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Transcriptional profiling reveals regulated genes in the hippocampus during memory formation - PubMed

Transcriptional profiling reveals regulated genes in the hippocampus during memory formation

Christine P Donahue et al. Hippocampus. 2002.

Abstract

Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-beta (NGF-beta), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampal-dependent associative memories.

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Figures

Figure 1
Figure 1

Acquisition of the classically conditioned eyeblink response. Graphic representation of the percentage of conditioned responses (CRs) that occurred during classical conditioning. Symbols designate groups of rats that were exposed to trace conditioning (●), unpaired stimuli (O), and delay conditioning (▾). Arrowheads indicate the number of trials (T) after which animals were sacrificed. Lowercase i denotes that sacrifice occurred immediately after training; its absence means that 24 h intervened. The number of animals used for each interval is as follows: for trace conditioning, T200i [n = 3, CRs = 65 ± 3.8], T200 [n = 5, CRs = 46.6 ± 7.1], T400 [n = 3], T600 [n = 3], T800i [n = 3, CRs = 69 ± 7.6], and T800 [n = 2, CRs = 56 ± 1.6]; for unpaired stimuli, T200i [n = 3, CRs = 5.3 ± 1.9], T200 [n = 4, CRs = 8.4 ± 2.9], T400 [n = 3], T600 [n = 3], T800i [n = 2, CRs = 12.5 ± 0.5], andT800 [n = 3, CRs = 9.0 ± 1.7]; and for delay conditioning, T200 [n = 5, CRs = 92.2 ± 3.1]. These group mean CRs are different from those depicted from the graph, since they represent on conditioning only in groups that were sacrificed at a specific time point after a specific number of trials.

Figure 2
Figure 2

Error analysis. A: Scatter plot of pooled total RNA from home caged control animals hybridized to two different arrays. The dashed box represents genes with an intensity of <1400. Genes with intensity values of >1400 correlate strongly between identical replicates. The two parallel lines represent the twofold change range from the correlation mean. This comparison is representative of scatter plots between other identically treated animals. B: Graphic representation of the total error in the array. The average ratio between identical genes was plotted versus expression threshold for genes whose expression was above the threshold cutoff (●). The standard error of the mean (SEM) was also calculated for this subset of genes at each expression threshold (O). This graph confirms the observed threshold cutoff value of 1400 observed in the gross analysis of the scatter plot, with a data plateau at an average ratio of 1.2; SEM = 0.03.

Figure 3
Figure 3

Percentage of genes regulated at each interval during the acquisition of a memory trace. Bars represent the percentage of the 1,186 genes on the array that changed in trace conditioning relative to the unpaired stimuli for that interval. Lowercase i denotes that animal was sacrificed immediately after training; its absence means that 24 h intervened.

Figure 4
Figure 4

Specificity of category 1 gene expression changes in trace conditioning compared with delay conditioning. Log2 gene expression values for trace conditioned rats, which acquired the conditioned response (CR) (trace), and for delay conditioned rats (delay) were each computed relative to rats exposed to unpaired stimuli and displayed using TreeView (Eisen et al., 1998). The color scale ranged from saturated green for log ratios of ≤2.0 to saturated red for log ratios of ≥2.0. Black indicates a value of 0 and no change between either trace or delay relative to unpaired.

Figure 5
Figure 5

Real-time polymerase chain reaction (PCR). A: Primers specific for 18S rRNA were used to normalize levels of total RNA added to the reactions. Relative to RNA isolated from caged controls, unpaired (white), trace (black), and delay (gray) data were corrected by a factor of 1.00, 1.13, and 1.03, respectively. B: Graphs represent fold change of real-time PCR products for delay (gray filled), trace (filled) relative to unpaired. Hatched boxes refer to fold change as calculated from the array data. Trace over unpaired ratios that were <1.0 are represented by the equation: −1/(trace/unpaired). Realtime PCR results shown are the averaged fold change calculated from three independent experiments, the error bar represents the standard error of the mean (SEM).

Figure 6
Figure 6

Hippocampal expression of growth hormone is enhanced during trace learning. In situ hybridization on a coronal section of a rat exposed to 200 trials of trace conditioning (A) or to the same number of unpaired stimuli (B). Digoxigenin-labeled RNA probes were used for growth hormone.

Figure 7
Figure 7

Layers of validation used to verify gene expression changes that correlate with the acquisition of trace eyeblink conditioning.

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