Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry

Abstract

Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early-use critical period.

Keywords: Activity-dependent; Autism spectrum disorder; Drosophila; Excitation vs. inhibition; Fragile X syndrome; Optogenetics.

Figure 1

Critical period manipulation of excitatory input and inhibitory output neurons of the mushroom body learning/memory circuit. A) Schematic illustrating developmental stages of analysis relative to the FMRP expression profile (Tessier and Broadie, 2008). B) Schematic depicting architecture of MB input excitatory cholinergic neuron AL-mPN2 (R65G01-Gal4 driver) and MB output inhibitory GABAergic neuron MBON-γ1pedc>α/β (MBON-11; R12G04-Gal4 driver). The membrane marker UAS-mCD8 is shown for MBON-11 (B1) and mPN2 (B2, B3) labeling soma, dendritic arbor and axon processes. Dashed boxes correspond to images. Abbreviations: MB, Mushroom body; AL, antennal lobe; ON, output neuron; SEZ, subesophogeal zone; IPm medial inferior protocerebrum; KC, Kenyon cell; LH, lateral horn; VL, vertical lobe; ML, medial lobe; mALT, medial antennal lobe tract; PN, projection neuron; VL1, ventrolateral glomerulus 1; AL-mPN2, antennal lobe medial projection neuron 2. Scale B1: 10μM, B2: 20μM, B3: 10μM.

Figure 2

Restricted critical period defect in AL-mPN2 Ca²⁺ dynamics in dfmr1 mutants. The UAS-GCamp5G fluorescent reporter driven by R65G01-Gal4 in excitatory mPN2 input neurons to assay depolarization-induced Ca²⁺ transients in both wildtype control (w¹¹¹⁸) and dfmr1 null mutants. Confocal fluorescent measurements done at the peak critical period of 1 day post-eclosion (A) compared to maturity at 7 days post-eclosion (B). Still frame heat map representations of baseline and peak fluorescence following acute K+ depolarization. The change of average fluorescence intensity over time (mean±SEM) is plotted on top, with histograms (minimum, median, maximum and quartiles) below depicting peak intensity and time to peak. n^WT-1dpe=19, n^dfmr1-1dpe=17, n^WT-7dpe=22, n^dfmr1-7dpe=18. Statistical significance indicated as ***p<0.001 or not significant (n.s.). Scale bars: 10μm.

Figure 3

Opposite critical period defect in MBON-11 Ca²⁺ dynamics in dfmr1 mutants. UAS-GCamp5G fluorescent reporter targeted by the R12G04-Gal4 driver to inhibitory MBON-11 output neurons to assay depolarization-induced Ca²⁺ transients in wildtype control (w¹¹¹⁸) and dfmr1 null mutants. Measurements at the 1 day post-eclosion critical period (A) compared to maturity at 7 days post-eclosion (B). Representative heat maps show baseline and peak fluorescence following acute K+ depolarization. The change of average fluorescence intensity over time (mean±SEM) is plotted on top, with histograms (minimum, median, maximum and quartiles) below depicting peak intensity and time to peak. n^WT-1dpe=20, n^dfmr1-1dpe=16, n^WT-7dpe=22, n^dfmr1-7dpe=23. Statistical significance indicated as **p<0.01, ***p<0.001 or not significant (n.s.). Scale bars: 10μm.

Figure 4

Conditional dfmr1 rescue/removal shows restricted critical period requirement. Gal80ts repressive paradigm for conditionally dfmr1 rescue (A) and knockdown (B) during critical period development for both excitatory input mPN2 and inhibitory output MBON-11 neurons. Animals raised at 18°C permissive temperature (Gal80 active, Gal4 inactive, red) until pupal day 4 (P4), then shifted to 29°C restrictive temperature (Gal80 inactive, Gal4 active, green) until 1 day post-eclosion (1 dpe). (C–F) K⁺ depolarization-induced Ca²⁺ transient GCamp5G fluorescence changes for genetic controls (grey line), constitutively active rescue/RNAi (black line), and conditional Gal80ts rescue/RNAi (red line). AL-mPN2 dfmr1 critical period rescue (dfmr1 control n=15, constitutive rescue n=22, conditional rescue n=25) (C) and RNAi knockdown (WT control n=16, constitutive RNAi n=16, conditional RNAi n=14) (D) shows restricted temporal FMRP requirement. Parallel, MBON-11 critical period rescue (dfmr1 control n=21, constitutive rescue n=23, conditional rescue n=17) (E) and removal (WT control n=15, constitutive RNAi n=21, conditional RNAi n=10) (F) of dfmr1 shows similar results. Each plot represents the change of average fluorescence intensity over time (mean±SEM), and includes an inset peak intensity histogram (minimum, median, maximum and quartiles). Significance determined by one-way ANOVA and indicated as ***p<0.001 or not significant (n.s.).

Figure 5

Critical period stimulation of mPN2 reveals FMRP-dependent Ca²⁺ dynamics. (A) Co-expression of UAS-GCamp5G Ca²⁺ reporter (green) and UAS-ChR2-H134R-mCherry optogenetic channel (red) driven by R65G01-Gal4 targeted to excitatory MB input mPN2 neurons. K⁺ depolarization-induced Ca²⁺ transients following 24 hours of blue light stimulation (5Hz, 20ms) in animals fed either vehicle alone or the all-trans retinal (ATR) essential cofactor for optogenetic stimulation in wildtype control (B) or dfmr1 null mutants (C). Left: Change of average fluorescence intensity over time ( F/F; mean±SEM). Right: Histograms (minimum, median, maximum and quartiles) depicting fluorescence peak intensity and time to peak fluorescence. WT^vehicle n=17, WT^ATR n=18, dfmr1^vehicle n=18, dfmr1^ATR n=14. Statistical significance determined via unpaired, two-tailed t-tests indicated as ***p<0.001 or not significant (n.s.). Scale bar: 0.5μm.

Figure 6

Critical period MBON-11 stimulation shows FMRP-dependent Ca²⁺ dynamics. (A) Dual expression of the UAS-GCamp5G Ca²⁺ reporter (green) and the UAS-ChR2-H134R-mCherry optogenetic channel (red) under control of the R12G04-Gal4 driver selectively targeted to inhibitory output MBON-11 neurons. K⁺ depolarization-induced Ca²⁺ transients following 24 hours of blue light stimulation (5Hz, 20ms) in animals fed either vehicle or all-trans retinal (ATR) for optogenetic stimulation in wildtype control (B) and dfmr1 null mutants (C). The change of average fluorescence intensity (mean±SEM) following acute K⁺ depolarization is shown on the left, with histograms on the right depicting the GCamp5G Ca²⁺ reporter fluorescence peak intensity and time to peak (minimum, median, maximum and quartiles). WT^vehicle n=20, WT^ATR n=19, dfmr1^vehicle n=21, dfmr1^ATR n=15. Statistical significance determined via unpaired, two-tailed t-tests indicated as ***p<0.001 or not significant (n.s.). Scale bar: 10μm.