Quantifying Dopaminergic Innervation in Rodents Using Unbiased Stereology

Quantitative microscopy techniques are valuable tools for neuroanatomical and psychiatric research. Qualitative methods are limited due to inherent biases and an inability to provide an absolute number of markers throughout an entire region of interest. Design-based stereologyDesign-based stereology provides an unbiased alternative, as it uses mathematical and statistical principles to form accurate predictions about the distribution of objects in three-dimensional brain tissue from two-dimensional images while avoiding assumptions about the distribution, orientation, size, or shape of objects. Stereological analysis requires careful a priori planning to provide accurate estimates and avoid bias. Here, we outline, from tissue collection to image processing, a complete stereologyStereology protocol to estimate the total number and density of varicosities and/or neurons throughout the mesocorticolimbic dopamineDopamine (DA) circuitry using StereoinvestigatorStereoInvestigator Software (MicroBrightField). These dopamineDopamine (DA) pathways continue to undergo profound developmentDevelopments during postnatal life, and disruptions to their maturation are implicated in a wide variety of psychiatric illnesses, including depressionDepression, schizophreniaSchizophrenia, and substance use disorderSubstance use disorder. Indeed, a stereological approach has allowed our group to identify and measure the impact of experiences in adolescenceAdolescence on dopamineDopamine (DA) neuroanatomy in adulthood. This protocol will prove useful for other groups studying the dopamineDopamine (DA) system and can be easily adapted to study countless other markers, cell types, and neurotransmitter systems.