A unit density method of grain analysis used to identify GABAergic neurons for electron microscopic autoradiographs

The distribution of electron microscopic autoradiographic grains over neurons in cerebellar cultures incubated with [3H]gamma-aminobutyric acid ([3H]GABA) was examined. With the unit density method of grain analysis, the number of grains over each structure was tested against the total grain density for the entire section. If an individual structure has a grain density higher than the expected grain density, it is considered one of the group of heavily labeled structures. The expected grain density for each structure is calculated based on the area for that structure, the total grain density and the Poisson distribution. A different expected grain density can be calculated for any P value required. The method provides an adequate population of structures for morphological analysis but excludes weakly labeled structures and thus may underestimate the number of labeled structures. The unit density method of grain analysis showed, as expected, a group of cell bodies and synapses that was labeled heavily. Cultures incubated with other [3H]amino acids did not have any heavily labeled synaptic elements. In addition, serial section analysis of sections showed that synapses heavily labeled with [3H]GABA are seen in adjacent section. The advantage of the unit density method of grain analysis is that it can be used to separate two groups of metabolically different neurons even when no morphological differences are present.