The innervation of the rat Harderian gland was studied using histochemical methods for catecholamines and acetylcholinesterase (AChE). Selective denervations were performed to investigate the neural connections of this gland with various ganglia. Light microscopically the AChE-positive nerves seemed to run as thick bundles in the intertubular connective tissue. These bundles sent finer branches around the acini. The blood vessels, localized in the connective tissue septa, were surrounded by a dense plexus of AChE-containing fibres. By electron microscopy, the AChE-positive fibres were seen to terminate near the myoepithelial cells surrounding secretory cells. These fibres were also observed in contact with the blood vessels and occasionally close to the secretory cells. Fluorescent adrenergic nerves surrounded the blood vessels. Some fibres were also observed in the interlobular tissue. All the AChE-containing nerves degenerated after cutting the zygomatic nerve. On the other hand, removal of the ciliary ganglion or the superior cervical ganglion, or stereotactic coagulation of the ophthalmic nerve did not affect these nerves. The fluorescent adrenergic fibres disappeared following both removal of the superior cervical ganglion and coagulation of the ophthalmic nerve. These fibres were intact after removal of the ciliary ganglion.

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The ophthalmic division of the trigeminal nerve was denervated stereotactically by electrocoagulation. Damage to the third nerve was avoided. Four days after denervation the homolateral iris was studied electron microscopically. There was Wallerian degeneration in the myelinated and also in part of the non-myelinated nerves of the iris. The ultrastructure of the iris nerves in the contralateral eye was normal. It is suggested that denervation of the ophthalmic division of the trigeminal nerve had caused interruption of the sympathetic nerves accompanying the trigeminal sensory nerves, and that the degenerated non-myelinated nerves of the iris are sympathetic in origin.

The thiocholine method for the demonstration of AChE-containing fibres and the formaldehyde-induced fluorescence technique for the visualization of adrenergic fibres were employed to study the innervation of the albino rat iris. The following denervations were performed in order to verify the origins of different nerve types: (1) extirpation of the ciliary ganglion, (2) extirpation of the superior cervical ganglion, (3) stereotactic coagulation of the ophthalmic division of the trigeminal nerve, and (4) all possible combinations of the above-mentioned procedures. The denervations disclosed three main types of AChE-containing nerves in the iris: (1) nerve fibres degenerating after ciliary ganglionectomy, (2) thick nerve bundles in the dilator region disappearing after trigeminal neurotomy, and (3) fibres remaining intact after any type of denervation. Cervical sympathectomy had no effect on AChE-positive fibres. Under electron microscope AChE activity could be seen in the axolemma both in unmyelinated and in myelinated fibres. All fluorescent fibres vanished after ipsilateral cervical sympathectomy. Most of these fibres also disappeared after trigeminal neurotomy and the remaining fibres degenerated after subsequent ciliary ganglionectomy. On the basis of the present findings, the following conclusions can be drawn: (1) Most AChE-containing fibres of the rat iris originate in the ciliary ganglion. (2) The majority of the myelinated sensory fibres of the rat iris also contain AChE. (3) There is no AChE in the adrenergic fibres of the rat iris. (4) All adrenergic fibres of the rat iris originate in the ipsilateral superior cervical ganglion, and (5) these fibres enter the iris along with both the long and short ciliary nerves.