Matt Peterson and Dr. Sterling Sudweeks, Physiology and Developmental Biology

Introduction

Hippocampal interneurons are a particularly diverse population of cells. Classifying these interneurons has historically been difficult using traditional schemes such as anatomical morphology, action potential spiking pattern, and receptor pharmacology. More recently however, classification of interneurons by examining expression of neuropeptide Y, somatostatin, and calcium-binding proteins (Ca-BPs: parvalbumin, calbindin, and calretinin) has been promoted as an effective means for identifying subpopulations within the hippocampus. Categorization of the expression of neuropeptide Y, somatostatin, and Ca-BPs with the various types of nicotinic acetylcholine receptors (nAChRs) could provide information valuable for characterizing how various interneuron subpopulations function to modulate the overall activity of the hippocampus. The studies presented here use single-cell quantitative real-time RT-PCR to characterize the mRNA expression of the a2, a3, a4, a5, a7, b2, b3 and b4 neuronal nAChR subunits together with the genes for neuropeptide Y, somatostatin, and the calcium-binding proteins parvalbumin, calbindin, and calretinin in hippocampal interneurons from the CA1 stratum radiatum and stratum oriens.

Methodology

Hippocampal slices (300 mm thick) were made from 8 to 18 day old Wistar rats. Individual hippocampal interneurons from the CA1 stratum radiatum and stratum oriens were visually identified using DIC optics, and aspirated into a standard whole-cell patch clamp pipette. A cDNA library was made by running a reverse transcription reaction.

A multiplex PCR reaction was run (15 cycles) for each aspirated neuron using all neuronal nAChR primers as well as primers for 18s rRNA, the Calcium-binding proteins parvalbumin, calbindin, and calretinin, and the neuropeptides NPY and somatostatin. A second round of PCR was run (60 cycles) for each specific target using real-time quantitative PCR.

CT values were calculated using the second derivative of the amplification curve. Relative quantification was performed using the DDCT method (see Livak and Schmittgen, Methods 2001. 4:402-8), comparing each individual subunit to the overall lowest target expression level. Gene co-expression analysis was performed looking for mRNA transcripts that were co-detected in a significant proportion of cells using a c2 test.

Results

Because of the difficulty of acquiring cells, we were only able to use four of the cells that were aspirated. The first cell is from the Stratum Radiatum. As can be seen from figure 1, the greatest concentration of mRNA found in calbindin. It had nearly a ten fold greater expression of its mRNA than expected. Parvalbumin was not seen in this cell.

The second cell was also pulled from the Stratum Radiatum and very different results are seen. Not only is parvalbumin observed in this cell, but it has an extremely high concentration within the cell. In this cell, however, calbindin was not seen. Neuropeptide Y, somatostatin, and calretinin were seen in both cells #1 and #2.

The third and fourth cells were taken from the Stratum Oriens rather than the Stratum Radiatum. From figure 2, we can see the oriens cells have less mRNA than the radiatum did. Oriens cell #1 does not have any NPY, or calretinin while cell #2 does not have NPY, calretinin, or parvalbumin. The highest concentration of mRNA in cells #1 and #2 were from parvalbumin and calbindin respectively.

Conclusions

With this little information, we are unable to make any solid conclusions. More data will need to be obtained, then conclusions can be made. According to the data we have seen, using neuropeptide Y, somatostatin, and the calcium-binding proteins may be an effective way of classifying nAChRs.