Fig 2: Neurons containing green fluorescent protein (GFP) isolated by fluorescence-activated cell sorting from a cell suspension of the dorsal striatum of transgenic mice, followed by assessment of the expression of specific genes in these neurons. (A) Characteristics of an isolated population of neurons stained with DRAQ5 (nuclear dye) and GFP. Histograms were plotted according to the density of all detected events. For each cell suspension, at least 1,000,000 events were analyzed. For each plot, n = 3. Data are presented as mean ± SEM. FSC, forward scatter; PE, phycoerythrin (detection channel); PI, propidium iodide; SSC, side scatter. (B) Fluorescent and differential interference contrast microscopy of isolated GFP neurons. Arrows show GFP-containing neurons. Scale bar: 50 µm. (C) Expression of genes for dopaminergic and non-dopaminergic phenotypes. (D) Expression of genes for neurotransmitter and hormone receptors. The leftmost lanes represent the DNA ladder (C,D). AADC, aromatic L-amino acid decarboxylase; α2C, alpha-2C adrenoceptor; D1, dopamine receptor 1; D2, dopamine receptors 2; DAT, dopamine transporter; ERA, endothelin receptor A; GPER1, G-protein coupled estrogen receptor 1; LAT1, L-amino acid transporter 1; LDR, L-DOPA receptor; MCR, mineralocorticosterone receptor; mPRα, membrane progesterone receptor alpha; Nurr1, nuclear receptor-related 1; SSR2, somatostatin receptor 2; TH, tyrosine hydroxylase; VMAT1, vesicular monoamine transporter 1; VMAT2, vesicular monoamine transporter 2.

Fig 3: Confocal microscopy of supraependymal and periventricular areas of the dorsal striatum of transgenic mice. (A) The neuronal cell body containing green fluorescent protein (GFP, blue) and stained with 4′,6-diamidino-2-phenylindole (DAPI) (gray) but immunonegative for tyrosine hydroxylase (TH, green) and aromatic L-amino acid decarboxylase (AADC, red) (arrowhead). (B) Supraependymal nerve fiber stained for AADC (red) but lacking GFP (blue) and TH (green) (dotted arrow) and nerve fibers between ependymal cells stained for GFP and TH but immunonegative for AADC (long arrow). (C) Supraependymal and subependymal nerve fibers stained for GFP (blue), TH (green), and AADC (red) (short arrow). Scale bars: 10 µm for (A–C). LV, lateral ventricle. Dotted line is the apical surface of the ependymal lining.

Fig 4: (A): expression of dopaminergic phenotype protein genes: tyrosine hydroxylase (TH), dopamine transporter (DAT), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), as well as the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH); (B): agarose phoresis for the AADC mRNA region between exons 2 and 5. Several forms of AADC are visible. The lower band corresponds to the gene for the enzymatically inactive form of AADC, which lacks exon 3, while the upper band corresponds to the gene for the enzymatically active form of AADC.

Fig 5: l-Dopa production in Daphnia magna gut. (a) Schematic structure of the midgut (37) analyzed with immunostaining (mv, microvillous layer; cc, columnar cells; gm, gut musculature; Ep, gut epithelium comprised of mv and cc). (b and d) Immunofluorescence localization of TH in the midgut epithelial cells; (c and e) Immunofluorescence localization of DDC in the midgut epithelial cells; the same region as for TH but in a different individual. Labeling with Alexa Fluorophore 488 is shown on the left, and the light microscopy of the same specimen is shown on the right; no labeling appeared in the unlabeled control (not shown). (f) The significant upregulation of Ddc gene expression in Daphnia gut observed during the postmolt decreasing during the intermolt and premolt; arrows indicate ecdysis. The data are shown as z scores (mean ± standard deviation [SD]; number of observations are shown on the bars for each stage) normalized to the cycle-specific average values for each of the replicate experimental runs.