Cytochemical and physiological evidence for cholinergic, neurogenic vasodilation of amphibian arterioles and precapillary sphincters

MICROVASCULAR

RESEARCH,

Cytochemical Neurogenic

3, 308-322 (1971)

and Physiological Evidence Vasodilation of Amphibian Precapillary Sphincters I. Light

for Cholinergic, Arterioles and

Microscopy

GEORGE ROBERT SIGGINS AND HOWARD A. WE~TSEN Laboratory of Neuropharmacology, DiGsion of Special Mental Health Research, National Institute of Mental Health, Saint Elizabeth’s Hospital, Washington, D.C. 20032 Received December 7. I970

Arterioles and precapillary sphincters of the frog retrolingual membrane exhibit pronounced vasodilation after microelectrode stimulation of associated terminal nerves. To establish if these vasomotor nerves are cholinergic, the thiocholine method of Karnovsky and the selective methylene blue (pH 6.5-7.0) technique of Richardson were applied to retrolingual membranes. With both methods, the identical nerves which were found to evoke vasodilation stained markedly. In general, both methods also stain scattered motor nerves and vasomotor nerves not immediately associated with blood vessels, and a fine tight-meshed perivascular plexus surrounding all arterioles and precapillary sphincters. With the thiocholine method, motor end-plates on striated muscle, precapillary sphincters, and occasional short segments of arteriolar walls stain most intensely for acetylcholinesterase. Methylene blue did not stain these areas, and preservation of the methylene blue staining product in nerves was much more capricious than the thiocholine stain. Destruction of adrenergic nerves by chronic treatment with 6-hydroxydopamine did not alter the staining of neuronal structures in retrolingual membranes stained by thiocholine or methylene blue techniques. These light microscopic findings supplement previous pharmacological and ultrastructural studies indicating a direct cholinergic innervation of arterioles and precapillary sphincters in the retrolingual membrane, and further establish the value of this membrane as a model for cholinergic innervation ofthe microvascular system.

INTRODUCTION Although adrenergic vasoconstrictor nerves are almost ubiquitous in vertebrates, the exact distribution of cholinergic vasodilator nerves remains to be established. This situation arises in part from the paucity of adequate histochemical methods specific for cholinergic nerves. As a consequence, the best evidence to date for cholinergic neurogenic vasodilation is derived from pharmacological studies (Katz and Jochim, 1939; Armin et al., 1953; Barcroft et al., 1960; Biilbring and Burn, 1936; Dale and Gaddum, 1930; Mchedlishvili and Nikolaishvili, 1970; Stinson et al., 1969; Uvnas, 1954). However, most autonomically active pharmacological agents have non specific actions (see Boyd et al., 1963; Moran and Perkins, 1958; Innes and Nickerson, 1965) and some cholinergic responses may actually be resistant to anticholinergic drugs such as atropine(Folkowet al., 1948; Gyermek, 1961). For studies on the microvascular system, the thin retrolingual membrane of the frog is a favorable model system for analysis of cholinergic vasodilation (Weitsen et al., 308

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1970). In early studies on this tissue Fulton and Lutz (1940, 1942) made direct microscopic documentation of a marked arteriolar dilation in response to stimulation by microelectrode of terminal nerves. Results of pharmacological tests (Siggins, 1967a; Berman and Siggins, 1968) indicate that this neurogenic response is cholinergic. In ultrastructural studies, we have observed periarteriolar nerve terminals containing predominantly small electronlucent vesicles which were distinct from histochemically characterized adrenergic nerves (Siggins, 1970; Siggins and Bloom, 1970). The nerve endings with clear vesicleswere tentatively classified as cholinergic (Siggins and Bloom, 1970). This communication, reported in preliminary form elsewhere (Weitsen et al., 1970),describesstudies combining electrophysiological, pharmacological, and recently developed histochemical techniques. This multidisciplinary approach was used to identify the chemical nature and distribution of vasodilator nerves in the retrolingual membrane. MATERIALS

AND METHODS

Physiology. Male and female frogs (Ranapipiens) of 25-55 g body wt were studied in spring, summer, and fall. The frogs were immobilized by immersion in MS-222 (Sandoz) 1 : 1000in tap water; single-pithing was occasionally used in acute studies.Neurogenic responsesvary little between arterioles of pithed or MS-222-treated animals (Siggins, 1967b). The thin retrolingual membrane was prepared with the aid of a dissecting microscope using a modification (Siggins, 1967b)of the Pratt and Reid (1930)technique. The tongue and retrolingual membrane were gently stretched over a Lucite block, transilluminated, and observed microscopically at 100 or 200 diameters. Arterioles, precapillary sphincters, and their associated vasomotor nerves were microscopically identified and their location and configuration mapped. The vasomotor nerves were stimulated with single rectangular pulses or trains of 3-4 pulses at a frequency of 1-2/set. Each pulse was 1 msecin duration, which allows separation of neurogenic vasodilation from the adrenergic vasoconstruction produced with shorter duration pulses (Berman and Siggins, 1968; Siggins and Bloom, 1970). The pulse was delivered by a monopolar microelectrode having a tip diameter of 10 p and constructed of platinum wire insulated to the tip with glass. Current was delivered by a Grass S-4 stimulator and monitored on a Tektronix oscilloscope. When vasodilation of arterioles and precapillary sphincters was observed in response to nerve stimulation, the control vessel diameter and the just-maximal vascular responsewere both photographed through a beam splitter in the microscope. After drug treatment or histochemical staining, the same nerve-arteriole unit was relocated using maps and photographs. Chronic 6-hydroxydopamine (6-HDA) treatment. After photographing neurogenic responses of control arterioles and precapillary sphincters, some retrolingual membranes were then chemically sympathectomized with 6-HDA. These retrolingual membranes were treated for 2 hr on 2 consecutive days with topical 6-HDA Hydrobromide (Regis), 0.5 mg/ml in Ringer’s solution (Siggins and Bloom. 1970). Sodium metabisulfite (about 0.001 mg/ml) was added to prevent 6-HDA oxidation. Neurogenic responsesof the same vesselspreviously studied were observed and photographed at least once within the 5-l 5-day period after the first 6-HDA treatment. The retrolingual membraneswere then prepared histochemically.

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Histochemical Procedure. To determine the presence of acetylcholinesterase-containing neuronal elements, the thiocholine method of Karnovsky and Roots (1964), as modified by El-Badawi and Schenk (1967) was used. Two staining procedures were used: first, the retrolingual membranes were surgically removed and prepared on a slide as a stretch mount. The membrane was dried in a current of dry air and placed in ice-cold buffered neutral formaldehyde for 20 min. The slide was washed with distilled water and transferred to the incubation medium which was maintained at 37” and contained 8 x low5 M iso-OMPA (tetraisopropylpyrophosphoramide) to insure complete inhibition of nonspecific cholinesterase (El-Badawi and Schenk, 1967). Optimal staining occurred after l-2 hr incubation, at which time the slides were briefly washed in distilled water and routinely dehydrated and mounted. In the second procedure, vital staining was accomplished by topical application of the incubation medium to the unfixed retrolingual membrane while it remained in the same position as used for electrophysiological experimentation. After 30-45 min of copious topical application, AChE-positive nerve staining could be clearly seen with the aid of the dissecting microscope. The retrolingual membranes were then removed as before, stretchmounted, fixed in cold neutral formaldehyde, and staining completed in vitro. The total incubation time again averaged 2 hr or less. For controls, several retrolingual membranes were incubated without the substrate acetylthiocholine. Previous studies (Brzin et al., 1966; Shen et al., 1955) have found no histochemical or manometric evidence for nonspecific cholinesterase in the frog. Thus, further controls such as the use of butyryl thiocholine as substrate or the inhibition of acetylcholinesterase were unnecessary. Due to the nature of the tissue and staining, no counterstain was needed. The slides were examined by bright field, dark field (Norvell et al., 1971), and phase contrast microscopy. A new modification of vital methylene blue staining (Ehinger et al., 1967; Richardson, 1968, 1969) which is reported to stain cholinergic nerves was also used. The methylene blue solution used in our study was prepared as described by Richardson (1968, 1969) or was modified by adding the methylene blue powder to buffered frog Ringer’s solution (2 mg/lOO ml). The pH of all staining solutions was standardized at 6.8 and osmolarity appropriately adjusted. Vital staining was carried out in situ via topical application for 20-30 min. The pH of the incubation medium bathing the tongue and retrolingual membrane was periodically monitored to insure maintenance within the range of pH 6.5-7.0. Although oxygenation of the solution was used on occasion it did not appear necessary, perhaps due to the topical application of the staining solution and maintenance of blood flow in the vessels of the membrane. The retrolingual membranes were immediately removed following staining and were dried on a slide as in the thiocholine technique. The slide was then placed in ammonium molybdate fixative for 1-24 hr followed by 1 hr in a solution of 90% absolute alcohol and 10% stock formaldehyde. The slides were dehydrated in absolute ethanol, cleared in xylene, and observed with bright field and phase contrast microscopy. RESULTS Normal animals-acetylcholinesterase (AChE). Whether incubated in vitro or in vivo, virtually all retrolingual membranes studied exhibited a profuse innervation which

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stained positively for acetylcholinesterase (Fig. 1). Although the greatest density of nerves was closely related to arterioles or precapillary sphincters, many highly stained nerveswere also observedin no apparent relationship to blood vessels.When the course of thesewidely dispersedfibers (the “primary plexus” of Lutz et aZ.,1950)were followed over sufficient distances, somewere seento merge eventually with arterioles or sphincters. Other primary nerves terminated at multiple en grappe type motor end-plates (Fig. 1) typical of the “slow” type of striated muscle (Hess, 1970)scatteredthroughout

FIG. 1. Overview of acetylcholinesterase-stained structures in the retrolingual membrane of the frog. Large arrows indicate the widely scattered nerves of the primary plexus of Lutz et al. (1950). The small arrows show the profuse fibers of the secondary and tertiary plexus surrounding an arteriole (a). Note heavy staining of motor end-plates (m) on the occasional striated muscle fibers (s) scattered throughout the retrolingual membrane. Incubation time, 2-hr. Bright field microscopy. Calibration bar in this and all subsequent figures = 100p.

the membrane (see Siggins et ai., 1968). Clearly, there appear to be two nerve fiber types staining positively for AChE in the retrolingual membrane, vasomotor nerves, and motor nervesinnervating striated muscle. When retrolingual membranes were incubated with the thiocholine medium in situ without initial fixation and observed in the dissecting microscope (50-100 x), motor end-plates were usually stained first (lo-15 min) and most intensely, followed by staining of precapillary sphincters and motor nerves (15-30 min). The periarteriolar nervesusually stained after 30-40 min of incubation (Figs. l-4). When membraneswere excised first, fixed 20 min in formalin, and then incubated in the thiocholine medium

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FIG. 2. Thiocholine staining of arterioles and associated nervous structures in the frog retrolingual membrane. Note the fine plexus of nerves around the arteriole (a) and the profound acetylcholinesterase localization at all four precapillary sphincters (arrows) issuing from this arteriole. S, striated muscle fibers. Bright field microscopy.

FIG. 3. Dark field micrograph of arteriole stained with the thiocholine method. All bright areas have high acetylcholinesterase activity. Note intense staining of precapillary sphincters (large arrows) and two arteriolar segments (brackets), compared to the more faint staining of primary nerve fibers (small arrows).

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(as per El-Badawi and Schenk, 1967) vasomotor nerves generally stained most intensely by 45 min to 2 hr. The intense staining of precapillary sphincters deserves special comment. These areas of high acetylcholinesterase activity are easily seen in normal light microscopy (Fig. 2) but are even more prominent when viewed with the dark field technique of Norvell et al. (1971) (Figs. 3 and 4). The appearance of acetylcholinesterase staining by this method may be compared with the catecholamine-containing nerves previously demonstrated in the retrolingual membrane (Berman and Siggins, 1968; Siggins and

FIG. 4. Dark field micrograph of retrolingual membrane stained for acetylcholinesterase. Intense staining is localized to precapillary sphincters (large arrows), two short arteriolar segments (brackets), and nerves of the primary plexus (small arrows).

Bloom, 1970) by the fluorescence method of Falck et al. (1962, 1963). Figures 3 and 4 illustrate an additional phenomenon seen in this study; occasional nonsphincter areas of arterioles (40-175 p in length) also displayed intense AChE activity similar to that seen inprecapillary sphincters. The precapillary sphincters and short arteriolar segments of intense staining were usually profusely innervated. However, these regions always exhibited a diffuse staining of the vessel wall, in addition to the more delimited staining of the associated nerves (Figs. 2-4). On occasion these regions of intense staining were not associated with obvious nerves. 12

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Although occasional single nerves (the “secondary plexus” of Lutz et al., 1950)were seen loosely associated with capillaries or venules, arterioles and precapillary sphincters exhibited the most profuse acetylcholinesterase-positive innervation. Furthermore, only arterioles and precapillary sphincters showed staining of the fine tertiary plexus closely overlying the vascular wall (Lutz et al., 1950; Berman and Siggins, 1968). No staining of nerves was seen in any of these structures when acetylthiocholine was omitted from the medium. Normal animals-methylene blue. When retrolingual membranes were incubated supravitally with methylene blue (2 mg/lOO ml) at pH 6.8, staining of vasomotor nerves

FIG. 5. Methylene blue (at pH 6.5) staining of a motor nerve (n) and terminals on motor end-plates (m) of striated muscle fibers (S) in the retrolingual membrane. Note discrete localization of staining of the terminals, with little apparent staining of postjunctional areas, compared to the broad staining of the whole end-plate apparatus by thiocholine (Fig. 1). Most often, methylene blue does not stain the motor nerve terminals. The striated muscle fiber(S) stains slightly. Bright field microscopy.

and motor nerves innervating striated muscle could be followed in the dissecting microscope. Motor nerves (Fig. 5) usually stained first (3-15 min), while vasomotor nerves on arterioles (Fig. 6) appeared by 7-20 min of incubation. Maximum staining of both types of nerves appeared by 20-25 min and then began to fade rapidly. In most respects the vasomotor nerves, especially before fixation, had the same plexiform appearance with methylene blue as with acetylcholinesterase staining. However, in contrast to the thiocholine method, walls of arterioles, precapillary sphincters, and postjunctional areas surrounding motor end-plates (Fig. 5) did not stain. Furthermore, the fine “tertiary plexus” of Lutz et al. (1950) closely applied to the arterioles or sphincters usually faded after excision of the retrolingual membrane and fixation with molybdate, followed by formal-alcohol (see Materials and Methods). Fixation of the methylene blue stain was always much more variable and less permanent than with the

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acetylcholinesterasestain. Methylene blue-stained nerveswere never seenin association with venulesor capillaries. An interesting finding was that the function of both vasomotor and skeletal muscle motor nerves was abolished, as determined by microelectrode stimulation, by lo-15 min of topical methylene blue treatment.

FIG. 6. Methylene blue staining (pH 6.8) of periarteriolar nerves: short incubation time (25 min). Note variable staining of nerves of primary and secondary plexus (arrows), which go in and out of view. Nerves of fine tertiary plexus on arteriole (A) stain very lightly, if at all. Striated muscle fibers (S) also stain, especially in areas (c)where contracture has injured them. Bright field microscopy.

Physiology and histochemistry of normal and 6-HDA-treated arterioles. Previous findings with respect to the effect of 6-HDA on neurogenic responses(Siggins and Bloom, 1970)were reconfirmed by the present investigation. While current thresholds for neurogenic vasodilation were the same(mean = 0.11 mA) before and after 6-HDA treatment, neurogenic vasoconstriction was abolished by 6-HDA. Furthermore, in most casesit was possible to photograph the neurogenic vasodilation in situ, incubate each membrane with thiocholine or methylene blue solutions, and photograph the same nerve-arteriole unit after staining, fixation, and dehydration. In all cases,with both normal (Fig. 7) and 6-HDA-treated membranes (Figs. 8 and 9), the same nerve which evoked vasodilation when stimulated by microelectrode showed marked staining by the thiocholine or methylene blue method. In all respects,staining of 6-HDA-treated retrolingual membranesappearedidentical to that of untreated membranes. Thus, motor nerves, motor end-plates, perivascular

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FIG. 7.

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VASODILATION

FIG. 8. Neurogenic vasodilation and thiocholine staining of vasodilator nerves 12 days after chronic 6-hydroxydopamine treatment. A. Control arteriole photographed in viuo just before stimulation of a vasomotor nerve (N) with a microelectrode (ME). B. Near maximal vasodilation evoked in same arteriole 21 set after nerve stimulation with a single shock of 0.17 mA, 1 msec. Note the dramatic twofold increase in outer diameter of the precapillary sphincter (arrows in A and B). C. Same arteriole after thiolcholine staining; bright field microscopy. Note intense staining of the same nerve which had previously evoked the vasodilation. The arrow in C shows the branch of the nerve probably responsible for producing dilation of the precapillary sphincter (PS) as shown in panels A and B. Calibration bar in panel A indicates 100 p for all three panels.

nerves and the diffusely stained precapillary sphincter, and arteriolar areas (described above) stained as profusely and intensely for acetylcholinesterase in 6-HDA-treated membranes as in normal ones. The same was true for 6-HDA-treated retrolingual

FIG. 7. Thiocholine staining of nerves which evoke vasodilation in a normal retrolingual membrane. Panels A and B are in viuo photomicrographs; C and D are micrographs of the same arteriole taken after excision, fixation, and staining of the retrolingual membrane. A. Control arteriole immediately before stimulation of a vasomotor nerve (out of the focal plane) by microelectrode (ME). B. Vasodilation evoked 18 set after nerve stimulation (producing a gas bubble at electrode tip) with a single just-maximal current pulse (1 msec duration, 0.20 mA intensity). Same magnification as in panel A. Note the nearly threefold increase in diameter of the precapillary sphincter (arrows in A and B) evoked by nerve stimulation. C. Bright field microscopy of stained arteriole and nerves. Note intense AChE staining of precapillary sphincter (PS) and associated nerves. Arrow indicates the locus of stimulation by microelectrode. D. Phase contrast micrograph which shows greater detail of the stained perivascular network, but also gives more background detail. Note especially the extremely fine nerve net (n). Excision and stretch-mounting of the retrolingual membrane have produced a slight distortion of the arteriole in C and D.

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membranes stained with methylene blue at pH 6.5-7.0: vasomotor nerves and motor nerves innervating striated muscle fibers appeared as before treatment, at a time after 6-HDA treatment (5-l 5 days) when adrenergic nerve terminals appear to be completely degenerated(Sigginsand Bloom, 1970).

FIG. 9. Methylene blue (pH 6.8) staining of the periarteriolar plexus 14 days after chronic 6-hydroxydopamine treatment. Arrow marks the primary nerve which was seen to evoke vasodilation of the arteriole (A) with stimulation. Note light staining of nerves around a precapillary sphincter (PS). Mast cells (spheroid bodies), red blood cells, and striated muscle fibers (S), especially at points of contracture (C), also stained with methylene blue. Bright field microscopy.

DISCUSSION Results of the present investigation support physiological studies showing a dramatic nerve-induced dilation of arterioles and precapillary sphincters of the frog retrolingual membrane (Fulton and Lutz, 1940,1942; Siggins, 1967a,b; Berman and Siggins, 1968). Sincethis dilation was mimicked by topical acetylcholine and blocked by administration of atropine, but not adrenergic blockers, antihistamines, or surgical sympathectomy, the response was judged to be cholinergic in nature (Siggins, 1967a; Berman and Siggins, 1968). The finding that neurogenic vasodilation and nerves containing predominantly clear vesicles persisted after “chemical sympathectomy” (Thoenen and Tranzer, 1968)with 6-HDA further confirmed that the dilator nerveswere not adrenergic (Siggins and Bloom, 1970). In view of the reported specificity of the acetylthiocholine (El-Badawi and Schenk, 1967) and pH 6.5-7.0 methylene blue (Richardson, 1969) techniques for cholinergic

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nerves, the present results demonstrate that cholinergic nerves are indeed abundant in the retrolingual membraneand are related to striated muscle,arterioles, and precapillary sphincters. Abundant innervation of arterioles and precapillary sphincters is suggested also by the ultrastructural studies of Rhodin (1967). A more direct demonstration of the cholinergic nature of the vasodilator nerves derives from the fact that the identical nerves which evoke marked vasodilation of arterioles and/or precapillary sphincters when stimulated by microelectrode also stain by the thiocholine and methylene blue techniques (Figs. 7-9). Furthermore, with the acetylcholinesterasemethod, staining is lost when the specific AChE reaction substrate, acetylthiocholine, is not added to the incubation medium. This finding, in addition to the presence of iso-OMPA (an inhibitor of nonspecific cholinesterase) in all incubation media used, suggests specific staining for “true” AChE. It has been suggestedthat nerves other than cholinergic nerves may show a positive acetylcholinesterase reaction with the thiocholine method (Jakobowitz and Koelle, 1965; Waterson et al., 1970). However, Fillenz (1970) points out that short incubation times (about 1 hr) such as those used in the current study have only been reported to stain cholinergic nerves. This proposal is strengthened by the ultrastructural studies of Esterhuizen et al. (1968) and Graham et al. (1968), which utilized a combined NE-3H autoradiographic and thiocholine marking method to show a nearly complete separation of AChE-staining axons from catecholamine-containing nerves. The methylene blue technique has been used extensively in the past for nonspecific staining of all nerve types (seeBusch, 1929; Hess, 1970; Richardson, 1969).Lutz et al. (1950) found a profuse innervation of retrolingual membrane arterioles when stained with methylene blue. However, high concentrations (0.1%) of this agent were used, and the specificity of the stain at pH 6.5-7.0 for cholinergic nerves was not known at the time. In recent ultrastructural studies utilizing pH 6.5-7.0 incubation media, Richardson (1968, 1969) found methylene blue deposits only in nerve profiles with agranular vesicles,and not in presumedadrenergic nerveswith small granular vesicles. As an additional control, the two staining techniques used in the present study were also applied to retrolingual membraneschemically sympathectomizedwith 6-HDA. One may assumethat most adrenergic nerve terminals and axons are extensively disrupted after 6-HDA treatment, since (1) all nerveswith large and small granular vesiclesshow numerous fine-structural signs of toxic degeneration; theseare the nervescharacterized as adrenergic by autoradiographic techniques, (2) the catecholamine uptake capability of perivascular nerves is lost, as shown by autoradiography and the formaldehydecondensation (fluorescence) technique, (3) normal fluorescence of adrenergic perivascular nerves is completely lost, and (4) neurogenic vasoconstriction is abolished (Siggins and Bloom, 1970). Despite the degeneration of the adrenergic nerves, the abundance and intensity of AChE and methylene blue positive nerves were not apparently diminished by 6-HDA treatment. In addition, neurogenic vasodilation persisted after 6-HDA, and the same nerves producing the arteriolar and sphincteric dilation were positively stained by the two techniques. Thus, on the assumption that sufficient degeneration of adrenergic nerves occurred, we propose that the surviving fibers are cholinergic. This conclusion is supported not only by our previous pharmacological and cytochemical studies (cited above), but also by preliminary ultrastructural findings which show that surviving

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nerve profiles with clear vesicles, but not those exhibiting 6-HDA-induced degeneration, stain positively for acetylcholinesterase (Weitsen et al., 1970). The intense staining of precapillary sphincters and small segments of arterioles by the thiocholine technique (see Figs. 2-4) raises intriguing possibilities. One might speculate on morphological grounds that these arteriolar segments represent a “nonbranch point sphincter,” capable of interrupting blood flow to several capillary beds, in contrast to the precapillary sphincters which could independently shunt blood from one capillary at a time. We find it interesting that these areas have several features in common with motor end-plates in the retrolingual membrane: (1) when treated with the thiocholine method, both structures stain in a much greater and more general area than bounded by the fine nerve terminals, (2) with the thiocholine method both structures stain more rapidly than their associated innervation, and (3) neither of these areas stain with the methylene blue technique, while the associated nerves stain positively. It is possible that these regions owe their intense thiocholine staining to diffusion of acetylcholinesterase from the associated nerve terminals to neighboring postjunctional regions (see Bloom and Barrnett, 1966; Hall, 1971). However, this explanation seems unlikely, since one would expect diffusion to all vascular regions. Finally, this possibility is not supported by the observation of a small number of precapillary sphincters with no obvious innervation, but which nonetheless stained intensely for AChE. One might thus speculate on the similarities between striated muscle end-plates and “sphincteric” vascular regions. Since cholinoceptive areas of skeletal muscle and other cholinergic systems appear to be closely related to areas of high cholinesterase activity (Riker, 1953; Roepke, 1937; but see Karlin, 1967; Podleski, 1967), it is possible that the intensely stained “sphincteric” regions represent postjunctional sites of high cholinergic receptivity. These areas could then possess an enhanced ability for dilation in response to acetylcholine released from nerve terminals. It would be interesting to know if similar regions of high AChE activity exist in mammalian arterioles.

ACKNOWLEDGMENTS The authors gratefully acknowledge the critical evaluation of this manuscript by Drs. F. E. Bloom, K. L. Sims, and B. J. Hoffer, and the technical assistance of Mrs. Elena Battenberg, Mrs. Jeannie Alwine, and Mrs. Odessa Calvin.

REFERENCES ARMIN, J., GRANT, R. T., THOMPSON, R. H. S., AND TICKNER, A. (1953). An explanation

for the heightened vascular reactivity of the denervated rabbits ear. J. Phvsiul. 121,603. BARCROFT, H., BROD, J., HEJL, Z., HIRSJARVI, E. A., AND KITCHIN, A. H. (1960). The mechanism of the vasodilation in the forearm muscle during stress (mental arithmetic). Clin. Sci. 19,577. BERMAN, H. J., AND SIGGINS, G. R. (1968). Neurogenic factors in the microvascular system. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 27,1384. BLOOM, F. E., AND BARRNETT, R. J. (1966). Fine structural localization of acetylcholinesterase in electroplaque of the electric eel. J. Cell. Biol. 29,475. BOLME, P., AND FUXE, K. (1967). Identification of sympathetic cholinergic nerve terminals in arterioles of skeletalmuscle. ActaPharmacoZ. Toxicol. 25 (SuppI. 4), 79. BOYD, H., BURNSTOCK, G., CAMPBELL, G., JOWETT, A., O’SHEA, J., AND WORD, M. (1963). The cholinergic blocking action of adrenergic blocking agents in the pharmacological analysis of autonomic innervation. Brit. J. Pharmacol. 20,418.

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321

BRZIN, M., TENNYSON, Y. M., AND DUFFY, P. E. (1966). Acetylcholinesterase in frog sympathetic and dorsalroot gang1ia.J. CeNBiol. 31 (2), 215. B~~LBRING, E., AND BURN, J. H. (1936). Sympathetic vasodilation in the skin and intestine of the dog. J. Physiol. 87,254. BUSCH, E. (1929). Studies on the nerves of the blood-vessels with special reference to periarterial sympathectomy. ActaPathol. Microbial. Stand. Suppl. 2,1. DALE, H. H., AND GADDUM, J. H. (1930). Reactions of denervated voluntary muscle, and their bearing on the mode of action of parasympathetic and related nerves. J. Physiol. 70,109. EHINGER, B., SPORRONG, B., AND STENEVI,U. (1967). Combining the catecholamine fluorescence and methylene blue staining methods for demonstrating nerve fibers. Life Sci. 6,1973. EL-BADAWI, A., AND SCHENK,E. A. (1967). Histochemical methods for separate, consecutive and

simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections. J. Histochem. Cytochem. 15,580. ESTERHUIZEN, A. C., SPRIGGS, T. L. B., AND LEVER,J. D. (1968). Nature of islet-cell innervation in the cat pancreas. Diabetes 17,33.

FALCK, B., HAGGENDAL,J., AND OWMAN, Ch. (1963). The localization of adrenaline in adrenergic nerves in the frog. Quart. J. Exp. Physiol. 48,253. FALCK, B., HILLARP, N. A., THIEME,G., and TORP, A. (1962). Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10,349. FILLENZ, M. (1970). Innervation of pulmonary and bronchial blood vesselsof the dog. J. Anat. 106,449. FOLKOW,B., HAEGAR,K., AND UVNAS, B. (1948). Cholinergic vasodilator nerves in the sympathetic outflow to the muscles of the hind limbs of the cat. Acta. Physiol. &and. 15,401. FULTON,G. P., AND LUTZ, B. R. (1940). The neuromotor mechanism of the small blood vessels of the frog. Science 92,223. FULTON, G. P., AND LUTZ, B. R. (1942). Smooth muscle motor units in small blood vessels. Amer. J. Physiol. 135,531.

GRAHAM, J. D. P., LEVER,J. D., AND SPRIGGS,T. L. B. (1968). An examination of adrenergic axons around pancreatic arterioles of the cat for the presence of acetylcholinesterase by high resolution autoradiographicand histochemicalmethods. Brit.J. Pharmacol. Chemother. 33,15. GYERMEK,L. (1961). Cholinergic stimulation and blockade on urinary bladder. Amer. J. Physiol. 201, 325.

HALL, Z. (1971). In “Structure and Function of Synapses” (G. D. Pappas, ed.). Appleton CenturyCrofts, New York, in press. HESS,A. (1970). Vertebrate slow muscle fibers. Physiol. Rev. 50,40. INNES, I. R., AND NICKERS~N,M. (1965). In “The Pharmacological Basis of Therapeutics” (L. S. Goodman and A. Gilman, eds.), pp. 521-545. Macmillan, New York. JACOBOWITZ,D., AND KOELLE, G. B. (1965). Histochemical correlations of acetylcholinesterase and catecholamines in postganglionic autonomic nerves of the cat, rabbit and guinea pig. J. Pharmacol. Exp. Ther. 148,225. KARLIN, A. (1967). Chemical distinctions between acetylcholinesterase and the acetylcholine receptor. Biochim. Biophys. Acta 139,358. KARNOVSKY, M. J., AND ROOTS,L. (1964). A direct-coloring thiocholine technique for cholinesterases. 1. Histochem. Cytochem. 12,219. KATZ, L. N., AND JOCHIM, K. (1939). Observations on the innervation of the coronary vesselsof the dog. Amer. J. Physiol. 126,395. LUTZ, B. R., FULTON,G. P., AND AKERS,R. P. (1950). The neuromotor mechanism of the small blood vesselsin membranes of the frog (Ranapipiens) and the hamster (mesocricetus auratus) with reference to normal and pathological conditions of blood flow. Exp. Med. Surg. 8,258.

MCHEDLISHVILI,G. I., AND NIKOLAISHVILI,L. S. (1970). Evidence of a cholinergic nervous mechanism mediating the autoregulatory dilatation of the cerebral blood vessels.Pfugers Arch. 315,27. MORAN,N. C., AND PERKINS,M. E. (1958). Adrenergic blockade of the mammalian heart by a dichloro analogue of isoproternol. J. Pharmacol. Exp. Ther. 124,223. NORVELL,J. E., HARRIS,T. M., AND WEITSEN,H. A. (1971). The use of dark-field microscopy for the visualization of acetylcholinesterase in cholinergic neurons. Stain Technol., 46,19. PODLESKI,T. R. (1967). Distinction between the active sites of acetylcholine-receptor and acetylcholinesterase. Proc. Nat. Acad. Sci. U.S.A. 58,268.

322

SIGGINS AND WEITSEN

PRAY, F. H., AND REID, M. A. (1930). A method for working on the terminal nerve-muscle unit, Science 12,43 1.

RHODIN, J. A. G. (1967). The ultrastructure of mammalian arterioles and precapillary sphincters. J. Ultrastruct.

Res. 18,181.

RICHARDSON,K. C. (1968). Cholinergic and adrenergic axons in methylene blue-stained rat iris: An electron-microscopical study. Life Sci. 7 (part l), 599. RICHARDSON,K. C. (1969). The fine structure of autonomic nerves after vital staining with methylene blue. Anat. Rec. 164,359. RIKER, W. F., JR. (1953). Excitatory and anti-curare properties of acetylcholine and related quaternary ammonium compounds at the neuromuscularjunction.Pharmacol. Rev. 5,l. ROEPKE,M. H. (1937). A study of cholinesterase. J. Pharmacol. Exp. 7’her. 59,264. SHEN, S. C., GREENFIELD, P., AND BOELL, E. J. (1955). The distribution of cholinesterase in the frog brain. J. Comp. Neural. 102,717. SIGGINS,G. R. (1967a). Evidence for reciprocal innervation of arterioles in the frog retrolingual membrane. Sib/. Anat. 9,328. SIGGINS, G. R. (1967b). Nervous control of the arterioles in the retrolingual membrane of the frog (Ranapipiens). Doctoral dissertation, Boston University Graduate School. SIGGINS, G. R. (1970). In “Microcirculation, Perfusion and Transplantation of Organs” (T. I. Malinin, ed.), pp. 59-74. Academic Press, New York. SIGGINS, G. R., BERMAN, H. J., AND MCKINNON, E. L. (1968). Striated muscle with unusual properties in the tongue of the frog. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 27,236. SIGGINS, G. R., AND BLOOM, F. E. (1970). Cytochemical and physiological effects of 6-hydroxydopamine on periarteriolar nerves of frogs. Circ. Res. 27,23. STINKIN, J. M., BARNES, A. B., ZAKHEIM, R. M., CHIMOSKEY, J. E., SPINELLI, F. R., AND BARGER, A. C. (1969). Reflex cholinergic vasodilation during renal artery constriction in the unanesthetized dog. Amer. J. Physiol. 217,239. H., AND TRANZER, J. P. (1968). Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Nauyn-Schmiedebergs Arch. Exp. Pharmakol. Exp.

THOENEN,

Pathol. 261,271. UVNAS, B. (1954). Sympathetic vasodilator outflow. Physiol. Rev. 34,608. WATERKIN, J. G., HUME, W. R., AND DE LA LANDE, I. S. (1970). The distribution

of cholinesterase in the rabbit ear artery. J. Histochem. Cytochem. 18,211. WEITSEN, H. A., SIGGINS, G. R., AND BLOCM, F. E. (1970). Cytochemical and neuropharmacological differentiation of peri-arteriolar nerves. Pharmacologist 12,262.