Number and nuclear morphology of TH+ and TH− neurons in the mouse ventral midbrain using epifluorescence stereology

The accurate and reliable counting of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH−) neurons in the ventral midbrain is an important measure in studies related to Parkinson's disease and many other disorders associated with this region. Despite recent advancements, the use of stereology remains limited due to a variety of challenges for many users. We implemented a real-time fluorescence detection method and the use of an antibody to the neuron specific nuclear antigen (NeuN) to overcome some challenges for users. We found that the regional value for the two different cell types (TH+ and TH−) varied with the method of detection (chromogenic versus fluorescence) and with different nuclear markers (Nissl, DAPI, or NeuN). The number of both TH+ and TH− neurons was higher using fluorescence detection. The number of TH− neurons was higher using NeuN as a neuronal nuclear marker compared to DAPI. We identified 3 types of neuronal nuclei using NeuN staining characteristics. The method is applicable for mouse and rat. We describe a practical approach for epifluorescence-based counting of these two types of neurons that may offer significant advantages over existing methods for potential users.

Research Highlights
► Epifluorescence detection for stereology is practical.
► Epifluorescence detection offers some advantages over chromogenic detection.
► NeuN detection of neurons offers advantages over nonselective nuclear stains.
► NeuN identifies 100% of TH− neurons in the mouse ventral midbrain.
► NeuN identifies 3 distinct types of neuronal nuclei in the mouse ventral midbrain.
► NeuN can be used in the rat ventral midbrain.