Characteristics of sensory neuronal groups in CGRP-cre-ER reporter mice: Comparison to Nav1.8-cre, TRPV1-cre and TRPV1-GFP mouse lines

Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRPcre-ER (CGRP-cre+); and compared them to DRG neurons expressing Nav1.8cre (Nav1.8-cre+), TRPV1cre (TRPV1-cre+) and TRPV1-GFP (V1-GFP+). Tamoxifen effectively induced CGRPcre-ER production in DRG. ≈87% of CGRPcre-ER-expressing neurons were co-labeled CGRP antibody. Three small and two medium-large-sized (5HT3a+/NPY2R- and NPY2R+) neuronal groups with unique electrophysiological profiles were CGRP-cre+. Nav1.8-cre+ neurons were detected in all CGRP-cre+ groups, as well as in 5 additional neuronal groups: MrgprD+/TRPA1-, MrgprD+/TRPA1+, TRPV1+/CGRP-, vGLUT3+ and ≈30% of trkC+ neurons. Differences between TRPV1cre and Nav1.8cre reporters were that unlike TRPV1-cre+, Nav1.8-cre+ expression was detected in non-nociceptive vGLUT3+ and trkC+ populations. Many TRPV1-cre+ neurons did not respond to capsaicin. In contrast, V1-GFP+ neurons were in 4 groups, each of which was capsaicin-sensitive. Finally, none of the analyzed reporter lines showed cre-recombination in trkB+, calbindin+, 70% of trkC+ or parvalbumin+ neurons, which together encompassed ≈20% of Nav1.8-cre- DRG neurons. The data presented here increases our knowledge of peptidergic sensory neuron characteristics, while showing the efficiency and specificity manipulation of peptidergic neurons by the CGRPcre-ER reporter. We also demonstrate that manipulation of all C- and A-nociceptors is better achieved with TRPV1-cre reporter. Finally, the described approach for detailed characterization of sensory neuronal groups can be applied to a variety of reporter mice.


Introduction
It is now recognized that somatosensory neurons are neurochemically, functionally and physiologically diverse [1,2,3]. This diversity allows for the detection of a wide range of sensory stimuli such as light touch, pressure, vibration, heat, cold and itch. With regards to pathological pain, it has been suggested that the roles of sensory neuronal groups are distinct [4]. Recent studies have utilized multiple markers such as MrgprA3, MrgprD, NPY2R, vGLUT3, trkB, trkC, calbindin (Calb) and parvalbumin (PV) for electrophysiological characterization of subsets of sensory neurons, and define their physiological functions [5,6,7,8,9,10,11]. Moreover, development of the next generation high-throughput and single-cell sequencing approaches has built transcription profiles for many sensory neuronal subsets/groups [1,7,12].
Sensory neurons involved in nociception and itch transmission are often divided into "peptidergic" and "non-peptidergic". Peptidergic neurons are defined as calcitonin gene-related peptide-positive (CGRP + ) [4]; and the majority express trkA [13]. Peptidergic neurons are important in triggering neurogenic inflammation [14,15,16]. The functional differences between peptidergic and non-peptidergic nociceptors and especially the myriad sub-groups of peptidergic nociceptors are not well known. In this respect, an effective transgenic mouse model and detailed characterization of peptidergic neuronal sub-groups could help us better understand the functions of peptidergic neurons in pain conditions and how they interact with immune and endocrine systems.
A mouse line with inducible cre-recombinase driven by the CGRP-α (CALCA) promotor (CGRP cre/+-ER ) has been generated [17]. The aims of the present study were to generate comprehensive electrophysiological profile and anatomical characterization of neuronal groups/ clusters visualized in DRG of CGRP cre-ER /TdTomato (labeled as CGRP-cre + ) mice and compare them with properties of neuronal groups detected in DRG of Nav1.8 cre /TdTomato (Nav1.8-cre + ) [18], TRPV1 cre /TdTomato (TRPV1-cre + ) [19] and TRPV1-GFP (V1-GFP + ) mice. Results of this study will advance our knowledge of peptidergic sensory neuron characteristic and offer an effective transgenic model for manipulation of peptidergic nociceptors. In addition, the described approach can be utilized in characterizing sensory neuronal groups expressing a variety of reporter mice; and sensory neuronal groups innervated by a range of tissues.

Mouse reporter lines
All animal experiments conformed to APS's Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training. We also followed guidelines issued by the National Institutes of Health (NIH) and the Society for Neuroscience (SfN) to minimize the numbers of animals used and their suffering. Protocols specifically used in these studies (20150100AR and 20150109AR) are approved by the University Texas Health Science Center at San Antonio (UTHSCSA) Animal Care and Use Committee (IACUC).

Primary DRG neuronal culture
Reporter mice expressing either GFP or TdTomato gene were deeply anaesthetized with isoflurane (0.3 ml in 1 liter administered for 60-90 sec) and sacrificed by cervical dislocation. L3-L5 DRG were quickly removed, and sensory neurons were cultured as previously described [20]. Briefly, DRG neurons were dissociated by treatment with a 1mg/ml collagenase-dispase (Roche, Indianapolis, IN) solution. Cells were maintained in DMEM supplemented with minimal serum percentage (i.e. 2% fetal bovine serum), 2mM L-glutamine, 100U/ml penicillin and 100μg/ml streptomycin. No growth factor was added to the media. The experiments were performed within 24 h after DRG neuron plating, since electrophysiological profile did not undergo changes within this period.
Cells were considered spherical, and therefore, diameter (d in μm) of cells was calculated from capacitance (C m in pF) values.
AP rise time (RT; time from V m to AP peak), fall time (FT; time from AP peak to V m level) and duration at the base (dB; time from V m starting point to V m levels at falling phase of AP) as well as 80% recovery time of after-hyper-polarization to baseline (AHP 80 ) were measured from data generated by protocol-1 (Fig 1B). Protocol-2 and -4 revealed algesic responses to ATP, CAP, 5-HT and/or MO as well as I ATP and I 5HT characteristics. I CAP and MO-induced response characteristics were not analyzed, since sequential recordings could have desensitized them leading to changes in kinetics and magnitude of these currents/responses. From protocol-3, the trace evoked by +20 mV was fit with a standard (i.e single or double) exponential function.
A1 exp½À ðt À kÞ=t þ C Fitting and decay tau (τ; ms) calculation was performed using pCLAMP10.2 software ( Fig  1C). Presence or absence of "spike-like" feature at steps to 0 and +20 mV was an important clustering variable. An approach for generation of clustering parameters described in details in "Results"; and these parameters outlining 16 sensory neuronal groups are presented in Table 1. Electrophysiology protocols. a. CGRP + neurons (red; marked with blue arrow) selected for recording were classified by size (C m in pF) and staining with IB4 (green; marked with yellow arrow). b. Stimulus waveform (1nA, 0.5 msec) indicated below trace generated a single AP in small DRG neuron. We analyzed AP and AHP parameters: resting membrane potential-V m ; duration at base-dB and the time required for the AHP (measured in mV) to decay by 80%-(AHP 80 ). In addition, we measured rise time (RT) and fall time (FT) of AP (Table 1). Characteristic AP "hump" is indicated by black arrow. c. Outward current was generated by the indicated waveforms found below traces. The decay constant τ was derived from standard single exponential fits between points indicated by arrows for the final outward current trace (+20 mV). Characteristic "spike-like" peak is shown by arrow on trace generated by stepping to +0 mV.
I ATP -current size and kinetic parameters are noted. Act is time to reach 95% of peak. Inact is time for 50% decline during drug delivery.
IB4-week expression is marked "+/-";trkCis trkC negative subset of medium-large-sized neurons Groups with clearly detected "spike-like" feature for outward current (I) is marked "+"; groups with no "spike-like" feature are marked "-". The τ obtained after fitting with single or double exponential equation is noted.

Statistics
Control IHC was performed on tissue sections processed as described but either lacking primary antibodies or lacking primary and secondary antibodies. Cell counts from IHC images acquired as Z-stuck were performed using Image J software. Total cells/section and cells positive for each marker as well as the combinations of markers were counted. Intensity of immunoreactivity or TdTomato labeling was also calculated with Image J software; subtractions of background intensity from signal levels were applied. We used 3 independent mice to generate sections and counted 3-5 sections per mouse. Mean values from n = 3-5 were generated per animal, and standard error of the mean was calculated on this basis. Data were presented on scatter plots with bars. GraphPad Prism 7.0 (GraphPad, La Jolla, CA) was used for statistical analyses. Data in the figures are mean ± standard error of the mean (SEM), with "n" referring to the number of recorded and analyzed cells. Differences between electrophysiologically characterized groups were assessed by unpaired t-test or regular 1-way ANOVA with Tukey's post-hoc tests, each column was compared to all other columns. A difference is accepted as statistically significant when p<0.05. Interaction F ratios, and the associated p values are reported.

Approaches to electrophysiological classification of sensory neuronal groups
Depending on the recording technique/approach, there are many accepted ways to classify sensory neurons [39]. One traditional classification is based on myelination status and fiber conduction velocities, which can be measured from recordings from ex vivo and in vivo preparations [40]. Each of these groups (i.e. un-myelinated and myelinated), in turn, forms a heterogeneous population of sensory neurons that have various functions and contain different fiber subtypes [4,22,41]. In addition, sensory neuronal clusters differ in their innervation target [42,43]. This type of classification of sensory neurons requires comprehensive in vivo extracellular or, preferably, intracellular recording and application of physiological stimuli to innervation sites [22,42,44]. Alternative approaches are the use of patch-clamp recording and classification according to AP properties, sensitivity to algesic agents and appearances of a variety of voltage-gated currents [21,45]. These approaches could become especially powerful after detailed characterization of sensory neuronal groups expressing defined markers [5,6,9,46]. The use of information from next generation sequencing for transcriptional profiling of different types of sensory neurons [1,12] would provide additional important details regarding patch-clamp recorded subsets.
Patch clamp recording from selected DRG neurons yielded data on 13 variables: cell size, dB, FT:RT ratio, AP kinetics, characteristic features of AP shapes, AHP 80 , IB4 staining, responsiveness to 5-HT, ATP, MO and CAP, τ (tau) from fitting of voltage-gated current, and presence or absence of a "spike-like" feature on outward portions of voltage-gated currents ( Table 1). IB4 signals were reported as non-detectable, weak or strong with clear plasma membrane staining [47]. Strongly stained IB4 + neurons have been classically considered non-peptidergic [48], while some of weakly stained IB4 + neurons (marked as +/-in Tables 1-5) are peptidergic [47]. ATP responses were classified according to their magnitude and kinetics. CAP, 5-HT and MO responses was qualified as positive (>50 pA) or negative. "Spike-like" feature was revealed by the presence of a sharp peak on outward portion of current generated by stepping to 0 and +20mV ( Fig 1C). It was noted that some myelinated A-fiber produce a "spike-like" feature ( Fig 1C) [21,45]. It worth to note that "spike-like" feature is likely not representative of only A-current as it was previously interpreted [21], but rather a depolarizing voltage-clamp error resulting from the proceeding of the large inward sodium current. Nevertheless, "spike-like" feature can be used as a clustering variable to distinguish some of A-fiber containing neurons [21]. In some neuronal groups, shape of outward current was biphasic (see Table 1). Before analyzing data from CGRP-cre + , Nav1.8-cre + , TRPV1-cre + and V1-GFP + neurons and assigning them to particular sensory neuronal groups, parameters for definitive and unambiguous clustering were created on basis of recording of DRG neurons from reporter mice, such as mrgD-GFP + , trkC-cre + , trkB-cre + , 5HT3a-GFP + , NPY2R + , PV-cre + and vGLUT3-cre + (Table 1). Mandatory variables (in bold) are essential for getting clear "separation" between some of sensory neuronal groups (Table 1). Clustering parameters generated on the basis of recording of sensory neurons from reporter mice allows clearer separation between groups to an extent that assigning a recorded neuron to particular group could be performed without software. Nevertheless, cluster analysis based on a distribution model (XLStat and NCSS) confirmed findings [21]. Analysis of recordings from >600 DRG neurons expressing or not expressing various markers (including CGRP-cre + , Nav1.8-cre + , TRPV1-cre + and V1-GFP + ) revealed 7 groups of small neurons (<35pF) and 7 groups of medium-large neurons (>35pF) ( Table 1; Figs 2, 3, 4 and 5). Two groups M1 and M3 contain sub-groups, M1a and M3a respectively. These sub-groups were only distinguished by responsiveness to ATP. Each neuron can be assigned to one of these groups based on the criteria that every parameter, with the possibility of one exception, should fit the characteristics summarized in Table 1. Moreover, variables in bold font were a mandatory fit for specified neuronal groups (Table 1). If neuron could not be assigned to either group, it was discarded from further analysis. Only 14 neurons from >600 recorded did not fit any group outlined in Table 1.
AP and AHP characteristic and shape, as well as I features of S2 and S3 CGRP-cre + DRG neurons were similar to those of S1 group neurons (Figs 2 and 4F; Tables 1 and 2). S2 and S3 neurons are responsive to CAP, but not MO (Fig 2). S2, unlike S3 CGRP + neurons had no IB4 I CAP magnitude scale is indicated. I 5-HT was recorded from S3, but not S2 neurons. I 5-HT time and magnitude scales are 1 sec and 50 pA, respectively, for each panel. An exception is the M1/M1a group that has a large I 5-HT current. MO responses were measured by Ca 2+ imaging. CGRP-cre + sensory neuronal groups are shown for each row. Drug application times are illustrated by horizontal bar above traces. More complete information on subgroups is presented in Table 2.  Table 2), while S3 was not gated by ATP. S2 did not have a distinct marker among those analyzed (Table 5). However, S3 neurons were weakly 5HT3a-GFP + (Table 5). Hence, tiny I 5HT was recorded from S3, but not S2 neurons (Fig 2).
Small-sized (<35pF) Nav1.8-cre + /CGRP-cre -DRG neurons. S4 and S5 neurons had strong staining with IB4, and did not respond to CAP (Table 3). They also displayed a unique Comparison of AP shapes generated in S1 CGRP-cre + /Nav1.8-cre + , S4 CGRP-cre -/Nav1.8-cre + and S6 CGRP-cre -/Nav1.8-cre + neurons. AP "hump" in S1 neurons and "bow" in S4 neurons are indicated with black and red arrows, respectively. S6 neurons' AP does not display any deflection during the falling phase of AP. b. Comparison of AP in S6 and S7 neurons. Deflection on the falling phase of S7 neuron AP is indicated by black arrow. c. Comparison of single AP in M1 and M3 CGRP-cre + neuronal group. "Hump" is marked with blue arrow, while "deflection" is indicated with black arrow. d. Comparison of AP in M3 and M4 Nav1.8-cre + / CGRP-cre -DRG neurons. M3 neuron's AP "deflection" is indicated with black arrow, and M4 neuron's AP with no "deflection" is shown with red arrow. e. Typical outward current (I) produced from Nav1.8-cre + /CGRP-cre -S6 and S7 neuronal groups. f. Typical I produced from the CGRP-cre + S1-S3 group neurons. g. Typical I produced from CGRP-cre + M1 and M3 group neurons. Names of neuronal groups are specified above traces. The time scale (horizontal bar) is 25 ms for each panel. https://doi.org/10.1371/journal.pone.0198601.g004 Characteristics of peptidergic sensory neuronal groups slow AP (i.e. high dB values) shape with "bow" during AP fall phase (Figs 3 and 4A). AHP was substantially faster in the S5 than S4 group (for S5 20.4±0.7 for S4 89.3±7.8; t-test; t = 6.5 df = 14; p<0.0001; n = 8-12; Table 3; Fig 3). Other distinctions between S4 and S5 were responses to ATP, MO and 5-HT. S4, but not S5 neurons, responded to all of these algesic agents (Table 5; Fig 3). Outward current shape and τ were similar between S1-S3 and S4-S5 groups ( Table 3). Analysis of recording from neurons expressing various reporter-markers revealed that S4 and S5 groups match clusters expressing MrgprD-GFP (Table 5). CGRP-cre-ER/TdTomato + groups are highlighted by yellow in the "Group" column and TRPV1-cre/TdTomato groups are highlighted by blue in the "N" column. 5-HT-large and fast 5-HT current is only noted in M1/M1a. 5-HT responses are small in S2, S3, M3 and M3a groups, and are marked as "+/-". MO responses are largest in S4 group, and are almost undistinguishable from current noise in S7 group. IB-4 staining in group S1 and S3 is weak; and are marked "+/-". Statistic is 1-way ANOVA, control column is S1, post-hoc analysis Bonferroni Ã p<0.05 ÃÃ p<0.01 ÃÃÃ p<0.001 ÃÃÃÃ p<0.0001. If difference is insignificant (P>0.05), then no sign is shown. CGRP-cre-ER/TdTomato + groups are highlighted by yellow in "Group" column. Sign "?" marks unknown or a candidate marker. Thus, S6 group marker could be somatostatin (SST). IB-4 staining in group S1 and S3 is weak, and marked "+/-". Statistic is 1-way ANOVA, control column is S1, post-hoc analysis Neurons of S6 and S7 groups were smallest sizes (i.e. <20pF), and not stained by IB4, nor do they respond to ATP or 5-HT (Table 3; Fig 3). Distinctively, S6 and S7 neurons have faster dB than other small neurons of S1-S5 groups (1-way ANOVA; F (6, 56) = 24.4; P<0.0001; Table 3; Fig 3). S6 and S7 were also unlike S1-S5 in regards to outward currents (I). Thus, I of S6 and S7 have higher than S1-S5 τ values (Table 3; Fig 4E vs Fig 4F). Unlike S6, neurons of the S7 group exhibited a slight deflection on the falling portion of AP (Fig 4B). AHP was relatively faster (compared to S1-S3) in S7 and especially S6 neurons (1-way ANOVA; F (2, 54) = 18.2; P<0.0001; Table 3; Figs 2 and 3). However, AP and AHP characteristics are not reliable in assigning neurons to S6 or S7 group. The definitive feature of S6 neurons that separates them from S7 was their CAP and MO sensitivity (Table 3; Fig 3). Moreover, I CAP in S6 neurons were largest compared to other TRPV1-GFP positive neuronal groups (Table 4; Figs 2 and 3). Properties of S7 neurons were matched to characteristics of vGLUT3-cre + DRG neurons from vGLUT3 cre /TdTomato mice (Table 5). It could be noted that vGLUT3-cre + neurons are completely CAP insensitive. It is also worth noting that %25% of vGLUT3-cre + neurons have C m of >50 pF, and their electrophysiological profiles distinct them from S7 neurons. These larger vGLUT3-cre + could be vGLUT3in the adult due to a fate map [49]. Data from larger vGLUT3-cre + neurons were not included in Table 5.
Medium-large-sized (>35pF) Nav1.8-cre + /CGRP-cre -DRG neurons. All but one medium-large Nav1.8-cre + neuronal group co-expressed CGRP-cre + ( Table 3). An exception was group M4. M4 are large IB4neurons (C m is >60pF), have a pronounced "spike-like" peak on their outward current, similar to M1 and M3 (Fig 4G), and do not respond to ATP, CAP, MO or 5-HT (Fig 3). The distinct feature of M4 neurons is a very fast dB of AP, but relatively slow AHP (Fig 3). M4 also differed from M1/M1a and M3/M3a in the falling phase of AP (Fig 4D). M4 has a very fast falling phase with no deflection; and FT:RT ratio is always <1 for M4, and >1 for M1/ M1a and M3/M3a ( Table 1). Analysis of properties of marker expressing neurons has unexpectedly shown that M4 is one of two prominent groups expressing trkC cre-ER /TdTomato (Table 5).

TRPV1 cre /TdTomato and TRPV1-GFP expressing sensory neuronal groups
Recording and analysis data from 88 DRG neurons of TRPV1 cre /TdTomato reporter mice showed that many TRPV1-cre + neuronal groups (highlighted with blue in Table 3) were not responsive to CAP. This phenomenon could be due to fate map and has been previously reported [19,49]. Moreover, TRPV1-cre + is present in all peptidergic CGRP + DRG neuronal groups (i.e. small and medium-large neurons), as well as non-peptidergic nociceptors (Table 3). Overall, TRPV1 cre /TdTomato slightly differs from the Nav1.8 cre /TdTomato reporter expression pattern in DRG neurons, since as TRPV1-cre + cells were not detected in S7 or M4 neuronal groups (Table 3).
Fifty nine TRPV1-GFP (V1-GFP + ) DRG neurons stained with IB4 were recorded with sequential protocols as described in "Methods". All but 2 V1-GFP + neurons could be assigned to one of 4 main clusters, each of which belonged to small-sized neuronal groups (Table 4). Every V1-GFP + neuron is responsive to CAP, and the strongest I CAP was found in S6 CGRPcreneurons. Generally, all V1-GFP + neuronal groups apart from S6 are peptidergic neurons and represented in CGRP-cre + DRG neurons from CGRP cre-ER /TdTomato mice. Moreover, all V1-GFP + neuronal groups are denoted in Nav1.8-cre + and TRPV1-cre + DRG neurons from Nav1.8 cre /TdTomato and TRPV1 cre /TdTomato mice, respectively.
Fifteen of 45 Nav1.8-creneurons fit into neuronal group M2 (Table 1). M2 is IB4and nonresponsive to ATP, CAP, MO and 5-HT. M2 has fast AP with FT:RT<1. %70% of M2 neurons exhibited an uncommon AHP shape that did not show the classical "overshoot" below V m (Fig 5). The remaining M2 neurons had a very small overshoot and a fast AHP (Fig 5). All M2 neurons also displayed a biphasic I with a combination of small "spike-like" peak and a distinctive "smooth I curve", which was typical for S1-S5 neurons (Figs 4F and 5). Analyses of electrophysiological profiles of trkB-cre + neurons from trkB cre-ER /TdTomato mice implied that M2 matches a profile of trkB-cre + neurons ( Table 5).
Ten of 45 Nav1.8-creneurons fit into neuronal group M5 (Table 1). Like M2, M5 is IB4and non-responsive to CAP or MO. However, some of M5 neurons show small (<0.3 nA) I 5HT . M5 had a fast AP with FT:RT<1 and the AHP quickly returned to V m level (Fig 5). The main distinctions of M5 from M2 are that M5 has medium-sized fast I ATP ; and "spike-like" peak from recording of I (Fig 5). TrkC-cre + neurons isolated from DRG of trkC cre-ER /TdTomato mice could be divided into two main and distinct groups. One of these trkC-cre + neuronal groups has matched M5, while another trkC-cre + group is M4 ( Table 5).
Four of 45 Nav1.8-creneurons were assigned to the M6 group, AP and AHP of which bears strong similarity to some of M2 neurons. M6 group neurons have fast AHP with shallow "overshoot" of AP below V m (Fig 5). However, the distinction between M2 and M6 is that the M6 outward current does not have a "spike-like" peak. Moreover M6 neurons exhibited I ATP , similar to those produced in M5 neurons (Fig 5). We could not identify the apparent marker for M6 neurons.
Sixteen of 45 Nav1.8-creneurons fit into neuronal group M7 (Table 1). M7 is IB4and non-responsive to ATP, CAP, MO or 5-HT. M7 have the fastest AHP and AP with FT:RT<1 compared to the other sensory neuron groups (Fig 5). Moreover, the "overshoot" below V m was largest for medium-large sensory neurons (Fig 5). M7 I has the characteristic "spike-like" peak, but it does not fit well with the exponential equation and yields high τ values (Fig 5). Analysis of parvalbumin (PV-cre + ) neurons from PV cre /TdTomato + mice showed that there are two PV-cre + groups. One of them is similar to M7 neuronal group (Table 5). Another PVcre + group has a profile matching M5 groups (see above). This suggests that M5 neurons are PV + /trkC + .
Despite dominat expression of CGRP and TRPV1 in sensory neurons, and many advantages of CGRP cre-ER/+ and TRPV1 cre/+ reporter mice in the manipulation of sensory neurons, TRPV1 and to lesser extent CGRP expression could be detected outside sensory neurons. Thus, TRPV1 is expressed at low-to-medium levels in the immune system [59], epithelial cells [60] and other neurons of the CNS [61]. Outside sensory neurons in naïve animals, CGRP medium-to-high levels of expression are localized to specific subsets of neuronal and non-neuronal cells [53,62]. Thus, pulmonary neuroendocrine epithelial cells express CGRP on very high levels [17], and there is CGRP on moderate levels in some brain regions [53]. Additionally, a variety of pathological conditions, including post-herpetic neuralgia and complex region pain syndrome type 1, could drastically up-regulate CGRP in non-sensory neurons [63].
Identification and determination of sensory neuron functional groups were carried out by several approaches. In vivo intracellular recording from DRG and TG neurons gives the utmost precision in determining sensory neuronal types [41,42], especially when this method is combined with post-recording IHC [47]. Extracellular recordings of "single fibers" from exvivo tissue-nerve preparations also produce reliable results [5]. However, these approaches have a problem in visualization of recorded neurons (or fibers) and therefore ex vivo electrophysiology approaches combined with ontogenetic are gaining popularity [5]. Accumulated information by in vivo intracellular recording makes clear the link between AP/AHP and the corresponding function of sensory neuronal groups disregarding whether neurons innervate paw, vibrissa pad or muscle [22,41,42]. Since AP/AHP parameters generated by patch clamp and intracellular recording are similar [64] (especially at the same recording temperature), whole-cell patch clamp data on isolated DRG (or TG) neurons could be correlated to the function of sensory neuronal groups. Moreover, recordings of responses to a variety of algesic agents, single-cell sequencing information [1,7,12] and detailed characterization of numerous sensory neuronal markers [5,6,7,9,46,47,56,58,65] allow for quick and precise identification of sensory neuronal groups (Table 1). Therefore, this reliable and relatively quick approach can be used in the identification of neuronal groups of molecules/targets that have an appropriate reporter mouse line.
In conclusion, our data show that CGRP cre-ER/+ is an effective mouse reporter line for manipulation of C-and A-peptidergic nociceptors. Our data also indicates that targeting nociceptors, but not LTMR neurons can be optimally achieved in TRPV1 cre/+ reporter mouse lines, while some LTMR neurons could potentially be affected in Nav1.8 cre/+ mice. Finally, presented here sensory neuron group identification approach could successfully be used for numerous other reporter mouse lines.