Unbiased counting of neurons in the cochlea of developing gerbils

Accurate counting of neurons in the cochlea has a significant impact on the interpretation of research and clinically relevant data. However, reports of numbers of neurons in the spiral ganglion are widely variable across studies, even within the same species. We suggest that the implementation of a more standardized, unbiased counting method will improve the consistency and accuracy of neuron counts and will impact scientific interpretations. To test this view, we compared, in different ways, the numbers of neurons in the spiral ganglia of developing gerbils, previously reported to decrease by 22–27% between birth and age 7 days. Cochleae from gerbils, aged newborn, 7 days, 20 days, 1.5 years and 2.5 years were embedded in Araldite and serially sectioned at 5 μm. A computer based stereological method was used to unambiguously count every neuron in serial sections, either throughout the entire cochlea, or in a 100-μm segment of the cochlea. No significant changes in neuron numbers during cochlear maturation were found. We demonstrate that in methods using sampling of sections, the identity of the starting section and the interval between sections impacts the variability of the estimate of neuron numbers. In addition, we show that packing density differs between the newborn and seven-day old animals. The data demonstrate that variability in counting methods and the comparison of non-uniform samples can lead to neuron number estimates that show differences where none exist. We propose that a standardized counting protocol be implemented across studies and suggest possible approaches to different types of comparisons between neurons of spiral ganglia from different sources. ► We have addressed unbiased counting of spiral ganglion cells. ► The actual number of spiral ganglion neurons in gerbils during development has been determined by unbiased counting of the entire cochleae and does not change drastically between newborn and postnatal day 7. ► Pitfalls of profile counting have been demonstrated in gerbil cochleae. ► Guidelines for unbiased counting have been presented.