All mice were housed under standard conditions under a 12 h dark/light cycle in the animal facility. Food and water were provided ad libitum.

The Thy1-h[A30P]αSyn transgenic mouse line (original publication: Kahle et al. [47] and Neumann et al. [48]) was originally generated by inserting the human SNCA gene coding sequence (CDS) carrying a point mutation in nucleotide position 88 (exchanging guanine to cysteine, causing a shift from alanine to proline at position 30 [9]) under control of the brain-specific Thy-1 promoter. Here, Thy1-h[A30P]αSyn homozygous transgenic mice on a C57BL/6J background were analyzed together with age- and sex-matched wild-type control mice of the same genetic background, as previously described [47–49]. Both male and female mice were analyzed; age span was 16–18 months. Previous studies showed no histological differences between sexes [48]. Here, no difference between male and female mice was observed at any stage of the analysis, and male and female mice were pooled to address groups by genotype and age only. n = 20 mice per genotype.

The Thy1-h[wt]αSyn transgenic mouse line [also known as line 61 (L61) and originally described in Chesselet et al. [50] and Rockenstein et al. [51]] was generated by inserting the CDS of wild-type human SNCA under control of the Thy-1 promoter. Here, heterozygous Thy1-h[wt]αSyn transgenic mice were generated by breeding heterozygous transgenic females with C57BL6/DBA2 males. Due to the transgenic insertion in the X-chromosome, only males were used in accordance with earlier publications [50, 52]. Adult mice were analyzed, aged 5 months (n = 8). Age-matched wild-type littermates for Thy1-h[wt]αSyn transgenic mice were used as controls (5 months, n = 7) in accordance with previous publications [50, 51].

To first prepare brain sections for mRNA analysis by fluorescent in situ hybridization (FISH), brains were quickly dissected from mice euthanized by cervical dislocation, snap-frozen in cold (–30℃ to –35℃) 2-methylbutane (≥ 99%, Honeywell) and kept at –80℃ until usage. Coronal serial sections were prepared on a cryostat at 16 µm thickness and placed onto SuperFrost^TM glass slides in a series of 8 slides (Thermo Fisher Scientific). Slides prepared for FISH analysis were stored at –80℃ until usage.

For detection of selected mRNAs in cryosections, mRNA-directed fluorescent riboprobes were prepared from cDNA (Oramacell). Probes were synthesized by PCR from DNA template containing T3 and T7 promotor sequences using promoter-attached primers (Table S1). Digoxigenin (DIG, Roche, 11207733910) and fluorescein (Roche, 11426346910) labeled RNA probes were made by a transcriptional reaction with incorporation of DIG or fluorescein labelled nucleotides following our previously described protocol [53]. The specificity of probes was verified using NCBI blast.

Probes detecting mouse αSyn mRNA [mouse alpha-synuclein (Snca), Figure 1]: Rat Snca DNA was used for generation of two different probes. A mouse-selective Snca probe (mSnca), referred to as mSnca, was generated to recognize endogenous Snca mRNA, designed in the untranslated region (UTR) 3’ region for high homology with the mouse Snca mRNA sequence and lacking counterpart in the transgenic construct (reference rat Snca NM_019169.3: sequence 639–1,035 specific for mouse Snca mRNA) (Figure 1A–B). Another probe detecting human and mouse SNCA (Snca) CDS (high sequence homology of mouse and human) was generated to recognize both endogenous mouse Snca and transgenic human SNCA (reference rat Snca NM_019169.3: sequence 59–1,037) mRNAs. This probe is referred to as probe detecting human and mouse alpha-synuclein mRNA mouse-selective alpha-synuclein probe (m/hSnca, Figure 1A–B).

Other probes were described previously, including probes detecting midbrain DA neuron markers, Aldh1a1, Girk2, Calb1, Calb2, gastrin-releasing peptide (Grp), Th, Vmat2, Dat, and vesicular glutamate transporter (Vglut2), which is also a marker of neurons with a glutamatergic phenotype [53, 54].

FISH experiments were performed following our previously described protocol [53]. To allow comparison between transgenic and control mice, each probe/probe pair was hybridized simultaneously on slides from transgene and control brain sections, and slides were treated in parallel throughout the protocol. Cryosections were air-dried, fixed in 4% paraformaldehyde (PFA, Merck Millipore, 1040051000), acetylated with 0.25% acetic anhydride (100 mmol/L, Sigma-Aldrich, A6404) in triethanolamine (pH 8, Sigma-Aldrich, T58300), hybridized in 100 µL formamide (Eurobio, GHYFOR01-01) buffer containing 1 µg/mL DIG and/or 1 µg/mL fluorescein-labeled probes at 65℃ for 18 h, followed by washes with sodium saline citrate (SSC, Invitrogen, AM9763) buffers of decreasing concentrations and maleic acid (Sigma-Aldrich, M0375) buffer, and blocked with 20% fetal bovine serum (FBS, Invitrogen, 10106-169) and 1% blocking solution. Riboprobe revelation was performed using horse-radish peroxidase (HRP) anti-fluorescein fab fragments (Sigma-Aldrich, 11426346910) diluted at 1/1,000, and biotin-labeled tyramide signal amplification (TSA) reagent (PerkinElmer, NEL749A001KT) followed by Neutravidin^TM-Oregon Green^TM (Invitrogen^TM, ThermoFisher, A6374), or anti-fluorescein fab fragments diluted at 1/5,000 and fluorescein-tyramide solution (ThermoFischer, 46410) diluted at 1/250. HRP-activity was stopped by incubation of sections in 0.1 mol/L glycine (Sigma-Aldrich, G7125) and 3% H₂O₂ (Sigma-Aldrich, 216763). DIG-epitopes were detected with HRP anti-DIG fab fragments diluted at 1/1,000 (Sigma-Aldrich, 11207733910) followed by cyanine 3 (Cy3)-labeled TSA reagent diluted at 1/150 (Akoya Biosciences, NEL744001KT) or Cy3-tyramide solution (Sigma Aldrich, GEPA13104) diluted at 1/100. DAPI (Sigma-Aldrich, D9542) was used as fluorescent nuclear counter-staining. Slides were covered and slipped using fluoromount (SouthernBiotech).

All images were acquired at 20× (0.46 µm/pixel) using a whole slide imaging digital slide scanner NanoZoomer S60 (Hamamatsu Photonics, C13210-01). Fluorescent images were acquired as multilayer images using filters for DAPI, fluorescein isothiocyanate (FITC), and tetramethylrhodamine isothiocyanate Cy3 (TRITC CY3) channels. Settings for optimized signal analysis (time exposure and gain of the camera) were set manually for each channel prior to scanning. Slides from control and transgenic mice for which the same probe/probe pair had been implemented in the FISH experiment were analyzed using the same settings under the same scanning session. Bright-field images were acquired as one-layer images. White-balance was performed prior to scanning and settings were kept throughout all scanning sessions. Slides from control and transgenic mice for which the same antibody was used in the immunohistochemical analysis were scanned in the same scanning session. The NDP.view2 software (Hamamatsu Photonics) was used for image visualization with high resolution and magnification, and analysis using multi-view function on original images with separate and simultaneous visualization of all channels (NanoZoomer Digital Pathology image set—NDPIS). Brain areas were defined using published atlas [55].

Analysis of m/hSnca and mSnca probe distribution was performed by thorough histological observations of sections from 3–5 mice per genotype (control and transgene), 6 slides per mouse, 10–19 sections per slide, between coordinates +3.56 mm and –5.52 mm from bregma. mSnca probe distribution in control and transgenic mice were considered as baseline. Elevated levels of m/hSnca probe distribution in a specific brain region of the transgenic mice were defined by increased density of labeling compared to baseline, as assessed by visual inspection.

Histological analysis of DA markers in the SNc and VTA subregions was performed by visual inspection of images from control and transgenic mice. For each probe/probe combination, 3–10 mice of each genotype were used. The distribution pattern of each DA marker in control and transgenic mice was compared to the distribution pattern of the Th probe and to reports from previous publications on the distribution pattern of the same DA markers in wild-type mice, including Th. Probe labeling was considered localized within a cell when five contiguous fluorescent puncta were present within the outline of the cell soma. Co-labeling/co-localization of two different probes/mRNAs was considered when both were detected within the same cell, as per the definition above. Cell nuclei counter-stained with DAPI were analyzed locally to control for one nucleus per cell soma.

Quantification was performed on 4 control and 4 transgenic mouse brains in the Thy1-h[A30P]αSyn transgenic mouse line in serial sections covering the SNc and VTA areas and analyzed for Alh1a1 and Th FISH-probe-positive signal (⁺). SNc was analyzed at three different coordinates: –3.08, –3.40, and –3.52 mm posteriorly from bregma. A total number of SNc cells was analyzed: control (2,060) and transgene (1,969). Paranigral nucleus (PN)/parainterfascicular nucleus (PIF) was analyzed at two levels –3.40 and –3.52 mm posteriorly from bregma; interfascicular nucleus (IF) was analyzed at two levels: –3.52 and –3.64 mm posteriorly from bregma. A total number of PN/PIF/IF cells was analyzed: control (1,131) and transgene (1,153). Caudal linear nucleus (CLi) was analyzed at two levels: –3.80 and –4.04 mm posteriorly from bregma. A total number of CLi cells analyzed: control (206) and transgene (201). No counting in rostral linear nucleus (RLi, as almost no Th⁺ cells are present in RLi). Counting was made manually using NDP.view2 and QuPath software [56]. Pie charts were generated to show the percentage of Th⁺/Aldh1a1⁺ cells vs. Th⁺/Aldh1a1-negative (^–) cells. All Aldh1a1 cells are Th⁺; thus, no percentage is indicated for Th^–/Aldh1a1⁺ cells. Quantification was made in an identical manner in Thy1-h[wt]αSyn transgenic mouse line (n = 2 of each genotype).

Two FISH probes were generated to detect Snca mRNA in brain sections of mice, a dual probe recognizing both endogenous mouse Snca mRNA and transgenic human SNCA mRNA, here referred to as the m/hSnca probe, and a second probe selectively recognizing the UTR 3’ region of mouse Snca mRNA, here referred to as the mSnca probe (Figure 1A–B, illustration of probe design). Due to high sequence homology between mouse and human CDS, it was not possible to generate a probe selectively recognizing human SNCA CDS, instead the dual m/hSnca probe was compared with the selective mSnca probe. In the brains of control mice, where no human SNCA transgene is present, the m/hSnca probe should thus only detect the endogenous Snca mRNA and show the same detection as the mSnca probe. In contrast, in the Thy1-h[A30P]αSyn and Thy1-h[wt]αSyn transgenic mice, the m/hSnca probe should detect both endogenous Snca and transgenic SNCA mRNA, and any detection beyond that of Snca is due to the transgene. This was verified using FISH analysis in coronal brain sections, which showed substantially more expression of the dual m/hSnca probe in each transgene than in respective controls, thereby validating the new probe (Figure 1C). Further, the mSnca probe appeared similar between genotypes (control vs. transgene) for both transgenic lines (Figure 1C).

To further investigate the extent of both endogenous Snca expression and that induced by the transgene, visual analysis in combination with FISH analysis in serial coronal sections throughout the brain of control and transgenic mice for both transgenic mouse lines was performed. Assessment of control mice for the Thy1-h[A30P]αSyn transgenic line showed m/hSnca probe distribution throughout the brain. Positive brain areas included the cerebral Ctx, anterior olfactory area (AO), hippocampus, amygdala, STN, ZI, dorsal raphe, pontine nucleus, median raphe nucleus, bed nucleus of the stria terminalis, ventral pallidum (VP), retrorubral field (RRF), and GP (Figure 2A–F, Table 1). In Thy1-h[A30P]αSyn transgenic mice, the m/hSnca probe was present in the same areas but with substantially stronger labeling (Figure 2G–L, Table 1). Further, in transgenic mice, m/hSnca probe labeling was also detected in additional areas not detected in control mice, such as the septal hippocampal nucleus, lateral habenula, interpeduncular nucleus, and sub-brachial nucleus (Figure 2G–L, Table 1). To selectively assess the impact of the transgene upon endogenous mouse Snca, the mSnca probe was analyzed and compared with the results of the m/hSnca probe (Figure 1C1–C18, Table 1). In both control and transgenic mice, mSnca labeling was detected throughout the same brain areas as listed above for the m/hSnca probe in control mice, thus not showing any detectable transgene-induced alterations (Figure 1C1–C18, Table 1).

Similar results were seen when addressing the Thy1-h[wt]αSyn transgenic mouse line. Analysis of control mice showed m/hSnca probe distribution throughout the brain, in the same areas as listed above for control mice of the Thy1-h[A30P]αSyn transgenic line, as well as in the glomerular layer of olfactory bulb, lateral habenula, and subbrachial nucleus of the midbrain (Figure 2M–R, Table 1). In Thy1-h[wt]αSyn transgenic mice, m/hSnca probe distribution was detected in the same areas as in controls. However, as seen with the Thy1-h[A30P]αSyn transgenic mice, labeling was substantially stronger in Thy1-h[wt]αSyn transgenic mice than in controls (Figure 2S–X, Table 1). As with Thy1-h[A30P]αSyn transgenic mice, also Thy1-h[wt]αSyn transgenic mice showed m/hSnca probe distribution in additional areas not identified in the control mice (Figure 2S–X, Table 1). These included the interpeduncular nucleus and the granular cell layer of the olfactory bulb (Table 1). In addition, similar results as above were obtained using the mSnca probe detecting endogenous Snca mRNA; FISH mSnca probe labeling was detected throughout the same brain areas in both control and transgenic mice as for the m/hSnca probe in control mice (Figure 1C19–C36, Table 1).

The results obtained by comparing the distribution and intensity of labeling between control and transgenic mice of both αSyn transgenic mouse lines suggest that expression seen in control mice with the dual m/hSnca probe, which is the same as seen with the mouse-selective mSnca probe in both control and transgenic mice, represents endogenous Snca. Further, expression beyond this is an effect of the SNCA transgene, which is detected as stronger and more wide-spread than the endogenous Snca in both mouse models of synucleinopathy.

Based on the verified expression of the human SNCA transgene throughout the brain, it was of interest to examine if SNc and VTA DA neurons were affected in the transgenic mice. First, m/hSnca and mSnca probes were analyzed in co-FISH analysis in combination with a Th probe. TH is the rate-limiting enzyme in DA synthesis, and TH protein as well as Th mRNA represent common general markers for midbrain DA neurons.

In control mice for the Thy1-h[A30P]αSyn transgenic mouse line, both the m/hSnca and mSnca probes co-localized almost entirely with the Th probe in both SNc and VTA, verifying expression in DA neurons (Figure 3A, A1, A1’–A1’’’, C, C’–C’’’). Notably, cells negative for Th were also negative for the m/hSnca and mSnca probes. The distribution in the Thy1-h[A30P]αSyn transgenic mice was different. Here, the distribution of the m/hSnca probe was substantially more widespread than in control mice. In fact, the m/hSnca probe labeled both Th⁺ and Th^– neurons in SNc and VTA of transgenic mice, showing that the transgene is expressed in non-DA neurons of the SNc and VTA (Figure 3B, B1, B’–B1’’’). In contrast, distribution of the mSnca probe showed no difference between Thy1-h[A30P]αSyn transgenic mice and controls, and co-localized completely with the Th probe (Figure 3C, C’–C’’’, D, D’–D’’’). These differences suggested that detection of the m/hSnca probe in Th^– neurons is due to presence of transgenic SNCA mRNA. Additionally, while an elevated level of m/hSnca probe detection was observed in SNc and VTA of Thy1-h[A30P]αSyn transgenic mice, the strength of m/hSnca probe detection in Th⁺ neurons did not seem increased compared to either controls or mSnca probe detection (Figure 3A1, A1’–A1’’’, B1, B1’–B1’’’, E, E’–E’’’, F, F’–F’’’). The results are in accordance with previous knowledge that the Thy-1 promoter does not drive transgenic expression strongly in the SNc and VTA [57]. Instead, the increase in labeling was due to m/hSnca probe detection in several Th^– neurons. Further, in Thy1-h[A30P]αSyn transgenic mice, but not control mice, substantial m/hSnca probe distribution, but not mSnca probe distribution, was observed in additional midbrain nuclei, including the periaqueductal gray, medial accessory oculomotor nucleus, medial geniculate nucleus, red nucleus (RN), SNr, and the interpeduncular nucleus, further emphasizing the strength of the transgene in the midbrain (Figure 3, Table 1).

When addressing SNc and VTA in the Thy1-h[wt]αSyn transgenic mouse line, it was found that the m/hSnca and mSnca probes were equally strong in control mice, representing endogenous Snca mRNA (Figure 4A, A1, A1’–A1’’’, C, C’–C’’’, E, E’–E’’’). Further, the mouse-selective mSnca probe was equally strong in control and transgenic mice (Figure 4C, C’–C’’, D, D’–D’’’, E, E’–E’’’, F, F’–F’’’). However, the m/hSnca and mSnca probes did not show perfect co-localization with the Th probe, instead a small number of Snca⁺ cells were negative for Th (Figure 4A, A1, A1’–A1’’’, C, C’–C’’’).

In SNc and VTA of Thy1-h[wt]αSyn transgenic mice, distribution of the m/hSnca probe was much stronger and more widespread than in control mice, and labeling was detected in both Th⁺ and Th^– neurons (Figure 4A, A1, A1’–A1’’’, B, B1, B1’–B1’’’). Further, Thy1-h[wt]αSyn transgenic mice showed substantial m/hSnca probe distribution in additional midbrain areas, with the same regional distribution as observed for the Thy1-h[A30P]αSyn transgenic mice (Figure 4B, B1, B1’–B1’’’, F, F’–F’’’, Table 1).

In summary, by comparing the distribution of the mSnca probe and the m/hSnca probe in the midbrain, it is evident that the m/hSnca probe showed more widespread distribution in transgenic mice than in control mice, for both αSyn transgenic mouse lines analyzed. In SNc and VTA of the Thy1-h[A30P]αSyn transgenic mouse line, no difference between control and transgenic mice was observed using the mouse-selective mSnca probe, while the m/hSnca probe showed more abundant distribution and was present in both Th⁺ and Th^– neurons in transgenic mice, but not in control mice, thus representing transgenic expression. Few such positive cells were detected in control mice of the Thy1-h[wt]αSyn mouse line. However, by far, most m/hSnca probe labeling in Th^– cells in SNc and VTA was seen in transgenic Thy1-h[wt]αSyn mice. Thus, in both mouse models of synucleinopathy, endogenous Snca is found normal (compared to controls) in SNc and VTA with detection in DA neurons. In contrast, transgenic mice of both models showed transgenic SNCA expression in Th^– cells in the midbrain, without elevation in Th⁺ cells. Thus, non-DA neurons of SNc and VTA showed SNCA mRNA labeling, giving rise to stronger overall labeling throughout SNc and VTA without specific elevation in DA neurons.

Following the observation of an almost ubiquitous presence of transgenic SNCA mRNA in the brain of both Thy1-h[A30P]αSyn and Thy1-h[wt]αSyn transgenic mice, including SNc and VTA neurons both positive and negative for Th mRNA, the integrity of DA neurons were further analyzed using general and subpopulation-specific markers.

First, Th mRNA was analyzed in serial sections along the anterior-posterior axis in control and transgenic mice of the two mouse lines. For ease of identification of cellular locations in the anatomically heterogeneous VTA, this area was divided into subareas in accordance with published atlas [55]: PBP, RLi, IF, PN, and PIF (Figure 5). Using a probe directed towards mouse Th mRNA, DA neurons of SNc and VTA were readily detected in control mice, including all subareas of the VTA (Figure 5D–F, J–L). The highest density of Th⁺ cells was seen in the SNc, PBP, PN, and PIF, followed by lower levels in the IF and RLi, in accordance with the literature [58, 59]. SNc and VTA of Thy1-h[A30P]αSyn and Thy1-h[wt]αSyn transgenic mice showed the same distribution pattern of Th mRNA as control mice for each respective transgene (Figure 5G–I, M–O). Visual analysis of mRNA in 8–10 mice per genotype did not reveal any detectable differences between control and transgenic mice of either the Thy1-h[A30P]αSyn and Thy1-h[wt]αSyn mouse lines.

This preserved pattern of Th mRNA suggests intact integrity of the distribution of DA neurons in SNc and VTA in the presence of abundant human SNCA transgene. To further validate the integrity of SNc and VTA DA neurons, heterogeneity was next analyzed by addressing subpopulation-specific markers.

Several molecular markers have been described to dissociate between subpopulations (also known as subtypes) of DA neurons within SNc and VTA [32, 33, 36]. To validate the integrity of SNc and VTA DA neurons, indicated by the finding above that Th mRNA appeared normal in transgenic compared to control mice, subpopulation-markers were next selected for analysis in co-FISH experiments on serial sections through midbrain of control and transgenic mice. Probes directed towards the following mRNAs were included in the analysis, and compared with general DA neuron markers Th, Vmat2, and/or Dat: Aldh1a1, Calb1, Calb2, Girk2, Grp, Vglut2 (Figure 6, Table S1). All probes were validated in previous studies [53, 54]. In both rodents and primates, several studies have used Girk2 and Calb2 as markers to separate and distinguish between SNc and VTA DA neurons, respectively. However, neither is distinct for SNc or VTA but is expressed at more or less high levels in both areas [26, 54, 60, 61]. Similar is true for most subpopulation markers, distribution patterns are rarely absolute but relative. For example, Aldh1a1 mRNA has been described to be mainly distributed in the ventral aspect of SNc and part of VTA, while Calb2, Vglut2, and Grp show more medial than lateral distribution within the VTA [26, 32, 33, 36].

SNc and VTA of the Thy1-h[A30P]αSyn transgenic mouse line were analyzed using Vmat2, Aldh1a1, Girk2, and Vglut2 probes in combination with the Th probe to validate the detection of DA neurons. In accordance with the literature [62], control mice showed distribution of Vmat2 mRNA in all Th⁺ neurons within both the SNc and VTA (Figure 6A1, A1’–A1’’’). Aldh1a1 mRNA was most prominently detected in the ventro-medial SNc and in the ventral aspect of the VTA, in the PN and PIF areas, and weaker in the dorsal SNc and PBP while modest in the IF and excluded from the RLi (Figure 6A3, A3’, A3’’’). Girk2 mRNA was detected in the SNc but also in the lateral VTA, where it was stronger in the PBP and modest in the PN and PIF, weak in the IF and excluded from the RLi (Figure 6A5, A5’, A5’’’). Vglut2 mRNA was most prominently detected in the IF and RLi, more modest in the PBP, PN, and PIF areas, and weak in the dorsal SNc (Figure 6A7, A7’, A7’’’). Calb1 mRNA was detected in the PN, PIF, and PBP areas, modest in the IF, weak in the RLi, modest in the ventral SNc and weak in the dorsal SNc (Figure 6B1, B1’–B1’’’). Calb2 mRNA was primarily detected in the IF, PIF, and PN, and was also found in the lateral PBP and ventral SNc, while modest in the medial PBP, RLi, and dorsal SNc (Figure 6B3, B3’–B3’’’).

In Thy1-h[A30P]αSyn transgenic mice, the results were identical to those of control mice. All mRNAs showed the same distribution pattern as in the control mice: Vmat2 (Figure 6A2, A2’, A2’’’), Aldh1a1 (Figure 6A4, A4’, A4’’’), Girk2 (Figure 6A6, A6’, A6’’’), Vglut2 (Figure 6A8, A8’, A8’’’), Calb1 (Figure 6B2, B2’–B2’’’), and Calb2 (Figure 6B4, B4’–B4’’’). Further, in accordance with literature [26, 30] and in addition to Vmat2 mRNA, also Aldh1a1 and Girk2 mRNAs co-localized extensively with Th mRNA in control mice (Figure 6A1, A1’–A1’’’, A3, A3’–A3’’’, A5, A5’–A5’’’). The same was observed for the Thy1-h[A30P]αSyn transgenic mice (Figure 6A2, A2’–A2’’’, A4, A4’–A4’’’, A6, A6’–A6’’’). Thus, when comparing Thy1-h[A30P]αSyn transgenic mice with control mice, the same level of intensity and the same distribution patterns were observed for each mRNA selected to represent DA cell groups within the SNc and VTA as a whole (Th and Vmat2) or subpopulations within these (Aldh1a1, Girk2, Calb1, Vglut2, and Calb2).

Next, SNc and VTA of Thy1-h[wt]αSyn transgenic mice were analyzed in the same way and compared with control mice. Here, probes targeting Vmat2, Aldh1a1, Calb1, and Calb2 mRNA were analyzed in co-FISH analysis in combination with the Th probe. Additionally, a probe toward Grp mRNA, a marker of medial VTA was also analyzed in combination with Th [26, 63]. Further, Girk2 was addressed in co-FISH analysis in combination with the Dat probe.

Similar as described above, and in accordance with the literature [62], control mice showed distribution of Vmat2 mRNA in all Th-positive neurons within both the VTA and SNc; the same observation was done for Thy1-h[wt]αSyn transgenic mice (Figure 7A1, A1’–A1’’’, A2, A2’–A2’’’). Aldh1a1 (Figure 7A3, A3’–A3’’’, A4, A4’–A4’’’) and Girk2 (Figure 7A5, A5’–A5’’’, A6, A6’–A6’’’) were similar in control and transgenic mice, and also similar as described above for the Thy1-h[A30P]αSyn mouse line. The highest levels of Dat mRNA in control and transgenic mice were detected in the SNc, PBP, PN, and PIF, followed by lower levels in the IF (Figure 7A5, A5’–A5’’’, A6, A6’–A6’’’), while Grp mRNA was primarily detected in the ventral aspect of the VTA, PN, PIF, and IF, and was weaker in the PBP and ventral SNc, and was excluded from the RLi, dorsal PBP, and dorsal SNc (Figure 7A7, A7’–A7’’’, A8, A8’–A8’’’), in accordance with literature [26, 63]. Calb1 mRNA was detected in the PN, PIF, and PBP areas, with more modest detection in the IF and ventral SNc and weak in the RLi and dorsal SNc (Figure 7B1, B1’–B1’’’, B2, B2’–B2’’’). Calb2 was detected primarily in the IF, PIF, and PN, but also in the lateral PBP and ventral SNc, while modest in the medial PBP, RLi, and dorsal SNc (Figure 7B3, B3’–B3’’’, B4, B4’–B4’’’). Similar expression was detected in control and transgenic mice. In addition to Vmat2, also Aldh1a1 and Grp mRNAs co-localized extensively with Th mRNA in control mice (Figure 7A3, A3’–A3’’’, A7, A7’–A7’’’), as expected based on literature [26, 30, 63], and the same was seen for Thy1-h[wt]αSyn transgenic mice (Figure 7A4, A4’–A4’’’, A8, A8’–A8’’’).

To further verify the findings, Aldh1a1 mRNA was selected for quantification, by counting cells positive for Aldh1a1 and Th, and their comparison. For this analysis, serial sections at the same levels posteriorly from bregma (–2.92, –3.28, –3.40, –3.64, –3.80, and –4.04 mm) covering the entire SNc and VTA region in an anterior-posterior manner from Thy1-h[A30P]αSyn transgenic mice and matching control mice were implemented which allowed careful quantitative assessment throughout SNc and VTA (PBP, IF, and PN/PIF) and also including CLi and RRF in four mice of each genotype. Th⁺/Aldh1a1⁺ cells (Figure 8A–L) and Aldh1a1⁺ cells (Figure 8A’–L’) were analyzed and quantified. The counts revealed that control and transgenic mice contained the same number of labeled cells (Th⁺/Aldh1a1⁺ and Th⁺/Aldh1a1^–), further confirming the visual inspection above (Th⁺/Aldh1a1⁺, control vs. transgene: SNc, 65% vs. 67%; IF/PN/PIF, 63% vs. 61%; PBP, 78% vs. 78%; CLi, 76% vs. 75%. Th⁺/Aldh1a1^–, control vs. transgene: SNc, 35% vs. 33%; IF/PN/PIF, 37% vs. 39%; PBP, 22% vs. 22%; CLi, 24% vs. 25%, Figure 8M).

A similar method of quantification was performed in Thy1-h[wt]αSyn mice and matching control mice, albeit in fewer mice, precluding relevant analysis. The results revealed a similar amount of Th⁺/Aldh1a1⁺ and Th⁺/Aldh1a1^– number of positive cells as listed above for Thy1-h[A30P]αSyn control vs. transgenic mice.

In summary, no differences between control mice and transgenic mice were observed for any of the mRNAs assessed, either for the Thy1-h[A30P]αSyn transgenic mouse line or the Thy1-h[wt]αSyn transgenic mouse line. In fact, all patterns of mRNAs analyzed were identical between control and transgenic mice of both mouse lines (summarized in Figure 9).