DMEM supplemented with 1 ng/mL NGF. Neurons were kept in suspension at 37°C with 5% CO 2 until needed. 40 µL of the cell suspension was added to the center of the recording chamber and, after a 20 min settling time, the recording chamber was flooded with the Na-based external recording solution. The external Na-based solution contained 135 mM NaCl, 2 mM CaCl 2 , 10 mM HEPES, 4 mM MgCl 2 , 2 mM KCl, adjusted to a pH of 7.4 with NaOH and to an osmolarity of 305 mOsm with NaCl; the TEA-based external solution used to isolate voltage-gated Ca 2+ currents contained 135 mM TEA-Cl, 2 mM CaCl 2 , 10 mM HEPES, 4 mM MgCl 2 adjusted to pH of 7.4 with TEA-OH and 305 mOsm with TEA-Cl. The Cs-based internal solution contained 126 mM CsCl, 4 mM Mg-ATP, 1 mM EDTA, 10 mM EGTA, and 10 mM HEPES, adjusted to a pH of 7.4 with CsOH and an osmolarity of 290 mOsm with CsCl as needed. The K-based internal solution contained 126 mM KCl, 4 mM Mg-ATP, 1 mM EDTA, 10 mM EGTA, and 10 mM HEPES (pH adjusted with NaOH), adjusted to a pH of 7.4 with KOH and an osmolarity of 290 mOsm with KCl as needed. We used a microperfusion system fed by gravity for rapid solution exchange during recording. This was comprised of 6-8 small diameter glass tubes aligned and adhered together and connected to inert tubing. Solution flow through each line was independently controlled via electronic valves. The glass tubes were immersed in the recording chamber using a micromanipulator. Once a stable recording was achieved, the cell is lifted from the bottom of the dish and carefully positioned at the outflow of the desired solution/pipe and solution exchange was achieved by manually moving the cell rapidly (within 1 sec) between pipes. There was a constant flow of the appropriate external solution applied via a larger outlet ensuring constant laminar flow. We applied 2 µM capsaicin in Na-based solution or 2 µM ω-Conotoxin GVIA (C-300, Alomone Labs) in a TEA-based solution to individual cells during recording. Our previous analyses of DRG neurons in mice revealed the presence of three types of neurons based on size and the presence or absence of a prominent low threshold Ca V 3 type current 18 . The smaller diameter cells (∼15 pF) included capsaicin responsive/TRPV1+ neurons. We biased recordings toward this cell population by primarily selecting smaller diameter neurons which were both positive and negative for tdTomato. We recorded neurons from Cacna1b fl/fl / Trpv1 Cre/- (cKO), Cacna1b fl/fl (WT-fl) littermate controls and Trpv1 Cre /Cre (WT-cre) mice. Neurons were also grouped for analysis post-recording based on capsaicin responsiveness (Supplemental Fig. 2). Whole cell voltage-gated calcium (Ca V ) currents were recorded in TEA-based extracellular solution with 2 mM Ca 2+ and elicited by depolarizing voltage steps form a holding potential of -80 mV. 2 µM ω-CgTx-GVIA was added to block Ca V 2.2 currents. Leak currents were subtracted online using a P/-4 protocol. Currents were recorded using an Axopatch 200B amplifier, filtered at 5 kHz and digitized at 10 kHz (Axon Instruments 1440A). For 2 µM capsaicin responses, cells were moved to a Na-based extracellular solution, held at -60 mV, and exposed to capsaicin for 1 second. Currents were analyzed with Clampex 10.7 and custom lab software using Python 3.7 and pyABF 63 and IPFX 64 packages. Recording electrodes were fire-polished to between 2 and 5 MΩ, the average series resistance was 7.7 MΩ, and average cell capacitance 15.86 ± 0.54 pF. All recordings were obtained at room temperature. Behavioral assessments All behavioral assessments were conducted by experimenters who were blind to the genotype of the mice. However, the hyperactivity phenotype of gKO mice and the edema and guarding associated with CFA injections were so consistent that the experimenter was able to distinguish these mice from WT or saline controls. Heat assessment . Paw withdrawal responses were elicited by radiant heat applied using a Hargreaves equipment (Plantar Analgesia Meter IITC Life Science). Mice were placed in Plexiglas boxes on an elevated glass plate and allowed to habituate for at least 30 min prior to testing. A radiant heat source was positioned beneath the mouse paw and aimed using low-intensity visible light to the plantar surface. For all trials, laser idle intensity was 5% and active intensity was 50% of maximum. Trials were terminated if the mouse withdrew, shook, and/or licked their hindpaw following stimulation or if the cut off time, 30 s, was reached. The light source was manually turned off if any of these criteria were met. The response latency was measured to the nearest 0.01 s for each trial using the built-in timer corresponding to the duration of the high-intensity beam, see 4 , 5 , 10 . Mechanical assessment. Mechanical responses were evoked and recorded with an automated von Frey plantar aesthesiometer (catalog #37550, Ugo Basile). Mice were placed in an elevated Plexiglas box with a wire mesh bottom and were allowed to habituate for at least 30 min prior to testing. The plantar surface of hind paws was assessed using a 15 s steady ramp of force ranging from 0 to 8 g for up to 90 s. The trial is automatically terminated when the paw is withdrawn or cutoff time is reached; force and reaction time are captured. Here, we report only the force associated with the withdrawal threshold 4 , 5 , 10 . For both heat and mechanical assessment, three trials were conducted on each hindpaw for each mouse, with at least 1 min rest between trials. The average of 3 trials was used for analysis, and N values are reported as the number of mice. After baseline measures, mice were anesthetized with isoflurane for intradermal injection of inflammatory agents and returned to the testing chamber to recover. Males and females were tested separately in different sessions. Open Field . Mice were tested a single time in an open field apparatus in dim light conditions. The apparatus consisted of an open field area of 20 x 20 x 14 in. Mice were recorded with an overhead video camera. Mice were tracked using Noldus EthoVision XT tracking software. After 30 min of habituation to the testing room, mice were placed in the center of the open field arena and allowed to freely explore for 5 min. Distance traveled was scored as a readout of activity. Males and females were tested separately in different sessions. Cold plate . Sensitivity to cold was assessed using the Hot/Cold Plate Analgesia Meter from IITC, Inc. After 30 min of habituation to the testing room, mice were placed on a cold surface kept at - 5°C. Mice were allowed to freely move on the cold plate. A 30 s cut off was used for this experiment. Two cameras were used to record the behavior of the animals on the cold plate. Latency to response with a paw flinching, licking, shaking, or jumping was scored post hoc using the recorded videos. At least two independent experimenters blind to the genotype scored the videos with an interrater variability <5%. Males and females were tested separately in different sessions. Intradermal administration of inflammatory agents For intradermal administration, mice were lightly anesthetized with isoflurane and a 30 G insulin syringe was used to inject 20 µL 0.1% w/v capsaicin in saline, 20 µL saline or 20 µL Complete Freund’s Adjuvant (CFA) into the plantar surface of the right hindpaw 4 , 5 , 10 . Statistical analysis Data were assessed for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. Based on these assumptions, appropriate parametric or non-parametric statistical tests were selected. For comparisons between two groups, Student’s t-test was used when data met assumptions of normality and equal variance. If variance was unequal, Welch’s t- test was performed. When data were non-normally distributed, the Mann-Whitney U test was used. For within-subject or paired comparisons, paired t-tests were used for normally distributed data, and the Wilcoxon signed-rank test was used when normality was violated. For multiple group comparisons, one-way or two-way ANOVA was used for normally distributed data, followed by Tukey’s HSD post hoc test when significant main effects or interactions were observed. If data violated normality or equal variance assumptions, the Kruskal-Wallis test with Dunn’s post hoc test was applied. For repeated measures designs, repeated measures ANOVA was used with Tukey’s HSD correction for post hoc pairwise comparisons. When random effects (e.g., repeated measures within subjects) needed to be accounted for, linear mixed-effects models were applied. All statistical analyses were performed using Python’s statsmodels and scipy packages. Data are presented as mean ± SEM, and we considered a p value of 0.05 as a measure of significance, but in all analyses, we show the actual p values together with information about the specific tests used. Funder Information Declared National Institute of Neurological Disorders and Stroke, https://ror.org/01s5ya894 , NS055251 , F31NS129281-02 REFERENCES 1. ↵ Dolphin , A. C. & Lee , A . Presynaptic calcium channels: specialized control of synaptic neurotransmitter release . Nat. Rev. Neurosci . 21 , 213 – 229 ( 2020 ). OpenUrl CrossRef PubMed 2. ↵ Catterall , W. A . Voltage-Gated Calcium Channels . Cold Spring Harb. Perspect. Biol . 3 , a003947 ( 2011 ). 3. ↵ Raingo , J. , Castiglioni , A. J. & Lipscombe , D . Alternative splicing controls G protein– dependent inhibition of N-type calcium channels in nociceptors . Nat. Neurosci . 10 , 285 – 292 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Salib , A.-M. N. et al. Interleukin-1α links peripheral CaV2.2 channel activation to rapid adaptive increases in heat sensitivity in skin . Sci. Rep . 14 , 9051 ( 2024 ). OpenUrl CrossRef PubMed 5. ↵ Salib , A.-M. N. , Crane , M. J. , Jamieson , A. M. & Lipscombe , D . Peripheral CaV2.2 Channels in the Skin Regulate Prolonged Heat Hypersensitivity during Neuroinflammation . eNeuro 11 , ( 2024 ). 6. ↵ White , D. M. & Cousins , M. J . Effect of subcutaneous administration of calcium channel blockers on nerve injury-induced hyperalgesia . Brain Res . 801 , 50 – 58 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 7. Nebe , J. , Vanegas , H. & Schaible , H.-G . Spinal application of ω-conotoxin GVIA, an N-type calcium channel antagonist, attenuates enhancement of dorsal spinal neuronal responses caused by intra-articular injection of mustard oil in the rat . Exp. Brain Res . 120 , 61 – 69 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 8. Lipscombe , D. & Andrade , A . Calcium Channel CaVα1 Splice Isoforms - Tissue Specificity and Drug Action . Curr. Mol. Pharmacol . 8 , 22 – 31 . 9. ↵ Zoidis , G. , Sandoval , A. , Pineda-Farias , J. B. , Granados-Soto , V. & Felix , R . Anti-allodynic effect of 2-(aminomethyl)adamantane-1-carboxylic acid in a rat model of neuropathic pain: A mechanism dependent on CaV2.2 channel inhibition . Bioorg. Med. Chem . 22 , 1797 – 1803 ( 2014 ). OpenUrl CrossRef 10. ↵ DuBreuil , D. M. et al. Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated Ca V 2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin . J. Neurosci . 41 , 7546 – 7560 ( 2021 ). OpenUrl Abstract / FREE Full Text 11. ↵ Beuckmann , C. T. , Sinton , C. M. , Miyamoto , N. , Ino , M. & Yanagisawa , M . N-Type Calcium Channel α1B Subunit (CaV2.2) Knock-Out Mice Display Hyperactivity and Vigilance State Differences . J. Neurosci . 23 , 6793 – 6797 ( 2003 ). OpenUrl Abstract / FREE Full Text 12. ↵ Gorman , K. M. et al. Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia . Am. J. Hum. Genet . 104 , 948 – 956 ( 2019 ). OpenUrl CrossRef PubMed 13. ↵ Saegusa , H. et al. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel . EMBO J . 20 , 2349 – 2356 ( 2001 ). OpenUrl Abstract / FREE Full Text 14. ↵ Nakagawasai , O. et al. Behavioral and neurochemical characterization of mice deficient in the N-type Ca2+ channel α1B subunit . Behav. Brain Res . 208 , 224 – 230 ( 2010 ). OpenUrl CrossRef PubMed 15. ↵ Caterina , M. J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway . Nature 389 , 816 – 824 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 16. Caterina , M. J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor . Science 288 , 306 – 313 ( 2000 ). OpenUrl Abstract / FREE Full Text 17. ↵ Cavanaugh , D. J. et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells . J. Neurosci. Off. J. Soc. Neurosci . 31 , 5067 – 5077 ( 2011 ). OpenUrl Abstract / FREE Full Text 18. ↵ Andrade , A. , Denome , S. , Jiang , Y.-Q. , Marangoudakis , S. & Lipscombe , D . Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing . Nat. Neurosci . 13 , 1249 – 1256 ( 2010 ). OpenUrl CrossRef PubMed 19. ↵ Marangoudakis , S. et al. Differential Ubiquitination and Proteasome Regulation of CaV2.2 N-Type Channel Splice Isoforms . J. Neurosci . 32 , 10365 – 10369 ( 2012 ). OpenUrl Abstract / FREE Full Text 20. ↵ Schiff , M. L. et al. Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel . Nature 408 , 723 – 727 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 21. ↵ ISH Data :: Allen Brain Atlas: Mouse Brain . https://mouse.brain-map.org/ . 22. ↵ Brain RNA-Seq – Brain RNA-Seq . https://brainrnaseq.org/ . 23. ↵ Zheng , Y. et al. Deep Sequencing of Somatosensory Neurons Reveals Molecular Determinants of Intrinsic Physiological Properties . Neuron 103 , 598 – 616 .e7 ( 2019 ). OpenUrl CrossRef PubMed 24. ↵ Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia | Science Advances . https://www.science.org/doi/10.1126/sciadv.adj9173 . 25. ↵ Gong , B. , Murray , K. D. & Trimmer , J. S . Developing high-quality mouse monoclonal antibodies for neuroscience research – approaches, perspectives and opportunities . New Biotechnol . 33 , 551 – 564 ( 2016 ). OpenUrl CrossRef 26. ↵ Bernal Sierra , Y. A. , Haseleu , J. , Kozlenkov , A. , Bégay , V. & Lewin , G. R . Genetic Tracing of Cav3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System . Front. Mol. Neurosci . 10 , ( 2017 ). 27. François , A. et al. The Low-Threshold Calcium Channel Cav3.2 Determines Low-Threshold Mechanoreceptor Function . Cell Rep . 10 , 370 – 382 ( 2015 ). OpenUrl CrossRef PubMed 28. Bourinet , E. et al. Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception . EMBO J . 24 , 315 – 324 ( 2005 ). OpenUrl Abstract / FREE Full Text 29. ↵ Lawson , J. J. , McIlwrath , S. L. , Woodbury , C. J. , Davis , B. M. & Koerber , H. R . TRPV1 Unlike TRPV2 is Restricted to a Subset of Mechanically Insensitive Cutaneous Nociceptors Responding to Heat . J. Pain Off. J. Am. Pain Soc . 9 , 298 – 308 ( 2008 ). OpenUrl CrossRef 30. ↵ An experimental investigation of rundown … | Wellcome Open Research . https://wellcomeopenresearch.org/articles/9-250 . 31. ↵ Hao , L.-Y. , Kameyama , A. , Kuroki , S. , Nishimura , S. & Kameyama , M . Run-Down of L-Type Ca2+Channels Occurs on the α1Subunit . Biochem. Biophys. Res. Commun . 247 , 844 – 850 ( 1998 ). OpenUrl CrossRef PubMed 32. ↵ Wilhelm , J. C. , Rich , M. M. & Wenner , P . Compensatory changes in cellular excitability, not synaptic scaling, contribute to homeostatic recovery of embryonic network activity . Proc. Natl. Acad. Sci . 106 , 6760 – 6765 ( 2009 ). OpenUrl Abstract / FREE Full Text 33. ↵ O’Leary , T. , Williams , A. H. , Caplan , J. S. & Marder , E . Correlations in ion channel expression emerge from homeostatic tuning rules . Proc. Natl. Acad. Sci . 110 , E2645 – E2654 ( 2013 ). OpenUrl Abstract / FREE Full Text 34. ↵ Dhaka , A. , Earley , T. J. , Watson , J. & Patapoutian , A . Visualizing Cold Spots: TRPM8- Expressing Sensory Neurons and Their Projections . J. Neurosci . 28 , 566 – 575 ( 2008 ). OpenUrl Abstract / FREE Full Text 35. ↵ Adams , D. J. & Berecki , G . Mechanisms of conotoxin inhibition of N-type (Cav2.2) calcium channels . Biochim. Biophys. Acta BBA - Biomembr . 1828 , 1619 – 1628 ( 2013 ). OpenUrl CrossRef 36. ↵ Pitake , S. , Middleton , L. J. , Abdus-Saboor , I. & Mishra , S. K . Inflammation Induced Sensory Nerve Growth and Pain Hypersensitivity Requires the N-Type Calcium Channel Cav2.2 . Front. Neurosci . 13 , 1009 ( 2019 ). OpenUrl CrossRef PubMed 37. ↵ Klotz , U . Ziconotide--a novel neuron-specific calcium channel blocker for the intrathecal treatment of severe chronic pain--a short review . Int. J. Clin. Pharmacol. Ther . 44 , 478 – 483 ( 2006 ). OpenUrl CrossRef PubMed 38. ↵ Wallace , M. S . Ziconotide: a new nonopioid intrathecal analgesic for the treatment of chronic pain . Expert Rev. Neurother . 6 , 1423 – 1428 ( 2006 ). OpenUrl CrossRef PubMed 39. ↵ Murakami , M. et al. Modified sympathetic regulation in N-type calcium channel null-mouse . Biochem. Biophys. Res. Commun . 354 , 1016 – 1020 ( 2007 ). OpenUrl CrossRef PubMed 40. Yang , J. et al. Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury . Brain. Behav. Immun . 71 , 52 – 65 ( 2018 ). OpenUrl CrossRef PubMed 41. Chai , Z. et al. CaV2.2 Gates Calcium-Independent but Voltage-Dependent Secretion in Mammalian Sensory Neurons . Neuron 96 , 1317 – 1326 .e4 ( 2017 ). OpenUrl CrossRef PubMed 42. ↵ Li , Y.-L . Stellate Ganglia and Cardiac Sympathetic Overactivation in Heart Failure . Int. J. Mol. Sci . 23 , 13311 ( 2022 ). 43. ↵ Renganathan , M. , Cummins , T. R. & Waxman , S. G . Contribution of Nav1.8 Sodium Channels to Action Potential Electrogenesis in DRG Neurons . J. Neurophysiol . 86 , 629 – 640 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 44. Blair , N. T. & Bean , B. P . Roles of Tetrodotoxin (TTX)-Sensitive Na+ Current, TTX-Resistant Na+ Current, and Ca2+ Current in the Action Potentials of Nociceptive Sensory Neurons . J. Neurosci . 22 , 10277 – 10290 ( 2002 ). OpenUrl Abstract / FREE Full Text 45. ↵ Yoshida , S. , Matsuda , Y. & Samejima , A . Tetrodotoxin-resistant sodium and calcium components of action potentials in dorsal root ganglion cells of the adult mouse . J. Neurophysiol . 41 , 1096 – 1106 ( 1978 ). OpenUrl CrossRef PubMed Web of Science 46. ↵ Turrigiano , G. , Abbott , L. F. & Marder , E . Activity-Dependent Changes in the Intrinsic Properties of Cultured Neurons . Science 264 , 974 – 977 ( 1994 ). OpenUrl Abstract / FREE Full Text 47. ↵ Pratt , K. G. & Aizenman , C. D . Homeostatic regulation of intrinsic excitability and synaptic transmission in a developing visual circuit . J. Neurosci. Off. J. Soc. Neurosci . 27 , 8268 – 8277 ( 2007 ). OpenUrl Abstract / FREE Full Text 48. ↵ Black , J. A. et al. Abnormal expression of SNS/PN3 sodium channel in cerebellar Purkinje cells following loss of myelin in the taiep rat . NeuroReport 10 , 913 ( 1999 ). 49. Waxman , S. G. , Dib-Hajj , S. , Cummins , T. R. & Black , J. A . Sodium channels and their genes: dynamic expression in the normal nervous system, dysregulation in disease states1 . Brain Res . 886 , 5 – 14 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis | PNAS . https://www.pnas.org/doi/abs/10.1073/pnas.97.21.11598 . 51. ↵ MacDonald , D. I. et al. A central mechanism of analgesia in mice and humans lacking the sodium channel NaV1.7 . Neuron 109 , 1497 – 1512 .e6 ( 2021 ). OpenUrl CrossRef PubMed 52. Jones , J. et al. Selective Inhibition of NaV1.8 with VX-548 for Acute Pain . N. Engl. J. Med . 389 , 393 – 405 ( 2023 ). OpenUrl CrossRef PubMed 53. Gingras , J. et al. Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain . PloS One 9 , e105895 ( 2014 ). OpenUrl CrossRef PubMed 54. Zakrzewska , J. M. et al. Safety and efficacy of a Nav1.7 selective sodium channel blocker in patients with trigeminal neuralgia: a double-blind, placebo-controlled, randomised withdrawal phase 2a trial . Lancet Neurol . 16 , 291 – 300 ( 2017 ). OpenUrl CrossRef PubMed 55. ↵ Hameed , S . Nav1.7 and Nav1.8: Role in the pathophysiology of pain . Mol. Pain 15 , 1744806919858801 ( 2019 ). 56. ↵ Omari , S. A. , Adams , M. J. & Geraghty , D. P . TRPV1 Channels in Immune Cells and Hematological Malignancies . Adv. Pharmacol. San Diego Calif 79 , 173 – 198 ( 2017 ). OpenUrl 57. Mariotton , J. , et al. TRPV1 activation in human Langerhans cells and T cells inhibits mucosal HIV-1 infection via CGRP-dependent and independent mechanisms . Proc. Natl. Acad. Sci . 120 , e2302509120 ( 2023 ). OpenUrl CrossRef PubMed 58. Bodó , E. et al. A Hot New Twist to Hair Biology: Involvement of Vanilloid Receptor-1 (VR1/TRPV1) Signaling in Human Hair Growth Control . Am. J. Pathol . 166 , 985 – 998 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 59. ↵ Bertin , S. et al. The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells . Nat. Immunol . 15 , 1055 – 1063 ( 2014 ). OpenUrl CrossRef PubMed 60. ↵ Cavanaugh , D. J. et al. Restriction of Transient Receptor Potential Vanilloid-1 to the Peptidergic Subset of Primary Afferent Neurons Follows Its Developmental Downregulation in Nonpeptidergic Neurons . J. Neurosci . 31 , 10119 – 10127 ( 2011 ). OpenUrl Abstract / FREE Full Text 61. ↵ Korobkin , A. A. et al. Developmental Changes in the Expression of TRPV1 Channels in Autonomic Nervous System Neurons . Neurosci. Behav. Physiol . 43 , 743 – 747 ( 2013 ). OpenUrl CrossRef 62. ↵ Li , D. , Li , Y. , Tian , Y. , Xu , Z. & Guo , Y . Direct Intrathecal Injection of Recombinant Adeno- associated Viruses in Adult Mice . JoVE J. Vis. Exp . e58565 ( 2019 ) doi: 10.3791/58565 . OpenUrl CrossRef 63. ↵ Pyabf: Python Library for Reading Files in Axon Binary Format (ABF) . 64. ↵ AllenInstitute/Ipfx . ( Allen Institute , 2024 ). View the discussion thread. Back to top Previous Next Posted May 10, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Trpv1-dependent Cacna1b gene inactivation reveals cell-specific functions of CaV2.2 channels in vivo Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Trpv1 -dependent Cacna1b gene inactivation reveals cell-specific functions of Ca V 2.2 channels in vivo Remy Y. Meir , Martin S. Sisti , Arturo Andrade , Diane Lipscombe bioRxiv 2025.05.09.652131; doi: https://doi.org/10.1101/2025.05.09.652131 Share This Article: Copy Citation Tools Trpv1 -dependent Cacna1b gene inactivation reveals cell-specific functions of Ca V 2.2 channels in vivo Remy Y. Meir , Martin S. Sisti , Arturo Andrade , Diane Lipscombe bioRxiv 2025.05.09.652131; doi: https://doi.org/10.1101/2025.05.09.652131 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17636) Bioengineering (13860) Bioinformatics (41847) Biophysics (21401) Cancer Biology (18536) Cell Biology (25424) Clinical Trials (138) Developmental Biology (13353) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24287) Genetics (15583) Genomics (22463) Immunology (17701) Microbiology (40300) Molecular Biology (17141) Neuroscience (88434) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9808) Zoology (2268)