Abnormal development of dendritic spines in FMR1 knock-out mice - PubMed (original) (raw)

Abnormal development of dendritic spines in FMR1 knock-out mice

E A Nimchinsky et al. J Neurosci. 2001.

Abstract

Fragile X syndrome is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that the disorder might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, we analyzed these properties in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increased 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice had longer spines than controls. At 1 week, spine length was 28% greater in mutants than in controls. At 2 weeks, this difference was 10%, and at 4 weeks only 3%. Similarly, spine density was 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences were not detectable. The spine abnormality was not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits.

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Figures

Fig. 1.

Fig. 1.

Two-photon laser scanning micrographs showing morphology of and spine development in EGFP-expressing layer V pyramidal neurons in the somatosensory cortex of wild-type (left) and mutant (right) mice transfected in vivo before fixation. Note the presence of long protrusions at 1 week, particularly in the mutant mice (arrows). Scale bars: A, B, 50 μm;C–H, 8 μm.

Fig. 2.

Fig. 2.

Developmental spine length changes in_FMR1_ knock-out mice and littermate controls. A, D, G, 1 week; B, E, H, 2 weeks; C, F, I, 4 weeks. A–C, Mean spine length in mutant and control mice. D–F, Cumulative frequency distribution of spine lengths. G–I, Mean spine length at different branching levels in the dendritic tree. In_D–F_, black areas indicate wild type and_white areas_ indicate mutant. In_G–I_, black bars indicate wild type and_gray bars_ indicate mutant. ap1–3, Primary through tertiary apical dendrites; bas1–3, primary through tertiary basal dendrites. Error bars indicate SEM. *p < 0.05; **p < 0.001; ***p < 0.0001.

Fig. 3.

Fig. 3.

Developmental spine density changes in_FMR1_ knock-out mice and littermate controls. A, D, 1 week; B, E, 2 weeks; C, F, 4 weeks. A–C, Mean spine density in mutant and control mice. D–F, Mean spine density at different branching levels in the dendritic tree. Means are shown only for primary and secondary dendrites, because the number of tertiary dendrites sampled was too inconsistent to provide reliable estimates. Black bars, Wild type; gray bars, mutant.ap1–2, Primary through tertiary apical dendrites; bas1–2, primary through tertiary basal dendrites. Error bars indicate SEM. *p < 0.05; **p < 0.001; ***p < 0.0001.

Fig. 4.

Fig. 4.

A, C, Summary of changes in spine length (A) and density (C) in wild-type mice during the first postnatal month. n = 5 animals in each group. Error bars indicate SEM. B, D, Summary of changes in spine length (B) and density (D) in mutant mice during the first postnatal month. The value for each parameter is normalized to the wild-type value. Error bars are computed as the sum of the SEM for wild-type and mutant animals.n = 5 animals in each group.

Fig. 5.

Fig. 5.

Two-photon laser scanning micrographs of biolistically transfected layer V neurons in neocortical organotypic cultures at 1 week from the date of birth. A, C, Wild type. B, D, Mutant. A, B, Low-magnification projections showing pyramidal morphology. C, D, High-magnification projections of dendritic segments. Note the increased presence of long protrusions in this preparation. Scale bar: A, B, 50 μm; C, D, 4 μm.E, Spine motility in neocortical cultures imaged using 2PLSM at 1 week from the date of birth. Micrographs taken at 2 min intervals show typical changes in spine morphology. One protrusion (arrow) changes shape several times during this interval, branching, extending, and retracting. Another (arrowhead) undergoes changes in the morphology of its head. Others change relatively little during this interval. This example was taken from a wild-type animal. Numbers indicate time in minutes. Scale bar, 2 μm.

Fig. 6.

Fig. 6.

Spine length and density in neocortical cultures analyzed 1 week from the date of birth. A, Mean spine length; n = 1754 and 1667 spines for wt and mutant animals, respectively. B, Spine length at different levels in the dendritic tree. C, Mean spine density; n = 247 and 255 dendritic segments for wt and mutant, respectively. D, Spine density at different levels in the dendritic tree. E–G, Spine motility in neocortical cultures analyzed 1 week from the date of birth.E, Mean motility per 2 min time interval.F, Mean length range over which spines vary over a 22 min time interval. G, Proportion-persistent spines for wild-type and mutant animals. Black, Wild type;gray, mutant. ap1–3, Primary through tertiary apical dendrites; bas1–3, primary through tertiary basal dendrites. Error bars indicate SEM. *p < 0.05; **p < 0.001; ***p < 0.0001.

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