GABAergic synapses made by a retinal dopaminergic neuron

Abstract

In the retina, dopaminergic amacrine (interplexiform) cells establish multiple synapses on the perikarya of AII amacrines, the neurons that distribute rod signals to on- and off-cone bipolars. We used triple-label immunocytochemistry and confocal microscopy to identify the receptors contained within the postsynaptic active zone of these synapses in both mouse and rat retinas. We found that at the interface between the dendrites of the dopaminergic neurons and the AII amacrine cell perikarya clusters of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors are situated in register with aggregates of presynaptic organelles immunoreactive for GABA, the GABA vesicular transporter, and the vesicular monoamine transporter-2. D1 and D23 dopamine receptors, on the other hand, do not form clusters on the surface of the perikarya of AII amacrine cells. We suggest that the synapses between retinal dopaminergic neurons and AII amacrine cells are GABAergic and that both GABA and dopamine are released by the presynaptic endings. GABA acts on the ionotropic receptors clustered at the postsynaptic active zone, whereas dopamine diffuses to more distant, slower-acting metabotropic receptors.

Figure 1

Connections of dopaminergic and AII amacrine cells. AII amacrine cells transfer rod signals from rod bipolars to the axonal endings of on- and off-cone bipolars. The synapses of DA cells onto the perikaryon of AII amacrines (arrowheads) are situated near the origin of the primary dendrite(s). Because there is no comprehensive morphological analysis of the rodent retina, neurons in the diagram are drawn in the style of Polyak (42).

Figure 2

Fine structure of DA-to-AII amacrine cell synapses. (a) DA cells were labeled in transgenic mice with PLAP, an enzyme that resides on the outer surface of the cell membrane. With the electron microscope, after staining with the histochemical reaction for phosphatase activity, a dense precipitate of lead phosphate occupies the intercellular spaces surrounding the processes of DA cells. In a vertical section of the retina, labeled DA cell dendrites form a plexus at the scleral margin of the inner plexiform layer. Bundles of labeled dendrites occur near the vitreal surface of the perikaryon of an AII amacrine cell. One of such processes is presynaptic (curved arrow). (b) In a horizontal section of the retina, numerous dendrites of DA cells (asterisks) surround the vitreal surface of an AII amacrine cell, forming a ring around the origin of its primary dendrite. Three of the labeled endings are presynaptic (curved arrows). The abundance of large mitochondria and the profusion of vesicle and tubules are typical of the cytoplasm of AII amacrine cells. A process belonging to a neuron that does not express PLAP is indicated by a star. (Bar = 1 μm.)

Figure 3

Distribution of pre- and postsynaptic markers at the DA-to-AII amacrine cell synapses. A 10-μm bar is included in all images of this figure. (a) Morphology of a DA cell and an AII amacrine in the rat retina. The two neurons were selected from PHOTOSHOP images of specimens stained for TH and PV and pasted into a single image. (b) Dab1, red; TH, blue; α3 subunits of the GABA_A receptor, green. In a radial section of the mouse retina, the perikarya of AII amacrine cells (red) are nested in the plexus formed by the dendrites of DA cells (blue) in the most superficial stratum (S1) of the IPL. The white spots at the periphery of the cell bodies of AII amacrines (arrowheads) represent clusters of α3 subunits. The white color indicates coincidence of the three labels; therefore, the clusters occur in the region of apposition between DA cell dendrites and cell body of AII amacrine cells. (c) PV, red; TH, blue; α3 subunits of the GABA_A receptor, green. In an oblique section through the rat retina, the dendrites of DA cells (blue) form a dense plexus whose meshes are predominantly occupied by the perikarya of AII amacrine cells (red). Clusters of α3 subunits (white, arrowheads) occur in the region of overlap between DA cell dendrites and AII amacrine cell bodies. One cell body is stained purple (black asterisk), because it contains both TH and PV. It probably belongs to a type 2 catecholaminergic amacrine. (d) PV, red; TH, blue; α1 subunits of the GABA_A receptor, green. In a horizontal section through the rat retina, clusters of α1 subunits (white, arrowheads) occur in the region of overlap between DA cell dendrites (blue) and AII amacrine cell bodies (red). Thus, the presence of clusters of GABA_A receptors at the interface between the two types of neurons is confirmed by the staining with the antibody to another subunit of the receptor. (e) TH, red; GABA, green. The cytoplasm of a DA cell (red) is intensely stained by antibody to GABA (yellow). The contrast of this micrograph was enhanced for clarity. (f) PV, red; TH, blue; GABA, green. Foci of immunoreactivity for GABA (white, arrowhead) occur at the site of apposition between DA cell dendrites (blue) and AII amacrine cell bodies (red). (g) PV, red; TH, blue; VGAT, green. In this high-contrast image, aggregates of VGAT-containing organelles (white, arrowhead) are situated on the surface of AII amacrine cell bodies (red), at the site where they are contacted by the dendrites of DA cells (blue). (h) PV, blue; VGAT, red; α3 subunits of the GABA_A receptor, green. Aggregates of VGAT-positive organelles are situated in register with clusters of α3 subunits of the GABA_A receptor (white, arrowheads) at the surface of AII amacrine cells (blue). (i) PV, red; TH, blue; VMAT2, green. Aggregates of VMAT2-positive organelles (white, arrowhead) are situated within DA cell dendrites (blue) at the site where they contact the cell bodies of AII amacrines (red). (j) PV, blue; VMAT2, red; α3 subunits of the GABA_A receptor, green. At the surface of the cell bodies of AII amacrines (blue), the aggregates of VMAT2-containing organelles are situated in register with clusters of α3 subunits of the GABA_A receptor (white, arrowhead).