Identification of upregulated SCG10 mRNA expression associated with late-phase long-term potentiation in the rat hippocampal Schaffer-CA1 pathway in vivo

Abstract

The maintenance of long-term potentiation (LTP) depends on alteration of gene transcription. By screening a subtracted cDNA library that is enriched in upregulated transcripts in rat hippocampus 3 hr after Schaffer-CA1 LTP induction in vivo, we identified a neural growth-associated protein SCG10 (superior cervical ganglia clone 10) gene. The semiquantitative reverse transcription-PCR and Northern blot experiments confirmed that SCG10 mRNA levels were elevated in tetanized rat hippocampi compared with those of sham controls that received only low-frequency stimulation. Both 1 and 2 kb forms of SCG10 mRNAs contributed to the increased expression. Using a riboprobe with a sequence specific to the 3'-untranslated region of rat SCG10 mRNA, in situ hybridization further revealed a significant increase of the SCG10 mRNA 2 kb form in the ipsilateral CA3 and CA1 regions of LTP animals. In addition, we systemically injected the competitive NMDA receptor antagonist d,l-3[(+/-)-2-carboxypiperazine-4-yl]-propyl-1-phosphonic acid (CPP) to determine whether the alteration of SCG10 expression depends on NMDA receptor activation or tetanus alone. Administration of CPP 1 hr before tetanus completely blocked LTP induction and the increase of SCG10 mRNA levels. Thus, these results suggest that the transcription of SCG10 in vivo is regulated by long-lasting synaptic activity and may contribute to the maintenance of long-term synaptic plasticity via a presynaptic remodeling mechanism.

Figure 1.

NMDA receptor-dependent LTP induction at the rat Schaffer-CA1 pathway in vivo. A, High-frequency stimulation induced LTP in the Schaffer-CA1 pathway. The plot shows field EPSP slope magnitudes of Schaffer-CA1 responses evoked at current intensities eliciting responses 50% of maximal. Values are group means ± SEM expressed as percentage of baseline. Sham control animals (n = 4) received only low-frequency stimulation (0.05 Hz). Delivery of trains of high-frequency stimulation (three 1 sec, 100 Hz trains with two 5 min intertrain intervals) to the Schaffer collaterals induced LTP (n = 4). B, High-frequency stimulation-induced LTP in the Schaffer-CA1 pathway was blocked by CPP. The potentiation was blocked when the competitive NMDA receptor antagonist CPP (10 mg/kg) was intraperitoneally injected 1 hr before tetanus (n = 4). C, High-frequency stimulation-induced LTP lasted for 3 hr. Each data point corresponds to the average of four consecutive neuronal responses recorded at 15 sec intervals (n = 3). Traces (averaged from five consecutive waveforms) are representative Schaffer-CA1 responses just before tetanus and 1 hr after. Calibration: 1.0 mV, 20 msec.

Figure 2.

Semiquantitative RT-PCR analysis of the SCG10 mRNA 2 kb isoform. A, Autoradiograph of the SCG10 and internal control 18S cDNA bands. The analysis of the gene expression levels by relative quantitative RT-PCR was performed using the primers specific to the nucleotide sequence of rat SCG10 mRNA 3′-UTR. Total RNAs were extracted from the ipsilateral hippocampi of individual sham (lanes 1-3), LTP (lanes 4-6), and CPP (lanes 7-9) animals. Simultaneously, the internal control 18S rRNA was also amplified in the same tubes with an optimal ratio of 18S primers and competimers (1:9). Top, The Southern blot of RT-PCR products was probed with a cDNA fragment corresponding to the rat SCG10 3′-UTR. Bottom, The RT-PCR products of 18S ribosomal RNA were also probed with 18S cDNA. B, These data were quantified using imaging densitometry and represented as ratios of SCG10 to 18S rRNA. The ratios were further normalized to the mean of the sham controls. LTP animals (n = 3; p = 0.017) have a significantly higher level of the 2 kb transcripts than do sham controls (n = 3). CPP (n = 3) blocked the increase of SCG10 expression.

Figure 3.

Semiquantitative RT-PCR analysis of SCG10 mRNA 1 and 2 kb isoforms. A, Autoradiograph of the SCG10 and internal control 18S cDNA bands. The analysis of the gene expression levels by RT-PCR was performed using the primers specific to the nucleotide sequence of the rat SCG10 mRNA coding region. Total RNAs were extracted from the ipsilateral hippocampi of individual sham (lanes 1-3), LTP (lanes 4-6), and CPP (lanes 7-9) animals. Simultaneously, a 324 bp fragment of 18S ribosomal RNA was amplified in the same tubes with the optimal ratio of 18S primers and competimers (1:19) as the internal control. Top, The Southern blot of RT-PCR products was probed with a cDNA fragment corresponding to the rat SCG10 coding region. Bottom, The RT-PCR products of 18S ribosomal RNA were also probed with 18S cDNA. B, These data were quantified using imaging densitometry and represented as ratios of SCG10 to 18S rRNA. The ratios were further normalized to the sham controls. LTP animals (n = 3; p=0.023) have a significantly higher level of both 1 and 2 kb transcripts than do sham controls (n = 3). CPP (n = 3) blocked the increase of the SCG10 expression.

Figure 4.

Northern analysis of SCG10 mRNA expression in the hippocampus. A, A Northern blot containing 10 μg of total RNA from individual rat hippocampi was hybridized with a riboprobe corresponding to the sequence of the rat SCG10 coding region. Top, Lanes 1-3, sham; lanes 4-6, LTP; lanes 7-9, CPP. Bottom, The Northern blot was stained with methylene blue. B, The data were quantified using imaging densitometry and represented as ratios of SCG10 to 28S rRNA. The ratios were further normalized to the sham controls. LTP animals (n = 3) have a significantly higher level of both the 2 kb (p = 0.007; top) and 1 kb (p = 0.001; bottom) transcripts than do sham controls (n = 3). CPP (n = 3) blocked the elevation of the SCG10 expression.

Figure 5.

Densitometric images of the in situ hybridization autoradiographs showing different subregional expression patterns of the SCG10 2 kb mRNA in the rat hippocampus. Coronal sections (8 μm) through the hippocampus from sham, LTP, and CPP were probed with the antisense nucleotide corresponding to the sequence of the SCG10 3′-UTR. Sense, Coronal brain sections were probed with the SCG10 sense riboprobe as a negative control. The relative hybridization level is indicated by the color scale on the right. The electrodes were placed in the right hippocampus.

Figure 6.

In vivo LTP induction elevated SCG10 2 kb mRNA expression in the ipsilateral CA3 and CA1 regions. The autoradiographs of in situ hybridization were quantified using imaging densitometry and represented as ROD ratios of hippocampal subregions to their surrounding cerebral cortex. A, B, LTP animals (n = 3) have a significantly higher level of 2 kb transcripts than do sham controls (n = 3) at the hippocampal CA3 (p < 0.001) and CA1 (p < 0.001) regions of the stimulated hemisphere. CPP (n = 3) blocked the increase of SCG10 mRNA expression. C, No significant alteration of SCG10 mRNA expression was detected in the dentate gyrus. L, Contralateral hippocampus (Fig. 5, left); R, ipsilateral hippocampus (Fig. 5, right).