Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ

The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology.

The single Fpr-rs gene cluster is adjacent to a stretch of more than 30 V1/2r genes. However, neither V1rs, nor V2rs share significant sequence homology with vomeronasal Fprrs genes. Liberles and coworkers suggested that vomeronasal Fprs evolved from recent gene duplications and positive selection in the rodent lineage (Liberles et al., 2009). Together with recent functional data obtained from recombinant FPR expression (Bufe et al., 2012), these considerations argue for a neofunctionalization of vomeronasal Fpr-rs genes. Their predicted seven-transmembrane topology, their selective, punctate and monogenic vomeronasal expression pattern, and their localization in microvillous dendritic VSN endings (Liberles et al., 2009;Rivière et al., 2009), however, strongly suggest a functional role of FPR-rs in vomeronasal chemosignaling. Interestingly, while Fpr-rs1 is coexpressed with G αo in basal sensory neurons, the remaining vomeronasal Fpr-rs genes all coexpress G αi2 in the apical layer of the VNO neuroepithelium (Liberles et al., 2009;Munger, 2009;Rivière et al., 2009). Vomeronasal sensory neurons are activated in situ by formylated peptides and various other antimicrobial/inflammatory modulators (Rivière et al., 2009;Chamero et al., 2011) and heterologously expressed FPRrs proteins retain agonist spectra that share some similarities to immune system FPRs (Rivière et al., 2009). However, the exact biological role of vomeronasal FPRs remains to be determined.
To address the neurobiological function of vomeronasal FPRs experimentally, a detailed physiological characterization of Fpr-rs neurons in their native environment is mandatory. Genetically modified animals in which the receptor identity of a given chemosensory neuron is marked by coexpression of a fluorescent reporter have proven particularly fruitful in the analysis of olfactory signaling (Boschat et al., 2002;Bozza et al., 2002;Grosmaitre et al., 2006Grosmaitre et al., , 2009Oka et al., 2006;Ukhanov et al., 2007;Leinders-Zufall et al., 2009;Pacifico et al., 2012). Here, we describe a transgenic mouse strain that expresses FPR-rs3 together with a fluorescent marker (Fpr-rs3-i-Venus). This mouse model allows optical identification and subsequent physiological analysis of FPR-rs3-expressing neurons in acute VNO tissue slices. Using single neuron patch-clamp recordings, we thus provide an in-depth electrophysiological characterization of the basic biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. Our analysis spans several types of voltage-activated conductances as well as action potential discharge parameters in both fluorescently labeled and control VSNs. Our data reveal a number of physiological similarities between FPR-rs3-expressing and non-expressing neurons. Together, these results confirm the suitability of Fpr-rs3-i-Venus mice for future studies of vomeronasal FPR neurobiology and, in addition, these findings indicate that the FPR expression does not confer a distinct biophysical phenotype to the subpopulation of FPR-positive VSNs.

ANIMALS
All animal procedures were in compliance with local and European Union legislation on the protection of animals used for experimental purposes (Directive 86/609/EEC) and with recommendations put forward by the Federation of European Laboratory Animal Science Associations (FELASA). Both C57BL/6 mice (Charles River Laboratories, Sulzfeld, Germany) and Fpr-rs3-i-Venus mice were housed in groups of both sexes at room temperature on a 12 h light/dark cycle with food and water available ad libitum. Experiments used young adults of either sex. We did not observe obvious gender-dependent differences.

TRANSGENIC MICE
The transgene (Fpr-rs3-i-Venus) contains the FPR-rs3 coding sequence followed by an internal ribosome entry site (IRES), and the coding sequence for tau-Venus, a fusion between the microtubule-associated protein tau and Venus yellow fluorescent protein (Nagai et al., 2002). These coding sequences are under the control of the H element followed by the MOR28 promoter (Serizawa et al., 2006; modified by and generously provided by P. Feinstein). The Fpr-rs3-i-Venus transgene was isolated on gel after BssHII digestion and purified using the QIAquick® Gel extraction kit (QIAGEN, Hilden, Germany). The transgene was injected into the pronuclei of fertilized C57BL6/DBA2 mouse oocytes following standard procedures. Four founders carrying the transgene were obtained. One of these founder animals expressed the transgene in VSNs and was, thus, used to start the colony. Backcrossed to C57BL/6J, mice were kept hemizygous. Wild type and transgenic mice had no obvious differences in size, weight, fertility, life expectancy or food consumption.

VIBRATOME SECTIONS
Acute vomeronasal tissue sections were prepared as previously described Spehr et al., 2009). Briefly, mice were sacrificed by brief exposure to CO 2 followed by decapitation using sharp surgical scissors. The lower jaw and the soft palate were removed allowing access to the vomeronasal capsule. After removal of the cartilage, the dissected VNO was embedded in 4% low-gelling temperature agarose and coronal slices (150-200 µm) were cut in ice-cold oxygenated extracellular solution (S 2 ) using a Leica VT1000S vibratome (speed: 3.5 a.u. = 0.15 mm/s; frequency: 7.5 a.u. = 75 Hz; amplitude: 0.6 mm; Leica Biosystems). Sections were transferred to a submerged, oxygenated (S 2 ) and chilled storage chamber until use.

DATA ANALYSIS
All data were obtained from independent experiments performed on at least 3 days using at least three different animals. Individual numbers of cells/experiments (n) are denoted in figure legends. If not stated otherwise, results are presented as means ± SEM. Statistical analyses were performed using paired or unpaired t-tests or one-way ANOVA with Tukey's HSD post hoc test. Tests and corresponding p-values that report statistical significance are individually specified in figure legends. Drug sensitivity of voltage-gated K + (Kv) currents was examined based on an "additive" drug exposure regime, i.e., TEA (1 mM), 4-AP (10 mM), and TEA (25 mM) were sequentially applied and the inhibitor-sensitive currents were isolated by subsequent "offline" subtraction from each preceding recording.
Electrophysiological data were analyzed offline using Patch-Master 2.67 (HEKA Elektronik), IGOR Pro 6.3 (WaveMetrics, Lake Oswego, OR) and Excel (Microsoft, Seattle, WA) software. Activation curves were fitted by the Hill equation to calculate the membrane potential of half-maximal activation (V 1/2 ). Current activation time constants (τ) were calculated by fitting individual traces to monoexponential functions I (t) = I 1 [exp (−t/τ)] + I 0 .

TRANSGENIC EXPRESSION OF Fpr-rs3-i-VENUS IN A SUBSET OF NEURONS IN THE MOUSE VNO
To analyze the biophysical properties inherent to a prototypical member of the FPR-expressing neurons, we engineered transgenic mice that express Fpr-rs3-i-Venus in a subset of olfactory sensory neurons (OSNs). Using standard transgenic techniques (see section materials and methods), we generated such a mouse strain in which FPR-rs3 is coexpressed with tau-Venus, an enhanced variant of yellow fluorescent protein (Nagai et al., 2002) fused to the microtubule-associated protein tau ( Figure 1A). Four founders were obtained. Two of them expressed the transgene in OSNs, and one of them in VSNs. We focused our attention on this latter line, given its exclusive vomeronasal expression pattern. Neither hemi-, nor homozygous Fpr-rs3-i-Venus mice from this line showed any obvious aberrant phenotype.

PASSIVE MEMBRANE PROPERTIES OF FPR-rs3 + VSNs
The passive membrane properties of a neuron determine its basic electrophysiological characteristics and, thus, control its individual stimulus-response function. For FPR-rs expressing vomeronasal neurons, these critical physiological parameters are unknown. Using Fpr-rs3-i-Venus mice, we performed whole-cell patch-clamp recordings from optically identified, fluorescently labeled FPR-rs3-expressing neurons in acute VNO tissue slices (Figures 2A,B). For quantitative comparison, we additionally performed a series of control experiments in randomly chosen VSNs from C57BL/6 wild type mice. Passive membrane properties (i.e., input resistance (R input ), membrane capacitance (C mem ), and membrane time constant (τ mem )) were obtained immediately after membrane rupture. Treated, to a first approximation, as a "biological constant" with a value of ∼1 µF/cm 2 (Gentet et al., 2000), C mem was determined using a square pulse (5 mV, 10 ms) routine. Transgene-positive (FPR-rs3 + ) neurons revealed an average C mem value of 5.96 ± 0.49 pF (n = 21), similar to data obtained from control VSNs (5.24 ± 0.38 pF; n = 21; Figure 2C). We next determined R input at the VSN soma by measuring the steadystate voltage response to a current step of defined amplitude. The average somatic R input of FPR-rs3 + neurons was 3.15 ± 0.49 G (n = 21; Figure 2D). This large value resembles R input measurements from control VSNs (3.29 ± 0.43 G ; n = 21), suggesting that FPR-rs3 + neurons share the extraordinary sensitivity of V1/2R-expressing VSNs (Liman and Corey, 1996;Shimazaki et al., 2006;Hagendorf et al., 2009). Linear passive voltage responses were also used to estimate τ mem from monoexponential fits to the voltage responses (from onset to steady state). We obtained relatively slow τ mem values of 26.79 ± 2.25 ms (n = 21) in FPR-rs3 + neurons vs. 24.29 ± 1.57 ms (n = 21) in control neurons ( Figure 2E).
Together, these results describe different passive membrane parameters of FPR-rs3 + neurons. Moreover, these data show that the passive electrical properties of FPR-rs3 expressing VSNs do not significantly differ from control neurons, suggesting (a) that FPR-rs expressing VSNs are not segregated or isolated from the "general" VSN population; and (b) that transgene expression per se does not perturb the passive biophysical properties of FPR-rs3 + neurons.

ACTIVE MEMBRANE PROPERTIES OF FPR-rs3 + NEURONS
Next, we examined the active membrane properties of FPR-rs3 + neurons. A hallmark of VSNs is that depolarizing current injection of only a few picoamperes triggers repetitive action potential discharge (Liman and Corey, 1996;Shimazaki et al., 2006). This also holds true for FPR-rs3 + neurons ( Figure 3A). Current-clamp recordings from fluorescently labeled VSNs show repetitive spiking in response to depolarizing current steps of 2-24 pA. Spontaneous activity (measured at 0 pA current injection) was 2.37 ± 0.54 Hz (n = 19) for FPRrs3 + neurons and 3.9 ± 1.08 Hz (n = 21) for control cells (Figure 3B, inset). By plotting mean instantaneous spike frequencies as a function of stationary current input (f -I curve; Figure 3B), response saturation at amplitudes >20 pA becomes apparent (maximum frequency f max = 14.5 ± 0.88 Hz (n = 19; FPR-rs3 + neurons) or 16.54 ± 1.17 Hz (n = 21; control VSNs)). Injection of negative current into FPR-rs3 + neurons revealed a hyperpolarizationactivated rebound depolarization ("sag"; Figure 3A iii ), indicative of I h currents and, thus, HCN channel expression (Robinson and Siegelbaum, 2003;Dibattista et al., 2008). Plotting the sag potential amplitude (∆V sag ; Figure 3A iii ) as a function of peak hyperpolarization reveals the threshold (< −75 mV) and voltage dependence of the sag (n = 5-23; Figure 3C), likely corresponding to an increase in HCN channel activation at more negative membrane potentials. A similar voltage dependence was observed for control cells (n = 5-32). In both FPR-rs3 + and control VSNs, we frequently observed rebound spikes upon repolarization (Figure 3A iii ).
Next, we examined action potential discharge of FPR-rs3 + neurons. Figure 3D i depicts an averaged spike waveform and shows schematically how different spike parameters were analyzed: spike amplitude was measured as the threshold-to-peak distance, spike duration was calculated as the full duration at half-maximum (FDHM), spike generating kinetics was measured as the time-to-peak (TTP). All analyses were based on the first spike of a given train of action potentials (see Figure 3A i ). Our results reveal an average amplitude of 72.24 ± 0.97 mV (n = 134) for FPR-rs3 + neurons and 73.92 ± 0.87 mV (n = 172) for control neurons (Figure 3D i ). Average TTP values were 2.29 ± 0.06 ms (FPR-rs3 + cells) and of 2.33 ± 0.09 ms (control neurons), while FDHM was 3.65 ± 0.08 ms (FPR-rs3 + neurons) and 3.67 ± 0.12 ms (control VSNs), respectively. These data show that FPR-rs3 expressing VSNs exhibit rather slow action potentials and, albeit an extraordinary sensitivity, show a relatively narrow spike frequency coding range. Together, these active membrane properties are shared by both FPR-rs3 expressing and control neurons.

VOLTAGE-GATED Na + CURRENTS OF FPR-rs3 + NEURONS
In excitable cells, voltage-gated Na + (Na V ) channels are primarily responsible for action potential initiation and impulse propagation. Upon membrane depolarization, Na V channels mediate the rapid Na + influx that underlies the upstroke of the action potential. However, the electrophysiological properties of the nine homologous members of the Na V channel family (Na V 1.1 to Na V 1.9) are not identical and even small differences in Na V channel expression can have profound effects on electrical excitability (Hille, 2001). Therefore, we next focused on macroscopic voltage-activated Na + currents (I Nav ) in FPR-rs3 + neurons. Stepwise depolarizations from −120 mV to +70 mV (30 ms duration; 5 mV increment) in absence and presence of tetrodotoxin (TTX; Figure 4A i-ii ;Narahashi et al., 1966;Wu and Narahashi, 1988) allowed pharmacological isolation of the TTX-sensitive I Nav (Figure 4A iii ). Plotting peak I Nav density as a function of membrane depolarization, the current-voltage relationship ( Figure 4B) reveals an activation threshold at approximately −65 mV and a maximum current density of −136.7 ± 14.1 pA/pF (n = 10). Similar values were recorded from control VSNs (maximum I Nav = −157.5 ± 17.4 pA/pF; n = 20). Figure 4C i illustrates the kinetics of channel gating during a single depolarizing step in membrane potential (−30 mV). As expected from relatively slow action potential firing in FPR-rs3 + neurons (Figure 3D), TTP analysis of I Nav reveals relatively slow activation kinetics (1.86 ± 0.10 ms; n = 10; Figure 4C ii ).
Next, we examined the voltage-dependence of TTXsensitive I Nav activation and inactivation in FPR-rs3 + neurons (Figures 4D,E). Fitting normalized peak I Nav amplitudes vs. voltage to a sigmoidal (Boltzmann) function demonstrates halfmaximal current activation upon depolarization to approximately −50 mV (V 1/2 = 48.6 mV; n = 9; Figure 4E i ). Steady-state I Nav inactivation was analyzed upon depolarization to +20 mV, preceded by prepulse steps to different potentials ranging from −120 mV to 0 mV (30 ms duration; 5 mV increment; Figure 4D). Again, offline subtraction of TTX-insensitive currents (Figure 4D ii ) from control recordings (Figure 4D i ) allowed pharmacological isolation of TTX-sensitive I Nav (Figure 4D iii ). Steady-state inactivation curves are derived from inverse sigmoidal fits to normalized peak I Nav amplitudes vs. prepulse voltage (Figure 4E ii ) and reveal half-maximal inactivation upon depolarization to V 1/2 = −25 mV (n = 10). Interestingly, at voltages ranging from approximately −60 mV to −5 mV, activation and inactivation curves overlap, suggesting coexpression of multiple Na V channel isoforms and/or a substantial "window current".
Together, these results demonstrate that FPR-rs3 + VSNs express one or more Na V channel isoform(s) that exhibit relatively slow activation upon membrane depolarization >−65 mV with half-maximal and complete activation at ∼−50 mV and −30 mV, respectively. Moreover, the slope of the steady-state inactivation curve is relatively shallow, revealing that full channel inactivation only occurs at positive potentials. Since all measured parameters are similar to data recorded from control VSNs, our data further substantiate the notion that that FPR-rs expressing neurons do not constitute a biophysically segregated "outgroup" of VSNs.

VOLTAGE-GATED K + CURRENTS OF FPR-rs3 + NEURONS
To a large extent, Kv channels control electrical signaling in excitable cells. Accordingly, the large and extended Kv channel family is functionally diversified by alternative splicing, oligomeric subunit assembly, and subcellular targeting (Jan and Jan, 2012). As Kv channels are involved in regulating a wide range of neuronal functions, such as setting the resting membrane potential, dictating the duration and/or frequency of action potentials, volume regulation, etc., we next characterized Kv channel-mediated currents (I Kv ) in FPR-rs3 + neurons. Activated by depolarization, outward flux of K + repolarizes the membrane and, thus, contributes to action potential termination and, in some neurons, afterhyperpolarization. To isolate different classes of I Kv we used a pharmacological toolkit of several welldescribed Kv channel inhibitors (Alexander et al., 2013). Depending on concentration, tetraethylammonium (TEA) functions as a relatively selective inhibitor of big conductance Ca 2+ -dependent K + (BK) channels at low millimolar concentrations (Yellen, 1984), whereas substantially higher concentrations (25 mM) serve as a nonselective "broadband" Kv channel blocker (Alexander et al., 2013). In addition, 4-aminopyridine (4-AP) specifically blocks A-type K + currents in various neurons (Mei et al., 1995;Amberg et al., 2003).
Under control conditions, stepwise depolarization from −100 to +85 mV (100 ms duration; 5 mV increment) triggered large outward currents that essentially showed no sign of inactivation (Figure 5A, inset). When steady-state currents were plotted as a function of depolarization, the resulting current-voltage relationship reveals I Kv activation at approximately −30 mV ( Figure 5A). Linear regression from data points corresponding to full activation (+60 mV -+85 mV) indicates I Kv reversal at ∼−65 mV. When drug-sensitive currents were isolated by digital subtraction of blocker-insensitive from respective "control" recordings ( Figures 5B-D, insets; see section materials and methods), the resulting current-voltage plots revealed no statistical differences between FPR-rs3 + neurons and control VSNs (Figures 5B-D). Somewhat surprisingly, currents isolated by 4-AP treatment did not show a pronounced transient component typical for A-type K + currents. Interestingly, summation of the individual drugsensitive I Kv components added up to almost 100% of control currents (276.5 ± 31.1 pA/pF at +85 mV; n = 13; Figure 5E) showing that a "cocktail" of 4-AP (10 mM) and TEA (25 mM) is sufficient to block essentially all Kv channels in FPR-rs3 + neurons. This pharmacological profile was statistically indistinguishable from control VSNs.
inhibitors affected the upstroke dynamics (Figure 5F iii ). However, while block of putative BK channels by TEA (1 mM) accelerated the upstroke, inhibition of A-type currents prolonged the average TTP. 4-AP treatment also prolonged the spike width (FDHM) and, consequently, spike duration ( Figure 5F iv-v ) whereas TEA did not elicit such effects. The effects of 4-AP are significantly more pronounced in FPR-rs3 expressing VSNs than in control neurons ( Figure 5F iii-v ).
In summary, these data demonstrate that multiple Kv channel subunits are expressed in FPR-rs3 + neurons. These different channel populations synergistically shape the firing properties of FPR-rs3 expressing VSNs. Moreover, with the notable exception of 4-AP-sensitive channel function during discharge, the Kv channel expression profile of FPR-rs3 + neurons is largely comparable to control VSNs.

VOLTAGE-GATED Ca 2+ CURRENTS OF FPR-rs3 + NEURONS
Voltage-gated Ca 2+ (Ca V ) channels are integral constituents of a neuron's Ca 2+ signaling toolkit (Berridge et al., 2003). As such, they are key signal transducers that transform electrical Frontiers in Neuroanatomy www.frontiersin.org November 2014 | Volume 8 | Article 134 | 8 impulses (depolarization) into a biochemically relevant signal (Ca 2+ influx) that regulates a wide variety of cellular events (Catterall, 2000b;Clapham, 2007). We therefore investigated Ca V currents (I Cav ) in FPR-rs3 + neurons. The ten functional vertebrate Ca V channel subunits are divided into three subfamilies (Ca V 1 to Ca V 3) that differ in function and regulation (Triggle et al., 2006). Both within and between subfamilies, individual Ca V channel isoforms are identified by their distinct biophysical properties and pharmacological profiles (Catterall, 2000b;Alexander et al., 2013). Thus, we isolated transient (T-type) currents mediated by members of the Ca V 3 subfamily by digital subtraction of I Cav recorded in response to depolarizing voltage steps (−100 mV to +45 mV; 100 ms duration; 5 mV increment) from two different prepulse potentials (−100 mV and −25 mV, respectively; Figure 6A i , inset). Based on steady-state inactivation of Ca V 3 channels at −25 mV (Catterall et al., 2005), the fraction of low voltage activated (LVA) Ca 2+ channels becomes readily apparent after subtraction (Figure 6A i ). As expected, these T-type currents rapidly inactivate and the underlying activation and inactivation kinetics become faster with increasing depolarization (Perez-Reyes et al., 1998). The resulting current-voltage relationship ( Figure 6A ii ) and normalized I Cav activation curve (sigmoidal fit; Figure 6A iii ) demonstrate an activation threshold of −60 mV and half-maximal current activation upon depolarization to −40 mV (V 1/2 = −40.27 mV; n = 9), values typical for T-type currents.
Next, we investigated functional expression of high voltage activated (HVA) Ca V channels in FPR-rs3 + neurons. All four members of the Ca V 1 subfamily are characterized by both longlasting and large (L-type) Ca 2+ currents and high sensitivity to dihydropyridines, such as nifedipine (Catterall et al., 2005). Therefore, to examine L-type I Cav , we recorded responses to depolarizing voltage steps (−100 mV to +85 mV; 100 ms duration; 5 mV increment) and isolated nifedipine-sensitive currents by digital subtraction (Figure 6B i ). As expected for L-type currents, isolated I Cav shows relatively slow, though lasting activation upon depolarization ≥−45 mV (Figure 6B ii ). Half-maximal activation is observed upon more pronounced depolarization (V 1/2 = −26.06 mV; n = 7; Figure 6B iii ).
A slight, though also significant difference between FPR-rs3 + and control neurons was observed for P/Q-type Ca 2+ currents that were pharmacologically isolated using ω-agatoxin IVA (Randall and Tsien, 1995;Catterall, 2011). P/Q-type currents revealed relatively slow activation and slight inactivation. Compared to control recordings, both the current-voltage relationship (Figure 6D ii ) and the sigmoidal activation curve (Figure 6D iii ) of P/Q-type I Cav in FPR-rs3 + neurons was left-shifted to more negative potentials. Maximum current density, however, did not significantly differ between FPR-rs3 + VSNs (−29.50 ± 3.31 pA/pF; n = 8) and control neurons (−31.46 ± 4.34 pA/pF; n = 5).
In summary, the above data show that FPR-rs3 + neurons exhibit a variety of Ca V currents, both LVA and HVA. Since both N-and P/Q-type currents show somewhat different properties in FPR-rs3 expressing VSNs, these two Ca V 2 channel isoforms might play distinct roles in FPR-rs3 + neurophysiology.

DISCUSSION
For most mammals, the VNO is crucial for intra-and interspecific chemical communication. While the basic biophysical properties of both V1R-and V2R-expressing vomeronasal neurons have been described (Liman and Corey, 1996;Trotier and Døving, 1996;Fieni et al., 2003;Shimazaki et al., 2006;Ukhanov et al., 2007;Hagendorf et al., 2009), VSNs that express members of the recently discovered family of vomeronasal FPR-rs proteins (Liberles et al., 2009;Rivière et al., 2009) remain physiologically unexplored. Here, we describe a transgenic mouse model  in which expression of one member of the FPR-rs family (FPR-rs3) is marked by Venus fluorescence. This mouse strain allows identification and electrophysiological analysis of FPR-rs3-expressing neurons in acute VNO tissue slices. Thus, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal a number of similarities, but also some differences in the basic (electro) physiological architecture of transgene-expressing vs. non-expressing neurons.
Vomeronasal transgene expression in Fpr-rs3-i-Venus mice faithfully recapitulates the punctate apical expression pattern of endogenous FPR-rs3 (Rivière et al., 2009;Dietschi et al., 2013). Furthermore, bicistronic expression of the tau-Venus fusion protein additionally targets the fluorescent marker to axons and axon terminals in the AOB. We therefore propose that Fpr-rs3-i-Venus mice not only provide a useful tool for physiological studies of FPR-rs3 + neurons in the VNO (as described here), but also for studies of axon targeting and glomerular innervation in the AOB. While, based on the experimental strategy used here, we cannot exclude that FPR-rs3 + VSNs additionally express other vomeronasal receptor genes, this appears unlikely since the negative feedback signal that ensures gene exclusion in apical VSNs is also maintained by exogenous expression of another receptor gene, even an OR (Capello et al., 2009).
The specific biophysical profile of FPR-rs3 + VSNs is a critical determinant of their sensory input-output function. Passive membrane properties, such as R input , C mem and τ mem , are therefore crucial functional descriptors of FPR-rs3 + neuron physiology. C mem and dendritic geometry together determine the amplitude of the receptor potential as well as, being inversely proportional, the speed of signal propagation along the dendrite (Gentet et al., 2000). C mem values obtained for FPR-rs3 + neurons are broadly consistent with previously reported data (Liman and Corey, 1996;Shimazaki et al., 2006;Ukhanov et al., 2007)  resistance previously reported for VSNs (Liman and Corey, 1996;Fieni et al., 2003;Shimazaki et al., 2006;Dibattista et al., 2008;Sagheddu et al., 2010) is shared by FPR-rs3 + neurons. Thus, FPR-rs3-dependent receptor currents of even a few picoamperes will be sufficient to trigger action potential discharge. We therefore propose that the primary signal transduction machinery in FPR-rs3 + neurons must be balanced by proper gain/offset control mechanisms to avoid false-positive output. In this context, the rather narrow tuning range of the input-output function of FPR-rs3 + neurons (and control VSNs) is noticeable. Frequency coding accommodates spike rates between 0 and ∼15 Hz that encode receptor currents ranging to a maximum of ∼25 pA (note that the "linear" dynamic range of the f -I curve is considerably more narrow). Similar values have previously been reported (Liman and Corey, 1996;Ukhanov et al., 2007). The relatively long τ mem values (∼25 ms) we obtained for both FPR-rs3 + and control neurons ensure that brief stimulatory events will not generate significant output, in line with the idea that stimulus exchange in the VNO is relatively slow probably allowing prolonged VSN receptor-ligand interaction. Detailed spike waveform analysis revealed rather slow and broad action potentials in line with previously published results (Shimazaki et al., 2006;Hagendorf et al., 2009). Moreover, hyperpolarizing current injection triggers rebound depolarizations resulting in a pronounced "voltage sag" (Robinson and Siegelbaum, 2003;Dibattista et al., 2008). Mediated by HCN channels, we and others observed increasing "sag" amplitudes with membrane potentials becoming more hyperpolarized (Ukhanov et al., 2007;Dibattista et al., 2008). Thus, active membrane properties of FPR-rs3 + neurons do not segregate these neurons from the "general" VSN population.
We used the pufferfish toxin TTX to isolate whole-cell currents mediated by voltage-gated Na V channels. FPR-rs3 + VSNs express one or more TTX-sensitive Na V channel isoform(s), i.e., Nav1.1, 1.2, 1.3, 1.4, or 1.7 (Hille, 2001), which exhibit relatively slow activation upon membrane depolarization >−65 mV with halfmaximal and complete activation at ∼−50 mV and −30 mV, respectively. Notably, the slope of the steady-state inactivation curve is relatively shallow, revealing that full channel inactivation only occurs at positive potentials and, in addition, resulting in a substantial "window current" that ranges from approximately −60 mV to −5 mV.
Similar pharmacological approaches were used to isolate currents mediated by K V and Ca V channels, respectively. At least three different and probably heterogeneous populations of K V channels were identified according to their sensitivity to 4-AP and different TEA concentrations, respectively (Liman and Corey, 1996). Interestingly, while 4-AP-sensitive currents lacked a prominent transient component typical for A-type K + currents (Mei et al., 1995;Amberg et al., 2003), this K V channel population exerted considerable effects on action potential waveform. Moreover, these effects on upstroke kinetics (TTP) and spike width (FDHM/duration) where different between FPR-rs3 + neurons and control VSNs. In addition to Na V and K V channels, several types of Ca V channels were identified in FPR-rs3 + neurons. T-, L-, N-, and P/Q-type I CaV was isolated, either pharmacologically (L-, N-, P/Q-type) or by prepulse inactivation (T-type). While T-and L-type I Cav in FPR-rs3 + VSNs did not significantly differ from control neurons, we find that both N-and P/Q-type currents show somewhat different properties in FPR-rs3 expressing VSNs. We can only speculate about the mechanisms that might link FPR-rs3 expression to altered expression and/or functionality of either N-or P/Q-type Ca V channels. The scope of possible explanations ranges from altered Cacna1a/Cacna1b transcription by random transgene insertion to direct binding of G β/γ to the α1 subunit of either Ca V 2 channel (Currie, 2010), complex co-regulation scenarios of, for example, accessory channel subunits (Neely and Hidalgo, 2014), or unknown intrinsic properties of a potential subpopulation of neurons that express FPR-rs3 instead of a "native" receptor. Whatever the mechanistic basis, the interpretation of future experiments will have to take potential physiological differences into account, which could arise from transgenic vs. endogenous expression.
The Fpr-rs3-i-Venus mouse model we introduce and the basic electrophysiological characterization we performed provide a foundation for future functional studies of FPR-rs neurophysiology. In analogy to FPR signaling in the immune system, current concepts of FPR-rs function suggest a role as chemoreceptors for inflammation-associated and pathogen-related compounds (Rivière et al., 2009;Chamero et al., 2011;Bufe et al., 2012). Immune system FPRs are broadly tuned detectors of either host-or pathogen-derived inflammatory signals (Le et al., 2002;Migeotte et al., 2006;He et al., 2014). Somewhat controversial results have been reported on the tuning profile(s) of recombinantly expressed vomeronasal FPR-rs proteins (Rivière et al., 2009;Bufe et al., 2012). Fpr-rs3-i-Venus mice will likely prove useful for studying FPR-rs3-ligand interaction in homologous cells.