Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

Transcription is a critical component for consolidation of long-term memory. However, relatively few transcriptional mechanisms have been identified for the regulation of gene expression in memory formation. In the current study, we investigated the activity of one specific member of the NF-kappaB transcription factor family, c-Rel, during memory consolidation. We found that contextual fear conditioning elicited a time-dependent increase in nuclear c-Rel levels in area CA1 and DG of hippocampus. These results suggest that c-rel is active in regulating transcription during memory consolidation. To identify the functional role of c-Rel in memory formation, we characterized c-rel(-/-) mice in several behavioral tasks. c-rel(-/-) mice displayed significant deficits in freezing behavior 24 h after training for contextual fear conditioning but showed normal freezing behavior in cued fear conditioning and in short-term contextual fear conditioning. In a novel object recognition test, wild-type littermate mice exhibited a significant preference for a novel object, but c-rel(-/-) mice did not. These results indicate that c-rel(-/-) mice have impaired hippocampus-dependent memory formation. To investigate the role of c-Rel in long-term synaptic plasticity, baseline synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses in c-rel(-/-) mice was assessed. c-rel(-/-) slices had normal baseline synaptic transmission but exhibited significantly less LTP than did wild-type littermate slices. Together, our results demonstrate that c-Rel is necessary for long-term synaptic potentiation in vitro and hippocampus-dependent memory formation in vivo.

An emerging theme in the field of neuroscience is that processes critical for neurodevelopment have been co-opted by the adult nervous system to subserve synaptic plasticity and cognition. In this review, we highlight a surprising intersection of two developmental processes that together play a critical role in synaptic plasticity, memory formation and cognition. Reelin, a large glycoprotein associated with the extracellular matrix, is crucial for cortical and cerebellar development. Recent data from several groups indicate that reelin plays a unique modulatory role in the induction of synaptic plasticity in the hippocampus, and that normal levels of reelin in the adult brain are essential for successful formation of certain forms of long-term memory. Given that both increases and decreases in reelin expression have significant effects on plasticity and memory, regulation of reelin expression is predicted to have significant effects on neural function. Epigenetic regulation of transcription is critical for differentiation of cellular phenotype in metazoans. Dozens of reports in the last few years have demonstrated that epigenetics is involved in modulating gene expression in the adult nervous system and subserves plasticity and memory formation. We review a series of studies that demonstrate that the reelin promoter is subject to differential DNA methylation in the adult nervous system, and that perturbations in reelin promoter methylation correlate with alterations in memory formation and cognition. Thus, two distinct developmental processes, reelin-mediated signaling and epigenetic-based transcriptional regulation, appear to have synergized in the adult nervous system to create a sensitive and robust system for modulation of synaptic plasticity, and ultimately provide a powerful set of tools to probe the molecular basis of cognition.

Integrins comprise a large family of heterodimeric, transmembrane cell adhesion receptors that mediate diverse neuronal functions in the developing and adult CNS. Recent pharmacological and genetic studies have suggested that beta1-integrins are critical in synaptic plasticity and memory formation. To further define the role of integrins in these processes, we generated a postnatal forebrain and excitatory neuron-specific knockout of alpha3-integrin, one of several binding partners for beta1 subunit. At hippocampal Schaffer collateral-CA1 synapses, deletion of alpha3-integrin resulted in impaired long-term potentiation (LTP). Basal synaptic transmission and paired-pulse facilitation were normal in the absence of alpha3-integrin. Behavioral studies demonstrated that the mutant mice were selectively defective in a hippocampus-dependent, nonmatch-to-place working memory task, but were normal in other hippocampusdependent spatial tasks. The impairment in LTP and working memory is similar to that observed in beta1-integrin conditional knockout mice, suggesting that alpha3-integrin is the functional binding partner for beta1 for these processes in the forebrain.

Does neuronal loss associated with dementia necessarily impair the ability to learn new information and recall old memories? In a recent report in Nature, Fischer et al.(2007) show that the ability to learn and remember can be reestablished in a mouse model of dementia through either environmental enrichment or chronic treatment with an inhibitor of histone deacetylase.

Abstract Regulation of glutamate transporters often accompanies glutamatergic synaptic plasticity. We investigated the mechanisms responsible for the increase in glutamate uptake associated with increased glutamate release at the Aplysia sensorimotor synapse during long-term sensitization (LTS) and long-term facilitation. An increase in the V(max) of transport, produced by LTS training, suggested that the increased glutamate uptake was due to an increase in the number of transporters in the membrane. We cloned a high-affinity, Na(+)-dependent glutamate transporter, ApGT1, from Aplysia central nervous system that is highly enriched in pleural sensory neurons, and in pleural-pedal synaptosome and cell/glial fractions. ApGT1, expressed in Xenopus oocytes, demonstrated a similar pharmacological profile to glutamate uptake in Aplysia synaptosome and cell/glial fractions (strong inhibition by threo-beta-benzyloxyaspartate and weak inhibition by dihydrokainate) suggesting that ApGT1 may be the primary glutamate transporter in pleural-pedal ganglia. Levels of ApGT1 and glutamate uptake were increased in synaptosomes 24 h after induction of LTS by electrical stimulation or serotonin. Regulation of ApGT1 during LTS appears to occur post-transcriptionally and results in an increased number of transporters in synaptic membranes. These results suggest that an increase in levels of ApGT1 is responsible, at least in part, for the long-term increase in glutamate uptake associated with long-term memory.

Originally discovered as epigenetic regulators of developmental gene expression, the Polycomb (PcG) and trithorax (trxG) group of proteins form distinct nuclear complexes governing post-translational modification of histone tails. The present study identified a novel, developmentally regulated interface between Eed and Mll, pivotal constituents of PcG and trxG pathways, respectively, in mouse brain. While the PcG proteins Eed and EzH2 engaged in a common complex during neurodevelopment, Eed associated with the trxG protein Mll upon brain maturation. Comprehensive analysis of multiple histone modifications revealed differential substrate specificity of the novel Eed/Mll complex in adult brain compared with the developmental Eed/EzH2 complex. Newborn brain from eed heterozygotes and eed;Mll double heterozygotes exhibited decreased trimethylation at lysine 27 of histone H3, as well as hyperacetylation of histone H4. In contrast, adult hippocampus from Mll heterozygotes was remarkable for decreased acetylation of histone H4, which restored to wild-type levels in eed;Mll double heterozygotes. A physiological role for the Eed/Mll complex in adult brain was evident from complementary defects in synaptic plasticity in eed and Mll mutant hippocampi. These results support the notion that developmental regulation of complex composition bestows the predominant Eed complex with the chromatin-remodeling activity conducive for gene regulation during neurodevelopment and adult brain function. Thus, this study suggests dynamic regulation of chromatin complex composition as a molecular mechanism to co-opt constituents of developmental pathways into the regulation of neuronal memory formation in adult brain.

The formation of enduring internal representation of sensory information demands, in many cases, convergence in time and space of two different stimuli. The first conveys the sensory input, mediated via fast neurotransmission. The second conveys the meaning of the input, hypothesized to be mediated via slow neurotransmission. We tested the biochemical conditions and feasibility for fast (NMDA) and slow (dopamine) neurotransmission to converge on the Mitogen Activated Protein Kinase signaling pathways, crucial in several forms of synaptic plasticity, and recorded its effects upon synaptic transmission. We detected differing kinetics of ERK2 activation and synaptic strength changes in the CA1 for low and high doses of neurotransmitters in hippocampal slices. Moreover, when weak fast and slow inputs are given together, they converge on ERK2, but not on p38 or JNK, and induce strong short-term synaptic depression. Surprisingly, pharmacological analysis revealed that a probable site of such convergence is the NMDA receptor itself, suggesting it serves as a detector and integrator of fast and slow neurotransmission in the mature mammalian brain, as revealed by ERK2 activation and synaptic function.

In this issue of Molecular Pharmacology, Kundakovic et al. present compelling evidence suggesting that the promoters for reelin and GAD67 are coordinately regulated. The regulation occurs at the level of DNA (cytosine-5) methylation. Moreover, the authors present evidence that suggests pharmacologic inhibition of DNA methyltransferase results in reversal of methylation, loss of methyl-DNA binding proteins and relief of repression. Repression of both reelin and GAD67 have been implicated in the pathogenesis of Schizophrenia. Therefore, these results suggest that the reelin and GAD67 promoters are subject to continuous repression by DNA methyltransferase, and that inhibitors of DNA methyltransferase represent a potential treatment for Schizophrenia.

This chapter explores some of the molecular events contributing to memory formation and how, when these events malfunction, disturbances in memory occur. After a brief discussion of signaling in the hippocampus, we will explore the topics of human mental retardation syndromes that involve disruption of these processes, including Angelman syndrome (AS), Neurofibromatosis 1 (NF1)-associated learning disorders, Coffin-Lowry syndrome (CLS), Rubinstein-Taybi syndrome (RTS), and Rett syndrome (RTT).