Stromblad, 1958]. Further, nerves stained for acetylcholinesterase were looked

Transcription

1 Quarterly Journal of Experimntal Phy8iology (1976) 61, UNCHARTED CHOLINERGIC NERVES IN THE RABBIT PAROTID GLAND. By P. ALM and J. EKSTROM. From the Institute of Anatomy and Histology, and the Institute of Physiology, University of Lund, Sweden. (Received for publication 16th October 1975) Cholinergic nerves are shown to be left in the rabbit parotid gland after avulsion of the auriculo-temporal nerve: a cholinesterase inhibitor injected through the duct caused secretion, thereby revealing leakage of acetylcholine from cholinergic nerve endings, and acetylcholinesterase positive nerves were found histochemically. The incomplete cholinergic denervation offers an explanation to the fact that some choline acetyltransferase activity remains in the 'denervated' glands. In the rabbit parotid glands a rapid and profound fall in the activity of the acetylcholine synthesizing enzyme, choline acetyltransferase, was found by Nordenfelt [1964] to follow a parasympathetic denervation, caused by section of the auriculo-temporal nerve. This finding probably indicates a neuronal localization for the enzyme. However, some enzyme activity usually remained suggesting either the presence of unsevered cholinergic neurones or other sources for the enzyme in the gland. In such denervated parotid glands of other species, i.e. cats [Nordenfelt, 1963; Ekstrom and Emmelin, 1974], dogs [Ekstrom and Holmberg, 1972] and rats [Ekstrom, 1974] a low choline acetyltransferase activity may be demonstrated also. In cats and dogs the residual enzyme activity seems to be accounted for, since several investigations, both physiological and histochemical, show that the denervation is incomplete leaving some cholinergic nerve fibres intact [Emmelin and Str6mblad, 1958; Emmelin and Holmberg, 1967; Garrett, 1966; Garrett and Holmberg, 1972; Holmberg, 1972; Ekstrom and Emmelin, 1974]. Accordingly, by including not only section of the auriculo-temporal nerve in the denervation procedure, but division of nerve strands on the internal maxillary artery [Holmberg, 1971] and the facial nerve the choline acetyltransferase activity of the parotid gland of dogs in most cases reached values below the limit of detection [Ekstrom and Holmberg, 1972]. The present experiments investigate whether in rabbits also there are some cholinergic neurones left following avulsion of the auriculo-temporal nerve. This was done by the use of a cholinesterase inhibitor injected through the duct into the gland; such a substance is known to cause secretion of saliva by preserving acetylcholine leaking from the nerve endings [Emmelin and Stromblad, 1958]. Further, nerves stained for acetylcholinesterase were looked for. METHODS Sixteen rabbits of both sexes weighing kg were used. Under ether and pentobarbitone (35 mg/kg i.p.) anaesthesia the following experimental procedures were 127

2 128 Alm and Ekstrom carried out on the left side: avulsion of the auriculo-temporal nerve, using the approach devised by Stromblad [1955] for cats, i.e. the nerve was traced under the zygomatic arch; avulsion of the superior cervical ganglion; a combination of the two procedures; or avulsion of the auriculo-temporal nerve combined with cutting the sympathetic cervical trunk. The animals were examined on the 7th to the 10th postoperative day. Physiological experiments. In preliminary experiments attempts were made to evoke secretion reflexly in a group of 5 animals. The anaesthesia was pentothal (30 mg/kg) given into an ear vein. The parotid duct on the left side was cannulated with a polyethene tube connected to a bottle via rubber tubing, where saliva displaced water. When the blinking reflex reappeared citric acid (100 mg/ml.) was poured on the tongue of the animals [see Holmberg, 1971]. The drops falling from the bottle were recorded on the smoked drum. In another group of 6 animals the anaesthesia was urethane (2 g/kg) given into an ear vein. A polyethene tube was inserted into the femoral vein; by this route additional anaesthesia and drugs were given. A cannula was inserted into the trachea. The parotid ducts were then cannulated using cannulae which delivered 67 drops of distilled water/ml. Drops of saliva falling from the tip of the cannula were recorded on the smoked drum by an electromagnetic pen operated manually. Hexamethonium 5 mg/kg was given in order to exclude activity from the central nervous system, and the sensitivity of the secretory cells to standard doses of 0 5 and 1,ug/kg of methacholine was estimated. The right parotid cannula was then replaced by another cannula of a similar type supplied with a three-way tap to which a syringe could be attached. Eserine sulphate, 200,ug in 0-2 ml saline solution, was injected into the gland during 5 sec; 5 sec later the tap was turned so as to allow saliva to flow out, and the drops of saliva were recorded for a period of 15 min [Emmelin, Muren and Str6mblad, 1954; Ekstr6m and Emmelin, 1974]. Thereafter an additional dose of 5 mg/kg hexamethonium was given and the same procedure was applied to the left parotid gland. Control injections of 0-2 ml saline and of cocaine were also made into the salivary ducts. Atropine was given intravenously. For statistical evaluation Student's t-test was used. Histochemical procedures. Eight rabbits were used, 3 of which had been used in the physiological studies. The rabbits were killed by air embolism. Pieces of parotid tissue were rapidly dissected out and frozen in liquid isopentane cooled by liquid nitrogen; the auriculo-temporal nerve removed bilaterally from one animal was treated in the same way. To demonstrate acetylcholinesterase activity, frozen sections, cut at 10,um (the nerves longitudinally cut), were treated according to the copper thiocholine method of Koelle and Friedenwald [1949] as modified by Holmstedt [1957]. For the inhibition of nonspecific cholinesterase MipafoxD (bismonoisopropylamide-phosphoryl fluoride) or Ro (Hoffman-La Roche, Switzerland) were used. Eosin was used as counterstain. To demonstrate adrenergic nerves the fluorescence method of Falck and Hillarp was used [Falck, Hillarp, Thieme and Torp, 1962; Bjorklund, Falck and Owman, 1972]. RESULTS Observations on secretory responses In the preliminary experiments, where citric acid was poured on the tongue of the animals, it was possible in some instances to obtain single drops or fractions of drops in 3 rabbits, one of which had not only its auriculo-temporal nerve avulsed but its sympathetic trunk cut also. However, it was difficult to standardize the experimental conditions, e.g. the depth of anaesthesia, and it was found to be difficult to obtain regular secretory responses even from 2

3 Uncharted nerves in rabbit parotids 129 rabbits with normally innervated glands. Instead the eserine test was used, prece(led by determining the senisitivity of the glands to methalcholine. In 5 animals in which the auriculo-temporal nerve had been avulsed on the left side the sensitivity to 1 ag/kg methacholine was much higher (P < 001) in the left thaii in the right parotid gland, as shown in Text-Fig. 1; a similar patterni wvas observed at a dose of 0 5 jg/kgr. All the glands responded with secretion to eserine, althouotoh the amiiount of saliva fromn the left glaind was much lower (P < 0-001) tlhan that from the right gland (Text-Fig. 1). Text-Fig. 2 shows the recordings from one experiment. It is obvious from the Figure that saline inijected through the duct did not initiate any secretion. It is also showin that the eserine is acting locally in the gland, as poilnted out earlier by Emmelin and Stro'mblad [1 958], since no secretion was obtained METHACHOLINE ESERINE TEXT-FIG. 1. Secretory respoinses in drops of saliva of the right control glaind (white columns) and of the left 'denervated' glaind (hatched columnis) in 5 rabbits to methacholine, 1,tg/kg and to eserine, 200,ug. V-alues are mean ±S.E.Al. MIN ( SAL 'E ESERINE RP _! LP SIG iniu LP TEXT-FIG. 2. Secretory effects of mothacholine, 1,ug/kg i.v., and of saline, 0-2 ml, and eserine, 200,g, injected through the salivary duct. Records from above: minute marks; drops of saliva from the right parotid gland; aind from the left gland, the auriculotemporal nerve of which was avulsed 7 days earlier; signal. In the upper panels the iintraductal injections w-ere made on the right side and in the lower panels on the left side. The iight parotid duct is not cannulated in the lower )anels. Before the start of the experiments hexamethonium 5 mg/kg was given i.v.; an additional dose of 5 nmg/kg was given before the saline injection into the left salivary duct.

4 130 Alm and Ekstrom from the sensitized gland when the substance was injected towards the contralateral gland. In one animal avulsion of the auriculo-temporal nerve was combined with avulsion of the superior cervical ganglion; such a gland still responded to eserine. The secretion obtained after eserine injection was abolished by atropine (1 mg/kg). As also shown in the experiments of Emmelin and Stromblad [1958] on cats, cocaine (10 or 20 mg) thought to paralyze the nerves, injected through the duct was found to stop the secretion evoked by eserine from the 'denervated' gland for a short time period during which methacholine (1 pg/kg) could still elicit secretion. Histochemical observations After avulsion of the left auriculo-temporal nerve a small number of acetyleholinesterase positive nerves, distributed around a few acini and ducts, and in the walls of some blood vessels (arteries and arterioles), was found in the glands on this side of 5 animals. In contrast, the picture of the right intact glands showed a very rich supply of acetylcholinesterase positive nerves around the acini and a few in relation to the secretory ducts. The walls of the blood vessels were moderately innervated. This picture of the innervated glands agrees with that described by Freitag and Engel [1970]. In Plate 1 the effect of avulsion of the auriculo-temporal nerve on the presence of acetylcholinesterase positive nerves is shown; the glands are from that rabbit, the secretory responses of which were demonstrated in Text-Fig. 2. The number and distribution of the acetylcholinesterase positive nerves were not changed after avulsion of the superior cervical ganglion, as studied in one animal. Consequently, nerve fibres were still found in the gland of an animal, where avulsion of the auriculo-temporal nerve was combined with avulsion of the sympathetic ganglion. In none of the examined glands (14 altogether) acetylcholinesterase positive ganglion cells were found; neither were any ganglia found in the auriculktemporal nerve. As to the presence of adrenergic nerves, the intact glands of 7 animals showed nerve terminals richly surrounding the acini, whereas no specific fluorescence occurred in relation to the secretory ducts. A plexus of adrenergic terminals surrounded the blood vessels (arteries and arterioles). The findings are in agreement with those of Ehinger, Garrett and Ohlin [1967]. As also shown by these authors, no specific adrenergic nerve fluorescence remained in the present study after avulsion of the superior cervical ganglion (one animal). Avulsion of the auriculo-temporal nerve did not change the fluorescence picture, as studied in 3 rabbits. DIsc-ussIoN The present findings of a secretion of saliva after injection of eserine and the presence of acetyleholinesterase positive nerves show that the parotid glands of rabbits are not completely devoid of cholinergic nerves after avulsion of

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6 Uncharted nerves in rabbit parotids 131 the auriculo-temporal nerve, aiming at a parasympathetic denervation. This view is also supported by the results of the preliminary experiments, in which the reflex secretion was studied. It should be pointed out that the time interval of 7-10 days between the operation and the examination of the glands was probably enough to allow degeneration of the severed nerves. In parotid glands of cats, Garrett [1966] observed that the loss of acetylcholinesterase positive nerves occurred within 6 days of the denervation procedure. It should be emphasized that the adrenergic nerves of the rabbit parotid gland were not stained by the acetylcholinesterase method: avulsion of the sympathetic ganglion did not reduce the number of acetylcholinesterase positive nerves although it caused a complete loss of the adrenergic nerves and further, avulsion of the auriculo-temporal nerve did not cause any reduction in the number of adrenergic nerves. The residual choline acetyltransferase activity, about 2% of the normal value, in the gland 3-5 days after section of the auriculo-temporal nerve made Nordenfelt [1964] suspect that some postganglionic neurones were left. However, he tried in vain to find any ganglion cells within the glands; nor could, in the present study, any ganglia be demonstrated in the glandular parenchyma or in the auriculo-temporal nerve. He suggested tentatively that the choline acetyltransferase activity left could be derived from the Schwann cells. This explanation is not excluded by the findings of the present experiments. However, it appears reasonable to attribute most of the activity of the enzyme to the presence of the unsevered cholinergic neurones. It should be observed that in the present study the approach to the auriculo-temporal nerve was the same as that used by Nordenfelt [1964] and further, instead of sectioning the nerve as Nordenfelt did the nerve was avulsed; this technique probably leads to severance of all branches of the nerve. It is apparent that the main nerve to the parotid gland in rabbits is the auriculo-temporal, the secretory nerve described in 1868 by Navrocki, and that the proportion of cholinergic nerves outside this 'classical' pathway is small. This is, for instance, shown by the large loss of choline acetyltransferase activity [Nordenfelt, 1964] and of acetylcholinesterase positive nerves, by the supersensitivity developed to methacholine and further, by the reduction in the response to eserine following severance of the nerve. It should be noted that the response to eserine is probably markedly exaggerated due to the sensitization of the gland cells. The anatomical routes for the remaining cholinergic nerves are unknown. The present study shows that they are not supplied from the sympathetic ganglion, since after combined avulsion of the auriculo-temporal nerve and the sympathetic ganglion secretion to eserine was still obtained and acetylcholinesterase positive nerves were still present. One possibility is that the facial nerve contributes to the innervation of the gland as is the case in dogs [Ekstr6m and Holmberg, 1972], another is that nerves travel along the blood vessels to the gland as is the case both in dogs [Holmberg, 1971] and in cats [EkstrOm and Emmelin, 1974]. In experiments by Nordenfelt and Ohlin [1957] the facial, lingual and chorda tympani nerves in rabbits were stimulated

7 132 Alm and Ekstr6m electrically; in one animal a small secretion from the parotid gland was obtained when the facial nerve was excited. REFERENCES BJORKLUND, A., FALCK, B. and OWMAN, CH. (1972). Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: S. A. Berson (ed.), Methods of investigative and diagnostic endocrinology, 1 (J. E. Rall and I. J. Kopin, eds.), The Thyroid and Biogenic amines, pp North-Holland Publication Company, Amsterdam. EHINGER, B., GARRETT, J. R. and OHLIN, P. (1967). Adrenergic nerves in the major salivary glands of the rabbit. Experientia, 23, 924. EKSTROM, J. (1974). Choline acetyltransferase activity in rat salivary glands after cellulose rich diet or treatment with an atropine-like drug. Quarterly Journal of Experimental Physiology, 59, EKSTR6M, J. and EMMELIN, N. (1974). The secretory innervation of the parotid gland of the cat: an unexpected component. Quarterly Journal of Experimental Physiology, 59, EKSTROM, J. and HOLMBERG, J. (1972). Choline acetyltransferase in the normal and parasympathetically denervated parotid gland of the dog. Acta Physiologica Scandinavica, 86, EMMELIN, N. and HOLMBERG, J. (1967). Impulse frequency in secretory nerves of salivary glands. Journal of Physiology, 191, EMMiLIN, N., MUREN, A. and STROMMBLAD, R. (1954). Secretory and vascular effects of various drugs injected into the submaxillary duct. Acta Physiologica Scandinavica, 32, EMMELIN, N. and STROMBLAD, B. C. R. (1958). The effect of anticholinesterases on the parotid gland after parasympathetic decentralization or denervation. British Journal of Pharmacology, 13, FALcK, B., HILLARP, N.-A., THIEME, G. and TORP, A. (1962). Fluorescence of catecholamines and related compounds with formaldehyde. Journal of Histochemistry and Cytochemistry, 10, FREITAG, P. and ENGEL, M. B. (1970). Autonomic innervation in rabbit salivary glands. Anatomical Record, 167, GARRETT, J. (1966). The innervation of salivary glands III. The effects of certain experimental procedures on cholinesterase-positive nerves in glands of the cat. Journal of the Royal Microscopical Society, 86, GARRETT, J. R. and HOLMBERG, J. (1972). Effects of surgical denervations on the autonomic nerves in parotid glands of dogs. Zeitschriftfiir Zellforschung und mikroskopische Anatomie, 131, HOLMBERG, J. (1971). The secretory nerves of the parotid gland of the dog. Journal of Physiology, 219, HOLMBERG, J. (1972). The secretory innervation of the dog's parotid gland. Thesis, pp Studentlitteratur, Lund. HOLMSTEDT, B. (1957). A modification of the thiocholine method for determination of cholinesterase. II. Histochemical application. Acta Physiologica Scandinavica, 40, KOELLE, B. G. and FRIEDENWALD, J. S. (1949). A histochemical method for localizing cholinesterase activity. Proceedings of the Society of Experimental Biology and Medicine, 70, NAvRoCKI, F. (1868). Die Innervation der Parotis. Studien des Physiologischen Instituts zu Breslau, 4, NORDENFELT, I. (1963). Choline acetylase in normal and denervated salivary glands. Quarterly Journal of Experimental Physiology, 48, NORDENFELT, I. (1964). Choline acetylase in denervated parotid glands of the rabbit. Acta Universitatis Lundensis, Sectio II, No. 9, 1-7. NORDENFELT, I. and OHLIN, P. (1957). Supersensitivity of salivary glands of rabbits. Acta Physiologica Scandinavica, 41, STROMBLAD, R. (1955). Sensitivity of the normal and denervated parotid gland to chemical agents. Acta Physiologica Scandinavica, 33,