Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

Asynchronic transmission in the CA3-CA1 hippocampal synapses in the neurological mutant taiep rat.

For the taiep rat, a neurological mutant with severe astrogliosis secondary to demyelination, we have described alterations in spinal cord synaptic transmission. Asynchronous responses result from phasic action potential-derived glutamate release in this mutant. To evaluate whether this anomalous transmission is also produced in other regions of the taiep CNS and whether its nature involves a presynaptic or postsynaptic disruption, we studied the CA3-CA1 hippocampal synapses. Excitatory postsynaptic currents (EPSC) evoked by stimulation of Schaffer collaterals were recorded from CA1 pyramidal cells on picrotoxin-treated slices. Initial fast and time-locked EPSCs were evoked by conventional stimulation in both control and taiep neurons, showing similar latency and amplitude values unimodally distributed. In a high percentage of taiep neurons (47%), the initial EPSC was frequently followed by additional asynchronous synaptic currents (EPSC(ASYN)) with latencies ranging from 10 to 300 msec. As with initial EPSCs, EPSC(ASYN) were action potential dependent, sensitive to tetrodotoxin, and blocked by D-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The occurrence probability of these events decayed monoexponentially as a function of poststimulus time. The elevation of extracellular Ca(2+) induced a reduction of amplitudes and a rate increase of EPSC(ASYN), in parallel with a reduction of paired pulse facilitation of initial EPSCs. The presynaptic fiber volley, extracellularly recorded, showed no significant differences between groups, with similar mean values of area and decay time. These findings in hippocampal circuitry suggest that, in taiep, the asynchronous evoked activity represents a rather generalized phenotype of the glutamatergic synapses and that EPSC(ASYN) seems to be determined by presynaptic alterations.