Table 1.
Experimental sample size and groups.
Fig 1.
Representative brain schematic.
Approximate location and size (2mm) of tissue samples collected for tissue processing (RT qPCR, ELISA, and western blotting). (A) Brain drawing adapted from Paxinos and Watson, 2005. Box shows approximate location of panel (B) representative Nissl stained brain section, 50μm thick section. Photomicrograph at 2.52x magnification. Circular holes were centered within the midbrain and illustrate the approximate location including the ventral tegmental area (approximately Bregma -4.80mm visible at the time of dissection) and adjacent tissue including substantia nigra. Scale bar indicates 200 μm; midline defined by dashed line. Abbreviations: ML = medial mammillary; PAG = periaqueductal gray; SN = substantia nigra; VTAR = Ventral tegmental area (Paxinos and Watson, 2005).
Table 2.
RT qPCR information.
Table 3.
Means and standard error of the means in the VTA.
Fig 2.
Spectrograms (x axis: time (sec), y-axis: frequency in (kHz)) of complex frequency-modulated USVs in representative samples from (A, B) Wildtype (WT), (C, D) Control Pink1-/-, (E, F) Sham-exercised (sham-ex) Pink1-/-, and (G, H) Vocal-exercised (vocal-ex) Pink1-/- rats at 8 months of age. Within black and white spectrograms: relative intensity (dB, loudness) is encoded by darkness of the signal; darker is louder. Legend for relative intensity for color spectrograms is at the bottom of the Fig Peak frequency (pf) is indicated above each vocalization per group. Scale bar (0.05 sec) is located in panel A.
Fig 3.
Average (A) bandwidth (kilohertz (kHz) and (B) peak frequency (kHz) +/- standard error of the mean (SEM). Frequency on the y-axis and group on the x-axis (white bar = wildtype (WT), light gray = control Pink1-/-, medium gray = sham-exercised (sham-ex) Pink1-/-, dark gray = vocal-exercised (vocal-ex) Pink1-/-). Statistical significance between groups indicated by bar and *** p<0.001, **p < 0.01, *p < 0.05. n.s. indicates no significant differences.
Fig 4.
Catecholamine protein concentrations in the ventral tegmental area.
Average (A) gene expression for tyrosine hydroxylase (Th), (B) dopamine protein concentration and (C) norepinephrine protein concentration +/- standard error of the mean (SEM). Concentration (pg per ug/total protein) on the y-axis and group on the x-axis (white bar = wildtype (WT), light gray = control Pink1-/-, medium gray = sham-exercised (sham-ex); dark gray = vocal-exercised (vocal-ex) Pink1-/-). n.s. = not significant. Sample size is located inside bars of respective panels.
Fig 5.
RT qPCR for GABAergic genes in the ventral tegmental area.
(A) Average relative mRNA expression values for Gad1, Gad2, Gls, and Glul +/- standard error of the mean (SEM). Relative expression on the y-axis and group on the x-axis (white bar = wildtype (WT), light gray = control Pink1 -/-, medium gray = sham-exercised (sham-ex) Pink1-/-, dark gray = vocal-exercised (vocal-ex) Pink1 -/-). All qPCR data were analyzed with the Pfaffl Method, normalized to reference genes (Ywaz, Gapdh) expression. (B) Quantification of GAD pixel intensity and representative GAD and β-actin bands on a western blot for each group (WT, control Pink1-/-, sham- & vocal-exercised Pink1-/-). Statistical significance between groups indicated by bar and *** p<0.001, **p < 0.01, *p < 0.05. Sample size is located inside bars of respective panels.
Fig 6.
Mu opioid receptor in the ventral tegmental area.
(A) relative expression of MuR mRNA expression +/- standard error of the mean (SEM). Relative expression on the y-axis and group on the x-axis (white bar = wildtype (WT), light gray = control Pink1-/-, medium gray = sham-exercise (sham-ex) Pink1-/-, dark gray = vocal-exercise (vocal-ex) Pink1-/-). All qPCR data were analyzed with the Pfaffl Method, normalized to reference genes (Ywaz, Gapdh) expression. (B) Quantification of MuR protein via western blot and representative MuR bands for each group. Statistical significance between groups indicated by bar and *** p<0.001, **p < 0.01, *p < 0.05. Sample size is located inside bars of respective panels.
Fig 7.
GABAergic gene expression in the substantia nigra.
(A) Average relative mRNA expression values for Gad1, Gad2, Gls, and Glul +/- standard error of the mean (SEM). Relative expression on the y-axis and group on the x-axis (white bar = wildtype (WT), light gray = control Pink1 -/-, medium gray = sham-exercised (sham-ex) Pink1-/-, dark gray = vocal-exercised (vocal-ex) Pink1 -/-). All qPCR data were analyzed with the Pfaffl Method, normalized to reference genes (Ywaz, Gapdh) expression. n.s. indicates no significant findings. Sample size is located inside bars of respective panels.
Table 4.
Means and standard error of the means in the SN.