Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas

Abstract

The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

Keywords: Gephyrin; Glycine; Midget; Parasol; Primate.

Fig. 1

Baboon retina labeled with rabbit antiserum against vGluT3. (A) A single optical section through the parafovea. Although the amacrine cell perikarya were unlabeled, there was robust labeling of the dendrites (green). The inner nuclear layer (INL) and ganglion cell layer (GCL) are labeled with DAPI (blue). (B) Stack of 7 optical sections. Most dendrites of vGluT3 cells (green) are located above the band of starburst amacrine cell dendrites in S4 labeled with antibody to choline acetyltransferase (ChAT, red), but there is some overlap between the two sets of dendrites. Note the vGluT3-positive primary dendrites (arrows) and the perikarya (arrowheads) of the starburst cells in the GCL. Scale bars = 10 μm.

Fig. 2

The depths of labeled varicosities in the IPL are plotted with 0% being the lower border of the INL and 100% being the upper border of the GCL. The data were grouped into bins of 5% each and fitted with Gaussian functions with means ± standard deviations for the two distributions of 30 ± 7 (red, outer) and 62 ± 11 (green, inner). To demonstrate that the distribution was bimodal, a likelihood ratio test (P < 0.0001) was used.

Fig. 3

A labeled primary dendrite of a vGluT3 amacrine cell receives a synapse from a large, electron-lucent amacrine cell (A) in the outer sublamina of the IPL. There are black arrowheads on either side of the synaptic density within the unlabeled profile.

Fig. 4

Two labeled amacrine cell dendrites; there are black arrowheads on either side of the synaptic density within the unlabeled profiles. (A) An unlabeled amacrine cell (A) makes a synapse (arrowheads) onto a vGluT3 dendrite. Note the accumulation of vesicles and the concavity on presynaptic side of the synapse. (B) A labeled amacrine cell dendrite is presynaptic to another amacrine cell (A). The density on the postsynaptic side of the synapse is labeled with arrowheads.

Fig. 5

(A) In the inner sublamina of the IPL, a vGluT3 cell dendrite receives a synapse from a bipolar cell axon (B) associated with a ribbon (white arrowhead). (B) In the outer sublamina of the IPL, a bipolar cell axon (B) receives a synapse from a labeled amacrine cell (black arrowheads). Note the synaptic ribbons (white arrowheads); these were not associated with labeled dendrites in this sublamina.

Fig. 6

(A) In the outer sublamina of the IPL, a labeled amacrine cell dendrite makes a synapse (arrowheads) onto a large ganglion cell dendrite (G). (B) In the inner sublamina of the IPL, a ganglion cell dendrite (G) receives a synapse (arrowheads) from a labeled amacrine cell.

Fig. 7

Labeled amacrine dendrites make two different types of synapses. (A) The postsynaptic density (black arrow) here is relatively thin, only an increase in electron density of the plasma membrane. This is typical of symmetric synapses in the central nervous system. (B) The postsynaptic density (black arrow) here extends approximately 40 nm into the cytoplasm, as in asymmetric synapses elsewhere in the central nervous system.

Fig. 8

(A) In the outer sublamina of the IPL, vGluT3-positive amacrine cell dendrites (green) make contacts with axon terminals of calbindin-positive DB3a bipolar cells (blue), and some of the contacts (aqua) are labeled with arrows. This is a single, 0.5 μm optical section, as are all the images that follow. (B) In the inner sublamina of the IPL, dendrites containing immunoreactive choline acetyltransferase (blue) make contacts (arrows) with dendrites of vGluT3 cells (green). (C) Dendrites containing immunoreactive calretinin (blue) make contacts (arrows) with dendrites of vGluT3 cells (green) in both halves of the IPL. Because AII cells do not make synapses onto other amacrine cells, the vGluT3 cells should be presynaptic there.

Fig. 9

Baboon retinal ganglion cells injected intracellularly with Neurobiotin. (A) Two ON parasol cells were injected and are illustrated in a flat mount preparation. In addition to the ganglion cells, perikarya of tracer-coupled amacrine cells (arrowheads) are visible. (B) An OFF midget ganglion cell was injected, and it is illustrated in a vertical, vibratome section. The axon is labeled with an arrow, and the boundaries of the IPL are marked with arrowheads. INL = inner nuclear layer and GCL = ganglion cell layer.

Fig. 10

Horizontal, optical section of the inner sublamina. (A) ON parasol ganglion cells (blue) are contacted (arrows) by vGluT3 cells (green), and puncta containing the inhibitory synapse marker gephyrin (red) are found there. (B and C) The contacts (arrowheads) are shown at higher magnification.

Fig. 11

Horizontal, optical section of the outer sublamina. (A) An OFF parasol ganglion cell (blue) is contacted (arrows) by vGluT3 cells (green), and puncta containing the inhibitory synapse marker gephyrin (red) are found there. (B and C) The contacts (arrowheads) are shown at higher magnification.

Fig. 12

An ON midget ganglion cell (blue) is contacted (arrow) by a vGluT3 cell (green), and a punctum containing the inhibitory synapse marker gephyrin (red) is found there. Inset: The contact is shown at higher magnification.

Fig. 13

An OFF midget ganglion cell (blue) is contacted (arrow) by a vGluT3 cell (green), and a punctum containing the inhibitory synapse marker gephyrin (red) is found there. The contact (arrowhead) is shown at higher magnification in the inset.

Fig. 14

(A) Amacrine cells were labeled with antibody to vesicular glutamate transporter 1 (red), but these did not include the vGluT3 cells (green). There were punctate contacts between the two types of cells (arrows). (B) Amacrine cells were labeled with antibody to GABA (red), but these did not include the vGluT3 cells (green). There were numerous punctate contacts between the two types of cells (yellow).