Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation methods
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A strategy for retrospective labelling of active synapses
We also used the ORN-PN synapse to test both the sensitivity (by using low odour concentrations) and specificity of the syb:GRASP responses. As expected, low odour concentrations activated fewer glomeruli, while increasing odour concentrations resulted in a dose-dependent boost in fluorescence for some glomeruli, while others showed no further increase, and yet others only responded to high concentrations ( ). This likely results from different OR affinities for the odour. Moreover, a small battery of odours and odour concentrations produced different patterns of glomerular activation in both an odour-dependent and concentration-dependent manner ( ), in general good agreement with glomerular patterns recorded acutely in live imaging studies. Interestingly, the dynamic range of GRASP responses for each glomerulus correlated well with the dynamic range of firing for the ORN type that i...
Labelling of active thermosensory and visual synapses
To further test the ability of our system to retrospectively report specific patterns of synaptic activation in vivo, we turned to the thermosensory system. In Drosophila, rapid temperature changes are detected by dedicated hot and cold temperature-receptor neurons (TRNs) in the last antennal segment, the arista. The projections of these sensory neurons innervate the base of the AL, where hot and cold TRNs form two distinct, adjacent glomeruli defining a simple map for temperature representation (; ref. ). For this experiment, we expressed syb:spGFP1-10 in both hot and cold TRNs (using IR93a-LexA ), while CD4:spGFP11 was directed to a subset of their targets: a unique population of 'broadly tuned' thermosensory-projection neurons (tPNs) that respond to both heating and cooling and are required for behavioural aversion to both hot and cold temperature ranges (labelled b...
Fly strains
All strains developed for this study are listed in. Aop-CD4:spGFP11 and UAS-CD4:spGFP1-10 were gifts of K. Scott. Drivers GH146-Gal4, GH146-LexA, GH146-QF, Orco-Gal4, Orco-LexA, Or92a-Gal4, GR21a-Gal4, VGlut-Gal4, MB247-Gal4 and MB247-LexA are available from the Bloomington Stock Center. IR84a-LexA was a gift from R. Benton. panR8-Gal4 (ref.; a combination of rh5-Gal4 + rh6-Gal4 ) and Tm5c-LexA have been previously published, as have Ir93a-LexA and VT40053-Gal4 (ref. ). Flies used for came from regular laboratory fly vials, were not sexed and were 2-6 days old. Flies used in were all females (due to constraints of the genetic cross), and 6-7 days old-this is because the tdTomato signal was difficult to detect in younger flies.
Odour induction of syb:X-RASP
For these experiments, we crossed flies with the genotype Orco-Gal4/UAS-syb:spGFP1-10 with GH146-LexA/Aop-CD4:spGFP11 to obtain syb:GRASP between olfactory sensory neurons and GH146 projection neurons. To minimize the exposure of these flies to odours, cleaned pupae were placed individually into eppendorf tubes containing sorbitol agarose substrate (1 M sorbitol, 2% agarose). These tubes were capped with aluminium foil with pinholes to allow air flow, and the flies were placed in a well-ventilated lab space. Within 24 h of eclosion, the flies were separated into control and odour-exposed groups and placed in glass beakers with foil on top. For the odour-exposed groups, the beakers were placed in the fume hood, a small strip of filter paper was dipped into odorant (either pure or diluted in mineral oil), and placed into the beaker for 10 min. Then the paper was...
Temperature induction of syb:GRASP
Flies of the following genotype w; Aop-syb-spGFP1-10; UAS-CD4:spGFP11/+; IR93a-LexA/VT40053-Gal4 were raised at 25 °C. One-day old adults were placed in either 15 or 35 °C incubators for 10 min. The control group was left at 25 °C. Flies were briefly anaesthetized with CO 2, their brains were dissected in PBS, and the syb:GRASP signal at the posterior antennal lobe (PAL) was imaged immediately on a Prairie Ultima SGS two-photon microscope. In monochrome images were rendered using a fire LUT to emphasize differences in intensity. syb:GRASP fluorescence was measured from ROIs of the cold and hot glomerulus from the maximum intensity Z -projection output image of the PAL.
Light induction of syb:GRASP
R8-Tmc5 syb:GRASP flies (genotype panR8-Gal4 /ort C1a -LexADBD, OK371-dVP16AD; UAS-syb:spGFP1-10, LexAop-spGFP11::CD4/+ ) were used for these experiments. Before eclosion, larvae and pupae were kept on a 12 h light/dark cycle, then after eclosion, the flies were raised in constant dark or constant light (50 lx (14 W cm -2 ), white light) condition for 3-5 days. The brains were dissected, fixed in 4% PFA, and imaged using a Zeiss LSM780 confocal microscope as described in Karuppudurai et al.. Note that, in all cases we tested (olfactory, temperature and light induction) a few days (3-5) of exposure to normal ambient stimuli is sufficient to produce adequate syb:GRASP signals for circuit mapping (which can often be easily boosted by brief application of KCl, see above).
Two-choice assay for temperature preference behaviour
Our rapid two-choice assay for temperature preference has been described in detail before. In brief, 20 3-5 days old flies are ice-anaesthetized and placed in an arena consisting of four 1 inch square, individually addressable Peltier tiles. In each trial, flies are presented for 3 min with a choice between 25 °C and a test temperature (TT) between 10 and 40 °C at 5 °C intervals. Trials are paired so that each TT is presented twice, each time in different opposing quadrants and the position of flies is recorded during each trial (by a BASLER A601FM camera) to calculate an avoidance index (AI) for each TT AI=number of flies at 25 °C - number of flies at TT)/total number of flies).
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We also used the ORN-PN synapse to test both the sensitivity (by using low odour concentrations) and specificity of the syb:GRASP responses. As expected, low odour concentrations activated fewer glomeruli, while increasing odour concentrations resulted in a dose-dependent boost in fluorescence for some glomeruli, while others showed no further increase, and yet others only responded to high concentrations ( ). This likely results from different OR affinities for the odour. Moreover, a small battery of odours and odour concentrations produced different patterns of glomerular activation in both an odour-dependent and concentration-dependent manner ( ), in general good agreement with glomerular patterns recorded acutely in live imaging studies. Interestingly, the dynamic range of GRASP responses for each glomerulus correlated well with the dynamic range of firing for the ORN type that innervates it (as compiled from literature, see and ); a notable exception was the DM1 glomerulus, characterized by a particularly large syb:GRASP dynamic range.
To further test the ability of our system to retrospectively report specific patterns of synaptic activation in vivo, we turned to the thermosensory system. In Drosophila, rapid temperature changes are detected by dedicated hot and cold temperature-receptor neurons (TRNs) in the last antennal segment, the arista. The projections of these sensory neurons innervate the base of the AL, where hot and cold TRNs form two distinct, adjacent glomeruli defining a simple map for temperature representation (; ref. ). For this experiment, we expressed syb:spGFP1-10 in both hot and cold TRNs (using IR93a-LexA ), while CD4:spGFP11 was directed to a subset of their targets: a unique population of 'broadly tuned' thermosensory-projection neurons (tPNs) that respond to both heating and cooling and are required for behavioural aversion to both hot and cold temperature ranges (labelled by VT40053-Gal4, ). First, we confirmed that the targeting of syb:GRASP at this synapse had no effect on the flies' temperature preference behaviour in our rapid two-choice assay ( ). Next, we exposed flies to a brief temperature shift from 25 °C to either 15 °C or 35 ...
All strains developed for this study are listed in. Aop-CD4:spGFP11 and UAS-CD4:spGFP1-10 were gifts of K. Scott. Drivers GH146-Gal4, GH146-LexA, GH146-QF, Orco-Gal4, Orco-LexA, Or92a-Gal4, GR21a-Gal4, VGlut-Gal4, MB247-Gal4 and MB247-LexA are available from the Bloomington Stock Center. IR84a-LexA was a gift from R. Benton. panR8-Gal4 (ref.; a combination of rh5-Gal4 + rh6-Gal4 ) and Tm5c-LexA have been previously published, as have Ir93a-LexA and VT40053-Gal4 (ref. ). Flies used for came from regular laboratory fly vials, were not sexed and were 2-6 days old. Flies used in were all females (due to constraints of the genetic cross), and 6-7 days old-this is because the tdTomato signal was difficult to detect in younger flies.
For these experiments, we crossed flies with the genotype Orco-Gal4/UAS-syb:spGFP1-10 with GH146-LexA/Aop-CD4:spGFP11 to obtain syb:GRASP between olfactory sensory neurons and GH146 projection neurons. To minimize the exposure of these flies to odours, cleaned pupae were placed individually into eppendorf tubes containing sorbitol agarose substrate (1 M sorbitol, 2% agarose). These tubes were capped with aluminium foil with pinholes to allow air flow, and the flies were placed in a well-ventilated lab space. Within 24 h of eclosion, the flies were separated into control and odour-exposed groups and placed in glass beakers with foil on top. For the odour-exposed groups, the beakers were placed in the fume hood, a small strip of filter paper was dipped into odorant (either pure or diluted in mineral oil), and placed into the beaker for 10 min. Then the paper was removed, and the beaker left open for 10 min, and then a new strip was dipped in odour, placed in the beaker, and recovered with foil. This was repeated for a total of four odour exposures. After the last exposure, flies were left in the fume hood, beaker uncovered for 1 h (or for 1, 4 a...
Flies of the following genotype w; Aop-syb-spGFP1-10; UAS-CD4:spGFP11/+; IR93a-LexA/VT40053-Gal4 were raised at 25 °C. One-day old adults were placed in either 15 or 35 °C incubators for 10 min. The control group was left at 25 °C. Flies were briefly anaesthetized with CO 2, their brains were dissected in PBS, and the syb:GRASP signal at the posterior antennal lobe (PAL) was imaged immediately on a Prairie Ultima SGS two-photon microscope. In monochrome images were rendered using a fire LUT to emphasize differences in intensity. syb:GRASP fluorescence was measured from ROIs of the cold and hot glomerulus from the maximum intensity Z -projection output image of the PAL.
R8-Tmc5 syb:GRASP flies (genotype panR8-Gal4 /ort C1a -LexADBD, OK371-dVP16AD; UAS-syb:spGFP1-10, LexAop-spGFP11::CD4/+ ) were used for these experiments. Before eclosion, larvae and pupae were kept on a 12 h light/dark cycle, then after eclosion, the flies were raised in constant dark or constant light (50 lx (14 W cm -2 ), white light) condition for 3-5 days. The brains were dissected, fixed in 4% PFA, and imaged using a Zeiss LSM780 confocal microscope as described in Karuppudurai et al.. Note that, in all cases we tested (olfactory, temperature and light induction) a few days (3-5) of exposure to normal ambient stimuli is sufficient to produce adequate syb:GRASP signals for circuit mapping (which can often be easily boosted by brief application of KCl, see above).
Our rapid two-choice assay for temperature preference has been described in detail before. In brief, 20 3-5 days old flies are ice-anaesthetized and placed in an arena consisting of four 1 inch square, individually addressable Peltier tiles. In each trial, flies are presented for 3 min with a choice between 25 °C and a test temperature (TT) between 10 and 40 °C at 5 °C intervals. Trials are paired so that each TT is presented twice, each time in different opposing quadrants and the position of flies is recorded during each trial (by a BASLER A601FM camera) to calculate an avoidance index (AI) for each TT AI=number of flies at 25 °C - number of flies at TT)/total number of flies).
Machine-readable layer
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