skin is the largest sensory organ of the body and is densely equipped with highly specialized sensory nerve endings capable of sensing a wide range of sensory stimuli such as  light touch , mechanical pressure , temperature , itch , and pain .
these cutaneous sensory afferents can be distinguished by many morphological , anatomical , electrophysiological , and molecular criteria ( liu and ma , 2011 , owens and lumpkin , 2014 ) .
for example , based on their cell body size , degree of myelination , and axonal conduction velocity , cutaneous fibers can be split into a , a , or c - fibers ( abraira and ginty , 2013 , bessou and perl , 1969 , li et  al . , 2011 , smith and lewin , 2009 , zimmermann et  al . , 2009 ) .
they can be split into free nerve endings , found both in glabrous and hairy skin where they are embedded between the keratinocytes of the epidermis , and a particular population of c - unmyelinated fibers , found exclusively in hairy skin where they form longitudinal lanceolate endings around hair follicles ( delfini et  al .
, 2013 , li et  al . , 2011 ) or terminate in large arborizations similar to c - fiber tactile afferent receptive fields ( liu et  al . , 2007 ,
cutaneous free nerve endings are tuned to respond to itch - inducing compounds and pain - evoking thermal , mechanical , and chemical stimuli ( zimmermann et  al . , 2009 ) .
mrgprd free nerve endings belong to a subset of non - peptidergic nociceptors that convey noxious mechanical and -alanin - induced itch stimuli ( cavanaugh et  al .
, 2009 , liu et  al . , 2012 ) , whereas c - low - threshold mechanoreceptors ( c - ltmrs ) have been proposed to contribute to light touch under normal conditions ( bessou et  al .
, 1971 , douglas and ritchie , 1957 , johansson et  al . , 1988 ,
lken et  al . , 2009 , maruhashi et  al . , 1952 ,
olausson et  al . , 2002 , seal et  al . , 2009 , zotterman , 1939 ) and to touch hypersensitivity after injury ( delfini et  al .
, 2013 , liljencrantz et  al . , 2013 , seal et  al . , 2009 ) .
our knowledge of the molecular contents that contribute to the functional specialization of these diametrically different subpopulations of cutaneous afferents is still at its infancy . in a recent study ( gaillard et  al . , 2014 )
, we identified a novel g inhibitory interacting protein ( ginip ) that marks two distinct subsets of non - peptidergic neurons : the cutaneous free nerve endings mrgprd neurons ( dong et  al .
, 2001 ) and the v - glut3- , th- , and tafa4-expressing c - ltmrs ( delfini et  al . , 2013 , li et  al . , 2011 , seal et  al . , 2009 ) . here , we took advantage of the ginip mouse model in combination with isolectin b4 ( ib4 ) cell surface staining and fluorescence - activated cell sorting ( facs ) .
we succeeded to purify three distinct populations of drg neurons and subjected their respective total rna contents to rna deep - sequencing technology ( rna - seq ) .
as expected , ginip / ib4 neurons showed a remarkable enrichment in mrgprd transcripts and ginip / ib4 neurons revealed a striking enrichment of the three known markers of c - ltmrs : tafa4 , th , and vglut3 .
deeper comparison of the rna - seq data revealed distinct transcriptional signatures between mrgprd - expressing neurons and c - ltmrs that were further confirmed by in  situ hybridization analysis of over 100 genes .
most importantly , in addition to providing the transcriptional signatures of two categories of primary sensory neurons , our study expands the molecular characterization of c - ltmrs and suggests that this particular subset of primary sensory neurons shares many molecular features of ltmrs . as a functional readout , we used electrophysiological recording to unravel the specific and exclusive functional expression of the low - voltage - gated ca channel cav3.3 in c - ltmrs , where it very likely plays a key role in shaping their functional specialization .
in a recent study , we generated a mouse model that expresses the fluorescent protein m - cherry from the ginip locus ( gaillard et  al . , 2014 ) .
double - labeling experiments using anti - ginip antibody in combination with ib4 staining showed that drg neurons can be split into four distinct categories : ginip / ib4 ; ginip / ib4 ; ginip / ib4 ; and ginip / ib4 neurons .
the ginip / ib4 double - positive ( dp ) population corresponds to the cutaneous free nerve endings mrgprd neurons ( figures 1a and 1b ) , the ginip / ib4 population corresponds to tafa4-expressing c - ltmrs ( figures 1a and 1b ) , the ginip / ib4 population contains the 20% remaining ib4 neurons ( figure  1a ) , and the ginip / ib4 double negative ( dn ) neurons represents a heterogeneous population of neurons composed of peptidergic nociceptors , a subset of ret neurons and trkb and trkc neurons ( figure  1a ) .
as ginip mouse model allows high - fidelity expression of m - cherry in ginip neurons , we sought to combine live m - cherry fluorescence with ib4 cell surface staining and facs to purify the four categories of drg cells ( figures 1c and 1d ) .
neurons with a cell body size above 70  m were eliminated by filtering the suspension , and axonal debris was removed by using a percoll gradient .
negative gating was used to exclude dead or dying cells that incorporated sytox blue dye and cells that were autofluorescent in v500 channel ( figures 1c and s1 ) .
next , we gated on m - cherry - positive and m - cherry - negative cells that we further subdivided into ib4 and ib4 subsets ( figures 1c and s1 ) .
facs - sorted ib4 and ib4 m - cherry - positive neurons gave rise to expected proportions of cells : about two - thirds were ib4 ( corresponding to ginip / ib4 ) and the remaining one - third was ib4 ( corresponding to ginip / ib4 ; figure  1d ) .
the m - cherry / ib4 subset was excluded from the transcriptional analysis , because it was highly contaminated by cd31 ( pecam)-expressing endothelial cells , consistent with ib4 binding on this cell type ( data not shown ) . for the remaining three sorted samples ( hereafter called dp for the ginip / ib4 , c - ltmrs for the ginip / ib4 , and dn for ginip / ib4 ) , we first confirmed by rt - pcr the accurate expression of ginip and m - cherry transcripts ( figure  s2b ) and then extended the purity evaluation by analyzing the expression of mrgprd , tafa4 , trka , and trpv1 in each sorted sample ( figure  s2c ) . as expected ,
mrgprd transcripts were highly detected in the dp sample but low in the two others , whereas tafa4 expression was high in c - ltmrs .
trka and trpv1 transcripts were enriched in the dn sample , consistent with the enrichment in peptidergic nociceptors in the dn fraction ( figure  s2c ) .
high - quality rna ( figure  s2a ) was extracted from the three purified populations of neurons , and their respective transcriptional profiles were generated using rna - seq .
after one round of rna amplification , libraries were built in biological duplicate and sequenced in 100-bp paired - end cycles using the illumina hiseq2000 system with sbs technology .
image analysis and base calling were performed using the hiseq control software and rta component from illumina .
this approach yielded between 40 and 50 millions of reads that were further submitted to sequence mapping on mouse reference genome mm10 with casava 1.8.2 software .
after removal of contaminants ( 28s , 18s , and 5s rrna ; mitochondrial chromosomes ; phix ; illumina controls ; and illumina adaptors ) , reads mapping to multiple splicing positions , and reads with no match , we obtained between 32 and 42 millions of reads that were used for the generation of the raw data file ( table s1 ) . among the first - ranked genes in the raw data
file , we found genes known to be related to structural organization of neurons such as tuba1a , prph , sncg , and mtap1b ( table s2 ) .
all these genes exhibited strong expression in the three sorted samples , as they generated at least 50,000 reads ( table s2 ) .
accordingly , they were highly and broadly expressed in drg neurons in allen - brain atlas database .
interestingly , genes associated with cellular stress such as hsp90ab1 and fos were also highly expressed in all samples , very likely due to the cell dissociation and facs process ( table s2 ) .
most importantly , quick analysis of the reads per kilobase per million of reads ( rpkm ) generated for genes expected to be enriched in each sorted subpopulation revealed high enrichment of mrgprd and p2rx3 in the dp subset , th and tafa4 in c - ltmrs , and cgrp and sp in the dn neurons ( figure  1e ) , providing the first proof of concept of the successful outcome of our experimental paradigm . to identify genes that were preferentially enriched in one sample
, we carried a pairwise comparative analysis ( 2-fold enrichment ; p  < 0.01 ) after libraries normalization with the trimmed mean of m values method ( tmm ) ( provided by edger statistical package ) .
then , for each subset , we extracted the list of genes that were upregulated in one subset as compared to the two others .
we found 486 genes enriched in dp subset , 549 in c - ltmrs , and 2,916 in dn subset ( tables s3 , s4 , and s5 ) .
these genes encode a variety of molecules including ion channels , receptors , signaling molecules , and previously uncharacterized genes . because not all neuronal subsets were included in our facs strategy and
because facs of acutely dissociated neurons may induce cellular stress that impacts on gene expression , the only follow - up strategy that could be employed to validate the specificity of our data sets was in  situ hybridization ( ish ) approach on fixed adult drg sections . knowing that we might miss genes that are expressed in subsets of our sorted neuronal subpopulations , we assumed that , below 1,000 number of reads , transcripts detection by ish would be difficult to monitor .
therefore , we carried out a second pairwise comparative analysis by considering only the genes with values above 1,000 reads ( figure  2a ) . as a result
, we obtained 156 transcripts preferentially expressed in the dp subset , 184 in c - ltmrs , and 784 in the dn population ( figure  2b ; tables s6 , s7 , and s8 ) .
we then monitored the expression of 48 dp- , 68 c - ltmrs- , and 13 dn - enriched genes using ish followed by ginip immunostaining and ib4 labeling ( for the dp and c - ltmrs candidate genes ) or ginip and trka immunolabeling ( for the dn candidate genes ) .
these triple - labeling experiments provided an unambiguous and accurate expression pattern of each tested gene .
the 13 dn - enriched tested genes revealed two different expression patterns : genes mainly expressed in neurons and genes exclusively expressed in non - neuronal cells that harbor satellite and/or glial cell morphology ( figure  s3 ) . in agreement with our rna - seq data , genes expressed in neurons were excluded from dp neurons and c - ltmrs .
furthermore , we found 6/13 genes expressed in trka peptidergic neurons and others ( 2/13 ) excluded from this neuronal population , revealing the heterogeneity of the dn subset ( figure  s3 ) . because the aim of this study consisted at deciphering the transcriptional signatures that confer the functional specializations of mrgprd - expressing cutaneous free nerve ending and c - ltmrs
, we analyzed the expression patterns of 48 dp - enriched genes and 68 c - ltmtrs - enriched genes .
in line with our rna - seq data sets , the 48 dp - enriched probes were highly expressed in the dp neurons and virtually excluded from c - ltmrs ( http://www.ibdm.univ-mrs.fr/equipes/reynders/dp-ishlibrary.pptx ) .
however , the vast majority of the dp - enriched  genes displayed an additional expression pattern outside dp neurons .
12 genes showed some expression in few ginip / ib4 neurons , 17 genes showed lower expression in discrete subsets of small ( 3/17 ) or medium - to - large size neurons ( 14/17 ) , and 19 genes were expressed in broader neuronal subsets in addition to intense expression in dp neurons ( figures 2c2e ; dp - ish library ) .
the same strategy applied to c - ltmrs - enriched genes showed that all 68 tested genes were expressed in c - ltmrs and massively excluded from dp neurons ( http://www.ibdm.univ-mrs.fr/equipes/reynders/cltmrs-ishlibrary.pptx ) .
one striking feature of c - ltmrs - enriched genes is that the vast majority was also expressed in medium - to - large  size drg neurons , whereas only two were expressed in small neurons .
indeed , out of the 68 tested genes , six were specifically expressed in c - ltmrs , as they only marked a subset of ginip neurons that do not bind ib4 ( figures 3a3f ) .
quantitative analyses showed that ceacam10 is expressed in 71.2%  6.3% of c - ltmrs , wfdc2 in 73.4%  1.7% , cacna1i in 89.2%  5.9% , c1ql4 in 70%  1.8% , fbp2 in 19.9%  4.7% , and bok in 62.7%  6.4% of c - ltmrs ( figure  3 g ) , further highlighting our previously suggested molecular heterogeneity of this particular class of neurons ( delfini et  al .
the remaining 60 genes could be split in three categories : 18 genes exhibited high expression in c - ltmrs but also marked a discrete subset of large - diameter neurons , 23 genes showed equal levels of expression in c - ltmrs and in  large - diameter neurons , and 19 genes exhibited a very high enrichment in larger - diameter neurons and a discrete expression in c - ltmrs ( figures 4a4c ; cltmrs - ishlibrary ) .
because 88% of c - ltmrs - enriched genes displayed an additional expression preferentially in large - diameter neurons , we sought to unravel the molecular identity of these neurons .
to do so , we performed double ish using nf200 antisense probe in combination with selected c - ltmrs - enriched probes followed by trka immunolabeling .
this triple - staining experiment showed that all the selected probes were massively excluded from trka peptidergic neurons and expressed in subsets of nf200 neurons ( figure  s4 ) . knowing that nf200 is a hallmark of proprioceptors and ltmrs ( bourane et  al . , 2009 , li et  al .
, 2011 , luo et  al . , 2009 , wende et  al . , 2012 ) , these data suggest that c - ltmrs share some molecular features of ltmrs .
mrgprd - expressing neurons and c - ltmrs exhibit fundamental differences both at anatomical and functional levels .
however , little is known about the molecular contents that instruct their respective functional specializations .
in line with this , we pooled dp and c - ltmrs - enriched genes according to their biological functions by searching for statistically over - represented associations with gene ontology ( go ) terms , using gorillla database ( figures 5 , 6 , and s5 ) .
this analysis yielded , respectively , 20 and 33 functional annotations for dp and cltmrs .
dp neurons exhibited a high enrichment in g - protein - coupled receptors as well as in transcripts encoding signaling molecules , such as gna14 , gna1 , gnaq , plcb3 , prkca , prkcd , and prkcq ( figures 5a and s5a ; table s9 ) , whereas c - ltmrs exhibited a striking enrichment in transcripts associated with voltage - gated ion channel activity ( figures 6a and s5b ; table s10 ) .
detailed analysis of genes associated with g protein receptor binding and signal transducer activity led to the identification of  two transcripts encoding for g - protein - coupled receptors , namely the angiotensin receptor type 1a ( agtr1a ) and the prostaglandin receptor d2 ( ptgdr ) ( figure  5a ) .
our ish experiments showed that agtr1a is expressed in dp neurons , but not in c - ltmrs or other subsets of drg neurons ( figure  5b ) , whereas ptgdr was found at higher intensity in dp neurons compared to c - ltmrs and large - size neurons ( figure  5c ) .
in addition to gpcrs , go analysis revealed a striking enrichment of transcripts encoding signaling molecules in dp neurons , including gna14 , gna1 , gnaq , plcb3 , prkca , prkcd , and prkcq ( table s9 ) .
our in  situ screen showed that all these transcripts were highly expressed in dp neurons and virtually absent in c - ltmrs ( figures 5d5 g ) .
the restricted enrichment of such number of signaling molecules likely endows dp neurons with the molecular machinery required to sense and transduce injury - induced pain hypersensitivity . regarding c - ltmrs , one of the most striking observations with respect to the go analysis was the high enrichment in transcripts associated with voltage - gated ion channel activity .
other physiologically pertinent functional annotations encompass substrate - specific channel , ion transmembrane transporter , and g - protein - coupled receptor activities ( figures 6a and s5b ) .
voltage - gated ion channels included potassium channels ( kcnq2 , kcnj12 , kcnv1 , kcna2 , and kcna4 ) , sodium channels and associated beta subunits ( scn1a , scn1b , scn3b , scn4b , scn7a , and scn8a ) , and low - voltage activated calcium channels ( cacna1h and cacna1i ; figures 6a and s5b ; table s10 ) .
ish screen showed that cacna1i is specifically expressed in c - ltmrs ( figure  3c ) and kcnj12 and cacna1h are expressed in c - ltmrs as well as in some large - diameter neurons but excluded from dp neurons ( figures 6b and 6c ) .
scn4b and scn8a exhibited low expression in c - ltmrs , very high expression in myelinated large neurons , and no detectable signal in dp neurons ( c - ltmrs - ish library , slides 67 and 68 ) .
the combinatorial enrichment of such channels in c - ltmrs is likely to explain the tonic and repetitive firing properties of this particular class of neurons ( delfini et  al .
interestingly , substrate - specific channels / ion transmembrane transporters encompassed anoctamin 6 ( ano6 ) and solute carrier 17a2 ( slc17a2 ) .
ano6 is part of outwardly rectifying chloride channels ( martins et  al . , 2011 ) , and slc12a7 encodes for the k / cl co - transporter kcc4 ( boettger et  al . , 2002 ) , suggesting that both channels could be involved in membrane excitability .
in situ analysis of ano6 and slc12a7 showed that both transcripts were expressed in c - ltmrs , but not in dp neurons , and exhibited some expression in large drg neurons ( figures 6d and 6e ) .
gpcr - activity - related transcripts were also represented in go analysis of c - ltmrs data set , including cholecystokinin receptor a ( cckar ) and purinergic receptor y1 ( p2ry1 ) .
our ish data showed that both cckar and p2ry1 transcripts were highly enriched in c - ltmrs , totally excluded from dp neurons , and displayed expression in some large - diameter neurons ( figures 6f , 6 g , and s4b ) .
together , our data provide a functional description of sets of  genes differentially expressed in dp versus c - ltmrs .
the differential expression of such clusters of genes likely contributes to differences in signal transduction , intrinsic membrane excitability , and the firing properties of c - ltmrs and mrgprd neurons .
our data also provide a predictive basis for deciphering the mechanisms by which specific external stimuli and downstream - associated signaling pathways fine - tune the activity of these two neuronal subsets .
our rna - seq data revealed high enrichment of cacna1h ( cav3.2 ) and cacna1i ( cav3.3 ) in c - ltmrs .
our ish showed that transcripts encoding both channels were expressed in c - ltmrs with cav3.3 being exclusively expressed in this neuronal subset ( figures 3e and 7a ) . to explore whether both channels were functional in c - ltmrs , we used whole - cell patch - clamp recordings on primary culture of heterozygous m - cherry - expressing neurons in the presence of alexa 488 ib4 live staining to distinguish between c - ltmrs and dp neurons . in ionic conditions designed to isolate calcium currents , current - voltage relationships showed inward low - voltage - activated ( lva ) and high - voltage - activated ( hva ) currents in all recorded c - ltmrs soma , confirming the systematic presence of t - type currents in these neurons ( figures 7b and 7c ) .
in sharp contrast , dp neurons did not express any lva current in agreement with the absence of cav3 transcripts in this population ( figures 7b and 7c ) . to test whether cav3.2 and cav3.3 were functional in c - lmtrs , we took advantage of cav3.2 high sensitivity to nickel ions ( ni ) ( lee et  al . , 1999 ) .
blockade of t - type calcium currents in c - ltmrs by low concentration of ni ( 30  m ) revealed a ni - insensitive current with slow kinetics of activation and inactivation typical of cav3.3 channel ( figure  7d ; chemin et  al . , 2002 ) .
in contrast , the ni - sensitive fraction obtained by subtraction revealed faster kinetics with an explicit criss - cross pattern typical of cav3.2 ( figure  7d ) . to date , zinc ( zn ) is the only available pharmacological tool that interferes with the gating kinetics of cav3.3 ( cataldi et  al . , 2007 , traboulsie et  al . , 2007 ) .
after blockade of cav3.2-like fraction by ni , subsequent application of zn ( 100  m ) slowed down the remaining native cav3.3-like current with a prominent slowing of the deactivation ( figure  7e ) .
analysis of several c - ltmr soma revealed that cav3.3-like currents account for a third of the total amplitude of c - ltmrs t - type calcium currents ( figure  7f ) .
the dramatic slowdown of the deactivation kinetics by zn further shows that cav3.3 is a unique feature in the portfolio of ion channels specific to c - ltmrs ( figure  7 g ) . to test whether these two t - type calcium channel isoforms impact c - ltmr excitability , we performed current - clamp experiments . in neurons ,
t - type calcium channels are well known to generate rebound low - threshold spikes ( lts ) following a transient hyperpolarization . in c - ltmrs ,
membrane depolarization triggered small lts when the membrane is held at more and more negative values ( figure  7h ) .
inhibition of cav3.2 by ni ( 50  m ) did not significantly alter lts and action potential ( ap ) threshold , whereas potentiation of cav3.3 by zn ( 100  m ) increased both lts amplitude and kinetics ( figure  7h , inset ) .
this resulted in an increased excitability with a lowering of ap activation threshold crowning the potentiated lts ( figures 7h and 7i ) . in two cells out of ten , the sole application  of zn at the resting potential of c - ltmrs elicited a depolarization sufficient to trigger spontaneous ap trains ( figure  7j ) .
application of ni in these conditions did not produce any change in membrane potential ( not shown ) . to exclude trpa1 channel contribution ,
also activated by zn , these experiments were performed in  presence of trpa1 blocker , hc030031 ( 10  m ) .
together , our electrophysiological data nicely corroborate the rna - seq  and  the ish expression data , demonstrating that both cav3.2 and cav3.3 channels are concomitantly expressed in c - ltmrs and that both channels are absent in the free nerve ending mrgprd - expressing neurons .
in this study , we used our recently generated mouse model in  which the cutaneous mrgprd - expressing neurons and c - ltmrs were genetically marked by the m - cherry protein , in combination with ib4 live staining followed by cell sorting and rna deep sequencing to gene profile three categories of primary sensory neurons : the free nerve ending mrgpd - expressing neurons , c - ltmrs , and a heterogeneous population of drg cells that  neither bind ib4 nor are labeled by the m - cherry protein .
we provide a digital  measure of the presence and prevalence of transcripts from known and previously unknown genes for each of the three neuronal subsets .
because the dn population is composed of a variety of neuronal subsets and glial cells , dn data set was used to identify genes specifically enriched in the free nerve ending mrgprd - expressing neurons and those specifically enriched in c - ltmrs .
this analysis yielded 156 genes specifically enriched in the dp neurons and 184 in c - ltmrs .
these genes encode a variety of molecules including ion channels , receptors , signaling molecules , and previously uncharacterized genes . a major concern with any gene - profiling experiment is the follow - up procedure .
which genes should one pursue , and what should be done with these lists of genes ?
the answer to this question was motivated by our objectives to expand the molecular characterization of c - ltmrs and to identify the combinatorial expression of the key genes that shape the functional specialization of the free nerve endings mrgprd neurons and c - ltmrs .
mrgprd neurons are required for acute and injury - induced mechanical pain as well as for the sensation of itch ( cavanaugh et  al .
2012 , shields et  al . , 2010 ) , whereas c - ltmrs were postulated to convey light touch under physiological conditions and mechanical and chemical pain after injury ( abraira and ginty , 2013 , delfini et  al . , 2013 ,
our rna - seq data in combination with ish and gene ontology analysis revealed a great number of genes expressed in dp neurons , but not in c - ltmrs , and vice versa , reflecting their anatomical and functional differences .
indeed , dp neurons showed very high enrichment in genes encoding gpcrs and signaling molecules in addition to a selective expression of three isoforms of the protein kinase c family .
the enrichment of such receptors and signaling molecules is in agreement with the involvement of dp neurons in acute and injury - induced mechanical pain and itch ( cavanaugh et  al .
we show that dp neurons express high levels of agtr1a and ptgdr , suggesting that the pro - nociceptive effect of their respective agonists , angiotensin ii and prostaglandin d2 , likely occurs through dp neurons activation .
pgd2 has been shown to enhance neuronal excitability by increasing the amplitude of ttx - sensitive currents ( ebersberger et  al . , 2011 , nakae et  al . ,
2005 ) , and continuous perfusion of angiotensin ii enhanced tactile and thermal hypersensitivity in the setting of nerve injury via the activation of agtr1a ( pavel et  al . , 2013 ) .
furthermore , dp neurons exclusively express mrgprd , a gpcr that is activated by -alanine , but not histamine ( liu et  al . , 2012 ) , suggesting that dp neurons are part of a histamine - independent itch neural circuit and that these neurons can be targeted for treating clinical itch that is resistant to antihistamines .
c - ltmrs on the other hand expressed different gpcrs than those expressed in dp neurons such as cckar and the metabotropic gq p2y1 receptor and showed a striking enrichment in voltage - gated channels including potassium channels ( kcnq2 , kcnj12 , kcnv1 , kcna2 , and kcna4 ) , sodium channels and associated beta subunits ( scn1a , scn1b , scn3b , scn4b , scn7a , and scn8a ) , and the low - voltage - gated calcium channels ( cacna1h and cacna1i ) .
it has been shown that cckar was restricted to 10% of neurons that do not overlap with peptidergic cgrp nociceptors ( broberger et  al . , 2001 ) .
our rna - seq and ish data demonstrate that cckar is expressed in c - ltmrs and excluded from dp neurons .
cholecystokinin octapeptide , which in its sulfated form binds the gq/11-coupled cckar , is involved in regulation of a large variety of physiological functions among which opioid - induced anti - nociception ( suh et  al . , 1995 ) .
electrophysiological studies in rat drg neurons have shown that activation of cckar evokes membrane depolarization , indicating that cckar mediates excitatory responses ( ma et  al . , 2006 ) .
psychophysiological studies in human using cholecystokinin octapeptide could lend new insights into the role of c - ltmrs in touch sensation under normal and pathological conditions .
c - ltmrs also exhibited high expression of the metabotropic gq p2y1 receptor , whereas dp neurons mainly expressed the ionotropic purinergic receptor p2x3 , demonstrating that , in dp neurons , atp / adp compounds are sensed through p2x3 , whereas in c - ltmrs , these signaling intermediates are likely detected though metabotropic gq p2y1 receptor .
our data also highlighted the specific and the functional enrichment of the lva t - type channels in c - ltmrs .
among the three t - type channels , we found cav3.2 and cav3.3 highly present in c - ltmrs , with cav3.3 being exclusively expressed in this particular class of neurons .
t - type currents with slow kinetics and resistance to nickel block have been described in a subset of small - sized rat drg and trigeminal neurons expressing slowly adapting mechano - activated currents ( coste et  al .
our rna - seq data in combination with ish and electrophysiological recordings provided a clear demonstration that cav3.3 is a specific marker of c - ltmrs and offers a new possible selective pharmacological target to  control c - ltmrs excitability in mice .
although zinc has plethoric effects , its use to investigate cav3.3 contribution to c - ltmr excitability shows specificity .
particularly , the slowing of lts kinetics is highly correlated to the massive changes in cav3.3 currents . in line with this , the depolarizing effects observed result , at least in part , from the persistent depolarizing zinc - modulated cav3.3 current . whereas the contribution of other zinc targets remains possible ,
trpa1 effect has been eliminated by the use of its specific blocker , and a possible inhibition of two pore potassium task3 channel ( kcnk9 ) is unlikely because the transcript is not present in c - ltmrs ( our study ; usoskin et  al . , 2015 ) .
mechanistically , our findings suggest a substantial contribution of cav3.3 to rebound firing reminiscent of those observed in nuclear reticularis thalamic neurons that , like c - ltmr , express both cav3.2 and cav3.3 and with a prominent cav3.3 effect on burst firing ( astori et  al . , 2011 ) .
finally , a recent elegant study using an unbiased large - scale single - cell rna sequencing revealed 11 molecularly distinct subsets of primary sensory neurons , including the th - expressing c - ltmrs population ( usoskin et  al . , 2015 ) .
very interestingly , genes enriched in c - ltmrs in our study were similar to those upregulated in the th population described in the usoskin and colleagues study .
however , our ish experiments showed that the vast majority of c - ltmrs - enriched genes were also expressed in other neurons , preferentially in the nf200 neurons , further supporting our strategy to validate the specific expression of the identified enriched genes using ish approach .
given that the majority of c - ltmrs - enriched genes were also expressed in nf200 neurons and that nf200 is a hallmark of proprioceptive and a and a ltmrs ( abraira and ginty , 2013 ) , we postulate that c - ltmrs share many molecular features with some a and a ltmrs . in line with this hypothesis ,
studies of  the last few years demonstrated that c - ltmrs share many functional and anatomical features of some a and a ltmrs ( abraira and ginty , 2013 ) .
c - ltmrs have been described to convey low - threshold mechanical stimuli ( bessou et  al . , 1971 , douglas and ritchie , 1957 , johansson et  al . , 1988 ,
, 1952 , seal et  al . , 2009 , zotterman , 1939 ) .
they are sensitive to skin indentation , respond to sustained mechanical stimuli with slow and intermediate adaptation rates , and form longitudinal lanceolate endings around hair follicles ( abraira and ginty , 2013 , delfini et  al . , 2013 , li et  al . , 2011 , lou et  al . , 2013 ,
cross comparison of our rna - seq data sets with those of the five nf subsets recently published by usoskin and colleagues ( usoskin et  al . , 2015 ) will provide a comprehensive view of the molecular signatures of c - ltmrs and a and a ltmrs . in conclusion , data presented in this study provide a wealth of  information about the presence and prevalence of transcripts  from known and previously unknown genes in three categories of  primary sensory neurons , they highlight the differential expression of a great number of genes in two diametrically opposite classes of cutaneous afferents , the ish screen followed by immunostaining and ib4 binding led to the generation of two open - access libraries that allow monitoring the cellular distribution of 115 genes , they provide the first electrophysiological recording of a low - voltage - gated ca channel exclusively present in c - ltmrs , and they suggest that c - ltmrs share many molecular features with a and a ltmrs .
the expression patterns of dn - enriched genes ; the co - expression of c - ltmrs - enriched genes with nf200 neurons ; the supplemental tables enclosing dp- , c - ltmrs- , and dn - enriched genes ; as well as the go analysis are provided in the supplemental information .
mice were maintained under standard housing conditions ( 23c , 40% humidity , 12  hr light cycles , and free access to food and water ) .
c57bl/6 mice were from charles river laboratories , and heterozygous ginip knockin mice were previously generated in the laboratory ( gaillard et  al . , 2014 ) .
special effort was made to minimize the number as well as the stress and suffering of mice used in this study .
all protocols are in agreement with european union recommendations for animal experimentation . ib4ginip , ib4ginip , ib4ginip , and ib4ginip cells were sorted on facs aria cell sorter ( bd bioscience ) by gating on sytox blue m - cherry for ginip cells and sytox blue m - cherry for ginip cells .
drgs cell suspensions from c57bl/6 mouse were used as a negative control of m - cherry fluorescence .
cells were sorted directly in ice - cold rlt lysis buffer from rneasy micro kit ( qiagen ) , supplemented with 10% ( v / v ) -mercaptoethanol ( sigma ) , and snap - frozen at 80c before rna extraction .
rna - seq libraries were constructed with the truseq rna sample preparation ( low - throughput protocol ) kit from illumina .
were performed using the hiseq control software and real - time analysis component provided by illumina .
the quality of the data was assessed with fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) .
demultiplexing was performed using illumina s sequencing analysis software ( casava 1.8.2 ) . the eland_rna module from casava
was used to align rna - seq reads to the mus musculus genome ( ucsc mm10 ) with a set of gene - model annotations ( refflat.txt file downloaded from ucsc on october 10 , 2012 ) in order to generate automatically the splice junctions .
reads were also aligned to a set of contaminants , including the rrnas , the mitochondrial chromosome , the phix genome ( illumina control ) , and the illumina adapters .
then , exon counts were summed by gene and divided by the length of reads ( i.e. , 100 ) to obtain the gene counts .
downstream statistical analysis was made on ( 1 ) genes that generated at least ten reads and ( 2 ) genes that generated at least 1,000 reads .
differentially expressed genes were identified using the bioconductor ( robinson et  al . , 2010 ) package edger ( alexa et  al . , 2006 ) version 2.6.2 .
data were normalized using the tmm ( robinson and oshlack , 2010 ) normalization factors .
genes with adjusted p value less than 1% ( according to the fdr method from benjamini - hochberg ) were declared differentially expressed .
genes were then classified using custom rules : ( 1 ) genes declared as differentially expressed in condition a relative to condition b and to condition c and having a fold - change of at least 2 ( absolute value ) in both cases are considered to be specific of condition a. ( 2 ) a gene will be specific of the two conditions a and b simultaneously if it is differentially expressed in a versus c and in b versus c with an absolute fold change > 2 and with an absolute fold change < 2 between a and b. ( 3 ) filtered genes are considered to be specific of none condition .
( 4 ) genes belonging to none of the previous categories are considered to be specific of the three conditions simultaneously .
free web access gorilla database was used to perform the go functional annotation of the resulting list of genes as described in eden et  al .
lumbar drgs were prepared and cultured as described previously ( francois et  al . ,
we incubated cultures with alexa-488-conjugated ib4 ( 1  g / ml ; 5  min at 37c ; invitrogen ) diluted in extracellular solution and washed before recordings .
conditions for recordings of voltage - gated calcium currents and of membrane potential were as described in delfini et  al .
patterns of expression of over 100 genes are enclosed in dp and c - ltmrs ish  libraries . for dp ,
and a. mantilleri performed the in  situ analyses , p.m. performed the pcrs for probe synthesis , s.l .
 summarycutaneous c - unmyelinated mrgprd+ free nerve endings and c - ltmrs innervating hair follicles convey two opposite aspects of touch sensation : a sensation of pain and a sensation of pleasant touch . 
 the molecular mechanisms underlying these diametrically opposite functions are unknown . here 
 , we used a mouse model that genetically marks c - ltmrs and mrgprd+ neurons in combination with fluorescent cell surface labeling , flow cytometry , and rna deep - sequencing technology ( rna - seq ) . cluster analysis of rna - seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in mrgprd - expressing neurons  and c - ltmrs , respectively . 
 we validated 48 mrgpd- and 68 c - ltmrs - enriched genes using a triple - staining approach , and the cav3.3 channel , found to be exclusively expressed in c - ltmrs , was validated using electrophysiology . 
 our study greatly expands the molecular characterization of c - ltmrs and suggests that this particular population of neurons shares some molecular features with a and a low - threshold mechanoreceptors . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions