Prominence of the dopamine D2 short isoform in dopaminergic pathways

Abstract

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.

Figure 1

Immonoblots of monkey brain tissues and crude membranes of recombinant Sf9 cell lines expressing dopaminergic human D1, D2S, D2L, D3_rat, D4.2, and D5 receptors are shown with D2S (a) and D2L (b) antibodies. Both antibodies immunoreacted with single polypeptide band and do not show cross-reactivity. The relative abundance of the D2S and D2L receptor isoforms in the same strip (c) is shown.

Figure 2

Immunoprecipitation of dopamine receptor binding sites by D2S and D2L antibodies. (a) Antibody concentration-dependent immunoprecipitation demonstrated by [³H]spiperone binding in striatal tissues. Saturation point at 20 μl shows that both antibodies are equally sensitive. (b) Quantitative immunoprecipitation from substantia nigra, motor cortex, and striatum showing different amounts of D2S and D2L receptors. The results were confirmed with two different dopamine D2 receptor antagonists.

Figure 3

Predominant expression of D2S protein in the substantia nigra (arrows) as shown with D2S (a, c, d) and D2L (b, e) antibodies. Double labeling immunofluorescence analysis shows colocalization of D2S (FITC, green) and TH immunoreactivity (Cy3, red) in dopaminergic neurons of substantia nigra (f, g), ventral tegmental area (h, i), and retrorubral field (j, k). (Scale bar: a and b, 400 μm; c, 50 μm; d, 11 μm; e, 50 μm; f–k, 50 μm.)

Figure 4

Electron micrographs of double labeled sections showing differential D2S and D2L receptor expression in dopaminergic neurons and axons. Dopaminergic markers, TH or dopamine transporter (DAT), are labeled by immunoperoxidase reaction and receptors by immunogold method. In dopaminergic neurons of substantia nigra, note the significantly higher number of immunogold particles (arrowheads) in the D2S (a) labeled cell body than D2L (b). D2S immunogold particles are frequently distributed at the cytoplasmic face of plasma membranes (arrowheads) and rough endoplasmic reticulum (arrows). D2L immunogold particles are only occasionally associated with plasma membranes (open arrow in b). TH immunolabeled axons (a) exhibit numerous D2S (c, d) immunogold particles (arrowheads), whereas D2L (e, h) immunoreactivity is not detected. In e, the immunogold particle is outside the TH-labeled axon (arrow). In h, D2L immunogold particles are localized along the cytoplasmic side of plasma membranes in dendritic spines (s; arrowheads), which receive asymmetric synapses (arrows). TH immunonegative axon (an) expresses D2L immunoreactivity (open arrow). (f–g) Dopaminergic axons in the area 6 labeled with DAT are strongly D2S positive. In f, D2S-labeled axon is apposed to the spine receiving an asymmetric input (arrows). N, nucleus. (Scale bars: a, 0.5 μm; b, 1 μm; c and d, 0.2 μm; e, 0.5 μm; f, 0.2 μm; g, 0.5 μm; h, 0.3 μm.)

Figure 5

Dense D2S immunopositive fiber network in the projection areas of dopaminergic neurons: striatum (a, c), nucleus accumbens (d, e), and cerebral cortex area 6 (f, darkfield micrograph; g). Immunopositive fibers bear numerous varicosities (arrows). (e) High-magnification micrograph depicted from square shown in d. D2L-positive neurons in striatum (b) and cerebral cortex area 6 (i, j) are shown. (g, h) Neurons labeled with D2S antibody in the area 6 of cerebral cortex. C, core; S, shell. (Scale bar: a and b, 25 μm; c, 8 μm; d, 400 μm; e, 25 μm; f, 80 μm; g and i, 67 μm; h and j, 10 μm.)