Table 1.
Primary Antibodies.
Table 2.
Secondary Antibodies.
Figure 1.
Characterization of tyrosine hydroxylase (TH) positive cells in A11.
A: Diagram showing the middle region of the A11 area in the mouse. The red frame represents the area where representative micrographs were taken. The diagram was adapted from [24] with permission. B: TH immunohistochemistry (green) of the middle region of A11. Scale bar 100 µm. 3 V = third ventricle, PF = parafascicular thalamic nucleus, fr = fasciculus retroflexus, PH = posterior hypothalamic nucleus, mt = mammillothalamic tract. C: Diameter of TH positive cells in three regions - rostral (AP −2.0 mm), middle (AP −2.3 mm) and caudal region (AP −2.5 mm). The mean cell diameter was 16.7±0.3 µm and the cell diameters between sections were similar.
Figure 2.
A11 TH positive neurons project to the spinal cord.
Example of retrograde labelling in the A11 caudal area following FluoroGold (FG) injections into the lumbar spinal cord between lumbar vertebral segments L4 and L5. Representative double-fluorescent immunostaining of tyrosine hydroxylase (TH; green) and FluoroGold (FG; magenta). The white arrows point to double-labeled cells. Most of the TH positive cells also contain FG (arrows), one cell shows labelling for TH but not FG. Several cells that contain FG but not TH are also seen (not co-localizing). Scale bar: 50 µm.
Figure 3.
YFP expression in A11 neurons following Cre-dependent viral expression and retrograde tracing with FG from the spinal cord.
Rostral region section of A11 from a TH-Cre mouse transduced with a Cre-dependent YFP AAV (green) and immunohistochemistry against FG (magenta) (A–D). Boxed inset in (B (b)) shows FG labeled cells in the motor cortex. Only cells in A11 were co-labeled with YFP and FG (C, D). Scale bars: 50 µm. D: Higher magnification of boxed area in A. Scale bar: 20 µm.
Figure 4.
YFP positive fibers in the lumbar spinal cord originating from A11.
Schematic of fiber localization in the lumbar spinal cord. Representative micrographs of YFP-labeled fibers in transverse (A, B) and parasagittal (C, D) lumbar spinal cord sections. Fibers were found in the dorsal (A) and ventral horn (B) as well as in the grey (C) and the white matter (D). Scale bar: 10 µm.
Figure 5.
A11 TH positive neurons are also expressing aromatic amino acid decarboxylase (AADC).
Immunohistochemistry targeted against TH (green) and AADC (magenta) shown in a representative middle region section of A11 (A, B) and the locus coeruleus (C). Note the co-localization between TH and AADC positive neurons (arrows) in A11 (B). Locus coeruleus served as a positive control (C). Scale bar 50 µm. A and B are derived from data shown in Figure 1. B: Higher magnification of boxed area in A.
Figure 6.
Vesicular monoamine transporter (VMAT-2) expression in A11 and locus coeruleus.
Immunohistochemistry targeted against TH (green) and VMAT-2 (magenta) in a representative micrograph of a middle region section of A11 (A, B). Note the co-localization between TH and VMAT-2 indicated by the arrows (B). Locus coeruleus served as a positive control (C). B: Higher magnification of boxed area in A. Scale bar: 50 µm.
Figure 7.
Absence of dopamine transporter (DAT) expression in A11.
Immunohistochemistry targeted against TH (green) and DAT (magenta) (A–D). Representative micrographs showing the absence of DAT labeling in the middle region of A11 (A, B). Note the DAT positive but TH negative fibers in A11 (B) (arrow). (C) As expected DAT is absent in the locus coeruleus (LC) which served as a negative control. Representative micrograph of TH and DAT labeled neurons and fibers in the ventral tegmental area (VTA) (D). Note the absence of DAT expression in the A11 and the LC in contrast with the intense expression in the VTA (positive control). B: Higher magnification of boxed area in A. Scale bar: 50 µm.
Figure 8.
YFP positive fibers in the lumbar spinal cord originating from A11 lack dopamine transporter (DAT).
Immunohistochemistry targeted against YFP (green) and DAT (magenta) (A–C). Representative micrographs showing YFP-labeled fibers in parasagittal lumbar spinal cord sections in the grey (A) and white (B) matter. (C) Positive control showing intense expression of DAT in the substantia nigra compacta. Scale bar: 10 µm.