A C T A O P H T H A L M O L O G I C A VOL. 5 4 1 9 7 6
From the Dekartment of Anatomy, University of Helsinki (Head: Professor 0 . Eriinko, M.D., Ph.D.) and the “Second Department of Surgery, University Hospital, Helsinki (Head: Professor M . Turunen, M.D.)
EFFECTS OF DENERVATIONS ON THE ACETYLCHOLINESTERASE-CONTAINING AND FLUORESCENT NERVES OF THE RAT IRIS BY
ANTERO HUHTALA, TIM0 TERVO, KAUKO T. HUIKURI‘i and ART0 PALKAMA
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 denervalions 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 Received October 24, 1975.
85
Antero Huhtala, Time Tervo, Kauko T . Huikuri and Art0 Palkama 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. K e y words: rat - iris - acetylcholinesterase - formaldehyde-induced fluorescence - autonomic nerves - sensory nerves - trigeminal neurotomy cervical sympathectomy - ciliary ganglionectomy.
The thiocholine method for histochemical demonstration of acetylcholinesterase (AChE) has been used by many investigators to study the innervation of the rat iris (Csillik & Koelle 1965; Eranko & Raisanen 1965; Ehinger 1966a,b; Ehinger & Falck 1966). It is generally accepted that the parasympathetic cholinergic fibres of the rat iris are rich in this enzyme. On the other hand, the results of previous investigations concerning the possible occurrence of AChE in the sympathetic adrenergic fibres of the iris are controversial. Csillik & Koelle (1965) reported that some AChE-containing fibres of the rat iris degenerated after extirpation of the superior cervical ganglion. This finding has been supported by the denervation experiments of Eranko et al. (1967). On the contrary, Ehinger & Falck (1966) found no changes in the AChEcontaining fibres of the rat iris after superior cervical ganglionectomy. More recently, however, Ivens et al. (1973) with the electron microscope observed AChE reaction in some iridic axons containing small dense cored vesicles characteristic of adrenergic nerves. Several investigations have been performed demonstrating that AChE is also present in various components of the sensory nervous system (Koelle 1955; Abrahhm 1956; Coupland & Holmes 1957; Gerebtzoff & Bertrand 1957). Recently the rat iris has been shown to have a rich supply of sensory nerves (Saari & Johansson 1974; Huhtala 1975). However, there are no studies concerning the AChE content of these nerves. All the fluorescent adrenergic fibres of the rat iris have been conclusively shown to degenerate after removal of the superior cervical ganglion (Falck 1962; Gsillik & Koelle 1965; Malmfors 1965a,b; Malmfors & Sachs 1965; Eranko et al. 1967). The ciliary ganglion of the rat also contains some fluorescent fibres (Huikuri 1966). However, it has been reported that extirpation of this ganglion does not affect the fluorescent nerves of the iris (Ehinger & Falck 1966; Eranko et al. 1967). Most adrenergic fibres seem to reach the cat iris via the ophthalmic division of the trigeminal nerve (Barlow & Root 1949; Johansson, Huhtala & Saari 1975). It is not known if the ocular adrenergic fibres of the rat have a similar route. 86
AChE-containing and Fluorescent N e ~ v e sof Iris
The present investigation was principally undertaken in order to following questions: ( 1 ) Do the adrenergic and/or sensory fibres of contain histochemically demonstrable AChE activity? (2) Do the postganglionic fibres to the rat iris run via the ophthalmic nerve the ciliary ganglion?
answer the the rat iris adrenergic and/or via
Material and Methods Denervation procedures. The material consisted of 40 adult albino rats of the Sprague-Dawley strain weighing between 180 and 370 g. The animals were subjected to ciliary or superior cervical ganglionectomy or to trigeminal neurotomy. All possible combinations of the above-mentioned denenrations were also used. In these experiments ciliary ganglionectomy was always the first and trigeminal neurotomy the last operation, the time between successive denervations being seven days. The denenrations were performed under ether anaesthesia on the right side, the left iris serving as a control. Atropine sulphate (0.02 mg/kg ip) was used as a pre-operative medication. Parasympathetic denervation of the iris was performed by removing the ciliary ganglion after dissecting the zygomatic arch and the lateral part of the Harderian gland as described by Huikuri (1966). Sympathetic denervation was carried out by extirpation of the superior cervical ganglion. The operation was always followed by ptosis and miosis on the denervated side. Preliminary denervations were controlled by histological examination of the removed ganglia. Sensory denervation was performed by an intracranial coagulation of the ophthalmic division of the trigeminal nerve. The stereotactic method for this operation has recently been described in detail by Huhtala (1975). In the initial stages of coagulation, signs of sympathetic stimulation could be seen in the ipsilateral eye: pupillary dilation, widening of the palpebral fissure and protrusion of the globe. After the operation the absence of blink reflex was tested as one control of the denervation. The eyelids on the denervated side were stitched together with silk sutures in order to reduce ocular lesions resulting from neuroparalytic keratitis. In order to avoid bacterial infection a single dose of procain penicillin (100 000 IU/kg) was injected intramuscularly after each operation, and chloramphenicol drops (0.5 O/O) were administered into the eyes once a day after the denervation. The coagulation point was confirmed mder a dissecting microscope at autopsy. The animals were killed 7-21 days after the first operation by decapitation. 87
Antero Huhtala, Tim0 Tervo, Kauko T . Huikuri and Art0 Palkama Demonstration of AChE-containing fibres. After killing the rat both eyes were enucleated and the irises dissected independently. Each iris was cut diagonally into two equal parts. One half of the iris was used to demonstrate AChE activity (Lewis & Shute 1966) and the other half was used for demonstration of one of the segments were immersed in liquid nitrogen and subsequently freeze The part of the iris subjected to the light microscopical demonstration of AChE activity was fixed in 2.5 O / o glutaraldehyde buffered with 0.2 M phosphate buffer, pH 7.2, for 1 h. For the electron microscopy the specimens were fixed in a similar solution for 3 h. After fixation the irises were rinsed overnight in the same buffer at 4 O C . Before incubation with the substrate (acetylthiocholine iodide, Fluka AG., Bucks) the preparations were pre-incubated for 30 min in the buffer solution without the substrate. This pre-incubation medium contained 0.02 mM tetraisopropylpyrophosphoramide (iso-OMPA). This inhibitor was also used in the same concentration in the substrate-containing incubation medium. The incubation was carried out at 4OC for 4 h. For the light microscopy, stretch preparations were mounted on slides in glycerol jelly. For the electron microscopy, specimens from control irises were postfixed in 1 o / o osmium tetroxide buffered with 0.1 M phosphate buffer at p H 7.2. After dehydration with ethyl alcohol the specimens were embedded in Epon-Araldite. Demonstration of the adrenergic fibres. T he adrenergic nerves were demonstrated by using the formaldehyde induced fluorescence technique (Falck & Torp 1961). Immediately after cutting the iris into two segments, small sections of one of the segments were immersed in liquid nitrogen and subsequently freeze dried for 7 days at -45°C in a vacuum of 10-5-10-4 mmHg. The freeze dried specimens were exposed to formaldehyde vapour generated from paraformaldehyde powder. The preparations were embedded in paraffin and 5 p m sections were viewed under a fluorescence microscope equipped with a HBO 200 mercury lamp (Osram) and a Ploem (1971) epi-illuminator. T he following filters were used: BG 38, two BG 3, a T A L 405 interference filter, the dichroic mirror TK 455 with protecting filter K 460, and a K 470 ultraviolet filter above the objective (all filters by Schott & Gen., Mainz). In some experiments the iris segment was treated as a stretch preparation. The tissue was dried with a piece of blotting paper and exposed to formaldehyde vapour without previous freeze drying. Controls f o r the histochemical methods. Non-specific cholinesterase of the normal iris was demonstrated using butyrylthiocholine iodide (Fluka AG., Bucks)
88
AChE-containing and Fluorescent Nerves of Iris
Figs. 1-5. Fig. 1 . Fluorescent (adrenergic) nerve fibres in the dilator area of a control iris. An air-dried stretch preparation. x 150. Fig. 2. Fluorescent (adrenergic) fibres in the dilator area of a control iris. Most fibres are localized in the posterior part (p) of the iris. A sagittal section from a freeze-dried specimen. x 160. Fig. 3. Fluorescent (adrenergic) fibres in the dilator area of the iris 14 days after trigeminal neurotomy. A clear decrease in the fluorescence activity can be seen. A sagittal section from a freeze-dried specimen. x 160. Fig. 4. An air-dried stretch preparation from the iris seven days after superior cervical ganglionectomy. All fluorescent fibres have disappeared. x 320. Fig. 5. Formaldehyde-induced fluorescence in the rat iris after combined ciliary ganglionectomy and trigeminal neurotomy. Almost all fluorescent fibres have disappeared. A sagittal section from a freeze-dried specimen. x 240.
89
Antero Huhtala, Tim0 Tervo, Kauko T. Huikuri and Arto Palkama
as a substrate and 0.02 mM 284 C 51 (1.5-bis-(allyl dimethylammoniumphenyl) pentan-3-onediiodide) as a selective inhibitor for acetylcholinesterase in the pre-incubation and incubation solutions. The incubation was performed a t 4°C for 4 h at pH 7.0. In order to control the specificity of the reactions, the inhibitor 284 C 51 was also combined with the substrate acetylthiocholine iodide, and iso-OMPA with butyrylthiocholine iodide. Moreover, both the inhibitors (iso-OMPA and 284 C 51) were used together, either with acetylthiocholine or butyrylthiocholine iodide as a substrate. The specificity of the bluegreen fluorescence due to the amines in the adrenergic fibres was controlled by means of the following pharmacological procedures: '( 1) reserpin (Serpasilm, Ciba) was injected intraperitoneally (10 mg/kg 12 h before killing) into four unoperated rats in order to deplete catecholamines from the adrenergic nerve fibres, (2) two normal rats were pretreated with a monoamine oxidase inhibitor, nialamide, (Pfizer) (100 mg/kg ip 1 h before killing) in order to increase the catecholamine content in the adrenergic nerves. Figs. 6-8. Fig. 6A. AChE-containing fibres in a stretch preparation from a control iris. Note the thick AChE-containing bundles (b) in the dilator area as well as the fine fibres (f). Separate fibres cannot be discerned in the intensely stained sphincter (s) area. x 23. Fig. 6B. A higher magnification from the sphincter (s) area of the same iris as in Fig. 6A. x 77. Fig. 6C. A higher magnification from the dilator area of the same iris as in Fig. 6A. showing a dense network of fine AChE-containing fibres (f) as well as the thick nerve bundles (b). x 77. Fig. 7A. AChE-containing fibres in the iris 21 days after ipsilateral ciliary ganglionectomy. The staining of the sphincter (s) is weaker than in the normal iris. There are dense intact nerve bundles (b) visible in the dilator region. x 23. Fig. 7B. A higher magnification from the sphincter (s) area of the same iris as in Fig. 7A. x 77. Fig. 7C. A higher magnification from the same iris as in Fig. 7A, showing intact thick nerve bundles (b) in the dilator area. x 77. Fig. 8A. AChE reaction in the iris 21 days after trigeminal neurotomy. Thick nerve bundles have disappeared from the dilator area but the staining of the sphincter (s) looks normal. x 23. Fig. 8B. A higher magnification of the same iris as in Fig. 8A, showing a normal staining in the sphincter (s). x 77. Fig. 8C. A higher magnification of the same iris as in Fig. 8A. There are no AChE-containing thick nerve bundles visible in the dilator area. x 77.
90
AChE-containing and Fluorescent Nerves of Iris
91
Antero Huhlula, Timo Tervo, Kauko T . Huikuri and Arto Palkama
Results Control (normal) iris. Under light microscope the AChE-containing nerves seemed to enter the ciliary root of the iris as thick bundles at several points. From a circularly arranged plexus in the ciliary border of the iris the bundles ran radially to the dilator region where they formed a loose circular plexus (Fig. 6A). A dense network of fine AChE-positive fibres could be seen throughout the dilator region. These fibres mainly ran radially, extending to the sphincter area (Figs. 6A-C). The AChE reaction was most intense in the sphincter region. Here single nerve fibres could rarely be discerned under low magnifications (Fig. 6B). Under electron microscopic examination, the AChE reaction was seen on the axolemma of unmyelinated fibres (Fig. 9). Also most myelinated fibres had intense staining for AChE on their axolemma (Fig. 9). Some myelinated fibres were stained only partially and a small number were not stained at all. No AChE-containing fibres were found around the blood vessels of the iris.
Fig. 9. An electron micrograph showing AChE reaction (arrows 1 and 2) on the axolemma of both myelinated (presumptive sensory) and unmyelinated (presumptive parasympathetic) fibres in the normal rat iris. x 28 000.
92
AChE-containing and Fluorescent Nerves
of Iris
Under light microscope the non-specific cholinesterase activity, like the AChE activity, seemed to be mainly localized in the nerves. The result was similar when 284 C 51 was used as an inhibitor and acetylcholine iodide as a substrate. When the incubation was performed at pH 7.0 and at 4OC for 4 h with butyrylthiocholine iodide as a substrate and iso-OMPA as an inhibitor, the localization of the reaction was identical to the AChE reaction but much weaker. When the irises were incubated with one of the two substrates in the presence of both inhibitors, only slight non-specific staining was seen inside the blood vessels. In the air-dried stretch preparations used for the demonstration of the adrenergic nerves, varicose fluorescent fibres were seen as a dense network in the dilator region (Fig. 1). Most fibres were oriented radially. Moreover, the spincter was supplied by the adrenergic fibres; here most fibres had a circular arrangement. There was a dense plexus of fluorescent fibres around the blood vessels. In tissue sections the adrenergic fibres were distributed most abundantly in the posterior part of the iris (dilator muscle) (Fig. 2). After reserpine pre-treatment, all fluorescent fibres disappeared from the iris. Treatment with nialamide strongly increased the intensity of the fluorescence reaction.
Ciliary ganglionectomy. Extirpation of the ciliary ganglion caused a clear decrease of the AChE reaction, especially in the sphincter area of the ipsilateral iris (Figs. 7A and B). This effect could be seen from the fourteenth day after the denervation. Thick AChE-positive bundles in the dilator region remained intact (Figs. 7A and C). In most experiments, some reduction in the number of adrenergic fibres could also be seen under the fluorescence microscope. Superior cervical ganglionectomy. No degeneration was observed in the AChEcontaining fibres of the iris after this operation. On the other hand, a total disappearance of adrenergic fibres was noted seven days after the denervation in the ipsilateral iris (Fig. 4).
Trigeminal neurotomy. After coagulation of the ophthalmic nerve, the AChEcontaining dense nerve bundles appearing in the dilator region of the control iris (Figs. 6A and C) were not observed in the denervated iris (Figs. 8A and C). The sphincter area stained for AChE with normal intensity after this denervation (Figs. 8A and B). 93
Antero Huhtala, Timo Terwo, Kauko T . Huikuri and Art0 Palkama
Most adrenergic fibres disappeared from the denervated iris after trigeminal neurotomy (Fig. 3). Effects o f combined denervations. The effects of all denervation combinations on AChE-containing nerves of the iris were simple summations of the separate denervations; when ciliarectomy was combined with trigeminal neurotomy both of the changes described above occurred, only few AChE-containing fibres remained visible (Table I). Cervical sympathectomy had no additional effect on the AChE-containing nerves when combined with either ciliarectomy and/or trigeminal neurotomy (Table I). When ciliary ganglionectomy was performed on the same animals together with coagulation of the ophthalmic nerve, a total disappearance of adrenergic fibres could be observed in the ipsilateral iris (Fig. 5, Table I). When cervical sympathectomy was combined with ciliary ganglionectomy and/or with trigeminal neurotomy the results were similar to those of sympathectomy alone (Table I).
Table I . Effects of denervations on different nerves of the iris.
I Denervations (explanation for codes in Fig. 10)
Types of nerves AChE-containing nerves Fluorescent (adrenergic) fibres
Fine fibres in sphincter area
Fine fibres in dilator area
Thick bundles in dilator area
The fluorescence intensity and the AChE activity were registered from to - (negative).
94
+++
(normal)
AChE-containing and Fluorescent Nerves
of
Iris
Discussion The present observation that superior cervical ganglionectomy did not affect the AChE-positive fibres of the rat iris, is in agreement with the findings of Ehinger & Falck (1966). Degeneration of most AChE-containing nerves in the iris after ciliary ganglionectomy confirms that majority of them are parasympathetic in character. The thick AChE-positive bundles remaining intact in the dilator region after ciliary ganglionectomy seem to contain sensory trigeminal fibres because they disappeared after ophthalmic neurotomy. A further evidence for the presence of AChE in the sensory fibres of the iris is the AChE precipitation seen with electron microscopy on the axolemma of myelinated nerves,
Fig. 10. Schematic illustration of adrenergic (preganglionic: - - - -, and postganglionic: -) and AChE-containing (parasympathetic preganglionic: -. - - -, parasympathetic postand sensory postganglionic: 1 ganglionic: . . . ., sensory pregang1ionic:-, nerve fibres of the iris. Denervations indicated by C. G . (ciliary ganglionectomy), S. C. G. (superior cervical ganglionectomy) and T. N. (trigeminai neurotomy).
- -
. .
95
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Antero Hulitala, Tima Tervo, Kauko T . Huikuri and Arto Palkama
which on the other hand, have been shown to originate exclusively from the trigeminal nerve (Huhtda 1975). The biological function 0: AChE in the sensory nerves is open to question. It may indicate that acetylcholine is a chemical mediator of sensory impulses in the rat iris as it seems to be in the rabbit cornea (Fitzgerald & Copper 1971). Some AChE activity seen in the thin iridic nerves after combined ciliary ganglionectomy and ophthalmic neurotomy may indicate that the rat iris receives some postganglionic parasympathetic fibres which do not originate from the ciliary ganglion. In the control experiments, carried out in order to specify the cholinesterase activity, it could be observed that the enzyme on the axonal membranes of both the unmyelinated and myelinated fibres was totally inhibited by 284 C 51. Thus the enzyme appeared to be acetylcholinesterase. However, this enzyme was also capable of hydrolyzing butyrylthiocholine iodide in the presence of iso-OMPA (0.02 mM) at pH 7.0, when the incubation time was 4 h. This finding supports the observations of Eranko & Eranko (1974). The majority of the fluorescent nerves seem to travel to the rat iris via ophthalmic nerve, since most of them disappeared after trigeminal neurotomoy. This observation is parallel to the previous reports concerning the sympathetic fibres of the cat eye (Barlow & Root 1949; Johansson, Huhtala & Saari 1975). All those fluorescent fibres of the iris which remained intact after trigeminal neurotomy disappeared after subsequent ciliary ganglionectomy. These fibres seem to constitute the sympathetic root of the ciliary ganglion, possible entering from the cavernous plexus along with blood vessels and leaving the ciliary ganglion via the short ciliary nerves (Fig. 10). Those fluorescent nerves which join the ophthalmic nerve evidently run to the eye with the long ciliary nerves and/or blood vessels (Fig. 10).
Acknowledgements This work is part of the research project carried out in the Eye Research Laboratory of the Department of Anatomy, University of Helsinki. The project has been supported
financially by a grant from S. JusClius Foundation, Helsinki, and by Star Ltd., Chemical Manufacturers, Tampere, Finland.
References Abrahlm A. (1956) Ober die Struktur und die Endigungen der Aorticusfasern im Aortenbogen des Menschen mit Beriicksichtigung der Cholinesterase-Aktivitat der Pressorrezeptoren. 2. mikr.-anat. Forsch. 62, 194-228.
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Iris
Barlow C. M. & Root W. S. (1949) The ocular sympathetic path between the superior cervical ganglion and the orbit in the cat. J. comp. Neurol. 91, 195-207. Coupland R. E. & Holmes K. L. (1957) The use of cholinesterase techniques for the demonstration of peripheral nervous structures. Quart. J. micr. Sci. 98, 327-330. Csillik B. & Koelle G. B. (1965) Histochemistry of the adrenergic and the cholinergic autonomic innervation apparatus as represented by the rat iris. Acta histochem. 22, 350-363. Ehinger B. (1966a) Cholinesterases in ocular and orbital tissues of some mammals. Acta Univ. Lund. 11, No. 2. Ehinger B. (1966b) Ocular and orbital vegetative nerves. Acta physiol. scand. 67, Suppl. 268. Ehinger B. & Falck B. (1966) Concomitant adrenergic and parasympathetic fibres in the rat iris. Acta physiol. scand. 67, 201-207. Eranko L. & Eranko 0. (1974) Histochemical observations on enzymatic hydrolysis of acetyl-, butyryl- and propionylthiocholine by rat sympathetic ganglion. Med. Biol. 52, 111-120. Eranko O., Huikuri K. & Raisanen L. (1967) The effect of denervation on the adrenergic and cholinergic innervation apparatus of the rat iris. Scand. /. clin. Lab. Invest. 19, Suppl. 95, 75. Eranko 0 . & Raisanen L. (1965) Fibres containing both noradrenaline and acetylcholinesterase in the nerve net of the rat iris. Acta physiol. scand. 63, 505-506. Falck B. (1962) Observations on the possibility of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand. 56, Suppl. 197. Falck B. & Torp A. (1961) A fluorescence method for histochemical demonstration of noradrenaline in the adrenal medulla. Med. exp. (Basel) 5, 429-432. Fitzgerald G. G. & Cooper J. R. (1971) Acetylcholine as a possible sensory mediator in rabbit corneal epithelium. Biochem. Pharmacol. 20, 2741-2748. Gerebtzoff M. A. & Bertrand J. (1957) Gradients d’activith cholinesterasique daus la muqueuse du tube digestif. Ann. Histochim. 2, 149-162. Huhtala A. (1975) Origin of myelinated nerves in the rat iris. Exp. Eye Res. Accepted for publication. Huikuri K. T. (1966) Histochemistry of the ciliary ganglion of the rat and the effect of pre- and postganglionic nerve division. Acta physiol. scand. 67, Suppl. 286. Ivens C., Mottram D. R., Lever J. D., Presley R. & Howells G. (1973) Studies on the acetylcholinesterase (Ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA) treated rat iris. 2. Zellforsch. 138, 21 1-222. Johansson G., Huhtala A. & Saari M. (1975) Observations on the nerves of the cat iris after denervation of the ophthalmic division of the trigeminal nerve. Ophthal. Res. 7, 315-319. Koelle G. B. (1955) The histochemical identification of acetylcholinesterase in cholinergic, adrenergic and sensory neurons. J. Pharmacol. exp. Ther. 114, 167-184. Lewis P. R. & Shute C. C. D. (1966) The simultaneous demonstration of catecholamines and cholinesterases with the electron microscope. /. Physiol. (Lond.) 186, 53-55. Malmfors T. (1965a) Studies on adrenergic nerves. The use of rat and mouse iris for direct observations on their physiology and pharmacology at cellular and subcellular levels. Acta physiol. scand. 64, Suppl. 248.
97 Acta ophthal. 51, I
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Antero Huhtala, Timo Tervo, Kauko T . Huikuri and Arto Palkama Malmfors T. (1965b) The adrenergic innervation of the eye as demonstrated by fluorescence microscopy. Acta physiol. scand. 65, 259-267. Malmfors T. & Sachs C; (1965) Direct studies on the disappearance of the transmitter and changes in the uptake-storage mechanism of degenerating adrenergic nerves. -4cta physiol. scand. 64, 21 1-223. Saari M. & Johansson G. (1974) Myelinated nerves of the rat iris. Acta anat. (Basel) 89, 159-144.
Author’s address: Dr. Antero Huhtala, Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, Helsinki, Finland.
98
From the Dekartment of Anatomy, University of Helsinki (Head: Professor 0 . Eriinko, M.D., Ph.D.) and the “Second Department of Surgery, University Hospital, Helsinki (Head: Professor M . Turunen, M.D.)
EFFECTS OF DENERVATIONS ON THE ACETYLCHOLINESTERASE-CONTAINING AND FLUORESCENT NERVES OF THE RAT IRIS BY
ANTERO HUHTALA, TIM0 TERVO, KAUKO T. HUIKURI‘i and ART0 PALKAMA
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 denervalions 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 Received October 24, 1975.
85
Antero Huhtala, Time Tervo, Kauko T . Huikuri and Art0 Palkama 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. K e y words: rat - iris - acetylcholinesterase - formaldehyde-induced fluorescence - autonomic nerves - sensory nerves - trigeminal neurotomy cervical sympathectomy - ciliary ganglionectomy.
The thiocholine method for histochemical demonstration of acetylcholinesterase (AChE) has been used by many investigators to study the innervation of the rat iris (Csillik & Koelle 1965; Eranko & Raisanen 1965; Ehinger 1966a,b; Ehinger & Falck 1966). It is generally accepted that the parasympathetic cholinergic fibres of the rat iris are rich in this enzyme. On the other hand, the results of previous investigations concerning the possible occurrence of AChE in the sympathetic adrenergic fibres of the iris are controversial. Csillik & Koelle (1965) reported that some AChE-containing fibres of the rat iris degenerated after extirpation of the superior cervical ganglion. This finding has been supported by the denervation experiments of Eranko et al. (1967). On the contrary, Ehinger & Falck (1966) found no changes in the AChEcontaining fibres of the rat iris after superior cervical ganglionectomy. More recently, however, Ivens et al. (1973) with the electron microscope observed AChE reaction in some iridic axons containing small dense cored vesicles characteristic of adrenergic nerves. Several investigations have been performed demonstrating that AChE is also present in various components of the sensory nervous system (Koelle 1955; Abrahhm 1956; Coupland & Holmes 1957; Gerebtzoff & Bertrand 1957). Recently the rat iris has been shown to have a rich supply of sensory nerves (Saari & Johansson 1974; Huhtala 1975). However, there are no studies concerning the AChE content of these nerves. All the fluorescent adrenergic fibres of the rat iris have been conclusively shown to degenerate after removal of the superior cervical ganglion (Falck 1962; Gsillik & Koelle 1965; Malmfors 1965a,b; Malmfors & Sachs 1965; Eranko et al. 1967). The ciliary ganglion of the rat also contains some fluorescent fibres (Huikuri 1966). However, it has been reported that extirpation of this ganglion does not affect the fluorescent nerves of the iris (Ehinger & Falck 1966; Eranko et al. 1967). Most adrenergic fibres seem to reach the cat iris via the ophthalmic division of the trigeminal nerve (Barlow & Root 1949; Johansson, Huhtala & Saari 1975). It is not known if the ocular adrenergic fibres of the rat have a similar route. 86
AChE-containing and Fluorescent N e ~ v e sof Iris
The present investigation was principally undertaken in order to following questions: ( 1 ) Do the adrenergic and/or sensory fibres of contain histochemically demonstrable AChE activity? (2) Do the postganglionic fibres to the rat iris run via the ophthalmic nerve the ciliary ganglion?
answer the the rat iris adrenergic and/or via
Material and Methods Denervation procedures. The material consisted of 40 adult albino rats of the Sprague-Dawley strain weighing between 180 and 370 g. The animals were subjected to ciliary or superior cervical ganglionectomy or to trigeminal neurotomy. All possible combinations of the above-mentioned denenrations were also used. In these experiments ciliary ganglionectomy was always the first and trigeminal neurotomy the last operation, the time between successive denervations being seven days. The denenrations were performed under ether anaesthesia on the right side, the left iris serving as a control. Atropine sulphate (0.02 mg/kg ip) was used as a pre-operative medication. Parasympathetic denervation of the iris was performed by removing the ciliary ganglion after dissecting the zygomatic arch and the lateral part of the Harderian gland as described by Huikuri (1966). Sympathetic denervation was carried out by extirpation of the superior cervical ganglion. The operation was always followed by ptosis and miosis on the denervated side. Preliminary denervations were controlled by histological examination of the removed ganglia. Sensory denervation was performed by an intracranial coagulation of the ophthalmic division of the trigeminal nerve. The stereotactic method for this operation has recently been described in detail by Huhtala (1975). In the initial stages of coagulation, signs of sympathetic stimulation could be seen in the ipsilateral eye: pupillary dilation, widening of the palpebral fissure and protrusion of the globe. After the operation the absence of blink reflex was tested as one control of the denervation. The eyelids on the denervated side were stitched together with silk sutures in order to reduce ocular lesions resulting from neuroparalytic keratitis. In order to avoid bacterial infection a single dose of procain penicillin (100 000 IU/kg) was injected intramuscularly after each operation, and chloramphenicol drops (0.5 O/O) were administered into the eyes once a day after the denervation. The coagulation point was confirmed mder a dissecting microscope at autopsy. The animals were killed 7-21 days after the first operation by decapitation. 87
Antero Huhtala, Tim0 Tervo, Kauko T . Huikuri and Art0 Palkama Demonstration of AChE-containing fibres. After killing the rat both eyes were enucleated and the irises dissected independently. Each iris was cut diagonally into two equal parts. One half of the iris was used to demonstrate AChE activity (Lewis & Shute 1966) and the other half was used for demonstration of one of the segments were immersed in liquid nitrogen and subsequently freeze The part of the iris subjected to the light microscopical demonstration of AChE activity was fixed in 2.5 O / o glutaraldehyde buffered with 0.2 M phosphate buffer, pH 7.2, for 1 h. For the electron microscopy the specimens were fixed in a similar solution for 3 h. After fixation the irises were rinsed overnight in the same buffer at 4 O C . Before incubation with the substrate (acetylthiocholine iodide, Fluka AG., Bucks) the preparations were pre-incubated for 30 min in the buffer solution without the substrate. This pre-incubation medium contained 0.02 mM tetraisopropylpyrophosphoramide (iso-OMPA). This inhibitor was also used in the same concentration in the substrate-containing incubation medium. The incubation was carried out at 4OC for 4 h. For the light microscopy, stretch preparations were mounted on slides in glycerol jelly. For the electron microscopy, specimens from control irises were postfixed in 1 o / o osmium tetroxide buffered with 0.1 M phosphate buffer at p H 7.2. After dehydration with ethyl alcohol the specimens were embedded in Epon-Araldite. Demonstration of the adrenergic fibres. T he adrenergic nerves were demonstrated by using the formaldehyde induced fluorescence technique (Falck & Torp 1961). Immediately after cutting the iris into two segments, small sections of one of the segments were immersed in liquid nitrogen and subsequently freeze dried for 7 days at -45°C in a vacuum of 10-5-10-4 mmHg. The freeze dried specimens were exposed to formaldehyde vapour generated from paraformaldehyde powder. The preparations were embedded in paraffin and 5 p m sections were viewed under a fluorescence microscope equipped with a HBO 200 mercury lamp (Osram) and a Ploem (1971) epi-illuminator. T he following filters were used: BG 38, two BG 3, a T A L 405 interference filter, the dichroic mirror TK 455 with protecting filter K 460, and a K 470 ultraviolet filter above the objective (all filters by Schott & Gen., Mainz). In some experiments the iris segment was treated as a stretch preparation. The tissue was dried with a piece of blotting paper and exposed to formaldehyde vapour without previous freeze drying. Controls f o r the histochemical methods. Non-specific cholinesterase of the normal iris was demonstrated using butyrylthiocholine iodide (Fluka AG., Bucks)
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Figs. 1-5. Fig. 1 . Fluorescent (adrenergic) nerve fibres in the dilator area of a control iris. An air-dried stretch preparation. x 150. Fig. 2. Fluorescent (adrenergic) fibres in the dilator area of a control iris. Most fibres are localized in the posterior part (p) of the iris. A sagittal section from a freeze-dried specimen. x 160. Fig. 3. Fluorescent (adrenergic) fibres in the dilator area of the iris 14 days after trigeminal neurotomy. A clear decrease in the fluorescence activity can be seen. A sagittal section from a freeze-dried specimen. x 160. Fig. 4. An air-dried stretch preparation from the iris seven days after superior cervical ganglionectomy. All fluorescent fibres have disappeared. x 320. Fig. 5. Formaldehyde-induced fluorescence in the rat iris after combined ciliary ganglionectomy and trigeminal neurotomy. Almost all fluorescent fibres have disappeared. A sagittal section from a freeze-dried specimen. x 240.
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as a substrate and 0.02 mM 284 C 51 (1.5-bis-(allyl dimethylammoniumphenyl) pentan-3-onediiodide) as a selective inhibitor for acetylcholinesterase in the pre-incubation and incubation solutions. The incubation was performed a t 4°C for 4 h at pH 7.0. In order to control the specificity of the reactions, the inhibitor 284 C 51 was also combined with the substrate acetylthiocholine iodide, and iso-OMPA with butyrylthiocholine iodide. Moreover, both the inhibitors (iso-OMPA and 284 C 51) were used together, either with acetylthiocholine or butyrylthiocholine iodide as a substrate. The specificity of the bluegreen fluorescence due to the amines in the adrenergic fibres was controlled by means of the following pharmacological procedures: '( 1) reserpin (Serpasilm, Ciba) was injected intraperitoneally (10 mg/kg 12 h before killing) into four unoperated rats in order to deplete catecholamines from the adrenergic nerve fibres, (2) two normal rats were pretreated with a monoamine oxidase inhibitor, nialamide, (Pfizer) (100 mg/kg ip 1 h before killing) in order to increase the catecholamine content in the adrenergic nerves. Figs. 6-8. Fig. 6A. AChE-containing fibres in a stretch preparation from a control iris. Note the thick AChE-containing bundles (b) in the dilator area as well as the fine fibres (f). Separate fibres cannot be discerned in the intensely stained sphincter (s) area. x 23. Fig. 6B. A higher magnification from the sphincter (s) area of the same iris as in Fig. 6A. x 77. Fig. 6C. A higher magnification from the dilator area of the same iris as in Fig. 6A. showing a dense network of fine AChE-containing fibres (f) as well as the thick nerve bundles (b). x 77. Fig. 7A. AChE-containing fibres in the iris 21 days after ipsilateral ciliary ganglionectomy. The staining of the sphincter (s) is weaker than in the normal iris. There are dense intact nerve bundles (b) visible in the dilator region. x 23. Fig. 7B. A higher magnification from the sphincter (s) area of the same iris as in Fig. 7A. x 77. Fig. 7C. A higher magnification from the same iris as in Fig. 7A, showing intact thick nerve bundles (b) in the dilator area. x 77. Fig. 8A. AChE reaction in the iris 21 days after trigeminal neurotomy. Thick nerve bundles have disappeared from the dilator area but the staining of the sphincter (s) looks normal. x 23. Fig. 8B. A higher magnification of the same iris as in Fig. 8A, showing a normal staining in the sphincter (s). x 77. Fig. 8C. A higher magnification of the same iris as in Fig. 8A. There are no AChE-containing thick nerve bundles visible in the dilator area. x 77.
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Results Control (normal) iris. Under light microscope the AChE-containing nerves seemed to enter the ciliary root of the iris as thick bundles at several points. From a circularly arranged plexus in the ciliary border of the iris the bundles ran radially to the dilator region where they formed a loose circular plexus (Fig. 6A). A dense network of fine AChE-positive fibres could be seen throughout the dilator region. These fibres mainly ran radially, extending to the sphincter area (Figs. 6A-C). The AChE reaction was most intense in the sphincter region. Here single nerve fibres could rarely be discerned under low magnifications (Fig. 6B). Under electron microscopic examination, the AChE reaction was seen on the axolemma of unmyelinated fibres (Fig. 9). Also most myelinated fibres had intense staining for AChE on their axolemma (Fig. 9). Some myelinated fibres were stained only partially and a small number were not stained at all. No AChE-containing fibres were found around the blood vessels of the iris.
Fig. 9. An electron micrograph showing AChE reaction (arrows 1 and 2) on the axolemma of both myelinated (presumptive sensory) and unmyelinated (presumptive parasympathetic) fibres in the normal rat iris. x 28 000.
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Under light microscope the non-specific cholinesterase activity, like the AChE activity, seemed to be mainly localized in the nerves. The result was similar when 284 C 51 was used as an inhibitor and acetylcholine iodide as a substrate. When the incubation was performed at pH 7.0 and at 4OC for 4 h with butyrylthiocholine iodide as a substrate and iso-OMPA as an inhibitor, the localization of the reaction was identical to the AChE reaction but much weaker. When the irises were incubated with one of the two substrates in the presence of both inhibitors, only slight non-specific staining was seen inside the blood vessels. In the air-dried stretch preparations used for the demonstration of the adrenergic nerves, varicose fluorescent fibres were seen as a dense network in the dilator region (Fig. 1). Most fibres were oriented radially. Moreover, the spincter was supplied by the adrenergic fibres; here most fibres had a circular arrangement. There was a dense plexus of fluorescent fibres around the blood vessels. In tissue sections the adrenergic fibres were distributed most abundantly in the posterior part of the iris (dilator muscle) (Fig. 2). After reserpine pre-treatment, all fluorescent fibres disappeared from the iris. Treatment with nialamide strongly increased the intensity of the fluorescence reaction.
Ciliary ganglionectomy. Extirpation of the ciliary ganglion caused a clear decrease of the AChE reaction, especially in the sphincter area of the ipsilateral iris (Figs. 7A and B). This effect could be seen from the fourteenth day after the denervation. Thick AChE-positive bundles in the dilator region remained intact (Figs. 7A and C). In most experiments, some reduction in the number of adrenergic fibres could also be seen under the fluorescence microscope. Superior cervical ganglionectomy. No degeneration was observed in the AChEcontaining fibres of the iris after this operation. On the other hand, a total disappearance of adrenergic fibres was noted seven days after the denervation in the ipsilateral iris (Fig. 4).
Trigeminal neurotomy. After coagulation of the ophthalmic nerve, the AChEcontaining dense nerve bundles appearing in the dilator region of the control iris (Figs. 6A and C) were not observed in the denervated iris (Figs. 8A and C). The sphincter area stained for AChE with normal intensity after this denervation (Figs. 8A and B). 93
Antero Huhtala, Timo Terwo, Kauko T . Huikuri and Art0 Palkama
Most adrenergic fibres disappeared from the denervated iris after trigeminal neurotomy (Fig. 3). Effects o f combined denervations. The effects of all denervation combinations on AChE-containing nerves of the iris were simple summations of the separate denervations; when ciliarectomy was combined with trigeminal neurotomy both of the changes described above occurred, only few AChE-containing fibres remained visible (Table I). Cervical sympathectomy had no additional effect on the AChE-containing nerves when combined with either ciliarectomy and/or trigeminal neurotomy (Table I). When ciliary ganglionectomy was performed on the same animals together with coagulation of the ophthalmic nerve, a total disappearance of adrenergic fibres could be observed in the ipsilateral iris (Fig. 5, Table I). When cervical sympathectomy was combined with ciliary ganglionectomy and/or with trigeminal neurotomy the results were similar to those of sympathectomy alone (Table I).
Table I . Effects of denervations on different nerves of the iris.
I Denervations (explanation for codes in Fig. 10)
Types of nerves AChE-containing nerves Fluorescent (adrenergic) fibres
Fine fibres in sphincter area
Fine fibres in dilator area
Thick bundles in dilator area
The fluorescence intensity and the AChE activity were registered from to - (negative).
94
+++
(normal)
AChE-containing and Fluorescent Nerves
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Iris
Discussion The present observation that superior cervical ganglionectomy did not affect the AChE-positive fibres of the rat iris, is in agreement with the findings of Ehinger & Falck (1966). Degeneration of most AChE-containing nerves in the iris after ciliary ganglionectomy confirms that majority of them are parasympathetic in character. The thick AChE-positive bundles remaining intact in the dilator region after ciliary ganglionectomy seem to contain sensory trigeminal fibres because they disappeared after ophthalmic neurotomy. A further evidence for the presence of AChE in the sensory fibres of the iris is the AChE precipitation seen with electron microscopy on the axolemma of myelinated nerves,
Fig. 10. Schematic illustration of adrenergic (preganglionic: - - - -, and postganglionic: -) and AChE-containing (parasympathetic preganglionic: -. - - -, parasympathetic postand sensory postganglionic: 1 ganglionic: . . . ., sensory pregang1ionic:-, nerve fibres of the iris. Denervations indicated by C. G . (ciliary ganglionectomy), S. C. G. (superior cervical ganglionectomy) and T. N. (trigeminai neurotomy).
- -
. .
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Antero Hulitala, Tima Tervo, Kauko T . Huikuri and Arto Palkama
which on the other hand, have been shown to originate exclusively from the trigeminal nerve (Huhtda 1975). The biological function 0: AChE in the sensory nerves is open to question. It may indicate that acetylcholine is a chemical mediator of sensory impulses in the rat iris as it seems to be in the rabbit cornea (Fitzgerald & Copper 1971). Some AChE activity seen in the thin iridic nerves after combined ciliary ganglionectomy and ophthalmic neurotomy may indicate that the rat iris receives some postganglionic parasympathetic fibres which do not originate from the ciliary ganglion. In the control experiments, carried out in order to specify the cholinesterase activity, it could be observed that the enzyme on the axonal membranes of both the unmyelinated and myelinated fibres was totally inhibited by 284 C 51. Thus the enzyme appeared to be acetylcholinesterase. However, this enzyme was also capable of hydrolyzing butyrylthiocholine iodide in the presence of iso-OMPA (0.02 mM) at pH 7.0, when the incubation time was 4 h. This finding supports the observations of Eranko & Eranko (1974). The majority of the fluorescent nerves seem to travel to the rat iris via ophthalmic nerve, since most of them disappeared after trigeminal neurotomoy. This observation is parallel to the previous reports concerning the sympathetic fibres of the cat eye (Barlow & Root 1949; Johansson, Huhtala & Saari 1975). All those fluorescent fibres of the iris which remained intact after trigeminal neurotomy disappeared after subsequent ciliary ganglionectomy. These fibres seem to constitute the sympathetic root of the ciliary ganglion, possible entering from the cavernous plexus along with blood vessels and leaving the ciliary ganglion via the short ciliary nerves (Fig. 10). Those fluorescent nerves which join the ophthalmic nerve evidently run to the eye with the long ciliary nerves and/or blood vessels (Fig. 10).
Acknowledgements This work is part of the research project carried out in the Eye Research Laboratory of the Department of Anatomy, University of Helsinki. The project has been supported
financially by a grant from S. JusClius Foundation, Helsinki, and by Star Ltd., Chemical Manufacturers, Tampere, Finland.
References Abrahlm A. (1956) Ober die Struktur und die Endigungen der Aorticusfasern im Aortenbogen des Menschen mit Beriicksichtigung der Cholinesterase-Aktivitat der Pressorrezeptoren. 2. mikr.-anat. Forsch. 62, 194-228.
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Barlow C. M. & Root W. S. (1949) The ocular sympathetic path between the superior cervical ganglion and the orbit in the cat. J. comp. Neurol. 91, 195-207. Coupland R. E. & Holmes K. L. (1957) The use of cholinesterase techniques for the demonstration of peripheral nervous structures. Quart. J. micr. Sci. 98, 327-330. Csillik B. & Koelle G. B. (1965) Histochemistry of the adrenergic and the cholinergic autonomic innervation apparatus as represented by the rat iris. Acta histochem. 22, 350-363. Ehinger B. (1966a) Cholinesterases in ocular and orbital tissues of some mammals. Acta Univ. Lund. 11, No. 2. Ehinger B. (1966b) Ocular and orbital vegetative nerves. Acta physiol. scand. 67, Suppl. 268. Ehinger B. & Falck B. (1966) Concomitant adrenergic and parasympathetic fibres in the rat iris. Acta physiol. scand. 67, 201-207. Eranko L. & Eranko 0. (1974) Histochemical observations on enzymatic hydrolysis of acetyl-, butyryl- and propionylthiocholine by rat sympathetic ganglion. Med. Biol. 52, 111-120. Eranko O., Huikuri K. & Raisanen L. (1967) The effect of denervation on the adrenergic and cholinergic innervation apparatus of the rat iris. Scand. /. clin. Lab. Invest. 19, Suppl. 95, 75. Eranko 0 . & Raisanen L. (1965) Fibres containing both noradrenaline and acetylcholinesterase in the nerve net of the rat iris. Acta physiol. scand. 63, 505-506. Falck B. (1962) Observations on the possibility of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand. 56, Suppl. 197. Falck B. & Torp A. (1961) A fluorescence method for histochemical demonstration of noradrenaline in the adrenal medulla. Med. exp. (Basel) 5, 429-432. Fitzgerald G. G. & Cooper J. R. (1971) Acetylcholine as a possible sensory mediator in rabbit corneal epithelium. Biochem. Pharmacol. 20, 2741-2748. Gerebtzoff M. A. & Bertrand J. (1957) Gradients d’activith cholinesterasique daus la muqueuse du tube digestif. Ann. Histochim. 2, 149-162. Huhtala A. (1975) Origin of myelinated nerves in the rat iris. Exp. Eye Res. Accepted for publication. Huikuri K. T. (1966) Histochemistry of the ciliary ganglion of the rat and the effect of pre- and postganglionic nerve division. Acta physiol. scand. 67, Suppl. 286. Ivens C., Mottram D. R., Lever J. D., Presley R. & Howells G. (1973) Studies on the acetylcholinesterase (Ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA) treated rat iris. 2. Zellforsch. 138, 21 1-222. Johansson G., Huhtala A. & Saari M. (1975) Observations on the nerves of the cat iris after denervation of the ophthalmic division of the trigeminal nerve. Ophthal. Res. 7, 315-319. Koelle G. B. (1955) The histochemical identification of acetylcholinesterase in cholinergic, adrenergic and sensory neurons. J. Pharmacol. exp. Ther. 114, 167-184. Lewis P. R. & Shute C. C. D. (1966) The simultaneous demonstration of catecholamines and cholinesterases with the electron microscope. /. Physiol. (Lond.) 186, 53-55. Malmfors T. (1965a) Studies on adrenergic nerves. The use of rat and mouse iris for direct observations on their physiology and pharmacology at cellular and subcellular levels. Acta physiol. scand. 64, Suppl. 248.
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Antero Huhtala, Timo Tervo, Kauko T . Huikuri and Arto Palkama Malmfors T. (1965b) The adrenergic innervation of the eye as demonstrated by fluorescence microscopy. Acta physiol. scand. 65, 259-267. Malmfors T. & Sachs C; (1965) Direct studies on the disappearance of the transmitter and changes in the uptake-storage mechanism of degenerating adrenergic nerves. -4cta physiol. scand. 64, 21 1-223. Saari M. & Johansson G. (1974) Myelinated nerves of the rat iris. Acta anat. (Basel) 89, 159-144.
Author’s address: Dr. Antero Huhtala, Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, Helsinki, Finland.
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