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. 1998 Jun 1;18(11):4305-13.
doi: 10.1523/JNEUROSCI.18-11-04305.1998.

Expression of integrin-associated protein gene associated with memory formation in rats

A M Huang  1 , H L Wang, Y P Tang, E H Lee
Affiliations

Expression of integrin-associated protein gene associated with memory formation in rats

A M Huang et al. J Neurosci. .

Abstract

The present study has adopted the PCR differential display method to identify cDNA clones associated with memory formation in rats. The one-way inhibitory avoidance learning task was used as the behavioral paradigm. Total RNA isolated from the hippocampus of poor-memory (<80 sec) and good-memory (600 sec) rats 3 hr after training was used for comparison. Three cDNA fragments corresponding to different spliced forms of integrin-associated protein (IAP) mRNA were found to be differentially expressed in the hippocampus of good-memory rats. Quantitative reverse transcription-PCR revealed approximately four fold higher of IAP mRNA level in good-memory rats. This result was confirmed further by in situ hybridization analysis, and the major difference was in the dentate gyrus. It has been demonstrated that this difference in IAP mRNA expression is not attributable to different sensitivities of individual rats to electric shock. Rapid amplification of cDNA ends obtained the full-length IAP cDNA, which is 1192 bp in length excluding the poly(A+) tail. The IAP mRNA expression was significantly upregulated by NMDA and amphetamine injections to the dentate gyrus of the hippocampus. On the other hand, injection of antisense oligonucleotide complementary to the IAP transcript markedly impaired memory retention in rats and decreased the amplitude and slope of EPSP in the in vivo long-term potentiation paradigm. These results together suggest that IAP gene expression plays an important role in memory formation and synaptic plasticity in rat hippocampus.

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Figures

Fig. 1.
Fig. 1.
PCR differential display of rat hippocampal RNA associated with retention performance of inhibitory avoidance learning. Total RNA isolated from the hippocampus of poor-memory (PM) and good-memory (GM) rats was subjected to differential display analysis using 5′ oligonucleotide Ldd8 (5′-AGC CAG CGA A-3′) and 3′ oligonucleotide T12VA as the primer set. Radiolabeled PCR products were analyzed with a 6.0% polyacrylamide gel. Differentially expressed cDNA fragments—A5, A6, and A7 cDNA bands, among three poor- and three good-memory rats—are illustrated.
Fig. 2.
Fig. 2.
Nucleotide and deduced amino acid sequence of rat IAP cDNA. A, Nucleotide sequence of rat IAP cDNA. Nucleotides are numbered relative to the start of the initiation codon. The putative poly(A+) addition site (AATAAA) isunderlined. The location of sites at which the 5′ [nucleotide (nt) 868–877] and 3′ (nt 1172–1185) PCR differential display primers hybridized are also shown. Gene-specific primers used for RACE cloning are shown. The deduced amino acid sequence is shown in a one-letter IUPAC code, starting with the initiator methionine. The splicing sites that generate form 2, form 3, and form 4 rat IAP are indicated by arrows. This sequence has been assigned accession number AF017437 by GenBank. B, Alignment of amino acid sequence among rat, mouse, and human IAP. Sequences are numbered starting with the initiator methionine. The amino acid sequences are given with gaps introduced during the alignment indicated by dots. Amino acids are shadowed where the residues of two or three sequences are identical at any given position. Potential N-linked glycosylation sites in the rat IAP are shown byasterisks above the sequence.
Fig. 3.
Fig. 3.
In situ hybridization showing a higher expression of IAP mRNA signal in the hippocampus of good-memory rats. Coronal sections through the hippocampus from (A) poor-memory rats (n = 4) and (B) good-memory rats (n = 6) were subjected to in situ hybridization analyses.CA1, CA1 cell body layer; CA3, CA3 cell body layer; DG, dentate gyrus. Scale bar, 500 μm.
Fig. 4.
Fig. 4.
Quantitative RT-PCR analysis of rat IAP mRNA.A, Autoradiograph of the IAP and HPRT cDNA bands. Serial quantities (6.25, 12.5, 25, 50, and 100 ng) of total hippocampal RNA were reverse-transcribed and amplified by PCR. The template of HPRT was used as an internal control. RT-PCR products were analyzed by a 9% polyacrylamide gel, visualized by a phosphoimager machine, and quantitated. B, Linear relationship between the optical density of the cDNA bands and the quantity of total RNA.C, Autoradiography of the IAP and HPRT cDNA bands from the poor-memory (PM) and good-memory (GM) rats. D, Higher expression of IAP mRNA level in good-memory rats (n= 11 in each group). **p < 0.01 by Student’st test. E, IAP mRNA expression in control animals and animals that received electric shock only. The control rats (n = 5) were placed in the chamber for 5 sec, returned to the home cage, and killed 3 hr later. Rats in the shocked group (n = 4) received a single electric shock, were returned to the home cage, and were killed 3 hr later.
Fig. 5.
Fig. 5.
Drug effects on IAP mRNA level. Saline (SAL), amphetamine (AMPH),NMDA, or carbachol (CARB) was infused into rat dentate gyrus bilaterally at a rate of 0.2 μg/min and a total volume of 0.8 μl on each side. The number in each column indicates the number of rats in each treatment group. Thirty minutes after drug infusion, rats were decapitated, and the bilateral dentate gyrus tissues were dissected out. Total RNA was isolated and subjected to quantitative RT-PCR analysis identical to the procedure in Figure 4. Both amphetamine and NMDA significantly increased IAP mRNA level in the hippocampus (*p < 0.05; Dunnett’s t test).
Fig. 6.
Fig. 6.
Effects of IAP antisense oligonucleotide on memory retention of one-way inhibitory avoidance learning in rats. The distribution of the retention score for each individual rat after saline (n = 11), IAP antisense oligonucleotide (n = 9), or random sequence (n= 11) treatment is shown. Four injections at intervals of 12 hr were given before the training procedure (for details, see Materials and Methods). For each injection, 1.0 μl of saline or the oligonucleotide (1 nmol) was injected directly into the dentate gyrus of the hippocampus bilaterally. There was a significant difference between the control group and the IAP antisense group when evaluated by the Mann–Whitney U test (p < 0.05).
Fig. 7.
Fig. 7.
Effects of IAP antisense oligonucleotide on LTP in rat hippocampus. Percentage change in the (A) mean amplitude and (B) slope of pEPSPs and SEMs was presented as a function of time after IAP antisense oligonucleotide injection (n = 5) and the random sequence injection (n = 5) versus the tetanization controls (n = 5). The tetanus stimulations that yielded LTP (indicated by arrows) were given at 30, 40, 50, and 60 min. Each stimulation contains five trains at 400 Hz. The duration of stimulation was 50 μ sec at 30 min, 100 μsec at 40 min, 150 μsec at 50 min, and 200 μsec at 60 min, respectively.
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