A novel Nav1.8-FLPo driver mouse for intersectional genetics to uncover the functional significance of primary sensory neuron diversity

Summary The recent development of single-cell and single-nucleus RNA sequencing has highlighted the extraordinary diversity of dorsal root ganglia neurons. However, the few available genetic tools limit our understanding of the functional significance of this heterogeneity. We generated a new mouse line expressing the flippase recombinase from the scn10a locus. By crossing Nav1.8Ires−FLPo mice with the AdvillinCre and RC::FL-hM3Dq mouse lines in an intersectional genetics approach, we were able to obtain somatodendritic expression of hM3Dq-mCherry selectively in the Nav1.8 lineage. The bath application of clozapine N-oxide triggered strong calcium responses selectively in mCherry+ neurons. The intraplantar injection of CNO caused robust flinching, shaking, and biting responses accompanied by strong cFos activation in the ipsilateral lumbar spinal cord. The Nav1.8Ires−FLPo mouse model will be a valuable tool for extending our understanding of the in vivo functional specialization of neuronal subsets of the Nav1.8 lineage for which inducible Cre lines are available.


INTRODUCTION
Primary somatosensory neurons are a highly diverse group of neurons that enable us to perceive and discriminate between diverse types of sensations from the outside world and the internal state of the body.2][3][4][5][6] Decades of research, including the recent development of single-cell RNA sequencing, have greatly increased our knowledge of the extraordinary diversity of DRG neurons.][9][10][11][12][13][14][15][16] However, despite this wealth of information, our understanding of the functional specialization of somatosensory neurons remains far from complete because of the lack of appropriate genetic tools selectively targeting these neuronal subsets with a very high degree of precision.
8][19][20][21][22] However, interpretation of the results is often complicated by the transient or dynamic developmental expression of the gene of interest in the somatosensory system and its expression outside the somatosensory system.1][32][33][34][35][36][37] Despite these advances in genome-editing techniques, it is becoming increasingly clear that the recombinase-mediated expression of a single gene cannot provide the necessary resolution for studies of the functional specialization of a single cell type.8][39][40][41][42][43][44] Intersectional genetics increases resolution through the use of a dual-recombinase system based on Cre and flippase (FLPo) to activate a conditional effector allele only in cells in which both recombinases are expressed.However, its use in the somatosensory system is hampered by the lack of mouse lines expressing the FLPo recombinase selectively in sensory neurons.In this study, we generated an FLPo-driver mouse line that expresses the FLPo recombinase from the 3 0 UTR of the Na v 1.8 locus.This approach ensures that the Na v 1.8 voltage-gated sodium channel remains intact and allows functional and selective expression of the FLPo recombinase in the Na v 1.8 lineage in DRG, TG, and jugular nodose ganglia (JNG) neurons.Crossing of Na v 1.8 IresÀFLPo with Advillin-Cre and RC::FL-hM3Dq reporter mice resulted in robust expression of the excitatory DREADD in a large number of DRG neurons.Bath applications of clozapine N-oxide (CNO) triggered strong calcium responses selectively in the intersecting DRG neurons in which both recombinases were expressed, demonstrating induction of the canonical Gq pathway and subsequent neuronal activation.Intraplantar injections of CNO caused robust flinching, shaking, and biting responses accompanied by strong cFos activation in the ipsilateral lumbar spinal cord.

Generation of Na v 1.8 IresÀFLPo mice
As expression of the tetrodotoxin (TTX)-resistant Na v 1.8 sodium channel is tightly restricted to somatosensory neurons, we sought to generate a new mouse model expressing the FLPo recombinase rather than the Cre recombinase in the Na v 1.8 lineage.Given the important physiological role of this channel in sensory neurons, we used CRISPR technology to knockin an Ires-FLPo cassette at the 3 0 UTR of the Scn10a locus (Figure 1A).With the design used, the resulting allele should produce a bicistronic mRNA encoding a functional Na v 1.8 channel and the FLPo recombinase in the Na v 1.8 lineage.For validation of our mouse model, we first crossed Na v 1.8 IresÀFLPo mice with RC::FL-hM3Dq mice 45 (Figure 1B).In the progeny of this cross, the FLPo excised the FRT-flanked transcription STOP cassette, allowing the expression of EGFP in a large number of DRG, TG, and JNG neurons (Figures 1C-1E; S1D for quantification).To determine the extent to which EGFP expression overlapped with the distribution of Na v 1.8 mRNA, we performed in situ hybridization for Na v 1.8 followed by immunostaining for EFGP (Figure S1A).We found that 85.2% G 0.6% of EGFP + neurons expressed Na v 1.8 mRNA, and 85.4% G 2.1% of Na v 1.8 + neurons expressed EGFP (Figures S1A and S1B).We were, thus, able to capture both persistent and transient Na v 1.8-expressing neurons, but the IRES-mediated translation of FLPo failed to trigger the excision of the FRT-flanked STOP cassette in a small subset of DRG neurons.Consistent with these findings, 82.9% G 0.4% of neurons were EGFP + in homozygous Na v 1.8 IresÀFLPo/IresÀFLPo :: RC::FL-hM3Dq mice, versus 71.0%G 1.9% in heterozygous Na v 1.8 IresÀFLPo/+ :: RC::FL-hM3Dq mice (Figures S1C and S1D).
Given the major functional role of the Na v 1.8 channel in sensory neurons, we checked that our genetic approach did not interfere with the function of this channel.We performed electrophysiological recordings of sodium currents in GFP + DRG neurons obtained from heterozygous Na v 1.8 IresÀFLPo/+ ::RC::FL-hM3Dq and homozygous Na v 1.8 IresÀFLPo/IresÀFLPo ::RC::FL-hM3Dq mice.We focused on putative Na v 1.8 currents by performing recordings at a holding potential of À40 mV, in the presence of 300 nM TTX, 0.1 mM external calcium, and 0.1 mM cadmium to inhibit voltage-gated calcium channels.Under these conditions, a 30 ms test pulse from À40 mV to +50 mV triggered robust TTX-resistant sodium currents, the maximal current being recorded at 0 mV (Figure S1E).Importantly, there was no difference in maximal current density between the two genotypes (Figure S1F), demonstrating that our targeting strategy allowed the expression of a functional Na v 1.8 channel in both heterozygous and homozygous mice.
We then performed a series of double-immunohistochemistry (IHC) experiments on DRG sections.Using anti-GFP and anti-RFP antibodies, we demonstrated the high-fidelity FLPo-mediated expression of EGFP, as no expression of mCherry was observed in DRG neurons (Figure 1F).We also found that FLPo-mediated recombination targeted C-fibers and Ad nociceptors expressing P2X3, GINIP, CGRP, TH, and TAFA4 (Figures 1G-1K).Our targeting strategy also captured a large number of large-diameter Ab neurons expressing NF200 and TrkC (Figures 1L and 1M).
For identification of the central and peripheral projections of EGFP + DRG neurons, we performed double-IHC on sections from the lumbar segment of the spinal cord and the skin.Consistent with the expression of EGFP predominantly in C-and Ad fibers, EGFP + central terminals were highly restricted to the dorsal horn of the spinal cord, particularly in laminae I (labeled with CGRP) and laminae II (labeled with IB4 and VGLUT3) (Figures 2B-2D).At the periphery, massive EGFP innervation of the skin was visualized through the colabeling of EGFP + nerve endings with the pan-neuronal marker PGP9.5 (Figure 2F).No RFP staining was observed in the central and peripheral endings of DRG neurons, further demonstrating an absence of leakage for the cassette expressing the -hM3Dq-mCherry fusion protein (Figures 2A and 2E).EGFP + terminals were also observed in several visceral organs, including the bladder (Figure S2A) and jejunum (Figure S2B), but not the liver (Figure S2C).Outside the primary sensory nervous system, our genetic design captured very strong expression of the Na v 1.8 allele in the cardiac conduction system (Figure S2D) and in a few EGFP + /PV À neurons in the limbic system (Figures S2E and S2F).Together, these data show that the mouse model generated drives FLPo recombinase activity with very high fidelity in the Na v 1.8 lineage and highlights several hotspots of Na v 1.8 gene activity in other organs, including the brain and heart.

The Na v 1.8 IresÀFLPo mouse is suitable for use in intersectional genetics
We assessed the suitability of our newly generated mouse model for use in intersectional genetics by crossing Na v 1.8 IresÀFLPo ::RC::FL-hM3Dq mice with Advillin-Cre (Adv ÀCre ) mice. 46In the triple-transgenic offspring, further exposure to Cre triggered the excision of EGFP and inverted the cassette mediating somatodendritic expression of the hM3Dq/mCherry fusion protein (Figure 3A).Indeed, double-IHC with antibodies against GFP and RFP showed that all DRG neurons expressed mCherry rather than EGFP (Figure 3 B).The double-recombination event occurred selectively in Na v 1.8 lineage because mCherry was co-expressed with markers of C-fibers and Ad nociceptive neurons labeled with P2X3 and CGRP (Figures 3C and 3D) and was excluded from large-diameter neurons expressing TrkC and TrkB (Figures 3E and 3F).Consistent with this finding, mCherry + terminals no longer expressed EGFP peripherally (Figure 3G) or centrally (Figure 3I), and they densely innervated the skin as free nerve endings, peripherally (Figure 3H), and were restricted to laminae I and II of the dorsal horn of the spinal cord labeled with IB4, CGRP, and VGLUT3 (Figures 3J-3L).CNO induced strong activation of the Na v 1.8 lineage both in vitro and in vivo Given that mCherry is fused to hM3Dq, the data suggest that the excitatory DREADD is expressed in the cell bodies in addition to the central and peripheral terminals of the Na v 1.8 lineage.CNO administration was therefore expected to induce the canonical G q pathway, leading to depolarization/activation of the targeted neurons.We tested this hypothesis by performing calcium imaging on cultured mCherry-positive and -negative neurons (Figure 4A left).We monitored hM3Dq-induced calcium responses to 25.5 mM CNO, followed by TRPV1-induced responses to 3 mM capsaicin (Figure 4A right).CNO triggered calcium responses in 31 of 52 mCherry + neurons, half of which also responded to capsaicin.By contrast, none of the mCherry À neurons responded to CNO.The overall activity of all neurons was assessed by monitoring their calcium responses in the presence of 140 mM KCl.
We then investigated the effect of CNO administration in vivo.We used two different readouts, one behavioral and the other molecular.Intraplantar injections of CNO triggered robust flinching, biting, and shaking responses in the injected paw (Figures 4B and 4C and Supplemental movies).This very pronounced behavior began immediately after CNO injection and lasted 30 min.This phenotype was selective, observed only in Cre + mice, with Cre À mice not displaying such behavior following CNO injection.As we also wanted to have a molecular readout of the effects of CNO injection, mice were killed 1 h after CNO administration, and sections of the lumbar segments of the spinal cord were prepared and subjected to double-IHC with anti-cFos antibody together with antibodies against CGRP, IB4, or VGLUT3.Very strong nuclear cFos immunostaining was observed selectively in the ipsilateral dorsal horn of the spinal cord in Cre + mice but not in Cre À mice (Figure 4D).Interestingly, cFos immunostaining was concentrated in the mediolateral part of the spinal cord, which is known to be innervated by lumbar DRG neurons projecting into the glabrous skin of the hind paw (Figure 4E).This spinal region is innervated by CGRP + and IB4 + central terminals but not by those labeled with VGLUT3, a specific marker of C-low threshold mechanoreceptors, which innervate exclusively hairy skin (Figure 4E).

DISCUSSION
In this study, we generated a new mouse model that will be extremely useful for use in elegant intersectional genetics approaches in mice. 38uch approaches make it possible to achieve a high degree of specificity in investigations of the biological functions of a specific subset of cells, identified by the expression of a particular gene, even if this gene of interest is also expressed in tissues other than the tissue of interest.Single-cell and single-nucleus RNA sequencing techniques have made it possible to identify a wide range of molecularly defined subpopulations of sensory neurons in the somatic sensory nervous system, 47 consistent with the broad range of sensory modalities that can be perceived by our bodies.Efforts to decipher the precise functional specialization of each of these neuronal subsets have been hampered by a lack of appropriate genetic tools for the selective and specific targeting of particular subsets.The mouse model developed here will facilitate such studies, through combination with appropriate mouse Cre driver lines.We show here that crossing the Na v 1.8 IresÀFLPo , the Advillin Cre , and RC::FL-hM3Dq reporter mice allows a the expression of a putatively functional excitatory DREADD throughout the entire Na v 1.8 lineage, as demonstrated by the expression of the mCherry.Using calcium imaging, we showed that 60% of the intersected neurons responded to bath applications of CNO, demonstrating that only a proportion of mCherry-expressing DRG neurons were competent to respond to bath application of CNO.However, from a behavioral point of view, a functional excitatory DREADD was fully validated in vivo as intraplantar injections of CNO triggered robust nocifensive behavior accompanied by strong cFos activation in the mediolateral part of the spinal dorsal horn, the spinal region in which the neurons innervating the glabrous skin of the hind paw terminate.Our results demonstrate that this new mouse model is a highly suitable tool for deciphering the functional specialization of a wide range of primary sensory neurons subsets by intersectional genetics.
Over the last two decades, the sensory biology community has generated a large number of mouse lines in which Cre-recombinase is expressed under the control of promoters of genes selectively expressed in particular subsets of DRG neurons.Unfortunately, many of these mouse lines lack temporal control over Cre recombination events, particularly for genes displaying dynamic expression during embryonic development and/or at postnatal stages.As a result, it is often not only the DRG neurons expressing the gene of interest that ends up being targeting, but also its lineage.For example, the NGF receptor TrkA is expressed in 90% of DRG neurons during development and at birth. 48uring the first 3 weeks of postnatal life, trkA expression is switched off in more than half the TrkA-expressing neurons, which instead express the glial cell-derived neurotrophic factor receptor Ret. 49 Therefore, in the adult DRG, trkA ends up being expressed in less than 30% of all adult DRG neurons.Likewise, MRGPRB4 + neurons account for fewer than half the neurons fate-mapped with the mrgprb4 Cre mouse. 31hus, targeting the lineages in which these genes are expressed does not provide any information about the functional specialization of the subsets of neurons actually expressing TrkA or MRGPRB4 in the adult mouse.
More mouse lines expressing inducible Cre will need to be generated to overcome these issues.Indeed, crossing the Na v 1.8 IresÀFLPo mouse with a mouse that expresses an inducible Cre in a small subset of cells of the Na v 1.8 lineage makes it possible to express effector molecules in a highly controlled manner.With such approaches, the subset of neurons of interest can be genetically marked, activated, (E) Hind-paw glabrous skin section co-immunolabeled for GFP (green) and RFP (red).(F) Hind-paw glabrous skin section co-immunolabeled for GFP (green) and PGP9.5 (magenta).Scale bar: 100 mm, n = 2 mice.inactivated, or ablated, at will.These manipulations can be achieved in vitro or in vivo, under physiological or pathological conditions.The high degree of spatial and temporal specificity is the key advantage of such systems.The Na v 1.8 IresÀFLPo mouse is an excellent model as it spatially restricts FLPo expression to a very small number of tissues, such as the DRG, TG, nodose ganglia (NG), and the conduction tissue of the heart.Temporal control over the expression and activation of the effector molecules depends on the induction of Cre recombination.Na v 1.8 IresÀFLPo restricts FLPo recombination to a small number of tissues, but there will inevitably be some circumstances in which a gene of interest is expressed in a subset of the Na v 1.8 DRG lineage, in the NG for example.This dual expression is necessarily problematic as manipulating the activity of NG neurons at the same time as that of DRG neurons may complicate interpretation of the results.It is possible to overcome this problem by using viral infection to express the dual-recombinase-dependent effector molecules in organs targeted by DRG but not by NG neurons.This strategy was recently used in an elegant study by the Ginty laboratory for selective visualization of the projection pattern and manipulation of the activity of two different somatosensory neuron subtypes innervating the Krause corpuscles of the mouse penis and clitoris. 36inally, the power of our approach lies in the fact that our mouse model allows expression of the FLPo recombinase without affecting the integrity of the scn10a locus, as it expresses the wild-type version of the Na v 1.8 channel.Our model, which does not impair the very important function of this channel in sensory neurons, can, therefore, be used in a wide range of intersectional genetic approaches with no potential confounding effects due to the absence of one copy of the Na v 1.8 allele.

Limitations of the study
In the present study, we generated and validated a new mouse model expressing the FLPo from the scn10a (Na v 1.8) locus.Using the IRES cassette to drive the translation of the FLPo does not target 100% of Na v 1.8 lineage.Therefore, depending on the subsets of DRG neurons to be targeted, one will need to use homozygous mice.
The second limitation of the study is related to the calcium imaging experiments.Only two mice and six plates were used to generate the data presented in Figure 4A.This part of the study was mainly performed to show that our newly generated mouse model allows the expression of a functional excitatory DREADD.It turns out that this experiment shows that CNO triggers calcium responses in only a fraction of DRG neurons, demonstrating that not all DRG neurons are competent to respond to CNO in the calcium imaging experiments.three washes for 5 minutes each in 1xPBS, sections were incubated for 1 h at room temperature with secondary antibodies diluted in the blocking solution described above.The corresponding donkey anti-chicken, anti-rat, anti-rabbit, anti-goat or anti-guineapig Alexa 488-, 555-, or 647-conjugated secondary antibodies (1:500, Thermo Fisher Scientific) were used for the detection of primary antibody binding.Isolectin B4 conjugates with AlexaFluorR 647 dye were used at a dilution of 1:200 (Thermo Fisher Scientific I32450).Tissues were washed (3 times in 1xPBS) and mounted in ImmuMount Reagent.Images were acquired with an AxioImager M2 (Zeiss) fluorescence microscope with a 20x/0,8 objective (or a 10x objective for spinal cord in Figure 4D) and contrast was adjusted with Fiji software.

In situ hybridization
RNA probes were synthesized with gene-specific PCR primers and cDNA templates from mouse DRG.In situ hybridization was performed with digoxigenin-labeled probes (Roche, cat# 11277073910).Probes were incubated with the slides overnight at 55 C and the slides were then incubated with the horseradish peroxidase-conjugated anti-digoxigenin antibody 1:500 (Roche, Cat#11207733910; RRID:AB_514500).Final detection was achieved with TSA-Cy3 at a dilution of 1:50 (Perkin Elmer Life Sciences, FP1170).The oligonucleotides used for the nested PCRs and for probe synthesis are listed in the key resources table.

Cell counts
For ISH experiments, a threshold was set and scn10A-positive cells were then classified as Na v 1.8 high or Na v 1.8 low according to this threshold.The total number of cells analyzed per animal and per marker (scn10A and GFP) ranged from 571 to 1506.
For Figure S1D, the autofluorescence of the neurons made it possible to determine the total number of neurons.The total number of cells analyzed per animal and per genotype ranged from 962 to 4432.

Electrophysiology recordings
Dorsal root ganglion (DRG) neurons were prepared as previously described. 23Briefly, adult male C57BL/6J mice were anesthetized by pentobarbital injection and transcardially perfused with HBSS (pH 7.4, 4 C).Lumbar DRGs with attached roots were dissected out and collected in cold HBSS supplemented with 5 mM HEPES, 10 mM D-glucose and 1% penicillin/streptomycin. DRGs were treated with 2 mg/ml collagenase II and 5 mg/ml dispase for 40 min at 37 C, washed in HBSS and resuspended in 1 ml neurobasal A medium supplemented with B27, 2 mM L-glutamine and 1% penicillin/streptomycin (Invitrogen, Thermo Fisher Scientific, France).Single-cell suspensions were obtained by five passages of the cell suspension through three needle tips of decreasing diameter (gauge 18, 21, and 26).The cells were then plated on polyornithine/laminin-coated dishes.After incubation for 2 hours, the medium was removed and replaced with neurobasal B27 supplemented with 12.5 ng/ml NGF.Patch-clamp recordings were performed 12-24 h after plating, on GFP-positive neurons of 20 to 25 mM in diameter.
Macroscopic currents were recorded at room temperature with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale CA).Borosilicate glass pipettes had a resistance of 1.5-2.5 MOhm when filled with an internal solution containing 100 mM CsCl, 40 mM TEA-Cl, 10 mM EGTA, 10 mM HEPES, 3 mM Mg-ATP, 0.6 mM GTP-Na, and 3 mM CaCl 2 (pH adjusted to 7.25 with TEA-OH, $300 mOsm, $100 nM free Ca 2+ according to Max-Chelator software, http://maxchelator.stanford.edu/).The extracellular solution contained 35 mM NaCl, 100 mM TEACl, 5 mM MgCl 2 , 0.1 mM CaCl 2 , 0.1 mM CdCl 2 , 5 mM KCl, 10 mM glucose and 10 mM HEPES (pH adjusted to 7.25 with TEA-OH, $300 mOsm).This extracellular solution also contained freshly prepared TTX (300 nM, Latoxan).Currents were elicited at 5 s intervals from a holding potential of -40 mV by test pulses of 30 ms duration from -40 mV to +50 mV with 5 mV increments.Recordings were filtered at 5 kHz.Data were analyzed with pCLAMP9 (Molecular Devices) and GraphPad Prism (GraphPad) software.Results are presented as the mean G SEM, and n is the number of cells.Statistical analysis was performed with Student's t-test.

Calcium imaging
Calcium imaging (Ca 2+ ) experiments were performed with an inverted microscope (Olympus IX73, 20X objective with a numerical aperture of 0.45) equipped with a LED illuminator (pE-300 white).Images were acquired at 10 Hz with an exposure time of 80 ms, with trueform waveform generators (33600B), and were recorded with a Basler acA4096 camera.Primary neuronal cultures were performed as described above and were stimulated with clozapine N-oxide (CNO) (25 mM), capsaicin (3 mM) and KCl (140 mM) solutions.They were then washed with Na-HEPES solution (containing 0.37 mg/ml KCl, 8.1 mg/ml NaCl, 2.38 mg/ml HEPES, 0.22 mg/ml CaCl 2 and 0.19 mg/ml MgCl 2 , pH 7.3).All solutions were applied with syringes controlled by a Peri-Star Pro pump.Calcium responses were recorded before, during and after stimulation.Before each experiment, DRG neurons were incubated with 400 ml Opti-MEM solution and 5 mM calcium dye (Fluo4-AM).Cells were imaged with Pylon Viewer software.All experiments were performed at room temperature (25 C).The data were analyzed with an in-house Matlab code (Matlab 2018A).Data are presented as the relative change in fluorescence (DF/F0) of each neuron, where F0 is basal fluorescence and DF = F À F0.All cells that did not respond to KCl or capsaicin were excluded from the analysis.
Regardless of their response to CNO, the cells retained for the final analysis were those that responded to capsaicin alone, KCl alone or to both compounds.

CNO preparation
CNO (6329, TOCRIS, batch no.: 4A/271069) was dissolved in water at a concentration of 10 mg/ml (w/v) and then aliquoted and stored at -20 C. CNO solution was freshly prepared at a concentration of 25.5 mM (for in vitro experiments) or 2 mg/ml (for in vivo experiments) by diluting the 10 mg/ml stock solution with Na-HEPES washing solution (for in vitro experiments) or 0.9% NaCl (for in vivo experiments).

Behavioral test
Behavioral assays were conducted on 8-to 12-week-old Na v 1.8 FLPo/+ ::Advilin +/+ ::R26 RC-FL-hM3Dq/+ (Cre -, control mice) and Na v 1.8 FLPo/+ :: Advilin Cre/+ ::R26 RC-FL-hM3Dq/+ (Cre + ) mice.Animals were allowed to get used to the experimenter before the tests were performed.Mice were placed individually into Perspex chambers and were allowed to acclimate to the testing environment for 30 min.We then injected 10 ml of a 2 mg/mL CNO solution subcutaneously into the plantar surface of the left hind paw.The animals were immediately placed in observation chambers and monitored for pain-related behavior (shaking, licking and biting of the injected paw) for 30 min.The cumulative duration of painrelated behavior was determined in seconds, at five-minute intervals.The experimenter was blind to the genotype of the mice.

QUANTIFICATION AND STATISTICAL ANALYSIS
The number of animals tested is indicated in the figure legends.The Shapiro-Wilk test was used to assess data normality.Values of p < 0.05 were considered to be statistically significant in unpaired t-tests for IF experiments or unpaired Mann-Whitney tests for in vivo experiments.Statistical analyses were performed with GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA).