The loss of Fragile X mental retardation protein (FMRP) causes Fragile X syndrome, the most common inherited mental retardation and single gene cause of autism. Although postsynaptic functions for FMRP are well established, potential roles at the presynaptic apparatus remain largely unexplored. Here, we characterize the expression of FMRP and its homologs, FXR1P and FXR2P, in the developing, mature and regenerating rodent nervous system, with a focus on presynaptic expression. As expected, FMRP is expressed in the somatodendritic domain in virtually all neurons. However, FMRP is also localized in discrete granules (Fragile X granules; FXGs) in a subset of brain regions including frontal cortex, hippocampal area CA3 and olfactory bulb glomeruli. Immunoelectron microscopy shows that FMRP is localized at presynaptic terminals and in axons within these FXG-rich regions. With the exception of the olfactory bulb, FXGs are prominent only in the developing brain. Experiments in regenerating olfactory circuits indicate that peak FXG expression occurs 2-4 weeks after neurogenesis, a period that correlates with synapse formation and refinement. Virtually all FXGs contain FXR2P, while region-selective subsets harbor FMRP and/or FXR1P. Genetic studies show that FXR2P is essential for FXG expression, while FMRP regulates FXG number and developmental profile. These findings suggest that Fragile X proteins play a distinct, presynaptic role during discrete developmental epochs in defined circuits of the mammalian CNS. We propose that the neurological defects in Fragile X syndrome, including the autistic features, could be due in part to the loss of FMRP function in presynaptic compartments.

We have studied the effects of GABAergic innervation on the clustering of GABA A receptors (GABA A Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 Ϯ 0.01 m diameter) GABA A R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 Ϯ 0.08 m breadth) GABA A R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA A R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA A R clusters of any neuron contained all GABA A R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA A R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA A R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA A R clusters at the synapse and disappearance of the small GABA A R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA A R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA A R clusters that remain juxtaposed to glutamate receptors at glutamatergic synapses.

The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.

We have used RNA interference (RNAi) to knock down the expression of the c2 subunit of the GABA A receptors (GABA A Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the c2 subunit, one targeting the coding region and the other one the 3¢-untranslated region (UTR) of the c2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA A receptor c2 subunit and the clustering of other GABA A R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the c2 shRNAs had no effect on the clustering of postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A c2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3¢-UTR targeted by c2 RNAi, rescued both the postsynaptic clustering of GABA A Rs and the GABAergic innervation. Decreased GABA A R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the c2 shRNAs. The results indicate that the postsynaptic clustering of GABA A Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts. Studies of c2 subunit-deficient mouse mutants have shown that the c2 subunit of the GABA A receptor (GABA A R) is necessary for the postsynaptic clustering of the GABA A Rs and for the maintenance of GABA A R clusters at GABAergic synapses (Essrich et al. 1998;Schweizer et al. 2003). The c2-/-mouse mutant shows a severe deficit in GABAergic synaptic transmission and dies soon after birth (Günther et al. 1995). GABA A Rs play a morphogenic role during embryonic development (Rudolph and Mohler 2004;Vicini and Ortinski 2004). Thus, some of the observed phenotypes in these and other mutant mice might result from developmental alterations, while the absence of phenotype might be because of compensatory mechanisms. RNA interference (RNAi, Dykxhoorn et al. 2003;Huppi et al. 2005) is a simpler alternative to the gene knockout technology that can also overcome some of the limitations inherent to the use of mouse mutants. In this study, we have used c2 RNAi to study GABA A R clustering in loss-of-function experiments, both in neuronal cultures and in the intact brain after in utero electroporation. Our results support the notion that the c2 subunit is necessary for the postsynaptic clustering and maintenance of GABA A Rs and gephyrin (a postsynaptic scaffolding protein that is present at inhibitory GABAergic and glycinergic synapses). More interesting, because we revealed it with RNAi technology but it had not been previously observed with the c2 mouse mutants, is the observation that the disruption of the postsynaptic clustering Abbreviations used: AIS, axon initial segmen...

We have studied gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R) clustering within the axon initial segment (AIS) in low-density cultures of hippocampal pyramidal cells following GABAergic and glutamatergic innervation of the AIS. Large, intensely fluorescent, and postsynaptic GABA(A)R clusters were present in the AIS. More than 95% of these clusters colocalized with presynaptic GABAergic or glutamatergic terminals, forming matched or mismatched synapses, respectively. Less than 5% of the GABA(A)R clusters of the AIS did not colocalize with GABAergic or glutamatergic terminals, suggesting that GABA(A)Rs normally do not form clusters unless the AIS received GABAergic or glutamatergic innervation. Few or no clusters of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors or the postsynaptic density-95 protein (PSD-95) were found in the AIS, even when the AIS was innervated by glutamatergic axons. Glutamatergic innervation of the AIS that formed mismatched synapses with postsynaptic GABA(A)R clusters mainly occurred when the AIS did not receive GABAergic innervation. However, when the AIS was innervated by GABAergic axons, the formation of matched GABAergic synapses predominated and coincided with large reductions in both the density of glutamatergic terminals from the AIS and the mismatching of GABA(A)R clusters. A similar effect was observed at axo-dendritic synapses, where GABAergic innervation also led to a large decrease in mismatched GABA(A)R clusters and a smaller, but significant, decrease in glutamatergic terminal density in dendrites that received GABAergic innervation. We hypothesize that competition between GABAergic and glutamatergic innervation of the AIS in the intact hippocampus leads to the exclusive presence of GABAergic inhibitory synapses in the AIS of pyramidal cells.

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We have used triple-label fluorescence immunocytochemistry to demonstrate that alpha5 subunit-containing GABA(A) receptors (GABA(A)Rs) form large clusters at GABAergic synapses in dendrites and axon initial segment of cultured hippocampal neurons. The large synaptic clusters of alpha5 subunit-containing GABA(A)Rs also contained alpha1, beta2/3, gamma2 GABA(A)R subunits and gephyrin. The alpha5 subunit-containing GABA(A)Rs also formed small clusters. The small clusters were not associated with GABAergic synapses and often did not co-localize with gephyrin.