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

Abstract

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.

Keywords: VNO; formyl peptide receptor; olfaction; sensory neurons; vomeronasal organ; vomeronasal receptor.

Figure 1

Generation and characterization of the Fpr-rs3-i-Venus transgenic mouse line. (A) Schematic of the transgene that includes an OR promoter/enhancer, followed by the coding sequence of FPR-rs3 and a polycistron that drives the tau-Venus fluorophore. (B) Confocal image of a coronal VNO cryosection showing sparsely distributed fluorescently labeled FPR-rs3⁺ VSNs (green) in the sensory neuroepithelium. (C) Confocal image displaying a coronal VNO cryosection immunostained with an antibody against V2R2 (red), a family-C V2R expressed in most basal VSNs. (D) Overlay of FPR-rs3 tau-Venus fluorescence and anti-V2R2 staining shows no co-localization of apically located FPR-rs3⁺ and basal V2R2 expressing neurons. (E) Higher magnification of the boxed area in (D) illustrating the absence of overlapping fluorescence. (F) Confocal image of a VNO cryosection showing distinct green fluorescent FPR-rs3⁺ sensory neurons (green). (G) Confocal image displaying the same area as in (F) stained against the FPR-rs3 protein (red). (H) Overlay of tau-Venus fluorescence and antibody staining against the FPR-rs3 protein. Note that all transgene-positive cells also express FPR-rs3 (yellow). Scale bars, 50 µm (B–D), 10 µm (E) and 20 µm (F–H).

Figure 2

Passive membrane properties of FPR-rs3⁺ VSNs. (A) Confocal image (maximum projection) of a 150 µm acute coronal VNO tissue slice showing the distribution of fluorescent FPR-rs3 tau-Venus⁺ neurons (green) in the vomeronasal sensory epithelium. Fluorescent axon bundles are visible within the basal lamina. (B) FPR-rs3 tau-Venus⁺ neurons exhibit a single apical dendrite ending in a knob-like structure at the luminal border. Whole cell patch-clamp recordings were performed from the VSN soma. (C) Membrane capacitance and (D) input resistance (R_input) are similar for both control and FPR-rs3⁺ neurons (n = 21). (E) Membrane time constant (τ_membrane) of control neurons compared to FPR-rs3⁺ cells shows no significant difference (n = 21). Data are mean ± SEM. Blood vessel (BV), lumen (L), patch pipette (PP), sensory epithelium (SE). Scale bars, 50 µm (A), 10 µm (B).

Figure 3

Active membrane properties of FPR-rs3⁺ VSNs. (A) Representative current clamp traces showing de- / hyperpolarization and trains of (rebound) action potentials generated upon stepwise current injection. Note the spontaneous activity measured at 0 pA current injection (A_ii). Injection of negative current produces a prominent voltage “sag” most likely mediated by activation of HCN channels (Aiii). (B) Firing frequency of control (n = 21) and FPR-rs3⁺ (n = 19) neurons as a function of the injected current (I_inject). The gradual increase in firing rate is comparable for control and FPR-rs3⁺ VSNs. Inset: Spontaneous spiking frequency at 0 pA current injection. Note that f-I curves have been “background-corrected” using these values. (C) Voltage “sag” (ΔV_sag, n = 3–32) as a function of the peak membrane hyperpolarization (10 mV bins). ΔV_sag values of control and FPR-rs3⁺ neurons show no statistical difference (p > 0.01, two-tailed Student’s t-test). (D) Average spike waveform illustrating analysis parameters (amplitude, time-to-peak (TTP), full duration at half-maximum (FDHM; D_i). Amplitude analysis of the first action potential for each current injection step shows no difference between control and FPR-rs3⁺ cells (D_ii). TTP analysis reveals values in the same range for both cell populations (D_iii). Spike width (FDHM) is not significantly different between control and FPR-rs3⁺ VSNs (D_iv). Data are mean ± SEM.

Figure 4

Voltage-gated Na⁺ currents. (A) Representative traces from whole-cell patch-clamp recordings of a TTX-sensitive fast activating Na⁺ current in FPR-rs3⁺ VSNs. (A_i) Voltage step recording under control conditions (extracellular solution S₁; intracellular solution S₉) reveals a voltage-dependent fast and transient inward current. (A_ii) TTX treatment (1 μM) strongly diminishes the current. Digitally subtracted trace (control—TTX (A_iii)) reveals the TTX-sensitive voltage-gated Na⁺ current. (B) Current-voltage relationships of TTX-sensitive Na⁺ currents isolated from control and FPR-rs3⁺ neurons (control, n = 20; FPR-rs3⁺, n = 10; p > 0.01, two-tailed Student’s t-test). (C) Example of a voltage-clamp recording showing the fast activating transient inward current used for upstroke kinetics analysis (C_i). TTP of the fast activating Na⁺ current upon depolarization to −30 mV (control, n = 20; FPR-rs3⁺, n = 10; C_ii). (D) Representative traces showing Na⁺ channel steady-state inactivation under control conditions (D_i), in presence of TTX (D_ii), and after digital subtraction (control—TTX (D_iii)). Prepulse steps from −120 mV to 0 mV were applied to analyze inactivation (D_iii, inset). (E) Normalized activation (E_i) and steady-state inactivation (E_ii) curves (peak current vs. pulse / prepulse voltage). Data points were fitted using a sigmoidal Boltzmann-type equation. Membrane voltage inducing half-maximal activation and inactivation (V_½) as indicated. Data are mean ± SEM.

Figure 5

Voltage-gated K⁺ currents and their role in action potential firing. (A) Voltage-activated outward K⁺ currents under control conditions (solution S₁ including TTX (1 µM) and Cd²⁺ (200 µM) to isolate K⁺ currents). Currents were induced by stepwise depolarization and measured during steady-state. Current densities were calculated and plotted against voltage (control, n = 10; FPR-rs3⁺, n = 13). (B) Outward currents sensitive to 1 mM TEA (control-TEA; digital subtraction; n = 10). (C) Outward currents sensitive to 10 mM 4-AP (control/TEA-4-AP; digital subtraction; n = 10). (D) Outward currents sensitive to 25 mM TEA (control/TEA/4-AP-TEA; digital subtraction; n = 10). (E) Quantification of outward currents. Maximum current densities under control conditions (left bars; 292.5 ± 22.4 pA/pF at +85 mV, n = 10 (ctrl); 276.5 ± 31.1 pA/pF, n = 13 (FPR-rs3⁺)) and added drug sensitive current densities (ctrl: 90.3 ± 9.1 pA/pF (1 mM TEA), 76.8 ± 7.9 pA/pF (10 mM 4-AP), 85.7 ± 11.2 pA/pF (25 mM TEA); FPR-rs3⁺: 78.9 ± 10.6 pA/pF (1 mM TEA), 77.5 ± 10.5 pA/pF (10 mM 4-AP), 99.8 ± 14.6 pA/pF (25 mM TEA)). Data are mean ± SEM. (F) Representative spike waveform under control conditions (solution S₁ (Extra)) and in presence of TEA (1 mM) and 4-AP (10 mM; F_i). Analysis parameters (amplitude, TTP, FDHM and spike duration) are depicted schematically. Bar graphs illustrate the quantification of discharge characteristics (F_ii–v). *p < 0.01; two-way ANOVA with Tukey’s multiple comparisons test. Data are mean ± SEM, number of cells as depicted inside the bars.

Figure 6

Voltage-gated Ca²⁺ currents. (A–D) Representative Ca²⁺ current traces isolated either biophysically (prepulse inactivation protocol; (A_i)) or pharmacologically (nifedipine (10 μM; B_i); ω-conotoxin-GVIA (2 μM; C_i); ω-agatoxin IVA (200 nM; D_i)). Step protocols as indicated. Absolute (A_ii–D_ii) and normalized (A_iii–D_iii) peak current densities are plotted as a function of membrane depolarization. Activation curves (A_iii–D_iii) are fitted according to a sigmoidal Boltzmann-type equation. Membrane voltage inducing half-maximal activation (V_½) as indicated. Data are mean ± SEM; *p < 0.01, two-tailed Student’s t-test.