Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines

Abstract

Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of functional fragile X mental retardation protein (FMRP). FXS is associated with elevated density and length of dendritic spines, as well as an immature-appearing distribution profile of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a morphology profile that, while essentially opposite of that described in the Fmr1 knockout neocortex, appears similarly immature. This finding strongly suggests that FMRP is required for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.

Introduction

A common inherited form of mental retardation, Fragile X syndrome (FXS) is characterized by the absence of functional FMRP (Hagerman and Hagerman, 2002). Postmortem tissue from the neocortex of FXS patients reveals pathology of dendritic spines (Hinton et al., 1991). Specifically, spines are longer and more dense along dendrites of cortical neurons in FXS patients than controls (Irwin et al., 2001). These neurons from FXS patients also exhibit a higher proportion of thin spines and a lower proportion of stubby or mushroom-shaped spines than controls. Similar cortical neuropathology has been observed in a murine model for FXS that also lacks FMRP, the Fmr1 knockout mouse (Bakker et al., 1994, Irwin et al., 2002, McKinney et al., 2005).

Across several species, maturation of the neocortex has been associated with dendritic spine overproduction followed by pruning of a subset of these spines and with a shift in distribution from a greater proportion of longer thinner spines to more shorter stubbier ones (Galofre and Ferrer, 1987, Horner, 1993, Murphy and Magness, 1984, Schuz, 1986). Recent findings indicate that, whereas dendritic spines in the somatosensory cortex of wildtype mice appear to follow this normal developmental course, adult Fmr1 knockout mice exhibit elevated spine density and a profile of spine lengths and morphologies similar to younger subjects (Galvez and Greenough, 2005). In light of these data and others, we have hypothesized that FMRP is required for the activity-dependent processes of spine shape maturation and pruning and that without FMRP dendritic spines that would normally either mature or be pruned tend to persist into adulthood (Bagni and Greenough, 2005, Churchill et al., 2002).

FMRP is highly expressed in the hippocampus, a brain region important for learning and memory (Eichenbaum and Cohen, 2001, Hinds et al., 1993). Patients with FXS exhibit bilaterally enlarged hippocampal volumes, and physiological abnormalities have been reported in hippocampal subfield CA1 of Fmr1 knockout mice, suggesting that the absence of FMRP may lead to structural and functional deficits in the hippocampus (Huber et al., 2002, Reiss et al., 1994). Several groups have examined hippocampal connectivity in Fmr1 knockout mice or have studied cultured hippocampal neurons from these animals (Braun and Segal, 2000, Ivanco and Greenough, 2002, Mineur et al., 2002), but there have been no reports of dendritic spine morphology in the Fmr1 knockout mouse hippocampus in vivo.

During normal development, dendritic spines on hippocampal pyramidal cells appear to follow a progression distinct from that observed in the neocortex. Harris et al. (1992), for example, have found by examining three-dimensional reconstructions of serial electron micrographs that the distribution profile of spines along apical shafts of pyramidal cells in hippocampal subfield CA1 shifts from an abundance of stubby or mushroom-shaped spines in young animals to predominantly thin spines by adulthood. If FMRP is involved in spine maturation, rather than associated with the development of a particular spine shape per se, one would expect to see more stubby or mushroom-shaped spines along these hippocampal neurons in adult Fmr1 knockout than control mice—essentially the opposite of the morphology profile in the neocortex of these animals and yet similarly suggestive of an immature synaptic state in the hippocampus.

To test this hypothesis, Golgi-impregnated dendritic spines were examined along apical shafts of hippocampal area CA1 pyramidal cells of adult Fmr1 knockout and control mice (wildtype with respect to the Fmr1 locus). Although there was no difference in the density of spines between the genotypes, there were more stubby, mushroom-shaped spines and fewer thin spines in knockout mice than in controls. Furthermore, Fmr1 knockout mice had more longer and fewer shorter dendritic spines than controls overall and for each spine morphology type analyzed. This phenotype suggests a context-dependent deficit in the brain-region-specific maturation of dendritic spines in Fmr1 knockout mice.