The modifications occurring in the brain during learning and memory are still poorly understood but may involve long-lasting changes in synaptic transmission (synaptic plasticity). In perirhinal cortex, a lasting decrement in neuronal responsiveness is associated with visual familiarity discrimination, leading to the hypothesis that long-term depression (LTD)-like synaptic plasticity may underlie recognition memory. LTD relies on internalization of AMPA receptors (AMPARs) through interaction between their GluR2 subunits and AP2, the clathrin adaptor protein required for endocytosis. We demonstrate that a peptide that blocks interactions between GluR2 and AP2 blocks LTD in perirhinal cortex in vitro. Viral transduction of this peptide in perirhinal cortex produced striking deficits in visual recognition memory. Furthermore, there was a deficit of LTD in perirhinal cortex slices from virally transduced, recognition memory-deficient animals. These results suggest that internalization of AMPA receptors, a process critical for the expression of LTD in perirhinal cortex, underlies visual recognition memory.

We established the importance of phosphorylation of cAMP responsive element-binding protein (CREB) to both the familiarity discrimination component of long-term recognition memory and plasticity within the perirhinal cortex of the temporal lobe. Adenoviral transduction of perirhinal cortex (and adjacent visual association cortex) with a dominant-negative inhibitor of CREB impaired the preferential exploration of novel over familiar objects at a long (24 h) but not a short (15 min) delay, disrupted the normal reduced activation of perirhinal neurons to familiar compared with novel pictures, and impaired long-term potentiation of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for involvement of CREB phosphorylation in synaptic plastic processes within perirhinal cortex necessary for long-term recognition memory.

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Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic celldeath. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.