Figures and data in Switch-like and persistent memory formation in individual Drosophila larvae

Figures
Tables
Additional files

4 figures, 5 tables and 2 additional files

Figures

Figure 1 with 3 supplements

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Y-maze assay to quantify innate and learned preference.

(A) Image sequence of a larva making two consecutive decisions in the Y-maze assay. White arrows indicate direction of air flow; red arrow shows direction of larva’s head. (B) Probability of choosing channel containing CO₂ without any training. (C) Schematic representation of experiments in (**D,E,F**). All larvae were tested in the Y-maze for 1 hr to determine initial preference and again following manipulation to determine a final preference. The manipulations were: Paired Training - reward in concert with CO₂ presentation, 15 s intervals, 20 repetitions; Offset After - reward presentation 7.5 s after CO₂ onset, 15 s intervals, 20 repetitions; Reverse-Paired Training - reward opposite CO₂ presentation, 15 s intervals, 20 repetitions; Offset Before - reward presentation 7.5 s before CO₂ onset, 15 s intervals, 20 repetitions; DAN Activation Without CO₂ - CO₂ is never presented, while reward is presented at 15 s intervals, 20 repetitions; no training - no manipulation between two testing periods; Forward Paired (extended spacing) - 15 s reward follows 15 s CO₂ presentation, followed by 60 s of air, 20 repetitions; Backwards Paired (extended spacing) - 15 s reward prior to 15 s CO₂ presentation, followed by 60 s of air, 20 repetitions; Reward Between CO₂ (extended spacing) - 15 s reward presentation between two 15 s CO₂ presentations, followed by 45 s of air, 20 repetitions. (D) Probability of choosing CO₂ containing channel before and after manipulation. All animals were fed ATR supplemented food, except those marked ATR-. (E) Probability of choosing CO₂ containing channel before and after training as a function of reward timing, in training protocols with extended air spacings. All animals were DANi1>CsChrimson and fed ATR. (F) Probability of choosing CO₂ containing channel before and after 20 cycles of paired training, as a function of CO₂ concentration, used both during training and testing. All animals were DANi1>CsChrimson and fed ATR. * p<0.05, ** p<0.01, *** p<0.001.

Figure 1—source data 1 Spreadsheet containing each individual animal’s decisions in temporal sequence.: https://cdn.elifesciences.org/articles/70317/elife-70317-fig1-data1-v1.xlsx
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Figure 1—video 1

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posterframe for video — Recording of a larva making 2 decisions within the Y-maze.

The direction of airflow and the larva’s decisions are noted. Video was recorded at 20 frames per second; the playback speed of 25 fps represents 1.25x real time.

Figure 1—video 2

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Figure 1—video 3

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Figure 2

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Dose dependence of learning DANi1>CsChrimson were given varying cycles of paired training (as in Figure 1C).

(A) Probability of choosing CO₂ containing channel before and after training, as a function of amount of training. *** p<0.001. (B) Histograms of individual larva preferences after training, grouped by number of training cycles. (C) Histogram of individual larva preference after training for all larvae. (D) Population average probability of choosing CO₂ following training vs. dose. (E) Fraction of larvae untrained vs. number of training cycles. Teal: fit parameters and error ranges from quantized model, purple lines, prediction and error ranges from memoryless model. Note logarithmic y-axis on insert. (**C–E**) Orange: graded model prediction - post-training preference is represented by a single Gaussian distribution whose mean and variance depend on amount of training; Teal: quantized model prediction - post-training preference is represented by two fixed Gaussian distributions and the fraction of larvae in each population depends on the amount of training; Purple: all-or-none model prediction - post-training preference is represented by two fixed Gaussian distributions and the effect of a single training cycle is to train a fixed fraction of the remaining untrained larvae.

Figure 2—source data 1 Spreadsheet containing each individual animal’s decisions in temporal sequence.: https://cdn.elifesciences.org/articles/70317/elife-70317-fig2-data1-v1.xlsx
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Figure 3 with 3 supplements

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Memory extinction (A) Testing and training protocols for B,C.

Training + Extinction: larvae were exposed to 18 cycles of alternating CO₂ and air following training. Habituation + Training: larvae were exposed to 18 cycles of alternating CO₂ and air prior to training. (B) Probability of choosing CO₂ containing channel (top) and fraction of larvae in trained group according to double Gaussian model fit (bottom) before and after training scheme. (C) Histograms of individual larva preference after training, for all larva and for larva trained with 2–4 training cycles. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3—source data 1 Spreadsheet containing each individual animal’s decisions in temporal sequence.: https://cdn.elifesciences.org/articles/70317/elife-70317-fig3-data1-v1.xlsx
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Figure 3—figure supplement 1

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After extinction, larvae can be trained again.

(A) Testing and training protocol for B,C. Larvae were trained with three cycles of paired training, followed by 18 extinction cycles of alternating CO₂ and air with no reward presented. After extinction, larvae were presented with three additional paired training cycles before testing. (B) Probability of choosing CO₂ containing channel before and after training scheme. (C) Fraction of larvae in trained group according to double Gaussian model fit before and after training scheme. All larvae were DAN-i1>CsChrimson and raised on ATR+ food. *** p<0.001.

Figure 3—figure supplement 2

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Larvae population average response following training.

(A) Larvae population average response in the first ten minutes following training (0–10 min), compared to the latter fifty minutes of testing (10–60 min), for larvae that had been given 2, 5, or 20 cycles of paired training. (B) Larvae population average response for the first five choices made by the larvae following training, compared to the remaining choices, for larvae that had been given 2, 5, or 20 cycles of paired training and made at least 10 decisions following training. (**C,D,E**) Larvae population average response over 15-min segments following training, for larvae trained with (C) 2 cycles, (D) 5 cycles, or (E) 20 cycles of training. All larvae were DAN-i1>CsChrimson and raised on ATR+ food.

Figure 3—figure supplement 3

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Larvae given additional training between testing periods.

(A) Testing and training protocols for experiments in B. All larvae are tested in the Y-maze for one hour to determine initial preference. Larvae were then trained with 10 cycles of paired training, followed by a 15-min test period. The 10 cycle train/15 min test was repeated four times. (B) Probability of choosing CO₂-containing channel before training, and during each of the four test periods. All larvae were DAN-i1>CsChrimson and raised on ATR+ food. *** p <0.001.

Figure 4

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Memory retention overnight.

(A) Testing and training protocols. Except where indicated, larvae were tested, trained immediately after testing, tested again, then placed on food overnight and tested the following day. For extinction experiments, larvae were trained three times, and then exposed to 18 cycles of alternating CO₂ and air either immediately following training or prior to testing the next day. (**B,C,D**) Probability of choosing CO₂ containing channel (top) and fraction of larvae in trained group according to double Gaussian model fit (bottom) prior to training, immediately following training, and the next day. When the center bar is missing, larvae were not tested immediately following training but instead removed immediately to food. M Nx = massed training, N repetitions, S 10x = spaced training 10 total pairings, RP = reverse paired (see Figure 1C), No Train = no training. Larvae in (**B,C**) were DANi1>CsChrimson. Larvae in (D) were DANi1>hs-dCREB2-b;CsChrimson. Larvae were raised on food containing ATR, except for ATR+/CXM-, ATR+/CXM+ larvae who were fed ATR supplemented yeast paste (without/with cycloheximide) for 4 hr prior to initial testing. For reverse-paired (RP) and no training schemes, see Figure 1B. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4—source data 1 Spreadsheet containing each individual animal’s decisions in temporal sequence.: https://cdn.elifesciences.org/articles/70317/elife-70317-fig4-data1-v1.xlsx
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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. melanogaster)	w[1118]; P{y[+t7.7]w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP2 (w;;UAS-CsChrimson)	Bloomington Stock Center	RRID:BDSC_55136
Genetic reagent (D. melanogaster)	SS00864 split-Gal4 (DAN-i1-Gal4)	Saumweber et al., 2018		Gift of Marta Zlatic, Janelia Research Campus
Genetic reagent (D. melanogaster)	w[*]; Gr63a[1]	Bloomington Stock Center	RRID:BDSC_9941
Genetic reagent (D. melanogaster)	w[1118]; P{y[+t7.7] w[+mC]=GMR58E02-GAL4}attP2 (GMR58E02-Gal4)	Bloomington Stock Center	RRID:BDSC_41347
Genetic reagent (D. melanogaster)	w;hs-dCREB2-b 17–2	Yin et al., 1995	FlyBase_ FBti0038019	Gift of Jerry Chi-Ping Yin, University of Wisconsin, Madison
Genetic reagent (D. melanogaster)	w[*]; P{w[+mW.hs]=GawB}ey[OK107]/In(4)ci[D], ci[D] pan[ciD] sv[spa-pol] (OK107-Gal4)	Bloomington Stock Center	RRID:BDSC_854
Genetic reagent (D. melanogaster)	w[*]; P{w[+mC]=UAS-Hsap\KCNJ2.EGFP}7 (UAS-kir2.1)	Bloomington Stock Center	RRID:BDSC_6595
Genetic reagent (D. melanogaster)	w[*]; P{w[+mC]=Gr21a-GAL4.C}133t52.1 (Gr21a-Gal4)	Bloomington Stock Center	RRID:BDSC_23890
Software, algorithm	livetracker	github.com/GershowLab/TrainingChamber (copy archived at URL swh:1:rev:e2a7ccc4e8d845e6cac59d3b2f344cca826c4727, Lesar, 2021)	This work

Table 1

Crosses used to generate larvae for experiments throughout this work.

For strain information, see key resource table.

Figure	Designation	Female parent	Male parent
1	Gr63a¹	w;Gr63a¹
1	OK107>Kir2.1	UAS-Kir2.1-GFP	OK107-Gal4
1	Gr21a>Kir2.1	UAS-Kir2.1-GFP	Gr21a-Gal4
1-4	DANi1>CsChrimson	w;;UAS-CsChrimson	SS00864
1	Driver ctrl	SS00864
1	Effector ctrl	w;;UAS-CsChrimson
1	58E02>CsChrimson	w;;UAS-CsChrimson	58E02-Gal4
4	hs-dcreb2-b;DANi1>CsChrimson	w;hs-dcreb2-b;UAS-CsChrimson	SS00864

Table 2

Data for experiments in Figure 1, Figure 2, Figure 3, and Figure 4.

# Larva: number of individual larvae tested for experiment type; # Approach Pre-Train: total number of times all larvae chose the channel containing air with CO₂ prior to training; # Avoid Pre-Train: total number of times all larvae chose the channel containing pure air prior to training; # Approach Post-Train: total number of times all larvae chose the channel containing air with CO₂ after the indicated training scheme; # Avoid Post-Train: total number of times all larvae chose the channel containing pure air after the indicated training scheme; # Approach Next Day: total number of times all larvae chose the channel containing air with CO₂ during testing approximately 20 hr after training; # Avoid Next: total number of times all larvae chose the channel containing pure air during testing approximately 20 hr after training. All tests were 1 hr (for each larva).

Experiment	Genotype	# Larva	# Approach Pre-Train	# Avoid Pre-Train	# Approach Post-Train	# Avoid Post-Train	# Approach Next Day	# Avoid Next Day
Figure 1B
Gr63a¹	Gr63a¹	44	831	745	-	-	-	-
DANi1> CsChrimson, ATR+	DANi1> CsChrimson	159	1714	4978	-	-	-	-
DANi1> CsChrimson, ATR-	DANi1> CsChrimson	16	256	614	-	-	-	-
Figure 1D
Paired	DANi1> CsChrimson	64	561	1760	936	868	-	-
Offset After	DANi1> CsChrimson	20	288	757	316	305	-	-
Reverse Paired	DANi1> CsChrimson	29	315	1022	154	530	-	-
Offset Before	DANi1> CsChrimson	19	218	512	136	315	-	-
Paired, ATR-	DANi1> CsChrimson	16	256	614	127	307	-	-
No Training	DANi1> CsChrimson	50	578	1599	479	1295	-	-
DAN w/o CO₂	DANi1> CsChrimson	16	260	597	161	354	-	-
Driver ctrl	SS00864	17	110	289	158	358	-	-
Effector ctrl	UAS-CsChrimson	18	214	516	114	294	-	-
58E02> CsChrimson	58E02> CsChrimson	21	380	912	493	501	-	-
Figure 1E	DANi1> CsChrimson
Forward Paired		22	181	496	350	337	-	-
Backwards Paired		18	181	438	124	320	-	-
Btw CO₂		23	272	652	165	283	-	-
Figure 1F	DANi1> CsChrimson
6.5%		19	361	568	319	290	-	-
8%		27	256	567	295	255	-	-
15%		19	170	368	249	233	-	-
18%		64	561	1760	936	868	-	-
Figure 2A	DANi1> CsChrimson
0 Cycles		50	578	1599	479	1295	-	-
1 Cycles		35	218	606	317	495	-	-
2 Cycles		87	840	2552	1081	1292	-	-
3 Cycles		31	310	930	686	686	-	-
4 Cycles		32	245	712	493	511	-	-
5 Cycles		63	863	2491	975	993	-	-
10 Cycles		14	100	287	154	144	-	-
20 Cycles		64	561	1760	936	868	-	-
Figure 3B	DANi1> CsChrimson
2 Cycles, Training		87	840	2552	1081	1292	-	-
2 Cycles, Habituation + Training		30	385	1127	422	554	-	-
2 Cycles, Training + Extinction		30	336	946	375	793	-	-
3 Cycles, Training		30	308	924	675	679	-	-
3 Cycles, Habituation + Training		18	222	591	260	294	-	-
3 Cycles, Training + Extinction		26	279	695	195	416	-	-
4 Cycles, Training		30	225	659	490	502	-	-
4 Cycles, Habituation + Training		18	239	701	372	352	-	-
4 Cycles, Training + Extinction		27	384	1074	394	475	-	-
5 Cycles, Training		63	863	2491	975	993	-	-
6 Cycles, Habituation + Training		19	266	758	367	324	-	-
6 Cycles, Training + Extinction		18	253	687	309	317	-	-
10 Cycles, Training		14	100	287	154	144	-	-
10 Cycles, Habituation + Training		30	406	1193	607	503	-	-
10 Cycles, Training + Extinction		30	426	1180	401	386	-	-
Figure 4B	DANi1> CsChrimson
20x		28	380	1172	509	499	459	409
20x (Only Test Next Day)		14	224	768	-	-	296	250
5x		29	472	1427	488	480	404	461
2x		42	514	1537	594	693	201	548
2x (Only Test Next Day)		22	209	696	-	-	213	283
No Train		20	316	889	187	544	104	337
RP 20x		21	282	905	121	430	109	361
Ext Post-Train		23	181	477	-	-	158	365
Ext Pre-Test		31	417	1002	-	-	385	429
Figure 4C	DANi1> CsChrimson
M 20x (CXM+/ATR+)		20	110	282	252	237	237	272
M 20x (CXM-/ATR+)		17	159	419	271	236	228	235
S 20x (CXM+/ATR+)		23	191	486	-	-	150	316
S 20x (CXM-/ATR+)		20	197	511	-	-	254	264
M 10x (CXM+/ATR+)		23	136	345	-	-	331	344
M 10x (CXM-/ATR+)		20	175	454	-	-	419	375
Figure 4D	DANi1> hs-dCREB2-b;CsChrimson
M 10x HS		21	175	434	392	370	253	246
M 10x No HS		22	248	656	367	353	451	490
S 10x HS		24	172	420	68	156	153	339
S 10x No HS		22	294	736	212	184	335	352

Table 3

p-Values for experiments in Figure 1, Figure 2, Figure 3, and Figure 4.

P-values for experiments were calculated: Bootstrap - p-values calculated as explained in Materials and methods; Fisher - p-values calculated using Fisher’s exact test; U-test - p-values calculated using two-sided Mann–Whitney U test. Unless otherwise noted, p-values are calculated between pre-train and post-train data. A shaded row indicates not all tests reach the same significance level (out of ns, p <0.05, p <0.01, p <0.001).

Experiment	Genotype	Hierarchical Bootstrap	Bootstrap Animal Only	Fisher	U-test
Figure 1B
Gr63a¹/DANi1> CsChrimson, ATR+		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Gr63a¹/DANi1> CsChrimson, ATR-		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 1D
Paired	DANi1> CsChrimson	<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Offset After	DANi1> CsChrimson	<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Reverse Paired	DANi1> CsChrimson	0.3429	0.2689	0.6166	0.9379
Offset Before	DANi1> CsChrimson	0.4479	0.4373	0.9479	0.9770
Paired, ATR-	DANi1> CsChrimson	0.4762	0.4315	1.000	0.2658
No Training	DANi1> CsChrimson	0.4066	0.3664	0.7726	0.9835
DAN w/o CO₂	DANi1> CsChrimson	0.3935	0.3102	0.7173	0.4852
Driver ctrl	SS00864	0.3106	0.0313	0.3411	0.3977
Effector ctrl	UAS-CsChrimson	0.3383	0.2361	0.6336	0.8366
58E02> CsChrimson	58E02> CsChrimson	<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 1C	DANi1> CsChrimson
Forward Paired		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Backwards Paired		0,3368	0.163	0.6801	0.1939
Btw CO₂		0.0107	0.0001	0.006543	0.0003257
Figure 1D	DANi1> CsChrimson
6.5%		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
8%		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
15%		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
18%		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 2A	DANi1> CsChrimson
0 Cycles		0.4132	0.3647	0.7726	0.9835
1 Cycles		0.0003	<10⁻⁴	<10⁻⁴	0.0591
2 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
3 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
4 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
5 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
10 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
20 Cycles		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 3B	DANi1> CsChrimson
2 Cycles, Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
2 Cycles, Habituation + Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
2 Cycles, Training + Extinction		0.0117	0.0020	0.001339	0.04743
3 Cycles, Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
3 Cycles, Habituation + Training		<10⁻⁴	<10⁻⁴	0.0007459	<10⁻⁴
3 Cycles, Training + Extinction		0.1133	0.0176	0.1763	0.03069
4 Cycles, Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
4 Cycles, Habituation + Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
4 Cycles, Training + Extinction		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
5 Cycles, Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
6 Cycles, Habituation + Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
6 Cycles, Training + Extinction		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
10 Cycles, Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
10 Cycles, Habituation + Training		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
10 Cycles, Training + Extinction		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 4B	DANi1> CsChrimson
20x Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
20x Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
20x (Only Test Next Day) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
5x Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
5x Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
2x Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
2x Pre-Test/Next Day		0.2086	0.0501	0.3524	0.07216
2x (Only Test Next Day) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
No Train Pre-Test/Post-Test		0.4035	0.3319	0.7893	0.2003
No Train Pre-Test/Next Day		0.1583	0.0530	0.3071	0.8884
RP 20x Pre-Test/Post-Test		0.2677	0.1507	0.4276	0.7396
RP 20x Pre-Test/Next Day		0.4205	0.3481	0.8474	0.3765
Ext Post-Train Pre-Test/Next Day		0.1801	0.0146	0.3315	0.01336
Ext Pre-Test Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 4C	DANi1> CsChrimson
M 20x (CXM+/ATR+) Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 20x (CXM+/ATR+) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 20x (CXM-/ATR+) Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 20x (CXM-/ATR+) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
S 10x (CXM+/ATR+) Pre-Test/Next Day		0.1099	0.014	0.1671	0.02985
S 10x (CXM-/ATR+) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 10x (CXM+/ATR+) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 10x (CXM-/ATR+) Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
Figure 4D	DANi1> hs-dCREB2-b;CsChrimson
M 10x, HS Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 10x, HS Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 10x, No HS Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
M 10x, No HS Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
S 10x, HS Pre-Test/Post-Test		0.3804	0.2830	0.7310	0.2750
S 10x, HS Pre-Test/Next Day		0.2645	0.08860	0.4650	0.3802
S 10x, No HS Pre-Test/Post-Test		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴
S 10x, No HS Pre-Test/Next Day		<10⁻⁴	<10⁻⁴	<10⁻⁴	<10⁻⁴

Table 4

Model fits to data in Figure 2.

Shifting Mean and $\tilde{σ}$ , shifting fraction, and exponential fraction models are presented in Figure 2. Model name: name of the model. Formula: expression for the probability of the data given the model and its parameters. # params: number of free parameters in the model. $Δ \log (P)$ logarithm of the probability of the data given best fit to this model minus logarithm of the probability of the data given the best fit model overall. A higher (less negative) value means the model better fits the data without regard to the number of parameters. $Δ A I C$ , $Δ B I C$ - Aikake and Bayes Information Criterion minus the lowest values over the models tested. Lower numbers indicate model is favored. According to both criterion, the exponential fraction model is strongly favored over the shifting fraction model, and the shifting fraction model is strongly favored over all models except the exponential fractional model.

Model name	Formula	# params	$Δ \log (P)$	$Δ AIC$	$Δ BIC$
Shifting Mean (fixed $\tilde{σ}$ )	$P \propto \prod_{n_{c} \in (0, 1, 2, 3, 4, 5, 10, 20)} \prod_{j} 𝒩 (p (n_{c}, j), μ (n_{c}), \tilde{σ} \sqrt{\frac{μ (n_{c}) * (1 - μ (n_{c}))}{n (n_{c}, j)}})$	9	−42.7	84.86	104.45
Shifting Mean and $σ$ (Graded learning)	$P \propto \prod_{n_{c} \in (0, 1, 2, 3, 4, 5, 10, 20)} \prod_{j} 𝒩 (p (n_{c}, j), μ (n_{c}), \frac{σ (n_{c})}{\sqrt{n (n_{c}, j)}})$	16	−12.9	39.3	86.3
Shifting Fraction (Quantized learning)	$P \propto \prod_{n_{c} \in (0, 1, 2, 3, 4, 5, 10, 20)} \prod_{j} f_{u} (n_{c}) 𝒩 (p (n_{c}, j), μ_{u}, \tilde{σ} \sqrt{\frac{μ_{u} * (1 - μ_{u})}{n (n_{c}, j)}}) +$ … $(1 - f_{u} (n_{c})) 𝒩 (p (n_{c}, j), μ_{t}, \tilde{σ} \sqrt{\frac{μ_{t} * (1 - μ_{t})}{n (n_{c}, j)}})$	11	−3.93	11.3	38.7
Shifting Fraction (3 clusters)	$P \propto \prod_{n_{c} \in (0, 1, 2, 3, 4, 5, 10, 20)} \prod_{j} f_{1} (n_{c}) 𝒩 (p (n_{c}, j), μ_{1}, \tilde{σ} \sqrt{\frac{μ_{1} * (1 - μ_{1})}{n (n_{c}, j)}}) +$ … $f_{2} (n_{c}) 𝒩 (p (n_{c}, j), μ_{2}, \tilde{σ} \sqrt{\frac{μ_{2} * (1 - μ_{2})}{n (n_{c}, j)}}) +$ … $(1 - f_{1} (n_{c}) - f_{2} (n_{c})) 𝒩 (p (n_{c}, j), μ_{3}, \tilde{σ} \sqrt{\frac{μ_{3} * (1 - μ_{3})}{n (n_{c}, j)}})$	20	0	21.4	84.1
Exponential Fraction (All-or-none)	$P \propto \prod_{n_{c} \in (0, 1, 2, 3, 4, 5, 10, 20)} \prod_{j} λ^{n_{c}} 𝒩 (p (n_{c}, j), μ_{u}, \tilde{σ} \sqrt{\frac{μ_{u} * (1 - μ_{u})}{n (n_{c}, j)}}) +$ … $(1 - λ^{n_{c}}) 𝒩 (p (n_{c}, j), μ_{t}, \tilde{σ} \sqrt{\frac{μ_{t} * (1 - μ_{t})}{n (n_{c}, j)}})$	4	−5.3	0	0

Symbol	Definition	Symbol	Definition
$n_{c}$	number of training cycles	$p (n_{c}, j)$	fraction of times $j^{t h}$ larva chose CO₂ after $n_{c}$ cycles
$μ (n_{c})$	mean probability of choosing CO₂ after $n_{c}$ training cycles	$n (n_{c}, j)$	# choices made by $j^{t h}$ larva after $n_{c}$ training cycles
$\tilde{σ}$	global adjustment to binomial standard deviation	$σ (n_{c})$	training dependent standard deviation
$μ_{u}$	probability of larva in untrained group choosing CO₂	$μ_{t}$	probability of larva in trained group choosing CO₂
$f_{u} (n_{c})$	fraction of larvae in untrained group after $n_{c}$ cycles	$μ_{1}, μ_{2}, μ_{3}$	probability of larva in group 1,2,3 choosing CO₂
$f_{1} (n_{c}), f_{2} (n_{c})$	fraction of larvae in groups 1,2 after $n_{c}$ cycles	$λ$	fraction of larvae not trained after one cycle
$𝒩 (x, μ, σ)$	normal cdf: $\frac{1}{\sqrt{2 π σ^{2}}} e^{- \frac{{(x - μ)}^{2}}{2 σ^{2}}}$	$Δ \log (P)$	relative log probability of data given model
AIC	Aikake Information Criterion: $2 k - 2 \log (P)$ , k = # params	$Δ AIC$	AIC - lowest AIC
BIC	Bayes Information Criterion: $k \log (n_{A}) - 2 \log (P)$ , k = # params, $n_{A}$ = # animals	$Δ BIC$	BIC - lowest BIC