Species differences in the synaptic membranes of the end bulb of held revealed with the freeze-fracture technique.код для вставкиСкачать
THE ANATOMICAL RECORD 20557-63 (1983) Species Differences in the Synaptic Membranes of the End Bulb of Held Revealed With the Freeze-Fracture Technique D. E. MATTOX, G. R. NEISES, AND R. L. GULLEY Department of Surgery, Division of Otorhinolaryngology, The Uniuersity of Texas Health Science Center at San Antonio, TX 78284 (D.E.M.), and Labomtory of Neurootolaryngology, NINCDS, National Institutes of Health, Bethesda, MD 20014 (GAN., RLGJ ABSTRACT Two morphological differences distinguish the membranes of the end bulb-spherical cell synapse in rats and mice from those in guinea pigs and chinchillas. First, in freeze-fracture replicas, the membranes of rat and mouse spherical cells lack perisynaptic aggregates which are present in the other species. Second, small gap junctions are present between the end bulb and spherical cell soma of rats and mice. These interspecies differences are not reflected in thinsectioned material. This observation points out the difficulty in attempting to generalize about the significance of intramembrane specializations in synaptic membranes. The freeze-fracture technique has been used in a variety of species to study the organization of synaptic membranes in different areas of the nervous system such as the cerebellum (Landis and Reese, 1974;Korte and Rosenbluth, 1980), the statoacoustic organ (Bagger-Sjoback and Flock, 1977; Gulley and Bagger-Sjoback, 1979; Bagger-Sjoback and Gulley, 19791, and the neuromuscular junction (Heuser et al., 1974; Rash and Ellisman, 1974). The distribution of intramembrane particles is remarkably similar at these different contacts. Those differences that have been identified correlate with differences in the shape of the presynaptic active zone (Bagger-Sjoback and Flock, 1977; Korte and Rosenbluth, 1980)and with whether or not the synapse is excitatory or inhibitory (Landis and Reese, 1974). No intra- or interspecies differences have been described in the organization of synaptic membranes. The anterior branch of auditory nerve fibers terminate in the rostral anteroventral cochlear nucleus (AVCN) as a large calyceal terminals on the soma of sperical cells. The ultrastructure of this terminal has been described in cat (Ibata and Pappas, 19761, guinea pig (Pirsig et al., 1969; Gulley et al., 1978a), chinchilla (Lenn and Reese, 1966; Gulley et al., 1978a), and rat (Gentschev and Sotelo, 1973). The internal organization of its membranes has been described in chinchilla and guinea pig using the freeze-fracture technique (Gulley et al., 1978a). Since the general cytological features of the terminals seen in thin sections are similar in these species, we assumed the organization of their membranes would also be similar. However, distinct species differences are present in the membranes of this synapse. This is the first description of species differences in the organization of synaptic membranes. METHODS AND MATERIALS The cochlear nuclei of ten adult albino rats and two brown rats were prepared for either thin section or freeze-fracture study. Freezefracture study of an albino guinea pig, two tricolor guinea pigs, two albino mice, and two brown mice were also included. All animals apparently were able to hear, at least as demonstrable by a startle response to auditory stimuli (Preyer’s reflex). Additional material was reviewed from cats, chinchillas, and guinea pigs used in previous studies. All animals were anesthetized with 60% urethane (0.25 mYlOO gm), perfused with 0.1 M sodium cacodylate with 20 mM CaC12 followed by 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate, and 20 mM CaC12. After perfusion, the head was removed and the skull was opened and placed in fixative. Freeze -Fracture The brain was dissected 2 hr after perfusion, and 250-km thick slices of the brain stem were cut on a vibratome. The rostral AVCN was 0003-276X/83/2051-0057$02.500 1983 ALAN R. LISS, INC h i v e d April 2,1981;accepted Sept. 29, 1982. 58 D.E. MATFOX, G.R. NEISES, AND R.L. GULLEY and chinchilla, each junctional aggregate is encircled by up to six perisynaptic aggregates (Fig. 6) on the spherical cell E-face opposite channels of enlarged extracellular space. These aggregates are not present in the rat on either the E-face or P-face. The number of nonaggregate particles on the E-face of rat spherical cells (Fig. 4,5) is similar to that in guinea pig and chinchilla;' thus it is unlikely that the particles constituting the aggregates in these species are merely dispersed in the membranes of the rat. Circular clusters of uniformly sized large particles are present on the P-face of the spherical cell associated with subadjacent subThin Sections surface cisternae. They do not resemble the The skull remained in fixative for 12 to 24 perisynaptic aggregate in size, shape, or parhr before dissection. After dissection, 250-pm ticle density. thick slices were cut through the brain stem, To ensure that the absence of the perisyand levels of the AVCN were selected which naptic aggregate is not an artifact of tissue were identical to those used for freeze-fracture. preparation or fracturing, a rat and guinea pig These were postfixed a t 4°C in 1.5%potassium were each perfused with one liter of fixative ferrocyanide and 1%OsOlin 0.05 M sodium taken from the same batch. The tissue was cacodylate buffer. All slices were dehydrated processed together through the same solutions in a graded series of methanol and embedded and was fractured and replicated simultanein Epon. Sections, 1 to 2-pm thick, from each ously on a multiple-specimen stage. Perisyblock were examined for orientation before thin naptic aggregates were present in the guinea sections were cut and stained with uranyl ace- pig, but were absent in the rat. Since some tate and lead citrate. Thin sections and repli- albino strains have hearing disorders which cas were examined in a Philips 201C electron microscope. Fig. 1. Thin section through an apposition of an end bulb Frozen sections, 5-10 pm thick, of rat brain (E) and spherical cell (S) from an albino rat. Active zones stem were cut and alternate sections stained (asterisks) appear as invaginations of the end bulb. Enwith cresyl-violet-luxolfast blue and the Bod- larged channels of extracellular space containing a glial ian silver stain. Rapid Golgi and Golgi-Kopsch process (arrows) separate some of the active zones. 17,000 material, from neonate, young adult, and adult X . rat used in an earlier study, was studied along Fig. 2. Thin section through a portion of an end bulb (El with the frozen sections, to obtain a general opposed to a spherical cell (S) in the albino rat. At least two understanding of the organization of the ros- somatic appendages (stars) are present and synapse with the end bulb (asterisks). Smaller, more irregular processes tral AVCN in the rat. around the appendages are probably from glial cells. 30,000 dissected from both sides and placed in 5% glycerol in 0.1 M cacodylate buffer. The tissue was transferred over a 1-hr period through 10% and 15%glycerol-buffer to 20% glycerol-buffer in which it remained for an additional hour. The pieces were then frozen in Freon 22 and fractured in a Balzers 301 freeze-fracture unit at -119°C. Platinum-carbon replicas were made with electron beam guns with the replica thickness standardized with a quartz crystal monitor. The replicas were cleaned in cold methanol, Chlorox bleach, and water before picking them up on uncoated grids. RESULTS In the rat, a single terminal or end bulb from a primary auditory fiber envelops the dentritic pole of each spherical cell in the dorsal portion of the rostra1 AVCN. From this end bulb, numerous synapticprocesses are distributed. Other non-primary terminals contact the remainder of the soma. In thin sections (Fig. 11, the end bulb and spherical cell resemble that seen in the guinea pig and chinchilla (GuIley et al., 1978a). In the rat, unlike the guinea pig or chinchilla, the spherical cell soma has occasional finger-like appendages located on the pole of the cell that is enveloped by the end bulb (Figs. 2,4). In freeze-fracture replicas, the E-face of the postsynaptic active zone has a junctional aggregate (Fig. 3) identical to that in guinea pig or chinchilla. In the guinea pig X. Fig. 3. Replica in which the fracture exposes a portion of a junctional aggregate on the E-face of a spherical cell membrane in an albino rat. The invagination of the presynaptic active zone is opposite the aggregate of large, tightly packed, irregular particles which appears to be coextensive with it. 62,000 x . Fig. 4. A replica of the P-face of an end bulb (El opposed to fragments of spherical cell E-face in a brown rat. The perspective is from the inside of the spherical cell. Several active zones (asterisks) and channels of enlarged extracellular space, one containing a glial process (arrow), are present. Cmfractured cytoplasm of aomatic appendages (stars) protrude around and into the end bulb .No perisynaptic aggregates are present on the fragments of the E-face of the spherical cell. 28,000 x . lln each species, particles were caunted on 20 wm2 of membrane Eface which had a aimilar shadow angle. SYNAPTIC MEMBRANES OF THE END BULB OF HELD 59 Fig. 5. A freeze-fracture replica of a spherical cell and end bulb in an albino rat. The fracture crosses the spherical cell cytoplasm (S) before exposing its E-face. Through windows in this E-face, the P-face of the end bulb (E)is seen. Three active mnes (asterisks) are present. An arrow points to an enlarged channel of extracellular space between two active zones. No perisynaptic aggregates are present on the spherical cell E-face (compare to Fig. 6). 40,000 X . Fig. 6. A replica of the P-face of a n end bulb (El viewed through windows in the E-face of a spherical cell (S) of tricolor guinea pig. Two active zones (asterisks) are present on the end bulb. The spherical cell E-face surrounding these active zones has numerous perisynaptic aggregates on the E-face (arrowheads). 40,000 x . SYNAPTIC MEMBRANES OF THE END BULB OF HELD 61 Fig. 7. A replica illustrating a gap junction between an end bulb (E) and spherical cell (S)in an albino rat. The fracture exposes the E-face of the end bulb (E) and a portion of the spherical cell P-face. A portion of crossfracture cytoplasm containing synaptic vesicles, some of which are clustered around a dome-shaped active zone (arrow), identifies the neuronal nature of the E-face membranes. The large size of the terminal and the multiple circular active zones identify it as an end bulb. A gap junction (arrowhead) is present on the spherical cell P-face adjacent to the fractured end bulb membrane and is illustrated at higher magnification in the inset. 21,000 x . Inset 73,000 x . conceivably could affect the perisynaptic aggregate, spherical cells in the AVCN of brown rats were also examined. They too lacked perisynaptic aggregates (Fig. 41, as did the spherical cells of both pigmented and unpigmented strains of mice. The spherical cells of the albino guinea pig have the aggregatesa2 Small gap junctions, about 0.1 pm or less in diameter, are present between the end bulbs and the spherical neurons of both rats and mice (Fig. 7). These junctions were usually found near active zones, and were only identified in freeze-fracture replicas. Their frequency is difficult to quantitate; however, they do not appear to be more common than one for every 25 active zones identified. DISCUSSION % an unpublished study by Drs. E. Kane and R. Gulley, perisynaptic aggregates were identified in the bushy cells of the cat. Perisynaptic aggregates were originally described at the spherical cell-end bulb contact in the AVCN of chinchillas and guinea pigs (Gulley et al., 1978a). They are also present in the medial nucleus of the trapezoid body of the guinea pig opposite the calyces of Held (Mattox, unpublished observations).At both of these synapses, these aggregates are on the E-face of the membrane opposed to channels of enlarged extracellular space surrounding the postsynaptic active zone. The aggregates are irregularly shaped and have a heterogeneous population of small and medium-sized particles. Both of the synapses where perisynaptic aggregates have been described are in auditory brain stem nuclei, and they both have a large area of apposition with many small active zones surrounded by channels of enlarged extracel- 62 D.E. MATTOX, G.R. NEISES, AND R.L. GULLEY lular space. The function of the perisynaptic aggregates is unknown; however, based upon their distribution in the membrane, Gulley et al. (1978a)suggested four possibilities: 1)Ionic pumps to maintain ionic concentrations in the face of sustained high rates of activity, 2) extrajunctional receptors, 3) enzymes for neurotransmitter degradation, or 4) sites of transient attachment for cisterns of endoplasmic reticulum. Subsequent studies eliminated some of these possibilities. After dederentation, perisynaptic aggregates are removed from the membrane (Gulley et al., 1977) but reappear when sprouting occurs in the nonprimary boutons (Collins and Gulley, 1979). The fact that these aggregates disappear after deafferentation argues that they are not extrajunctional receptors for neurotransmitters since, in other systems, this type of extrajunctional receptor increases in response to deafferentation. Either aspartate or glutamate is the neurotransmitter for primary auditory terminals (Wenthold and Gulley, 1978;Wenthold, 1980).Since postsynaptic enzymes are not believed to play a major role in degrading these neurotransmitters, it seems unlikely that these aggregates are enzymes involved in transmitter degradation. Cisternae of endoplasmic reticulum are common in the rat where perisynaptic aggregates are not present; thus, these aggregates are probably not exclusively sites for transient attachment for these structures. There is evidence that the function of these aggregates may be related to the activity of the synapse, which might be the case if they were ionic pumps. In the waltzing guinea pig, a genetically induced, postnatal loss of hair cells is accompanied by the cessation of VIII nerve firing. In these animals, the number of perisynaptic aggregates decrease in the spherical cell membrane after the period ofhair cell loss (Gulley et al., 1978b). Yet, if these particles do represent structures involved in synaptic activity, their function must not be related strictly to VIII nerve activity, since they are not present in rats and mice with apparently normal hearing. Thus we are left with no compelling informationon which to base a hypothesis on the function of the perisynaptic aggregate. The freeze-fracture technique is a valuable morphological tool for learning about synaptic specializations. Nonetheless, as we learn more about synaptic organization with this tool, we also learn more about the limitations of the technique. Through this process we are being forced to rethink some of the earlier general- izations derived from this technique (cf. Landis and Reese, 1974 to Gulley and Reese, 1981). The discussion of the perisynaptic aggregate in this manuscript is an example of the devolution of ideas about function of intramembrane specialization which were based solely on morphological observations. The fact that these aggregates appear in the membranes of spherical neurons of some species but not others may ultimately be an important clue as to their function, but it also should serve as a caution against speculating too freely about the significance of intramembrane specializations in synaptic membranes. ACKNOWLEDGMENTS This work was supported by the Laboratory of Neuro-otolaryngology,National Institute of Neurological and Communicative Disorders and Stroke, the National Institute of General Medical Sciences, and by grants from the Deafness Research Foundation and NIH (NSRO115058). We wish to acknowledge Marianne Parakkal for her assistance in the preparation of the manuscript. LITERATURE CITED Bagger-Sjoback, D. and A. 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