Calbindin-Positive Neurons Co-express Functional Markers in a Location-Dependent Manner Within the A11 Region of the Rat Brain

The A11 region plays a role in numerous physiological functions, including pain and locomotor activity, and consists of a variety of neurons including GABAergic, calbindin positive (Calb + ), and dopaminergic (DA) neurons. However, the neurochemical nature of Calb + neurons and their regulatory role in the A11 region remain largely unknown. In this study, we examined the kind of functional markers co-expressed in the Calb + neurons using sections from 8-week-old rats. To examine a marker related to classical neurotransmitters, we performed in situ hybridization for vesicular glutamate transporter 2 (vGluT2) or glutamate decarboxylase (GAD) 65 and 67, in conjunction with Calb immunohistochemistry. We found cellular co-expression of Calb with vGluT2 or GAD65/67 throughout the A11 region. Nearly all Calb + /GAD65/67 + neurons were found in the rostral-middle aspect of the A11 region. In contrast, Calb + /vGluT2 + neurons were found predominantly in the middle-caudal aspect of the A11 region. For receptors and neuropeptides, we performed immunohistochemistry for androgen receptor (AR), estrogen receptors (ERα and ERβ), and calcitonin gene-related peptide (CGRP). We found that Calb + neurons co-expressed AR in the rostral aspect of the A11 region in both male and female rats. However, we rarely find cellular co-expression of Calb with ERα or ERβ in this region. For CGRP, we found both Calb + neurons with or without CGRP expression. These results demonstrate that Calb + neurons co-express many functional markers. Calb + neurons have a distinct distribution pattern and may play a variety of regulatory roles, depending on their location within the A11 region.