Heterogeneous expression of dopaminergic markers and Vglut2 in mouse mesodiencephalic dopaminergic nuclei A8–A13

Abstract

Co-transmission of glutamate by brain dopaminergic (DA) neurons was recently proposed as a potential factor influencing cell survival in models of Parkinson's disease. Intriguingly, brain DA nuclei are differentially affected in Parkinson's disease. Whether this is associated with different patterns of co-expression of the glutamatergic phenotype along the rostrocaudal brain axis is unknown in mammals. We hypothesized that, as in zebrafish, the glutamatergic phenotype is present preferentially in the caudal mesodiencephalic DA nuclei. Here, we used in mice a cell fate mapping strategy based on reporter protein expression (ZsGreen) consecutive to previous expression of the vesicular glutamate transporter 2 (Vglut2) gene, coupled with immunofluorescence experiments against tyrosine hydroxylase (TH) or dopamine transporter (DAT). We found three expression patterns in DA cells, organized along the rostrocaudal brain axis. The first pattern (TH-positive, DAT-positive, ZsGreenpositive) was found in A8-A10. The second pattern (TH-positive, DAT-negative, ZsGreen-positive) was found in A11. The third pattern (TH-positive, DAT-negative, ZsGreen-negative) was found in A12-A13. These patterns should help to refine the establishment of the homology of DA nuclei between vertebrate species. Our results also uncover that Vglut2 is expressed at some point during cell lifetime in DA nuclei known to degenerate in Parkinson's disease and largely absent from those that are preserved, suggesting that co-expression of the glutamatergic phenotype in DA cells influences their survival in Parkinson's disease.

Freely behaving mice can brake and turn during optogenetic stimulation of the Mesencephalic Locomotor Region

“…After exposure to Cre-recombinase, the floxed STOP cassette is removed, and this results in the expression of the ZsGreen fluorescent protein under control of CAG promoter. Cells display intense labelling with ZsGreen as demonstrated by us [1] and others [32, 23]. In our previous study, we compared ZsGreen-lox mouse (Cre-negative) and Vglut2-ZsGreen (Cre-positive) brain sections and confirmed that prior to introduction of Cre-recombinase, only a very low baseline level of fluorescence was present in brain slices of homozygous ZsGreen-lox mice [1].…”

“…We crossed Vglut2-Cre with ChR2-EYFP-lox mice. In the offspring (Vglut2-ChR2-EYFP), if Vglut2 is expressed during cell lifetime, ChR2 is expressed permanently under control of the CAG promoter even if Vglut2 is not expressed anymore [53, 54]. Mainly glutamatergic and GABAergic neurons are present in the CnF (for review [3]).…”

“…Mice were genotyped as previously described [1]. Briefly, DNA was extracted from ear punches using the Taq DNA polymerase (NEB, Ipswich, MA, United States).…”

“…Procedures were as previously reported [1]. Briefly, mice were anaesthetized using isoflurane (5%, 2.5 L per minute) and transcardially perfused with 50 mL of a phosphate buffer solution (0.1M) containing 0.9% NaCl (PBS, pH = 7.4), followed by 40 to 75 mL of a PBS solution containing 4% (wt/vol) of paraformaldehyde (PFA 4%).…”

“…The AB177487 anti-NeuN has been widely used to label the neuronal marker NeuN (also called Fox-3, see [4, 5]) in mouse brain tissues by us [1] and others [6, 7]. According to the supplier, this monoclonal purified antibody (clone EPR12763) is directed towards a synthetic peptide of the residues 1-100 of the human NeuN.…”