The LAS studies the molecular biophysical, and integrative bases of associative memory in brain networks. LAS observations have related learning and memory behavior of living animals to signal processing in neuronal networks and to subcellular molecular cascades. Our data have implicated molecular and biophysical mechanisms that are conserved in molluscan and mammalian species and thus could have relevance for human learning and memory. Cellular analyses of associative memory in the snail Hermissenda (Pavlovian/classical conditioning), the rabbit (classical conditioning), and the rat (spatial maze learning, olfactory discrimination) revealed a cascade of cellular and subcellular events during memory formation. These events include: long-term synaptic transformation of GABAergic inhibition into excitation (LTT); elevation of intracellular calcium and DAG; translocation of PKC; PKC-mediated phosphorylation of the Ca2+ and GTP-binding protein, cp20 (also called Calexcitin): inactivation of voltage-dependent K+ channels; learning-specific regulation of gene transcription; and rearrangement of synaptic terminal branches. Other signaling proteins such as map kinase and ras have also recently been implicated in longer time domains of memory storage. Other molecular biologic tools such as antisense have helped to identify specific ionic channels on neuronal dendrites that participate in long-term memory. State-of-the-art molecular biologic screening techniques in the LAS have recently implicated new biochemical steps in memory storage. Recent observations have uncovered specific late genes that undergo prolonged activation well into the period of memory consolidation. These genes have been confirmed with Northern blot analyses, reverse transcriptase-PCR, and in situ hybridization techniques. One of these memory-related genes encodes the type II ryanodine receptor. The ryanodine receptor (RR) is a 450 k calcium channel that has 10 membrane-spanning domains. This receptor is responsible for calcium-mediated calcium release (CICR) from the endoplasmic reticulum. Very recently LAS studies identified the first known signaling protein that activates the neuronal RR in a calcium-dependent manner. This protein is Calexcitin (cp20) which was previously shown to be phosphorylated by the alpha-isozyme of PKC during associative learning and memory. Calexcitin has also recently been shown by LAS scientists to potently activate the Ca2+-ATPase on the ER. Other analyses revealed that a single coding nucleotide is responsible for the presence of a P-loop domain on the C-terminal end of calexcitin. The P-loop causes a 4-fold increase of the Ca2+-ATPase activation effect of calexcitin. Other recent studies have linked the calexcitin-ryanodine receptor cascade to long term synaptic modifications such as LTT (long-term transformation) and LTP. Activation of this cascade can induce LTT for hours but prevent the occurrence of LTP. The latter finding is consistent with LAS research that has clearly dissociated rat spatial maze learning from short and long-term LTP. RT-PCR and Western blot analysis with specific antibodies showed that antisense oligodeoxyribonucleotide to Kv1.4 microinjected intraventricularly into rat brains obstructed hippocampal Kv1.4 mRNA, knocking-down the protein in the hippocampus. This antisense knockdown had no effect on rat spatial maze learning, memory or exploratory behavior, but eliminated both early and late phase LTP and reduced paired-pulse facilitation ( a pre-synaptic effect) in CA1 pyramidal neurons without affecting dentate gyrus LTP. This presynaptic Kv1.4 knockdown together with previous post-synaptic Kv1.1 knockdown demonstrates that CA1 LTP is neither necessary nor sufficient for rat spatial memory. Major insights into the synaptic basis of attention-gated learning of the rat spatial maze have been uncovered during the past year. In brief, during attentional focusing a cholinergic-mediated theta rhythm is induced in the rat hippocampus. The theta rhythm is accompanied by long-term transformation (LTT) of GABAergic synapses received by the hippocampal pyramidal cells. Blocking the underlying conductance (bicarbonate) of LTT blocks the theta rhythm and prevents acquisition of the maze learning. Within a CA1 subset or ensemble, memory of the maze is accompanied by long-term shifts of GABAergic synaptic reversal potentials toward that of bicarbonate. These and other findings form the basis of a plausible molecular cascade for repeated and prolonged mobilization of intracellular calcium during consolidation of associative memory. A conceptual synthesis (TINS, 1998) has emerged recently from LAS discovery of the above molecular events demonstrated to occur during learning in the mammalian brain. Time domains of memory correspond to time domains of enhanced calcium signaling. Associated training stimuli translocate PKC, activate Calexcitin, inactivate voltage-dependent K+ channels on the outer membrane and activate the ryanodine receptor and Ca2+-ATPase to amplify intraneuronal, and as LAS studies have implicated, intradendritic calcium waves. These sequential molecular events could participate, therefore, in making memory representations in the brain more permanent for later recall. Implication of these events in memory of diverse species suggests conservation during evolution. Such conservation across species suggests that comparable associative memory mechanisms in humans may provide targets of dysfunction in Alzheimers disease. Recent corroboration of Alzheimers diagnostics developed in the LAS supports the relevance of the above Ca++ signaling cascade for human memory. Based on this cascade, the LAS predicted that function of specific molecules such as the a isozyme of PKC, the voltage-dependent IA and I Ca2+-K+ channels, calexcitin, and intracellular calcium release receptors on the ER will be compromised in Alzheimers disease. These predictions have been borne out so consistently by numerous studies (including those recently conducted at extramural laboratories) that physiologic Alzheimers diagnostic measures have been identified and patented. Thus, this calcium signaling cascade is implicated as important for human memory dysfunction and possibly as a target for AD therapeutics. Finally, theoretical constructs derived from brain-based memory networks have also been mathematically described and incorporated into computer-based artificial networks which have demonstrated significant pattern recognition capabilities. Future directions will include further molecular characterization of events within this cascade such as determining the precise active sites in calexcitin for ryanodine receptor and Ca2+-ATP-ase activation, full characterization of mammalian calexcitin, induction of dendritic, synaptic, and morphologic transformations by the calexcitin-ryanodine receptor cascade, involvement of cascade steps in Alzheimers disease and mental retardation, as well as continued correlations of relevant subcellular events with memory storage.
