A neurotrophin functioning with a Toll regulates structural plasticity in a dopaminergic circuit

Jun Sun
Francisca Rojo-Cortés
Suzana Ulian-Benitez
Manuel G Forero
Guiyi Li
Deepanshu Singh
Xiaocui Wang
Sebastian Cachero
Marta Moreira
Dean Kavanagh
Gregory Jefferis
Vincent Croset
Alicia Hidalgo author has email address

Structural Plasticity & Regeneration Lab, Birmingham Centre for Neurogenetics, University of Birmingham, UK
Buck Institute for Research on Aging, California, US
Semillero Lún, Grupo D+Tec, Universidad de Ibagué, Colombia
MRC LMB, Cambridge, UK
Institute of Biomedical Research, University of Birmingham, UK
Department of Biosciences, Durham University, UK

https://doi.org/10.7554/eLife.102222.1

Open access
Copyright information

Figures and data

Neurons expressing DNT-2 and its receptors Toll-6 and kek-6 in the adult brain
(A) DNT-2A expressing neurons (DNT-2>FlyBow1.1 in green; anti-Brp in magenta) have cell bodies in SOG and project to FLA/PRW and SMP. (B,D) Pre-synaptic (green) and post-synaptic (magenta) terminals of DNT-2A neurons seen with DNT-2>DenMark::RFP, Dsyd1::GFP, higher magnification in (D), different specimens from (B). DNT-2A projections at SMP and PRW have both pre- and post-synaptic sites. (C) Single neuron DNT-2A>MCFO clones. (E) Left: DNT-2A neurons have the vesicular glutamate transporter vGlut (arrows). Right: Colocalization between Dop2RLex>AopCD8-GFP and DNT2Gal4>UASCD8-RFP in cell bodies of DNT-2A neurons (arrows). (F) Terminals of dopaminergic neurons (TH>mCD8GFP) abut and overlap those of DNT-2A neurons (DNT2>CD8-RFP, magenta), arrows; magnified projections on the right. (G) Illustration of neurons expressing DNT-2 (magenta) and KCs, DAN PAM and PPL1, and DAL neurons (H) Toll-6>FlyBow1.1 is expressed in Kenyon cells, PPL1, PPL2 and PAM DANs, as revealed by co-localization with anti-TH. (I) kek-6>FlyBow1.1 co-localises with TH in MB vertical lobes, dopaminergic PALs, VUMs, PPL1, PPM2 and PPM3. SMP: superior medial protocerebrum, PRW: Prow, FLA: Flange, SOG: sub-aesophageal ganglion. Scale bars: (A-C, F-left, H, I) 50µm; (D,E,F-right) 25µm.

DNT-2 neurons are functionally connected to dopaminergic neurons
(A) TransTango revealed out-puts of DNT-2 neurons. All neurons express TransTango and expression of the TransTango ligand in DNT-2 neurons identified DNT-2 out-puts with Tomato (anti-DsRed). TransTango identified as DNT-2 outputs KC α’ ’ MB lobes (anterior brain, arrow, top left); Kenyon and possibly DAN cell bodies (posterior, arrows top right); DAL neurons (bottom left, arrows) and the dorsal layer of the fan shaped body (bottom right, arrow). See also Supplementary Figure S3 for further controls. (B) BAcTrace tests connectivity to a candidate neuron input visualised with LexAop>sybGFP by driving the expression of a ligand from DNT-2GAL4 that will activate QUASTomato in the candidate input neuron (Cachero et al. 2020). Candidate PAM neurons visualised at SMP with GFP (green): Control: R58E02LexA>BAcTrace 806, no GAL4. Test: R58E02LexA, DNT-2GAL4>BAcTrace 806 revealed PAMs are inputs of DNT-2A neurons at SMP (Tomato, bottom). Magenta shows QUAS-Tomato. (C) qRT-PCR showing that TH mRNA levels increased with DNT-2 over-expression at 30°C (tubGAL80^ts, DNT-2>DNT-2FL). One way ANOVA, P=0.0085; post-doc Dunnett’s multiple comparison test. (D) FRET cAMP probe Epac1 revealed that DNT-2>Dop2R-RNAi knockdown decreased YFP/CFP ratio over time in DNT-2A neurons, meaning that cAMP levels increased. Two-way ANOVA, genotype factor p<0.0001, time factor p<0.0001; post-doc Dunnett’s. (E,F) Summary: DNT-2 neurons and DANs are functionally connected and modulate each other. (E) DNT-2 can induce TH expression in DANs; (F) this is followed by negative feedback from DANs to DNT-2 neurons. TH: tyrosine hydroxylase. Scale bars: (A) 50µm; (B) 30µm; (D) 20µm. P values over graphs in (C) refer to group analyses; stars indicate multiple comparisons tests. *p<0.05,**p<0.01, ***p<0.001. For sample sizes and further statistical details, see Supplementary Table S2.

DNT-2 and Toll-6 maintain PAM neuron survival in the developing and adult brain.
(A) Illustration of PAM neuronal cell bodies and experimental temporal profile. DANs are shown in green, DNT-2A neurons in magenta and MB in dark grey. The left hemisphere shows the anterior brain with PAL and PAM DAN neurons (green) and DNT-2 neurons (magenta); the right shows the posterior brain, with the calyx and other DAN neurons (PPM1, PPM2, PPM3, PPL1, PPL2, green). (B-D) Fruit-flies were kept constantly at 25°C, from development to adult. Analyses done in adult brains. (B) DNT-2³⁷/DNT-2¹⁸ mutants had fewer histone-YFP+ labelled PAM neurons (THGAL4, R58E02-GAL4>hisYFP). Un-paired Student t-test. (C) Toll-6 RNAi knock-down in all DANs (THGAL4, R58E02-GAL4>hisYFP, Toll-6 RNAi) reduced Histone-YFP+ labelled PAM cell number. Un-paired Student t-test. (D) DNT-2³⁷/DNT-2¹⁸ mutants had fewer PAMs stained with anti-TH antibodies. Over-expressing Toll-6^CY in DANs (THGAL4, R58E02 GAL4>hisYFP, Toll-6^CY) rescued TH+ PAM neurons in DNT-2³⁷/DNT-2¹⁸ mutants, demonstrating that DNT-2 functions via Toll-6 to maintain PAM cell survival. Welch ANOVA p<0.0001, post-hoc Dunnett test. (E-H) Adult specific restricted over-expression or knock-down at 30°C, using the temperature sensitive GAL4 repressor tubGAL80^ts. (E) Adult specific DNT-2 RNAi knockdown in DNT-2 neurons decreased Tomato+ PAM cell number (tubGAL80^ts, R58E02-LexA, DNT-2 GAL4>LexAOP-Tomato, UAS DNT-2-RNAi). Un-paired Student t-test, p= 0.005. (F) Adult specific Toll-6 RNAi knock-down in Toll-6³¹ heterozygous mutant flies in DANs, reduced Histone-YFP+ PAM cell number (tubGAL80^ts; THGAL4, R58E02-GAL4>hisYFP, Toll-6 RNAi/Toll6³¹). Un-paired Student t-test. (G) PAMs were visualised with anti-TH. Left: tubGAL80^ts, Toll-6>Toll-6-RNAi knock-down decreased TH+ PAM cell number. Un-paired Student t-test. Right: tubGAL80^ts, DNT-2>DNT-2-RNAi knock-down decreased TH+ PAM cell number. Kruskal-Wallis ANOVA, p=0.0001, post-hoc Dunn’s test. (H) Adult-specific tubGAL80^ts, DNT-2>DNT-2RNAi knock-down increased the number of apoptotic cells in the brain labelled with anti-DCP-1. Dcp-1+ cells co-localise with anti-TH at least in PAM clusters. One Way ANOVA, p<0.0001, post-doc Bonferroni’s multiple comparisons test. DANs>histone-YFP: all dopaminergic neurons expressing histone-YFP, genotype: THGal4 R58E02Gal4>UAS-histoneYFP. PAMsLexA>tomato: restricted to PAM DANs: R58E02LexA>LexAop-nlstdTomato. Controls: GAL4 drivers crossed to wild-type Canton-S. Scale bars: (B-G) 30µm; (H) 20µm. Graphs show box-plots around the median. P values over graphs in (D, G right, H) refer to group analyses; stars indicate multiple comparisons tests. *p<0.05, **p<0.01, ***p<0.001. For further genotypes, sample sizes and statistical details, see Supplementary Table S2.

DNT-2, Toll-6 and Kek-6 are required for arborisations and synapse formation.
(A) Illustration showing the ROIs (dashed lines) used for the analyses, corresponding to dendrites and axonal endings of PAMs and axonal terminals of PPL1ped neurons. (B-F) Fruit-flies were kept constantly at 25°C. (B) Complete loss of PAM synapses (PAM>syt-GFP) onto α, MB lobe in DNT-2 null mutants. (C) Toll-6 RNAi knockdown in PAM 2 ’2 neurons (MB301B>FB1.1, Toll-6-RNAi) decreased dendrite complexity. Un-paired Student t-test. (D) Over-expression of cleaved DNT-2CK in PAM 2 ’2 neurons (MB301B>CD8-GFP, DNT-2CK) increased dendrite complexity. Un-paired Student t-test. (E,E’,F) PPL1ped axonal misrouting was visualised with split-GAL4 MB320CGal4>FlyBow1.1. Images show PPL1-ψped neurons and some PPL1-α2α’2. (D,D’) RNAi knock-down of Toll-6, kek-6 or both (e.g. MB320CGal4>FlyBow1.1, Toll-6RNAi) in PPL1-ψped neurons caused axonal terminal misrouting (arrows). (E’) Higher magnification of (E, dotted squares). Chi-square for group analysis: p = 0.0224, and multiple comparisons Bonferroni correction control vs Toll-6RNAi p<0.01; vs kek-6RNAi p<0.05; vs Toll-6RNAi kek-6RNAi p<0.01, see Table S2. (F) PPL1 misrouting was also induced by over-expressed DNT-2CK or DNT-2FL (e.g. MB320CGal4>FlyBow1.1, DNT-2FL). Chi-square for group analysis: p<0.05, Bonferroni correction control vs DNT-2CK ns, vs DNT-2FL *p<0.05, see Table S2. (G) Adult-specific Toll-6 kek-6 RNAi knockdown in PAM neurons decreased size of post-synaptic density sites (PAM>Homer-GCaMP and anti-GFP antibodies). Temperature regime shown on the right. (C,D) Graphs show box-plots around the median; (G) are box-plots with dot plots. (C,D,G) *p<0.05, **p<0.01, ****p<0.0001. Scale bars: (B,C,D,E) 30µm; (E’,F,G) 20µm. For genotypes, sample sizes and statistical details, see Supplementary Table S2.

DNT-2 neuron activation and DNT-2 over-expression induced synaptogenesis.
(A) Thermo-activation of DNT-2 neurons at 30°C altered DNT-2 arborisations, here showing an example with smaller dendrites and enlarged axonal arbours (magenta shows 3D-rendering of axonal arborisation done with Imaris, merged with raw image in green). (Genotype: DNT-2>FlyBow1.1, TrpA1). (B) Thermogenetic activation of DNT-2 neurons increased the number of Homer+ PSDs in DNT-2 neurons, at SMP (DNT-2>Homer-GCAMP3, TrpA1, anti-GFP). Test at 30°C 24h: Unpaired Student t test. See Table S2. (C) Thermogenetic activation of DNT-2 neurons induced synaptogenesis in PAM target neurons at SMP, but not at MB lobe. (Genotype: PAM(R58E02)LexA/LexAOP-sytGCaMP; DNT-2GAL4/UASTrpA1). At SMP: No. Syt+ synapses: Mann Whitney-U; Syt+ synapse volume: Mann Whitney-U. At MB lobe: No. Syt+ synapses: Unpaired Student t ns; Syt+ synapse volume: Mann Whitney-U ns. (D) Over-expression of DNT-2FL in DNT-2 neurons increased synapse volume at SMP dendrite and induced synaptogenesis at MB lobe. (Genotype: PAM(R58E02)LexA/LexAopSytGcaMP6; DNT-2Gal4/UAS-DNT-2FL). At SMP: No. Syt+ synapses: Unpaired Student t ns. Syt+ synapse volume: Mann Whitney-U. At MB lobe: No. Syt+ synapses: Unpaired Student t; Syt+ synapse volume: Mann Whitney-U ns. Graphs show box-plots around the median, except for PSD volume data that are dot plots. *p<0.05, ****p<0.0001; ns: not significantly different from control. Scale bars: 30µm. For Genotypes, sample sizes, p values and other statistical details, see Supplementary Table S2.

DNT-2-induced circuit plasticity modified dopamine-dependent behaviour.
(A) DNT-2 mutants (left, DNT-2³⁷/DNT-2¹⁸) and flies with adult-specific RNAi knock-down of DNT-2 in DNT-2 neurons (right, tubGAL80^ts; DNT-2>DNT-2RNAi), had impaired climbing. Left Mann Whitney U; Right: One Way ANOV, post-hoc Dunnett. (B) Adult-specific Toll-6 and kek-6 RNAi knock-down in Toll-6 (tubGAL80^ts; Toll-6>Toll-6RNAi, kek-6RNAi, left) or PAM (tubGAL80^ts; R58E02>Toll-6RNAi, kek-6RNAi, right) neurons impaired climbing. Left: Welch ANOVA, post-hoc Dunnett. Right: Welch ANOVA, post-hoc Dunnett. (C) The climbing impairment of DNT-2 mutants could be rescued with the over-expression of Toll-6^CY in dopaminergic neurons (Rescue genotype: UASToll-6^CY/+; DNT-2¹⁸THGAL4 R58E02GAL4/DNT-2³⁷). Welch ANOVA, post-hoc Dunnett. (D) Adult specific over-expression of Toll-6^CY in DANs increased locomotion in DNT-2³⁷/DNT-2¹⁸mutants. (Test genotype: UASToll-6^CY/+; DNT-2¹⁸THGAL4 R58E02GAL4/DNT-2³⁷). Walking speed: Kruskal Wallis ANOVA, post-hoc Dunn’s. Distance walked: Kruskal Wallis ANOVA, post-hoc Dunn’s. Time spent immobile: Kruskal Wallis ANOVA, post-hoc Dunn’s. (E) Adult specific DNT-2FL overexpression in DNT-2 neurons increased fruit-fly locomotion speed in an open arena at 30°C (see also Supplementary Figure S6A for further controls). (Test genotype: tubGAL80^ts, DNT-2>DNT-2FL) Kruskal-Wallis, post-hoc Dunn’s test. (F) Thermogenetic activation of DNT-2 neurons at 30°C (DNT-2>TrpA1) increased fruit-fly locomotion speed. (See also Supplementary Figure S6B for further controls). One Way ANOVA p<0.0001, post-hoc Dunnett’s test. (G) Over-expression of DNT-2-FL in DNT-2 neurons increased long-term memory. (Test genotype: tubGAL80^ts, DNT-2>DNT-2FL). Left: 23°C controls: One Way ANOVA p=0.8006. Right 30°C: One Way ANOVA, post-hoc Dunnett’s test. Graphs show box-plots around the median, under also dot plots. P values over graphs refer to group analyses; asterisks indicate multiple comparisons tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns: not significantly different from control. For further genotypes, sample sizes, p values and other statistical details, see Supplementary Table S2.

Sign up for email alerts