Overview
Our primary research interests are the molecular, cellular, and neuronal circuitry mechanisms underlying acquisition, consolidation, and retrieval of hippocampus-dependent memory in rodents. To study these problems, we produce conditionally engineered (i.e., spatially targeted and/or temporally regulated) mice and analyze these mice by multifaceted methods, including molecular and cellular biology, in vitro and in vivo electrophysiology, and behavioral studies. We attempt to identify deficits at each of the multiple levels of complexity in specific brain areas or cell types and to determine which deficits underlie a specific aspect or type of learning or memory in conditionally engineered mice. Conditionally engineered mice provide not only powerful tools for studying the fundamental mechanisms underlying cognition and behavior but also excellent small animal models for neurological and psychiatric diseases.

Research Summary
Memory acquisition. Using the Cre/loxP system, we previously targeted a knockout of the obligatory N-methyl-D-aspartate (NMDA) receptor (NR) subunit, NR1, to the CA1 pyramidal cells of young adult mice. (Tsien et al., 1996b) These mice displayed impairments in the Schaffer collateral (SC) CA1 long-term potentiation (LTP) and in spatial learning in the standard Morris water maze task. That this mutant mouse is defective in the acquisition rather than the retrieval of the memory was suggested by its inability to form normal memory representations as CA1 place cells (McHugh et al., 1996). These findings remain the most cogent single evidence for the hypothesis that synaptic plasticity underlies memory.

During the past year, we investigated the function of the recurrent network with robust synaptic modifiability present in area CA3 of the hippocampus with respect to the rapid encoding of a novel event. For this purpose, we generated a knockout mouse (CA3-NR1 KO) in which the deletion of the NR1 gene is restricted to the CA3 pyramidal cells of an adult mouse. These mice are impaired in the delayed-matching-to-place (DMP) version of the Morris water maze task in which the platform is placed in a novel location each day. However, the performance of these mutant mice are normal when the platform location employed a few days earlier is reused. This behavioral deficit is highly specific, in that the mutants’ behavioral deficits are restricted to a rapid acquisition of a novel location of the platform, rather than an incremental learning of the platform location through multiple trials (reference memory). We monitored the activities of the pyramidal cells in CA1, the area downstream of CA3 and the site for the hippocampal output, before and after the animals entered a novel space from a familiar space (a collaboration with Matthew Wilson). The specificity of tuning in the mutants was reduced during the first 15 minutes of exploration in the novel space compared to the same period in the familiar space. In contrast, no space shift–associated change of spatial tuning was observed when the mutant mice were returned one day later to the pair of spaces experienced on the previous day. The spatial tuning of CA1 place cells of control animals did not exhibit any space shift–associated changes. These results suggest that CA3 NRs, most probably those in the recurrent network, play a crucial role in rapid hippocampal encoding of a novel encounter and in one-trial- or one-experience-based rapid learning (episodic memory).

Memory Consolidation. A critical feature of both memory consolidation and the formation of long-lasting synaptic plasticity is a requirement for new mRNA and protein synthesis. Previous studies of memory consolidation have largely focused on the regulation of gene expression, establishing an important role for a transcription factor, the cyclic AMP–response element–binding protein (CREB), in this process. For instance, we previously generated transgenic mice in which a dominant-negative form of the Ca²⁺/calmodulin-dependent protein kinase IV (dn CaMKIV) inhibits endogenous Ca²⁺-stimulated CaMKIV activity in the postnatal forebrain. Analysis of these transgenic mice demonstrated that the CaMKIV signaling pathway plays a crucial role in the transcription-dependent late long-term potentiation (L-LTP) in the hippocampus and in the consolidation of long-term memory through its function in a neural activity-induced CREB phosphorylation (Kang et al., 2001).

In a more recent study, we examined a role of another kinase cascade involving ERK (extracellular signal-regulated kinase), which is activated in response to calcium influx and neurotrophin stimulation. Although previous studies relying on the use of pharmacologic inhibitors have implicated ERK activation in LTP and memory, the underlying cellular and molecular mechanisms remain unclear. We generated transgenic mice in which ERK activation is inhibited by a dominant-negative ERK kinase (dnMEK1) transgene only in the postnatal forebrain. The mutant mice exhibited selective deficits in hippocampal memory retention and in the translation-dependent, transcription-independent phase of hippocampal L-LTP. In hippocampal neurons, ERK inhibition blocked neuronal activity-induced translation as well as phosphorylation of the translation factors elF4E, 4EBP1, and ribosomal protein S6. Correspondingly, protein synthesis and translation factor phosphorylation induced in control hippocampal slices by L-LTP-generating tetanization were significantly reduced in mutant slices. Translation factor phosphorylation induced in the control hippocampus by memory formation was similarly diminished in the mutant hippocampus. These results suggest a crucial role for translational control by MAPK signaling in long-lasting forms of synaptic plasticity and memory. It thus appears that upon strong neuronal activation, up-regulation of protein synthesis that occurs directly at the translational level and indirectly through gene activation at the transcriptional level are at work. Differential roles of these two mechanisms as well as whether and how synapse-specificity is accomplished under each of these mechanisms remain important questions.

Remote memory (very long term memory) is thought to be stored in the cortex and, once stored, its maintenance and retrieval are thought to be independent of the integrity of the hippocampus. However, the mechanisms underlying the formation of this type of cortical memory is poorly known although it has been hypothesized that structural alterations of cortical synapses may be crucially involved. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.

Memory Recall. In day-to-day life, recall of associative memory almost always occurs under the constraints of limited cues. For instance, recalling the rich content of interesting conversations with someone can be triggered by the mere subsequent sighting of that person. In the past, a study of the mechanism underlying this fundamental feature of memory recall, referred to as "pattern completion," has been limited to computational modeling. These theoretical studies hypothesized that a recurrent network with modifiable synaptic strength such as that in hippocampal area CA3 could provide this pattern completion capability. We addressed this issue with the CA3-NR1 KO mice. The mutant mice were normal in the acquisition and retrieval of spatial memory tested in the standard hidden platform version of the Morris water maze. However, when the memory of the location of the hidden platform was tested following removal of three of the four major extramaze cues (partial cue conditions), the mutants exhibited a clear deficit of retrieval compared to the control animals.

To investigate the neural mechanisms that might underlie the specific recall deficit, we examined the neurophysiological consequences of the CA3-NR1 deletion by analyzing CA1 place cell activity (a collaboration with Matthew Wilson). We found that spatial information within CA1 is relatively preserved, despite the loss of CA3 NRs, providing a physiological correlate of the intact spatial performance of the CA3-NR1 KO mice in the Morris water maze under full-cue conditions. To investigate the effect of partial-cue removal on CA1 output, we allowed mice to explore a familiar arena for 20 to 30 minutes under full-cue conditions and then removed them to their home cage. Following a 2-hour delay, mice were returned to the arena with either the same four major extramaze cues present (full-cue conditions) or with three of the four cues removed (partial-cue conditions). In the control mice, there were no significant changes in place field properties associated with the change in the cue conditions, while mutant CA1 cells showed significant reduction in spatial tuning properties. These physiological impairments may underlie the inability of mutants to recall the location of the hidden platform when only partial distal cues are available.

This study, along with our previous study with CA1-NR1 KO mice, illustrates the power of cell-type-restricted, adult-onset gene manipulations in the study of molecular, cellular, and neuronal circuitry mechanisms underlying cognition. This degree of spatial targeting—not to mention the cell-type specificity—is difficult to accomplish by pharmacological manipulation.

Mouse Model of Schizophrenia. Calcineurin (CN), a calcium- and calmodulin-dependent protein phosphatase, plays a significant role in the central nervous system. Previously, we reported that forebrain-specific CN knockout mice (CN mutant mice) have impaired working memory (Zeng et al., 2001). To further analyze the behavioral effects of CN deficiency, we subjected CN mutant mice to a comprehensive behavioral test battery. Mutant mice showed increased locomotor activity, decreased social interaction, and impairments in prepulse inhibition and latent inhibition. In addition, CN mutant mice displayed an increased response to the locomotor stimulating effects of MK-801, an NMDA receptor blocker. Collectively, the abnormalities of CN mutant mice are strikingly similar to those described for schizophrenia. These results suggest that alterations affecting CN signaling could comprise a contributing factor in schizophrenia pathogenesis.

Schizophrenia is a severe psychiatric disorder characterized by a complex mode of inheritance. To examine whether calcineurin dysfunction is involved in schizophrenia etiology, we undertook studies of an initial subset of calcineurin-related genes, prioritizing ones that map to loci previously implicated in schizophrenia by linkage studies. Transmission disequilibrium studies in a large sample of affected families detected association of the PPP3CC gene, which encodes the calcineurin _ catalytic subunit, with disease. Our results identify PPP3CC, located at 8p21.3, as a potential schizophrenia susceptibility gene and support the proposal that alterations in calcineurin signaling contribute to schizophrenia pathogenesis.
This work received support from the National Institute of Neurological Disorders and Stroke, the National Institute of Mental Health, the National Institute on Aging, and RIKEN (Institute of Physical and Chemical Research, Japan).

Selected Publications
Tsien, J.Z., Huerta, P.T., and Tonegawa, S. The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory.  Cell 87, 1327-1338 (1996).

Nakazawa, K., Quirk, M.C., Chitwood, R.A., Watanabe, M., Yeckel, M.F., Sun, L.D., Kato, A., Carr, C.A., Johnston, D., Wilson, M.A., and Tonegawa, S. Requirement for hippocampal CA3 NMDA receptors in associative memory recall.  Science 297:211-218 (2002).

Miyakawa, T., Leiter, L.M., Gerber, D.J., Gainetdinov, R.R., Sotnikova, T.D., Zeng, H., Caron, M.G., and Tonegawa, S. Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia.  Proc. Natl. Acad. Sci. USA 100:8987-8992 (2003).

Kelleher, R.J., Govindarajan, A., Jung, H.-Y., Kang, H., and Tonegawa, S. Translational control by MAPK signaling in long-term synaptic plasticity and memory.  Cell 115:467-479 (2004).

Nakazawa, K., McHugh, T.J., Wilson, M.A., and Tonegawa, S. NMDA receptors, place cells and hippocampal spatial memory.  Nature Neuroscience Reviews 5:361-372 (2004).

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