The hippocampus is composed of anatomically heterogeneous subregions, including CA1, CA3, dentate gyrus and subiculum. A major feature of the hippocampal area CA3 is its recurrent collateral circuitry, by which the CA3 pyramidal cells make excitatory synaptic contacts with each other, while CA1 pyramidal cells are not extensively interconnected. These anatomical differences have inspired David Marr, conceptualizing the hippocampus, in particular area CA3, as an autoassociator network that performs pattern storage and retrieval. McClelland et al. proposed that this autoassociator network could mediate a dynamic competition between pattern completion and pattern separation, two complementary processes of associative memory. Pattern separation and completion are considered to be the two main complementary associative memory processes. Pattern completion is the process by which the network can recall an entire memory using only a degraded input, while pattern separation is the ability of the network to distinguish similar inputs to make dissimilar. The use of pattern completion and separation is important for hippocampus dependent contextual learning to decrease the possibility of error in memory recall. While Nakazawa et al. demonstrated that area CA3 is crucial for spatial pattern completion using CA3 NMDA receptor 1 (NR1) KO mice, the evidence for the involvement of CA3 in pattern separation is scarce. Further, there are few relevant tasks that can test pattern separation. First, Catherine Cravens developed a new contextual pattern separation behavioral paradigm using C57BL/6N (B6) mice. A mouse was placed in a fear conditioning chamber A for 3 min. At the end of exposure in chamber A as the conditional stimulus (CS), the mouse received a single foot shock as the unconditioned stimulus (US) to learn association between context A and aversive foot shock. Three hours or 24 hours later, freezing of mice was observed in chamber A where mice were conditioned, or in chamber B, in which the contextual cues were totally changed. Under this condition, both B6 wild type mice and the floxed-NR1 control mice showed robust freezing to context A, but not to B 3 hr later. However, while CA3-NR1 KO mice showed a comparable level of freezing to A, she found that the mutants also showed freezing to context B to the same level, suggesting that they cannot distinguish the two contexts. Furthermore, the onset of mutants' freezing to context A was significantly slower than that of controls. Since the mutant impairment in the context specificity and rapid recall was not observed when they were tested 24 hr later or after 3 CS-US pairings, it was concluded that the mutants' impairment is due to the one-trial acquisition deficit, suggesting that CA3-NR1 KO mice are impaired in contextual pattern separation (unpublished). Kimberly Christian has also spent time developing behavioral research strategies to further delineate the role of CA3 in the acquisition and retrieval of context specificity in an associative memory. It has been shown that the context specificity acquired during extinction of conditioned fear depends on the hippocampus. Animals show renewed fear to a conditioned cue presented in a context different from that used during extinction. However, while this phenomenon, called contextual renewal, has been reported repeatedly in rats, there are very limited data available for this effect in mice. Pilot data showed significant variability in the mouse behavior, while some data suggested that there may be a phenotype in the mutant mice in which expression of context specificity during renewal is delayed when compared with wild-type mice. This paradigm may not be the most appropriate means of examining hippocampal-dependent contextual learning in the B6 strain of mice. The second research focus of this CA3 project is to investigate the behavioral and physiological consequence of CA3 NR ablation in nucleus accumbens (NAcc). It is well known that hippocampus sends a strong efferent signal to NAcc, presumably providing the context information for the process of reinforcement learning in the NAcc. However, since NAcc also receives inputs from the cortex, amygdala, and the ventral tegmental area (VTA), how the hippocampal input modulates the NAcc activity and its physiological importance has not been clarified. Juan Belforte, under direction of Kazu Nakazawa, set up the recording environment with in vivo multi-electrodes from awake behaving mice last year. Beginning this year, he redesigned a microdrive that was developed at MIT to be used in a more sophisticated manner at NIMH. Our new microdrive is hand-made and allows the independent advancement of 7 electrodes once implanted into the skull of the mouse. He has successfully implanted several drives, and recording activity in the cortex and nucleus accumbens neurons (NAcc) is in progress. Using this approach we plan to characterize the neuronal response of NAcc induced by contextual changes in CA3-NR1 KO mice. We have also needed to establish relevant behavioral tasks, which could measure the function of NAcc and can be combined with in vivo physiology. While Melissa Tanner tried to establish the behavioral task called """"""""novel object to place"""""""" task, which has been reported to depend on the hippocampus, our protocol needs to be shaped for the use of B6 mice. This project has been undertaken by new fellow, Angela Miracle. In parallel, Juan Belforte is trying to establish the behavioral paradigm, called the prepulse inhibition (PPI) of the startle reflex for simultaneous in vivo recording. This task evaluates sensorimotor gating function, which is impaired by the disruption of many brain areas, such as prefrontal cortex, hippocampus and NAcc, and this deficit is also observed in schizophrenic patients. While in humans it is measured by electromyography, the PPI is measured in rodents as a whole body startle, which has not been compatible with any simultaneous neuronal recordings. Juan Belforte has worked to establish a new recording system that allows us to record the PPI in mice by electromyography (EMG). He is now able to evaluate the result of EMG recording with the data which will be obtained from conventional settings using startle chambers, and further plans to record neural activity from NAcc simultaneously with the EMG PPI in the CA3-NR1 KO mice. Finally, Melissa Tanner has engaged in developing CA3 pyramidal cell-restricted inducible transgenic mice. Last year, we planned to suppress CA3 firing, including sharp waves, by creating genetically engineered mice, in which membrane hyperpolarzation could be induced only in CA3 cells. We used macrolide-inducible system, in which a trans-activator ET4 induces ETR-element dependent transgene transcription in the presence of erythromycin. To test this system in vivo, we needed to generate two mouse strains, one is a CA3-restricted ET4 transgenic line and another is a transgenic line in which Kir2.1 fused with GFP (green fluorescent protein) is under the control of ETR-minimal promoter. Kir2.1 is an inwardly rectifying potassium channel, overexpression of which has shown to hyperpolarize the neurons, leading to the electrical shut down. We established 5 independent ET4 transgenic lines and over 10 ETR-Kir2.1-GFP lines. We then crossed two ET4 lines, which showed discrete expression pattern, with Kir2.1-GFP lines, and tested inducibility of GFP-immunoreactivity following intra-peritoneal injection of erythromycin daily for a week. However, as far as 2 ET4 lines were tested with 3 Kir2.1-GFP lines each, robust induction of GFP expression was hardly observed in any combination of two lines, suggesting macrolide-inducible system does not work well in the brain cells. We plan to use tetracycline-dependent inducible system as an alternative method.
| Kishimoto, Yasushi; Nakazawa, Kazu; Tonegawa, Susumu et al. (2006) Hippocampal CA3 NMDA receptors are crucial for adaptive timing of trace eyeblink conditioned response. J Neurosci 26:1562-70 |
| Nakazawa, Kazu; McHugh, Thomas J; Wilson, Matthew A et al. (2004) NMDA receptors, place cells and hippocampal spatial memory. Nat Rev Neurosci 5:361-72 |
