Gene expression and the diversity of identified neurons.

Nervous systems consist of diverse populations of neurons that are anatomically and functionally distinct. The diversity of neurons and the precision with which they are interconnected suggest that specific genes or sets of genes are activated in some neurons but not expressed in others. Experimentally, this problem may be considered at two levels. First, what is the total number of genes expressed in the brain, and how are they distributed among the different populations of neurons? Second, can we identify specific genes expressed in individual neurons and relate the expression of these genes to the unique functional properties of these neurons? A number of investigators have attempted to determine how many genes are expressed in the brain. Recent data suggest that the mRNA population from the entire mammalian brain may be far more diverse than mRNA populations present in homogeneous populations of cells in other, nonneural tissues (Chikaraishi 1979; Van Ness et al. 1979; Chaudhari and Hahn 1983). The brain, however, consists of a large number of heterogeneous cells or cell groupings that are anatomically or biochemically distinct. This immediately raises the question as to whether the complexity of brain mRNA populations is a reflection of a large number of diverse cell types, each expressing a small number of unique neuron-specific sequences. On the other hand, this complexity may result from a novel requirement that all neurons express a significantly greater number of different mRNAs than nonneural cells. The examination of the pattern of gene expression in individual neurons may allow the distinction between these alternatives. How does one begin to characterize the genes expressed in individual neurons in nervous systems composed of from 102 to 10 ~2 cells? In a direct approach, it is now possible to identify and clone specific gene sequences expressed in individual neurons and attempt to relate the activation of specific genes to the unique functions of individual neurons. A second, indirect approach involves the isolation of genes whose transcription is restricted to the nervous system. The site of expression of these genes can then be explored by in situ hybridization to mRNA in tissue sections through the developing and adult nervous systems. The simple nervous system of the invertebrate Aplysia californica, a marine snail, is particularly suitable for analyzing specific gene expression because it contains only about 20,000 central nerve cells, which are collected into four pairs of symmetric ganglia and a single asymmetric abdominal ganglion. Moreover, several of the neurons in these ganglia may be recognized by highly reproducible characteristics such as size, shape, position, pigmentation, and function (for review, see Kandel 1979). This numerical simplification has made it possible to relate the function of particular cells to specific patterns of behavior and may permit us to attribute neuronal function to the expression of specific genes. In addition to being few in number, neurons in Aplysia can be quite large, up to 1 mm in diameter. Most of these large cells are polyploid and contain as much as 2 #g of DNA, more than 105 times the content of the haploid genome (Coggeshall et al. 1971; Lasek and Dower 1971). Furthermore, our data indicate that mRNA content is proportional to cell size such that the largest of cells contains up to 5 ng of mRNA. It is therefore possible to dissect out single neurons and examine the activity of individual genes in a single cell. We have constructed both a genomic library from sperm DNA and a library of eDNA clones from the abdominal ganglion of Aplysia and have screened these libraries with probes synthesized from the mRNA of individual neurons. These procedures now permit the identification of genes whose expression is restricted to unique and identified nerve cells of known function. At the limit, with the largest neurons, screening for prevalent sequences can be performed with the eDNA synthesized from a single cell. When these procedures are applied to nerve cells that express prevalent gene products such as peptidergic neurons, clones encoding multiple behavioral neuropeptides can be isolated. In this manner, we have isolated the genes enc6ding a set of egg laying neuropeptides and, more recently, a gene that encodes a peptide involved in water balance.