( Additional tools for neuroscience res...)Gene-Specific Primers for NMDA Receptor Subunits

Additional tools for neuroscience researchers

Robert Buchner Peter L. Pingerelli Michael D. Browning Mary Ellen Simcox
Stratagene Cloning Systems, Inc.

Stratagene is now offering gene-specific primer sets for the rat and human NMDA receptor. In this article, we describe using these primers in reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays to show expression of NMDA subunits with Stratagenes rat cDNA, human cDNA and hNT neuron cDNA libraries as templates. By using these primers, researchers will be able to study rat and human NMDAR subunit-specific expression in a variety of different cell types and regions of the brain.

The ion channels activated by glutamate are typically divided into two classes. The ion channels that are sensitive to N-methyl-D-aspartate (NMDA) are designated NMDA receptors (NMDAR), while those activated by kainate and a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) are known as kainate/AMPA receptors (K/AMPAR). The NMDAR assumes a critical role in long-term potentiation (LTP), a putative cellular substrate of memory.¹ This receptor has also been linked to neuronal development and has been implicated in several disorders of the central nervous system, including epilepsy and ischemic neuronal cell death.

The rat NMDAR1 (NR1) was the first subunit of the NMDAR to be cloned.² The NR1 protein can form NMDA-activated ion channels when expressed in Xenopus oocytes, but the currents in such channels are much weaker than those seen in situ. Channels with more relevant physiological characteristics are produced when the NR1 subunit is combined with one or more of the NMDAR2 (NR2A-D) subunits.³ In addition, there are a number of different splice variants of the NR1.⁴ Significant functional diversity has been seen in recombinant systems with heterologous coexpression of various NR2 subunits and/or NR1 splice variants.⁵ However, little is known about the expression of the NMDAR in situ.

Table 1

figure 1

RT-PCR assays with gene-specific primer sets can be used to study gene expression of the NMDA receptor subunits.⁶ Stratagenes cDNA library templates and amplification primer sets⁶ for rat NMDA subunits were used for RT-PCR reactions to identify cell populations expressing these specific NMDA receptor subunit genes. Briefly, RT-PCR was conducted using species-specific primer sets for various NMDA receptor subunits (table 1) with cDNA constructed from whole rat brain (figure 1). (Data from reactions using human brain and human hNT cells as templates are not shown). PCR products resulting from amplification of rat NMDA receptor subunit genes were electrophoresed on an agarose gel (figure 1), and their sizes were visualized by ethidium bromide staining. Amplification products with the gene-specific primers for the NMDA receptor subunits resulted in products with correct sizes for all cDNAs tested. For NR1, amplification using primers flanking the C1 or N1 splice-variant exons from rat resulted in two products of different sizes, corresponding to mRNAs either containing or lacking these exons.

Conclusions

Investigators can now use the extremely sensitive RT-PCR method to study expression of NMDA receptor subunit genes. Stratagenes RT-PCR primers for rat and human NMDA receptor genes have been tested using cDNA templates derived from whole brain or cultured hNT cells. These new RT-PCR primers will allow further studies of the subunit composition of NMDA receptors.

REFERENCES

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2. Moriyoshi, K., et al. (1991) Nature 354: 31-37.

3. Monyer, H., et al. (1992) Science 256: 1217-1221.

4. Laurie, D.J., Seeburg, P.H. (1994) J. Neurosci. 14: 3180-3194.

5. Monyer, H., et al. (1994) Neuron 12: 529-540.

6. Sheng, M., et al. (1994) Nature 368: 144-147.