Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Abstract

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-beta-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and alpha-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.

Fig. 1

a NADPH-diaphorase cytochemistry (light microscopy): ICN are closely associated with choroidal blood vessels (asterisks). b Immunohistochemistry for α-smooth muscle actin (green) and galanin (red): ICN-forming processes with boutons are enmeshed within the smooth-muscle framework of the choroid (confocal microscopy, extended focus mode). c–e Double immunohistochemistry for nNOS (red, c) and GAL (green, d) reveals a colocalization of both markers in ICN (e, mixed yellow color). f–h Double immunohistochemistry for GAL (red, f) and VIP (green, g) reveals a colocalization of both markers in ICN (h, mixed yellow color). i–k Double immunohistochemistry for VIP (red, i) and nNOS (green, j) reveals a colocalization of both markers in ICN (k, mixed yellow color). l Double immunohistochemistry for ChAT (red) and VIP (green) reveals no co-localization in ICN; CHAT-immunoreactive nerve fibers forming boutons closely approached ICN (arrowhead, mixed yellow color). m Double immunohistochemistry for GAL (green) and SOM (red) reveals no co-localization in ICN, but ICN show close association with boutons immunoreactive for SOM (arrowheads). b–m Confocal microscopy, b extended focus mode, c–m single optical sections

Fig. 2

a, b Double immunohistochemistry for GAL (red) and VIP (green) reveals a co-localization of both markers in the perivascular plexus (a, asterisks) as well as in nerve fibers in the choroidal stroma (b) as indicated by mixed yellow color. c, d Double immunohistochemistry for ChAT(red) and VIP (green) reveals no co-localization of both markers within the perivascular plexus (c, asterisks) as well as none in nerve fibers within the choroidal stroma (d) as indicated by the absence of mixed yellow color. e Double immunohistochemistry for ChAT (red) and SOM (green) reveals a co-localization of both markers in nerve fibers within the choroidal stroma as indicated by mixed yellow color. All images: confocal images; a, b extended focus mode; c–e single optical sections

Fig. 3

Ciliary ganglion. a NADPH-d cytochemistry (light microscopy): numerous structures and processes display the blue NADPH-d reaction product. b, c Double immunohistochemistry for nNOS (b) and SOM (c): numerous nNOS-positive neurons with cap-like endings (arrowheads, representing ciliary neurons) and bouton-like endings (arrows, representing choroid neurons) are visible. While all choroid neurons show co-localization for SOM (c, arrows), this is not the case for the ciliary neurons (c, arrowheads; confocal image, single optical section). d Double immunohistochemistry for GAL (green) and SOM (red): GAL is not present in SOM-positive choroid neurons (confocal image, extended focus mode)

Fig. 4

a Trigeminal ganglion: double immunohistochemistry for CGRP (green) and GAL (red). Neurons of the trigeminal ganglion show no co-localization of GAL (confocal image, extended focus mode). b Choroid: double immunohistochemistry for CGRP (green) and SOM (red): primary afferent nerve fibers in the choroid do not co-localize for SOM as indicated by absence of mixed yellow color (confocal image, single optical section) c, d Pterygopalatine ganglion: double immunohistochemistry for ChAT (c) and nNOS (d) revealed a complete colocalization of bothmarkers (confocal images, single optical sections). e, f Pterygopalatine ganglion: double immunohistochemistry for GAL (red) and nNOS (green, e), and VIP (red) andGAL (green, f), revealed a co-localization of the aforementioned markers as indicated by mixed yellow color (arrowheads; confocal images, single optical sections)

Fig. 5

a, b Double immunohistochemistry of the superior cervical ganglion for GAL (green) and TH (red): out of confocal images (a), plots were created (b) to define co-localization rates (red TH only, green GAL only, yellow co-localization of TH and GAL). c In the superior cervical ganglion, 21% of all neurons are nitrergic as revealed by NADPH-diaphorase cytochemistry combined with eosin counterstaining. d Triple immunohistochemistry for nNOS (green), VIP (blue), and TH (red) in the superior cervical ganglion: co-localization of nNOS and VIP was amounted to 76.9% as indicated by mixed cyan color. e Triple immunohistochemistry for TH (red), VIP (blue), and GAL (green) in the superior cervical ganglion: colocalization of VIP and GAL was amounted to 54.4% as indicated by mixed cyan color. f Double immunohistochemistry for GAL (green) and TH (red): nerve fibers in the choroid show no co-localization (single optical section)

Fig. 6

a Immunohistochemistry for VMAT2 in the superior cervical ganglion reveals immunoreactivity in neurons as well as nerve fibers (single optical section). b Triple immunohistochemistry for VMAT2 (red), TH (blue), and VIP (green): nerve fibers co-localizing VMAT2/TH (mixed pink color) forming bouton-like appositions (cyan and white mixed colors, arrowheads) onto VIP-positive ICN (single optical section). c Double immunohistochemistry for VMAT2 (green) and TH (red): nerve fibers in the choroid show a co-localization as depicted by mixed yellow color (single optical section). d Double immunohistochemistry for CGRP (green) and VIP (red): primary afferent nerve fibers forming bouton-like appositions onto VIP-positive ICN (single optical section)

Fig. 7

Colocalization rates of ICN and subsets of neurons in ganglia supplying the choroid (in percentages; SCG superior cervical ganglion, PPG pterygopalatine ganglion, CIL ciliary ganglion, TRIGEM trigeminal ganglion). Possible interaction of corresponding ganglia onto ICNs is indicated by arrows