Jan Klooster

The peripheral and central projections of the Edinger-Westphal nucleus in the rat. A light and electron microscopic tracing study

The peripheral and central efferent projections of the rostral part of the Edinger-Westphal nucleus in the rat were investigated at the light and electron microscopic level by means of iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and retrograde tracer injections of Fast blue and Nuclear yellow into the facial nucleus and into the principal olive. Two pathways leaving the rostral part of the Edinger-Westphal nucleus were studied, a peripheral and a central descending pathway. Fluorescent experiments demonstrated that the central pathway fibers originated from distinct individual Edinger-Westphal neurons. These neurons were mainly distributed throughout the rostral part of the Edinger-Westphal nucleus and had fusiform cell bodies. The neurons rarely form collateral projections. The central descending pathway left the Edinger-Westphal nucleus medially and terminated bilaterally in the principal olive, in the subnuclei A, B and C of the inferior olive and ipsilaterally in the medial accessory olive. The central pathway also terminated contralaterally in the lateral parabrachial nucleus, the facial nucleus, the trigeminal brainstem nuclear complex, the lateral reticular nucleus and the rostroventral reticular nucleus. The projection to the facial nucleus provides evidence for the existence of a polysynaptic loop forming the central part of the corneal blink reflex. Projections from the Edinger-Westphal nucleus to the cerebellar cortex or the deep nuclei, as described in cat and primate, could not be confirmed. The peripheral pathway left the Edinger-Westphal nucleus ventrally and terminated on dendrites of ciliary ganglion cells, along smooth muscle cells of ciliary ganglion associated arterioles and in the proximity of ciliary ganglion associated venules. The central and peripheral terminals that originate in the Edinger-Westphal nucleus all had similar ultrastructural features: clear, round vesicles and electron dense mitochondria. The terminals originating from the central descending pathway were often found to be arranged in glomerular-like structures. The central and peripheral terminals made asymmetric synaptic membrane specializations (Gray type one), except terminals innervating the ciliary ganglion associated vessels, which showed no synaptic contacts.

Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes a light microscopic tracing study

The olivary pretectal nucleus is a primary visual centre sensitive to luminance changes. It is involved in the pupillary light reflex, the consensual pupillary light reflex and related reflexes, such as the lid closure reflex whereby pupillary constriction takes place. Since the olivary pretectal nucleus is a small nucleus, previous studies using degeneration, horseradish peroxidase and radioactive amino acid tracing were limited regarding to the exclusiveness of the projections from the olivary pretectal nucleus. In the present study the position of the olivary pretectal nucleus in the rat was first localized by physiological recording of the neurons upon luminance stimulation. Subsequently, an anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically. This allows a much more precise localization of the olivary pretectal nucleus projections. Ascending and descending pathways originating from the olivary pretectal nucleus were observed. Ascending fibres project bilaterally to the intergeniculate leaflet, the ventral part of the lateral geniculate nucleus and ipsilaterally to the anterior pretectal nucleus. In addition, contralateral projections were observed to the zona incerta and the fields of Forel. Descending fibres project bilaterally to the periaqueductal gray, the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, the Edinger-Westphal nucleus and the intermediate gray layer of the superior colliculus. Also a contralateral projection to the oculomotor nucleus and an ipsilateral projection to the pontine nucleus and the nucleus of the optic tract were found. Furthermore, the contralateral olivary pretectal nucleus received a small projection. Retrograde tracing experiments using two fluorescent dyes revealed that the fibres projecting to the contralateral olivary pretectal nucleus and to the contralateral interstitial nucleus of Cajal are collaterals. The projection from the olivary pretectal nucleus to the facial nucleus which has been described to receive an input in cats could not be confirmed for the rat. The fact that the Edinger-Westphal nucleus, the interstitial nucleus of Cajal and the superior colliculus receive an input from the olivary pretectal nucleus suggests that this primary visual centre is not only involved in the pupillary light reflex, but also in controlling eye and head position and saccadic eye movements. Although visual acuity largely depends on receptive field sizes of retinal ganglion cells and their central connections, the stronger sympathetic influence during the pupillary light reflex in animals with frontally placed eyes compared to animals with laterally placed eyes may also contribute to the higher visual acuity in animals with frontally placed eyes.

Pre- and post-ganglionic nerve fibres of the pterygopalatine ganglion and their allocation to the eyeball of rats

The origin, course and distribution of pre- and postganglionic neurons of the pterygopalatine ganglion (PPG) in the rat were studied using acetylcholinesterase staining, wheat germ agglutinin coupled to horseradish peroxidase (WGA-HRP) histochemistry and autoradiography. These methods were used in a selected and planned fashion to reveal details concerning the innervation of the lacrimal gland and portions of the eye. The PPG in rats consists of a rostral triangular portion and additional perikarya surrounding the distal part of the major petrosal nerve. Fibres from the superior cervical ganglion (SCG) reach the PPG via the inferior petrosal sinus. Application of WGA-HRP was made after transections: (1) rostral to the PPG; and (2) caudal to the PPG. The first of these applications labelled mainly fibres in the PPG; the second application labelled preganglionic parasympathetic brainstem neurons dorsolateral to the facial nucleus (i.e. the lacrimal nucleus), rostral cells in the SCG and trigeminal sensory fibres. WGA-HRP injections of the lacrimal gland, the conjunctiva and the anterior chamber of the eye all labelled cells in different parts of the PPG. This means that the PPG contains sensory and sympathetic nerve fibres and that the PPG has a topographical organisation along the rostrocaudal axis. Isotope injections of the PPG anterogradely labelled fibres passing through the ciliary ganglion that innervated the conjunctiva, the limbus and parts of the choroid.

The allocation of nerve fibres to the anterior eye segment and peripheral ganglia of rats. II. The sympathetic innervation.

The sympathetic innervation of the peripheral ganglia related to the eye, i.e. the trigeminal ganglion, the ciliary ganglion and the pterygopalatine ganglion, and of the anterior eye segment was studied in rats. Selective labelling of sympathetic nerves was obtained by means of injection of [3H]leucine into the superior cervical ganglion. Bundles of sympathetic nerve fibres were found in the trigeminal ganglion and the pterygopalatine ganglion but were absent in the ciliary ganglion. In addition individual sympathetic nerve fibres, which may have contacts with trigeminal ganglion cells, were found between the ganglion cell bodies all over the trigeminal ganglion indicating a sympathetic innervation of this ganglion. In the anterior eye segment, there appeared to be a sympathetic innervation of the ciliary cleft, the ciliary body and the iris. Within the ciliary body sympathetic nerve fibres innervate the central stroma and the stroma of the ciliary processes. Labelled sympathetic nerve fibres were also observed in the stroma of the iris and were most abundant in its periphery. Most sympathetic fibres reach the iris and ciliary body by way of the base of the ciliary body. Only few sympathetic fibres are present in the ciliary cleft. No sympathetic innervation of the cornea was found.

Satellite cells as blood-ganglion cell barrier in autonomic ganglia.

In a preliminary study a difference in the uptake and transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) between the trigeminal ganglion and the superior cervical ganglion was observed. After injection of WGA-HRP and HRP into the trigeminal ganglion, peroxidase was found in the space between the satellite cell processes and the ganglion cells. The ganglion cells showed pinocytosis and uptake of WGA-HRP and HRP. In the superior cervical ganglion WGA-HRP and HRP were found alongside the satellite cells but were absent in the space between satellite cells and ganglion cells. Intravenous injection revealed the presence of HRP in the space between sensory ganglion cells and their satellite cells of the trigeminal and nodose ganglion whereas HRP was absent in the space between autonomic ganglion cells and their satellite cells of the superior cervical, medial cervical and pterygopalatine ganglion although HRP lined the satellite cell membranes. By means of electron microscopy, satellite cell processes in the superior cervical ganglion were found to enwrap ganglion cells very tightly with a marginal space between both cell types. Satellite cells and their processes were mutually anchored by numerous tight junctions. In the trigeminal ganglion the extracellular space between ganglion cells and satellite cells was larger and satellite cells were found to be more loosely arranged around the ganglion cells. Satellite cell processes were only occasionally linked by tight junctions. It is concluded that satellite cells in autonomic ganglia comprise an effective barrier for WGA-HRP and HRP and probably large molecules in general. This barrier is absent in sensory ganglia.

The innervation of the rabbit's anterior eye segment: A retrograde tracing study

The differential innervation of the distinct elements of the anterior segment of the rabbit eye, i.e. cornea, conjunctiva, ciliary body, iris, and trabecular meshwork, was studied using horseradish peroxidase-wheatgerm agglutinin (HRP-WGA) as a tracer. HRP-WGA was injected into the anterior chamber, the conjunctiva, or the cornea, and the animals were killed after 6 to 96 hr. The HRP-WGA localization was studied at the light and electron microscopic level. Injection into the anterior chamber led to an ubiquitous spread of the tracer in all elements of the anterior segment, resulting in a consistent retrograde labelling of neurons in the trigeminal (sensory), superior cervical (sympathetic) and ciliary (parasympathetic) ganglion. Neurons in the proximal part of the pterygopalatine ganglion (parasympathetic) were labelled only when the injected tracer was present in the conjunctiva. Varying survival times after corneal injection were used to establish restricted corneal uptake. Up to 16 hr after corneal injection, HRP-WGA was limited to the cornea and led to the exclusive labelling of neurons in the trigeminal ganglion, indicating that the central cornea has an almost exclusive sensory innervation. The trigeminal sensory neurons projecting to the cornea were restricted to a small antero-medial part of the trigeminal ganglion. Electron microscopy revealed four different cell types in the trigeminal ganglion. However, distinct elements of the anterior segment did not project to specific cell types.

Ultrastructural identification of trigeminal nerve terminals in the pterygopalatine ganglion of rats : an anterograde tracing and immunohistochemical study

Trigeminal nerve terminals in the rat pterygopalatine ganglion (PPG) were ultrastructurally identified using anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L). Electron microscopic immunohistochemistry was used to demonstrate the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) in nerve terminals of the PPG. Adjacent to the rostral part of the PPG an additional minor area was described. Perikarya in this minor rostral part were more spherical and had irregular outlines. Ultrastructurally, the glial enwrapment of the nerve terminals seemed to be more loosely arranged in comparison to that in the major rostral part of the PPG. With PHA-L, numerous labelled nerve fibres and terminals were found in all parts of the PPG. The ultrastructure of these terminals was uniform, many of them showing synaptic contacts. Numerous terminals in the PPG were SP-positive, whereas only a few were CGRP-positive. Fibres stained positive for both neuropeptides. The PPG is shown to be synaptically innervated by sensory fibres arising in the trigeminal ganglion, with the strong suggestion of SP and CGRP acting as neurotransmitters. A modulatory interaction between the autonomic and sensory system, resembling an axon reflex mechanism in the peripheral nervous system is endorsed.

Sympathetic Innervation of the Rat Choroid: An Autoradiographic Tracing and Immunohistochemical Study

The sympathetic innervation of the choroid was investigated by means of the anterograde tracer 3H-leucine, injected into the rostral part of the superior cervical ganglion. The tracer was autoradiographically visualized at the light- and electron-microscopic levels. Labelled unmyelinated fibres were found in the choroid and labelled terminals were observed in close proximity to the smooth muscle cells of arterioles. The labelled terminals contain granular vesicles of different size and clear vesicles; the mitochondria were of the electron-dense type and no cell-to-cell synaptic contacts were observed. In addition, tyrosine hydroxylase immunocytochemistry was performed. Immunostained terminals found in the choroid show ultrastructural features similar to those found in the tracing experiments: granular vesicles of different size, clear vesicles and electron-dense mitochondria. On the basis of previous studies and the present observations, it is concluded that the choroid has a dual innervation, a parasympathetic innervation from the pterygopalatine ganglion and a sympathetic innervation from the superior cervical ganglion. The dual innervation is most likely involved in maintaining the homeostasis of vasodilatation and vasoconstriction in the choroid.

Peripheral neural circuits regulating IOP? A review of its anatomical backbone.

The peripheral nervous system is classically separated into a somatic division containing both afferent and efferent pathways and an autonomic division composed of efferents only. The somatic afferent division is divided in A- and B-neurons. The B-neurons are supposed to be autonomic afferents as part of a reflex system involved in homeostasis. Recent data obtained by neuronal tracing and immunohistochemical experiments concerning the eye related peripheral nervous system endorse the existence of these peripheral reflex systems. Somatic afferents of trigeminal origin synaptically innervate parasympathetic neurons in the pterygopalatine ganglion. This probably represents a pathway mediating autonomically regulated ocular activity in response to sensory stimulation. In addition, it has been hypothesized that trigeminal sensory nerve fibres have an efferent function in response to noxious stimuli e.g. the ocular injury response. Sympathetic nerve fibres originating in the superior cervical ganglion course through the trigeminal and pterygopalatine ganglion without forming direct synaptic contacts. These fibres, however, contain clusters of vesicles suggesting some kind of interneural communication. Parasympathetic nerve fibres of pterygopalatine origin course through the ciliary ganglion. These nerve fibre terminals also contain clusters of vesicles without direct synaptic contacts. Experimental data concerning the distribution of neuropeptides revealed a more detailed knowledge of the anterior eye segment innervation. These experimental data are subject to some debate. The pros and cons of different techniques are discussed. Neural circuits regulating IOP have long been postulated. The possible role of peripheral reflex systems in the regulation of IOP is discussed.

Elimination of immune precipitates from the rat corneal stroma: a histological study.

The pathogenesis of peripheral corneal lesions of immune aetiology, like Moorens ulcer and catarrhal infiltrates, has been related to the formation or deposition of immune compkexes. The present investigation was undertaken to study the mechanisms involved in the elimination of immune precipitates from the cornea. Immune precipitates were induced by injecting human serum albumin (HSA) and rabbit anti-HSA serum into opposite sites of the rat corneal stroma. This resulted in a line-shaped opacity in the stroma, which remained visible by slit-lamp for 7 days, and disappeared without clinical signs of keratitis and uveitis. At the ultrastructural level, the immune precipitates were clearly visible. Keratocytes in the vicinity of the immune precipitates appeared activated, as suggested by their less flattened appearance and well-developed rough endoplasmic reticulum. The arrangement of the collagen fibrils was not affected. Cells with a macrophage-like morphology were also present and contained electron-dense material, closely resembling the precipitate, suggesting phagocytosis. Separate corneas were injected with latex beads, which is known to induce migration of Langerhans cells into the cornea. Immunohistochemical analysis revealed that both latex beads and immune precipitates induced migration of macrophages (ED1+) into the rat corneal stroma. However, differences were observed with regard to the expression of MHC class II antigens by these ED1+ cells and the presence of complement deposits in the corneal stroma. ED1+ cells in corneas injected with latex beads were all MHC class II positive (OX4+), whereas most of the ED1+ cells at the site of the immune precipitates were negative (OX4-).(ABSTRACT TRUNCATED AT 250 WORDS)