Transmission Electron microscopy (TEM)

Analysis was performed in fields employed from a previous study [50]. Details of tissue preparation, post-embedding procedure, ultrathin cutting, and TH staining are fully reported in [50]. Briefly, following perfusion, brains were cut on a vibratome in 50 μm horizontal sections containing the midbrain and collected in PB until processing [74]. Sections were obtained through the dorso-ventral extent of the VTA, resulting in 12–13 consecutive sections and processed with an osmium-free embedding method [74]. VTA chips were sectioned with an ultramicrotome (60–80 nm), mounted on 300 mesh nickel grids and processed for immunogold labeling [74, 75], with primary antibody anti-TH (1:200; Abcam; #AB1542; RRID: AB_90755), and secondary antibody conjugated to 18 nm gold particles (1:20; Jackson, 713–215-147; RRID: AB_2340734).

Ultrathin sections were examined with a Philips CM10 electron microscope coupled to a MegaView-II high resolution CCD camera (Soft Imaging System). Identification of ultrastructural profiles was based on established morphological criteria [76].

Mitochondria morphology was analyzed in the same TH⁺ neurons recognized and studied in [50], and identification of normal and swollen/vacuolated mitochondria was performed according to established morphological criteria [77–79]. In more detail: normal-appearing rounded and oval-elongated-shaped mitochondria were characterized by a smooth outer membrane and an inner folded membrane that formed the cristae, which was filled with dense matrix. In normal mitochondria, the cristae were well preserved, and the electron density of mitochondrial matrix appeared as regular (representative examples of normal mitochondria (n) are illustrated in the upper, middle, and lower panels from WT and Tg mice). Swollen and vacuolated-appearing mitochondria exhibited a dilatation of the intermembrane space with a splitting of the outer and inner membranes including micro­vacuolization of the inner compartment and an irregular appearance of matrix and cristae (examples of swollen mitochondria (s) are illustrated in the upper, middle, and lower panels from WT and Tg mice). Vacuolated mitochondria included vacuoles deriving from the dilatation of the intermembrane space localized between the outer and inner membranes, vacuoles in the matrix or cristae (see (v) in examples illustrated in Fig. 3), and finally large vacuoles (referring to (V) in representative panels) containing granular or amorphous substance or characterized by empty spaces of various sizes.

VTA DA neurons in the Tg2576 mice show mitochondrial alterations at 3 months of age. A Representative confocal images of AIF labeling in TH⁺ neurons from WT and Tg2576 mice at 3 months of age (scale: 10 μm). B The graph shows the % of AIF⁺ nuclei in DA neurons of the VTA (n = 4 mice per genotype; Unpaired t-test: **p = 0.0018). C TEM representative images of normal, swollen, and vacuolated mitochondria (upper row; original magnification 92.000x) from osmium-free TH-stained post-embedded VTA of 3-month-old WT (panels of middle row) and Tg2576 mice (lower row; original magnification 24.000x). Normal-appearing mitochondria (n in all panels) display a smooth outer membrane and well-preserved cristae filled with a regular electron density of the mitochondrial matrix. Swollen (s) and vacuolated-appearing (v) mitochondria exhibit dilation of the intermembrane space, vacuolization of the inner compartment, and an irregular appearance of matrix and cristae. Entirely vacuolized mitochondria (V) contain an amorphous substance or appear as empty vacuoles of various sizes. Although mitochondrial swelling and vacuolization are detectable in WT neurons, Tg2576 neurons show an increase of mitochondria at different stages of swelling and/or vacuolization (plots D-G). In all TEM panels, blue arrowheads point to TH-coding gold particles (scale bar: 30 nm for upper panels, and 130 nm for middle and lower panels; WT: n = 53 TH⁺ neurons; from 3 mice; Tg2576 mice: n = 51 TH⁺ neurons, from 3 mice. D Total density of mitochondria (0.68/μm² ± 0.05 and 0.76/μm² ± 0.04); E density of normal mitochondria (0.55/μm² ± 0.04 and 0.34/μm² ± 0.02; Mann-Whitney test: ***p = 0.0003), F density of swollen/vacuolated mitochondria (0.12/μm² ± 0.01 and 0.42/μm² ± 0.03; Mann-Whitney test: ****p < 0.0001), and G ratio of swollen-vacuolated/normal mitochondria (Mann-Whitney test: ****p < 0.0001)