Light responses and morphology of bNOS‐immunoreactive neurons in the mouse retina

Abstract

Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography and light responses of NOS-containing amacrine cells (NOACs) in dark-adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole-cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark-adapted conditions bNOS-immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS-IR somas were negative for LY, thus they were identified as ACs; nearly 6 % of the cells were labeled by NB but not by LY, indicating that they were dye-coupled with GCs. Three morphological subtypes of NOACs (NI, NII and displaced) were identified. The cell density, inter-cellular distance and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON-OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1 to 2 Hz) or abrupt change of light-intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed.

Survey on Amacrine Cells Coupling to Retrograde-Identified Ganglion Cells in the Mouse Retina

“…A more reliable way to distinguish GCs from ACs, however, is retrograde labeling of GCs. [35][36][37] In the retrogradely labeled mouse retina, the specificity of GABA, glycine, ChAT, and nNOS antibodies in labeling retinal ACs has been recently confirmed, 28 but similar investigations have not been done for TH, 38 CR, [32][33][34] and PV. [29][30][31] Of the multiple subtypes of ACs, GABA ACs are the only subtype that have been confirmed to couple with GCs.…”

“…8 GCs and ACs may both reside in the GC layer (GCL) and inner nuclear layer (INL), and they are not obviously distinguishable by soma size, 15,16 axons, 17,18 or dendritic tree shape. 6,15,19 Amacrine cells are often identified immunologically by antibodies against GABA, glycine, 20,21 Choline acetyltransferase (ChAT), 20,22 tyrosine hydroxylase (TH), [23][24][25] and neuronal or brain-type nitric oxide synthase (nNOS or bNOS), [26][27][28] while antibodies against calcium-binding proteins, such as parvalbumin (PV) [29][30][31] and calretinin (CR), 32-34 may label GCs. A more reliable way to distinguish GCs from ACs, however, is retrograde labeling of GCs.…”

“…Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 NB is widely used to study gap junctions in the central nervous system 1,9,10,28 and Lucifer yellow (LY) is a fluorescent dye that is commonly used to reveal individual cell morphology. 21,43,44 Neuronal gap junctions are known to be impermeable to LY.…”

“…45 Using these two dyes, we have previously established a retrograde labeling method for identification of the entire retinal GC population and simultaneous visualization of all the coupling partners. 11,28 The labeling specificity and efficiency on the whole GC population have been examined and confirmed. The two dyes are actively transported into GC somas by GC axonal transportation and NB is freely passed into coupled ACs via gap junctions.…”

Decreased vascular supply leads to higher numbers of NADPH diaphorase amacrine cells in the degenerating mouse retina

“…Retinae with increase of vascular supply like in diabetes show reduced numbers of NAC [11]. Since blood supply is correlated with the activity of the retina, light-dependent changes in NAC [12] might explain their modulating role for both, the neuronal aspects of the retina and the retinal vasculature. Further studies have to address the functional connection between the named systems.…”