Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation during an Early-Life Critical Period

Randall M. Golovin; Jacob Vest; Kendal Broadie

doi:10.1523/JNEUROSCI.2167-20.2020

Abstract

Critical periods are developmental windows during which neural circuits effectively adapt to the new sensory environment. Animal models of fragile X syndrome (FXS), a common monogenic autism spectrum disorder (ASD), exhibit profound impairments of sensory experience-driven critical periods. However, it is not known whether the causative fragile X mental retardation protein (FMRP) acts uniformly across neurons, or instead manifests neuron-specific functions. Here, we use the genetically-tractable Drosophila brain antennal lobe (AL) olfactory circuit of both sexes to investigate neuron-specific FMRP roles in the odorant experience-dependent remodeling of the olfactory sensory neuron (OSN) innervation during an early-life critical period. We find targeted OSN class-specific FMRP RNAi impairs innervation remodeling within AL synaptic glomeruli, whereas global dfmr1 null mutants display relatively normal odorant-driven refinement. We find both OSN cell autonomous and cell non-autonomous FMRP functions mediate odorant experience-dependent remodeling, with AL circuit FMRP imbalance causing defects in overall glomerulus innervation refinement. We find OSN class-specific FMRP levels bidirectionally regulate critical period remodeling, with odorant experience selectively controlling OSN synaptic terminals in AL glomeruli. We find OSN class-specific FMRP loss impairs critical period remodeling by disrupting responses to lateral modulation from other odorant-responsive OSNs mediating overall AL gain control. We find that silencing glutamatergic AL interneurons reduces OSN remodeling, while conversely, interfering with the OSN class-specific GABA_A signaling enhances remodeling. These findings reveal control of OSN synaptic remodeling by FMRP with neuron-specific circuit functions, and indicate how neural circuitry can compensate for global FMRP loss to reinstate normal critical period brain circuit remodeling.

SIGNIFICANCE STATEMENT Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD), manifests severe neurodevelopmental delays. Likewise, FXS disease models display disrupted neurodevelopmental critical periods. In the well-mapped Drosophila olfactory circuit model, perturbing the causative fragile X mental retardation protein (FMRP) within a single olfactory sensory neuron (OSN) class impairs odorant-dependent remodeling during an early-life critical period. Importantly, this impairment requires activation of other OSNs, and the olfactory circuit can compensate when FMRP is removed from all OSNs. Understanding the neuron-specific FMRP requirements within a developing neural circuit, as well as the FMRP loss compensation mechanisms, should help us engineer FXS treatments. This work suggests FXS treatments could use homeostatic mechanisms to alleviate circuit-level deficits.

Introduction

Critical periods are time windows when brain circuitry is particularly susceptible to initial sensory input driving activity-dependent remodeling (Hubel and Wiesel, 1970; Hensch, 2005). This refinement is impaired in a range of heritable neurologic disorders (Dölen et al., 2007; Greenhill et al., 2015; Krishnan et al., 2015; Meredith, 2015). Fragile X syndrome (FXS) patients exhibit profound developmental delays (Bailey et al., 1998; Roberts et al., 2016), and FXS disease models display disrupted critical periods (Dölen et al., 2007; Contractor et al., 2015). This leading monogenic cause of both intellectual disability and autism spectrum disorder is characterized by hypersensitivity to sensory stimuli and childhood activity-dependent seizures (Crawford et al., 2001; Hersh et al., 2011; Contractor et al., 2015). The causal fragile X mental retardation protein (FMRP) regulates activity-dependent protein synthesis enabling experience-dependent synaptic plasticity (Brown et al., 2001; Darnell et al., 2001; Zalfa et al., 2003; Dölen et al., 2007), especially during critical periods (Bureau et al., 2008; He et al., 2014; Doll et al., 2017). Building evidence suggests specific FMRP roles in different brain circuits and cell types (Dahlhaus, 2018). Neuron class-specific FMRP genetic manipulations reveal striking differences controlling activity-dependent connectivity remodeling (Doll and Broadie, 2015), channel-binding (Brandalise et al., 2020), and translational control (Sawicka et al., 2019). Thus, it is crucial to test FMRP functions within specific neurons of defined brain circuits, and particularly FMRP roles during activity-dependent neural circuit remodeling in early sensory critical periods.

The Drosophila brain antennal lobe (AL) olfactory circuit provides an excellent model to study odorant sensory experience-dependent critical period remodeling (Devaud et al., 2003). Numerous studies have shown that exposing young animals to selected odorants changes AL circuit structure and function (Devaud et al., 2003; Sachse et al., 2007; Doll and Broadie, 2015; Golovin et al., 2019; Chodankar et al., 2020). Early work established that critical period odor exposure alters olfactory sensory neuron (OSN) connectivity in activated AL regions (Devaud et al., 2003; Sachse et al., 2007). Subsequent work showed that odorant exposure or optogenetic activity stimulation during just the first day following eclosion drives FMRP-dependent PN structural and functional remodeling (Doll and Broadie, 2015, 2016). Recently, we discovered a new form of experience-dependent OSN remodeling (Golovin et al., 2019), which reduces Or42a-expressing OSN innervation of a specific AL synaptic glomerulus following ethyl butyrate (EB) odorant exposure during the first 2 days of life, but not a week later. Furthermore, this remodeling requires functional odorant receptors, but not OSN output, and is reversed following prolonged removal from the odorant (Golovin et al., 2019). Together, these studies demonstrate that temporally restricted critical period odor experience refines AL glomeruli innervation and activity-dependent function. However, it remains unclear how reversible critical period OSN remodeling employs FMRP, and whether neuron-specific FMRP actions operate in AL circuit mechanisms.

Based on the key roles of FMRP in regulating critical period remodeling within the AL, we hypothesized an FMRP requirement in OSNs. To test this hypothesis, we assayed Or42a OSN innervation of the VM7 glomerulus following EB exposure during the well-mapped critical period (Golovin et al., 2019). Surprisingly, we find FMRP null mutants manifest normal OSN innervation refinement following EB exposure, whereas Or42a-targeted FMRP RNAi strongly attenuates experience-dependent remodeling. Consistently, global FMRP RNAi mimics null mutants by not affecting synaptic remodeling, whereas Or42a-targeted FMRP overexpression (OE) strongly enhances remodeling following EB exposure. These results indicate that balanced FMRP levels across EB-activated OSNs are required to tune LN input. We find that Or42a-targeted FMRP RNAi does not affect OSN remodeling after Or42a-specific optogenetic activation, but still attenuates circuit remodeling when Or42a OSN synaptic output is blocked. Importantly, we find that blocking glutamatergic AL interneuron neurotransmission as well as GABA_AR signaling disrupts Or42a OSN remodeling, suggesting that imbalanced FMRP levels perturb LN to Or42a OSN activity. Together, these results reveal neuron-specific FMRP functions in AL circuit critical period remodeling, and demonstrate that this circuit can restore normal function in the absence of FMRP.

Materials and Methods

Drosophila genetics

All animals were reared at 25°C before odor/light exposure. Animals were reared on a 12/12 h light/dark cycle except for light exposure experiments, for which animals were kept in darkness. All animals were fed on the standard Drosophila cornmeal molasses food. Initial experiments used animals of both sexes, but males show reduced critical period OSN innervation remodeling compared with females. Therefore, later experiments were conducted using only females to control the remodeling variability, and ensure a robust response to the odorant-dependent critical period OSN remodeling across the many experimental genotypes. All genotypes were confirmed with visible markers and/or PCR. Transgenic controls include w^-; UAS-mCD8::GFP/+; Or42a-Gal4/+, w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP, w^-; Or42a-mCD8::4xGFP/+; Or42a-mCD8::4xGFP/+ and w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP. The genetic lines used for each figure are listed in Table 1.

View this table:

Table 1.

List of experimental genotypes

Odorant exposure

Critical period odorant exposure was done as we previously reported (Golovin et al., 2019). Briefly; Animals were staged as dark pupa (4 d after puparium formation at 25°C), separated based on both the sex and genotype. Fine wire mesh caps were secured onto the animal vials to allow good airflow, and the vials were then placed within larger airtight containers (3700 ml, Glasslock). In 1.5-ml microcentrifuge tubes, 1 ml of mineral oil (Sigma-Aldrich) was placed alone (vehicle control), or with 10%, 15%, 20%, or 25% EB (% v/v in mineral oil; Sigma-Aldrich). Containers were placed in humidified 23°C incubators with a 12/12 h light/dark cycle. After 24 h, the adult animals were rapidly transferred to new vials in clean chambers with a fresh odorant supply. Animals were then kept in the odorant chambers for another 24 h. The entire odorant exposure period was 2 d; 0–2 d posteclosion (dpe).

Light exposure

The optogenetic light exposure matched the above critical period odorant exposure paradigm. Dark-reared animals were staged as dark pupa, separated based on sex and genotype, and then transferred to a Petri dish (35 × 10 mm Falcon) with 3 ml of food. The Petri dish was then placed in the same containers used above (3700 ml, Glasslock) in dark, humidified 23°C incubators. Light was supplied through a custom-built cyan LED array (515 nm) controlled by an Arduino Uno (Arduino) using a custom script. The light exposure was 5-Hz 50-ms pulses (337 μW/mm²). After 24 h, animals were rapidly transferred to a new dish in a clean chamber. The entire adult animal light exposure period was 2 d; 0–2 dpe.

Confocal imaging

Staged animals were anesthetized on ice for at least 1–2 min, and then brains were dissected using fine forceps (Dumont #5) in physiological saline (128 mm NaCl, 2 mm KCl, 4 mm MgCl₂, 1.8 mm CaCl₂, 64.6 mm sucrose, and 5 mm HEPES, pH 7.2; Sigma-Aldrich). Dissected brains were fixed for 30 min at room temperature (RT) in 4% PFA (EMS)/4% sucrose in PBS, pH 7.4 (Invitrogen). Brains were washed 3× with PBS and then blocked for 1 h in 1% BSA (Sigma-Aldrich) in PBS-T (0.2% Triton X-100 in PBS; Fisher Chemical). Brains were then incubated with primary antibodies diluted in 0.2% BSA in PBS-T at 4°C overnight (O/N). Primary antibodies used were as follows: rabbit anti-GFP (Abcam 290; 1:3000), rat anti-RFP (Chromotek 5F8; 1:500), mouse anti-dFMRP (Abcam a10299 [6A15]; 1:125), and mouse anti-Bruchpilot [BRP; Developmental Studies Hybridoma Bank (DSHB), nc82; 1:50]. Brains were washed 3× for 20 min with PBS-T and then incubated O/N with secondary antibodies. Secondary antibodies used were as follows: Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 555 donkey anti-mouse, and Alexa Fluor 546 goat anti-rat (all at 1:250). Brains were then washed in PBS-T 3× for 20 min, followed by PBS 1× for 20 min. Brains were then rinsed with dH₂O and mounted in Fluoromount (EMS 17984-25) on a glass slide (ProbeOn Microscope Slides, Fisherbrand) with a glass coverslip (No. 1.5H, Carl Zeiss). Double-sided adhesive tape (Scotch) was used to raise the coverslips above the brains, and clear nail polish (Sally Hansen) was used to seal the coverslip to the slide. For maxillary palp studies, whole proboscises were dissected and processed as above, except with longer fixation (45 min), longer primary/secondary antibody incubations (38–42 h) and no double-sided tap used for mounting. The whole head image (see Fig. 1) was taken using an iPhone 6 (Apple) through the oculars of a Leica dissecting scope with both white light and filtered mercury lamp light for illumination. Confocal images were collected on a 510 META laser-scanning confocal microscope (Carl Zeiss) with 40× and 63× oil-immersion objectives. Images taken with the 40× lens were collected at 1024 × 1024 resolution with a Z-slice of 1-μm thickness. Images taken with the 63× lens were collected at 2048 × 2048 resolution with a Z-slice thickness of 0.8 μm. The microscope and imaging settings were kept constant within every experiment.

Quantification

AL glomeruli measurements were done as previously reported (Golovin et al., 2019). Briefly, blinded brain images were visualized in ImageJ (NIH) with the quantification channel isolated. A maximum intensity projection that captured the whole VM7 glomerulus was used to generate a region of interest (RoI). This RoI was used with the FIJI plugin 3D Object Counter (Schindelin et al., 2012) to quantify the RoI volume. For every experiment, the threshold was set at a constant value that minimized the noise. In controls, the 3D Object Counter output typically contained two RoIs, which were summed. In experimental conditions, when the glomerular RoI was more discontinuous, all the regions were summed. To control for variations of signal across the different labeling constructs, antibody aliquots and experimental days, glomerulus volumes were normalized to the control mean for each experimental replicate. For the maxillary palp, blinded Z-stack images were analyzed using ImageJ. A maximum intensity projection was used to capture all the Or42a OSNs. For the fluorescence intensity measurements, one RoI for each Or42a-positive soma was generated using the GFP signal. ImageJ was then used to quantify the mean intensity for each soma in both the GFP and FMRP channels.

Statistics

All tests were done using Prism 8 (GraphPad). For comparisons with more than or equal to two genotypes, a two-way ANOVA was used with odorant/light and genotype as independent variables. Follow-up pairwise comparisons were done using t tests, with Sidak's multiple comparisons correction. Interactions between genotype and odorant/light exposure were tested with the two-way ANOVA interaction term. A ROUT outlier test was done for data with Q set to 1%. Some genotypes showed altered basal glomerulus innervation volumes in the vehicle-exposed control animals. While the ANOVA post hoc test can distinguish whether the EB-treated means are different, it does not account for differences in glomerulus volume of vehicle-treated controls. With only two independent variables, an ANOVA (2 × 2) interaction term can be attributed to the specific genotype/odorant exposure. However, if more than the independent variables occur, the interaction term cannot be used for pairwise comparisons between genotypes. Therefore, in all the cases with more than two independent variables, a linear regression with t tests comparing pairwise interaction terms was used to assess EB treatment effects while controlling for any changes in the basal glomerulus innervation volume. Regression coefficients for each genotype interaction term represent the differences in the treatment effect on the experimental genotype compared with the control genotype. If the interaction term of an experimental genotype were zero, this would indicate the effect of treatment had not changed in the genotype compared with the matched control (glomerular innervation changed by the same amount). If the interaction term were significantly greater than zero, this would indicate that the treatment had not reduced innervation as much in the experimental condition as in control. If the interaction term were significantly less than zero, this would indicate that the treatment had reduced the innervation by more in the experimental condition compared with the control. To display regression analyses, bar values of each genotype show the difference between vehicle and treatment conditions. Error bars are the sum of the error of the EB effect regression plus the error of the genotype effect regression. The sample size (n) is the number of brains or maxillary palps. When comparing data with only two conditions, Sidak's corrected t-tests were employed. Line graphs are of linear regression analyses. ANOVAs are displayed as scatter plots with mean ± SEM. The EB effects from the linear regression analyses are displayed as bar graphs with mean ± standard error of the regression (SER). Significance is shown as not significant (N.S.; p > 0.05) or significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Results

OSN-specific FMRP loss disrupts odor experience critical period remodeling of AL innervation

The well-mapped and genetically tractable neuron classes of the Drosophila AL olfactory circuit make it a very powerful system to study the mechanisms of sensory experience-dependent remodeling. The AL is the first brain synaptic node of the olfactory circuit (Fig. 1A,B; Wilson, 2013). Odorants transduced by OSNs in both the antenna and maxillary palps (Fig. 1C) project axons along antennal and labial nerves (Fig. 1B, bottom arrow), respectively, to innervate OSN-specific AL synaptic glomeruli (Fig. 1D,E, top left; Wilson, 2013). Each OSN generally expresses a single olfactory receptor (e.g., Or42a; Fig. 1A–D), in addition to the pan-OSN Orco co-receptor (Larsson et al., 2004; Couto et al., 2005). Each AL glomerulus receives convergent OSNs that express only that single olfactory receptor (Fig. 1B, boxed region, D). Within each AL glomerulus, OSNs synapse onto projection neurons (PNs; Fig. 1D,E, top right). PNs subsequently send olfactory information to the central brain mushroom body (MB) and lateral horn (Fig. 1B; Jefferis et al., 2002; Marin et al., 2002). Within the AL, multiple local interneurons (LNs) synapse broadly to provide modulation of OSN-PN excitatory (cholinergic) connections. The LNs release a variety of neuromodulators that can both inhibit and excite this olfactory neurotransmission, including GABA (Fig. 1E, bottom left) and glutamate (Fig. 1E, bottom right; Chou et al., 2010; Wilson, 2013). In addition, LNs can also signal through gap junction mediated electrical synapses (Huang et al., 2010). OSN-AL synaptic connectivity is strongly altered by critical period olfactory experience, providing an ideal system to test circuit level FMRP roles in different neuron classes during synaptic remodeling.

Figure 1.

Maxillary palp (MP) to antennal lobe (AL) olfactory circuitry and neuron class-specific drivers A, Whole Drosophila head showing Or42a olfactory sensory neuron (OSN) innervation pattern (Or42a-Gal4>UAS-GtACR1-eYFP; green). OSN cell bodies in the maxillary palp (MP) project to the antennal lobe (AL). B, Or42a OSN innervation (Or42a-Gal4>UAS-CsChrimson::mVenus; green) of central brain (larger box in A) co-labeled for presynaptic Bruchpilot (BRP; magenta). Or42a OSNs extend axons via the labial nerve (bottom arrow) to terminate in the VM7 glomeruli (top arrow) of each AL. AL glomeruli postsynaptic projection neurons (PNs) send axons to the mushroom body (MB) and lateral horn (LH) in each brain hemisphere. C, Or42a OSN cell bodies (Or42a-mCD8-4xGFP; green) in MP (smaller box in (A) co-labeled for all OSN somata (Pebbled (Peb)-Gal4>UAS-mCD8::RFP; magenta). D, Or42a OSN axonal termini (Or42a-mCD8-4xGFP; green) and postsynaptic PNs (NP3481-Gal4>UAS-mCD8::RFP; magenta) in the AL VM7 (white box in (B). E, Neuron class-specific Gal4 drivers expressing UAS-mCD8::GFP in presynaptic OSNs (Peb-Gal4; top left), postsynaptic PNs (GH146-Gal4; top right), GABAergic local interneurons (LNs, NP1227-Gal4; bottom left) and Glutamatergic LNs (OK107-Gal4; bottom right) of the brain AL.

We have reported that odorant exposure to EB during the critical period (0–2 dpe) causes a large-scale reduction of Or42a OSN innervation of the AL VM7 glomerulus (Fig. 1A–D; Golovin et al., 2019). Based on previous work from our lab and others on the role of FMRP in mediating AL circuit remodeling (Sudhakaran et al., 2014; Doll and Broadie, 2015), and FMRP roles in regulating the critical period plasticity in other neural circuits (Dölen et al., 2007; Bureau et al., 2008; Gonçalves et al., 2013), we hypothesized that FMRP has a role in OSN remodeling. To test this idea, we assayed Or42a OSN innervation of the AL VM7 glomerulus following 0- to 2-dpe critical period EB exposure in dfmr1 null mutants (dfmr1^50M), Or42a OSN-targeted dfmr1 RNAi, and matched genetic background controls. To visualize Or42a OSN innervation of the VM7 glomerulus, the membrane marker UAS-mCD8::GFP was driven with Or42a-Gal4 (Fig. 2). Innervation volume was assayed between animals exposed to the mineral oil vehicle compared with EB dissolved in the oil at lower concentrations (15% EB; Fig. 2A) or higher concentrations (25% EB; Fig. 2B) during the 0- to 2-dpe critical period. The three genotypes compared were the transgenic control (Or42a-Gal4>mCD8::GFP; Fig. 2A,B, top panels), dfmr1 null mutant (dfmr1^50M; Fig. 2A,B, middle panels), and Or42a OSN-targeted dfmr1 knock-down (Or42a-Gal4>dfmr1 RNAi; Fig. 2A,B, bottom panels). Sample images of Or42a OSN AL innervation, quantified VM7 glomerular innervation volumes, and quantified genotype effect comparisons are all shown in Figure 2.

Figure 2.

Or42a OSN-specific FMRP loss impairs VM7 innervation critical period remodeling A, B, Representative confocal maximum intensity projections of AL innervation by Or42a OSNs (Or42a-Gal4>UAS-mCD8::GFP; green) co-labeled for presynaptic BRP (magenta). Exposure to mineral oil vehicle (oil; left) or odorant (EB) during the critical period (0–2 dpe) at either (A) 15% EB or (B) 25% EB (%V/V). Three genotypes are shown: Or42a-Gal4>mCD8::GFP transgenic control (top), dfmr1 null (dfmr1^50M; middle), and Or42a-Gal4 targeted dfmr1 RNAi (A, 1-1-7; B, TriP GL00075). C, Quantification of Or42a-OSN AL VM7 glomerulus innervation volumes comparing oil vehicle and 15% EB, normalized to vehicle control. D, The difference between oil and EB exposure for each genotype. E, Quantification of the Or42a-OSNs VM7 innervation at 25% EB for all three genotypes. F, The difference between oil and EB exposure for each genotype. The scatter plots show all data points and the mean ± SEM for each assay. The bar graphs show mean ± SER for each assay. The significance is indicated as not significant (N.S.; p > 0.05), or significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Control animals exposed to EB during the 0- to 2-dpe critical period show striking reduction of Or42a OSN innervation of the VM7 glomerulus (Fig. 2A,B, top). In contrast to our working hypothesis, animals completely lacking FMRP (dfmr1^50M null) exhibit a similar large reduction in VM7 innervation following critical period EB odorant exposure, despite an increase in the basal glomerulus innervation (Fig. 2A,B middle). In stark contrast, Or42a OSN-targeted FMRP RNAi strongly suppresses the EB odorant-dependent loss of Or42a OSN innervation, supporting the hypothesis (Fig. 2A,B, bottom). An ANOVA (3 × 2) was used to compare the effects on the three genotypes exposed to vehicle control versus 15% EB (Fig. 2C). Quantification of the Or42a OSN innervation volume shows significant effects of both genotype (F_(2,123) = 17.20, p = 2.59e-7) and EB exposure (F_(1,123) = 14, p = 0.0003), with a significant interaction between genotype and odorant exposure (F_(2,123) = 12.21, p = 1.45e-5). The remodeling is EB concentration-dependent, as increasing the odorant concentration (25% EB) causes a larger innervation reduction (Fig. 2A vs B). An ANOVA (3 × 2) comparing transgenic control, dfmr1 null, and Or42a OSN-targeted FMRP RNAi shows significant effects of both genotype (F_(2,170) = 29.09, p = 1.363e-11) and EB exposure (F_(1,170) = 329.7, p = 3.113e-59), with a significant interaction between the genotype and the odorant (F_(2,170) = 33.20, p = 6.732e-13; Fig. 2E). Quantitative analyses with pairwise comparisons as well as linear regression models further indicate that Or42a OSN-specific FMRP loss suppresses critical period remodeling.

Unpaired t tests with Sidak's corrections were done to compare the ANOVA conditions. In controls, VM7 innervation is significantly reduced following 15% EB critical period exposure [normalized vehicle control (oil) 1.0 ± 0.046 (n = 24 brains) vs 15% EB 0.592 ± 0.065 (n = 23); t₍₁₂₃₎ = 3.331, p = 0.017; Fig. 2C, bottom left bar]. A stronger reduction (91.2% vs 40.8%) occurs with 25% EB [oil control 1.0 ± 0.033 (n = 37 brains) vs 25% EB 0.088 ± 0.019 (n = 32); t₍₁₇₀₎ = 12.47, p = 1.38e-24; Fig. 2E, bottom left]. Compared with vehicle control animals, dfmr1 nulls have significantly greater VM7 basal innervation in both 15% EB [oil dfmr1 null 1.698 ± 0.2 (n = 12); t₍₁₂₃₎ = 4.705, p = 0.0001; Fig. 2C, middle bar] and 25% EB experiments [oil dfmr1 null 1.41 ± 0.098 (n = 28); t₍₁₇₀₎ = 5.41, p = 3.18e-6; Fig. 2E, middle bar]. Nevertheless, there is still a strong reduction in innervation following critical period EB odorant exposure (Fig. 2A,B, middle). Following 15% EB, null mutants show a significant loss of innervation volume [oil dfmr1 null vs 15% EB dfmr1 null 1.03 ± 0.12 (n = 17); t₍₁₂₃₎ = 4.224, p = 0.0007; Fig. 2C, bottom center bar]. Likewise, null dfmr1 VM7 glomerular innervation is significantly reduced following 25% EB exposure compared with the oil-exposed controls [25% EB dfmr1 null 0.12 ± 0.041 (n = 19); t₍₁₇₀₎ = 14.33, p = 7.12e-30; Fig. 2E, bottom center]. Similar to control animals, the effect of the higher 25% EB exposure is greater than the lower 15% EB exposure in the dfmr1 null mutants (91.5% vs 39.3%). These results indicate FMRP loss has an experience-independent function regulating basal glomerulus innervation, but little impact on experience-dependent remodeling.

In direct contrast to dfmr1 nulls, FMRP RNAi targeted only to Or42a OSNs using two different constructs (1-1-7, Bolduc et al., 2008; TRiP GL00075, Flockhart et al., 2006; Greenblatt and Spradling, 2018) strongly impairs critical period remodeling (Fig. 2A,B, bottom). Innervation volume in vehicle knock-down animals is not significantly different from controls [15% EB: oil Or42a OSN-targeted FMRP RNAi 1.01 ± 0.069 (n = 27); t₍₁₂₃₎ = 0.1197, p > 0.9999; 25% EB: oil Or42a OSN-targeted FMRP RNAi 1.12 ± 0.053 (n = 32); t₍₁₇₀₎ = 1.663, p = 0.79]. Moreover, 15% EB critical period exposure does not significantly alter VM7 innervation [15% EB 1.23 ± 0.09 (n = 26); t₍₁₂₃₎ = 1.841, p = 0.6527; Fig. 2C, bottom right bar]. At 25% EB, innervation remodeling is also suppressed with Or42a OSN-targeted FMRP knock-down compared with vehicle controls [25% EB 0.78 ± 0.064 (n = 28); t₍₁₇₀₎ = 4.335, p = 0.0004; Fig. 2E, bottom right]. Since targeted FMRP removal in Or42a OSNs significantly reduces odorant experience-dependent remodeling of VM7 innervation, EB exposure in the knock-down condition was directly compared with the EB control to show a significantly reduced effect on VM7 innervation, at both 15% and 25% EB concentrations [15% EB control vs 15% EB Or42a OSN-targeted FMRP RNAi; t₍₁₂₃₎ = 5.279, p = 8.52e-6 (Fig. 2C, top bar); 25% EB control vs 25% EB Or42a OSN-targeted FMRP RNAi; t₍₁₇₀₎ = 8.847, p = 1.69e-14 (Fig. 2E, top)]. The inability of FMRP RNAi to completely block OSN remodeling after 25% EB exposure could indicate that rather than impairing the mechanism directly it alters the OSN response to the odorant, thereby increasing the stimulus threshold needed for innervation loss. These results suggest that Or42a OSN-specific FMRP removal within the AL circuit has a significant impact on odorant experience critical period remodeling, but required further analyses to account for variations in basal innervation volume.

Differences in basal OSN innervation between genotypes complicates comparing the EB-exposed conditions. Therefore, a linear regression model was used to analyze EB exposure compared with vehicle, while controlling for genotype differences (see Materials and Methods). Unpaired t tests of the null hypothesis that genotype does not affect the treatment relationship were done compare regression coefficients for each interaction term. Regression analyses show dfmr1 null interaction with 15% EB exposure is not significant (dfmr1x15% EB β = −0.26 ± 0.2; t₍₁₂₃₎ = 1.302, p = 0.1953; Fig. 2D, top), indicating no significant impact from global FMRP loss on EB exposure effects. With 25% EB, the dfmr1 null regression coefficient becomes significant (β = −0.38 ± 0.12; t₍₁₇₀₎ = 3.267, p = 0.0013; Fig. 2F, top), indicating FMRP removal augments innervation loss from odorant exposure. Consistent with the pairwise comparisons between the control genotype and Or42a OSN-targeted FMRP RNAi, regression analyses show significant interactions at both 15% and 25% EB (Or42a OSN-targeted FMRP RNAix15% EB β = 0.62 ± 0.17; t₍₁₂₃₎ = 3.687, p = 0.0003; Or42a OSN-targeted FMRP RNAix25% EB β = 0.57 ± 0.11; t₍₁₇₀₎ = 5.334, p = 4.54e-6; Fig. 2D,F, bottom). Together, these results indicate that FMRP has two roles: (1) a cell non-autonomous role regulating basal Or42a OSN-VM7 innervation, with dfmr1 nulls displaying experience-independent increased glomeruli innervation; and (2) an OSN cell autonomous role regulating critical period olfactory experience-dependent remodeling, with Or42a-specific FMRP removal, but not global FMRP loss, limiting innervation refinement.

Sex-specific differences in early odorant experience critical period remodeling of AL innervation

In experiments testing the role of FMRP in critical period OSN remodeling, we observed that much of the variation between animal responses to EB exposure could be attributed to sex. To quantify this sex difference, we compared male and female animals following exposure to vehicle control and 20% EB from 0 to 2 dpe. To further validate our finding that Or42a-targeted RNAi against FMRP disrupts EB-dependent AL innervation remodeling, we used a third RNAi targeting FMRP (2–1; Bolduc et al., 2008). After odorant exposure, the volume of the Or42a OSN innervation of the VM7 glomerulus was assessed by quantifying Or42a-Gal4>UAS-mCD8::GFP. Animals exposed to EB during the critical period show pronounced changes in VM7 glomeruli, with sparser innervation and the appearance of OSN puncta (Fig. 3A,B). In EB-exposed males, OSN puncta occur in both controls and dfmr1 RNAi animals, but the exposure has relatively little impact on the glomerulus innervation volume (Fig. 3A). Or42a-targeted dfmr1 RNAi males lack any VM7 innervation loss but produce more OSN puncta (Fig. 3A, bottom right). In EB-exposed females, there is a much more pronounced response to odorant experience during the critical period, with a strong shift toward greater innervation loss but with fewer OSN puncta (Fig. 3B). As with the males, females expressing Or42a-targeted dfmr1 RNAi exhibit very altered critical period remodeling, with the appearance of greater numbers of OSN puncta and much less loss of the overall VM7 glomerulus innervation compared with control females (Fig. 3B, bottom right). Representative images for both the sexes and both the genotypes, together with the quantitative innervation measurements, are shown in Figure 3.

Figure 3.

Sex-specific differences in EB-dependent critical period remodeling of AL innervation. A, Representative confocal maximum intensity projections of Or42a OSNs innervating the male AL VM7 glomerulus (Or42a-Gal4>UAS-mCD8::GFP; white). B, Representative images from females under identical conditions. All animals were exposed to mineral oil vehicle (top), or 20% EB odorant (bottom) during the 0- to 2-dpe critical period. The paired genotypes shown are the following: the transgenic control (Or42a-Gal4>mCD8::GFP; left column) and the same transgenic line with dfmr1 RNAi expression (Or42a-Gal4>dfmr1 RNAi 2-1; right column). C, Quantification of the Or42a OSN VM7 innervation volumes for both genotypes, treatment conditions, and sexes. Scatter plots show all data points and the mean ± SEM. The significance is indicated as not significant (N.S.; p > 0.05), or significant at **p < 0.01 and ***p < 0.001.

A three-way ANOVA (2 × 2 × 2) comparing innervation volume reveals significant effects of EB exposure (F_(1,206) = 17.47, p = 4.313e-5), genotype (F_(1,206) = 21.80, p = 5.45e-6) and sex (F_(1,206) = 57.26, p = 1.251e-12; Fig. 3C). There are significant interactions between odorant and genotype (F_(1,206) = 26.87, p = 5.179e-7) as well as sex (F_(1,206) = 63.83, p = 9.485e-14), but not genotype and sex (F_(1,206) = 0.005, p = 0.9427). EB-exposed control males show only a small loss in innervation volume [oil 1 ± 0.036 (n = 26) vs EB 0.91 ± 0.097 (n = 25); multiple comparisons with Sidak's correction, t₍₂₀₆₎ = 0.9129, p > 0.9999; Fig. 3C, bottom left bar] compared with dfmr1 RNAi males with significantly greater innervation relative to vehicle exposure [oil 0.955 ± 0.1 (n = 26) vs EB 1.412 ± 0.099 (n = 25); t₍₂₀₆₎ = 4.636, p = 0.0002; Fig. 3C, second bottom bar]. EB-exposed control females show a much greater loss of innervation [oil 1.0 ± 0.037 (n = 28) vs EB 0.187 ± 0.029 (n = 28); t₍₂₀₆₎ = 8.634, p = 4.55e-14; Fig. 3C, third bottom bar], with Or42a-Gal4>dfmr1 RNAi causing severely attenuated remodeling [RNAi oil 0.996 ± 0.063 (n = 28) vs EB 0.635 ± 0.053 (n = 28); t₍₂₀₆₎ = 3.831, p = 0.0047; Fig. 3C, right bottom bar]. Quantification reveals that Or42a-targeted dfmr1 RNAi causes significantly altered innervation in both sexes (control EB male vs RNAi EB Male; t₍₂₀₆₎ = 5.042, p = 2.81e-5; control EB female vs RNAi EB female; t₍₂₀₆₎ = 4.756, p = 0.0001; Fig. 3C, second bars). EB-exposed females have significantly less innervation than males in both controls and with dfmr1 RNAi (control EB male vs control EB female; t₍₂₀₆₎ = 7.457, p = 6.78e-11; RNAi EB male vs RNAi EB female; t₍₂₀₆₎ = 8.02, p = 2.2e-12; Fig. 3C, top two bars). Together, these results indicate critical period EB exposure causes a more robust loss of glomerulus innervation in control females, with Or42a-targeted dfmr1 RNAi strongly attenuating this OSN remodeling. For consistency and clarity in dissecting the causal mechanisms, females were employed in subsequent experiments.

Null dfmr1 mutants and global FMRP RNAi animals both maintain critical period remodeling

Because of the above surprising difference between dfmr1 null mutants and Or42a OSN-targeted dfmr1 RNAi, we wanted to test the conclusion that neuron class-specific differences (Or42a OSN-targeted vs global) rather than technical differences (null mutant vs all three RNAi knock-downs) accounts for the result. We differentiated between these two possibilities by testing olfactory experience-dependent remodeling of Or42a OSN VM7 innervation with a global FMRP knock-down (UH1-Gal4>dfmr1 RNAi; Wodarz et al., 1995) and transgenic control lacking dfmr1 RNAi, compared with a second dfmr1 null mutant (dfmr1^B55; Inoue et al., 2002). Animals of these three genotypes expressing a membrane-bound GFP under direct control of the Or42a promoter (Or42a-mCD8::4xGFP; Stephan et al., 2012) were exposed to either oil vehicle or 20% EB during the 0–2 dpe critical period. If all three genotypes show the strong reduction in Or42a OSN VM7 innervation following EB exposure relative to the vehicle control, then the result supports neuron class-specific FMRP roles within the AL olfactory circuit. On the other hand, if the UH1-Gal4>dfmr1 RNAi animals show an impaired response to odorant experience during the critical period, then this would suggest an important difference between the FMRP null mutant and RNAi knock-down in mediating the olfactory experience-dependent remodeling phenotype. Representative images of brain FMRP levels and VM7 glomerulus innervation, as well as quantifications for all conditions, are shown in Figure 4.

Figure 4.

Neither dfmr1 mutants nor global dfmr1 RNAi impair OSN critical period remodeling A, Representative confocal maximum intensity projections of the entire central brain labeled with anti-FMRP (white) in the w^-;Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP;UH1-Gal4/+ transgenic control (top), with ubiquitous dfmr1 RNAi (w^-;Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP;UH1-Gal4/UAS-dfmr1 RNAi TriP GL00075; middle) and in a dfmr1 null mutant (dfmr1^B55; bottom). B, Representative confocal maximum intensity projections of Or42a OSNs innervating the female AL VM7 glomerulus (Or42a-mCD8-4xGFP; white). The same genotypes above exposed to mineral oil vehicle (left) or 20% EB odorant (right) from 0 to 2 dpe. The bright puncta following EB exposure are labeled by white arrows. C, Quantification of Or42a-OSN AL VM7 glomerulus innervation volumes comparing oil vehicle and 20% EB of all three genotypes. D, The difference between oil and EB exposure for each genotype. The scatter plots show all data points and the mean ± SEM for each assay. The bar graphs show mean ± SER for each assay. The significance is indicated as not significant (N.S.; p > 0.05), or significant at *p < 0.05, **p < 0.01, ***p < 0.001.

In first testing our genetic tools, both global dfmr1 RNAi and the homozygous dfmr1^B55 mutation led to an indistinguishable complete loss of brain FMRP expression compared with the robust FMRP levels in the matched transgenic controls (Fig. 4A, top vs middle and bottom). This agrees with previous reports on these lines (Inoue et al., 2002; Greenblatt and Spradling, 2018), showing a loss of detectable FMRP. As in the above experiments, exposing control animals to EB during the 0- to 2-dpe critical period causes a stark reduction of Or42a OSN VM7 glomerulus innervation (Fig. 4B, top). Importantly, the global UH1-Gal4>dfmr1 RNAi animals look remarkably like the null mutants (Fig. 4B, middle). Global FMRP removal leads to an increase in the basal oil-exposed Or42a OSN innervation volume, while maintaining robust olfactory experience-dependent remodeling. As in the dfmr1^50M null mutant above, the alternate dfmr1^B55 null mutant shows an indistinguishable response to both oil vehicle and EB exposure, with vehicle-treated animals showing a larger innervation volume compared with controls, but still maintaining the robust innervation loss following critical period EB odorant exposure (Fig. 4B, bottom). Note also the characteristic Or42a OSN puncta in the VM7 glomeruli of both the global dfmr1 knock-down and null mutant following critical period EB exposure (Fig. 4B, middle and bottom arrows) suggesting a dynamic stage in the OSN remodeling process, as we reported previously (Golovin et al., 2019). Together, these results support an OSN-specific FMRP role in olfactory experience-dependent critical period remodeling.

ANOVA (3 × 2) analyses support the qualitative conclusion with significant effects of odor (F_(1,161) = 487.5, p = 1.411e-50) and genotype (F_(2,161) = 18.08, p = 8.252e-8), but no significant interaction (F_(2,161) = 2.57, p = 0.0797; Fig. 4C). Pairwise t tests with Sidak's correction indicate EB exposure significantly reduces innervation in all 3 genotypes [control oil 1.0 ± 0.05 (n = 34) vs EB 0.008 ± 0.006 (n = 35); t₍₁₆₁₎ = 13.54, p = 3.4e-27; UH1-Gal4>dfmr1 RNAi oil 1.377 ± 0.08 (n = 31) vs EB 0.156 ± 0.04 (n = 29); t₍₁₆₁₎ = 15.52, p = 1.27e-32; dfmr1^B55 oil 1.328 ± 0.06 (n = 19) vs EB 0.318 ± 0.10 (n = 19); t₍₁₆₁₎ = 10.23, p = 4.62e-18; Fig. 4C, bottom bars]. Vehicle null and RNAi animals both have larger basal innervation volumes (oil control vs oil UH1-Gal4>dfmr1 RNAi; t₍₁₆₁₎ = 4.989, p = 2.3e-5; oil control vs dfmr1^B55 oil; t₍₁₆₁₎ = 3.76, p = 0.0036; Fig. 4C, middle and top bars). Multiple linear regression to compare vehicle and EB exposure while controlling for this innervation difference shows UH1-Gal4>dfmr1 animals have a slightly larger change (UH1-Gal4>dfmr1 RNAi x 20% EB β = −0.23 ± 0.11; t₍₁₆₁₎ = 2.127, p = 0.035; Fig. 4D, bottom bar) and dfmr1 nulls have no significant difference (dfm1^B55 x 20% EB β = −0.02 ± 0.12; t₍₁₆₁₎ = 0.147, p = 0.8833; Fig. 4D, top bar). Despite this small difference, both the ubiquitous FMRP knock-down and dfmr1^B55 mutant largely resemble the dfmr1^50M mutant. These findings indicate that Or42a OSN-targeted FMRP removal selectively disrupts olfactory experience-dependent critical period remodeling. Together, these results further demonstrate that global FMRP loss in the AL circuit can be compensated for, despite the Or42a OSN-specific FMRP function.

Or42a OSN-specific FMRP OE enhances critical period odorant remodeling

The above results suggest that a FMRP balance between Or42a OSNs and other circuit neurons is required for proper critical period remodeling. If so, then elevating FMRP levels in OR42a OSNs should also alter experience-dependent remodeling. We have found bidirectional FMRP regulation within other neural circuits (Zhang et al., 2001; Sears et al., 2019). To test whether targeted FMRP OE impacts critical period remodeling, transgenic controls (Or42a>mCD8::GFP) were compared with UAS-FMRP 9557–3 (Or42a>FMRP OE; Zhang et al., 2001) after exposure to either the oil vehicle or 20% EB during 0–2 dpe. As above, the controls show a striking reduction in Or42a OSN innervation (Fig. 5A, left panels, top vs bottom). In agreement with the FMRP balance hypothesis, Or42a OSN-targeted FMRP OE greatly enhances this olfactory experience-dependent remodeling (Fig. 5A, right panels, top vs bottom). Note that the directional disruption of the Or42a OSN innervation volume change is the opposite to Or42a OSN-targeted FMRP knock-down (Fig. 2), indicating a clear bidirectional consequence of FMRP imbalance within the circuit. Both the control and FMRP OE conditions show the OSN puncta characteristic of remodeling (Fig. 5A, arrows), but remnant Or42a OSN innervation is much sparser in the FMRP OE condition, similar to the consequence of increasing EB odorant concentration (Fig. 2). The quantitative assessment of Or42a OSN VM7 innervation volume further supports the role in FMRP balance in regulating olfactory experience-dependent synaptic remodeling during the early-use critical period.

Figure 5.

Or42a OSN-specific FMRP OE increases VM7 innervation remodeling A, Representative confocal maximum intensity projections of Or42a OSNs innervating the AL VM7 glomerulus (Or42a-Gal4>UAS-mCD8::GFP; white). Females were exposed to mineral oil vehicle (top) or 20% EB odorant (bottom) from 0 to 2 dpe. Two genotypes are shown: transgenic control (Or42a-Gal4>mCD8::GFP; left), and the same transgenic line overexpressing FMRP (Or42a-Gal4>FMRP 9557-3; right). The bright puncta following EB exposure are labeled by white arrows. B, Quantification of the Or42a-OSN VM7 innervation volume for both genotypes and treatment conditions. Scatter plots show all data points and the mean ± SEM. The significance is indicated as ***p < 0.001.

ANOVA (2 × 2) analyses to compare these conditions show significant effects of both the genotype (F_(1,71) = 11.03, p = 0.0014) and odorant exposure (F_(1,71) = 186.7, p = 1.471e-21), with a significant interaction between them (F_(1,71) = 13.96, p = 0.0004; Fig. 5B). Unpaired t tests with Sidak's correction pairwise comparisons show EB exposure in controls significantly reduces Or42a OSN innervation [control oil 1.0 ± 0.037 (n = 17) vs 20% EB 0.48 ± 0.054 (n = 17); t₍₇₁₎ = 6.714, p = 2.35e-8; Fig. 5B, bottom left], with a stronger effect following targeted FMRP OE [oil 1.022 ± 0.07 (n = 21) vs EB 0.11 ± 0.032 (n = 20); t₍₇₁₎ = 12.92, p = 1.53e-19; Fig. 5B, bottom right]. FMRP OE does not impact the basal innervation (t₍₇₁₎ = 0.2955, p = 0.9998), so we directly compared EB-exposed control and FMRP OE conditions. FMRP OE in the Or42a OSNs significantly enhances critical period remodeling from the EB exposure (t₍₇₁₎ = 4.963, p = 2.75e-5; Fig. 5B, top bar). This confirms the ANOVA analyses indicating a significant interaction between FMRP OE and odorant exposure. These results support the conclusion that FMRP balance within the AL circuit determines olfactory experience-dependent synaptic remodeling during the critical period. More specifically, the opposite effects of targeted FMRP decrease and increase only in Or42a OSNs indicates a bidirectional regulation of remodeling. When FMRP levels are lower in Or42a OSNs compared with other neurons, remodeling is diminished, and conversely increasing FMRP levels in Or42a OSNs enhances critical period remodeling.

Pan-OSN FMRP knock-down does not impact olfactory experience critical period remodeling

The above results indicate FMRP works cell autonomously and cell non-autonomously in opposition to regulate critical period olfactory experience OSN synaptic remodeling. Given the Or42a OSN-specific FMRP functions, the next goal was to identify the neurons providing the cell non-autonomous counterbalance. To begin this new pursuit, Or42a OSN remodeling was tested after altering FMRP levels in all OSNs. The Orco-Gal4 line drives expression in all OSNs (Larsson et al., 2004) but comes on relatively late in pupation. Pebbled-Gal4 (Peb-Gal4) is also expressed in all OSNs (Fig. 1E; Sweeney et al., 2007) and comes on earlier. Since both drivers include the Or42a OSNs, the prediction is that if only the Or42a OSNs are involved in the critical period remodeling, then altering FMRP levels should phenocopy the Or42a OSN-specific driver. Alternatively, if other OSNs contribute to Or42a OSN remodeling, then this should phenocopy the global UH1-Gal4, without affecting Or42a OSN remodeling. To test these two possibilities, Peb-Gal4 and Orco-Gal4 were used to eliminate and overexpress FMRP throughout the OSN population while assaying specifically for Or42a OSN olfactory experience-dependent critical period remodeling. Peb-Gal4 was first used to drive dfmr1 RNAi, while labeling Or42a OSNs using Or42a-mCD8::4xGFP. Transgenic controls (lacking the RNAi) and experimental animals were again exposed to oil vehicle or 20% EB from 0 to 2 dpe. Sample images of FMRP expression in maxillary palp OSNs and Or42a OSN innervation in VM7 glomeruli, as well as quantified glomerular innervation values, are all shown in Figure 6.

Figure 6.

Pan-OSN FMRP knock-down/OE does not impact the VM7 remodeling A, Maxillary Palp (MP) anti-FMRP (magenta; top row), co-labeled with Or42a OSNs (Or42a-mCD8-4xGFP, green; second row) in transgenic control (w^-;Peb-Gal4/+;Or42a-mCD8::4xGFP/+;Or42a-mCD8::4xGFP/+; left) and Peb-Gal4>dfmr1 RNAi (w^-;Peb-Gal4/+; Or42a-mCD8::4xGFP/+; Or42a-mCD8::4xGFP/UAS-dfmr1 RNAi; right). Bottom two rows, Or42a OSN innervation of VM7 glomerulus after exposure to oil vehicle or 20% EB from 0 to 2 dpe. BRP labeling (magenta) shows VM7 and surrounding glomeruli (dotted white outlines). B, Quantification of Or42a OSN VM7 innervation volume with Peb-Gal4 dfmr1 RNAi. C, The same MP labeling of transgenic control (w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP; Orco-Gal4/+; left) and Orco-Gal4 FMRP OE (w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP; Orco-Gal4/UAS-FMRP 9557-3; right). Bottom two rows, The same Or42a OSN-VM7 innervation exposed to either oil vehicle or 20% EB from 0 to 2 dpe. D, Quantification of the Or42a-OSN VM7 innervation volume for Orco-Gal4 FMRP OE. Scatter plot shows all data points and the mean ± SEM. The significance is indicated as not significant (N.S.; p > 0.05), or significant at ***p < 0.001.

All OSN cell bodies in the maxillary palp express FMRP, and can be co-labeled for the Or42a OSN population (Fig. 6A, top). Peb-Gal4 driven FMRP RNAi (Peb-Gal4>dfmr1 RNAi) strongly suppresses FMRP in all OSNs, including Or42a OSNs (Fig. 6A, right). Following EB exposure, transgenic controls exhibit a near complete loss of Or42a OSN innervation (Fig. 6A, bottom). Because of strong loss of Or42a innervation (GFP signal) in the VM7 glomeruli, presynaptic active zone BRP labeling is also shown to outline the AL glomeruli (Fig. 6A, bottom, magenta). Three distinct glomeruli are thus demarcated, including the central VM7 glomerulus innervated by Or42a OSNs (Fig. 6A, dotted white lines). In the oil-exposed animals, typical innervation is observed. After 20% EB critical period exposure, both transgenic controls and the Peb-Gal4>dfmr1 RNAi animals show near complete loss of VM7 innervation (Fig. 6A, third row vs bottom). Importantly, Or42a OSN innervation loss corresponds with the loss of synaptic BRP within VM7 glomeruli, and therefore an inability to clearly define the VM7 border with BRP labeling (Fig. 6A, bottom, white dotted regions). This loss of BRP synapse after critical period EB exposure is consistent with our previous report that strong olfactory experience during the critical period diminishes BRP volume and intensity within Or42a OSN presynaptic active zones (Golovin et al., 2019). These results indicate that global OSN-targeted FMRP removal does not impact Or42a OSN innervation remodeling because of EB exposure during the critical period, a conclusion next confirmed by quantitative assessment.

ANOVA (2 × 2) analyses to compare genotypes versus odorant treatments show a significant effect of odorant (F_(1,57) = 378.3, p = 7.734e-27), but not genotype (F_(1,57) = 1.148, p = 0.288), with no significant interaction between genotype and odorant (F_(1,57) = 0.7329, p = 0.396; Fig. 6B). Unpaired t tests with Sidak's correction evaluating pairwise comparisons indicate EB-exposed control animals have significantly reduced Or42a OSN innervation [control oil 1.0 ± 0.032 (n = 17) vs 20% EB 0.002 ± 0.001 (n = 17); t₍₅₇₎ = 14.02, p = 4.64e-26; Fig. 6B, bottom left]. FMRP OSN knock-down (Peb-Gal4>dfmr1 RNAi) also causes significantly reduced OSN innervation volumes following EB exposure [Peb-Gal4> dfmr1 RNAi oil 1.103 ± 0.102 (n = 15) vs EB 0.013 ± 0.009 (n = 12); t₍₅₇₎ = 13.56, p = 1.042e-18; Fig. 6B, bottom right]. FMRP removal does not impact the basal extent of VM7 innervation (t₍₅₇₎ = 1.408, p = 0.6603), and we therefore can directly compare the two EB-exposed genotypes. Peb-Gal4>dfmr1 RNAi in all the OSNs does not significantly impact the Or42a OSN innervation following critical period EB exposure compared with transgenic controls (t₍₅₇₎ = 0.1478, p > 0.9999; Fig. 6B, top bar). This confirms the above ANOVA analyses indicating no significant interaction between EB exposure and the Peb-Gal4 FMRP knock-down. These results bolster the hypothesis that balanced FMRP within the AL circuit acts to regulate Or42a OSN critical period remodeling, and predict that OE of FMRP in all OSNs should also have no effect, thus mirroring the results of pan-OSN knock-down.

We next examined FMRP in transgenic control and Orco-Gal4>FMRP maxillary palps to find strong FMRP OE in all OSNs, including the marked Or42a OSNs (Fig. 6C, top). Similar to global OSN FMRP knock-down, FMRP OE throughout OSNs does not impact Or42a OSN critical period remodeling following EB exposure (Fig. 6C, third and bottom rows, compare left and right). ANOVA (2 × 2) quantification confirms the similar EB effect between transgenic controls and Orco-Gal4>FMRP OE animals, with a significant main effect of odorant exposure (F_(1,71) = 907.8, p = 3.473e-42), but not genotype (F_(1,71) = 0.518, p = 0.474), and no significant interaction between EB exposure and genotype (F_(1,71) = 1.506, p = 0.224; Fig. 6D). Pairwise comparisons using unpaired t tests with Sidak's correction reveal that EB exposure significantly reduces VM7 glomerular innervation in both controls and with Orco-Gal4>FMRP OE compared with the oil-exposed animals [control oil 1.0 ± 0.05 (n = 16) vs 20% EB 0.049 ± 0.03 (n = 19); t₍₇₁₎ = 19.77, p = 8.28e-30; Orco-Gal4>FMRP oil 1.06 ± 0.04 (n = 19) vs 20% EB 0.033 ± 0.01 (n = 21); t₍₇₁₎ = 22.98, p = 7.91e-34; Fig. 6D, bottom bars]. In addition, these tests show that Orco-Gal4>FMRP OE does not alter VM7 innervation after oil or EB exposure (control oil vs Orco-Gal4>FMRP oil; t₍₇₁₎ = 1.331, p = 0.712; control 20% EB vs Orco-Gal4>FMRP 20% EB; t₍₇₁₎ = 0.372, p = 0.999; Fig. 6D, top bar). These results, along with the results from global OSN FMRP RNAi, all point to a FMRP role in Or42a OSNs and other OSN classes controlling VM7 innervation remodeling driven by critical period olfactory experience.

Or42a OSN-specific optogenetic activation is sufficient to drive critical period remodeling

The FMRP role across the OSN population suggests a function mediating Or42a OSN innervation remodeling in response to EB odorant exposure in the critical period. We previously showed that the functional Or42a receptor is required for Or42a OSN critical period remodeling (Golovin et al., 2019). However, EB activates other OSN classes (DoOR v2.0; Münch and Galizia, 2016), and higher EB levels may activate more OSNs (Semmelhack and Wang, 2009). Therefore, it remains possible that critical period remodeling requires both Or42a receptors and EB-sensitive receptors in other OSN classes. To test this hypothesis, parallel approaches were employed. First, CsChrimson::mVenus channelrhodopsin (Klapoetke et al., 2014) was targeted to Or42a OSNs (Or42a-Gal4>CsChrimson::mVenus) for the specific activation of Or42a OSNs with timed cyan (515 nm) light stimulation (Fig. 7A,B). Second, removal of the essential olfactory Orco co-receptor required to mediate OSN responses (Larsson et al., 2004) was used to compare orco null mutants and orco nulls with Orco re-expressed only within Or42a OSNs, compared with transgenic driver controls (Fig. 7C). These tests allow the assessment of (1) the requirement of OSN activity in general, and (2) the sufficiency of Or42a OSN activity specifically, to mediate critical period remodeling. As above, Or42a OSN innervation was imaged within the VM7 glomerulus following 0- to 2-dpe critical period exposure to either the oil vehicle or 20% EB. Representative images and innervation quantifications for both channelrhodopsin and orco mutant experiments are shown in Figure 7.

Figure 7.

Or42a OSN-targeted neuronal activation drives VM7 critical period remodeling. A, Representative confocal maximum intensity projections of Or42a-OSN VM7 innervation, with Or42a-Gal4 driven expression of fluorescently tagged channelrhodopsin Cschrimson::mVenus. Females were reared in complete darkness (dark, top) or with 515 nm cyan light (light, bottom) during the 0- to 2-dpe critical period. B, Quantification of Or42a-OSN VM7 innervation volume without activation (dark) and with optogenetic stimulation (light). C, Representative images of Or42a OSN VM7 innervation (Or42a-Gal4>UAS-mCD8::GFP; white). Females were exposed to the oil vehicle (top) or 20% EB (bottom) during the 0- to 2-dpe critical period. Three genotypes are shown: transgenic control (w^-; UAS-mCD8::GFP/+; Or42a-Gal4/+; left), orco null mutant (orco¹/orco²; middle), and the orco null with Or42a OSN-targeted Orco rescue (orco¹/orco², Or42a-Gal4>UAS-Orco; right). D, Quantification of VM7 innervation for all genotypes and conditions. E, The difference between the oil vehicle and EB exposures. Scatter plots show all data points and mean ± SEM. Bar graphs show mean ± SER. The significance is indicated as significant at *p < 0.05, **p < 0.01, ***p < 0.001.

To mimic the effects of EB odorant exposure during the critical period, Or42a-Gal4> CsChrimson::mVenus animals were staged and exposed to 515 nm light as closely as possible to the manner of EB odorant exposure. However, there were two differences: (1) the animals were kept in constant darkness before exposure to cyan light stimulus; and (2) the animals were raised in Petri dishes to allow for stronger optogenetic light stimulation (see Materials and Methods). Critical period light exposure of the targeted Or42a OSN CsChrimson-expressing animals drives a striking reduction of the Or42a innervation of the VM7 glomerulus, which qualitatively resembles the remodeling driven by the critical period EB olfactory experience (Fig. 7A, top vs bottom). Changes include a reduction in intensity, sparse and reduced glomerulus coverage, and appearance of the characteristic OSN puncta. This qualitative assessment is supported by quantitative measurements, which show that Or42a-Gal4>CsChrimson::mVenus animals exposed to cyan light during the critical period have significantly reduced innervation compared with dark-reared control animals (dark 1.0 ± 0.058 vs light 0.08 ± 0.02; t₍₂₆₎ = 15.04; p = 2.421e-14, unpaired t test; Fig. 7B). The ability of channelrhodopsin-driven activity to reduce Or42a OSN innervation similar to olfactory EB exposure indicates that Or42a activity is sufficient for critical period synaptic remodeling of VM7 glomerulus innervation. However, because light and odorant driven activity levels in the Or42a neurons could be different, it is possible that the magnitude of innervation remodeling is different when only Or42a OSNs are activated.

Or42a OSN-specific odorant activation is essential for critical period innervation remodeling

To test whether Or42a OSN-specific EB receptor activation produces the same effect as global OSN EB activation, orco null mutants were compared with orco nulls with targeted Orco rescue only in Or42a OSNs (Or42a-Gal4>UAS-Orco; Fig. 7C). As in all the above experiments, the transgenic controls show the normal striking reduction in VM7 innervation caused by 20% EB odorant exposure during the 0- to 2-dpe critical period, compared with the robust innervation characterizing the oil vehicle alone (Fig. 7C, left, top vs bottom). Consistent with the role of olfactory reception mediating critical period remodeling (Golovin et al., 2019), orco null mutants lack any reduction in VM7 innervation following critical period EB exposure, compared with the matched oil-exposed animals (Fig. 7C, middle, top vs bottom). In agreement with the above optogenetic studies, when Orco is re-expressed only in Or42a OSNs (Or42a-Gal4>Orco) there is again a strong reduction in VM7 innervation following EB olfactory experience during the critical period, compared with the oil-exposed animals (Fig. 7C, right, top vs bottom). Interestingly, the reduction of VM7 innervation with targeted Or42a-Gal4>Orco rescue appears even more extreme than the matched control animals (Fig. 7C, left bottom vs right bottom). In the targeted Or42a-Gal4>Orco rescue animals, critical period EB exposure generates sparser and less intensely labeled VM7 glomeruli innervation. Taken together, these results suggest a highly specific Or42a OSN activity requirement in critical period innervation remodeling, a conclusion supported and expanded by quantitative analyses.

ANOVA (3 × 2) quantification comparing the controls and orco null mutants exposed to either EB or oil vehicle alone during the 0- to 2-dpe critical period shows significant effects of genotype (F_(2,105) = 76.63 p = 3.016e-21) and odorant exposure (F_(1,105) = 266.3, p = 1.455e-30), with a significant interaction between the two (F_(2,105) = 142.3, p = 1.273e-30; Fig. 7D). Unpaired t tests with Sidak's correction evaluating pairwise comparisons show EB-exposed transgenic controls (Or42a-Gal4>mCD8::GFP) have significantly reduced innervation volumes [control oil 1.0 ± 0.025 (n = 20) vs EB 0.365 ± 0.045 (n = 17); t₍₁₀₅₎ = 12.69, p = 9.46e-22; Fig. 7D, bottom left bar]. Unexpectedly, the oil-exposed orco null mutants (orco¹/orco², Larsson et al., 2004) exhibit significantly lower innervation volumes compared with matched controls [control oil vs orco oil 0.842 ± 0.032 (n = 19); t₍₁₀₅₎ = 3.245, p = 0.0234; Fig. 7D, second from bottom bar]. Although the orco null AL has been reported to have grossly normal overall morphology (Larsson et al., 2004), this basal difference could mark the beginnings of the later glomerular degeneration that later occurs in orco null mutants (Chiang et al., 2009). Also surprisingly, orco null mutants exposed to 20% EB during the critical period have significantly larger glomerulus innervation volumes than the oil-exposed orco mutants [orco oil vs orco EB 1.028 ± 0.041 (n = 20); t₍₁₀₅₎ = 3.814, p = 0.0035; Fig. 7D, bottom middle bar]. These results show that complete loss of olfaction in all the OR-expressing OSNs has striking impacts on the olfactory circuitry and may shift the AL circuit connectivity in unexpected ways.

Since the oil-exposed control animals and EB-exposed orco mutants show no significant difference in VM7 glomerulus innervation (t₍₁₀₅₎ = 0.577, p > 0.9999), one possible explanation is that EB exposure prevents the glomerular degeneration starting to appear in orco mutants via the lateral excitation onto Or42a OSNs triggered by other OSN classes (Huang et al., 2010). Despite the above unanticipated results, the orco null mutants clearly show the expected lack of VM7 innervation remodeling after critical period EB exposure (Fig. 7C,D). There is a significantly increased innervation volume in orco nulls exposed to EB, compared with EB-exposed controls (t₍₁₀₅₎ = 13.25, p = 5.71e-23; Fig. 7D, third from bottom bar). Targeted restoration of Orco only in Or42a OSNs in otherwise orco null mutants prevents the innervation loss occurring in the orco nulls alone, leading to innervation volumes that are not significantly different compared with the oil-exposed controls [control oil = vs Or42a-Gal4>Orco oil 0.983 ± 0.043 (n = 18); t₍₁₀₅₎ = 0.3539, p > 0.9999; Fig. 7D]. The Or42a-Gal4>Orco rescue condition also restores the VM7 innervation remodeling caused by critical period EB exposure, compared with the oil vehicle condition alone [EB 0.02 ± 0.015 (n = 17); t₍₁₀₅₎ = 18.77, p = 3.74e-34; Fig. 7D, bottom right bar]. Moreover, the VM7 innervation reduction is significantly greater than within EB-exposed control animals (t₍₁₀₅₎ = 6.636, p = 2.17e-8; Fig. 7D, top bar). These results demonstrate that Orco-dependent Or42a OSN activation is the main driver of critical period AL innervation remodeling, but that other OSNs can fine-tune the response.

To directly compare interactions between critical period EB exposure and each genotype separately, a linear regression model was used to generate interaction terms interrogated to determine whether genotypes significantly alter innervation following EB exposure (Fig. 7E). There is a significant interaction between the critical period EB exposure and the orco null mutant genotype, with a significant regression coefficient (20% EB x orco β = 0.82 ± 0.07; t₍₁₀₅₎ = 11.76, p = 7.082e-21; Fig. 7E, left bar). This result indicates that introduction of the orco null prevents the olfactory experience-dependent innervation loss after critical period EB exposure, compared with the matched controls. The interaction of the odorant exposure with the Or42a-Gal4>Orco rescue in an otherwise orco null mutant is in the opposite direction, with another very significant regression coefficient (20% EB x Or42a>Orco β = −0.328 ± 0.071; t₍₁₀₅₎ = 4.574, p = 1.315e-5; Fig. 7E, right bar). This result indicates that the critical period EB odorant exposure leads to a greater reduction of VM7 innervation volume in the orco null mutants with Orco re-expressed only in the Or42a OSNs, compared with the transgenic control animals. Taken together, these results provide very strong evidence that Or42a OSN-specific activity is sufficient to drive critical period remodeling of the VM7 glomerulus innervation. However, although only Or42a OSN activity is required for the innervation remodeling, other OSNs appear to modulate the level of EB experience remodeling through lateral inhibition, as the innervation reduction seen when Orco is only targeted to Or42a OSNs is greater than in the matched transgenic control animals.

Or42a OSN-targeted activation is not affected by Or42a OSN-targeted FMRP knock-down

The Or42a OSN presynaptic terminals innervating the VM7 glomerulus receive lateral inhibition from AL GABAergic LNs (Fig. 1E, bottom left; Olsen and Wilson, 2008). This inhibition scales with OSN olfactory activation and is effectively blocked by removing or shielding from external odorant stimuli (Olsen and Wilson, 2008). Based on the role of balanced OSN FMRP levels (Fig. 6), and the impact of silencing many OSNs (Fig. 7) in mediating the Or42a OSN critical period remodeling, we next hypothesized that FMRP might regulate the local lateral modulation downstream of broad EB-driven OSN activation. To test this hypothesis, we took advantage of Or42a OSN-targeted CsChrimson::mVenus channelrhodopsin (Klapoetke et al., 2014) to specifically activate just the target OSNs (as in Fig. 7), while also targeting FMRP removal (Or42a-Gal4>UAS-CsChrimson::mVenus, UAS-dfmr1 RNAi; Fig. 8). These animals were compared with animals raised in total darkness, and to transgenic control animals (lacking the RNAi) raised in either darkness or in the same light conditions. As further controls, we examined the effect of oil vehicle alone or 20% EB on the same genotypes raised in complete darkness. All light and odorant treatments were done in the 0- to 2-dpe critical period. Representative images of Or42a OSN terminals in the VM7 glomerulus, and the innervation quantifications for all genotypes and treatments, are shown in Figure 8.

Figure 8.

Or42a-targeted optogenetic activation is not affected by Or42a-targeted FMRP RNAi All images show confocal maximum intensity projections of Or42a-OSN VM7 innervation, with Or42a-Gal4 driven expression of fluorescently tagged channelrhodopsin Cschrimson::mVenus. A, B, Females were reared in total darkness (dark, top) or with 515 nm cyan light (light, bottom). A, Or42a>Cschrimson::mVenus transgenic controls and (B) Or42a-targeted dfmr1 RNAi (TriP GL00075). C, Quantification of VM7 innervation in control and Or42a>dfmr1 RNAi animals following dark and light treatment. D, E, Females were reared with oil vehicle (top) or 20% EB odorant (bottom) during the 0- to 2-dpe critical period. D, Or42a>Cschrimson::mVenus transgenic controls and (E) Or42a-targeted dfmr1 RNAi (TriP GL00075). F, Quantification of VM7 innervation in control and Or42a>dfmr1 RNAi animals following oil and 20% EB exposure. Scatter plots show all data points and the mean ± SEM. The significance is indicated as not significant (N.S.; p > 0.05) and significant at ***p < 0.001.

Transgenic controls show the expected strong reduction in Or42a OSN innervation of the VM7 glomerulus following critical period light stimulation, compared with animals raised in total darkness (Fig. 8A). Or42a OSN-specific optogenetic activation in Or42a-Gal4>UAS-dfmr1 RNAi animals causes a similar decrease in VM7 innervation compared with the dark-reared animals (Fig. 8A vs B), indicating the EB-driven OSN activity is an important driver of the FMRP effect. ANOVA (2 × 2) analyses of glomeruli innervation for each condition show a significant light stimulation effect (F_(1,73) = 114.3, p = 1.368e-16), but no FMRP genotype effect (F_(1,73) = 0.05, p = 0.8158), with no significant interaction between stimulation and genotype (F_(1,73) = 1.71, p = 0.195). Pairwise comparisons using t tests with Sidak's correction show that critical period EB exposure significantly reduces innervation in both transgenic controls and Or42a-Gal4>UAS-dfmr1 RNAi animals [control dark 1.0 ± 0.04 (n = 21) vs light 0.41 ± 0.06 (n = 21); t₍₇₃₎ = 8.9, p = 1.71e-12; RNAi dark 0.947 ± 0.02 (n = 18) vs light 0.486 ± 0.07 (n = 17); t₍₇₃₎ = 6.35, p = 9.7e-8; Fig. 8C, bottom bars]. Further comparisons show that transgenic controls raised in darkness or light-stimulated during the 0- to 2-dpe critical period have VM7 glomerulus innervation volumes not statistically different from Or42a-Gal4>UAS-dfmr1 RNAi animals under the same conditions (control dark vs RNAi dark; t₍₇₃₎ = 0.766, p = 0.9712; control light vs RNAi light; t₍₇₃₎ = 1.08, p = 0.8641; Fig. 8C, top bar). Together, these results indicate that broad OSN activation is required for the effect of Or42a OSN-targeted FMRP removal.

Consistent with previous results, transgenic controls exposed to 20% EB show reduced Or42a OSN VM7 innervation compared with oil-exposed animals (Fig. 8D, top vs bottom). The effect of Or42a OSN-targeted FMRP RNAi also agrees with previous experiments, showing impaired innervation remodeling compared with the transgenic controls (Fig. 8E). ANOVA (2 × 2) quantification of the effects of oil versus 20% EB exposure on the two genotypes show a significant effect of both odorant (F_(1,75) = 284.1, p = 3.211e-27) and genotype (F_(1,75) = 17.3, p = 8.4e-5), with a significant interaction between exposure and genotype (F_(1,75) = 5.02, p = 0.028). Pairwise comparisons using t tests with Sidak's correction show that EB exposure significantly reduces VM7 glomerulus innervation volume for both transgenic controls and Or42a-Gal4>UAS-dfmr1 RNAi animals [control oil 1.0 ± 0.03 (n = 21) vs 20% EB 0.359 ± 0.04 (n = 21); t₍₇₅₎ = 13.95, p = 9.38e-22; RNAi oil 1.064 ± 0.04 (n = 19) vs 20% EB 0.574 ± 0.02 (n = 18); t₍₇₅₎ = 10.02, p = 1.05e-14; Fig. 8F, bottom bars]. Or42a-Gal4>UAS-dfmr1 RNAi animals exposed to the oil vehicle have statistically similar glomerulus innervation volumes to the transgenic controls (control oil vs RNAi oil; t₍₇₅₎ = 1.37, p = 0.687). In line with our previous experiments, Or42a-Gal4>UAS-dfmr1 RNAi animals had significantly greater VM7 innervation compared with transgenic controls after critical period EB exposure (control EB vs RNAi EB; t₍₇₅₎ = 4.49, p = 0.0002; Fig. 8F, top bar). Overall, these results suggest lateral connections from broadly branching AL LNs are the likely mediators of critical period remodeling.

Or42a OSN synaptic output is not required for Or42a-targeted FMRP RNAi remodeling effects

Results up to this point indicate that Or42a OSN-specific activity is needed for critical period remodeling and that refinement is impaired if FMRP levels are not balanced between OSNs. We previously reported that Or42a OSN synaptic output does not drive critical period remodeling, but rather serves to limit the effect of EB odorant experience (Golovin et al., 2019). Because of the known FMRP roles controlling trans-synaptic signaling that coordinates with neurotransmitter release (Friedman et al., 2013), we hypothesized that FMRP roles in critical period remodeling require synaptic output. To block synaptic output in Or42a OSNs, we used a targeted tetanus toxin light chain (Or42a-Gal4>UAS-TeTxLc), while unbalancing FMRP levels using targeted FMRP RNAi (Or42a-Gal4>UAS-dfmr1 RNAi), with Or42a-Gal4>UAS-mCD8::GFP labeling. The combined Or42a-Gal4>UAS-TeTxLc, UAS-dfmr1 RNAi animals were compared with the controls (Or42a-Gal4>UAS-mCD8::GFP), as well as TeTxLc (Or42a-Gal4>UAS-TeTxLc) and FMRP RNAi (Or42a-Gal4>UAS-dfmr1 RNAi) alone. Animals of each genotype were exposed to either the oil vehicle or 10% EB during the 0- to 2-dpe critical period. The lower EB concentration was used for this experiment because our previous work with Or42a-Gal4>UAS-TeTxLc animals showed that Or42a OSN innervation of the VM7 glomerulus was completely eliminated at higher EB concentrations (Golovin et al., 2019). Representative images of Or42a OSN terminals in the VM7 glomerulus, innervation quantifications, and EB effect quantifications for all genotypes and treatments are shown in Figure 9.

Figure 9.

Or42a OSN synaptic output is not required for Or42a-targeted FMRP RNAi effect. A, Representative confocal maximum intensity projections of Or42a OSN VM7 innervation (Or42a-Gal4>UAS-mCD8::GFP; white) following exposure to oil vehicle (left) or 10% EB (right) during the 0- to 2-dpe critical period. Four genotypes are shown: the transgenic control (Or42a-Gal4>mCD8::GFP; top), with Or42a-targeted dfmr1 RNAi (TriP GL00075; second), with Or42a-targeted tetanus toxin light chain (TeTxLc; third), and with both Or42a-targeted dfmr1 RNAi and TeTxLc (bottom). The bright puncta following EB odorant exposure are labeled by white arrows. B, Quantification of the Or42a OSN VM7 innervation volume for each genotype and condition. C, Difference between oil vehicle and EB odorant shown for each genotype. Scatter plots show all data points and mean ± SEM. Bar graphs show mean ± SER. The significance is indicated as not significant (N.S.; p > 0.05), or significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Unlike the higher EB exposures, 10% EB from 0 to 2 dpe has little effect on Or42a OSN innervation in transgenic control animals (Fig. 9A, top), although characteristic OSN punctae still occur after odorant exposure. The 10% EB-exposed controls show some regions of thinner VM7 innervation, with other areas containing the OSN puncta often occurring in EB-exposed animals (Fig. 9A, top, right arrows). Comparing Or42a-Gal4>UAS-dfmr1 RNAi animals exposed to oil vehicle or EB odorant also reveals similar OSN puncta (Fig. 9A, second row, right arrows). However, unlike the control animals, Or42a-target FMRP RNAi animals have more widespread OSN puncta with little thinning of the innervation, which leads to a small expansion of the overall glomerulus innervation (Fig. 9A, second row). Consistent with our previous experimental report (Golovin et al., 2019), Or42a-Gal4>UAS-TeTxLc causes both more expansive basal innervation and a stronger EB odorant-induced reduction than matched controls (Fig. 9A, third row). These TeTxLc-expressing animals show a response to critical period 10% EB exposure that appears similar to the control response to 20% EB. In addition, examining animals expressing both Or42a-targeted dfmr1 RNAi and TeTxLc blockade shows basal glomerulus innervation even further increased compared with controls (Fig. 9A, bottom row). Moreover, EB-exposed animals with both dfmr1 RNAi and TeTxLc synapse blockade show impaired remodeling compared with TeTxLc alone (Fig. 9A, bottom right). These results indicate that blocking Or42a OSN synaptic output does not impair the effect of Or42a-targeted FMRP RNAi.

ANOVA (2 × 2 × 2) quantitative analyses of VM7 glomerulus innervation for each condition strongly support the above conclusions (Fig. 9B,C). Comparisons show a significant effect of the EB odorant exposure (F_(1,316) = 335.9, p = 1.272e-51), TeTxLc synaptic transmission blockade (F_(1,316) = 34.04, p = 1.336e-8) and targeted dfmr1 RNAi (F_(1,316) = 102.4, p = 4.924e-21), with significant two-way interactions between odorant and TeTxLc (F_(1,316) = 428.4, p = 9.46e-61), odor and dfmr1 RNAi (F_(1,316) = 15.9, p = 8.3e-5), and TeTxlc and dfmr1 RNAi (F_(1,316) = 7.96, p = 0.0051), but no significant three-way interaction (F_(1,316) = 0.0014, p = 0.9697). Pairwise comparisons using t tests with Sidak's correction show that critical period EB exposure does not significantly change VM7 glomerulus innervation for the transgenic controls compared with the oil-exposed animals [control oil 1.0 ± 0.03 (n = 42) vs 10% EB 0.959 ± 0.03 (n = 41); t₍₃₁₆₎ = 0.8375, p > 0.9999; Fig. 9B, bottom left bar]. Interestingly, Or42a-Gal4>UAS-dfmr1 RNAi animals have significantly larger VM7 glomerulus innervation volumes after EB exposure compared with the oil vehicle [RNAi oil 1.082 ± 0.04 (n = 39) vs 10% EB 1.242 ± 0.04 (n = 41); t₍₃₁₆₎ = 3.179, p = 0.0445; Fig. 9B, second bottom bar]. Critical period 10% EB exposure in Or42a OSN-targeted TeTxLc animals, with or without dfmr1 RNAi, leads to a significant reduction in VM7 innervation compared with the oil-exposed animals [TeTxLC oil 1.3 ± 0.03 (n = 41) vs 10% EB 0.226 ± 0.03 (n = 38); t₍₃₁₆₎ = 21.22, p = 2.69e-61; RNAi+TeTxLc oil 1.525 ± 0.04 (n = 42) vs 10% EB 0.648 ± 0.05 (n = 40); t₍₃₁₆₎ = 17.65, p = 1.5e-47; Fig. 9B, bottom third and right bars].

Further comparisons show that oil-exposed transgenic controls have similar Or42a OSN VM7 innervation compared with Or42a-Gal4>UAS-dfmr1 RNAi, but significantly smaller than with Or42a-Gal4>UAS-TeTxLc (control oil vs RNAi oil; t₍₃₁₆₎ = 1.636, p = 0.9519; control oil vs TeTxLc oil; t₍₃₁₆₎ = 6.079, p = 9.75e-8; Fig. 9B, second and third from bottom left bars). Combining both dfmr1 RNAi and TeTxLc significantly increases the glomerulus innervation of oil-exposed animals, possibly indicating an odor experience-independent interaction (TeTxLc oil vs dfmr1 RNAi + TeTxLc oil; t₍₃₁₆₎ = 4.553, p = 0.0002; Fig. 9B, second from bottom bar right). As expected, following 10% EB exposure, Or42a OSN-targeted dfmr1 RNAi impairs and Or42a OSN-targeted TeTxLc enhances VM7 glomerulus innervation, compared with matched controls (control 10% EB vs RNAi 10% EB; t₍₃₁₆₎ = 5.7, p = 7.69e-7; control 10% EB vs TeTxLc 10% EB; t₍₃₁₆₎ = 14.47, p = 2.58e-35; Fig. 9B, fourth from bottom and top left bars). Glomerulus innervation was also compared in Or42a-Gal4>UAS-TeTxLc animals after 10% EB exposure, with or without dfmr1 RNAi. Similar to Or42a OSNs with intact synaptic output, transmission-blocked animals with dfmr1 RNAi show significantly increased VM7 innervation, implying the remodeling impairment from imbalanced FMRP levels does not require synaptic transmission from Or42a OSNs (TeTxLC 10% EB vs RNAi + TeTxLc 10% EB; t₍₃₁₆₎ = 8.289, p = 9.265e-14; Fig. 9B, right, fourth bar from bottom). The ANOVA quantification indicates that FMRP effects Or42a OSN critical period remodeling independent of OSN synaptic output.

To compare interactions between the critical period EB exposure and each of the genotypes, a linear regression model was generated to test for significant interactions (Fig. 9C). There is significant interaction between critical period EB experience and targeted dfmr1 RNAi, with a significant regression coefficient (10% EB x dfmr1 RNAi β = 0.22 ± 0.07; t₍₃₁₆₎ = 3.108, p = 0.0021; Fig. 9C, bottom left bar), indicating that targeted dfmr1 RNAi significantly mitigates EB-induced innervation remodeling. The interaction of critical period EB exposure and TeTxLc is in the opposite direction, with another very significant regression coefficient (10% EB x TeTxLc β = −1.009 ± 0.07; t₍₃₁₆₎ = 13.96, p = 7.849e-35; Fig. 9C, top bar), indicating that EB exposure causes a greater reduction of VM7 innervation in Or42a-targeted TeTxLc animals. The interaction between EB exposure and targeted dfmr1 RNAi is not altered by the TeTxLc blockade (10% EB x dfmr1 RNAi x TeTxLc β = −0.027 ± 0.1; t₍₃₁₆₎ = 0.26, p = 0.795). To test more specifically for an effect of Or42a-targeted dfmr1 RNAi on EB exposure in the TeTxLc animals, we built a second linear regression model using only the TeTxLc blockade data. The regression coefficient for the interaction between critical period EB exposure and Or42a OSN-targeted dfmr1 RNAi in the TeTxLc model is still significant (10% EB x dfmr1 RNAi β = 0.1973 ± 0.07; t₍₁₅₇₎ = 2.77, p = 0.0063; Fig. 9C, bottom right bar). Taken together, these findings indicate that the cell autonomous FMRP role on odorant experience-dependent innervation remodeling does not rely on the Or42a OSN synaptic output.

It is possible that OSN-targeted TeTxLc synaptic transmission blockade might be having effects by modifying FMRP levels in these neurons. In the above FMRP OE studies, elevating FMRP levels in the Or42a OSNs enhances the effect of EB experience-dependent critical period remodeling (Fig. 4). Therefore, rather than TeTxLc blockade acting independently from FMRP, OSN-targeted TeTxLc could possibly increase FMRP expression and thus mimic the effects of FMRP OE. In order to test this possibility, we used FMRP antibody labeling to compare controls to animals expressing TeTxLc in Or42a OSNs. Silencing synaptic output of Or42a OSNs does not detectably alter FMRP levels in these neurons (Fig. 10). Comparisons of Or42a OSN somata in maxillary palps reveal no differences in the intensity or extent of FMRP labeling (Fig. 10A). Quantification shows that Or42a OSN FMRP levels do not significantly differ between the controls and Or42a-Gal4>TeTxLc animals [control 2723 ± 161.4 FMRP intensity (A.U.; n = 20 palps) vs Or42a-Gal4>TeTxLc 2445 ± 417.8 arbitrary units (A.U.) (n = 12); unpaired t test, t₍₃₀₎ = 0.7262, p = 0.4733; Fig. 10B]. Moreover, the ratio between Or42a OSN FMRP and total FMRP (i.e., FMRP in all maxillary palp OSNs) is not different between controls and TeTxLc animals [control 1.151 ± 0.03487 (n = 20) vs Or42a-Gal4>TeTxLc 1.176 ± 0.2347 (n = 12); unpaired t test, t₍₃₀₎ = 0.3627, p = 0.7193; Fig. 10C]. Together, these results suggest silencing Or42a OSN synaptic output does not detectably alter FMRP levels, again implicating the importance of lateral connections from other neurons rather than a direct feedback mechanism.

Figure 10.

Or42a OSN-targeted TeTxLc neurotransmission block does not alter FMRP levels. A, Representative confocal maximum intensity projections of maxillary palp OSNs labeled with anti-FMRP (magenta) and Or42a-Gal4>mCD8::GFP (green), showing both channels (merged, top) or the FMRP channel alone (bottom). The maxillary palps are from the transgenic controls (Or42a-Gal4>Or42a-mCD8::GFP, left) and with TeTxLc expression (Or42a-Gal4>TeTxLc, right). B, Quantification of the mean FMRP fluorescence intensity levels within the GFP-positive Or42a OSNs comparing the controls and TeTxLc-expressing animals. C, Quantification of the ratio of mean FMRP fluorescence intensity in Or42a OSNs compared with all OSNs. Scatter plots show all data points and mean ± SEM. The significance is indicated as not significant (N.S.; p > 0.05).

Critical period olfactory experience selectively remodels presynaptic OSN innervation

Previous work from our lab and others has shown that critical period odorant experience can drive dendritic arbor changes in postsynaptic PNs downstream of OSN glomerular innervation (Sachse et al., 2007; Doll and Broadie, 2015; Chodankar et al., 2020). However, a recent study has suggested that Or42a OSN presynaptic remodeling is a completely separable mechanism (Chodankar et al., 2020). This recent study, combined with Or42a OSN remodeling in absence of synaptic output to VM7 PNs, made it unclear whether presynaptic innervation changes would be mirrored in a comparable PN postsynaptic refinement. We therefore next jointly assayed both the presynaptic OSNs and the postsynaptic PNs following the critical period odorant exposure. To simultaneously image presynaptic and postsynaptic partners within the VM7 glomerulus, we labeled the Or42a OSNs with Or42a-mCD8::4xGFP (Fig. 11A, left, green), while using NP3481-Gal4 (Olsen and Wilson, 2008) to drive UAS-mCD8::RFP (NP3481>mCD8::RFP) in the PNs (Fig. 11A, middle, magenta). The co-labeling shows the RFP-marked PNs overlap with the GFP-marked Or42a OSNs within the VM7 glomerulus in merged single slice confocal images (Fig. 11A, right, merged). These animals were exposed to either oil vehicle or 20% EB during the 0- to 2-dpe critical period. Representative images of the presynaptic and postsynaptic processes in the VM7 glomerulus, and the innervation quantifications for both Or42a OSNs and VM7 PNs following critical period treatments, are shown in Figure 11.

Figure 11.

Odorant exposure selectively remodels presynaptic OSNs in the VM7 glomerulus. A, Representative confocal slices showing presynaptic Or42a OSNs (Or42a-mCD8::4xGFP, green; left) and postsynaptic PNs (NP3481-Gal4>mCD8::RFP, magenta; middle), with the merged image (right). B, Representative VM7 merged images after exposure to the oil vehicle alone (left) or 20% EB odorant (right) during the 0- to 2-dpe critical period. C, Quantification of VM7 glomerulus volume of Or42a OSNs (green) and VM7 PNs (magenta), normalized to the vehicle control. Data shown as a scatter plot of all data points with mean ± SEM. D, Quantification of the relationship between the presynaptic Or42a OSN volume and postsynaptic PN volume within the VM7 glomerulus. Data shown as a scatter plot with lines fit to vehicle (magenta) and EB (green) conditions. R² values given for each condition. Significance is presented as not significant (N.S; p > 0.05), and significant at ***p < 0.001.

Following 20% EB critical period exposure, the control animals show the typical strong reduction of Or42a OSN innervation (Fig. 11B, green, left vs right). Despite loss of presynaptic innervation, postsynaptic PNs are largely unchanged between oil-exposed and EB-exposed conditions (Fig. 11B, magenta, left vs right). Presynaptic and postsynaptic volumes were quantified and compared with t tests with Sidak's correction and a simple linear regression. Compared with oil-exposed animals, EB-exposed animals show significantly reduced Or42a OSN innervation, but no significant difference in the PN volume in the VM7 glomerulus [Or42a OSN oil 1.0 ± 0.07 (n = 18) vs 20% EB 0.15 ± 0.06 (n = 18); t₍₃₄₎ = 9.43, p = 1.04e-10; VM7 PN oil 1.24 ± 0.04 (n = 18) vs 20% EB 1.1 ± 0.11 (n = 18) PN volumes normalized to Or42a OSN oil volume mean; t₍₃₄₎ = 1.12, p = 0.47; Fig. 11C]. Note that there is some increased variability in the VM7 PN volumes in the EB-exposed animals compared with vehicle controls (SEM oil 0.04 vs 20% EB 0.11; Fig. 11C). We therefore tested whether Or42a OSN innervation and PN dendritic arborization might correlate, and possibly account for some of the odorant-induced variability. A simple linear regression was used to compare the presynaptic and postsynaptic volumes, but only a very weak, EB-independent relationship is apparent (oil R² = 0.1591 vs EB R² = 0.1387; Fig. 11D). In summary, these results show that critical period odorant exposure drives striking remodeling in the presynaptic Or42a OSNs, but not the postsynaptic PNs, and therefore separates this process from similar processes that have mirrored presynaptic and postsynaptic alterations.

Silencing AL glutamatergic interneurons reduces Or42a OSN critical period remodeling

AL LNs are prime candidates to regulate critical period remodeling. LNs interconnect OSNs for lateral modulation of OSN activity (Chou et al., 2010; Fig. 8) receive broad synaptic output from many OSNs (Olsen and Wilson, 2008; Fig. 9), and innervate OSN presynaptic terminals (Wilson, 2013; Fig. 11). LNs release neuromodulators including GABA and glutamate (Jackson et al., 1990; Das et al., 2010). We previously found that Or42a-targeted knock-down of the glutamate receptor NMDAR1 subunit strongly impairs Or42a OSN critical period remodeling (Golovin et al., 2019). Glutamatergic LNs (GluLNs; Fig. 1E, bottom right) provide the major source of glutamate neurotransmission in the AL circuit (Liu and Wilson, 2013). Therefore, we next tested the contribution of the GluLNs to Or42a OSN critical period remodeling. GluLN synaptic output was silenced as above, using OK107-Gal4 to drive tetanus toxin (OK107-Gal4>TeTxLc), with Or42a OSNs labeled by Or42a-mCD8::4xGFP. Since the OK107-Gal4 driver has extensive expression in the MB (Connolly et al., 1996), we also used the MB-restricted driver MB247-Gal4 (Zars et al., 2000) as a control to express TeTxLc (MB-247-Gal4>TeTxLc) and assess the effect of MB silencing on Or42a critical period remodeling. The same transgenic lines lacking TeTxLc were used as controls. Staged animals from all genotypes were exposed to either the oil vehicle or 20% EB during the 0- to 2-dpe critical period. Representative VM7 images and glomerulus innervation quantifications for all genotypes and conditions are shown in Figure 12.

Figure 12.

Silencing AL glutamatergic neurons reduces Or42a OSN critical period remodeling. A, Representative confocal maximum intensity projections of Or42a OSN VM7 innervation (two copies of Or42a-mCD8::4xGFP; white). Transgenic control (w^-; Or42a-mCD8::4xGFP/+; Or42a-mCD8::4xGFP/+; OK107-Gal4/+; top) and with OK107-Gal4 driving UAS-tetanus toxin (OK107>TeTxLc; bottom). Females exposed to oil vehicle alone (left) or 20% EB odorant (right) during the 0- to 2-dpe critical period. B, Quantification of VM7 innervation for the OK107-Gal4 control and TeTxLc blocked animals exposed to either oil or EB. C, Imaging as above in A, transgenic control (w^-/w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP; MB247-Gal4/+; top) and with MB247-Gal4 driving UAS-TeTxLc (bottom). Females exposed to oil (left) or 20% EB (right) during the critical period. D, Quantification of VM7 innervation for MB247-Gal4 control and TeTxLc animals exposed to oil or EB. Scatter plots show all data points and the mean ± SEM. Significance is indicated as *p < 0.05 and ***p < 0.001.

As above, transgenic control animals show a striking reduction in VM7 glomerulus innervation with 20% EB critical period exposure (Fig. 12A, top). In contrast, animals with GluLN silencing by TeTxLc (OK107-Gal4>TeTxLc) have impaired Or42a OSN remodeling following the EB odorant exposure (Fig. 12A, top vs bottom). Quantification of glomerulus innervation and ANOVA (2 × 2) analyses show a significant effect of the odor (F_(1,74) = 367.9, p = 1.946e-30) and the GluLN TeTxLc silencing (F_(1,74) = 10.24, p = 0.002), with a significant interaction between experience and genotype (F_(1,74) = 25.61, p = 2.95e-6; Fig. 12B). Pairwise comparisons using the t tests with Sidak's corrections show that EB-exposed animals have significantly reduced innervation compared with oil-exposed transgenic controls and OK107-Gal4>TeTxLc animals [control oil 1.0 ± 0.03 (n = 26) vs 20% EB 0.003 ± 0.001 (n = 25); t₍₇₄₎ = 20.61, p = 1.31e-31; TeTxLc oil 0.923 ± 0.06 (n = 13) vs 20% EB 0.343 ± 0.07 (n = 14); t₍₇₄₎ = 8.73, p = 3.23e-12; Fig. 10B, bottom bars]. Silencing GluLNs does not alter the basal glomerulus innervation under control oil-exposed conditions (control oil vs TeTxLc oil; t₍₇₄₎ = 1.304, p = 0.7303), but does significantly increase Or42a OSN innervation following the critical period EB odorant exposure (control 20% EB vs TeTxLc 20% EB; t₍₇₄₎ = 5.893, p = 6.27e-7; Fig. 12B, top bar). Together, these results suggest a role for GluLN glutamatergic signaling in Or42a OSN olfactory-experience-dependent critical period remodeling, but it is possible that some or all of this effect is because of OK107-Gal4 expression within the downstream MB learning/memory center.

MB-restricted silencing with MB247-Gal4 slightly enhances the Or42a OSN remodeling (Fig. 12C). Like transgenic controls, MB247-Gal4>TeTxLc animals exposed to 20% EB show reduced VM7 innervation, but the effect is even stronger than in the controls. Quantification of innervation and ANOVA (2 × 2) analyses show a significant effect of both odor (F_(1,64) = 136.9, p = 1.513e-17) and genotype (F_(1,64) = 4.459, p = 0.0386), but no significant interaction between them (F_(1,64) = 0.743, p = 0.3919; Fig. 12D). Pairwise comparisons using t tests with Sidak's corrections show that EB-exposed animals have significantly reduced innervation compared with both oil-exposed transgenic controls and MB247-Gal4>TeTxLc animals [control oil 1.0 ± 0.03 (n = 25) vs 20% EB 0.357 ± 0.07 (n = 20); t₍₆₄₎ = 9.287, p = 1.07e-12; TeTxLc oil 0.926 ± 0.07 (n = 12) vs 20% EB 0.181 ± 0.08 (n = 11); t₍₆₄₎ = 7.736, p = 5.65e-10; Fig. 12D, bottom bars]. Silencing the MB does not significantly alter basal glomerulus innervation under oil-exposed control conditions (control oil vs TeTxLc oil; t₍₆₄₎ = 0.9146, p = 0.3639), but does significantly decrease Or42a OSN innervation following critical period EB exposure (control 20% EB vs TeTxLc 20% EB; t₍₆₄₎ = 2.036, p = 0.0459; Fig. 12D, top bar). Despite the significant decrease in innervation volume in the MB247>TeTxLc EB condition compared with the transgenic control, the lack of a significant interaction term between genotype and odorant exposure in the ANOVA complicates interpretation. Taken together, these results indicate GluLNs role in critical period OSN remodeling, but do not clearly demonstrate a potential MB role.

Based on the above findings, we hypothesized that GluLN-released glutamate binds to Or42a OSN NMDARs to modulate critical period remodeling. To further test this hypothesis, we next set forth to assess whether NMDAR1 specifically regulates critical period remodeling. Transgenic controls with Or42a OSNs labeled using Or42a-mCD8::GFP were compared in two Nmdar1 mutant combinations: (1) Nmdar1 null mutants homozygous for a Mi{MIC} insertion (Nmdar1^{MI11796/MI11796}; Nagarkar-Jaiswal et al., 2015); and (2) heterozygous mutants with a copy of Nmdar1^MI11796 over a hypomorphic mutation (Nmdar1^{EP331/MI11796}; Rorth, 1996; Xia et al., 2005). Control and mutant animals were exposed to either oil vehicle or 20% EB in the 0- to 2-dpe critical period. Representative images of the Or42a OSN VM7 glomerular innervation, as well as innervation quantifications and the effect of EB exposure for all genotypes and conditions, are all shown in Figure 13. Transgenic control animals show the characteristic strong reduction in VM7 innervation following 20% EB critical period exposure (Fig. 13A, left). Surprisingly, we found that both mutants also show a very similar response to the EB exposure. Mutants exhibit a strong reduction in Or42a OSN axon terminal innervation and the characteristic OSN bright puncta (Fig. 13A, middle and right). This suggests two possibilities: (1) the effect of GluLNs silencing on Or42a OSN critical period remodeling does not rely on NMDAR1-dependent signaling; or (2) Or42a-targeted NMDAR1 knock-down may have a similar effect as with FMRP with only circuit imbalance having an impact on critical period remodeling.

Figure 13.

NMDAR1 signaling is not required for OSN critical period innervation remodeling. A, Representative confocal maximum intensity projections of Or42a OSN VM7 innervation (two copies of Or42a-mCD8::4xGFP; white) following exposure to oil vehicle (top) or 20% EB (bottom) during the 0- to 2-dpe critical period. Three genotypes are shown: transgenic control (w^-; Or42a-mCD8::4xGFP/Or42a-mCD8::4xGFP; left), NMDAR1 mutant (NMDAR1^MI11796; middle) and a second NMDAR1 mutant (NMDAR1^EP331/NMDAR1^MI11796; right). Remnant puncta following EB exposure are labeled by white arrows. B, Quantification of VM7 innervation volume for each genotype and condition. C, The difference between oil and EB conditions for each genotype. Scatter plots show all data points and the mean ± SEM. Bar graphs show mean ± SER. Significance is indicated as not significant (N.S.; p > 0.05) and ***p > 0.001.

ANOVA (3 × 2) quantification shows a significant effect of both odorant (F_(1,128) = 567.7, p = 6.876e-49) and genotype (F_(2,128) = 6.084, p = 0.003), but no significant interaction between them (F_(2,128) = 2.089, p = 0.128; Fig. 13B). Pairwise comparisons using Sidak's corrected t tests show that both control and mutants significantly reduce innervation volumes with critical period EB exposure [control oil 1.0 ± 0.03 (n = 32) vs 20% EB 0.085 ±0.03 (n = 33); t₍₁₂₈₎ = 17.73, p = 4.25e-35; Nmdar1^{MI11796/MI11796} oil 0.873 ± 0.06 (n = 13) vs 20% EB 0.078 ± 0.03 (n = 18); t₍₁₂₈₎ = 10.5, p = 8.29e-18; Nmdar1^{EP331/MI11796} oil 1.149 ± 0.08 (n = 19) vs 20% EB 0.147 ± 0.05 (n = 19); t₍₁₂₈₎ = 14.85, p = 1.9e-28; Fig. 13B, bottom bars], but there is no significant difference in the basal innervation with vehicle exposure (control oil vs Nmdar1^{MI11796/MI11796} oil; t₍₁₂₈₎ = 1.858, p = 0.6376; control oil vs Nmdar1^{EP331/MI11796} oil; t₍₁₂₈₎ = 2.467, p = 0.202) or in odor-dependent remodeling (control EB vs Nmdar1^{MI11796/MI11796} EB; t₍₁₂₈₎ = 0.113, p > 0.9999; control EB vs Nmdar1^{EP331/MI11796} EB; t₍₁₂₈₎ = 1.029, p = 0.9958; Fig. 13B, middle and top bars). Quantification of EB effects using multiple linear regression shows that all genotypes have similar responses without significantly different regression coefficients (20% EB x Nmdar1^{MI11796/MI11796} β = 0.1203 ± 0.09; t₍₁₂₈₎ = 1.312, p = 0.1918; 20% EB x Nmdar1^{EP331/MI11796} β = −0.08,699 ± 0.08; t₍₁₂₈₎ = 1.024, p = 0.3079; Fig. 13C). Together, these results suggest that NMDAR1 signaling functions in an Or42a OSN-specific pattern similar to FMRP to mediate the olfactory experience-dependent critical period remodeling of Or42a OSN presynaptic terminals.

Or42a OSN-targeted GABA_AR knock-down enhances critical period innervation remodeling

Inhibitory GABAergic LNs oppose excitatory inputs, with the excitatory and inhibitory LN balance controlling AL circuit output (Acebes et al., 2011, 2012). Excitatory/inhibitory imbalance characterizes FXS disease models (Contractor et al., 2015). Therefore, we hypothesized altering inhibitory LN signaling might imbalance the AL circuit and impair Or42a OSN refinement. We showed above that remodeling depends on neural activity specifically within the Or42a OSNs. In addition, we showed that Or42a OSN critical period remodeling is augmented when other OSNs cannot transduce odorants. Since the overall impact of OSN population interactions on Or42a OSNs is lateral inhibition (Olsen and Wilson, 2008), we hypothesized that reducing inhibition should mimic the effects of the orco mutant with Or42a-Gal4>Orco rescue (Fig. 7). Since a major component of lateral inhibition onto Or42a OSNs comes from ionotropic GABA receptors (Olsen and Wilson, 2008), we used Or42a-targeted RNAi to knock-down resistant to dieldrin (RDL), a principle GABA_AR subunit (Aronstein and Ffrench-Constant, 1995; Okada et al., 2009). In order to examine the role of RDL in the critical period remodeling of Or42a OSNs, we used Or42a-Gal4>mCD8::GFP to label the neurons and compared the RDL RNAi expressing animals (Or42a-Gal4>Rdl RNAi) to a transgenic control expressing only the membrane-bound GFP. Staged animals were exposed to either oil vehicle or 20% EB during the 0- to 2-dpe critical period. Representative images of Or42a OSN innervation of the VM7 glomerulus as well as innervation quantifications for all conditions are shown in Figure 14.

Figure 14.

Or42a OSN-targeted GABA_AR knock-down enhances critical period remodeling. A, Representative confocal maximum intensity projections of Or42a OSN VM7 innervation (Or42a-Gal4>UAS-mCD8::GFP; white) following exposure to oil vehicle (top) or 20% EB odorant (bottom) during the 0- to 2-dpe critical period. Two genotypes are shown; transgenic control (Or42a-Gal4>mCD8::GFP; top) and Or42a-targeted Rdl RNAi (Rdl RNAi 8-10J; bottom). B, Quantification of VM7 innervation for the two genotypes and conditions. Scatter plots show all data points with the mean ± SEM. The significance is indicated as ***p < 0.001.

As in all studies above, control animals EB-exposed during the critical period exhibit a strong reduction in Or42a OSN innervation of the VM7 glomerulus (Fig. 14A, top). When GABA_A signaling is reduced with Or42a OSN-targeted Rdl RNAi, basal innervation is similar to controls following oil exposure (Fig. 14A, left, top vs bottom). However, when Or42a-Gal4>Rdl RNAi animals are exposed to EB during the critical period, they have a larger reduction in Or42a OSN innervation compared with controls (Fig. 14A, right, top vs bottom). ANOVA (2 × 2) quantification shows significant effects of odor (F_(1,98) = 259.6, p = 2.671e-29) and Rdl RNAi (F_(1,98) = 19.78, p = 2.3e-5; with a significant interaction between the two (F_(1,98) = 17.11, p = 7.5e-5); Fig. 14B]. Pairwise comparisons with t tests and Sidak's correction show both control and Rdl RNAi animals have significantly reduced Or42a OSN innervation after EB exposure [control oil 1.0 ± 0.03 (n = 26) vs 20% EB 0.538 ± 0.04 (n = 28); t₍₉₈₎ = 8.729, p = 4.14e-13; Rdl RNAi oil 0.988 ± 0.04 (n = 23) vs 20% EB 0.206 ± 0.04 (n = 25); t₍₉₈₎ = 14.57, p = 1.73e-25; Fig. 14B, bottom bars]. Although the two genotypes had comparable innervation with vehicle (control oil vs Rdl RNAi oil; t₍₉₈₎ = 0.2161, p > 0.9999), Or42a-Gal4>Rdl RNAi significantly decreases the VM7 glomerulus innervation following critical period EB exposure (control EB vs Rdl RNAi EB; t₍₉₈₎ = 6.193, p = 8.32e-8; Fig. 14B, top bar). These results reveal a role of GABA_AR-mediated inhibition in regulating Or42a OSN synaptic remodeling, and provide a mechanism by which FMRP acts to modulate olfactory experience-dependent critical period refinement.

Discussion

The first days of Drosophila adulthood mark a critical period for the remodeling of brain olfactory circuitry (Devaud et al., 2003; Sachse et al., 2007; Tessier and Broadie, 2009; Doll and Broadie, 2015; Golovin et al., 2019; Chodankar et al., 2020). During this developmental window, OSNs manifest heightened adaptability to the new odorant sensory environment. Vertebrates show similarly heightened responsiveness to early odorant exposure. Rodents exposed to odors during development show increased or decreased effects dependent on conditions examined (Dalland and Døving, 1981; Geramita and Urban, 2016; Liu and Urban, 2017). Zebrafish also exhibit olfactory imprinting from developmental odorant exposure (Gerlach et al., 2019). We previously described OSN innervation remodeling that is restricted to an early-life critical period (0–2 dpe; Golovin et al., 2019). Here, we expand our knowledge of this remodeling by testing the role of FMRP, a protein strongly implicated in activity-dependent critical periods (Dölen et al., 2007; Tessier and Broadie, 2009; Contractor et al., 2015). Based on this study, we propose that FMRP functions to regulate the lateral interactions between OSNs mediated by LNs (Acebes et al., 2011). Previous studies have shown that each OSN receives lateral presynaptic GABAergic inhibition that scales with the total activity of all OSNs, and serves as a gain control mechanism by reducing OSN activity to odorants that activate multiple OSN classes (Olsen and Wilson, 2008). High EB concentrations likely activate multiple OSN classes, in addition to the Or42a OSNs, and should therefore recruit presynaptic inhibition. We would expect this inhibition to limit activity in response to EB exposure and reduce remodeling. Indeed, when we block lateral inhibition either through (1) specifically activating Or42a OSNs or (2) selectively removing Or42a OSN GABA_A receptors, EB odorant exposure has a greater effect on critical period remodeling.

In addition, our previous work showed that Or42a-targeted NMDAR1 knock-down impairs Or42a OSN innervation remodeling, likely via reduced lateral excitation (Golovin et al., 2019). However, NMDAR signaling on OSNs has not been directly shown. Or42a-targeted FMRP RNAi impairs the critical period remodeling of Or42a OSNs. Or42a-specific FMRP loss likely alters the OSN response to circuit lateral inputs both by enhancing inhibition and also reducing excitation. This role represents a novel FMRP function for regulating acute OSN remodeling specifically on the presynaptic side (Sudhakaran et al., 2014; Doll and Broadie, 2015, 2016; Franco et al., 2017). Previous studies have found that FMRP is required for mediating long-term habituation (LTH), a form of structural and functional adaption that leads to a reduction in innate avoidance behavior (Das et al., 2011). More specifically, FMRP interacts with a second RNA-binding protein (Ataxin2), contributing to LTH by acting in both the PNs and LNs (Sudhakaran et al., 2014). FMRP-dependent PN remodeling likewise occurs following a single day of activation within a cell-autonomous mechanism (Doll and Broadie, 2015, 2016). The results presented here extend beyond this earlier work in three ways: (1) Or42a OSN remodeling involves lateral inhibition mechanisms; (2) Or42a OSN remodeling is a purely presynaptic process; and (3) Or42a OSN remodeling manifests acute reversibility (Chodankar et al., 2020). The neuron-specific FMRP functions in the AL olfactory circuit highlight an increasingly appreciated FMRP role specificity within different neuron classes.

FMRP is widely expressed in the nervous system (Khandjian et al., 1995; Zhang et al., 2001), where it binds multiple different target mRNAs, including neuron class-specific transcripts (Darnell et al., 2011). For example, a recent study in mice showed that FMRP binds to circadian protein-encoding mRNAs preferentially in hippocampal CA1 pyramidal neurons compared with cerebellar granule neurons (Sawicka et al., 2019). Therefore, it is important to identify how FMRP loss perturbs the function of specific neuron classes to affect particular neural circuits. The current study describes how unbalancing FMRP levels between neurons of the Drosophila olfactory circuit can alter odorant experience-dependent remodeling, revealing how dissecting FMRP functions at a fine cellular resolution can uncover FXS circuit-level impairments (Contractor et al., 2015; Franco et al., 2017; Goel et al., 2018; Lovelace et al., 2020). Neuron class-specific FMRP functions are also revealed by activity-dependent dissection, with optogenetic stimulation causing opposite phenotypes in the olfactory PN and MB output neurons, but both effects failing when FMRP is absent (Doll and Broadie, 2015). Functionally, recent work on the FMRP role in binding to HCN cation channels demonstrates opposing excitability consequences in hippocampal CA1 compared with layer 5 prefrontal cortex (Brandalise et al., 2020). Together with the results presented here, this work underscores the importance of neuron-specific FMRP mechanisms, and the need to understand how these altered roles combine to generate circuit-level FXS phenotypes.

One intriguing result from our experiments is that despite the important FMRP role in Or42a remodeling, FMRP null mutants maintain normal remodeling capacity. We suggest that this compensation is because of AL LN circuitry that allows for OSN activity to be modified based on the total input to the system (Olsen and Wilson, 2008). LNs excitation scales with the total OSN activity, so when FMRP is altered (LOF/GOF) only in Or42a OSNs there is little change to LN output. However, when FMRP is manipulated in all OSNs equally, the output of LNs is adjusted based on the responsiveness of all the OSNs, thereby balancing the circuit (Olsen et al., 2010; Mohamed et al., 2019). Although this result was unexpected by us, it is not unprecedented for homeostatic mechanisms to be able to overcome FMRP loss (Antoine et al., 2019; Domanski et al., 2019). For instance, in a recent analysis of four monogenic mouse autism models, including FXS, there was an increase in the excitatory-to-inhibitory ratio of pyramidal neurons within the primary somatosensory cortex, which in general served as a homeostatic mechanism to maintain the overall network activity, but there was not increased excitatory spiking (Antoine et al., 2019). Another example from mouse barrel cortex demonstrates that although the development of NMDA-dependent LTP is disrupted in the FXS disease model, there is no apparent defect in lesion-induced plasticity owing to homeostatic compensation (Harlow et al., 2010). In order to more fully understand the FXS condition, we must grasp not only neuron-specific FMRP functions, but also how these functions balance across circuits.

Our results indicate the balanced roles of inhibitory GABA_AR and excitatory NMDAR signaling in OSN remodeling. In our previous report on critical period remodeling, we found that Or42a-targeted NMDAR1 RNAi impairs the innervation loss from early-life EB odorant exposure (Golovin et al., 2019). Here, we find that targeted tetanus toxin synaptic silencing of GluLNs, the major source of AL glutamatergic transmission (Das et al., 2010), causes a similar impairment to Or42a OSN-targeted NMDAR1 knock-down. However, global Nmdar1 mutants lack a detectable phenotype. One explanation is that NMDAR signaling acts in a similar fashion to FMRP function, with targeted removal putting Or42a OSNs out of balance with the rest of the circuit, but global loss not generating this imbalance. Since the AL has both metabotropic glutamate receptors (Devaud et al., 2008) and glutamate-gated chloride channels (Liu and Wilson, 2013), GluLNs could mediate their effect on Or42a remodeling by also altering signaling through these receptors. Another unlikely possibility is that since the OK107-Gal4 driver expresses in a few neurons outside the olfactory circuitry (Aso et al., 2009) these distant populations might be mediating the effects. Future experiments testing broader NMDAR1 functions, as well as studies of possible mGLuR-mediated glutamate signaling in AL, will be important to fully elucidate circuit mechanisms that regulate OSN remodeling.

In conclusion, we discover here that unbalanced neuron class-specific FMRP functions can alter lateral OSN interactions and impact critical period OSN remodeling. The findings show that FMRP acts in Or42a OSNs as well as other EB-responsive OSNs to control the response to lateral input. When FMRP is removed only in Or42a OSNs, they have a lower responsiveness to EB exposure and therefore manifest impaired critical period remodeling. However, when FMRP is removed from all OSNs, balance is restored to reinstate the normal remodeling. The FMRP requirement is bidirectional as targeted FMRP elevation in Or42a OSNs also causes circuit imbalance to enhance the effect of odorant experience on critical period remodeling. Furthermore, unlike other forms of activity-dependent remodeling in the AL circuit, this reversible remodeling occurs only in the presynaptic OSN terminals and requires OSN activity, but not OSN synaptic output. Or42a OSN-targeted optogenetic activation drives critical period remodeling, but this activity-dependent mechanism does not require FMRP function in the Or42a OSNs. Despite the clear involvement of LN glutamatergic and GABAergic signaling in regulating OSN critical period remodeling, their exact circuit connectivity remains to be fully elucidated. The innervation loss and retraction characteristics following critical period odorant experience resemble developmental pruning during Drosophila metamorphosis, which suggests similar underlying mechanisms of cytoskeleton disassembly and glial phagocytosis (Yu and Schuldiner, 2014). In addition, the mechanism(s) by which FMRP regulates OSN responses to lateral inputs will be an important avenue for future research. Overall, this work provides a new example of neuron class-specific FMRP function, neural circuit compensation for FMRP loss, and an avenue to inform therapies addressing FXS circuit-level symptoms.

Footnotes

This work was supported by the National Institutes of Health Grant MH084989 (to K.B.). We thank Francois Bolduc for UAS-dfmr1 RNAi lines (1-1-7, 2-1; University of Alberta, Canada), Ron Davis for UAS-Rdl RNAi (8-10J; The Scripps Research Institute Florida), Ilona Grunwald-Kadow for Or42a-mCD8:GFP (Technical University of Munich, Germany), Craig Montell for GtACR1-eYFP (University of California Santa Barbara), Mani Ramaswami for NP1227(LN1)-Gal4 (Trinity College Dublin, Ireland), and Bloomington Drosophila Stock Center (Indiana University) for many lines. We also thank Dominic Vita, Danielle Kopke, and Jim Sears for their input.
The authors declare no competing financial interests.
Correspondence should be addressed to Kendal Broadie at kendal.broadie{at}vanderbilt.edu

SfN exclusive license.

References

↵
2. Acebes A,
3. Martín-Peña A,
4. Chevalier V,
5. Ferrús A
(2011) Synapse loss in olfactory local interneurons modifies perception. J Neurosci 31:2734–2745. doi:10.1523/JNEUROSCI.5046-10.2011 pmid:21414896
OpenUrl Abstract/FREE Full Text
↵
2. Acebes A,
3. Devaud J,
4. Arnés M,
5. Ferrús A
(2012) Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe. J Neurosci 32:417–422. doi:10.1523/JNEUROSCI.2921-11.2012 pmid:22238078
OpenUrl Abstract/FREE Full Text
↵
2. Antoine M,
3. Langberg T,
4. Schnepel P,
5. Feldman D
(2019) Increased excitation-inhibition ratio stabilizes synapse and circuit excitability in four autism mouse models. Neuron 101:648–661.e4. doi:10.1016/j.neuron.2018.12.026 pmid:30679017
OpenUrl CrossRef PubMed
↵
2. Aronstein K,
3. Ffrench-Constant R
(1995) Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster. Invert Neurosci 1:25–31. doi:10.1007/BF02331829 pmid:9372130
OpenUrl CrossRef PubMed
↵
2. Aso Y,
3. Grübel K,
4. Busch S,
5. Friedrich A,
6. Siwanowicz I,
7. Tanimoto H
(2009) The mushroom body of adult Drosophila characterized by GAL4 drivers. J Neurogenet 23:156–172. doi:10.1080/01677060802471718 pmid:19140035
OpenUrl CrossRef PubMed
↵
2. Bailey D,
3. Hatton D,
4. Skinner M
(1998) Early developmental trajectories of males with fragile X syndrome. Am J Mental Retard 103:29. doi:10.1352/0895-8017(1998)103<0029:EDTOMW>2.0.CO;2
OpenUrl CrossRef PubMed
↵
2. Bolduc F,
3. Bell K,
4. Cox H,
5. Broadie K,
6. Tully T
(2008) Excess protein synthesis in Drosophila Fragile X mutants impairs long-term memory. Nat Neurosci 11:1143–1145. doi:10.1038/nn.2175
OpenUrl CrossRef PubMed
↵
2. Brandalise F,
3. Kalmbach B,
4. Mehta P,
5. Thornton O,
6. Johnston D,
7. Zemelman B,
8. Brager D
(2020) Fragile X mental retardation protein bidirectionally controls dendritic Ih in a cell-type specific manner between mouse hippocampus and prefrontal cortex. J Neurosci 40:5327–5340. doi:10.1523/JNEUROSCI.1670-19.2020 pmid:32467357
OpenUrl Abstract/FREE Full Text
↵
2. Brown V,
3. Jin P,
4. Ceman S,
5. Darnell J,
6. O'Donnell W,
7. Tenenbaum S,
8. Jin X,
9. Feng Y,
10. Wilkinson K,
11. Keene J,
12. Darnell R,
13. Warren S
(2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107:477–487. doi:10.1016/S0092-8674(01)00568-2
OpenUrl CrossRef PubMed
↵
2. Bureau I,
3. Shepherd G,
4. Svoboda K
(2008) Circuit and plasticity defects in the developing somatosensory cortex of Fmr1 knock-out mice. J Neurosci 28:5178–5188. doi:10.1523/JNEUROSCI.1076-08.2008 pmid:18480274
OpenUrl Abstract/FREE Full Text
↵
2. Chiang A,
3. Priya R,
4. Ramaswami M,
5. VijayRaghavan K,
6. Rodrigues V
(2009) Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons. Development 136:1273–1282. doi:10.1242/dev.031377 pmid:19304886
OpenUrl Abstract/FREE Full Text
↵
2. Chodankar A,
3. Sadanandappa M,
4. VijayRaghavan K,
5. Ramaswami M
(2020) Glomerulus-selective regulation of a critical period for interneuron plasticity in the Drosophila antennal lobe. J Neurosci 40:5549–5560. doi:10.1523/JNEUROSCI.2192-19.2020 pmid:32532889
OpenUrl Abstract/FREE Full Text
↵
2. Chou Y,
3. Spletter M,
4. Yaksi E,
5. Leong J,
6. Wilson R,
7. Luo L
(2010) Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe. Nat Neurosci 13:439–449. doi:10.1038/nn.2489 pmid:20139975
OpenUrl CrossRef PubMed
↵
2. Contractor A,
3. Klyachko V,
4. Portera-Cailliau C
(2015) Altered neuronal and circuit excitability in fragile X syndrome. Neuron 87:699–715. doi:10.1016/j.neuron.2015.06.017 pmid:26291156
OpenUrl CrossRef PubMed
↵
2. Connolly J,
3. Roberts I,
4. Armstrong J,
5. Kaiser K,
6. Forte M,
7. Tully T,
8. O'Kane C
(1996) Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274:2104–2107. doi:10.1126/science.274.5295.2104 pmid:8953046
OpenUrl Abstract/FREE Full Text
↵
2. Couto A,
3. Alenius M,
4. Dickson B
(2005) Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15:1535–1547. doi:10.1016/j.cub.2005.07.034 pmid:16139208
OpenUrl CrossRef PubMed
↵
2. Crawford D,
3. Acuña J,
4. Sherman S
(2001) FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med 3:359–371. doi:10.1097/00125817-200109000-00006 pmid:11545690
OpenUrl CrossRef PubMed
↵
2. Dahlhaus R
(2018) Of men and mice: modeling the fragile X syndrome. Front Mol Neurosci 11:41. doi:10.3389/fnmol.2018.00041 pmid:29599705
OpenUrl CrossRef PubMed
↵
2. Dalland T,
3. Døving K
(1981) Reaction to olfactory stimuli in odor-exposed rats. Behav Neural Biol 32:79–88. doi:10.1016/S0163-1047(81)90302-2
OpenUrl CrossRef PubMed
↵
2. Darnell J,
3. Jensen K,
4. Jin P,
5. Brown V,
6. Warren S,
7. Darnell R
(2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107:489–499. doi:10.1016/S0092-8674(01)00566-9
OpenUrl CrossRef PubMed
↵
2. Darnell J,
3. Van Driesche S,
4. Zhang C,
5. Hung K,
6. Mele A,
7. Fraser C,
8. Stone E,
9. Chen C,
10. Fak J,
11. Chi S,
12. Licatalosi D,
13. Richter J,
14. Darnell R
(2011) FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146:247–261. doi:10.1016/j.cell.2011.06.013 pmid:21784246
OpenUrl CrossRef PubMed
↵
2. Das A,
3. Chiang A,
4. Davla S,
5. Priya R,
6. Reichert H,
7. VijayRaghavan K,
8. Rodrigues V
(2010) Identification and analysis of a glutamatergic local interneuron lineage in the adult Drosophila olfactory system. Neural Syst Circuits 1:4.
OpenUrl
↵
2. Das S,
3. Sadanandappa M,
4. Dervan A,
5. Larkin A,
6. Lee J,
7. Sudhakaran I,
8. Priya R,
9. Heidari R,
10. Holohan E,
11. Pimentel A,
12. Gandhi A,
13. Ito K,
14. Sanyal S,
15. Wang J,
16. Rodrigues V,
17. Ramaswami M
(2011) Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci USA 108:E646–E654. doi:10.1073/pnas.1106411108
OpenUrl Abstract/FREE Full Text
↵
2. Devaud J,
3. Acebes A,
4. Ramaswami M,
5. Ferrús A
(2003) Structural and functional changes in the olfactory pathway of adult Drosophila take place at a critical age. J Neurobiol 56:13–23. doi:10.1002/neu.10215 pmid:12767029
OpenUrl CrossRef PubMed
↵
2. Devaud J,
3. Clouet-Redt C,
4. Bockaert J,
5. Grau Y,
6. Parmentier M
(2008) Widespread brain distribution of the Drosophila metabotropic glutamate receptor. Neuroreport 19:367–371.
OpenUrl CrossRef PubMed
↵
2. Doll C,
3. Broadie K
(2015) Activity-dependent FMRP requirements in development of the neural circuitry of learning and memory. Development 142:1346–1356. doi:10.1242/dev.117127 pmid:25804740
OpenUrl Abstract/FREE Full Text
↵
2. Doll C,
3. Broadie K
(2016) Neuron class-specific requirements for fragile X mental retardation protein in critical period development of calcium signaling in learning and memory circuitry. Neurobiol Dis 89:76–87. doi:10.1016/j.nbd.2016.02.006 pmid:26851502
OpenUrl CrossRef PubMed
↵
2. Doll C,
3. Vita D,
4. Broadie K
(2017) Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement. Curr Biol 27:2318–2330. doi:10.1016/j.cub.2017.06.046 pmid:28756946
OpenUrl CrossRef PubMed
↵
2. Dölen G,
3. Osterweil E,
4. Rao B,
5. Smith G,
6. Auerbach B,
7. Chattarji S,
8. Bear M
(2007) Correction of fragile X syndrome in mice. Neuron 56:955–962. doi:10.1016/j.neuron.2007.12.001 pmid:18093519
OpenUrl CrossRef PubMed
↵
2. Domanski A,
3. Booker S,
4. Wyllie D,
5. Isaac J,
6. Kind P
(2019) Cellular and synaptic phenotypes lead to disrupted information processing in Fmr1-KO mouse layer 4 barrel cortex. Nat Commun 10:4814. doi:10.1038/s41467-019-12736-y
OpenUrl CrossRef PubMed
2. Fishilevich E,
3. Vosshall L
(2005) Genetic and functional subdivision of the Drosophila antennal lobe. Curr Biol 15:1548–1553. doi:10.1016/j.cub.2005.07.066 pmid:16139209
OpenUrl CrossRef PubMed
↵
2. Flockhart I,
3. Booker M,
4. Kiger A,
5. Boutros M,
6. Armknecht S,
7. Ramadan N,
8. Richardson K,
9. Xu A,
10. Perrimon N,
11. Mathey-Prevot B
(2006) FlyRNAi: the Drosophila RNAi screening center database. Nucleic Acids Res 34:D489–D494. doi:10.1093/nar/gkj114
OpenUrl CrossRef PubMed
↵
2. Franco L,
3. Okray Z,
4. Linneweber G,
5. Hassan B,
6. Yaksi E
(2017) Reduced lateral inhibition impairs olfactory computations and behaviors in a Drosophila model of fragile X syndrome. Curr Biol 27:1111–1123. doi:10.1016/j.cub.2017.02.065 pmid:28366741
OpenUrl CrossRef PubMed
↵
2. Friedman S,
3. Dani N,
4. Rushton E,
5. Broadie K
(2013) Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila. Dis Model Mech 6:1400–1413. doi:10.1242/dmm.012229 pmid:24046358
OpenUrl Abstract/FREE Full Text
↵
2. Geramita M,
3. Urban N
(2016) Postnatal odor exposure increases the strength of interglomerular lateral inhibition onto olfactory bulb tufted cells. J Neurosci 36:12321–12327. doi:10.1523/JNEUROSCI.1991-16.2016 pmid:27927952
OpenUrl Abstract/FREE Full Text
↵
2. Gerlach G,
3. Tietje K,
4. Biechl D,
5. Namekawa I,
6. Schalm G,
7. Sulmann A
(2019) Behavioural and neuronal basis of olfactory imprinting and kin recognition in larval fish. J Exp Biol 222:jeb189746. doi:10.1242/jeb.189746
OpenUrl Abstract/FREE Full Text
↵
2. Goel A,
3. Cantu D,
4. Guilfoyle J,
5. Chaudhari G,
6. Newadkar A,
7. Todisco B,
8. Alba D,
9. Kourdougli N,
10. Schmitt L,
11. Pedapati E,
12. Erickson C,
13. Portera-Cailliau C
(2018) Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible. Nat Neurosci 21:1404–1411. doi:10.1038/s41593-018-0231-0 pmid:30250263
OpenUrl CrossRef PubMed
↵
2. Golovin R,
3. Vest J,
4. Vita D,
5. Broadie K
(2019) Activity-dependent remodeling of Drosophila olfactory sensory neuron brain innervation during an early-life critical period. J Neurosci 39:2995–3012. doi:10.1523/JNEUROSCI.2223-18.2019
OpenUrl Abstract/FREE Full Text
↵
2. Gonçalves J,
3. Anstey J,
4. Golshani P,
5. Portera-Cailliau C
(2013) Circuit level defects in the developing neocortex of fragile X mice. Nat Neurosci 16:903–909. doi:10.1038/nn.3415 pmid:23727819
OpenUrl CrossRef PubMed
↵
2. Greenblatt E,
3. Spradling A
(2018) Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins. Science 361:709–712. doi:10.1126/science.aas9963 pmid:30115809
OpenUrl Abstract/FREE Full Text
↵
2. Greenhill S,
3. Juczewski K,
4. Haan A,
5. Seaton G,
6. Fox K,
7. Hardingham N
(2015) Adult cortical plasticity depends on an early postnatal critical period. Science 349:424–427. doi:10.1126/science.aaa8481
OpenUrl Abstract/FREE Full Text
↵
2. Harlow E,
3. Till S,
4. Russell T,
5. Wijetunge L,
6. Kind P,
7. Contractor A
(2010) Critical period plasticity is disrupted in the barrel cortex of FMR1 knockout mice. Neuron 65:385–398. doi:10.1016/j.neuron.2010.01.024 pmid:20159451
OpenUrl CrossRef PubMed
2. Hayashi S,
3. Ito K,
4. Sado Y,
5. Taniguchi M,
6. Akimoto A,
7. Takeuchi H,
8. Aigaki T,
9. Matsuzaki F,
10. Nakagoshi H,
11. Tanimura T,
12. Ueda R,
13. Uemura T,
14. Yoshihara M,
15. Goto S
(2002) GETDB, a database compiling expression patterns and molecular locations of a collection of gal4 enhancer traps. Genesis 34:58–61. doi:10.1002/gene.10137 pmid:12324948
OpenUrl CrossRef PubMed
↵
2. He Q,
3. Nomura T,
4. Xu J,
5. Contractor A
(2014) The developmental switch in GABA polarity is delayed in fragile X mice. J Neurosci 34:446–450. doi:10.1523/JNEUROSCI.4447-13.2014 pmid:24403144
OpenUrl Abstract/FREE Full Text
↵
2. Hensch T
(2005) Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6:877–888. doi:10.1038/nrn1787 pmid:16261181
OpenUrl CrossRef PubMed
↵
2. Hersh J,
3. Saul R
; Committee on Genetics (2011) Health supervision for children with fragile X syndrome. Pediatrics 127:994–1006. doi:10.1542/peds.2010-3500 pmid:21518720
OpenUrl Abstract/FREE Full Text
↵
2. Huang J,
3. Zhang W,
4. Qiao W,
5. Hu A,
6. Wang Z
(2010) Functional connectivity and selective odor responses of excitatory local interneurons in Drosophila antennal lobe. Neuron 67:1021–1033. doi:10.1016/j.neuron.2010.08.025 pmid:20869598
OpenUrl CrossRef PubMed
↵
2. Hubel D,
3. Wiesel T
(1970) The period of susceptibility to the physiological effects of unilateral eye closure in kittens. J Physiol 206:419–436. doi:10.1113/jphysiol.1970.sp009022 pmid:5498493
OpenUrl CrossRef PubMed
↵
2. Inoue S,
3. Shimoda M,
4. Nishinokubi I,
5. Siomi M,
6. Okamura M,
7. Nakamura A,
8. Kobayashi S,
9. Ishida N,
10. Siomi H
(2002) A role for the Drosophila fragile X-related gene in circadian output. Curr Biol 12:1331–1335. doi:10.1016/S0960-9822(02)01036-9
OpenUrl CrossRef PubMed
↵
2. Jackson F,
3. Newby L,
4. Kulkarni S
(1990) Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxylase. J Neurochem 54:1068–1078. doi:10.1111/j.1471-4159.1990.tb02359.x pmid:1689376
OpenUrl CrossRef PubMed
↵
2. Jefferis G,
3. Marin E,
4. Watts R,
5. Luo L
(2002) Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies. Curr Opin Neurobiol 12:80–86. doi:10.1016/S0959-4388(02)00293-3
OpenUrl CrossRef PubMed
↵
2. Khandjian EW,
3. Fortin A,
4. Thibodeau A,
5. Tremblay S,
6. Côté F,
7. Devys D,
8. Mandel JL,
9. Rousseau F
(1995) A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture. Hum Mol Genet 4:783–789. doi:10.1093/hmg/4.5.783 pmid:7633436
OpenUrl CrossRef PubMed
↵
2. Klapoetke N,
3. Murata Y,
4. Kim S,
5. Pulver S,
6. Birdsey-Benson A,
7. Cho Y,
8. Morimoto T,
9. Chuong A,
10. Carpenter E,
11. Tian Z,
12. Wang J,
13. Xie Y,
14. Yan Z,
15. Zhang Y,
16. Chow B,
17. Surek B,
18. Melkonian M,
19. Jayaraman V,
20. Constantine-Paton M,
21. Wong G, et al
. (2014) Independent optical excitation of distinct neural populations. Nat Methods 11:338–346. doi:10.1038/nmeth.2836 pmid:24509633
OpenUrl CrossRef PubMed
↵
2. Krishnan K,
3. Wang B,
4. Lu J,
5. Wang L,
6. Maffei A,
7. Cang J,
8. Huang Z
(2015) MeCP2 regulates the timing of critical period plasticity that shapes functional connectivity in primary visual cortex. Proc Natl Acad Sci USA 112:E4782–E4791. doi:10.1073/pnas.1506499112
OpenUrl Abstract/FREE Full Text
↵
2. Larsson M,
3. Domingos A,
4. Jones W,
5. Chiappe M,
6. Amrein H,
7. Vosshall L
(2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43:703–714. doi:10.1016/j.neuron.2004.08.019 pmid:15339651
OpenUrl CrossRef PubMed
2. Lee T,
3. Luo L
(1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461. doi:10.1016/S0896-6273(00)80701-1
OpenUrl CrossRef PubMed
↵
2. Liu A,
3. Urban N
(2017) Prenatal and early postnatal odorant exposure heightens odor-evoked mitral cell responses in the mouse olfactory bulb. eNeuro 4:ENEURO.0129-17.2017. doi:10.1523/ENEURO.0129-17.2017
OpenUrl Abstract/FREE Full Text
↵
2. Liu W,
3. Wilson R
(2013) Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system. Proc Natl Acad Sci USA 110:10294–10299. doi:10.1016/j.cub.2013.05.016 pmid:23770187
OpenUrl Abstract/FREE Full Text
2. Liu X,
3. Krause W,
4. Davis R
(2007) GABAA receptor RDL inhibits Drosophila olfactory associative learning. Neuron 56:1090–1102. doi:10.1016/j.neuron.2007.10.036 pmid:18093529
OpenUrl CrossRef PubMed
↵
2. Lovelace J,
3. Rais M,
4. Palacios A,
5. Shuai X,
6. Bishay S,
7. Popa O,
8. Pirbhoy P,
9. Binder D,
10. Nelson D,
11. Ethell I,
12. Razak K
(2020) Deletion of Fmr1 from forebrain excitatory neurons triggers abnormal cellular, EEG, and behavioral phenotypes in the auditory cortex of a mouse model of fragile X syndrome. Cereb Cortex 30:969–988. doi:10.1093/cercor/bhz141 pmid:31364704
OpenUrl CrossRef PubMed
↵
2. Marin E,
3. Jefferis G,
4. Komiyama T,
5. Zhu H,
6. Luo L
(2002) Representation of the glomerular olfactory map in the Drosophila brain. Cell 109:243–255. doi:10.1016/S0092-8674(02)00700-6
OpenUrl CrossRef PubMed
↵
2. Meredith R
(2015) Sensitive and critical periods during neurotypical and aberrant neurodevelopment: a framework for neurodevelopmental disorders. Neurosci Biobehav Rev 50:180–188. doi:10.1016/j.neubiorev.2014.12.001 pmid:25496903
OpenUrl CrossRef PubMed
↵
2. Mohamed A,
3. Retzke T,
4. Chakraborty S,
5. Fabian B,
6. Hansson B,
7. Knaden M,
8. Sachse S
(2019) Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe. Nat Commun 10:1201. doi:10.1038/s41467-019-09069-1 pmid:30867415
OpenUrl CrossRef PubMed
2. Mohammad F,
3. Stewart J,
4. Ott S,
5. Chlebikova K,
6. Chua J,
7. Koh T,
8. Ho J,
9. Claridge-Chang A
(2017) Optogenetic inhibition of behavior with anion channelrhodopsins. Nat Methods 14:271–274. doi:10.1038/nmeth.4148 pmid:28114289
OpenUrl CrossRef PubMed
↵
2. Münch D,
3. Galizia C
(2016) DoOR 2.0–comprehensive mapping of Drosophila melanogaster odorant responses. Sci Rep 6:21841. doi:10.1038/srep21841 pmid:26912260
OpenUrl CrossRef PubMed
↵
2. Nagarkar-Jaiswal S,
3. DeLuca S,
4. Lee P,
5. Lin W,
6. Pan H,
7. Zuo Z,
8. Lv J,
9. Spradling A,
10. Bellen H
(2015) A genetic toolkit for tagging intronic MiMIC containing genes. Elife 4:e08469. doi:10.7554/eLife.08469
OpenUrl CrossRef PubMed
↵
2. Okada R,
3. Awasaki T,
4. Ito K
(2009) Gamma-aminobutyric acid (GABA)-mediated neural connections in the Drosophila antennal lobe. J Comp Neurol 514:74–91. doi:10.1002/cne.21971 pmid:19260068
OpenUrl CrossRef PubMed
↵
2. Olsen S,
3. Wilson R
(2008) Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452:956–960. doi:10.1038/nature06864
OpenUrl CrossRef PubMed
↵
2. Olsen S,
3. Bhandawat V,
4. Wilson R
(2010) Divisive normalization in olfactory population codes. Neuron 66:287–299. doi:10.1016/j.neuron.2010.04.009 pmid:20435004
OpenUrl CrossRef PubMed
↵
2. Roberts J,
3. McCary L,
4. Shinkareva S,
5. Bailey D
(2016) Infant development in fragile X syndrome: cross-syndrome comparisons. J Autism Dev Disord 46:2088–2099. doi:10.1007/s10803-016-2737-1 pmid:26864160
OpenUrl CrossRef PubMed
↵
2. Rorth P
(1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci USA 93:12418–12422. doi:10.1073/pnas.93.22.12418
OpenUrl Abstract/FREE Full Text
↵
2. Sachse S,
3. Rueckert E,
4. Keller A,
5. Okada R,
6. Tanaka N,
7. Ito K,
8. Vosshall L
(2007) Activity-dependent plasticity in an olfactory circuit. Neuron 56:838–850. doi:10.1016/j.neuron.2007.10.035 pmid:18054860
OpenUrl CrossRef PubMed
↵
2. Sawicka K,
3. Hale C,
4. Park C,
5. Fak J,
6. Gresack J,
7. Driesche S,
8. Kang J,
9. Darnell J,
10. Darnell R
(2019) FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory. Elife 8:e46919. doi:10.7554/eLife.46919
OpenUrl CrossRef
↵
2. Schindelin J,
3. Arganda-Carreras I,
4. Frise E,
5. Kaynig V,
6. Longair M,
7. Pietzsch T,
8. Preibisch S,
9. Rueden C,
10. Saalfeld S,
11. Schmid B,
12. Tinevez J,
13. White D,
14. Hartenstein V,
15. Eliceiri K,
16. Tomancak P,
17. Cardona A
(2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi:10.1038/nmeth.2019 pmid:22743772
OpenUrl CrossRef PubMed
↵
2. Sears J,
3. Choi W,
4. Broadie K
(2019) Fragile X mental retardation protein positively regulates PKA anchor rugose and PKA activity to control actin assembly in learning/memory circuitry. Neurobiol Dis 127:53–64. doi:10.1016/j.nbd.2019.02.004 pmid:30771457
OpenUrl CrossRef PubMed
↵
2. Semmelhack J,
3. Wang J
(2009) Select Drosophila glomeruli mediate innate olfactory attraction and aversion. Nature 459:218–223. doi:10.1038/nature07983 pmid:19396157
OpenUrl CrossRef PubMed
↵
2. Stephan D,
3. Sánchez-Soriano N,
4. Loschek L,
5. Gerhards R,
6. Gutmann S,
7. Storchova Z,
8. Prokop A,
9. Kadow I
(2012) Drosophila psidin regulates olfactory neuron number and axon targeting through two distinct molecular mechanisms. J Neurosci 32:16080–16094. doi:10.1523/JNEUROSCI.3116-12.2012 pmid:23152593
OpenUrl Abstract/FREE Full Text
2. Stocker R,
3. Heimbeck G,
4. Gendre N,
5. Belle J
(1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J Neurobiol 32:443–456. doi:10.1002/(SICI)1097-4695(199705)32:5<443::AID-NEU1>3.0.CO;2-5
OpenUrl CrossRef PubMed
↵
2. Sudhakaran I,
3. Hillebrand J,
4. Dervan A,
5. Das S,
6. Holohan E,
7. Hülsmeier J,
8. Sarov M,
9. Parker R,
10. VijayRaghavan K,
11. Ramaswami M
(2014) FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control. Proc Natl Acad Sci USA 111:E99–E108. doi:10.1073/pnas.1309543111
OpenUrl Abstract/FREE Full Text
↵
2. Sweeney L,
3. Couto A,
4. Chou Y,
5. Berdnik D,
6. Dickson B,
7. Luo L,
8. Komiyama T
(2007) Temporal target restriction of olfactory receptor neurons by semaphorin-1a/plexinA-mediated axon-axon interactions. Neuron 53:185–200. doi:10.1016/j.neuron.2006.12.022 pmid:17224402
OpenUrl CrossRef PubMed
2. Sweeney S,
3. Broadie K,
4. Keane J,
5. Niemann H,
6. O'Kane C
(1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14:341–351. doi:10.1016/0896-6273(95)90290-2
OpenUrl CrossRef PubMed
↵
2. Tessier C,
3. Broadie K
(2009) Activity-dependent modulation of neural circuit synaptic connectivity. Front Mol Neurosci 2:8. doi:10.3389/neuro.02.008.2009 pmid:19668708
OpenUrl CrossRef PubMed
2. Wang J,
3. Beck E,
4. McCabe B
(2012) A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila. PLoS One 7:e42102. doi:10.1371/journal.pone.0042102 pmid:22848718
OpenUrl CrossRef PubMed
↵
2. Wilson R
(2013) Early olfactory processing in Drosophila: mechanisms and principles. Annu Rev Neurosci 36:217–241. doi:10.1146/annurev-neuro-062111-150533 pmid:23841839
OpenUrl CrossRef PubMed
↵
2. Wodarz A,
3. Hinz U,
4. Engelbert M,
5. Knust E
(1995) Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82:67–76. doi:10.1016/0092-8674(95)90053-5
OpenUrl CrossRef PubMed
↵
2. Xia S,
3. Miyashita T,
4. Fu T,
5. Lin W,
6. Wu C,
7. Pyzocha L,
8. Lin I,
9. Saitoe M,
10. Tully T,
11. Chiang A
(2005) NMDA receptors mediate olfactory learning and memory in Drosophila. Curr Biol 15:603–615. doi:10.1016/j.cub.2005.02.059 pmid:15823532
OpenUrl CrossRef PubMed
↵
2. Yu F,
3. Schuldiner O
(2014) Axon and dendrite pruning in Drosophila. Curr Opin Neurobiol 27:192–198. doi:10.1016/j.conb.2014.04.005 pmid:24793180
OpenUrl CrossRef PubMed
↵
2. Zalfa F,
3. Giorgi M,
4. Primerano B,
5. Moro A,
6. Penta A,
7. Reis S,
8. Oostra B,
9. Bagni C
(2003) The Fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell 112:317–327. doi:10.1016/S0092-8674(03)00079-5
OpenUrl CrossRef PubMed
↵
2. Zars T,
3. Fischer M,
4. Schulz R,
5. Heisenberg M
(2000) Localization of a short-term memory in Drosophila. Science 288:672–675. doi:10.1126/science.288.5466.672 pmid:10784450
OpenUrl Abstract/FREE Full Text
↵
2. Zhang Y,
3. Bailey A,
4. Matthies H,
5. Renden R,
6. Smith M,
7. Speese S,
8. Rubin G,
9. Broadie K
(2001) Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function. Cell 107:591–603. doi:10.1016/S0092-8674(01)00589-X
OpenUrl CrossRef PubMed

In this issue

View Full Page PDF

Citation Tools

Respond to this article

Request Permissions

Keywords

Cited By...

Research Articles

Show more Research Articles

Development/Plasticity/Repair

Show more Development/Plasticity/Repair

[1] ↵

Acebes A,
Martín-Peña A,
Chevalier V,
Ferrús A
(2011) Synapse loss in olfactory local interneurons modifies perception. J Neurosci 31:2734–2745. doi:10.1523/JNEUROSCI.5046-10.2011 pmid:21414896
OpenUrl Abstract/FREE Full Text

[3] Acebes A,

[4] Martín-Peña A,

[5] Chevalier V,

[6] Ferrús A

[7] ↵

Acebes A,
Devaud J,
Arnés M,
Ferrús A
(2012) Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe. J Neurosci 32:417–422. doi:10.1523/JNEUROSCI.2921-11.2012 pmid:22238078
OpenUrl Abstract/FREE Full Text

[9] Acebes A,

[10] Devaud J,

[11] Arnés M,

[12] Ferrús A

[13] ↵

Antoine M,
Langberg T,
Schnepel P,
Feldman D
(2019) Increased excitation-inhibition ratio stabilizes synapse and circuit excitability in four autism mouse models. Neuron 101:648–661.e4. doi:10.1016/j.neuron.2018.12.026 pmid:30679017
OpenUrl CrossRef PubMed

[15] Antoine M,

[16] Langberg T,

[17] Schnepel P,

[18] Feldman D

[19] ↵

Aronstein K,
Ffrench-Constant R
(1995) Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster. Invert Neurosci 1:25–31. doi:10.1007/BF02331829 pmid:9372130
OpenUrl CrossRef PubMed

[21] Aronstein K,

[22] Ffrench-Constant R

[23] ↵

Aso Y,
Grübel K,
Busch S,
Friedrich A,
Siwanowicz I,
Tanimoto H
(2009) The mushroom body of adult Drosophila characterized by GAL4 drivers. J Neurogenet 23:156–172. doi:10.1080/01677060802471718 pmid:19140035
OpenUrl CrossRef PubMed

[25] Aso Y,

[26] Grübel K,

[27] Busch S,

[28] Friedrich A,

[29] Siwanowicz I,

[30] Tanimoto H

[31] ↵

Bailey D,
Hatton D,
Skinner M
(1998) Early developmental trajectories of males with fragile X syndrome. Am J Mental Retard 103:29. doi:10.1352/0895-8017(1998)103<0029:EDTOMW>2.0.CO;2
OpenUrl CrossRef PubMed

[33] Bailey D,

[34] Hatton D,

[35] Skinner M

[36] ↵

Bolduc F,
Bell K,
Cox H,
Broadie K,
Tully T
(2008) Excess protein synthesis in Drosophila Fragile X mutants impairs long-term memory. Nat Neurosci 11:1143–1145. doi:10.1038/nn.2175
OpenUrl CrossRef PubMed

[38] Bolduc F,

[39] Bell K,

[40] Cox H,

[41] Broadie K,

[42] Tully T

[43] ↵

Brandalise F,
Kalmbach B,
Mehta P,
Thornton O,
Johnston D,
Zemelman B,
Brager D
(2020) Fragile X mental retardation protein bidirectionally controls dendritic Ih in a cell-type specific manner between mouse hippocampus and prefrontal cortex. J Neurosci 40:5327–5340. doi:10.1523/JNEUROSCI.1670-19.2020 pmid:32467357
OpenUrl Abstract/FREE Full Text

[45] Brandalise F,

[46] Kalmbach B,

[47] Mehta P,

[48] Thornton O,

[49] Johnston D,

[50] Zemelman B,

[51] Brager D

[52] ↵

Brown V,
Jin P,
Ceman S,
Darnell J,
O'Donnell W,
Tenenbaum S,
Jin X,
Feng Y,
Wilkinson K,
Keene J,
Darnell R,
Warren S
(2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107:477–487. doi:10.1016/S0092-8674(01)00568-2
OpenUrl CrossRef PubMed

[54] Brown V,

[55] Jin P,

[56] Ceman S,

[57] Darnell J,

[58] O'Donnell W,

[59] Tenenbaum S,

[60] Jin X,

[61] Feng Y,

[62] Wilkinson K,

[63] Keene J,

[64] Darnell R,

[65] Warren S

[66] ↵

Bureau I,
Shepherd G,
Svoboda K
(2008) Circuit and plasticity defects in the developing somatosensory cortex of Fmr1 knock-out mice. J Neurosci 28:5178–5188. doi:10.1523/JNEUROSCI.1076-08.2008 pmid:18480274
OpenUrl Abstract/FREE Full Text

[68] Bureau I,

[69] Shepherd G,

[70] Svoboda K

[71] ↵

Chiang A,
Priya R,
Ramaswami M,
VijayRaghavan K,
Rodrigues V
(2009) Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons. Development 136:1273–1282. doi:10.1242/dev.031377 pmid:19304886
OpenUrl Abstract/FREE Full Text

[73] Chiang A,

[74] Priya R,

[75] Ramaswami M,

[76] VijayRaghavan K,

[77] Rodrigues V

[78] ↵

Chodankar A,
Sadanandappa M,
VijayRaghavan K,
Ramaswami M
(2020) Glomerulus-selective regulation of a critical period for interneuron plasticity in the Drosophila antennal lobe. J Neurosci 40:5549–5560. doi:10.1523/JNEUROSCI.2192-19.2020 pmid:32532889
OpenUrl Abstract/FREE Full Text

[80] Chodankar A,

[81] Sadanandappa M,

[82] VijayRaghavan K,

[83] Ramaswami M

[84] ↵

Chou Y,
Spletter M,
Yaksi E,
Leong J,
Wilson R,
Luo L
(2010) Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe. Nat Neurosci 13:439–449. doi:10.1038/nn.2489 pmid:20139975
OpenUrl CrossRef PubMed

[86] Chou Y,

[87] Spletter M,

[88] Yaksi E,

[89] Leong J,

[90] Wilson R,

[91] Luo L

[92] ↵

Contractor A,
Klyachko V,
Portera-Cailliau C
(2015) Altered neuronal and circuit excitability in fragile X syndrome. Neuron 87:699–715. doi:10.1016/j.neuron.2015.06.017 pmid:26291156
OpenUrl CrossRef PubMed

[94] Contractor A,

[95] Klyachko V,

[96] Portera-Cailliau C

[97] ↵

Connolly J,
Roberts I,
Armstrong J,
Kaiser K,
Forte M,
Tully T,
O'Kane C
(1996) Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274:2104–2107. doi:10.1126/science.274.5295.2104 pmid:8953046
OpenUrl Abstract/FREE Full Text

[99] Connolly J,

[100] Roberts I,

[101] Armstrong J,

[102] Kaiser K,

[103] Forte M,

[104] Tully T,

[105] O'Kane C

[106] ↵

Couto A,
Alenius M,
Dickson B
(2005) Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15:1535–1547. doi:10.1016/j.cub.2005.07.034 pmid:16139208
OpenUrl CrossRef PubMed

[108] Couto A,

[109] Alenius M,

[110] Dickson B

[111] ↵

Crawford D,
Acuña J,
Sherman S
(2001) FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med 3:359–371. doi:10.1097/00125817-200109000-00006 pmid:11545690
OpenUrl CrossRef PubMed

[113] Crawford D,

[114] Acuña J,

[115] Sherman S

[116] ↵

Dahlhaus R
(2018) Of men and mice: modeling the fragile X syndrome. Front Mol Neurosci 11:41. doi:10.3389/fnmol.2018.00041 pmid:29599705
OpenUrl CrossRef PubMed

[118] Dahlhaus R

[119] ↵

Dalland T,
Døving K
(1981) Reaction to olfactory stimuli in odor-exposed rats. Behav Neural Biol 32:79–88. doi:10.1016/S0163-1047(81)90302-2
OpenUrl CrossRef PubMed

[121] Dalland T,

[122] Døving K

[123] ↵

Darnell J,
Jensen K,
Jin P,
Brown V,
Warren S,
Darnell R
(2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107:489–499. doi:10.1016/S0092-8674(01)00566-9
OpenUrl CrossRef PubMed

[125] Darnell J,

[126] Jensen K,

[127] Jin P,

[128] Brown V,

[129] Warren S,

[130] Darnell R

[131] ↵

Darnell J,
Van Driesche S,
Zhang C,
Hung K,
Mele A,
Fraser C,
Stone E,
Chen C,
Fak J,
Chi S,
Licatalosi D,
Richter J,
Darnell R
(2011) FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146:247–261. doi:10.1016/j.cell.2011.06.013 pmid:21784246
OpenUrl CrossRef PubMed

[133] Darnell J,

[134] Van Driesche S,

[135] Zhang C,

[136] Hung K,

[137] Mele A,

[138] Fraser C,

[139] Stone E,

[140] Chen C,

[141] Fak J,

[142] Chi S,

[143] Licatalosi D,

[144] Richter J,

[145] Darnell R

[146] ↵

Das A,
Chiang A,
Davla S,
Priya R,
Reichert H,
VijayRaghavan K,
Rodrigues V
(2010) Identification and analysis of a glutamatergic local interneuron lineage in the adult Drosophila olfactory system. Neural Syst Circuits 1:4.
OpenUrl

[148] Das A,

[149] Chiang A,

[150] Davla S,

[151] Priya R,

[152] Reichert H,

[153] VijayRaghavan K,

[154] Rodrigues V

[155] ↵

Das S,
Sadanandappa M,
Dervan A,
Larkin A,
Lee J,
Sudhakaran I,
Priya R,
Heidari R,
Holohan E,
Pimentel A,
Gandhi A,
Ito K,
Sanyal S,
Wang J,
Rodrigues V,
Ramaswami M
(2011) Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci USA 108:E646–E654. doi:10.1073/pnas.1106411108
OpenUrl Abstract/FREE Full Text

[157] Das S,

[158] Sadanandappa M,

[159] Dervan A,

[160] Larkin A,

[161] Lee J,

[162] Sudhakaran I,

[163] Priya R,

[164] Heidari R,

[165] Holohan E,

[166] Pimentel A,

[167] Gandhi A,

[168] Ito K,

[169] Sanyal S,

[170] Wang J,

[171] Rodrigues V,

[172] Ramaswami M

[173] ↵

Devaud J,
Acebes A,
Ramaswami M,
Ferrús A
(2003) Structural and functional changes in the olfactory pathway of adult Drosophila take place at a critical age. J Neurobiol 56:13–23. doi:10.1002/neu.10215 pmid:12767029
OpenUrl CrossRef PubMed

[175] Devaud J,

[176] Acebes A,

[177] Ramaswami M,

[178] Ferrús A

[179] ↵

Devaud J,
Clouet-Redt C,
Bockaert J,
Grau Y,
Parmentier M
(2008) Widespread brain distribution of the Drosophila metabotropic glutamate receptor. Neuroreport 19:367–371.
OpenUrl CrossRef PubMed

[181] Devaud J,

[182] Clouet-Redt C,

[183] Bockaert J,

[184] Grau Y,

[185] Parmentier M

[186] ↵

Doll C,
Broadie K
(2015) Activity-dependent FMRP requirements in development of the neural circuitry of learning and memory. Development 142:1346–1356. doi:10.1242/dev.117127 pmid:25804740
OpenUrl Abstract/FREE Full Text

[188] Doll C,

[189] Broadie K

[190] ↵

Doll C,
Broadie K
(2016) Neuron class-specific requirements for fragile X mental retardation protein in critical period development of calcium signaling in learning and memory circuitry. Neurobiol Dis 89:76–87. doi:10.1016/j.nbd.2016.02.006 pmid:26851502
OpenUrl CrossRef PubMed

[192] Doll C,

[193] Broadie K

[194] ↵

Doll C,
Vita D,
Broadie K
(2017) Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement. Curr Biol 27:2318–2330. doi:10.1016/j.cub.2017.06.046 pmid:28756946
OpenUrl CrossRef PubMed

[196] Doll C,

[197] Vita D,

[198] Broadie K

[199] ↵

Dölen G,
Osterweil E,
Rao B,
Smith G,
Auerbach B,
Chattarji S,
Bear M
(2007) Correction of fragile X syndrome in mice. Neuron 56:955–962. doi:10.1016/j.neuron.2007.12.001 pmid:18093519
OpenUrl CrossRef PubMed

[201] Dölen G,

[202] Osterweil E,

[203] Rao B,

[204] Smith G,

[205] Auerbach B,

[206] Chattarji S,

[207] Bear M

[208] ↵

Domanski A,
Booker S,
Wyllie D,
Isaac J,
Kind P
(2019) Cellular and synaptic phenotypes lead to disrupted information processing in Fmr1-KO mouse layer 4 barrel cortex. Nat Commun 10:4814. doi:10.1038/s41467-019-12736-y
OpenUrl CrossRef PubMed

[210] Domanski A,

[211] Booker S,

[212] Wyllie D,

[213] Isaac J,

[214] Kind P

[215] Fishilevich E,
Vosshall L
(2005) Genetic and functional subdivision of the Drosophila antennal lobe. Curr Biol 15:1548–1553. doi:10.1016/j.cub.2005.07.066 pmid:16139209
OpenUrl CrossRef PubMed

[217] Fishilevich E,

[218] Vosshall L

[219] ↵

Flockhart I,
Booker M,
Kiger A,
Boutros M,
Armknecht S,
Ramadan N,
Richardson K,
Xu A,
Perrimon N,
Mathey-Prevot B
(2006) FlyRNAi: the Drosophila RNAi screening center database. Nucleic Acids Res 34:D489–D494. doi:10.1093/nar/gkj114
OpenUrl CrossRef PubMed

[221] Flockhart I,

[222] Booker M,

[223] Kiger A,

[224] Boutros M,

[225] Armknecht S,

[226] Ramadan N,

[227] Richardson K,

[228] Xu A,

[229] Perrimon N,

[230] Mathey-Prevot B

[231] ↵

Franco L,
Okray Z,
Linneweber G,
Hassan B,
Yaksi E
(2017) Reduced lateral inhibition impairs olfactory computations and behaviors in a Drosophila model of fragile X syndrome. Curr Biol 27:1111–1123. doi:10.1016/j.cub.2017.02.065 pmid:28366741
OpenUrl CrossRef PubMed

[233] Franco L,

[234] Okray Z,

[235] Linneweber G,

[236] Hassan B,

[237] Yaksi E

[238] ↵

Friedman S,
Dani N,
Rushton E,
Broadie K
(2013) Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila. Dis Model Mech 6:1400–1413. doi:10.1242/dmm.012229 pmid:24046358
OpenUrl Abstract/FREE Full Text

[240] Friedman S,

[241] Dani N,

[242] Rushton E,

[243] Broadie K

[244] ↵

Geramita M,
Urban N
(2016) Postnatal odor exposure increases the strength of interglomerular lateral inhibition onto olfactory bulb tufted cells. J Neurosci 36:12321–12327. doi:10.1523/JNEUROSCI.1991-16.2016 pmid:27927952
OpenUrl Abstract/FREE Full Text

[246] Geramita M,

[247] Urban N

[248] ↵

Gerlach G,
Tietje K,
Biechl D,
Namekawa I,
Schalm G,
Sulmann A
(2019) Behavioural and neuronal basis of olfactory imprinting and kin recognition in larval fish. J Exp Biol 222:jeb189746. doi:10.1242/jeb.189746
OpenUrl Abstract/FREE Full Text

[250] Gerlach G,

[251] Tietje K,

[252] Biechl D,

[253] Namekawa I,

[254] Schalm G,

[255] Sulmann A

[256] ↵

Goel A,
Cantu D,
Guilfoyle J,
Chaudhari G,
Newadkar A,
Todisco B,
Alba D,
Kourdougli N,
Schmitt L,
Pedapati E,
Erickson C,
Portera-Cailliau C
(2018) Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible. Nat Neurosci 21:1404–1411. doi:10.1038/s41593-018-0231-0 pmid:30250263
OpenUrl CrossRef PubMed

[258] Goel A,

[259] Cantu D,

[260] Guilfoyle J,

[261] Chaudhari G,

[262] Newadkar A,

[263] Todisco B,

[264] Alba D,

[265] Kourdougli N,

[266] Schmitt L,

[267] Pedapati E,

[268] Erickson C,

[269] Portera-Cailliau C

[270] ↵

Golovin R,
Vest J,
Vita D,
Broadie K
(2019) Activity-dependent remodeling of Drosophila olfactory sensory neuron brain innervation during an early-life critical period. J Neurosci 39:2995–3012. doi:10.1523/JNEUROSCI.2223-18.2019
OpenUrl Abstract/FREE Full Text

[272] Golovin R,

[273] Vest J,

[274] Vita D,

[275] Broadie K

[276] ↵

Gonçalves J,
Anstey J,
Golshani P,
Portera-Cailliau C
(2013) Circuit level defects in the developing neocortex of fragile X mice. Nat Neurosci 16:903–909. doi:10.1038/nn.3415 pmid:23727819
OpenUrl CrossRef PubMed

[278] Gonçalves J,

[279] Anstey J,

[280] Golshani P,

[281] Portera-Cailliau C

[282] ↵

Greenblatt E,
Spradling A
(2018) Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins. Science 361:709–712. doi:10.1126/science.aas9963 pmid:30115809
OpenUrl Abstract/FREE Full Text

[284] Greenblatt E,

[285] Spradling A

[286] ↵

Greenhill S,
Juczewski K,
Haan A,
Seaton G,
Fox K,
Hardingham N
(2015) Adult cortical plasticity depends on an early postnatal critical period. Science 349:424–427. doi:10.1126/science.aaa8481
OpenUrl Abstract/FREE Full Text

[288] Greenhill S,

[289] Juczewski K,

[290] Haan A,

[291] Seaton G,

[292] Fox K,

[293] Hardingham N

[294] ↵

Harlow E,
Till S,
Russell T,
Wijetunge L,
Kind P,
Contractor A
(2010) Critical period plasticity is disrupted in the barrel cortex of FMR1 knockout mice. Neuron 65:385–398. doi:10.1016/j.neuron.2010.01.024 pmid:20159451
OpenUrl CrossRef PubMed

[296] Harlow E,

[297] Till S,

[298] Russell T,

[299] Wijetunge L,

[300] Kind P,

[301] Contractor A

[302] Hayashi S,
Ito K,
Sado Y,
Taniguchi M,
Akimoto A,
Takeuchi H,
Aigaki T,
Matsuzaki F,
Nakagoshi H,
Tanimura T,
Ueda R,
Uemura T,
Yoshihara M,
Goto S
(2002) GETDB, a database compiling expression patterns and molecular locations of a collection of gal4 enhancer traps. Genesis 34:58–61. doi:10.1002/gene.10137 pmid:12324948
OpenUrl CrossRef PubMed

[304] Hayashi S,

[305] Ito K,

[306] Sado Y,

[307] Taniguchi M,

[308] Akimoto A,

[309] Takeuchi H,

[310] Aigaki T,

[311] Matsuzaki F,

[312] Nakagoshi H,

[313] Tanimura T,

[314] Ueda R,

[315] Uemura T,

[316] Yoshihara M,

[317] Goto S

[318] ↵

He Q,
Nomura T,
Xu J,
Contractor A
(2014) The developmental switch in GABA polarity is delayed in fragile X mice. J Neurosci 34:446–450. doi:10.1523/JNEUROSCI.4447-13.2014 pmid:24403144
OpenUrl Abstract/FREE Full Text

[320] He Q,

[321] Nomura T,

[322] Xu J,

[323] Contractor A

[324] ↵

Hensch T
(2005) Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6:877–888. doi:10.1038/nrn1787 pmid:16261181
OpenUrl CrossRef PubMed

[326] Hensch T

[327] ↵

Hersh J,
Saul R
; Committee on Genetics (2011) Health supervision for children with fragile X syndrome. Pediatrics 127:994–1006. doi:10.1542/peds.2010-3500 pmid:21518720
OpenUrl Abstract/FREE Full Text

[329] Hersh J,

[330] Saul R

[331] ↵

Huang J,
Zhang W,
Qiao W,
Hu A,
Wang Z
(2010) Functional connectivity and selective odor responses of excitatory local interneurons in Drosophila antennal lobe. Neuron 67:1021–1033. doi:10.1016/j.neuron.2010.08.025 pmid:20869598
OpenUrl CrossRef PubMed

[333] Huang J,

[334] Zhang W,

[335] Qiao W,

[336] Hu A,

[337] Wang Z

[338] ↵

Hubel D,
Wiesel T
(1970) The period of susceptibility to the physiological effects of unilateral eye closure in kittens. J Physiol 206:419–436. doi:10.1113/jphysiol.1970.sp009022 pmid:5498493
OpenUrl CrossRef PubMed

[340] Hubel D,

[341] Wiesel T

[342] ↵

Inoue S,
Shimoda M,
Nishinokubi I,
Siomi M,
Okamura M,
Nakamura A,
Kobayashi S,
Ishida N,
Siomi H
(2002) A role for the Drosophila fragile X-related gene in circadian output. Curr Biol 12:1331–1335. doi:10.1016/S0960-9822(02)01036-9
OpenUrl CrossRef PubMed

[344] Inoue S,

[345] Shimoda M,

[346] Nishinokubi I,

[347] Siomi M,

[348] Okamura M,

[349] Nakamura A,

[350] Kobayashi S,

[351] Ishida N,

[352] Siomi H

[353] ↵

Jackson F,
Newby L,
Kulkarni S
(1990) Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxylase. J Neurochem 54:1068–1078. doi:10.1111/j.1471-4159.1990.tb02359.x pmid:1689376
OpenUrl CrossRef PubMed

[355] Jackson F,

[356] Newby L,

[357] Kulkarni S

[358] ↵

Jefferis G,
Marin E,
Watts R,
Luo L
(2002) Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies. Curr Opin Neurobiol 12:80–86. doi:10.1016/S0959-4388(02)00293-3
OpenUrl CrossRef PubMed

[360] Jefferis G,

[361] Marin E,

[362] Watts R,

[363] Luo L

[364] ↵

Khandjian EW,
Fortin A,
Thibodeau A,
Tremblay S,
Côté F,
Devys D,
Mandel JL,
Rousseau F
(1995) A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture. Hum Mol Genet 4:783–789. doi:10.1093/hmg/4.5.783 pmid:7633436
OpenUrl CrossRef PubMed

[366] Khandjian EW,

[367] Fortin A,

[368] Thibodeau A,

[369] Tremblay S,

[370] Côté F,

[371] Devys D,

[372] Mandel JL,

[373] Rousseau F

[374] ↵

Klapoetke N,
Murata Y,
Kim S,
Pulver S,
Birdsey-Benson A,
Cho Y,
Morimoto T,
Chuong A,
Carpenter E,
Tian Z,
Wang J,
Xie Y,
Yan Z,
Zhang Y,
Chow B,
Surek B,
Melkonian M,
Jayaraman V,
Constantine-Paton M,
Wong G, et al
. (2014) Independent optical excitation of distinct neural populations. Nat Methods 11:338–346. doi:10.1038/nmeth.2836 pmid:24509633
OpenUrl CrossRef PubMed

[376] Klapoetke N,

[377] Murata Y,

[378] Kim S,

[379] Pulver S,

[380] Birdsey-Benson A,

[381] Cho Y,

[382] Morimoto T,

[383] Chuong A,

[384] Carpenter E,

[385] Tian Z,

[386] Wang J,

[387] Xie Y,

[388] Yan Z,

[389] Zhang Y,

[390] Chow B,

[391] Surek B,

[392] Melkonian M,

[393] Jayaraman V,

[394] Constantine-Paton M,

[395] Wong G, et al

[396] ↵

Krishnan K,
Wang B,
Lu J,
Wang L,
Maffei A,
Cang J,
Huang Z
(2015) MeCP2 regulates the timing of critical period plasticity that shapes functional connectivity in primary visual cortex. Proc Natl Acad Sci USA 112:E4782–E4791. doi:10.1073/pnas.1506499112
OpenUrl Abstract/FREE Full Text

[398] Krishnan K,

[399] Wang B,

[400] Lu J,

[401] Wang L,

[402] Maffei A,

[403] Cang J,

[404] Huang Z

[405] ↵

Larsson M,
Domingos A,
Jones W,
Chiappe M,
Amrein H,
Vosshall L
(2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43:703–714. doi:10.1016/j.neuron.2004.08.019 pmid:15339651
OpenUrl CrossRef PubMed

[407] Larsson M,

[408] Domingos A,

[409] Jones W,

[410] Chiappe M,

[411] Amrein H,

[412] Vosshall L

[413] Lee T,
Luo L
(1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461. doi:10.1016/S0896-6273(00)80701-1
OpenUrl CrossRef PubMed

[415] Lee T,

[416] Luo L

[417] ↵

Liu A,
Urban N
(2017) Prenatal and early postnatal odorant exposure heightens odor-evoked mitral cell responses in the mouse olfactory bulb. eNeuro 4:ENEURO.0129-17.2017. doi:10.1523/ENEURO.0129-17.2017
OpenUrl Abstract/FREE Full Text

[419] Liu A,

[420] Urban N

[421] ↵

Liu W,
Wilson R
(2013) Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system. Proc Natl Acad Sci USA 110:10294–10299. doi:10.1016/j.cub.2013.05.016 pmid:23770187
OpenUrl Abstract/FREE Full Text

[423] Liu W,

[424] Wilson R

[425] Liu X,
Krause W,
Davis R
(2007) GABAA receptor RDL inhibits Drosophila olfactory associative learning. Neuron 56:1090–1102. doi:10.1016/j.neuron.2007.10.036 pmid:18093529
OpenUrl CrossRef PubMed

[427] Liu X,

[428] Krause W,

[429] Davis R

[430] ↵

Lovelace J,
Rais M,
Palacios A,
Shuai X,
Bishay S,
Popa O,
Pirbhoy P,
Binder D,
Nelson D,
Ethell I,
Razak K
(2020) Deletion of Fmr1 from forebrain excitatory neurons triggers abnormal cellular, EEG, and behavioral phenotypes in the auditory cortex of a mouse model of fragile X syndrome. Cereb Cortex 30:969–988. doi:10.1093/cercor/bhz141 pmid:31364704
OpenUrl CrossRef PubMed

[432] Lovelace J,

[433] Rais M,

[434] Palacios A,

[435] Shuai X,

[436] Bishay S,

[437] Popa O,

[438] Pirbhoy P,

[439] Binder D,

[440] Nelson D,

[441] Ethell I,

[442] Razak K

[443] ↵

Marin E,
Jefferis G,
Komiyama T,
Zhu H,
Luo L
(2002) Representation of the glomerular olfactory map in the Drosophila brain. Cell 109:243–255. doi:10.1016/S0092-8674(02)00700-6
OpenUrl CrossRef PubMed

[445] Marin E,

[446] Jefferis G,

[447] Komiyama T,

[448] Zhu H,

[449] Luo L

[450] ↵

Meredith R
(2015) Sensitive and critical periods during neurotypical and aberrant neurodevelopment: a framework for neurodevelopmental disorders. Neurosci Biobehav Rev 50:180–188. doi:10.1016/j.neubiorev.2014.12.001 pmid:25496903
OpenUrl CrossRef PubMed

[452] Meredith R

[453] ↵

Mohamed A,
Retzke T,
Chakraborty S,
Fabian B,
Hansson B,
Knaden M,
Sachse S
(2019) Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe. Nat Commun 10:1201. doi:10.1038/s41467-019-09069-1 pmid:30867415
OpenUrl CrossRef PubMed

[455] Mohamed A,

[456] Retzke T,

[457] Chakraborty S,

[458] Fabian B,

[459] Hansson B,

[460] Knaden M,

[461] Sachse S

[462] Mohammad F,
Stewart J,
Ott S,
Chlebikova K,
Chua J,
Koh T,
Ho J,
Claridge-Chang A
(2017) Optogenetic inhibition of behavior with anion channelrhodopsins. Nat Methods 14:271–274. doi:10.1038/nmeth.4148 pmid:28114289
OpenUrl CrossRef PubMed

[464] Mohammad F,

[465] Stewart J,

[466] Ott S,

[467] Chlebikova K,

[468] Chua J,

[469] Koh T,

[470] Ho J,

[471] Claridge-Chang A

[472] ↵

Münch D,
Galizia C
(2016) DoOR 2.0–comprehensive mapping of Drosophila melanogaster odorant responses. Sci Rep 6:21841. doi:10.1038/srep21841 pmid:26912260
OpenUrl CrossRef PubMed

[474] Münch D,

[475] Galizia C

[476] ↵

Nagarkar-Jaiswal S,
DeLuca S,
Lee P,
Lin W,
Pan H,
Zuo Z,
Lv J,
Spradling A,
Bellen H
(2015) A genetic toolkit for tagging intronic MiMIC containing genes. Elife 4:e08469. doi:10.7554/eLife.08469
OpenUrl CrossRef PubMed

[478] Nagarkar-Jaiswal S,

[479] DeLuca S,

[480] Lee P,

[481] Lin W,

[482] Pan H,

[483] Zuo Z,

[484] Lv J,

[485] Spradling A,

[486] Bellen H

[487] ↵

Okada R,
Awasaki T,
Ito K
(2009) Gamma-aminobutyric acid (GABA)-mediated neural connections in the Drosophila antennal lobe. J Comp Neurol 514:74–91. doi:10.1002/cne.21971 pmid:19260068
OpenUrl CrossRef PubMed

[489] Okada R,

[490] Awasaki T,

[491] Ito K

[492] ↵

Olsen S,
Wilson R
(2008) Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452:956–960. doi:10.1038/nature06864
OpenUrl CrossRef PubMed

[494] Olsen S,

[495] Wilson R

[496] ↵

Olsen S,
Bhandawat V,
Wilson R
(2010) Divisive normalization in olfactory population codes. Neuron 66:287–299. doi:10.1016/j.neuron.2010.04.009 pmid:20435004
OpenUrl CrossRef PubMed

[498] Olsen S,

[499] Bhandawat V,

[500] Wilson R

[501] ↵

Roberts J,
McCary L,
Shinkareva S,
Bailey D
(2016) Infant development in fragile X syndrome: cross-syndrome comparisons. J Autism Dev Disord 46:2088–2099. doi:10.1007/s10803-016-2737-1 pmid:26864160
OpenUrl CrossRef PubMed

Main menu

User menu

Search

Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation during an Early-Life Critical Period

Abstract

Introduction

Materials and Methods

Drosophila genetics

Odorant exposure

Light exposure

Confocal imaging

Quantification

Statistics

Results

OSN-specific FMRP loss disrupts odor experience critical period remodeling of AL innervation

Sex-specific differences in early odorant experience critical period remodeling of AL innervation

Null dfmr1 mutants and global FMRP RNAi animals both maintain critical period remodeling

Or42a OSN-specific FMRP OE enhances critical period odorant remodeling

Pan-OSN FMRP knock-down does not impact olfactory experience critical period remodeling

Or42a OSN-specific optogenetic activation is sufficient to drive critical period remodeling

Or42a OSN-specific odorant activation is essential for critical period innervation remodeling

Or42a OSN-targeted activation is not affected by Or42a OSN-targeted FMRP knock-down

Or42a OSN synaptic output is not required for Or42a-targeted FMRP RNAi remodeling effects

Critical period olfactory experience selectively remodels presynaptic OSN innervation

Silencing AL glutamatergic interneurons reduces Or42a OSN critical period remodeling

Or42a OSN-targeted GABA_AR knock-down enhances critical period innervation remodeling

Discussion

Footnotes

References

In this issue

Citation Manager Formats

Keywords

Responses to this article

Jump to comment:

Related Articles

Cited By...

More in this TOC Section

Research Articles

Development/Plasticity/Repair

Main menu

User menu

Search

Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation during an Early-Life Critical Period

Abstract

Introduction

Materials and Methods

Drosophila genetics

Odorant exposure

Light exposure

Confocal imaging

Quantification

Statistics

Results

OSN-specific FMRP loss disrupts odor experience critical period remodeling of AL innervation

Sex-specific differences in early odorant experience critical period remodeling of AL innervation

Null dfmr1 mutants and global FMRP RNAi animals both maintain critical period remodeling

Or42a OSN-specific FMRP OE enhances critical period odorant remodeling

Pan-OSN FMRP knock-down does not impact olfactory experience critical period remodeling

Or42a OSN-specific optogenetic activation is sufficient to drive critical period remodeling

Or42a OSN-specific odorant activation is essential for critical period innervation remodeling

Or42a OSN-targeted activation is not affected by Or42a OSN-targeted FMRP knock-down

Or42a OSN synaptic output is not required for Or42a-targeted FMRP RNAi remodeling effects

Critical period olfactory experience selectively remodels presynaptic OSN innervation

Silencing AL glutamatergic interneurons reduces Or42a OSN critical period remodeling

Or42a OSN-targeted GABAAR knock-down enhances critical period innervation remodeling

Discussion

Footnotes

References

In this issue

Citation Manager Formats

Jump to section

Keywords

Responses to this article

Jump to comment:

Related Articles

Cited By...

More in this TOC Section

Research Articles

Development/Plasticity/Repair

Or42a OSN-targeted GABA_AR knock-down enhances critical period innervation remodeling