Fragile X Mental Retardation Protein Requirements in Activity-Dependent Critical Period Neural Circuit Refinement

Abstract

Activity-dependent synaptic remodeling occurs during early-use critical periods, when naive juveniles experience sensory input. Fragile X mental retardation protein (FMRP) sculpts synaptic refinement in an activity sensor mechanism based on sensory cues, with FMRP loss causing the most common heritable autism spectrum disorder (ASD), fragile X syndrome (FXS). In the well-mapped Drosophila olfactory circuitry, projection neurons (PNs) relay peripheral sensory information to the central brain mushroom body (MB) learning/memory center. FMRP-null PNs reduce synaptic branching and enlarge boutons, with ultrastructural and synaptic reconstitution MB connectivity defects. Critical period activity modulation via odorant stimuli, optogenetics, and transgenic tetanus toxin neurotransmission block show that elevated PN activity phenocopies FMRP-null defects, whereas PN silencing causes opposing changes. FMRP-null PNs lose activity-dependent synaptic modulation, with impairments restricted to the critical period. We conclude that FMRP is absolutely required for experience-dependent changes in synaptic connectivity during the developmental critical period of neural circuit optimization for sensory input.

Keywords: Drosophila; activity-dependent; critical period; mushroom body; olfactory; optogenetics; synapse.

Figure 1. FMRP controls mPN2 innervation of MB calyx during the critical period

(A) Schematic of AL-mPN2 connectivity within the olfactory learning and memory circuit. mPN2 receives Ir75 olfactory sensory neuron dendritic input in the antennal lobe VL1 glomerulus and projects an axon to 1) MB calyx and 2) lateral horn. In the calyx (inset, right), mPN2 projects ventral branches innervating Kenyon cells within microglomeruli. AL-mPN2, antennal lobe medial projection neuron 2; MB, mushroom body; LH, lateral horn; iACT, inner antennal cerebral tract; Ir75d, ionotropic receptor 75d. (B) mPN2 dendrites (top, left) and Ir75d axons (bottom, left) connect in the VL1 glomerulus. mPN2 axons in the MB calyx, showing presynaptic Brp (top, right) and postsynaptic Dlg (bottom, right) forming microglomeruli. (C) Genetic background control (w¹¹¹⁸) and dfmr1 null (dfmr1^50M) mPN2 innervation of MB calyx, showing synaptic branches (brackets) and boutons (arrows) with R65G01-Gal4>UAS-mCD8::GFP expression. (D) Quantification (mean ± standard error) of synaptic branch length (left) and bouton area (right) in both genotypes over developmental time at 0, 1 and 7 days post-eclosion (dpe). Significance determined from two-tailed Mann-Whitney tests: p***<0.001.

Figure 2. FMRP promotes mPN2 synaptic differentiation during the critical period

(A) Genetic background (w¹¹¹⁸) control mPN2 synaptic boutons at 1 day post-eclosion, labeled by R65G01-Gal4>UAS-mCD8::GFP (green) and the presynaptic marker Brp (red). (B) Null dfmr1 (dfmr1^50M) mutant mPN2 boutons display a developmental delay in expression of Brp relative to genetic controls. (C) Brp expression within individual mPN2 axonal boutons over developmental time (mean ± standard error). Five boutons were selected from each neuron per developmental time point. Expression levels normalized to genetic controls. Significance determined through two-tailed unpaired t-test (0 dpe) or two-tailed Mann-Whitney test (1 and 7 dpe). p***<0.001.

Figure 3. FMRP regulates mPN2-KC synaptic connectivity during critical period

(A) GFP reconstitution across synaptic partners (GRASP) between control (w¹¹¹⁸) mPN2 neuron (presynaptic axon, red) and mb247-expressing Kenyon cells in MB calyx. GFP (green) is restricted to MB calyx (dashed line, left) and not evident in the adjacent lateral horn (LH). Right: High magnification reveals GRASP contact points (arrows) distributed through innervating boutons. (B) GRASP analysis in dfmr1 null (dfmr1^50M) animals. GRASP contacts remain confined to the MB calyx (left panel), yet mPN2-KC contacts occur clustered proximally in axon (bar, right panel), are reduced, and do not appear to occur in all boutons (arrowhead). (C) Schematic showing GRASP experimental design: KCs express spGFP11 (mb247>spGFP11) and mPN2 expresses spGFP1-10 (R65G01-Gal4>UAS-spGFP1-10). Reconstitution of intact GFP molecule (green signal) occurs only with immediate contact proximity in the calyx. (D) Quantification (mean ± standard error) of total GRASP contact number per calyx (left) and area (right) in control and dfmr1 at 0, 1 and 7 days post-eclosion (dpe). Significance determined through two-tailed unpaired t-test or two-tailed Mann-Whitney tests: p***<0.001.

Figure 4. FMRP limits synaptic bouton growth and promotes synapse formation

(A) MB calyx transmission electron micrographs of genetic background control (w¹¹¹⁸) and dfmr1 null mutant (dfmr1^50M) animals, pseudocolored to show cellular architecture: MB calyx neuropil (green), Kenyon cell bodies (red), and antennocerebral tract (ACT, yellow) carrying mPN2 axons. (B) Higher magnification micrographs showing PN-KC synaptic contacts within the MB calyx in control (left) and dfmr1 null (right). Individual synaptic boutons containing synaptic vesicle (SV) pools, t-bar active zones (arrows) and mitochondria (M). (C) High magnification images showing examples of single t-bar active zones in the control (top) and dfmr1 null mutant (bottom). (D) Quantification of PN bouton area (left) and T-bar active zone number per unit area (right), comparing control and dfmr1 null mutants. All micrographs taken from 0 day post-eclosion brains. Significance determined through two-tailed Mann-Whitney tests: p***<0.001.

Figure 5. FMRP required for critical period sensory remodeling of mPN2 synapses

(A) mPN2 innervation of the MB calyx during the 1 dpe critical period for w¹¹¹⁸ control (left) and dfmr1 null (right). Arrows indicate synaptic boutons in the two genotypes. Images show R65G01-Gal4>UAS-mCD8::GFP expression in inverted black-and-white to better reveal synaptic architecture. (B) Comparable mPN2 images after 24-hours of pyrrolidine odor exposure during the early critical period (0–1 dpe) in control (left) and dfmr1 null (right) animals. The dramatically shortened control synaptic branches often possess foot-like protrusions (arrowheads). (C) Quantification of synaptic branch length and bouton area, with and without 24-hour pyrrolidine odor exposure. Left panels: critical period at 1 dpe comparing control (no odor) and odor exposed (0–1 dpe) w¹¹¹⁸ and dfmr1 null. Right panels: maturity at 7 dpe comparing control (no odor) and odor exposed (6–7 dpe) for both genotypes. Significance determined from Dunn’s multiple comparisons tests: p*<0.05, p***<0.001. See also Figures S1 and S2.

Figure 6. FMRP- and activity-dependent bidirectional changes in mPN2 synapses

(A) mPN2 neurons innervating MB calyx expressing R65G01-Gal4>UAS-CsChrimson, but lacking the essential ATR cofactor required for channelrhodopsin function (control), display the usual synaptic differences between w¹¹¹⁸ and dfmr1 null mutant at 1 dpe after 24 hours of blue light stimulation. (B) Stimulated (ATR fed) neurons display reduced synaptic branch lengths and enlarged boutons in w¹¹¹⁸ (left), but no effect in dfmr1 (right) after 24-hours of blue light stimulation (0–1 dpe). (C) Quantification of branch length (left) and bouton area (right) from critical period light stimulation. Significance from Dunn’s multiple comparisons tests: p***<0.001. (D) mPN2 innervating MB calyx expressing R65G01-Gal4>UAS-eNpHR3.0 but lacking ATR (control) display typical w¹¹¹⁸ vs. dfmr1 null synaptic differences at 1 dpe after 24-hours of amber light. (E) Activity repressed (ATR fed) neurons exhibit the opposing consequence of increased MB calyx innervation in w¹¹¹⁸ (left), but no effect in dfmr1 nulls (right) after 24-hours of amber light (0–1 dpe). (F) Quantification of synaptic branch length (left) and bouton area (right) following critical period optogenetic activity repression. Significance determined from Dunn’s multiple comparisons tests: p*<0.05, p**<0.01, p***<0.001. See accompanying data on optogenetics at maturity in Figures S3 and S4.

Figure 7. FMRP enables neurotransmission-dependent mPN2 synaptic refinement

(A) Temporally-controlled, conditional expression of tetanus neurotoxin (TNT) in mPN2 neurons innervating the MB calyx (Gal80^ts; R65G01-Gal4>UAS-TNT; UAS-GCaMP5G) in the w¹¹¹⁸ genetic background. Genetic control neurons without tetanus toxin (control, left) display typical mPN2 innervation, whereas neurotransmission blockade (UAS-TNT, right) causes a dramatic expansion. (B) The same experiment in dfmr1 null (dfmr1^50M) mutants. mPN2 innervation of the calyx in the absence (control, left) and presence of tetanus toxin (UAS-TNT, right). (C) Quantification of synaptic branch length (left) and bouton area (right) in all four conditions. All animals were raised at restrictive 18°C and shifted to permissive 29°C at pupal day 4, leading to expression of TNT. All analyses were performed at 1 dpe. Significance determined from Dunn’s multiple comparisons tests: p*<0.05, p**<0.01, p***<0.001.