Single-cell RT-PCR following Ca²⁺ imaging and cell isolation.

<p>(<b>A</b>) Representative intracellular Ca²⁺ increase (F340/380 ratio, arbitrary units) of a VSN (cell “A”) loaded with fura-2 in response to the sulfated steroid E1050 (chemical structure shown on the side), but not to urine high molecular weight fraction (HMW). (B) Ratiometric (340/380) imaging of the cell shown in A during stimulation with control buffer (DMSO) and E1050. Responsive cells are later picked using a glass capillary micropipette. Arrowhead points to the cell before and after (inside of the micropipette) picking. Scale bar, 10 μm. (C) Ethidium-bromide stained agarose gels of RT-PCR products generated from 5 cells (A to E) showing Ca²⁺ responses to E1050, a single cell lacking responses (n/r), and two water controls (w1 and w2). cDNA collected from pooled whole VNOs was used as positive control (VNO). PCR amplification of cDNA collected from single cells was performed using gene specific primers for <i>Omp</i>, <i>Gnao1</i> (Gαo), <i>Gnai2</i> (Gαi2) and degenerate primers for three members of the <i>V1rj</i> family.</p

Functional overexpression of <i>V1rj2</i> in HSV-infected VSNs.

<p>(A) Diagram of the HSV-1 expression cassettes. In control amplicons <i>GFP</i> is expressed under the HSV-1 IE4/5 promoter, whereas in V1rj2 amplicons <i>V1rj2</i> cDNA is inserted in the multi cloning site (MCS) upstream of IRES and GFP. (B) Bright field (BF), GFP and pseudocolor fura-2 images of a representative VSN infected with HSV-<i>V1rj2</i>-IRES-<i>GFP</i> and activated by the mix of four sulfated estrogens (E mix). Time course of intracellular calcium is shown on the right. Stimulations were 30 s long. Scale bar, 10 μm. (C) Color-coded heat map of normalized Δratio responses (Δratio_norm) from 26 GFP+ VSNs infected with <i>V1rj2</i>-IRES-<i>GFP</i> which showed responses to the estrogen mix (E mix). These cells lacked responses to other stimuli except for high K⁺. (D) Comparison of response amplitudes expressed as normalized Δratio responses to E mix and high K of 26 individual cells shown in C. Each neuron is shown as a separate circle. Red line represents linear fit (slope = 0.55). Responses to E mix and high K⁺ are not significantly different (p = 0.92, Mann-Whitney test). (E) Summary of GFP+ cell activation for every stimulus on VSNs infected either with V1rj2-GFP (green) or with GFP control amplicons (grey). Values are expressed as percentage (%) of activated cells from the total of GFP+ cells. VSNs expressing <i>V1rj2-GFP</i> show increased cell responsivity to E mix (p < 0.005) but not to E2734, HMW, DMSO or high K⁺ (p = 0.1–0.4, Student’s t test). Responses to DMSO, E2734 or HMW did not overlap with responses to E-mix. N = 436 <i>V1rj2-GFP</i> cells and 563 control-<i>GFP</i> cells, in 8 and 16 experiments, respectively.</p

Herpes simplex virus type 1 (HSV-1) expression system in HEK cells and VSNs.

<p>(A) HEK cells transfected with pHSV-<i>V1rj2</i>-IRES-<i>GFP</i> expression vector do not show responses to a mix of sulfated steroids (E mix: E1100, E0893, E0588, and E1050, each 100 μM), HMW nor E2734 in Ca²⁺ imaging. (B) Infection of HEK cells (top panels) and freshly prepared VSNs (bottom panels) with HSV-GFP amplicon virus monitored at three different time points (6 h, 24 h and 48 h). Scale bars, 50 μm (C) Left and center, measures of fluorescence intensity (in arbitrary units, a.u.) on infected single HEK cells and VSNs. Right, normalized abundance of infected VSNs (GFP+) at each time point. (D) Single VSNs infected with HSV-GFP virus for 20 h and loaded with fura-2. Bright field (BF), GFP and F340/380 ratio images of an infected cell are shown. Average rate of infection was 23% (N = 16 402 cells in 69 infections). Infection rate for specific batches of HSV: GFP, 19% (N = 10 171); V1rj2, 15% (N = 3109); V2r1b, 27% (N = 2675); Fpr3, 39% (N = 447). (E) VSNs prepared from OMP-GFP mice infected with a HSV-mCherry virus. Of all mCherry-positive cells 76% were also positive for GFP. N = 978 mCherry+ cells in 14 infections. Scale bars, 10 μm.</p

Viral transduction of <i>V2r1b</i> and <i>Fpr3</i> receptors in VSNs.

<p>(A) Bright field (BF), GFP and pseudocolor fura-2 images of a VSN infected with HSV-<i>V2r1b</i>-IRES-<i>GFP</i> (arrowhead) and activated by the MHC binding peptide SYFPEITHI (SYF). The neighboring non-infected GFP-negative cell (arrow) does not show any calcium increase during peptide stimulation. (B) Summary of cell responses to different stimuli. A 6-fold increase of responsivity to SYF is observed in <i>V2r1b-GFP</i> cells (p < 0.005, Student t test), but not to HMW fraction, the mitochondria-derived peptide ND1, or high K⁺. (C) Enhanced responsivity to SYF is not observed in Gαo-deficient mice (cGαo^-/-) VSNs infected with HSV-<i>V2r1b</i>-IRES-<i>GFP</i>. (D) A single HSV-<i>Fpr3</i>-IRES-<i>GFP</i> infected VSN activated by the synthetic hexapeptide W-peptide (w-pep) is shown. (E) <i>Fpr3-GFP</i> cells show a significantly enhanced number of responses to W-peptide versus GFP control cells (p < 0.001, Student t test), but not to HMW, ND1 or high K⁺. N = 469 <i>V2r1b-GFP</i>, 163 <i>Fpr3-GFP</i> and 1224 control-GFP cells, in 12, 8 and 27 experiments, respectively. Scale bars, 10 μm.</p