The hypo-function of the GABAergic system and the glutamate toxicity found in autism have been implicated as possibly having a causal role for autism 
. Memantine is an uncompetitive antagonist at NMDA receptors 
and is widely used for the treatment of AD. Memantine has been demonstrated to have neuroprotective effects and promote neurogenesis 
. Most recently, several clinical trials have reported that memantine can significantly improve language function and social behavior of patients with autism and fragile X syndrome (FXS) 
. Neuroanatomical studies on autistic individuals have revealed loss of purkinje and granule cells of the cerebellum 
and cerebellar dysfunction in animal models can induce autism-like behavioral deficits 
. Granule cells of the cerebellum constitute the largest homogeneous neuronal population of mammalian brain. Due to their postnatal generation and the feasibility of well characterized primary in vitro cultures, cerebellar granule cells (CGCs) are a model of choice for the study of neural development, function and pathology 
. In this study, using cultured CGCs from Fmr1
-KO, we first examined how memantine affects the cell properties. We found that memantine significantly promoted the adhesion of CGCs, while having little effect on the cell migration.
Integrin-mediated cell adhesion has been shown to be essential in the regulation of neuronal migration, which is a critical process that determines the final location of neurons, and thus establishes the basis for neural circuits 
. In addition, the complexity and specificity of synaptogenesis relies upon regulation of cell adhesion molecules that mediate contact initiation, synapse formation and plasticity. Disruption of adhesion could result in an imbalance in the structure and function of synapses 
. Recently, several lines of studies have shown that mutations in the synaptic adhesion molecules Neurexin 1 and Neuroligins 3 and 4 are associated with autism 
. Mutations in scaffolding molecule Shank3 that interacts with Neuroligins have also been detected in some autistic subjects 
. These observations imply that a defect in synaptic cell adhesion may alter synapse formation, maturation and plasticity in autistic subjects. Since memantine promotes CGC adhesion, we reason that it could play a role in the regulation of neural synaptogenesis. Thus, memantine might counteract some of the defects in synaptic cell adhesion molecules found in autism. In our study, we found that the adhesion of Fmr1
-KO CGCs was increased as compared with controls. Whether there is a defect in adhesion-associated molecules in Fmr1
-KO mice or FXS patients is unknown and our results suggest that it is of important significance to further investigate synaptic cell adhesion and migration properties in FXS in the future.
Our study further demonstrated that memantine stimulated synapse formation. In particular, memantine restored the number of excitatory synapses found in Fmr1
-KO CGCs to the level of that in the WT CGCs. Synapses critically mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries 
. Recently, Yun and Trommer 
reported that that the peak amplitude of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) was smaller in Fmr1
-KO mice as compared with controls, while AMPA receptor-mediated EPSCs were comparable in the two groups. In addition, they found diminished medial perforant path-granule cell long-term potentiation (LTP), complementing previous findings that demonstrated impaired LTP in CA1, neocortex, and amygdala and exaggerated long-term depression in CA1 of Fmr1
-KO mice 
. We suggest that the stimulatory effect of memantine on excitatory synapse formation could counter the smaller EPSCs found in Fmr1
-KO CGCs and exert a therapeutic role. However whether memantine can actually promote excitatory synaptic transmission remains to be further studied.
In addition, we found that memantine also stimulated the formation of inhibitory synapses. Thus memantine treatment did not change the ratio of excitatory to inhibitory synapses. This leaves open the question of whether memantine affects the neural circuit balance. Future studies will be needed to answer this question. The stimulatory effect of memantine on both excitatory and inhibitory synapse formation suggests that memantine may have a general role in promoting neurogenesis. Recently, several studies have shown that memantine promotes cell proliferation and production of mature granule neurons in the adult hippocampus 
. Memantine has also been demonstrated to stimulate the proliferation of hippocampal progenitor cells 
. All these findings imply that memantine can play a role in neurogenesis.
To further elucidate the role of memantine on neural development and function, we examined dendritic spines. We showed that memantine treatment promotes the development of mature (mushroom-shaped) dendritic spines and can restore the dendritic spine to normal levels in Fmr1
-KO CGCs. Several categories of spines have been identified based on their shape and size, including thin, stubby, cup, and mushroom shaped 
. Spine morphology is linked to synapse function and the mushroom-shaped spines considered to represent the most mature and stable spine morphology 
. Recent studies suggest that excitatory synapses mainly connect to mushroom-shaped dendritic spines 
. Thus, the increase in mushroom-shaped spines in response to memantine treatment is consistent with our findings that memantine stimulates the formation of excitatory synapses. The effect of memantine on spines in Fmr1
-KO CGCs also suggests a role of memantine in restoring synaptic transmission.
A number of studies have reported defects of dendritic spines in Fmr1
-KO mice 
. It has been reported that the length of dendritic spines, a measure of immaturity, is increased in Fmr1
-KO mice, suggesting a delay in the development of dendritic spines. In our study, we found that the length of dendritic spines was significantly impaired in Fmr1
-KO CGCs, which is supportive of the findings from Nimchinsky et al 
. The effect of memantine in enhancing dendritic spine maturation could contribute to its observed therapeutic effects in autistic and FXS patients. We reckon there could be two ways memantine exert its restoring role in FMRP deficient neurons. It could specifically counteract the loss of FMRP and therefore only acting on those dendritic spines that are immature as a result of FMRP loss. Alternatively memantine could have a more general effect on the development and maturation of dendritic spines and not directly target those neurons altered by the loss of FMRP. Studies will be carried out on wild type mice to further elucidate the mechanism through which memantine affects the neuronal properties, synapse formation and dendritic spine maturation.
In summary, we have shown that Fmr1-KO CGCs exhibit delayed development of dendritic spines and reduced excitatory synapse formation. Memantine treatment of Fmr1-KO CGCs promoted cell adhesion. Memantine also stimulated the development of mushroom-shaped dendritic spines and restored the dendritic spine to normal levels in Fmr1-KO CGCs. Furthermore, we demonstrated that memantine treatment can enhance synapse formation, particularly excitatory synapses and it restored the excitatory synapses to a normal range in Fmr1-KO CGCs. These findings suggest that the stimulatory effect of memantine on dendritic spines development and excitatory synapse formation, as well as the adhesion promoting effect could be mechanisms by which memantine exerts its therapeutic effect in autism and FXS.