Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A and C) Representative images of midbrain (A) and striatum (C) 2 weeks after bilateral injections of AAV1-EF1α-eGFP- Rosa26 gRNAs (left) and AAV1-EF1α-eGFP- Th gRNAs (right) into the midbrain of Dat-iCre x LSL-Cas9 rats. GFP fluorescence (green) served as a marker for gRNA delivery. (B, D,F, H) Quantification of TH immunoreactivity was assessed and shown as integrated density. ROIs were drawn using GFP fluorescence. (B and D) Each data point represents 1 animal, where 3 Rosa26 or Th gRNA hemispheres from coronal sections were averaged for each animal (n=6 animals, 3M, 3F). Paired t-tests were performed: B) t=20.38, df=5, ****p<0.0001. D) t=1.219, df=5, p = 0.2772. TH immunoreactivity is significantly reduced in midbrain (B) but not striatum (D) two weeks post gRNA injection. (E and G) Representative images of midbrain (E) and striatum (G) 4 weeks after bilateral injections of AAV1-EF1α-eGFP- Rosa26 gRNAs (left) and AAV1-EF1α-eGFP- Th gRNAs (right) into the midbrain of Dat-iCre x LSL-Cas9 rats. Each data point represents 1 animal, where 3 Rosa26 or Th gRNA hemispheres from coronal sections were averaged for each animal (n=5 animals, 3M, 2F). Pair t-tests were performed: F) t=4.167, df=4, ***p<0.0141. H) t=9.625, df=4, ***p<0.0007. TH immunoreactivity is significantly reduced in midbrain (F) and striatum (H) four weeks post gRNA injection. Scale bars = 1mm

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques: Fluorescence, Marker, Injection

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A) Mean responses are shown for both the Rosa26 gRNA (black) and Th gRNA (red) hemisphere. Individual recordings are shown in light gray. (B) Mean response for the control recordings is scaled to the peak amplitude of the mean Th gRNA response. Note the decay of the Rosa26 gRNA response is slightly prolonged, relative to the Th gRNA response. (C) Effect of 4-AP (30 µM) on the DA signal. Inset shows the baseline response scaled to the peak amplitude of the 4-AP response. (D) DA release under baseline conditions (gray) and in the presence of 4-AP (orange). Under both baseline conditions and in the presence of 4-AP, release did not significantly differ between control and Th gRNA (two way RM-ANOVA, F 1,36 = 3.44, p =0.07; Baseline control vs. baseline TH-, p = 0.68; 4-AP control vs. 4-AP TH-, p = 0.08, Sidak post-hoc). 4-AP significantly increased DA release in both Rosa26 and Th gRNA (two way RM-ANOVA, F 1,36 = 74.05, p <0.001; ***p <0.001, Th gRNA baseline vs. Th gRNA 4-AP; Rosa26 baseline vs. Rosa26 4-AP, Sidak-post hoc). (E) DA uptake summary: Under baseline conditions, uptake was slightly but significantly increased in Th gRNA slices, relative to control slices (two way RM-ANOVA, F 1,36 = 5.64, p =0.02; *p=0.04, baseline Rosa26 gRNA vs. baseline Th gRNA, Sidak post-hoc). This increase was absent following 4-AP treatment (4-AP Rosa26 gRNA vs. 4-AP Th gRNA, p =0.30, Sidak post-hoc). Note that 4-AP significantly decreased uptake in both Rosa26 gRNA and Th gRNA slices (two way RM-ANOVA, F 1,36 = 76.18, p <0.001; ***p< 0.001, Rosa26 gRNA baseline vs. Rosa26 gRNA 4-AP; Th gRNA baseline vs. Th gRNA 4AP, Sidak post-hoc). (F-I) HPLC measuring dopamine and dopamine metabolites from Dat-iCre x Cas9 brain punches two weeks post gRNA injection. (F) DA concentration (ng/g) from midbrain punches (paired t test, t = 9.35, df = 4, ***p = 0.0007). (G) DA concentration (ng/g) from striatal punches (paired t test, t = 0.07, df = 4, p = 0.95). (H) DOPAC concentration (ng/g) from striatal punches (paired t test, t = 0.55, df = 4, p = 0.61). (I) HVA concentration (ng/g) from striatal punches (paired t test, t = 0.55, df = 4, p = 0.61).

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques: Control, Injection, Concentration Assay

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A)UMAP representing cell clusters across integrated midbrain dataset. (B) Number of DEGs identified in each cell cluster when comparing Th gRNA dataset to Rosa26 gRNA dataset within clusters. Blue bars on left represent downregulated genes and red bars on right represent upregulated genes. (C – E) Lollipop plots of enriched GO: Biological Processes pathways from significantly differentially expressed genes identified through ShinyGo gene enrichment tool. Color indicates -log 10 (FDR), dot size represents number of DEGs identified in pathway, and x-axis indicates fold enrichment. Cell clusters shown are (C) astrocytes, (D) GABAergic neurons, and (E) glutamatergic neurons.

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques: Cell-Signaling

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A)UMAP representing cell clusters across integrated striatum datasets. (B) Number of DEGs identified in each cell cluster when comparing Th gRNA dataset to Rosa26 gRNA dataset within clusters. Blue bars on left represent downregulated genes and red bars on right represent upregulated genes (C-E) Lollipop plots of enriched GO: Biological Processes pathways from significantly differentially expressed genes identified through ShinyGo gene enrichment tool. Color indicates -log 10 (FDR), dot size represents number of DEGs identified in pathway, and x-axis indicates fold enrichment. Cell clusters shown are (C) PV+ interneurons, (D) DRD1 MSNs, and (E) DRD2 MSNs.

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques:

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A)Significant DEGs ( Th gRNA vs Rosa26 gRNA) in midbrain dopaminergic cluster. y-axis is average log2FoldChange and color depicts adjusted p-value. (B) Representative images of midbrain 2 weeks post 2 weeks after bilateral injections of AAV1-EF1α-eGFP- Rosa26 gRNAs (top) and AAV1-EF1α-eGFP- Th gRNAs (bottom). Images include individual channels of GFP (green), TH (red), NRG1 (magenta). Scale bar = 50µm. (C) Quantification of NRG1 immunoreactivity from 20x whole section scans (not shown) of same tissue used in high magnification images in B, shown as integrated density (n=6, 3M, 3F). ROIs were drawn using GFP fluorescence. Each data point represents 1 animal, where 3 Rosa26 or Th gRNA hemispheres from coronal sections were averaged for each animal (n=6 animals, 3M, 3F). Pair t-tests were performed: t=2.610, df=5, *p = 0.048.

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques: Tissue, Fluorescence

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: (A)Relative cell numbers within each cell cluster from midbrain snRNAseq Rosa26 gRNA (Black) and Th gRNA (red) datasets presented as percentage of total cells in dataset (B) Lollipop plots of enriched GO: Biological Processes pathways from significantly differentially expressed downregulated genes in mature oligodendrocyte midbrain clusters. Pathways identified through ShinyGo gene enrichment tool. Color indicates -log 10 (FDR), dot size represents number of DEGs identified in pathway, and x-axis indicates fold enrichment.

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques:

Journal: bioRxiv

Article Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

doi: 10.64898/2026.10.09.757761

Figure Lengend Snippet: Recordings were performed in striatal brain slices from 5 rats. Analyses were performed on 14 neurons from the control hemisphere and 12 neurons from the Th gRNA hemisphere. Whole-cell current-clamp recordings revealed no significant differences between Rosa26 and Th KO neurons in number of action potentials (A-B), resting membrane potential (C), input resistance (D), rheobase (E), half-width (F), fast afterhyperpolarization amplitude (G), adaptation ratio (H), or mean interspike interval (I) (all p > 0.12; ). Analysis of firing frequency-current relationships using a REML mixed-effects model demonstrated a significant effect of injected current (F(12,285)=124.6, p<0.0001), but no main effect of treatment (F(1,24)=1.877, p=0.183) and no treatment × current interaction (F(12,285)=0.954, p=0.493), indicating similar excitability across current injection levels in Control and TH KO neurons .

Article Snippet: Animals were injected with pAAV Rosa26 gRNA A+B EF1a EGFP (Addgene 113156) and pAAV TH gRNA A+B pair EF1a EGFP (Addgene 113155) previously described in Bӓck et al 2019 at a viral titer of 1.95e12 viral genomes/mL.

Techniques: Control, Membrane, Analysis, Injection