J Korean Ophthalmol Soc.  2008 Feb;49(2):340-351. 10.3341/jkos.2008.49.2.340.

The Calretinin Immunoreactive Ganglion Cell Postsynaptic to the ON-Cholinergic Amacrine Cell in the Guinea Pig

• Kim HC
• Suh W
• Moon JI
• Choi KR

• KMID: 2127127
• DOI: http://doi.org/10.3341/jkos.2008.49.2.340

Abstract

PURPOSE: To demonstrate the characterization calretinin-immunoreactive displaced amacrine cells in the ganglion cell layer using immunohistochemistry and electron microscopy.
METHODS
For immunohistochemistry, sections from guinea pig retina were incubated with mouse monoclonal antibody directed against calretinin. For double label studies, sections were incuated in mixture of mouse monoclonal anti-calretinin or rabbit polyclonal anti-calretinin with following antibodies: goat polyclonal anti-ChAT, rabbit polyclonal anti-GABA, mouse monoclonal anti-GABAA receptor alpha1, beta2/3. Sections were analyzed using Bio-rad Radiance Plus confocal scanning microscope. Stained sections from three guinea pig were observed with transmission electron microscope.
RESULTS
Calretinin immunoreactivity was present in displaced amacrine cells and ganglion cells gaving rise to processes ramified in the inner part of the inner plexiform layer in stratum 4. The same stratum was also occupied by the dendrites of ON-cholinergic amacrine cells. Double-labeling demonstrated that dendrites and cell bodies of displaced amacrine cells colocalized with ON-cholinergic amacrine cells and dendrites of ganglion cells directly overlapped with dendrites of ON-cholinergic amacrine cells. The synaptic connectivity was identified by electron microscopy. Ganglion cell dendrites received synaptic input from ON-cholinergic amacrine cell. GABAA receptor beta2/3 subunit bands cofaciculates the dendrites of displaced amacrine cell and ganglion cell that are juxtapose to the alpha1 subunit of GABAA receptor.
CONCLUSIONS
These results indicate that ON-cholinergic amacrine cells modulate calretinin-labeled ganglion cell via GABAA receptor beta2/3 in the guinea pig retina.

Keyword

Amacrine cell; Calretinin; Ganglion cell; Immunohistochemistry

MeSH Terms

Amacrine Cells
Animals
Calcium-Binding Protein, Vitamin D-Dependent
Dendrites
Electrons
Ganglion Cysts
Goats
Guinea
Guinea Pigs
Immunohistochemistry
Mice
Microscopy, Electron
Retina
Calcium-Binding Protein, Vitamin D-Dependent

Figure

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  Figure 1. Light micrographs taken from 50 µm thick vertical vibratome sections processed for calretinin immunoreactivity. (A) A low-power micrograph shows dense calretinin immunoreactivity in the inner half of the retina. Number of amacrine cells in the INL and cells in the GCL express calretinin. Inner plexiform layer contains immunoreactive fibers deriving from amacrine and cells in the ganglion cell layer. (B) A high-power micrograph of (A) shows calretinin immunnoreactivity in displaced amacrine cell (arrowheads) and ganglion cell (arrows) that showed axon-like processes entering into the optic fiber, ramifies in stratum 4 of the IPL. (C) Calretinin immunoreactive ganglion cell showing their dendrites in strata 3 and 4 of the IPL is also seen. (D) Higher magnification of (C). Number 1, 2, 3, 4, 5 indicate stratum of IPL. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars in A, C=50 µm in B, D=10 µm.

• 

  Figure 2. Light micrographs taken at different focal planes in the same field of a whole-mount guinea pig retina processed for calretinin immunoreactivity. (A) A micrograph focused at the calretinin immunoreactive cells in the GCL. Two subpopulations of calretinin immunoreactive cells can be distinguished by their soma size and presence of axon entering optic fiber, medium cells (arrowheads) with no axon and large cells (arrows) with axon. (B) The focus is on the different focal plane of (A) within the outer aspect of the IPL. Calretinin immunoreactive ganglion cell (arrow) shows several stout primary dendrites emerging from the soma (arrowheads) and branched dendrites. (C) The focus is on the IPL slightly different focal plane as (B). Bushy processes of calretinin immunoreactive cells in GCL are visible. Scale bar=10 µm.

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  Figure 3. Confocal micrographs taken from 50 µm thick vertical vibratome sections (A-D) and whole mount (E-M) processed for calretinin (A, E, H, K) and ChAT (B, F, I, L) immunoreactivities. Calretinin immunoreactivity was visualized using a Cy3-conjugated secondary antibody. ChAT immunoreactivity is visualized using anFITC-conjugated secondary antibody. (A) Calretinin immunore-activity in amacrine cell, displaced amacrine cells, and ganglion cell is seen (red). (B) ChAT immunoreactivity shows in mirror symmetric populations of amacrine cells in the INL and displaced amacrine cells in the GCL (green). (C) Double exposure of A and B. Colocalization (yellow) of calretinin and ChAT immunoreactivities is visible in sublamina b of the IPL and displaced amacrine cells in the GCL. (D) The high-magnification view of the inset rectangle in (C). E-G: The focuse is on the GCL. (E) Calretinin immunoreactivity shows in displaced amacrine cells (arrowheads) and ganglion cells (arrows). (F) ChAT immunoreactivity shows in displaced amacrine cells (arrowheads). (G) Double exposure of E and F. Colocalization (yellow) of calretinin and ChAT immunoreactivities is visible in the displaced amacrine cells (arrowheads). H-M: The focuse is on the different focal plane of (E-G) within the outer aspect of the IPL. (H, K) Dendrites of calretinin immunoreactive ganglion cell (arrows) and displaced amacrine cell are seen. (I, L) ChAT immnoreactive dendrites are seen (arrowheads). (J, M) Double exposure of H and I; K and L, shows colocalization (yellow) of ChAT immunoreactive dendrites and calretinin immunoreactive ganglion cell dendrites (arrowheads). Scale bar in A-C=50 µm; in D-M=10 µm.

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  Figure 4. An electron micrograph taken from a vertical ultrathin section in stratum 4 of the IPL of the guinea pig retina processed for calretinin immunoreactivity. A calretinin immunoreactive displaced amacrine cell (A) makes chemical synapse onto the calretinin immunoreactive ganglion cell dendrite (G). Arrow indicates direction of impulse transmission. Scale bar=0.5 µm.

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  Figure 5. Confocal micrographs taken from 50 µm thick vertical vibratome sections processed for calreteinin (A), ChAT (D), and GABA (B, E) immunoreactivities. Calretinin and ChAT immunore-activities were visualized using a FITC-conjugated secondary antibody. GABA immunoreactivity was visualized using an Cy3-conjugated secondary antibody. (A) Calretinin immunoreactive displaced amacrine cell, ganglion cell and their dendrites are visible (green). (B, E) Several GABA immunoreactive cell bodies (red) in INL and GCL are visible. (C) Double exposure of A and B. Colocalization (yellow) of calretinin and GABA within the same cell (arrows) is clear in the GCL. (D) ChAT immunoreactive cell bodies (green) in INL and GCL are visible. (F) Double exposure of D and E shows colocalization (yellow) of ChAT and GABA within the same cells in the INL and GCL (arrows). Scale bar=50 µm.

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  Figure 6. Confocal micrographs taken from 50 µm thick vertical vibratome sections processed for ChAT [A, D], 1 [B, H] and 2/3 [E, K] of GABA A receptors, and calretinin [G, J] immunoreactivities. ChAT and calretinin immunoreactivities are visualized using an FITC-conjugated secondary antibody. The α1 and β2/3 of GABAA receptors, immunoreactivities were visualized using a Cy3-conjugated secondary antibody. (A, D) ChAT immunoreactivity shows in mirror symmetric populations of amacrine cells in the INL and displaced amacrine cells in the GCL (green). (G, J) Calretinin immunoreactivity in amacrine cell, displaced amacrine cells, and ganglion cell is seen (green). (B, E, H, K) GABA A receptor, α1 (red) and β2/3 (red), immunoreactivities are visible in cells in the INL and two bands in the IPL. (C, F) Double exposure of ChAT [A, D] and GABAA receptor, α1 [B] and β2/3 [E], shows ChAT immunoreactive cells completely devoid of GABA A receptor, α1, immunoreactivity, wherease, dendrites of ChAT immunoreactive cells show very weak GABA A receptor, β2/3, immunoreactivity (yellow; arrows). (I, L) Double exposure of calretinin [G, J] and GABA A receptor, α1 [H] and β2/3 [K], shows calretinin immunoreactive cells completely devoid of GABA A receptor, α1, immunoreactivity, wherease, dendrites of calretinin immunoreactive cells show GABA A receptor, β2/3, immunoreactivity (yellow; arrows) in the sublamina b of the IPL. (M-O) Confocal micrographs taken at a horizontal section processed for calretinin [M] and GABAA receptor, β2/3 [N], immunoreactivities. [O] Double exposure of M and N, dendrites of calretinin immunoreactive ganglion cells show GABA A receptor, β2/3, immunoreactivity (yellow; arrows). [P] A micrograph taken at different focal planes in the same field as [O], GABA A receptor, β2/3, immunoreactivitive bands cofasiculating calretinin immunoreative dendrites are visible. Scale bar=50 µm.

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