Dopamine D1 Receptor Immunoreactivity on Fine Processes of GFAP-Positive Astrocytes in the Substantia Nigra Pars Reticulata of Adult Mouse

Abstract

Substantia nigra pars reticulata (SNr), the major output nucleus of the basal ganglia, receives dopamine from dendrites extending from dopaminergic neurons of the adjacent nucleus pars compacta (SNc), which is known for its selective degeneration in Parkinson's disease. As a recipient for dendritically released dopamine, the dopamine D1 receptor (D1R) is a primary candidate due to its very dense immunoreactivity in the SNr. However, the precise location of D1R remains unclear at the cellular level in the SNr except for that reported on axons/axon terminals of presumably striatal GABAergic neurons. To address this, we used D1R promotor-controlled, mVenus-expressing transgenic mice. When cells were acutely dissociated from SNr of mouse brain, prominent mVenus fluorescence was detected in fine processes of glia-like cells, but no such fluorescence was detected from neurons in the same preparation, except for the synaptic bouton-like structure on the neurons. Double immunolabeling of SNr cells dissociated from adult wild-type mice brain further revealed marked D1R immunoreactivity in the processes of glial fibrillary acidic protein (GFAP)-positive astrocytes. Such D1R imunoreactivity was significantly stronger in the SNr astrocytes than that in those of the visual cortex in the same preparation. Interestingly, GFAP-positive astrocytes dissociated from the striatum demonstrated D1R immunoreactivity, either remarkable or minimal, similarly to that shown in neurons in this nucleus. In contrast, in the SNr and visual cortex, only weak D1R immunoreactivity was detected in the neurons tested. These results suggest that the SNr astrocyte may be a candidate recipient for dendritically released dopamine. Further study is required to fully elucidate the physiological roles of divergent dopamine receptor immunoreactivity profiles in GFAP-positive astrocytes.

Keywords: basal ganglia; dendritic release; glia; striatum; visual cortex.

Figure 1

Immunoreactivity of anti-dopamine D1 and anti-dopamine D2 receptor antibodies used in wild-type mouse substantia nigra. (A) Dense dopamine D1 receptor (D1R) immunoreactivity was detected in substantia nigra pars reticulata (SNr), but not in pars compacta (SNc). (B) Abundant GABAergic neurons were shown in the SNr by parvalbumin (Parv) immunoreactivity. (C) Dopamine D2 receptor (D2R) immunoreactivity was remarkable in the SNc, but only sparse in the SNr. (D) Tyrosine hydroxylase (TH) immunoreactivity was well correlated with D2R immunoreactivity in the substantia nigra. Dashed lines indicate the outer boundary for dissecting out SNr tissue with a customized needle having 200 μm-thick wall. The orientation and scale are common to A–D.

Figure 2

Expression of mVenus signal in a coronal slice dissected from the Drd1-mVenus mouse brain and that in cells dissociated from the SNr of the mouse. (A–C) Images of a 500 μm-thick coronal section, in which both SNr at the oculomotor nerve and visual cortex were included, revealing a prominent mVenus signal in the SNr (arrowheads). (A), (B), and (C) show a bright field image, mVenus fluorescence, and a merged image, respectively. Note the considerable difference in mVenus fluorescence between SNr and visual cortex. (D–F) Cells acutely dissociated from the SNr of adult Drd1-mVenus mouse. (D) Differential interference contrast image. (E) A glia-like cell bearing mVenus-positive fine processes (filled arrow) and a neuron exhibiting a very weak mVenus fluorescence (empty arrow). (F) Merged image of (D,E). Synaptic bouton-like structures showing mVenus signal (see inset for a magnified view). Nuclear staining with DAPI (blue) was overlaid in (E,F). (G–I) Similar to (D–F), but a typical glia-like cell bearing bushy mVenus-positive processes in a magnified view (filled arrow).

Figure 3

Double immunolabeling of SNr cells dissociated from the adult wild-type mouse brain with anti-D1R and anti-GFAP antibodies. (A–D) A typical D1R-positive/GFAP-positive SNr astrocyte (filled arrow). A typical D1R-negative (GFAP-negative) SNr neuron is shown in the same field of view (empty arrow). (A), (B), (C), and (D) represent represent D1R immunofluorescence, GFAP immunofluorescence, differential interference contrast image, and a merged image of these, respectively. Nuclear staining with DAPI (blue) was overlaid. Inset in (D) depicts a magnified view of D1R-positive synaptic bouton-like structures on the SNr neuron. D1R-negative small cells bearing no fine process were excluded from the analysis due to too much digestion and/or mechanical damage.

Figure 4

Double immunolabeling of visual cortical cells dissociated from the adult wild-type mouse brain with anti-D1R and anti-GFAP antibodies. (A-D) Similar to Figure 3, but for cells dissociated from the visual cortex. A typical D1R-negative/GFAP-positive visual cortical astrocyte (filled arrow). A typical D1R-negative (GFAP-negative) visual cortical neuron is shown in the same field of view (empty arrow). Nuclear staining with DAPI (blue) was overlaid in (A,B,D).

Figure 5

Double immunolabeling of striatal cells dissociated from the adult wild-type mouse brain with anti-D1R and anti-GFAP antibodies. (A–D) Similar to Figure 4, but for cells dissociated from the striatum. A typical D1R-positive/GFAP-positive striatal astrocyte (filled arrowhead) is shown. A typical D1R-negative (GFAP-negative) striatal neuron is also shown in the same field of view (empty arrow). In contrast to (A–D), (E–H) show a typical D1R-negative/GFAP-positive striatal astrocyte (empty arrowhead). A typical D1R-positive (GFAP-negative) striatal neuron is also shown in the same field of view (filled arrow). Nuclear staining with DAPI (blue) was overlaid except for (C,G).

Figure 6

Divergence in D1R immunoreactivity of cells acutely dissociated from SNr, visual cortex, and striatum of the adult wild-type mouse brain. (A–D) The intensity profiles of D1R immunofluorescence, which were measured for isolated cells from SNr (A), visual cortex (B), and striatum (C), were summarized against the longitudinal diameter of the cell body. Red triangles and black circles represent astrocytes and neurons, respectively. (D) Fluorescence intensity of cells acutely dissociated from SNr, visual cortex, and striatum, to which only Alexa Fluor 488 donkey anti-goat IgG and Cy3-conjugated donkey anti-rabbit IgG were applied, without using primary antibodies such as goat polyclonal anti-mouse D1R antibody and polyclonal rabbit anti-GFAP antibody.