Research reportParasympathetic varicosity proliferation and synaptogenesis in rat eyelid smooth muscle 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].
Section snippets
Experimental preparations
Nine Sprague–Dawley rats (Harlan) aged 50–60 days post-natal were anesthetized with a mixture of ketamine (27.5 mg kg−1), xylazine (2.5 mg kg−1) and atropine (0.24 mg kg−1). Using aseptic technique, a ventral midline incision was made in the neck and the right superior cervical ganglion was excised; unilateral superior cervical ganglionectomy in adult rats produces complete sympathetic denervation of ipsilateral anterior orbital targets that persists through at least 1 yr post-surgery 27, 64, 66
Smooth muscle with intact innervation
In STMs with intact innervation, neural elements were observed frequently throughout the muscle 15, 82. Neural structures that contained microtubules but lacked accumulations of vesicles were characterized as axons. Varicosities lacked microtubules but contained accumulations of small spherical vesicles (SSVs) with either clear or dense cores and diameters of approximately 50 nm, and frequently large dense-cored vesicles (LDVs) with diameters of approximately 100 nm. Varicosities also contained
Autonomic innervation of the intact superior tarsal muscle
Short-term denervation studies show that the innervation of the rat STM is derived almost entirely from the ipsilateral superior cervical ganglion. Two days after ganglionectomy, only 3% of varicosities remained, indicating that 97% derive from sympathetic neurons. This is consistent with findings in the mouse, though the percentage of sympathetic varicosities in that rodent (84%) [82]appears to be somewhat lower than that of the rat. Because only a small proportion of the total varicosities is
Conclusions
These findings, together with previous physiological studies, demonstrate that rat superior tarsal muscle normally contains excitatory sympathetic innervation that is closely apposed to smooth muscle cells. Also present is a much smaller number of parasympathetic varicosities that are distant to the smooth muscle cells but appear to make axo–axonal synaptic contacts with sympathetic nerves. Following long-term sympathectomy, parasympathetic varicosities increase in number and form neuroeffector
Acknowledgements
This work was supported by grant HD33025. The R.L. Smith MRDDRC provided core facilities. The authors gratefully acknowledge the technical assistance of Donna Millard, Barbara Fegley, and Karen Grantham.
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Present address: Dept. of Anatomical Sciences, Parker College, Dallas, TX 75229.