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

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

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

NCX is critical for reducing elevated intracellular Ca²⁺ levels in OSN knobs.

<p>A, Comparison of the kinetic properties of the Ca²⁺ transients (normalized responses) in OSN knobs evoked by a 1-s pulse of KCl (80 mM) before (control) and after thapsigargin (200 nM) treatment. B, Ca²⁺ response (ΔF/F) of a single knob stimulated with a 5-s pulse of caffeine (10 mM) followed by a 1-s pulse of KCl (80 mM), confirming that thapsigargin (200 nM) pretreatment depleted Ca²⁺ from intracellular stores. C, Comparison of the kinetic properties of the Ca²⁺ transients (normalized responses) in OSN knobs evoked by a 1-s pulse of IBMX (100 µM) before (control) and after treatment with the PMCA inhibitor carboxyeosin (10 µM). D, Comparison of the kinetic properties of IBMX-induced Ca²⁺ transients (normalized responses) in OSN knobs before (control) and after treatment with the NCX inhibitor 3,4-dichlorobenzamil hydrochloride (DCB; 10 µM). E, Bar graphs showing collected decay time constants from control (gray) and treated (black) OSN knobs. For thapsigargin (Tg), control: τ = 21.1±1.9 s (<i>n</i> = 13, <i>N</i> = 4), Tg: τ = 19.0±1.9 s (<i>n</i> = 14, <i>N</i> = 4). For carboxyeosin (CE), control: τ = 8.5±1.9 s (<i>n</i> = 25, <i>N</i> = 8), CE: τ = 13.3±1.4 s (<i>n</i> = 16, <i>N</i> = 4). For 3,4-dichlorobenzamil hydrochloride (DCB), control: τ = 11.0±1.4 s (<i>n</i> = 12, <i>N</i> = 4), DCB: τ = 35.1±4.4 s (<i>n</i> = 12, <i>N</i> = 4). NS, not significant; *<i>p</i><0.05; **<i>p</i><0.0001. The Ca²⁺ transients in A,C, and D were rescaled to give the same peak amplitude.</p

Evidence that OMP regulates NCX1 through interaction with CaM and Bex.

<p>A, NCX1 and OMP are highly expressed and co-localized in cell bodies, dendrites, dendritic knobs (long arrows) and cilia (short arrows) of mature OSNs of WT mice. Scale bar, 5 µm. B, Sensorgrams for interaction of XIP peptide immobilized on a CM5 sensorchip and CaM (10, 25, 50,100, 250 nM) using a BIAcore 3000 biosensor. Running buffer used during recording contained 0.1 mM Ca²⁺. Data were fitted with a 1∶1 Langmuir binding model using BIAevaluation software, giving K_d = 20 nM. C, Sensorgram for interaction of immobilized Bex1 (50–75) peptide and CaM (31.25, 62.5, 125, 250, 500 nM) on a CM5 chip in presence of 0.1 mM Ca²⁺. D, Because of the rapid on/off kinetics of the interaction between CaM and Bex1 peptide, saturation curves of equilibrium response versus CaM concentration were analyzed using a nonlinear 1∶1 binding isotherm model, giving K_d = 280 nM.</p

Recovery of elevated intracellular Ca²⁺ levels is compromised in OMP^−/− mice.

<p>A–C, Comparison of fluorescence intensity changes (ΔF/F) of Ca²⁺ responses in WT and OMP^−/− knobs to a 1-s pulse of 100 µM IBMX (A, B) or 80 mM KCl (C) using 1 mM external Ca²⁺. Recovery time course of the signals was fitted with single exponential functions (dashed lines). The decay time constants (τ) of the fitted curves are indicated. These results demonstrate an apparent defect in the kinetics of removal of elevated Ca²⁺_i from the dendritic knobs of the OSNs of OMP^−/− mice. D, Ca²⁺ response of a single WT knob stimulated with a 1-s pulse of IBMX (100 µM) followed by a 5-s pulse of caffeine (10 mM), both in low external Ca²⁺ solution (0.6 µM), confirming that the caffeine-induced Ca²⁺ transient depends on an intracellular source. E, Comparison of Ca²⁺ transients in WT and OMP^−/− knobs evoked by a 5-s pulse of caffeine (10 mM) in low extracellular Ca²⁺ solution (0.6 µM). Recovery time course of the signals was fitted with single exponential functions (dashed lines). The decay time constants (τ) of the fitted curves are indicated. F, Bar graphs showing collected results from OSN knobs of WT and OMP^−/− mice. For stimulation with IBMX, WT: τ = 8.5±1.3 s (<i>n</i> = 25, <i>N</i> = 8), OMP^−/−: τ = 21.3±2.2 s (<i>n</i> = 13, <i>N</i> = 4). For stimulation with KCl, WT: τ = 20.1±1.7 s (<i>n</i> = 13, <i>N</i> = 4), OMP^−/−: τ = 28.7±3.2 s (<i>n</i> = 13, <i>N</i> = 4). For stimulation with caffeine in 1 mM Ca²⁺_o, WT: τ = 13.9±1.6 s (<i>n</i> = 15, <i>N</i> = 5), OMP^−/−: τ = 26.8±1.9 s (<i>n</i> = 15, <i>N</i> = 4), **<i>p</i><0.0001 and in low external Ca²⁺ (0.6 µM), WT: τ = 13.1±1.8 s (<i>n</i> = 19, <i>N</i> = 6), OMP^−/−: τ = 27.3±1.9 s (<i>n</i> = 15, <i>N</i> = 5), *<i>p</i><0.001; **<i>p</i><0.0001.</p

OMP facilitates NCX activity.

<p>A, The activity of NCX was probed in reverse mode by monitoring the rise of Ca²⁺_i in response to a stepwise reduction of Na⁺_o (by substituting Na⁺ by Li⁺). Ca²⁺ responses are reversibly inhibited by the NCX inhibitor KB-R7943 (10 µM) as well as by dichlorobenzamil (DCB, 10 µM). This confirms that the rise in Ca²⁺ induced by low Na⁺_o is a result of Ca²⁺ entry through NCX. B, Bar histogram of the Ca²⁺ responses obtained after treatment with pharmacological agents that modulate NCX activity. Concentrations are given below each bar (in µM). Number of knobs tested are indicated in parentheses above each bar. C, Averaged Ca²⁺ responses due to reverse mode activity of NCX from WT (gray) and OMP^−/− knobs (black) show a 2.5-fold increase (p<0.0001) in time-to-peak in OMP^−/− (<i>n</i> = 27; <i>N</i> = 5) <i>vs</i>. WT mice (<i>n</i> = 26; <i>N</i> = 4). D, Averaged decay time courses of the same signal in WT (gray) and OMP^−/− knobs (black) reveal an approximately 2-fold increase in the recovery rate in OMP^−/− mice (p<0.03). Data were normalized to the value obtained immediately at the end of the 20-s low Na⁺ stimulus.</p

Newborn interneurons in the accessory olfactory bulb promote mate recognition in female mice

<p>In the olfactory bulb of adult rodents, local interneurons are constantly replaced by immature precursors derived from the subventricular zone. Whether any olfactory sensory process specifically relies on this cell renewal remains largely unclear. By using the well known model of mating-induced imprinting to avoid pregnancy block, which requires accessory olfactory bulb (AOB) function, we demonstrate that this olfactory memory formation critically depends on the presence of newborn granule neurons in this brain region. We show that, in adult female mice, exposure to the male urine compounds involved in mate recognition increases the number of new granule cells surviving in the AOB. This process is modulated by male signals sensed through the vomeronasal organ and, in turn, changes the activity of the downstream amygdaloid and hypothalamic nuclei involved in the pregnancy block response. Chemical depletion of newly generated bulbar interneurons causes strong impairment in mate recognition, thus resulting in a high pregnancy failure rate to familiar mating male odors. Taken together, our results indicate that adult neurogenesis is essential for specific brain functions such as persistent odor learning and mate recognition.</p

Additional file 2: of Pregnancy and estrogen enhance neural progenitor-cell proliferation in the vomeronasal sensory epithelium

Excel workbook containing the mapping statistics of the RNAseq data for each sample, along with the accession numbers for the raw data; the normalized counts for the whole transcriptome; the differential expression analysis between the pregnant and control samples; the gene ontology categories significantly overrepresented in the differentially expressed genes; the normalized counts for the VR genes, accounting for total VSN number; and the differential expression analysis on the VR genes only, after normalization for VSN number, between the pregnant and control samples. The column ‘length’ corresponds to the total exonic bases in all transcripts from the gene. For the differential expression sheets, the columns contain the following data: ‘baseMean’, corresponds to the mean normalized expression value for the gene across all samples; ‘log2FoldChange’, is the fold change between the pregnant and control samples, log2 transformed; ‘lfcSE’, corresponds to the standard error associated with the fold change estimation; ‘stat’, is the Wald statistic; ‘pvalue’ is the P value of the test; and ‘padj’ is the P value after adjusting for multiple testing (Benjamini-Hochberg). Genes that have both their ‘pvalue’ and ‘padj’ set to NA contain outliers; genes with only their ‘padj’ set to NA were filtered prior to the test because their normalized counts were too low. (XLSX 11150 kb