Inverse correlation between Synaptic Э ribbon number and the density of adrenergic nerve endings in the pineal gland of various mammals.код для вставкиСкачать
THE ANATOMICAL RECORD 205:93-99 (1983) Inverse Correlation Between “Synaptic” Ribbon Number and the Density of Adrenergic Nerve Endings in the Pineal Gland of Various Mammals MICHAL KARASEK, THOMAS S. KING, JAMES BROKAW, JOHN T. HANSEN, LARRY J. PElTERBORG, AND RUSSEL J. REITER Department of Anatomy, The University of Texas Health Science Center at Sun Antonio, J.B., J.T.H., L.J.P.,R.J.R.) and Labomtory of San Antonio, TX 78284 ( M X . , T.S.K., Electron Microscopy, Department of Pathological Anatomy, Institute of Pathology, Medical School, Look, Poland ( M X . ) ABSTRACT The number of “synaptic” ribbons was inversely correlated with the density of the adrenergic nerve endings of the pineal gland compared among a diverse group of species including the fox, cat, rat, cotton rat, white-footed mouse, Djungarian hamster, ground squirrel, and chipmunk. The concentration of norepinephrine paralleled the number of adrenergic nerve terminals in the pineal glands of the cotton rat, rat, and ground squirrel, the only species in which norepinephrine concentrations were measured. The number of ribbon fields paralleled numbers of “synaptic” ribbons in all species examined. Adrenergic nerve endings were observed primarily within the perivascular spaces, although some endings also were found among parenchymal cells. Adrenergic nerve endings forming synaptic junctions with pinealocytes were not observed in any of these species, nor was there any physical association between these nerve endings and “synaptic” ribbons. The functionally enigmatic pineal “synaptic” ribbon (SR) has been described in every mammalian species studied thus far (for review, see Pevet, 1979). A circadian rhythm in SR numbers, high at night and low during the daytime, has been reported in many of these species (guinea-pig: Vollrath, 1973; rat: Kurumado and Mori, 1977; King and Dougherty, 1980; hamster: Hewing, 1979; baboon: Theron et al., 1979). Because the gland’s adrenergic innervation regulates most, if not all pineal circadian rhythms (for review, see Vollrath, 1981), the rhythms in SR formation may similarly be regulated by the adrenergic innervation. This hypothesis has been examined with a variety of experimentally altered conditions including acute (Vollrath and Howe, 1976)and chronic (Vollrath and HUSS,1973; Lues, 1971; Hewing, 1980; King and Dougherty, 1982a; Vollrath, 1975)changes in the 1ight:dark cycle, blinding (Kurumado and Mori, 1980), pineal sympathectomy (Romijn, 1975,1976; King and Dougherty, 1982b) as well as pharmacological manipulation of the adrenergic function (Karasek, 1974; Vollrath and Howe, 1976; Romijn and Gelsema, 1976; King and Dougherty, 1982a,b). Common to each of these studies is 0003-276W83/2051-0093$02.500 1983 ALAN R. LISS, IN(: the conclusion that the adrenergic innervation of the pineal gland influences the level of SR formation. A comparison between previously reported qualitative descriptions of the density of pineal adrenergic nerve endings (e.g. Wolfe, 1965; Ito and Matsushima, 1967; Matsushima and Reiter, 1977; Matsushima et al., 1979; Karasek and Hansen, 1982) and quantitative analyses of SR numbers suggest a potential relationship between these two pineal components. This relationship is apparent in experimental studies concerning SR formation following the destruction of the adrenergic innervation to the pineal gland (e.g., Romijn, 1975; Karasek, 1976; Romijn and Gelsema, 1976; King and Dougherty, 1982b). Synaptic ribbon numbers increase in the absence of adrenergic nerve fibers in the pineal glands. In order to examine this potential relationship under nonexperimental natural conditions, we attempted to correlate morphometrically the number of adrenergic nerve endings with the density of “synaptic” ribbons in a diverse number of mammalian species. Norepinephrine concentrations also Received June 1,1982;accepted September 20, 1982. 94 M. KARASEK ET AL were determined in three of these species, each having either high, medium, or low numbers of adrenergic nerve endings. The norepinephrine content then was correlated with the density of the adrenergic innervation in the pineal gland. MATERIALS AND METHODS The pineal gland of rats (four males), foxes (two males and two females), cats (four females), Djungarian hamsters (two males and two females), ground squirrels (two males and two females), chipmunks (two males and two females), cotton rats (two males and two females), and white-footed mice (two males and two females) were used for ultrastructural analysis. Rats and Djungarian hamsters were kept in a 1ight:dark cycle of 14:lO h (lights on at 0600 h), whereas the other species were kept under natural lighting conditions. All animals were sacrificed by decapitation between 1200 h and 1400 h. The pineal glands were immediately removed and immersion-fixed in 3.5% glutaraldehyde-2% formaldehyde in 0.067 M cacodylate buffer (pH 7.2 at 4°C). All glands were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a graded series of acetones and embedded in Spurr’s (1969) low vicosity epoxy resin. One thin section from a series of thin sections exhibiting light gold interference colors (70-90 nm) was taken from the largest transverse diameter of each gland, stained with uranyl acetate and lead citrate and examined with a Siemens 1A electron microscope. Five grid squares (200 mesh; total area of 45,125 p.m2)containing pineal parenchyma were chosen without prior examination and at random for analysis. The numbers of “synaptic” ribbon (SR),ribbons fields (RF), and adrenergic nerve terminals (NT) were counted by using a consecutive series of parallel scans across each grid square at 12,000 to 14,000 x magnification. Ribbon fields were defined as one or more spatially related SR (Vollrath and Huss, 1973; King and Dougherty, 1982a,b). Data was expressed as the number of SR, RF, or NT & SEM per 20,000 p.m2. For biochemical estimation of norepinephrine (NE) the pineal glands of rats (six males), cotton rats (three males and three females), and ground squirrels (four males and two females) were used. Norepinephrine in the pineal gland was assayed by high performance liquid chromatography with electrochemical detection (LCEC) as previously described (Hansen and Christie, 1981). Essentially, this procedure involved the extraction of pineal NE with 0.1 M perchloric acid, absorption onto acidwashed alumina and elution from the alumina with 0.1 M perchloric acid. The internal stan- dard dihydroxybenzylamine (DHBA) (2 ng/20 p1) was added to each sample before processing. Aliquots (20 pl) of the alumina eluate were valve loaded (Altex) onto a reverse phase U1trasphere ODS 5 pm column (Beckman).ARer elution from the column, the sample passed through a thin-layer flow cell containing a glassy carbon electrode (Bioanalytical Systems) at a preset potential of + 0.72 V. The oxidation current, which was directly proportional to the concentration of NE passing by the electrode, was monitored on a strip chart recorder (Houston Omniscribe). For calibration purposes, a sample consisting of proper strength (2 ng/20 p.1) synthetic NE and DHBA was eluted along with the pineal samples. To calculate NE concentration, the peak height ratios relative to the internal standard DHBA for unknown pineal samples was compared to that of the synthetic standard whose concentration was known. Protein determinationswere performed by the method of Lowry et al. (1951) as adapted for use with a Technicon Autoanalyzer. Results were expressed as ng of NE per mg protein. Data were analyzed first by a one-way analysis of variance. Significant differences between means were determined using the Newman-Keuls multiple range test. RESULTS “Synaptic” ribbons in the pineal glands of all species examined were morphologically similar. The structure of the SR core varied in appearance from a trilaminar (Fig. 1)to platelike (Fig. 2) configuration with a variety of intermediate forms (Figs. 2, 31, depending on the plane of section. There were significant differences between the numbers of both SR and RF among the species examined (Fig. 6). Low numbers of SR and RF were observed in the cotton rat, white-footed mouse, fox, cat, and rat. In contrast, significantly higher numbers (p < 0.001 vs other species) of SR and RF were present in the chipmunk (Fig. 4) and ground squirrel, whereas the pineal gland of DjunFigs. 1,Z. Rat. Typical appearance of “synaptic”ribbons relative to the plane of section from trilaminar (TIto platelike (P) with an intermediate form (I). Figure 1, x 47,000; Figure 2, x 36,000. Fig. 3. Rat. Plate-like and intermediate form of “synaptic”ribbons adjacent to plasmalemma. NT, nerve ending. x 11,000. Fig. 4. Chipmunk. Numerous ribbon fields farrows). Pi, pinealocyte;G, glial cell. x 16,000. Fig. 5. Chipmunk. Large ribbon field. x 49,000. PINEAL “SYNAPTIC” RIBBONS AND NERVE ENDINGS 95 96 M. KARASEK ET AL. 3001 250 - N 5 8 200- 0 Ribbon Fmlda “Synaptic” Ribbona Nerve Terminala 9 h i f z 150- 100- 50 - 0Mouse Ground Squirrel Homster Chipmunk Fig. 6. Inverse correlation between numbers of “synaptic” ribbons (ribbon fields) and adrenergic nerve terminals in eight mammalian species. Half brackets refer to SEM. TABLE 1 . Relation of ribbon fields (RF) and distribution Species A. Vulpes uulpes domesticus (Fox) B. Sigmodon hispidw (Cotton rat) C. Rattus mttus (Sprague-Dawley rat) D. Felix domesticus (Cat) E. Perornyscus leucopus (White-footed mouse) F. Phoabpus sungorus (Djungarian hamster) G. Spernwphilus richardsonii (Ground squirrel) H. T a m h striatus (Chipmunk) RF adjacent to cell membrane 95.5 ? 3.2 of ? 2.6” 54.5 * 4.3b NT in perivascular spaces RF NT in parenchyma 4.4 i 2.8 39.5 t 2.8C 60.5 0 17.0 ? 0.4d 83.0 t 0.4h 2.4 t 1.4 11.2 ? 3.1 88.8 t 3.1’ 0 19.6 ? 2.8‘ 80.4 0 21.8 ? 3.3‘ 78.2 t 3.3k “Paired” 93.8 t 2.2 85.1 nerve endings (NT) in the pineal parenchyma 100 ? ? 2.8% 2.g 92.4 t 0.8 4.2 ? 1.2 13.0 f 1.6 87.0 94.1 t 0.6 2.1 ? 0.5 7.1 f 0.8 92.9 t 0.8 94.2 2.4 ? 0.6 5.6 ? 1.2 94.4 t 1.2 ? 0.8 ? 1.6 Data are percents and represent means f SEM.Letters indicate statistically significant differences(SD) at level p < 0.05 vs species noted. a: SD vs A, E, F; b: SD vs A-H; c: SD vs B-H; d: SD vs G , H; e: SD vs G , H; E SD vs C, G , H; g: SD vs B-H h SD vs G , H,i: SD vs E; j: SD vs G , H, k SD vs G , H. garian hamsters exhibited intermediate numbers of SR and RF (p < 0.001 vs other species). Most RF in the species studied were associated with the plasmalemma (Fig. 3), with the exception of those in the cat (Table 1). In the chipmunk and ground squirrel, RF frequently consisted of large numbers of SR (Fig. 5). “Synaptic” ribbons did not appear to have any consistant relationship to nerve fibers or their endings, to endothelial cells, to glial cells or to other pinealocytes. So-called “paired’ RF, i.e., RF lying opposite one another in adjacent pinealocytes, constituted only a small percent of the total number of RF in all species studied (Table 1). The adrenergic nerve terminals in all species studied were typical morphologically of previously described adrenergic nerve terminals (Fig. 7) (see Vollrath, ’81).The number of NT varied significantly among the species studied (Fig. 6). The highest numbers (p < 0.001 vs other species) of NT were observed in the cot- PINEAL “SYNAPTIC” RIBBONS AND NERVE ENDINGS Fig. 7. Fox. High density of nerve endings (asterisks) found in the pineal parenchyma. Pi, pinealocyte: GP, glial 97 process. x 16,000.Inset, Morphologically typical adrenergic nerve ending. x 48,000. 98 M. KARASEK ET AL. ton rat and fox, whereas in the chipmunk and ground squirrel they were lowest (p < 0.005 vs other species). The majority of NT in all species studied were located in the perivascular spaces (Table 1).A lower number of NT was present in the parenchyma of the pineal glands of the chipmunk and ground squirrel when compared to other species examined (Table 1).An inverse correlation was observed between the numbers of either SR or RF and the numbers of NT (r = -0.6247 and -0.6942, respectively; p < 0.05). Norepinephrine concentrations were measured in three species, one having low (ground squirrel), another intermediate (rat), and a third, high (cotton rat) NT numbers. Concentrations of NE were highest in the cotton rat (67.0 10.0 ng/mg protein), intermediate in the rat (21.5 2 5.1 ng/mg protein), and lowest in the ground squirrel (9.1 5 1.0 ng/mg protein). Concentration of NE in the pineal gland of the cotton rat was significantly greater (p < 0.001) than that in the rat and ground squirrel. Although the concentrationof NE in the ground squirrel was 137%lower than that in the rat, the difference was not statistically significant because of variation between animals. gan culture of the rat pineal gland which essentially can be considered a form of denervation, likewise results in an increase in SR numbers (Karasek, 1976; Romijn and Gelsema, 1976). Our results suggest a similar relationship between SR formation and the density of adrenergic innervation of the pineal gland, The pineal glands of those species having few nerve endings exhibit the greatest populations of SR. In turn, those species where pineal glands are richly innervated by adrenergic nerves contain the fewest number of SR. Despite the relationship between a high density of adrenergic innervation and low SR numbers, the addition of norepinephrine to cultured rat pineal glands (Karasek, 1974) or the administration of a synthetic adrenergic agonist (isoproterenol)to rats which are acutely sympathectomized by superior cervical ganglionectomy (King and Dougherty, 1982b)leads to an increase in SR formation. King and Dougherty (1982a) have offered a working hypothesis which may explain this apparent paradox. They found that SR formation decreases during continuous light but can be increased with the administration of isoproterenol. The density of beta-adrenergic receptors increases during continuous light in apparent compenDISCUSSION sation for reduced levels of norepinephrine Although a relationship between the adre- (Cantor et al., 1981). On the other hand, SR nergic innervation of the pineal gland and SR formation increases during continuous darkformation has been hypothesized (Vollrath, ness when the density of these receptors is low. 1973;Vollrath and Howe, 1976; Karasek, 1976; Thus, King and Dougherty (1982a)suggest that King and Dougherty, 1980),the nature of this the function of SR formation is related to a relationship has been speculative. Our study decrease in the density of these receptors along demonstrates an inverse correlation between the plasmalemma of the pinealocyte, presumthe density of adrenergic nerve endings and ably by membrane turnover associated with SR or RF numbers in the pineal gland of a SR vesicles. The potential association between diverse number of mammalian species. The membrane turnover and SR vesicle function concentration of NE paralleled the density of has been proposed by various investigators NT in three species (cotton rat, rat, and ground (Spadaroet al., 1978;King and Dougherty, 1979, squirrel-the only species in which NE con- 1980; McNulty, 1980). The inverse relationship between adrenergic centration have been measured). These results indicate that SR numbers are inversely pro- nerve endings and SR numbers observed among portional not only to NT numbers but also to diverse mammalian species may offer a collecNE concentrations. These results represent the tive model system for further study of the regfirst evidence for such a relationship under ulation and function of SR formation in the normal conditions. However, this relationship mammalian pinealocyte. Assuming that SR has been suggested by various other investi- formation is related to changes in the density gators using experimentally altered conditions of adrenergic receptors along the plasmato affect SR formation in the pineal gland of lemma of pinealocytes, then the inverse correlation between fewer adrenergic nerve endrodent species. Denervation of the mammalian pineal gland ings and higher numbers of SR in the pineal by superior cervical ganglionectomy (Romijn, gland of some mammalian species may be as1975; Romijn and Gelsema, 1976; King and sociated with a compensatory increase in the Dougherty, 1981b) or by chemical sympathec- density of beta-adrenergic receptors in these tomy (6-hydroxydopamine:Romijn, 1976) pro- species. Our study may present further eviduce consistently elevated numbers of SR. Or- dence for the working hypothesis that the for- * PINEAL “SYNAPTIC” RIBBONS AND NERVE ENDINGS mation of pineal SR serves to regulate the density of beta-adrenergic receptors along the plasmalemma and, therefore, adrenergic control of pinealocyte metabolism in general. ACKNOWLEDGMENTS The authors wish to thank Dr. William W. Morgan for his help with the present work, Ms Gwynne Duke for her excellent technical assistance, and Mrs. Nancy Elms for typing the manuscript. This study was supported by NSF grant #PCM 8003441to R. J. R. and by a grant from the Polish Academy of Sciences, within project 10.4 to M.K. T.S.K. is a postdoctoral fellow in the Center for Training in Reproductive Biology HD 07139. J.T.H. is the recipient of NIH RCDA KO4 HL-00680. This paper was presented in part at Ninety Fifth Annual Session of the American Association of Anatomists, April 4-8, 1982 (abstract published in Anatomical Record 1982, 202: 93A). LITERATURE CITED Cantor, E.H., L.H. Greenberg, and B. 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