Calcium‐binding proteins typify the dopaminergic neuronal subtypes in the ventral tegmental area of zebra finch, Taeniopygia guttata

Calcium‐binding proteins (CBPs) regulate neuronal function in midbrain dopamine (DA)‐ergic neurons in mammals by buffering and sensing the intracellular Ca2+, and vesicular release. In birds, the equivalent set of neurons are important in song learning, directed singing, courtship, and energy balance, yet the status of CBPs in these neurons is unknown. Herein, for the first time, we probe the nature of CBPs, namely, Calbindin‐, Calretinin‐, Parvalbumin‐, and Secretagogin‐expressing DA neurons in the ventral tegmental area (VTA) and substantia nigra (SN) in the midbrain of zebra finch, Taeniopygia guttata. qRT‐PCR analysis of ventral midbrain tissue fragment revealed higher Calbindin‐ and Calretinin‐mRNA levels compared to Parvalbumin and Secretagogin. Application of immunofluorescence showed CBP‐immunoreactive (‐i) neurons in VTA (anterior [VTAa], mid [VTAm], caudal [VTAc]), SN (compacta [SNc], and reticulata [SNr]). Compared to VTAa, higher Calbindin‐ and Parvalbumin‐immunoreactivity (‐ir), and lower Calretinin‐ir were observed in VTAm and VTAc. Secretagogin‐ir was highly localized to VTAa. In SN, Calbindin‐ and Calretinin‐ir were higher in SNc, SNr was Parvalbumin enriched, and Secretagogin‐ir was not detected. Weak, moderate, and intense tyrosine hydroxylase (TH)‐i VTA neurons were demarcated as subtypes 1, 2, and 3, respectively. While subtype 1 TH‐i neurons were neither Calbindin‐ nor Calretinin‐i, ∼80 and ∼65% subtype 2 and ∼30 and ∼45% subtype 3 TH‐i neurons co‐expressed Calbindin and Calretinin, respectively. All TH‐i neuronal subtypes co‐expressed Parvalbumin with reciprocal relationship with TH‐ir. We suggest that the CBPs may determine VTA DA neuronal heterogeneity and differentially regulate their activity in T. guttata. Graphical : Dopamine (DA) neurons residing in the ventral tegmental area (VTA) and substantia nigra (SN) in the midbrain of zebra finch demonstrate distinct differences in size and tyrosine hydroxylase immunoreactivity (TH‐ir). The DA neurons in VTA were smaller than those in SN. Based on TH‐ir, VTA neurons have been demarcated as subtype 1, 2, and 3. The further study probed the nature of the calcium binding proteins (CBPs), namely, Calbindin (CB)‐, Calretinin (CR)‐, Parvalbumin (PV)‐, and Secretagogin (SCGN)‐expressing DA neurons in the VTA and SN. CBPs mRNA and protein showed differential expression in VTA subdivisions and SN. While the subtype 1 TH neurons in the VTA were CB‐ or CR‐negative, subtype 2 and 3 TH neurons co‐ex