Parasympathetic varicosity proliferation and synaptogenesis in rat eyelid smooth muscle after sympathectomy

Abstract

Parasympathetic innervation to eyelid smooth muscle inhibits sympathetic neurotransmission pre-junctionally without appreciable direct post-junctional effects. However, 5 weeks after sympathectomy, parasympathetic stimulation elicits substantial cholinergically mediated contractions. This study examined ultrastructural changes accompanying the conversion to parasympathetic excitation. In intact muscles, 64±9 nerve varicosities were encountered per 10⁴μm². Most were close to muscle cells and not fully enclosed by supporting cells. Axo–axonal synapses were observed occasionally. Two days following sympathectomy, varicosity numbers were reduced by 97% and, relative to controls, remaining varicosities were farther from muscle cells and more frequently fully enclosed by supporting cells, but contained greater numbers of small spherical and large dense vesicles. By 6 weeks post-sympathectomy, numbers of varicosities per unit muscle volume increased to 14% of controls. These varicosities differed from those at 2 days in being closer to smooth muscle cells, less frequently enclosed, and having fewer small vesicles. These findings indicate that intact eyelid smooth muscle varicosities are predominantly sympathetic, but a small number of parasympathetic varicosities are present, some of which may form pre-junctional synapses with sympathetic nerves. Between 2 days and 6 weeks post-sympathectomy, varicosities increased in number and established appositions with smooth muscle cells. This suggests that parasympathetic nerves are capable of re-innervating an atypical smooth muscle target after sympathectomy, and that parasympathetic synaptogenesis is likely to contribute to conversion from pre-junctional inhibition to post-junctional excitation after sympathectomy.

Introduction

Injury to one population of nerves can affect other similarly distributed populations that are not damaged directly. Uninjured heterologous nerves can undergo sprouting 29, 51, 73as well as alterations in expression of neurotransmitters, enzymes, and peptides 12, 26, 49, 76. However, the extent to which these changes influence neural control of target function remains largely uncertain.

A system where functional alterations are well documented is the parasympathetic innervation to rat the superior tarsal muscle (STM) following sympathetic nerve degeneration. This smooth muscle contracts to elevate the eyelid when excited by sympathetic nerves releasing norepinephrine 27, 63, 64, 66, 67. Sympathetic neurons residing within the ipsilateral superior cervical ganglion 62, 66, 71form a dense ground plexus within the STM as revealed by catecholamine histofluorescence, dopamine β-hydroxylase immunoreactivity (ir) 27, 62, 66, 68, 69, 70, and electron microscopy [82]. The rat STM also receives sparser parasympathetic innervation from the ipsilateral pterygopalatine ganglion 56, 61, 62. Stimulation of these nerves has little or no effect on resting tarsal muscle tone 4, 65. However, when tarsal muscle is contracted by electrical stimulation of the sympathetic nerves, parasympathetic activation elicits significant relaxation. This is blocked by atropine and mimicked by bethanechol, and therefore is mediated by cholinergic muscarinic receptors. Although muscarinic receptors are present on STM smooth muscle cells, these do not mediate relaxation, as they elicit contractions in response to bethanechol [4]. Accordingly, parasympathetic nerves appear to act pre-junctionally to inhibit norepinephrine release from the excitatory sympathetic nerves, as occurs in other systems 34, 46, 57, 74, 78.

Although parasympathetic actions are predominantly pre-junctional in the intact STM, these neurons elicit direct excitation of sympathectomized muscles. Five weeks following superior cervical ganglionectomy (SCG-X), parasympathetic stimulation evokes sizable contraction [65]. This is blocked by atropine and therefore is mediated by parasympathetically released acetylcholine acting on STM muscarinic receptors. Sympathectomy thus initiates changes in tarsal muscle parasympathetic innervation that result in the conversion of function from pre-junctional inhibition to post-junctional excitation.

The mechanisms responsible for parasympathetic excitation after sympathectomy are not known. However, this does not appear to be due either to muscarinic receptor supersensitivity [65]or diminished acetylcholinesterase activity [5]. One possibility is that, in the absence of sympathetic innervation, parasympathetic nerves establish neuroeffector contacts with the denervated smooth muscle cells. This possibility was evaluated in the present study using quantitative ultrastructural analyses. An abstract of some of this work has appeared [45].