Long-term effects of gustatory neurectomy on fungiform papillae in the young rat.код для вставкиСкачать
THE ANATOMICAL RECORD 225:224-231 (1989) Long-Term Effects of Gustatory Neurectomy on Fungiform Papillae in the Young Rat JUDITH R. GANCHROW AND DONALD GANCHROW Department of Oral Biology, The Hebrew Uniuersity-HadassahFaculty of Dental Medicine Founded by The Alpha Omega Fraternity, Jerusalem 91 -010 (J.R.G.),and Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69-978, Tel - Aviv (D.G .),Israel ABSTRACT Recent evidence from mature hamster fungiform papillae indicates that following denervation taste buds are present from 21 to 330 days in the absence of discernible intragemmal nerve fibers. In contrast, most prior taste bud degeneration studies focused on shorter survival times. The present inquiry in young rats examined the issue of postneurectomy buds, in which regeneration of the resected chorda tympani or facial nerves was prevented and anterior tongue tissue examined over a range of relatively long survival times (30-90 days). Conditions for observing potential taste buds used three histologic stains and a definition of the taste bud not necessarily requiring pore identification. In each case, serial section examination of the anterior-most 2-3 mm of lingual epithelium revealed 29-56 bud-containing fungiform papillae on the unoperated side. In contrast, ipsilateral to the neurectomy, only zero-7 medially-placed, mature-looking buds were observed per case, as well as zero-3 more laterally situated fungiform papillae containing small clusters of cells in basal epithelium that lacked the vertical organization and cytoplasmic staining intensity of mature taste buds. These cell aggregates were distributed evenly across survival time and stain used. Therefore, in young rats following gustatory neurectomy, longer survival times, per se, would not appear to be a prerequisite for sustaining fungiform taste buds. The appearance of “midline” buds postsurgery may be attributed to either normal contralateral or a net bilateral innervation, andlor ipsilateral denervation and bud loss inducing neural sprouting from the contralateral side. For more than a century it has been maintained that taste bud survival is critically nerve-dependent (e.g., von Vintschgau and Honigschmied, 1877; also see Table 1). It is typically reported that damage to a gustatory nerve results in degeneration and disappearance of taste buds in the relevant oral sensory epithelium and should appropriate reinnervation of the taste target tissue occur, the buds, in turn, regenerate. Only gustatory or arterial chemosensory neurons seem capable of supporting taste bud regeneration in the oral cavity, and not cutaneous sensory or motor neurons (e.g., Dinger et al., 1984; Guth, 1958; Oakley, 1974; State, 1977; State et al., 1982; Zalewski, 1969b, 1970). Recently, the concept of taste bud neural dependence has been challenged. Whitehead et al. (1987) reported that taste buds in fungiform papillae of adult hamsters persisted for as long as 330 days following chorda tympani resection. Care was taken to prevent neural regeneration to the denervated area, and electron microscopic analysis failed to reveal any neuron profiles in the budlike configurations observed. Both dark and light cells could be visualized in buds on the operated and unoperated sides. In addition, buds on the operated side seemed smaller and had no easily stainable taste pore communication with the oral cavity (see also Hou et al., 1985). 0 1989 ALAN R. LISS. INC In contrast, for example, the present authors reported that chicken taste buds are completely absent 11-21 days following chorda tympani resection (Ganchrow et al., 1986).Epithelial organization in the denervated tissue was devoid of incipient, developing, or mature taste buds during the postoperative period studied by serial section analysis. However, oral taste buds in chickens are not contained within specialized papillae (e.g., Berkhoudt, 1985; Ganchrow and Ganchrow, 1987; Gentle, 1971; Gentle and Hunter, 1983; Saito, 1966) that, conceivably, could provide some maintenance function for degenerating or newly forming taste buds. Furthermore, on the operated side, Whitehead et al. (1987) saw buds in an apparently degenerative phase at 8 days, but did not examine materials in the 9-20 postoperative-day interval. Perhaps taste buds do completely disappear in this later interval, as is accordingly recorded by various authors, and then show limited regeneration over more extended periods of time. Received March 10, 1988; accepted January 5, 1989. Address reprint requests t o Dr. Judith R. Ganchrow, The Hebrew University-Hadassah Faculty of Dental Medicine, P.O. Box 1172, Jerusalem 91-010, Israel. 225 CHORDA TYMPANI NEURECTOMY TABLE 1. Results of gustatory nerve damage in vertebrates as related to age,’ target tissue, survival time, and staining technique Species Rat2 Rat2 Rat Rat3 Rat4 Rat4 Rat4 Rat2 Nerve Survival damaged time (days) 7 VII 24-25 VII VII 7 IX 7-87 7-10 IX IX 7 IX 35 1-5 IX Rat2 IX IX IX Rat Rat IX IX Rat IX + VII Rat4 Gerbi12,4 VII VII Gerbil2 VII Hamster2 Hamster VII IX Rabbit4 Rat2 Rabbit2 Rabbit’ Rabbit Rabbit Rabbit Cat Cat‘ Monkey’ Monkey2 Chicken3 Chicken4 Duck Frog Catfish2 Catfish Catfish Catfish Catfish IX IX IX IX IX VII VII IX VII VII IX VII IX VII IX VII IX VII VII VII VII VII Tissue examined FP FP FP CP FOP CP CP CP 7-2 1 14-100 21 21 10-30 14-140 7-9 15 1-8 21-330 82-143 FOP CP CP CP CP FOP FP FP FP FP CP and FOP 6-10 21-120 10-21 17-35 14-18 8-15 13 28 16-19 24-27 42-140 8 21-30 11-21 10-46 7-10 42-300 15-37 18-33 14-17 12 10-15 CP and FOP CP and FOP CP FOP FOP FP FP CP FP FP CP FP CP ALB Bud cells remain (x) X X X X X X - PT - P FP B B B B B X X X - - Stain EM (electron microscopy) Iron hematoxylin Not listed Iron hematoxylin EM Iron hematoxylin Iron hematoxylin Picro-fuchsin; Weigert hematoxylin Immunostaining, substance P Enzyme reactions Iron hematoxylin Iron hematoxylin Enzyme reactions Methyl green; enzyme reactions Iron hematoxylin; silver Iron hematoxylin EM EM Osmic acid Hematoxylin & eosin (H & E) H&E EM EM EM; paraphenylendiamin Phosphotungstic hematoxylin Phosphotungstic hematoxylin Enzyme reactions Delafield‘s hematoxylin Methylene blue Iron hematoxylin; protargol EM H & E; silver H&E H&E Silver Iron hematoxylin Silver Silver Iron hematoxvlin Iron hematoxylin; H & E Iron hematoxylin; H & E Author(s) Farbman, 1969 Whiteside, 1927 Beidler, 1963 Hosley et al., 1987 Chelyshev et al., 1982 Guth, 1957 Guth, 1963 Kennedy, 1972 Nishimoto et al., 1985 Zalewski, 1981 Oakley, 1970 Oakley, 1974 Zalewski, 1970 Zalewski, 1969a Cheal and Oakley, 1977 Sloan et al., 1983 Whitehead et al., 1985 Whitehead et al., 1987 von Vintschgau and Honigschmied, 1877 State and Dessouky, 1977 Naga et al., 1970 Fujimoto and Murray, 1970 Iwayama, 1970 Jeppsson, 1969 Olmsted, 1921 Olmsted, 1922 State et al., 1982 Hayes and Elliott, 1942 Langworthy, 1924 Guth, 1958 Zahm and Munger, 1985 Vij et al., 1972 Ganchrow et al., 1986 Gentle, 1971 Krol and Dubbeldam, 1979 Robbins, 1967 Kamrin and Singer, 1953 May, 1925 Olmsted. 1920 Torrey, 1934 Torrey, 1936 Abbreviations: ALB-anterior lower beak oral epithelium; B-barbels; CP-circumvallate papillae; FP-fungiform papillae; FOP-foliate apillae; P-palate; PT-posterior tongue. ‘Age, puberty, breeding, and weight relationships were obtained from Holmes (1984),Koch (1973),and Weisbroth (1984)if not specifically indicated in the study referenced. 2Adults (at surgery). 3Prepubertal. 4Peripubertal. Table 1 summarizes some previous studies on budnerve dependency in order to ascertain which factors may have been critically linked to the conclusions drawn. An “x” in the column titled “Bud cells remain” indicates that complete buds, or some fusiform cells or other budlike structures, could be discerned in the epithelial position where one would expect to find a bud. The symbol “-” indicates no buds were discovered after gustatory nerve damage. Footnotes 2,3, and 4 indicate maturity of each species for studies that mention that variable. As can be seen from this table, there is no obvious relation between reported surviving “buds” and species, age, stain, o r taste bud locus. Of the 41 studies presented, 54% claimed the taste buds totally disappeared, and 82% of these latter reports based their results on tissue examined in the postoperative interval between 1 and 5 weeks. An additional 29% reported total bud disappearance with the exception of a few buds o r budlike figures still remaining. Likewise, most (75%)of these latter studies examined the 1-5 week postoperative interval. Some authors account for the scant buds remaining during the degenerative period either as midline-region buds receiving contralateral innervation (e.g., Cheal and Oakley, 1977; Hayes and Elliott, 1942; Guth, 1958; State et al., 1982; Whiteside, 1927) or by the incompleteness of J.R. GANCHROW AND D. GANCHROW 226 Wallerian degeneration in the sectioned nerve (e.g., Kamrin and Singer, 1953) or by the coincidental occurrence of regeneration when nerve regrowth could not be ruled out (e.g., Hosley et al., 1987). In addition, of ten studies reporting presence of buds or bud cells (Cheal and Oakley, 1977; Chelyshev et al., 1982; Farbman, 1969; Fujimoto and Murray, 1970; Kennedy, 1972; Krol and Dubbeldam, 1979; Olmsted, 1921,1922; Sloan et al., 1983; Whitehead et al., 1985), nine examined tissue between 1and 15 postoperative days, which may be early for complete degeneration to have occurred, under their experimental conditions. In one instance of longer survival, von Vintschgau and Honigschmied (1877) reported that the place where the buds had been located was noticeable, suggesting some kind of nonstratified epithelial cell organization lacking typical bud features. In the rabbit (Jeppsson, 19691, rat (Zalewski, 1969a; 1981), and chicken (Gentle, 1971) after survival times of up to 6 months, 3-5 months, and 6 weeks, respectively, glossopharyngeal-innervated taste buds unequivocally disappeared: “The permanent loss of buds in chronically denervated papillae verifies that buds do not regenerate spontaneously in the absence of appropriate innervation” (Zalewski, 1981, p. 310). However, the latter author used cholinesterase and ATPase stains to mark the reactions, and conceivably, potentially remaining buds could have physiological characteristics refractory to these stains. In summary, most studies reporting total bud disappearance in fungiform papillae used survival times up to 27 days. Following glossopharyngeal neurectomy, total bud loss occasionally has been sustained beyond 100 days, but, in general, mammalian examples using a postneurectomy time frame beyond 1 month are noticeably lacking. The present study in the young rat extended the survival time period to include 30-90 days postneurectomy to see whether bud presence could be identified within this relatively longer postoperative interval. Also, the majority of previous reports (Table 1) used hematoxylin stains. The present study incorporated this stain plus two others so as to decrease the possibility that postneurectomy buds in these young animals would be refractory to a single epithelial stain and escape identification. Surgery was performed in the fifth postnatal week, after weaning was complete. This relatively young prepubertal age may highlight a critical taste bud-neural dependency or, conversely, optimize the potential for plasticity and regenerative capacity since gustatory system maturation progresses gradually over the first 90 postnatal days in rats (e.g., Farbman, 1965; Ferrell et al., 1981, 1985; Hill and Almli, 1980; Hosley and Oakley, 1987; Mistretta, 1972; Yamada, 1980). Furthermore, criteria for bud counting did not rely exclusively on the presence of a taste pore. Thus, even if the pore region were obscured by oblique sectioning, or resisted staining after denervation (Hou et al., 19851,the bud would still be counted if identified by other characteristics (see below). MATERIALS AND METHODS Surgery was performed on six female SpragueDawley rats ranging in age from 28 to 35 days (mean age = 30 days; mean weight = 86 g). The rats were anesthetized (Equithesin) and placed in a supine position for a ventral approach to the nerve. The head was stabilized via a rubber band extending from just behind the incisor teeth to a cardboard box containing the body warmer. A skin incision was made just lateral t o the midline and parallel to the mandible. Blunt dissection exposed the posterior belly of the digastricus, styloglossus, internal pterygoid, and masseter muscles (see Greene, 1955) that were then retracted. The chorda tympani nerve was visualized as it passed ventral to the lateral pterygoid plate and into the tympanic bulla. A length of nerve was exposed, cut distally, and evulsed proximally (n = 2) with jeweler’s forceps. No muscles were cut. The chorda tympani is still undergoing the process of myelinization at this age (Ferrell et al., 1985) and is delicate, and difficult to locate, thereby necessitating an alternative surgical approach. Since the rat chorda tympani has its origin in the facial nerve canal (Green 19551, about a 6 mm length of facial nerve was evulsed (n = 4) as it exited the adjacent stylomastoid foramen. This portion of extirpated nerve included the chorda tympani and remnants of geniculate ganglion cells (verified histologically in some cases). In all cases, a 10% (wt/vol) solution of methyl methacrylate polymer (Howe & French, Boston) was applied to the distal cut end of the nerve, petrotympanic fissure, andlor stylomastoid foramen. At this concentration, transected nerves coated with methyl methacrylate fail to regenerate even after 1 year (Edds, 1945). After postsurgical intervals of 30, 45, 60, 75, and 90 days, animals were deeply anesthetized and perfused transcardially with 0.9% saline followed by 10% neutral formol-saline. The tongues were removed and stored in formol-saline, and the success of surgery was confirmed by dissection. The anterior-most 2-3 mm of the tongue was dehydrated, embedded in paraffin, and serially sectioned in the frontal plane (7-10 pm). Successive 100 km distances of tissue were alternately stained with hematoxylin and eosin, cresyl violet, and Pollak‘s trichrome stain (Humason, 1972). Iron hematoxylin was used in one additional case, but found to be the least useful and discontinued. All sections were examined serially by two independent observers, who were unaware on which side the surgery was performed or of the survival times. Rat fungiform papillae typically contain a single taste bud. In light microscopy, these buds have four main characteristics: 1)dorsally, a pore opens into the oral cavity; 2) the cytoplasm of bud cells stains dif- Fig. 1. Comparison of taste papillae contralateral and ipsilateral to gustatory neurectomy a t various survival times. Calibration bar: A = 100 pm, B = 16 pm, C = 27 pm, D = 41 pm, E = 26 pm. A: Two fungiform papillae (arrows) in the midline region of the anterior dorsal surface of the tongue. Asterisk is located within the lingual central sulcus. Papilla on the right is contralateral to neurectomy and contains a taste bud. Surgery, 35 days; survival time, 90 days; cresyl violet stain. B: Intact fungiform taste bud, contralateral to gustatory neurectomy. Surgery, 31 days; survival time, 75 days; cresyl violet stain. C: A budless fungiform papilla ipsilateral to gustatory neurectomy. Surgery, 35 days; survival time, 90 days; cresyl violet stain. D: Fungiform papilla, ipsilateral to gustatory neurectomy, whose dorsal surface is keratinized and bears a filiform-like peak. Surgery, 35 days; survival time, 90 days; Pollak trichrome stain. E: Fungiform papilla ipsilateral to gustatory neurectomy, with a n ovoid, cell aggregate (arrow) in its dorsal epithelium. Surgery, 28 days; survival time, 45 days; hematoxylin and eosin stain. CHORDA TYMPANI NEURECTOMY Fig. 1 227 228 J.R. GANCHROW AND D. GANCHROW ferently from the surround (usually lighter); 3) the orientation of bud cells is mainly perpendicular to the surface in contrast to the horizontal layering of surrounding epithelial cells; on the lateral edges of the buds-in-longitudinal-section, cellular organization is less well-defined, but still not horizontal; and 4) the basal region of the bud invaginates the underlying papillary connective tissue core. The occasional budless papilla has an uninterrupted basal cellular layer. In order not t p overlook buds on the operated side, observation of any one of these signs on either side was accepted as a potential bud, the structure traced serially, and counted. RESULTS About 80% of the contralateral (unoperated side), as compared with 12% of the ipsilateral (operated side), fungiform papillae contained buds or budlike formations. The number of taste buds counted on the unoperated side ( n = 247) greatly exceeded that on the operated side (n = 26) regardless of age or surgical approach (see Table 2). In the midline region, ipsilaterally, 18 structures mostly displayed all four bud characteristics as described in the Materials and Methods section. The remaining eight, more laterally situated budlike structures on the operated side appeared as small cell clusters in the basal epithelium of the dorsal surface of the papilla (see below). Figure 1compares taste buds on the unoperated and operated sides. Figure 1A shows two fungiform papillae (arrows) flanked by several stalklike filiform papillae in the lingual midline region. The fungiform papilla on the unoperated side (right) contains a normal, onion-shaped taste bud that, when traced serially, exhibited all four defining bud features (see Materials and Methods). In contrast, the fungiform papilla on the operated side (left) is budless. At a higher magnification, Figure 1B demonstrates that the fungiform bud in nonneurectomized tissue is easily discernible from the stratified epithelial surround in that the bud contains lighter-staining, vertically oriented cells with large, round nuclei. Moreover, a taste pore opens from the apical bud region, and a sheath of darker-staining cells surrounds the basal two-thirds of the bud, which in turn, invaginates the connective tissue core. None of these features are observed in most fungiform papillae ipsilateral to gustatory neurectomy. Figure 1C illustrates a typical fungiform papilla deprived of gustatory innervation. The basal epithelial cell layer overlaying the connective tissue core is uniform and uninterrupted. Some darkly stained cells appear to lie above the most basal stratum in the central apical region of the papilla, but this epithelial organization does not resemble that of a taste bud. Following surgery on the operated side, some broad fungiform papillae reflect filiform structure. For example, in Figure lD, papillary width suggests a fungiform characteristic but for a miterlike protuberance near the dorsal center of the papillar epithelium. Moreover, the oral surface of this papilla is unusually well keratinized. Though noted several times, the frequency of this combining papillar form was not specifically counted. Figure 1E shows a fungiform papilla ipsilateral to gustatory neurectomy and presents the best of eight examples of structures counted as budlike from the lateral edge of the tongue (Table 2). Near the dorsal center of the papilla, in the basal epithelium, a cell aggregate (arrow) with large darkly staining nuclei appears to barely invade the connective tissue ventrally and dorsally extends toward but does not penetrate through overlying stratified epithelium t o reach the papillar surface. Cytoplasm of this cell cluster stained similarly to that of cells in the epithelial surround, and there was no particular vertical cell organization characteristic of mature buds. Keratin covering the papillar dorsal surface was similar to that in adjacent tissue. This aggregate, at best, resembles the embryonic “spherical stage” of (presumptive) taste bud development prior to pore communication with the oral cavity (cf., Bradley and Stern, 1967; Bradley et al., 1980; Mistretta, 1972; Ganchrow and Ganchrow, 1987) and was equally discernible with each of the histologic stains used. The slight bulge into the connective tissue core was a consistent feature of these budlike structures. Lastly, some fungiform papillae ipsilateral to neurectomy appeared to be smaller than those contralaterally. Furthermore, ipsilaterally, the number of clearly definable fungiform papillae diminished by about 30%. Atrophic signs in foliate and circumvallate papillae have also been reported (e.g., Guth, 1963; Naga et al., 1970, von Vintschgau and Honigschmied, 1877; Zalewski, 1969a) following gustatory nerve transection. DISCUSSION Surgical destruction of the chorda tympani nerve in 1month-old rats nearly eliminated all taste buds in the ipsilateral fungiform papillae. With the most lenient of bud definitions, the operated side of the anterior-most lingual surface contained about one-tenth as many buds as seen contralaterally. More than half of this small, ipsilateral bud population was located near the lingual midline and displayed mature bud features. This raises the question of whether fungiform papillae near the median plane are normally bilaterally or contralaterally innervated. Normal bilateral innervation is evident for taste buds in the rat circumvallate papilla (e.g., Guth, 1963; Hosley et al., 1987; Oakley, 1974; Whiteside, 1927). Zander (18971, using gross dissection techniques in human cadavers, reported that fibers of the lingual branch of the trigeminal nerve (in which the distal chorda tympani passes) also cross the midline, especially in the anterior-most 5 mm of lingual tissue. In gerbils, Cheal and Oakley (1977) unilaterally removed the chorda tympani nerve, producing ipsilateral taste bud degeneration, and then performed a contralateral chorda tympani neurectomy. They concluded that “with the possible exception of a few fungiform papillae on or adjacent to the midline, all taste bud innervation in the foliate and fungiform papillae is ipsilateral” (p. 613). Likewise, following lingual neurectomy in the kitten (Hayes and Elliott, 1942) and adult rat (Whiteside, 19271, taste buds were seen in fungiform papillae 229 CHORDA TYMPANI NEURECTOMY TABLE 2. Taste bud counts for various survival times following chorda tympani (CT) or facial nerve (FN)neurectomy Neurectomv CT CT FN FN FN FN Age at surgery (davs) 25 34 28 29 31 35 Survival time (days) 30 60 45 60 75 90 Tongue length sampled (mm) 2.0 2.0 2.6 2.6 2.4 2.9 located in the ipsilateral “midregion,” especially a t the tongue tip. Whether this is the normal situation, or contralateral sprouting induced by denervation had occurred, is a research question that to date has not been adequately addressed. Recently, Hou et al. (1985) recorded from the hamster intact chorda tympani following unilateral chorda tympani removal. They concluded that “no chorda tympani contralateral to neurectomy responded to chemical stimulation localized to the side of the tongue ipsilateral to neurectomy, indicating numerous fibers had not sprouted across the midline” (p. 444). It is unclear whether 1) the most medial fungiform papillae of the anterior tongue were stimulated in this study or 2) a critical number of midline papillae must be stimulated to assure adequate responsiveness of the intact chorda tympani. What of that small population of cell aggregates seen after ipsilateral neurectomy that lie in basal epithelium of fungiform papillae, lateral to the midline region? As reported here in the young rat, these aggregates appear substantially less well organized than the buds described in adult hamster after gustatory nerve transection (Whitehead et al., 1987). Possibly, the cell aggregates may constitute the remnants of the last degenerating buds following denervation or simply are thickenings of the dorsal, central fungiform epithelium whose cells are oriented in nonlaminar fashion. If the first hypothesis was correct, progressive degenerative changes across survival times might have been expected, but were not observed. Alternatively, some cellular aggregates observed in the dorsal epithelium of fungiform papillae deprived of gustatory innervation could represent an epithelial reorganization, not unlike that in filiform papillae (Farbman, 1970). Two of the eight budlike structures were found in papillae exhibiting a slight peak a t the dorsal surface (cf. Fig. 1D). Finally, recent evidence from adult rats has suggested that lingual nerve collateral sprouting may be able to reinnervate some taste buds that have sustained chronic chorda tympani resection (Kinnman and Aldskogius, 1988). With the exception of a few midline-region buds, the present study using relatively long survival times in young rats failed to reveal any mature taste buds ipsilateral to the neurectomies that were comparable to the well-developed examples reported for the mature hamster (Whitehead et al., 1987). This difference may have been related to bud-nerve ontogenetic factors occurring coincident with the experimental time frame of Unoperated side 29 41 56 37 34 50 Taste bud counts Operated side Midline area More lateral 1 2 3 1 7 3 0 1 3 0 4 1 our prepubertal animals. Hosley et al. (1987) examined the ability of circumvallate taste buds to be induced following glossopharyngeal nerve transections in rats whose ages included those used in the present study. While the greatest retardation to induction was observed following nerve transections in the first 10 postnatal days, the capacity for taste bud induction was still impaired when transection was carried out at 30 postnatal days. Possibly, complete replacement of a degenerated bud is dictated by a site previously occupied by a mature bud. Mature circumvallate taste buds increase their numbers up to 90 postnatal days (Hosley and Oakley, 1987). To our knowledge, a complete developmental time course for fungiform taste bud maturation in the rat has not been quantified, although Hill (1987) suggests it probably continues through the first postnatal month since maturation of chorda tympani nerve responsiveness extends well beyond this period. Perhaps the capacity to form or maintain a bud following sustained neurectomy may require animals older than those used in the present study. It might be pointed out that the population of hamster fungiform taste buds does not increase between 19 and 140 postnatal days (Miller and Smith, 1984). In conclusion, the postlesion persistence or development de novo of the peripheral gustatory receptor in some adult vertebrate forms may indicate the strength of local factors guiding the expression of taste neuroepithelium in mature animals. Taste bud ontogenesis in younger animals may be more critically neurally dependent. A longer survival period per se does not appear to be the major determining factor of taste bud survival. ACKNOWLEDGMENTS This research was supported, in part, by a grant from the United States-Israel Binational Science Foundation (B.S.F.),Jerusalem, Israel. 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