Supplementary Figure 1. Schematic illustrating major conclusions of this study.

Transcription

1 ORNs GABA A GABA B glomeruli LN PNs Supplementary Figure 1. Shemati illustrating major onlusions of this study. This study represents the most diret evidene to date of inhiitory interations etween olfatory glomeruli in vivo. Our results show that the odor responses of a Drosophila antennal loe projetion neuron (PN) an e sustantially inhiited y ativity in the rest of the antennal loe. Muh of this inhiition appears to our at a presynapti lous, via the suppression of neurotransmitter release from ORN axons. Our results suggest that oth GABA A and GABA B reeptors are present on the same presynapti terminals. We find that the amount of lateral inhiition evoked y an odor stimulus is proportional to the total numer of ORN spikes produed y that stimulus, suggesting that inhiition reflets pooled input from all ORN types. We propose that this form of spatially diffuse presynapti lateral inhiition serves to ontrol the gain of olfatory proessing. When stimuli are weak, ORN-PN synapses are strong, ut when stimuli are strong, ORN-PN synapses are weakened. This should prevent a stimulus from saturating the dynami range of many PN types simultaneously. Beause strong and redundant stimuli are preferentially suppressed, this may tend to derease ross-orrelations etween the output of different glomeruli, and thus promote a more effiient neural ode for odors.

2 glomerulus VM7 ORNs PNs antennae intat PNs antennae removed 2 enzaldehyde utyri aid 1-utanol ylohexanone 2 3-hydroxy fenhone geranyl 2-heptanone 2 linalool 4-m ylohexanol m saliylate 3-methythiol- 1-propanol otanal 2 1-oten-3-ol paraffin oil pentyl 4-m phenol trans-2- hexenal enzaldehyde utyri aid 1-utanol ylohexanone 2 3-hydroxy fenhone geranyl 2-heptanone 2 linalool 4-m ylohexanol m saliylate 3-methythiol- 1-propanol otanal 2 1-oten-3-ol paraffin oil pentyl 4-m phenol trans-2- hexenal Supplementary Figure 2. Peri-stimulus time histograms for glomerulus VM7 PSTHs for ORNs (green) and PNs either with (magneta) or without (lue) antennal input. The odor stimulus in eah panel is from to.5. All panels use the same x and y-axes. All PSTHs are averaged aross 4-12 experiments, ± s.e.m in pastel. In some PSTHs the pastel error ars are too small to e visile.

3 glomerulus VC1 ORNs PNs antennae intat PNs antennae removed 2 enzaldehyde utyri aid 1-utanol ylohexanone 2 3-hydroxy fenhone geranyl 2-heptanone 2 linalool 4-m ylohexanol m saliylate 3-methythiol- 1-propanol otanal 2 1-oten-3-ol paraffin oil pentyl 4-m phenol trans-2- hexenal enzaldehyde utyri aid 1-utanol ylohexanone 2 3-hydroxy fenhone geranyl 2-heptanone 2 linalool 4-m ylohexanol m saliylate 3-methythiol- 1-propanol otanal 2 1-oten-3-ol paraffin oil pentyl 4-m phenol trans-2- hexenal Supplementary Figure 3. Peri-stimulus time histograms for glomerulus VC1 PSTHs for ORNs (green) and PNs either with (magneta) or without (lue) antennal input. The odor stimulus in eah panel is from to.5. All panels use the same x and y-axes. All PSTHs are averaged aross 4-12 experiments, ± s.e.m in pastel. In some PSTHs the pastel error ars are too small to e visile.

4 a antenna palp (5 ms) V m (mv) antenna removed palp V m (mv) antenna palp removed -4 V m (mv) -6-8 d antenna palp -4 V m (mv) -6-8 Supplementary Figure 4. Sample reords for glomerulus VM7. Odor stimulus for all reords is (5 ms). (a) A whole-ell path lamp reording from a VM7 PN, reorded in the normal onfiguration (oth antennae and palps attahed, no shielding of palps). Note aundant spontaneous suthreshold input prior to odor stimulation. This mainly reflets spike-driven spontaneous EPSPs (sepsps) arising from ORN-PN synapses. () Reording from a VM7 PN with antennae removed. Note that sepsps prior to odor stimulation are similar to aove, as expeted. Removing the antennae disinhiits the odor response, implying that antennal glomeruli are a soure of lateral inhiition for this glomerulus. () Reording from a VM7 PN with palps removed (same trae as in Fig. 2a). Note that sepsps are now asent, as expeted. We and others 7,11,12 have interpreted the depolarizing odor response as lateral postsynapti exitatory input from the antennae. Lateral postsynapti inhiitory input may also e present, ut during the odor response period it is evidently weaker than the lateral exitation. (d) Reording from a VM7 PN with palps shielded (same trae as in Fig. 2). Note that sepsps are visile again, implying that ORNs fire spontaneously even when they are shielded from odors. The shield is learly preventing VM7 ORNs from sensing, eause the strong exitation visile in () is asent. Now, lateral input is mainly hyperpolarizing. The only differene etween () and (d) is the asene versus presene of spontaneous ORN-PN EPSPs, and this implies that the differene etween these traes reflets lateral inhiition of ORN-PN synapses.

5 a pentyl -2 Vm (mv) Vm (mv) pentyl no antagonists pirotoxin pentyl Vm (mv) -2 Vm (mv) -4-6 m saliylate -4-6 Supplementary Figure 5. Evidene for lateral postsynapti inhiition. In this experiment we tested whether the lateral postsynapti input to a PN has an inhiitory omponent. To isolate the lateral postsynapti input, the maxillary palps were removed, and reordings were made from PNs in the palp glomerulus VM7 (see Fig. 2a). Current pulses were applied to the ell so that odor responses ould e reorded at different memrane potentials. Raw data from one experiment are shown here. Similar results were oserved in other experiments (n = 5). The odor duration for all reords is 5 ms. (a) The odor pentyl eliited depolarizations when the memrane potential was more hyperpolarized than aout -4 mv. At potentials more depolarized than -4 mv, pentyl produed a small hyperpolarization and an interruption of the spiking ativity evoked y urrent injetion. This result suggests that odors an eliit oth lateral postsynapti exitation and lateral postsynapti inhiition. Moreover, the alane etween exitation and inhiition depends on memrane potential: pentyl ould either evoke a spike (orange trae) or inhiit spikes (green, lue, purple traes). () The odor m saliylate evoked postsynapti exitation ut did not hyperpolarize the ell at any memrane potential. This indiates that the alane of lateral postsynapti exitation and inhiition is odor-dependent. () Bloking GABAA and GABAB reeptors with a omination of pirotoxin and did not eliminate the postsynapti inhiition evoked y pentyl. Beause the postsynapti response to GABA iontophoresis is ompletely loked y pirotoxin and , this suggests that GABA may not mediate this postsynapti inhiition. Moreover, this result indiates that the disinhiition produed y pirotoxin and in Fig. 5 is not due to the lok of postsynapti inhiition. Rather, the disinhiition in Fig. 5 is more onsistent with the removal of presynapti inhiition, whih is shown in Figs. 3-4 to e ompletely sensitive to the omination of pirotoxin and

6 olfatory stimulation eletrial stimulation odor efore odor during odor ontrol EPSC (pa) a + PCT + PCT 2 2 pa 1 minutes 2 reord eletrial GABA stimulation iontophoresis e ontrol f * + PCT reord efore GABA after GABA EPSC (pa) d + PCT 4 4 pa 15 minutes 3 Supplementary Figure 6. GABA reeptor antagonists do not lok lateral postsynapti exitation, ut do lok lateral inhiition. (a) Experimental design for measuring odor-evoked lateral suppression of EPSCs. () Voltage-lamp traes from the ell shown in Fig. 3(). Antennal nerve stimulation (arrowheads) evokes EPSCs. Odor stimulation suppresses EPSCs and evokes an inward urrent. This inward urrent reflets odor-evoked lateral exitatory input and pirotoxin lok the EPSC suppression ut do not lok the odor-evoked inward urrent. This demonstrates that EPSC suppression is not due to postsynapti shunting y lateral exitatory input. () Time ourse for antagonist lok of odor-evoked EPSC suppression. Symols show the average EPSC amplitude immediately efore odor stimulation ( ) or during odor stimulation ( ) for eah trial. (d) Experimental design for measuring GABA-evoked suppression of EPSCs. (e) Voltage-lamp traes from the ell shown in Fig. 3(e). Antennal nerve stimulation (arrowheads) evokes EPSCs. GABA iontophoresis (indiated y asterisk) suppresses EPSCs and evokes an outward urrent. This outward urrent is due to the diret postsynapti ation of GABA (see ref. 8) and pirotoxin together lok oth the EPSC suppression and the GABA-evoked outward urrent. (f) Time ourse for antagonist lok of GABA-evoked EPSC suppression. Symols show the average EPSC amplitude immediately efore GABA iontophoresis ( ) or following GABA iontophoresis ( ) for eah trial.

7 a ontrol Cd 2+ 1 pa 5 ms paired-pulse ratio 1.. Cd 2+ d ontrol GABA 1 pa 5 ms paired-pulse ratio 1.. GABA GABA + GABA +PCT GABA + +PCT odor Supplementary Figure 7. Inreased paired-pulse ratio indiates a presynapti lous for EPSC inhiition. We performed paired-pulse experiments to test whether either GABA A and/or GABA B reeptors mediate EPSC suppression at least partially presynaptially. Presynapti inhiition generally inreases the paired-pulse ratio (PPR), defined as the the amplitude of EPSC2/EPSC1. Reduing presynapti release proaility dereases vesiular depletion, so relatively more vesiles are availale for release on the seond stimulus. Although PPR hanges generally indiate the involvement of a presynapti mehanism, they do not exlude the involvement of additional postsynapti mehanisms. As a positive ontrol, we first verified that a purely presynapti manipulation (inhiiting voltage-dependent alium hannels with Cd 2+ ) produes a detetale inrease in PPR. (a) Representative responses to paired-pulse stimulation efore and after adding a su-saturating dose of Cd 2+ to the saline perfusate. () Summary of PPR hanges in five experiments measured in ontrol saline (lak) and after the addition of Cd 2+ (gray) (n = 5, p <.1, paired t-test). () Representative responses to paired-pulse stimulation efore and after GABA iontophoresis. (d) Summary of PPR hanges during either GABA iontophoresis (see Fig. 3d) or olfatory stimulation of lateral input (see Fig. 3a). For GABA: n = 7 without antagonists, p <.5; n = 5 in, p <.5; n = 5 in PCT, p <.5, paired t-tests. In the presene of oth antagonists, GABA auses a very small suppression of EPSCs (see Fig. 3), assoiated with a small ut nonsignifiant hange in PPR (n = 4, p =.22, paired t-test). This may reflet inomplete antagonism or a minor role for a pharmaologially distint GABA reeptor. For olfatory stimulation: n = 7, p <.1, paired t-test. The smaller PPR hanges oserved with odor ompared to GABA likely reflets the smaller EPSC suppression produed y olfatory stimulation. GABA suppressed EPSC1 to 15 ± 2% of the ontrol value, while odor suppressed EPSC1 to 46 ± 4% (mean ± s.e.m).

8 a eletrial GABA stimulation iontophoresis reord Evoked EPSC (% of aseline) Evoked EPSC (% of aseline) * Gal4 only (Or83-Gal4) 1 2 * 1 2 no antagonists PCT UAS only (UAS-PTX) no antagonists PCT Supplementary Figure 8. Negative ontrol for Gal4 driver and pertussis toxin transgene. In ontrol flies GABA iontophoresis suppresses EPSCs, and this suppression is insensitive to pirotoxin (PCT) (Fig. 3f). In ontrast, flies that express pertussis toxin speifially in ORNs display GABA-evoked EPSC suppression that is ompletely loked y pirotoxin (Fig. 4). Here we show that this phenotype requires oth the Gal4 driver and toxin transgene. All panels show mean ± s.e.m averaged aross experiments. Asterisk indiates GABA pulse (3-2 ms). (a) Experimental onfiguration. () Like ontrol flies, flies with only the Gal4 driver (Or83-Gal4/Or83-Gal4) are insensitive to PCT (n = 5). () Similarly, flies with only the UAS transgene (UAS-PTX/+) are also insensitive to PCT (n = 5).