FLUORESCENCE HISTOCHEMICAL DEMONSTRATION OF ADRENERGIC NERVE FIBERS IN THE VAGUS NERVE OF CATS AND DOGS

FLUORESCENCE OF

HISTOCHEMICAL

ADRENERGIC

NERVE

NERVE

TAKASHI

OF

MURYOBAYASHI,

DEMONSTRATION

FIBERS

CATS

AND

IN

THE

VAGUS

DOGS

JYO MORI, MOTOHATSU

FUJIWARA

AND KIRO SHIMAMOTO Department of Pharmacology, Faculty of :'Medicine,Kyoto University, Sakyo-ku, Kyoto Received

for

publication

February

21,

1968

The cervial vagus nerves of the mammalians are known to be composed of the fol lowing five, functionally different fibers: 1) general somatic afferent fibers, 2) general visceral afferent fibers, 3) specific visceral afferent fibers, 4) general visceral efferent fibers and 5) specific visceral efferent fibers (1). The general visceral efferent fibers originating from the dorsal motor nucleus of the vagus in the medulla oblongata are regarded to be cholinergic in nature. But fluorescence histochemical studies showed that green-fluores cent noradrenaline terminals became visible in the part of this nucleus after giving 1-dopa to rats (2). Cholinergic impulses conducted in the vagus nerve give rise to the inhibition of cardiac rate and contractility, the relaxation of gastrointestinal sphincters and also to the increase of gastrointestinal motility and tone. However, since the demonstration by McEwen (3) that electrical stimulation of the vagus nerve to the isolated rabbit atria resulted in the increase in cardiac rate and contractility under the presence of hexamethonium, evi dence to confirm the dual responses of the heart to vagal stimulation is accumulating in cats (4), rabbits (5-7) and guinea pigs (8) under various experimental conditions. Jellinek et al. (9) have shown that bilateral cervical vagosympathectomy results in a significant reduction of cardiac catecholamine if dogs survived surgery. The results are rather indicative of the contribution of the vagus nerves to retaining the endogenous noradrenaline in the heart. The demonstration by Hamberger and Norberg (10) that the pelvic nerve of the cat, known to be pure cholinergic in nature, contained the nora drenaline-fluorescent fibers stimulated the authors to perform the present experiments. The vagus nerve of the cat, readily separable from the cervical sympathetic nerve, and also the same nerve of the dog constituting the vagosympathicus were found to contain the noradrenali ne-fluorescent fibers. METHODS 1. Animals Cats 無 謎林

of both 尭 ・森

sexes

weighing

裏 ・潔 原

1.5

元始 ・島本

to 3.0 kg and 暉朗

dogs

of both

sexes

weighing

5 to

10 kg

were used. Animals anesthetized with ether were sacrificed by exsanguination. The right cervical vagus was quickly exposed at the level directly below the epiglottis, and the stomach branch of the right vagus was at the portion just before entry to the cardia or at the beginning of the lesser curvature of the stomach. They were dissected out in pieces of 0.5 to 1.0 cm' for the fluorescent histochemical procedures. In control studies, the right sciatic nerve was dissected from the middle portion of the femoral region. In order to confirm the presence of adrenergic component in these nerves, the following procedures were also adopted. Ligation of nerve: The vagus and sciatic nerves were exposed under ether anesthesia as described above and then ligated tightly with silk thread at the middle portion of the exposed nerve trunks. Two or five days after ligation, nerve pieces involving the ligature were dissected out for the histochemical examination. Infusion of noradrenaline: It has been confirmed that the adrenergic nerves take up circulating noradrenaline and the catecholamine-fluorescence is intensified (11). For this purpose, the dose of 100 itg/kg dl-noradrenaline hydrochloride was injected into the external jugular vein, taking 5 minutes. Fifteen minutes after the end of injection, the animals were sacrificed by exsanguination for dissecting the nerve pieces. Superior cervical ganglionectom), : In the cat and dog, a firm adhesion of the superior cervical ganglion with the nodose ganglion often occurs, suggesting the communication between both ganglia. In order to know whether the adrenergic postganglionic nerve fibers originated from the superior cervical ganglion enter the vagus nerve, the right superior cervical ganglion was removed aseptically. Ten days after surgery the vagus nerves were dissected as described. 2. Fluorescenthistochemicalprodedure The isolated nerve specimens were frozen in an isopentane solution cooled to -35 to 40 C by liquid nitrogen and were dehydrated in vacuoat 30 to 35 C for 6 days. Ac cording to Falck and Hillarp (12, 13), the freeze-dried tissues were exposed to formaldehyde gas at 80 C for one hour, infiltrated with paraffin at 60-C for 10 minutes, sectioned to 6-8 /1 thick and mounted on the non-fluorescent glass i 14). Embedding was made in a mixture of equal amount of Entellan and xylene. The monoamine-fluorescence developed by the exposure to formaldehyde gas was observed under a Zeiss fluorescence microscope and photographed on Kodak Tri-X film. The exciting light was delivered from the Osram HBO 200 high-pressure mercury lamp and was filtered through Schott BG 12 and Zeiss 50 as the primary and secondary filters. RESULTS 1.

Cervical vagus nerve The

nerve

vagus

fibers ranging

impulses fibers

cervical

of the vagus

of the

from

nerve

is composed

of bundles

1 to 14 It in diameter.

to the heart

2-4 It diameter

and

group

bronchial and

of mvelinated

According musculatures

by unmyelinated

and

unmyelinated

to Daly and Evans (15), efferent are conveyed fibers.

Afferent

by myelinated functions

are

FiG. 1.

Fluorescence

Cervical in

FiG. 2.

the

microphotograph

trunk. nerve

x 128.

green

sectioned

fluorescent

vagus material

nerve

of

cat.

is scattered

trunk.

Longitudinally

Green

of cross

Weakly

fluorescent

sectioned slender

vagus fiber

nerve

is seen

of cat. in

the

nerve

Cervical

trunk.

x 128.

trunk.

served by myelinated fibers found in all diameter group (1-14P) and by unmyelinated fibers. Many of the small-sized myelinated fibers did not develop green fluorescence representing catecholamines. On the other hand, many of the small-sized unmyelinated fibers in the cervical vagus nerve showed the specific green fluorescence. However, owing to the faintness of the fluorescence, the fibers were not always readily detectable (Figs. 1 and 2). The fluorescent fibers were sometimes bound into bundles or otherwise scat tered among the myelinated fibers. At the portion where the fluorescent fibers were pre sent in groups, the varicose or net-structures of the communicating branches were seen.

FiG. 3.

Longitudinally

tensely above

FiG. 4.

green the

sectioned fluorescent

ligation,

Longitudinally

tensely

green

within

swollen

vagus material

made

2 days

sectioned fluorescent axons

In contrast to the abundant

above

the

distribution

cal vagus, there was few fluorescent

before

vagus material

nerve.

Cervical

is accumulated

x 128. swollen

In axons

killing.

nerve.

Cervical

is more ligation,

trunk. within

trunk.

prominently

made

5 days

x 128.

In

accumulated before

of the specific fluorescent

fiber in the nodose ganglion.

killing.

fibers in the cervi But the fluorescence

was intense and it seemed to originate from the relatively small-sized myelinated fibers. Two days after ligation of the cervical vagus, the intensity of green fluorescence in creased markedly of fluorescence,

above but not below the ligation (Fig. 3). there were several patterns

2) with some intensely fluorescent

of nerve bundles

As regards to the development as follows : 1) no fluorescent,

fibers, 3) intensely fluorescent in the half, 4) relatively

small-sized but filled diffusely with the intensely fluorescent fibers, and 5) diffusely but relatively weak fluorescent. In additon to the vagus nerve fibers proper, there were spe cific fluorescent fibers in the adventitia of the small arteries and arterioles in the interfascicu lar tissues. The specific fluorescence above ligature became more prominent 5 days after surgery (Fig. 4). The intravenous injection of 100 i g/kg of noradrenaline intensified the specific fluores cence of the unmyelinated fibers in control as well as in ligated vagus (Fig. 5). However,

FIG.5. Cross sectioned vagus nerve. Cervical trunk. x 126. intensely green fluorescent material is scattered in the normal nerve trunk, 15 minutes after the intravenous injection of noradrenaline, 100 ug/kg.

FIG. 6.

Cross

rescent made

sectioned material

2 days

vagus

nerve.

is accumulated before

killing.

Gastric within

branch. swollen

x 50. axons

above

Intensely the

fluo ligation

,

capillary

hemorrhage

structure

in the

2.

often

vicinity

occurred

at the

of hemorrhage

site of intensified

was

made

fluorescence

and

the

fine

obscure.

Gastric branch of the vagus nerve Most of the myelinated

or small-sized

in diameter,

sized unmyelinated

FIG. 7.

FIG. 8.

Longitudinally

sectioned

fluorescent

ligation,

Cross

rescent the

and they did not show the specific fluorescence.

fibers with the relatively

tensely the

fibers in the gastric branch of the vagus nerve were medium

made

2 days

sectioned material,

ligation

cervical

material

vagus which

made

ganglionectomy

2 days

intense fluorescence

vagus

nerve.

is accumulated before

nerve.

before

branch.

within

swollen

x 128. axons

In above

killing.

Gastric

is otherwise

7 days

Gastric

The small

were found to be dis

branch.

x 128.

accumulated killing,

before.

is not

within seen

under

Green axons the

fluo above superior

seminated around the non-fluorescent myelinated fibers. Two or five days after ligation, the fluorescent fibers above ligature increased in number and density, but not below it (Figs. 6 and 7). The injection of noradrenaline resulted in the increases in number and intensity of fluorescence more than that in the cervical vagus. Capillary hemorrhage in the gastric branch was also more severe than in the cervical vagus. 3. Effects of the superior cervical ganglionectomy Ten days after the removal of the superior cervical ganglion the specific fluorescence in the cervical vagus and its gastric branch disappeared completely. Neither ligation of the nerve nor the infusion of noradrenaline did cause the appearance of the specific flu orescence even above the ligature (Fig. 8). However, the specific fluorescent fibers in the nodose ganglion was unaffected by the ganglionectomy. 4.

Sciatic nerve The sciatic nerve is mainly composed of the relatively large or medium-sized, mye linated fibers. The small-sized unmyelinated fibers were present disseminatedly among the myelinated fibers. Both fibers did not develop the specific fluorescence. Three or five days after ligation of the nerve, a marked degree of accumulation of fluorescent ma terial was observed above the ligature but not below it. Unlike the vagus nerve, the in jection of noradrenaline usually did not cause the appearance of the specific fluorescent material in small-sized unmyelinated fibers of the non-ligated sciatic nerve. The fluores cence in the ligated nerve also did not increase after injection of noradrenaline. Capillary hemorrhage was not porduced by the injection of noradrenaline even at the site of ligation. DISCUSSION Physiological evidence to support the presence of the adrenergic component in the cervical vagus nerve has been accumulating. The present experiments were designed in an attempt to demonstrate histochemically the presence of adrenergic fibers in the vagus nerve of the cat and dog. The catecholamine-fluorescent fibers were found in the vagus of the cat and dog. They were mainly unmyelinated and of small diameter. The inten sity of the fluorescence was much less than that of the adrenergic fibers innervating the salivary glands (11, 14, 16,), heart (17) and stomach (18). The absence of the specific fluorescence from many of the myelinated fibers of the vagus nerve indicates that the preg anglionic fibers of the vagus nerve contain few if any adrenergic component. However, the presence of the specific fluorescent material in the myelinated fibers of the nodose gan glion does not exclude the possibility that there exist the preganglionic, adrenergic fibers in the vagus, probably originating from the dorsal motor nucleus of the vagus in the me dulla oblongata. Dahlstrom and Fuxe (19) have demonstrated that green-fluorescent materials accumu late above ligature of the great splanchnic and sciatic nerves. The ligation of the cervical vagus nerve and its gastric branch resulted in a marked intensification of the specific fluo rescence in the small-sized unmyelinated fibers above the ligature. This fact suggests that

the endogenous catecholamines synthesized in the adrenergic ganglionic cells are trans ported in the axon down to the terminals. The intensification of the specific fluorescence after ligation made the adrenergic components in the vagus more apparent than usual. Some of the bundles were totally or partially occupied by the fluorescent fibers, while others were not or diffusely fluorescent. The mode of innervation of the adrenergic nerve fibers in the vagus to the visceral organs remains to be settled in the further study. The results of the uptake of circulating noradrenaline also support the view that the fluorescent fibers in the vagus is adrenergic in nature. The increase in intensity of the specific fluorescence after the injection of noradrenaline was more readily observed in the gastric branch of the vagus than in the cervical vagus or sciatic nerve. The sciatic nerve did not respond to the injection with the intensification of fluorescence. This might be due to a difficult access of injected noradrenaline across the blood-tissue barrier in the scia tic nerve. This assumption is supported by the fact that noradrenaline causes capillary hemorrhage above the site of ligation in the vagus but not in the sciatic. About 10 days after the superior cervical ganglionectomy the specific fluorescent ma terial in the vagus nerve disappeared totally. Intensification of the fluorescence did not occur after the injection of noradrenaline or after the ligation of the nerve. These facts suggest that the adrenergic fibers in the vagus nerve originate from or pass through the superior cervical ganglion and are postganglionic in nature. SUMMARY In the cervical vagus nerve and its gastric branch of the cat and dog, green-fluorescent materials representing catecholamines were seen among the myelinated fibers. The fluo rescence originated from the small-sized unmyelinated fibers. The ligation of the nerve resulted in an intensification of the fluorescence above ligature. Injection of noradrenaline increased the number and intensity of fluorescence in control as well as ligated vagi. Re moval of the superior cervical ganglion resulted in a complete disappearance of the specific fluorescence in the cervical vagus and its gastric branch. Neither ligation of nerve nor injection of noradrenaline did cause the appearance of the specific fluorescence after gan glionectomy. It is concluded that the vagus nerve of the cat and dog contains the adrener gic component, which is mainly small-sized unmyelinated fibers and originated from or pass through the superior cervical ganglion. REFERENCES

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2) 3)

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