Although long-term memory is thought to require a cellular program of gene expression and increased protein synthesis, the identity of proteins critical for associative memory is largely unknown. We used RNA fingerprinting to identify candidate memory-related genes (MRGs), which were up-regulated in the hippocampus of water maze-trained rats, a brain area that is critically involved in spatial learning. Two of the original 10 candidate genes implicated by RNA fingerprinting, the rat homolog of the ryanodine receptor type-2 and glutamate dehydrogenase (EC 1.4.1.3), were further investigated by Northern blot analysis, reverse transcription-PCR, and in situ hybridization and confirmed as MRGs with distinct temporal and regional expression. Successive RNA screening as illustrated here may help to reveal a spectrum of MRGs as they appear in distinct domains of memory storage.Identifying the mechanisms responsible for memory formation and consolidation has long been a goal of behavioral neuroscience. Many experiments over the past few decades have demonstrated that inhibitors of transcription or translation interfere with long-term memory formation, indicating the requirement of de novo gene expression (1-4). Despite the importance of this finding, little is known about the identity and specificity of the required proteins. Changes in early inducible genes, for example, are known to occur not only during learning and memory, but also during a broad range of behaviors, including motor activity and sensory discrimination (5-10). Changes in the expression of late effector genes, such as those encoding BiP and calreticulin, have been described during long-term sensitization in Aplysia but not in associative memory (11,12). To our knowledge, no changes in late effector genes have been previously demonstrated during associative memory.To identify memory-related genes (MRGs) we have used a new and sensitive approach, RNA fingerprinting by arbitrarily primed PCR (13,14), to compare gene expression in control swimming rats with water maze-trained rats. The Morris water maze is a learning paradigm in which a rodent learns to locate a submerged island in a large pool by creating a spatial map using extra-pool cues (15)(16)(17). This learning ability represents a complex faculty involving input from different senses including visual, olfactory, auditory, and somatosensory information (18)(19)(20). The hippocampus has been shown to be a brain locus for spatial memory (21). Pyramidal cells in the rat hippocampus discharge selectively at specific locations of a spatial environment (22, 23) and maintain their receptive field when the relevant cues are removed (24) or when the light is turned off (25). Lesions of the hippocampus result in impaired acquisition of tasks that depend on spatial strategies (26-28) and spatial memory impairment parallels the magnitude of dorsal hippocampal lesions (29). MATERIALS AND METHODSWater Maze Learning. Male Wistar rats, 60-90 days old (200-300 g) were housed individually in plastic cages wi...

A previously uncharacterized 22-kDa Ca 2؉ -binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EFhands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca 2؉ , calexcitin bound GTP and possessed GTPase activity. Calexcitin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca 2؉ and the absence of endogenous Ca 2؉ ͞cal-modulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 ؎ 6 pS K ؉ channels. Calexcitin thus appears to directly regulate K ؉ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca 2؉ -activated signaling molecule that increases cellular excitability, which would in turn increase Ca 2؉ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca 2؉ .In neuronal cells, ion channel conductance is regulated by ligand binding, direct interaction with G proteins, or phosphorylation (1). K ϩ and Ca 2ϩ channels, for example, can be phosphorylated by Ca 2ϩ ͞calmodulin-dependent kinase (2, 3) and͞or protein kinase C (PKC) (4-8); however, other elements of Ca 2ϩ signaling cascades might also regulate ion channels directly. Such a protein was suggested by a previous study in which a low molecular weight protein, designated cp20, reduced two voltage-dependent K ϩ currents, i A and i Ca-Kϩ , in identified molluscan neurons (9-11). These same currents were reduced in the same neurons in molluscs (Hermissenda crassicornis) exposed to a Pavlovian conditioning paradigm (9, 12). To investigate this protein, we purified calexcitin (CE) from squid optic lobe and used microsequencing and PCR techniques to obtain a DNA probe which was used to screen a cDNA library. The cloned protein had separate regions that are homologous to Ca 2ϩ -binding proteins and to GTPbinding proteins. Functionally, this protein binds both Ca 2ϩ and GTP, is phosphorylated by PKC, and regulates K ϩ channels in human fibroblasts and membrane excitability in mammalian and molluscan neurons. CE, therefore, offers a molecular link between PKC, intraneuronal Ca 2ϩ , and persistent changes of membrane excitability that correlate with associative memory storage. METHODSPurification of CE. Squid optic lobes were homogenized in a high speed homogenizer with 1 ml of buffer (10 mm Tris⅐HCl, pH 7.4͞20 g/ml leupeptin͞50 g/ml pepstatin͞50 mM NaF͞1 mM EDTA͞1 mM EGTA) containing 1 M DTT and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication with a 20-W probe sonicator. The homogenate was centrifuged at ...

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