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.

Fragile-X syndrome is a common form of mental retardation resulting from the inability to produce the fragile-X mental retardation protein. The specific function of this protein is unknown; however, it has been proposed to play a role in developmental synaptic plasticity. Examination of human brain autopsy material has shown that fragile-X patients exhibit abnormalities in dendritic spine structure and number, suggesting a failure of normal developmental dendritic spine maturation and pruning in this syndrome. Similar results using a knockout mouse model have previously been described; however, it was noted in retrospect that the mice used in that study may have carried a mutation for retinal degeneration, which may have affected cell morphology in the visual cortex of those animals. In this study, dendritic spines on layer V pyramidal cells of visual cortices, taken from fragile-X knockout and wild-type control mice without the retinal degeneration mutation and stained using the Golgi-Cox method, were investigated for comparison with the human condition. Quantitative analyses of the lengths, morphologies, and numbers of dendritic spines, as well as amount of dendritic arbor and dendritic branching complexity, were conducted. The fragile-X mice exhibited significantly more long dendritic spines and significantly fewer short dendritic spines than control mice. Similarly, fragile-X mice exhibited significantly more dendritic spines with an immature-like morphology and significantly fewer with a more mature type morphology. However, unlike the human condition, fragile-X mice did not exhibit statistically significant dendritic spine density differences from controls. Fragile-X mice also did not demonstrate any significant differences from controls in dendritic tree complexity or dendritic arbor. Long dendritic spines with immature morphologies are characteristic of early development or a lack of sensory experience. These results are similar to those found in the human condition and further support a role for the fragile-X mental retardation protein specifically in normal dendritic spine developmental processes. They also support the validity of these mice as a model of fragile-X syndrome.

Few studies have been conducted comparing the FMR1 mutation in multiple tissues of individuals affected with fragile X syndrome. We report a postmortem study of the FMR1 mutation in multiple tissues from a high-functioning male with fragile X syndrome. This man was not mentally retarded and had only a few manifestations of the disorder such as learning disabilities and mild attention problems. Southern blot analysis of leukocytes demonstrated an unmethylated mutation with a wide span of sizes extending from the premutation to full mutation range. A similar pattern was seen in most regions of the brain. In contrast, a methylated full mutation of a single size was seen in the parietal lobe and in most non-brain tissues studied. Therefore, there were striking differences in both FMR1 mutation size and methylation status between tissues. Lack of mental retardation in this individual may have been due to sufficient expression of FMR1 protein (FMRP) in most areas of the brain. Immunocytochemistry showed FMRP expression in regions of the brain with the unmethylated mutation (superior temporal cortex, frontal cortex, and hippocampus) and no expression in the region with the methylated full mutation (parietal). Neuroanatomical studies showed no dendritic spine pathology in any regions of the brain analyzed.