Fig. 5
(A-C, left panels) DAPI and PI immunohistochemistry conducted in LDTF and LDTM from brain slices exposed to α-synM under 3 different treatment protocols is shown in representative fluorescent images. The first column represents living cells visualized by DAPI (blue). The second column represents dead cells visualized by PI (red), and the last column is a merged image of DAPI and PI labeled cells. (A) The presence of DAPI and PI in the LDTF following incubation of one half of a slice in control solution (ACSF) and the other half in ACSF containing α-synM for 7 h is shown and indicates relatively lower cell survival in the half of the slice exposed to α-synM, which was reflected in the population data. The bar graph to the right shows that following α-synM exposure, cell survival in LDTF was significantly greater than that seen in LDTM (Cell Survival Female: na = 183/n40 = 48, Cell Survival Male: na=168/n40 = 66; p < 0.0001; Unpaired Student’s T-test). In this and subsequent panels, red points represent observations from LDTF, blue represent data from LDTM, and cell counts within each area (na) represent one data point or observation in the bar chart columns. (B) Fluorescent images showing DAPI and PI presence in LDTF cells treated for 7 h with α-synM or with α-synM in presence of GABAA, GABAB and glycine receptors antagonists (G-ANT). As shown in the bar graph to the right, a reduced cell survival indicative of greater cell mortality was observed in the population of LDTF slices exposed to α-synM when GABA and glycine receptor antagonists were present (Cell survival: α-synM: na = 199/n40 = 44, Cell survival α-synM + G-ANT: na = 169/n40 = 35; p = 0.0001; Mann-Whitney Test). To compare the population data, bisected slices were used, and the proportion of surviving cells observed in the half of the bisected slice exposed to α-synM was considered the baseline, and the number of surviving cells in the other half of the bisected slice exposed to α-synM + G-ANT was normalized to this baseline. (C) Fluorescent images of LDTM slices exposed to ⍺-synM or to α-synM in presence of 7 h of GABAA and GABAB receptor agonists (G-AGO). As can be seen from the population data shown in bar graphs to the right, the presence of the GABA and glycine receptor agonists in the LDTM was associated with significantly greater cell survival following exposure to α-synM (Cell Survival α-synM: na = 168/n40 = 66, Cell Survival α-synM + G-AGO: na = 127 /n40 = 48; p = 0.0334; Mann-Whitney Test). In this protocol, the proportion of surviving cells observed in the half of the bisected slice exposed to α-synM was considered the baseline, and the number of surviving cells in the other half of the bisected slice exposed to α-synM + G-AGO was normalized to this baseline. LDTF: the laterodorsal tegmental nucleus of female; LDTM: the laterodorsal tegmental nucleus of male. G-ANT: contains SR-95,531 (gabazine, 10 µM), CGP 55,845 (10 µM) and strychnine (2.5 µM) to block GABAA, GABAB, and glycine receptor-mediated responses, respectively. G-AGO: contains muscimol (30 µM) and baclofen (10 µM), which are agonists of GABAA and GABAB receptors, respectively. The scale bar in all images corresponds to 50 μm under 40x magnification. Contrast has been added equally across all the images. *p < 0.05, **** p < 0.0001