No abstract

In patients suffering from temporal lobe epilepsy (TLE), increased extracellular glutamate levels in the epileptogenic hippocampus both during and after clinical seizures have been reported. These increased glutamate levels could be the result of malfunctioning and/or downregulation of glutamate transporters (also known as EAATs; excitatory amino acid transporters). In this study, the distribution of protein and mRNA of EAAT subtypes was examined in the hippocampus of TLE patients with hippocampal sclerosis (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls without neurological disorders. EAAT protein localization was studied by immunohistochemistry on paraffin sections using specific poly- and monoclonal antibodies against the glial glutamate transporters EAAT1 and EAAT2 and the neuronal glutamate transporter EAAT3. Antibody specificity was shown by immunoblotting. In the HS group, a small decrease in EAAT1-immunoreactivity (IR) was observed in CA4 and in the polymorphic and supragranular layer of the dentate gyrus, compared with the control group. The strongest changes were found for EAAT2 levels. In the non-HS group, increased EAAT2-IR was detected in the CA1 and CA2 field, compared with non-epileptic controls. EAAT2-IR was decreased in the HS compared with the non-HS group. Fewer EAAT3-positive cells were found in the HS group than in the non-HS and control group. In both TLE groups, increased EAAT3 levels were observed in individual neurones. In the HS group, the percentage of EAAT3-IR neurones was increased in CA2 and in the granule cell layer of the dentate gyrus. Radioactive in situ hybridization for EAAT1-3 confirmed our immunohistochemical results. Non-radioactive in situ hybridization showed that not only astrocytes, but also neurones express EAAT2 mRNA. Taken together, differences in both mRNA and protein levels of glutamate transporter subtypes were found in specific regions in the TLE hippocampus, with most severe changes found for EAAT2 and EAAT3 levels. The results indicate an upregulation of EAAT2 protein expression in CA1 and CA2 in neurones in the non-HS group. This is in line with decreased EAAT2 protein levels in the HS group, since these hippocampi are characterized by severe neuronal cell loss. The functional consequences (glutamate transport capacity) of the reported changes in EAAT2 and EAAT3 remain to be determined.

Behavioural experience (e.g. chronic stress, environmental enrichment) can have long-lasting effects on cognitive functions. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as hippocampus, we tested whether behaviour has also long-lasting effects on synaptic plasticity by examining the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices of hippocampal CA1 obtained from rats either 7-9 months after social defeat (behavioural stress) or 3-5 weeks after 5-week exposure to environmental enrichment. Compared with age-matched controls, defeated rats showed markedly reduced LTP. LTP was even completely impaired but LTD was enhanced in defeated and, subsequently, individually housed (during the 7-9-month period after defeat) rats. However, increasing stimulus intensity during 100-Hz stimulation resulted in significant LTP. This suggests that the threshold for LTP induction is still raised and that for LTD lowered several months after a short stressful experience. Both LTD and LTP were enhanced in environmentally enriched rats, 3-5 weeks after enrichment, as compared with age-matched controls. Because enrichment reduced paired-pulse facilitation, an increase in presynaptic release, facilitating both LTD and LTP induction, might contribute to enhanced synaptic changes. Consistently, enrichment reduced the number of 100-Hz stimuli required for inducing LTP. But enrichment may also actually enhance the range of synaptic modification. Repeated LTP and LTD induction produced larger synaptic changes in enriched than in control rats. These data reveal that exposure to very different behavioural experiences can produce long-lasting effects on the susceptibility to synaptic plasticity, involving pre- and postsynaptic processes.

NMDA receptor, Ca 2ϩ /calmodulin-dependent protein kinase II (␣CaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both ␣CaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant ␣CaMKII and PSD-95. was able to compete with the binding of both native and recombinant ␣CaMKII to the NR2A C-tail. Accordingly, ␣CaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native ␣CaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of ␣CaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, ␣CaMKII, and PSD-95 in hippocampal synaptic plasticity. Key words: ␣CaMKII; LTP; NMDA; postsynaptic density; PSD-95; synaptic plasticityA mature synapse is capable of modulating its efficacy by means of activity-dependent plasticity events. Such a process might in turn modulate the structural organization of specific synaptic compartment, i.e., the postsynaptic density (PSD), in which clustering of ligand-gated receptors to scaffolding proteins and to enzymes can be dynamically regulated (Ziff, 1997). PSD consists of a complex network of interacting proteins involved in the regulation of synaptic function and modulation of postsynaptic responses. PSDs are enriched in ionotropic AMPA and NMDA glutamate receptors Kennedy, 1997Kennedy, , 1998. NMDA receptors are of major interest because they are involved in synaptogenesis, neuronal circuitry formation, synaptic plasticity, and learning and memory, as well as in the molecular pathogenesis of neurological disorders (Hollmann and Heinemann, 1994;During et al., 2000). NMDA receptors are oligomeric complexes formed by the coassembly of members of three receptor subunit families: NR1, NR2 subfamily (NR2A-D; Hollmann and Heinemann, 1994), and NR3A (Das et al., 1998). Among NR2 subunits, whose expression is developmentally regulated, NR2A is expressed in the adult rat brain in the large majority of synapses (Monyer et al., 1994). Because of its anatomical localization and expression onset, NR2A is likely to play a major role in synaptic plasticity modulating long-term potentiation (LTP) and long-term depression. In fact, animals with C-terminal truncation of postnatally expressed NR1/NR2A heteromeric receptors, but with an intact NR1/NR2B complex, exhibit impaired hippocampal LTP (Sprengel et al., 1998). In PSD, NR2A and NR2B subunits directly interact w...

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