Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may ref lect impaired developmental organizational processes of synapse stabilization and elimination or pruning.Fragile X syndrome is the most common inherited form of human mental retardation after Down Syndrome. It is an X-linked genetic trait with an incidence of 1͞2,000 in males (1-3). Phenotypic abnormalities include moderate to severe mental retardation, autistic behavior, macroorchidism, and facial abnormalities (1). The FMR1 gene contains a trinucleotide repeat [(CGG) n ] in the 5Ј untranslated region that is expanded (Ն200 repeats) in fragile X patients (2, 4, 5), resulting in hypermethylation of the FMR1 promoter region and absence of the encoded fragile X mental retardation protein (FMRP). Studies prior to the characterization of fragile X syndrome associated immature dendritic spine morphology (long and thin) with some forms of mental retardation (6-8). Similarly, fragile X cerebral cortical autopsy material exhibits thin, elongated spines and small synaptic contacts (9, 10).Recently, transgenic Fmr1 mice were produced in which the Fmr1 gene was disrupted in embryonic stem cells using a targeting vector to exon 5 and homologous recombination (11). The resultant homozygous knockout mice express no FMRP. Phenotypically, these mice exhibit increased testicular size and mildly impaired performance on the Morris water maze (11), paralleling human symptoms. To explore further parallels to the human syndrome, we examined the effect of the Fmr1 knockout on dendritic spines in the visual cortex using the Golgi-Cox impregnation technique. MATERIALS AND METHODSMale mutant (n ϭ 4) and wild-type FVB strain (n ϭ 4) adult (16-week-old) mice were prepared for Golgi-Cox impregnation. Mice were anesthetized with sodium pentobarbital (85 mg͞kg) and perfused transcardially with 60 ml of 0.9% NaCl (pH 7.3). Brains were removed and were cut sagittally along the midline. Tissue blocks were then immersed in 100 ml of Golgi-Cox solution (1% potassium dichromate͞1% mercuric chloride͞0.8% potassium chromate in distilled water) for 18 days. Tissue blocks were then immersed in 30% sucrose in 0.9% saline for 2 days and subsequently sectioned (200 m) using a vibratome. Sections were mounted o...

Fragile-X syndrome is a common form of mental retardation resulting from the inability to produce the fragile-X mental retardation protein. Qualitative examination of human brain autopsy material has shown that fragile-X patients exhibit abnormal dendritic spine lengths and shapes on parieto-occipital neocortical pyramidal cells. Similar quantitative results have been obtained in fragile-X knockout mice, that have been engineered to lack the fragile-X mental retardation protein. Dendritic spines on layer V pyramidal cells of human temporal and visual cortices stained using the Golgi-Kopsch method were investigated. Quantitative analysis of dendritic spine length, morphology, and number was carried out on patients with fragile-X syndrome and normal age-matched controls. Fragile-X patients exhibited significantly more long dendritic spines and fewer short dendritic spines than did control subjects in both temporal and visual cortical areas. Similarly, fragile-X patients exhibited significantly more dendritic spines with an immature morphology and fewer with a more mature type morphology in both cortical areas. In addition, fragile-X patients had a higher density of dendritic spines than did controls on distal segments of apical and basilar dendrites in both cortical areas. Long dendritic spines with immature morphologies and elevated spine numbers are characteristic of early development or a lack of sensory experience. The fact that these characteristics are found in fragile-X patients throughout multiple cortical areas may suggest a global failure of normal dendritic spine maturation and or pruning during development that persists throughout adulthood.

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.Changes in synaptic connectivity are likely to be a key mechanism by which nervous system organization is permanently changed by experience. Local translation of some proteins in dendrites is increasingly considered to be important for changes in synaptic structure and receptor composition. Certain mRNAs are known to be targeted to dendrites (1, 2), and polyribosomal aggregates are observed in or near dendritic spines, more frequently at newly forming synapses (3-5). Dendrites have been shown to be equipped with components necessary for protein synthesis (6), and synthesis of proteins has been demonstrated directly in synaptoneurosomes (7,8) and in preparations of dendrites isolated from hippocampal neurons in culture (9). Local translation of transfected reporter-tagged mRNA has been demonstrated in transected dendrites (10).Protein translation induced by metabotropic receptor stimulation has previously been proposed to play a role in longterm potentiation (LTP), a model for synaptic plasticity (1,11,12). LTP induction also alters levels of specific mRNAs in tissue slices (13), and isotope-tagged leucine is taken up in dendritic regions of hippocampal slices in response to stimulation (14). We have demonstrated that phosphoinositidelinked metabotropic glutamate receptors (mGluR1), known to trigger a phosphorylation cascade, cause certain mRNAs to associate rapidly with protein translation complexes in synaptoneurosomes (15); this process is modulated by ionotropic receptors (16). Furthermore, we have shown that depolarization by 40 mM K ϩ or stimulation by phosphoinositide receptorspecific mGluR agonists increases [ 35 S]methionine incorporation into trichloroacetic acid-precipitable polypeptides (15, 17), indicating that de novo protein synthesis at the synapse occurs as a result of t...