Neural Circuits: Reduced Inhibition in Fragile X Syndrome

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Neural Circuits: Reduced Inhibition in Fragile X Syndrome Randall M. Golovin1 and Kendal Broadie1,2,* 1Vanderbilt

Brain Institute, Vanderbilt University, Nashville, TN 37232, USA of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2017.03.011 2Department

The Drosophila Fragile X Syndrome model has long generated insights into this devastating neurological disease state. A recent study of olfactory neural circuitry shows that decreased lateral inhibition onto projection neurons relaying sensory input into higher brain centers causes impaired behavior. Fragile X Syndrome (FXS) is the leading heritable cause of intellectual disability and autism spectrum disorders. Loss of Fragile X Mental Retardation Protein (FMRP) causes FXS, which typically results from CGG repeat expansion, hyper-methylation and gene silencing. The canonical role of FRMP is as an RNA-binding protein that inhibits translation, although the mechanism through which it targets specific RNA transcripts remains controversial, and the identity of those targets remains indeterminate [1,2]. For the past 15 years, impaired metabotropic glutamate receptor (mGluR) function in excitatory synaptic circuitry has been the posterchild FMRP mechanism. Both mouse and Drosophila FXS models show elevated mGluR1/5 signaling, with correction providing therapeutic benefits [3,4]. Far less prominently, however, it has long been known that FXS models also display reduced function in inhibitory GABAergic circuits [5]. Current models suggest that impaired excitatory/ inhibitory synaptic balance, particularly during early-use critical periods, underlie

FXS symptoms [6]. Interest in inhibitory dysfunction drove Franco et al. to explore the role of GABAergic signaling in the beautifully mapped Drosophila olfactory circuit, as reported in this issue of Current Biology [7]. GABAergic impairments have been reported in mouse, fish and fly FXS models [5,8,9]. However, it has been challenging to link GABAergic signaling defects to impaired behavioral outputs. Franco and colleagues tackle this problem by investigating circuit level changes in the Drosophila Antennal Lobe (AL), the first olfactory relay station, analogous to the mammalian olfactory bulb [10]. Peripheral olfactory receptor neurons transduce odorant information by synapsing on projection neurons (PNs) and local interneurons (LNs) within AL glomeruli. Different odorants produce characteristic activity in specific glomeruli, and LNs are thought to sharpen the PN response by increasing contrast through interglomerular inhibition, a process termed lateral inhibition [11]. This circuitry helps distinguish odorants, allowing for the

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appropriate behavioral response (e.g. attractive vs. aversive). The exact connectivity between LNs, PNs and olfactory receptor neurons is not known, but it is thought to depend on the complex connections of inhibitory GABAergic LNs that mediate lateral inhibition, and excitatory cholinergic LNs and gap junctions that mediate lateral excitation [10]. Franco and colleagues employ this genetically tractable system with a combination of calcium imaging, optogenetics, electrophysiology and quantitative behavioral studies to show that defective LN GABAergic signaling underlies impairments in olfactory behavior in the Drosophila FXS disease model. Drosophila FMRP null mutants (dfmr1) in a four-chamber behavioral arena show reduced attraction and aversion, with FMRP genomic rescue restoring the normal responses (Figure 1A). The impaired olfactory behavior was partially reproduced by targeted FMRP loss in projection neurons or local interneurons, suggesting FMRP acts in tandem in both circuit elements to regulate

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function. Following the footsteps of two other recent publications [12,13], calcium imaging with a geneticallyencoded GCaMP reporter was done in conjunction with a battery of 24 odorants, as a means to explore in vivo circuit activity in the FXS disease state. As is typical for FXS neurons, PNs were hyperexcitable with more excitatory and fewer inhibitory responses. Null dfmr1 mutants display a greater number of weakly excited glomeruli, but many fewer greatly excited glomeruli (Figure 1B), suggesting odorant responses are broader and more difficult to distinguish. Interestingly, a very recent study found that inhibitory olfactory maps show a broadening over the course of development, which could be the process altered in FXS [14]. To test this hypothesis, the SuperClomeleon chloride sensor could monitor population PN inhibitory responses to odorants in vivo, complementing the work of Franco and colleagues. The calcium imaging done here provides a circuit level explanation for olfactory behavioral abnormalities. In the FXS disease state, odors produce more similar responses, making it more difficult to select the appropriate behavioral response (Figure 1A,B). To more specifically address how FMRP loss might impair odorant differentiation, Franco and colleagues next mixed together two odorants, holding one constant and varying the concentration of the second. Null dfmr1 animals showed impaired lateral (i.e. interglomerular) inhibition with similar responses even with increasing odorant concentrations. Interestingly, the lateral inhibition defects did not carry over to lateral excitation, which persisted normally and therefore represented the major concentration-dependent change. Using electrophysiological recording to assay the major inhibitory inputs onto projection neurons, Franco and colleagues show that PNs fire at a basal rate, even without odorant presentation. The blue light-gated cation channel channelrhodopsin was then used to optically excite the LNs, while also simultaneously recording spontaneous activity in PNs using whole-cell patch-clamp. Consistent with the above GCaMP calcium

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Figure 1. Reduced inhibition broadens circuit and behavior responses in an FXS model. (A) The four-chamber behavioral arena from [7]. Attractive odorants are loaded in one chamber (air in remaining three) and repellant odorants in three chambers (air in other chamber). Air movement flow via a vacuum port produces a movement response that appears identical between attraction and aversion. Right: a theoretical heat plot of position probability for wild-type and FXS model flies. The mutant animals show both impaired olfactory attraction and aversion. (B) Schematic of wild-type and FXS model antennal lobe with a theoretical calcium response to odorant presentation. Left: wild-type response is narrow, with sharp activation near the strongly activated glomeruli and rapidly decaying signal farther away. Right: mutant response is broader, with weaker activation of the most strongly activated glomeruli and more signal area of spread. (C) Schematic of the antennal lobe circuitry showing how decreased inhibition might arise. Left: wild-type with strongly activated glomeruli (orange) also activate GABAergic local interneurons inhibiting surrounding glomeruli (blue) to sharpen odor representation. Right: FXS model circuit displays less strongly activated glomeruli (yellow), proposed due to decreased lateral excitation (not demonstrated). Mutant circuits have reduced projection neuron (green) inhibition response, such that even strongly activated GABAergic local interneurons provide little effective inhibition. This change acts to broaden the odor response by limiting lateral inhibition between glomeruli. LN, lateral interneurons; PN, projection neurons.

imaging results, FXS model LNs were less able to reduce the response of PNs (Figure 1C), as indicated by a

smaller change in membrane potential and a smaller reduction in firing rate. Together, the calcium imaging

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Dispatches and electrophysiology support the conclusion that reduced lateral inhibition decreases the contrast between odors leading to broader responses and impaired behavior in the FXS disease state. Franco and colleagues next asked whether reduced FXS inhibition results from less active LNs or less responsive PNs (Figure 1C). Direct recordings from optogenetically activated LNs showed a greater response in dfmr1 null mutants. Since LNs are coupled to PNs and other LNs, this could mean that LN–LN connectivity might be strengthened at the expense of LN–PN connections. The study took advantage of incomplete channelrhodopsin expression in LNs to test LN–LN connectivity by recording from a channelrhodopsin-negative LN while stimulating the channelrhodopsinpositive LNs. Similar to the LN–PN connection, the LN–LN connection was also reduced in the FXS model, suggesting reduced inhibition might be a general impairment throughout the circuit. This left only one option: a diminished response to LN inhibition by PNs. PN GABAA receptors were targeted with rdl-RNAi to mimic the reduced inhibition characterizing the FXS model. The rdl-RNAi animals performed more poorly than controls in behavioral tests, consistent with reduced PN sensitivity to inhibition causing broader circuit connectivity (Figure 1C). Thus, at least at the circuit level, reduced lateral inhibition can explain the impaired olfactory behavior. Overall, Franco et al. provide strong evidence that reduced neural circuit inhibition impairs behavior in the FXS disease state. This study paves the way for many interesting avenues of research. First, more work is needed to reveal the molecular causes of impaired inhibition in FXS. In the Drosophila model, Franco and colleagues demonstrate that a GABAA receptor reduction can lead to behavioral impairments. However, other research from both mouse and Drosophila FXS models indicate the mechanism is likely more complicated and possibly indirect [8,15]. A second intriguing idea is that impaired inhibition stems from altered activity-dependent plasticity during developmentally critical

periods [16]. Both mouse and Drosophila FXS models show impaired critical period plasticity [3,17,18], and early activity is critical for shaping E/I synaptic balance [19,20]. A third fascinating area to expand would be to ask how impaired neural circuit computations at the first olfactory center (AL) alter computations in higher olfactory centers such as the central brain mushroom body and lateral horn. Since projection neurons link the AL with higher brain centers, a reduction in the inhibitory response of projection neurons may affect presynaptic inhibition in higher order brain regions. The altered behavioral output in dfmr1 null animals is likely due to impaired neural circuits in many brain areas. Overall, Franco et al. provide much needed in vivo evidence for inhibitory circuit impairments in FXS, and set the foundation for future work linking molecular to circuit level to behavioral changes. Addressing altered GABAergic circuit function should lead to more effective treatments for FXS patients. REFERENCES 1. Darnell, J.C., Van Driesche, S.J., Zhang, C., Hung, K.Y., Mele, A., Fraser, C.E., Stone, E.F., Chen, C., Fak, J.J., Chi, S.W., et al. (2011). FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261. 2. Anderson, B.R., Chopra, P., Suhl, J.A., Warren, S.T., and Bassell, G.J. (2016). Identification of consensus binding sites clarifies FMRP binding determinants. Nucleic Acids Res. 44, 6649–6659. 3. Do¨len, G., Osterweil, E., Rao, B.S.S., Smith, G.B., Auerbach, B.D., Chattarji, S., and Bear, M.F. (2007). Correction of fragile X syndrome in mice. Neuron 56, 955–962. 4. Pan, L., Woodruff, E., Liang, P., and Broadie, K. (2008). Mechanistic relationships between Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A signaling. Mol. Cell. Neurosci. 37, 747–760. 5. El Idrissi, A., Ding, X.-H.H., Scalia, J., Trenkner, E., Brown, W.T., and Dobkin, C. (2005). Decreased GABA(A) receptor expression in the seizure-prone fragile X mouse. Neurosci. Lett. 377, 141–146. 6. Contractor, A., Klyachko, V.A., and PorteraCailliau, C. (2015). Altered neuronal and circuit excitability in Fragile X Syndrome. Neuron 87, 699–715. 7. Franco, L.M., Okray, Z., Linneweber, G.A., Hassan, B.A., and Yaksi, E. (2017). Reduced lateral inhibition impairs olfactory computations and behaviors in a Drosophila

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