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Abstract

One of the major limitations in the use of genetically modified mice for studying cognitive functions is the lack of regional and temporal control of gene function. To overcome these limitations, a forebrain-specific promoter was combined with the tetracycline transactivator system to achieve both regional and temporal control of transgene expression. Expression of an activated calcium-independent form of calcium-calmodulin-dependent kinase II (CaMKII) resulted in a loss of hippocampal long-term potentiation in response to 10-hertz stimulation and a deficit in spatial memory, a form of explicit memory. Suppression of transgene expression reversed both the physiological and the memory deficit. When the transgene was expressed at high levels in the lateral amygdala and the striatum but not other forebrain structures, there was a deficit in fear conditioning, an implicit memory task, that also was reversible. Thus, the CaMKII signaling pathway is critical for both explicit and implicit memory storage, in a manner that is independent of its potential role in development.
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The CaMKIIα promoter consisted of 8.5 kb of genomic DNA upstream of the transcription initiation site of the mouse CaMKIIα gene, as well as 84 base pairs of the 5′ noncoding exon. Genomic DNA was isolated from a C57 B16/J mouse spleen cosmid library with a rat genomic probe consisting of a 0.4-kb Ava I fragment comprising the transcription-initiation region of rat CaMKIIα (40). The tTA gene from plasmid pUHD 15-1 (10) was flanked by an artificial intron and splice sites at the 5′ end (41) and by a polyadenylation signal from SV40 at the 3′ end. The cDNA with intron and polyadenylation signal was placed downstream of the 8.5-kb CaMKII promoter fragment. The cDNAs for Escherichia coli lacZ and mouse CaMKIIα were similarly flanked by the hybrid intron and polyadenylation signal and placed downstream of the tet-O promoter element of plasmid pUHD 10-3 (10). The CaMKIIα gene was a full-length cDNA (4.8 kb) isolated from a C57B16/J mouse brain cDNA library. The lacZ gene carried an SV40 large T antigen nuclear localization signal as well as the 3′ untranslated region (UTR) of CaMKIIα, which targets the mRNA to dendrites (42).
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RT-PCR was performed essentially as described (7). Total forebrain RNA (100 ng) was used in each reaction with oligonucleotide primers to amplify a region of the transcript that includes the Thr286→Asp mutation. Equal amounts of amplified cDNA (both wild-type and mutant sequences) were separated on a 3% agarose gel, transferred to nylon membranes, and hybridized with a 32P-labeled oligonucleotide probe specific for the Asp286 mutation (oligonucleotide sequence: 5′-CTTCAGGCAGTCGACGTCCTCCTGTCTGTG-3′). Blots were washed under conditions in which only the Asp286 mutant cDNA was detected (2′ 15 min, 60°C, 0.2′ standard saline citrate). A Northern (RNA) blot of total forebrain mRNA revealed expression of a shorter-than-expected CaMKII-Asp286 transcript (∼3.4 kb). As shown in Fig. 3, this shorter CaMKII-Asp286 transcript did not localize to dendrites, presumably as a result of the loss of a sequence element in the 3′ UTR that is necessary for mRNA targeting to dendrites (42).
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It would also be useful to suppress transgene expression during development and then activate the gene only in the adult animal. However, we found that treatment of wild-type mice with doxycycline (1 mg/ml) during development impaired adult spatial memory and memory for fear conditioning. This result suggests that doxycycline itself produces a defect in neuronal development. We therefore used transgene suppression only in the adult animal in which the doxycycline treatment did not affect memory. Given the activation of the transgene throughout development, it is possible that the LTP and memory phenotypes observed with the transgene active in the adult animal result from a synergistic interaction between developmental and adult expression rather than a direct acute effect of transgene expression in the adult animal.
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On the Barnes circular maze (8), the mice (2.5 to 6 months of age) were tested once a day until they met the criterion (five out of six sessions with three or fewer errors) or until 40 days had elapsed. The order of holes searched was recorded by an observer who was blind to genotype and doxycycline condition, and from these data the number of errors was determined. Errors were defined as searches of any hole that did not have the tunnel beneath it. Searches included nose pokes and head deflections over the hole. At the end of each session the search strategy used was recorded by the observer. The spatial search strategy was operationally defined as reaching the escape tunnel with both error and distance scores ≤3. Distance was calculated by counting the number of holes between the first hole searched within a session and the escape tunnel. A one-factor analysis of variance (ANOVA) (gender) revealed no significant effect of gender for either transgenic or wild-type mice, so the data were collapsed across this variable. For the error data, a three-factor ANOVA (genotype, doxycycline, and session block) with one repeated measure was used. For the spatial search strategy data, the two groups of B22 transgenic mice were compared with a two-way ANOVA (doxycycline and session block) with one repeated measure.
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We thank R. Axel and T. Jessell for critically reading this manuscript; J. Finkelstein for maintaining and genotyping the mice; R. Shih, V. Winder, and L. Varshavsky for help with behavioral experiments; C. Lam for help with figures; H. Ayers and I. Trumpet for typing the manuscript; and M. Osman for animal care. This research was supported by the Howard Hughes Medical Institute and the National Institute of Mental Health.

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Science
Volume 274Issue 52936 December 1996
Pages: 1678 - 1683

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Received: 8 August 1996
Accepted: 23 October 1996
Published online: 6 December 1996

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Mark Mayford
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.
Mary Elizabeth Bach
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.
Yan-You Huang
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.
Lei Wang
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.
Robert D. Hawkins
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.
Eric R. Kandel*
The authors are at the Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, and Howard Hughes Medical Institute, 722 West 168 Street, New York, NY 10032, USA.

Notes

* To whom correspondence should be addressed.

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Science
Volume 274|Issue 5293
6 December 1996
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Received:8 August 1996
Accepted:23 October 1996
Published in print:6 December 1996
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