Li Z et al. (2014), Requirement for Drosophila SNMP1 for rapid acti...

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PLoS Genet 2014 Sep 25;109:e1004600. doi: 10.1371/journal.pgen.1004600.

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Requirement for Drosophila SNMP1 for rapid activation and termination of pheromone-induced activity.

Li Z , Ni JD , Huang J , Montell C .

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Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). Here, we found the snmp1(1) mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported "spontaneous activity," which represented continuous activation following transient exposure to environmental cVA. We introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp1(1) flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). We expressed the bombykol receptor complex in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP) and found that addition of BmSNMP accelerated receptor activation and deactivation. Our results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.

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Species referenced: Xenopus
Genes referenced: cd36 lgals4.2

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	Figure 2. High cVA levels elicited weak responses in snmp11, which displayed slow activation and deactivation kinetics.The SSRs were from female flies of the indicated genotypes. (A) Representative SSRs obtained from OR67d ORNs stimulated with cVA for 1 sec (indicated by the horizontal black bar) using the conventional odorant delivery approach. (B) Action potential frequencies as a function of the concentration of the applied cVA using the conventional odorant delivery method. (C) Representative traces showing the responses from OR67d ORNs evoked by close-range application of 100% cVA (indicated by the horizontal bar above the trace). The cVA was puffed onto the antenna through a pipette tip placed 3 mm away from the fly antenna. The flies expressing UAS-snmp1 under the control of the snmp1-Gal4 transgene were in a snmp11 background. (D) Quantification of peak firing rates following close-range application of cVA. n = 17–20. (E) Quantification of the onset delays of the responses to close-range stimulation with cVA. (F) The upper graph shows the duration of the firing of OR67d neurons after close-range application of 100% cVA. The estimated times required for a 50% reduction of the evoked firing rates (t1/2) are shown (n = 17–20). The frequencies (spikes/sec) were binned every 0.5 sec. Therefore, the t1/2 were rounded to the nearest 0.5 sec. The traces in the lower graph plot were derived from the upper panel, and were normalized to their respective peak firing rates. Means ±S.E.M. The asterisks indicate significant differences from wild-type and rescue flies (**p<0.01) based on unpaired Student t-test for comparing pairs of data (B and D) and ANOVA with the Bonferroni-Holm post hoc test for comparing multiple samples (E).
	Figure 3. OR67d ORNs in snmp11 females showed high neuronal activity long after transient stimulation with cVA.(A) Close-range application of cVA elicited long-lasting responses in OR67d ORNs from snmp11 flies. The upper, middle and lower traces show the traces recorded at different times relative to the cessation of the cVA stimulation The upper trace indicates the spontaneous activity of the OR67d ORNs before cVA stimulation. The middle trace shows the point of application of 100% cVA applied for 1 sec (indicated by the black bar). The start of the cVA puff is defined as time 0. The lower trace demonstrates that the response persisted 10 min after the cVA stimulation. (B) Quantification of the firing rates immediately after cessation of the cVA stimulation (0 min) and 10 min later (n = 5). (C) Close range application of the cVA solvent (paraffin oil) did not elicit responses in OR67d ORNs. (D) Duration of the responses of OR67d ORNs to close-range application of 0.01% (wild-type) and 100% cVA (snmp11, regenerated from Fig. 2F). (E) The dynamics of the firing rates after close-range application of with either 100% or 0.01% cVA for 12 sec as indicated by the black bar. Means ±S.E.M.
	Figure 4. SNMP1 affects the response to bombykol in OR67d ORNs ectopically expressing BmOR1.We expressed UAS-BmOr1 under control of the snmp1-Gal4 in either a snmp1+ or snmp11 background. We applied bombykol using the conventional odorant delivery approach, and performed SSRs from T1 sensilla from males and females. (A) Representative traces of bombykol-evoked responses from OR67d ORNs. We applied 100% bombykol for 1 sec as indicated by the horizontal black bar above the traces. (B) Dose-response curve for bombykol. n = 12–20 for each data point. (C) The upper graph shows the response dynamics of BmOR1-expressing OR67d neurons to bombykol. n = 14–15. The estimated t1/2 values are indicated. The lower graph shows the same traces from the upper panel normalized to their respective peak spiking rates. (D) Two-electrode voltage clamp recordings of Xenopus oocytes expressing BmOR1 and BmORCO with or without BmSNMP. 10 µM bombykol was applied as indicated by the black bar, and then removed (Wash) as indicated by the open bar. (E) Quantification of the times to reach 50% of the maximum activation (t1/2) during the 10 µM bombykol application. The results from cells with and without BmSNMP are indicated. n = 5–6. (F) Quantification of the times required for the decline to 50% of the maximum current (t1/2) after the washout of bombykol. n = 5–6. Means ±S.E.M. The asterisks indicate significant differences between groups (p<0.05, *p<0.01). Unpaired Student's t-tests.
	Figure 5. Close application of 100% cVA elicited responses in the lush1,snmp11 double mutants.(A) Representative traces of OR67d ORNs from the indicated genotypes in response to stimulation with 100% cVA applied at close range. The 1 sec application of cVA is indicated by the bar above the traces. (B) Response dynamics of OR67d ORNs from the indicated genotypes in response to 100% cVA applied using the close range approach (n = 14–18). snmp11 data were regenerated from Figure 2. Means ±S.E.M.
	Figure 6. Proposed model for SNMP1 function in pheromone sensation.We suggest that SNMP1 promotes rapid activation and deactivation of the pheromone responses by lowering the energy barrier for the pheromone to associate and dissociate from the pheromone receptors (ORX).
	Figure 1. Effects of prior exposure either to males or to cVA on “spontaneous” spiking activity.Single sensillum recordings (SSRs) were from trichoid sensilla (T1), which contain an OR67d-expressing ORN. Neither the wild-type nor the snmp11 flies were exposed to cVA during the recordings. (A) Representative traces showing firing activities from wild-type and snmp11 females, which were either reared in isolation or in groups with males, as indicated. (B) Mean firing rates elicited by grouped or isolated wild-type and snmp11 females. n = 15–18. (C) Average spiking activities of OR67d neurons from isolated male or female snmp11 flies. The ages of the flies are indicated. n = 8–10. (D) Wild-type and snmp11 females were exposed to cVA or the vehicle (paraffin oil) for 24 hrs immediately prior to the recordings. n = 16–18. Mean ±S.E.M. The asterisks indicate significant differences between groups (p<0.05, *p<0.01) using ANOVA with Bonferroni-Holm post hoc test to compare multiple samples (B) and the unpaired Student t-test for comparing pairs of data (C and D). NS, no significant difference.

References [+] :

BUTENANDT, [On the sexattractant of silk-moths. I. The biological test and the isolation of the pure sex-attractant bombykol]. 1961, Pubmed