Received 29 March 1982; revised 16 June 1982; accepted 2 July 1982

Summary

The sensory epitheltum hning the ampulla o f Lorenzml in the skate was examlned bv the freeze-fracture technique Anastolnoslng tight junctions (zonula occludens) completely encircle the apex o f each receptor cell joining it to neighbouring support cells The t~gh t junctions separate two distinctly dtfferent regions o f the receptor-cell surface The apical P-face has numerous large partlcles while just below the ttght junctions o f the lateral surface have many smaller partlcles O n ~ t s basal surface each receptor cell makes several evaginatlng rlbbon synapes w t h an afferent nerve Three regions o f the synaptic evagination can be distinguished on the basis o f membrane speciahzations 1 At the tip o f the evagination a regular array o f large parttcles is found on the P-face o f the receptor cell directly opposite a sim~lar regular array o f large partrcles on the P-face o f the afferent nerve, 2 just above the tip at a narrow constriction, below which vesicles are not found, a population o f large particles on the P-face o f the receptor cell opposes a well-defined strip o f large particles that cleaves with the E-face o f the nerve hbre, 3 at the arch o f the synaptlc evagination randomly occurring dimples are found on the P-face and protrus~ons on the E-face o f the receptor cell The density o f these protruslons increased m ~kates that were electrically stimulated W e suggest that the to-extenswe arrays o f particles at the tip o f the ribbon synapse is an ~ntercellular junction, that the active zone o f the synapse is at or above the constriction, and that membrane retrieval occurs in the synaptic arch region

Introduction

In 1678 Lorenzini first described the long jelly-filled canals that open on the surface of sharks, rays and skates, and terminate in ampullae. It has only recently become clear that the ampullae of Lorenzini endow elasmobranchs with an acute sensitivity for ambient electric fields; for example, skates respond to field gradients as low as 10 nV/crn

' P r e s m f nifd~ess Department of Biophysics, The Johns Hopkms Universitv, Balt~more, MD 2121 8, USA

=Preseizf nrld1eqs Department of Neurob~ologv, Harvard Medical School, 25 Shattuck S t , Boston, MA 02115, USA

0300-48641 82/060897-16$04.42/0 .L. 1982 Chapman and Hall Ltd.

kinociiium and is specialized for sensory transduction; the basal surface makes several synapses on an afferent fibre of cranial nerve VIII. A typical ampulla may contain around 10 000 receptor cells and receive innervation from about 10 nerve fibres.

The afferent synapses of electroreceptors contain ribbons of fuzzy material. Ribbon synapses are also found in vertebrate photoreceptors and collections of dense presynaptic material are associated with synaptic vesicles in a variety of acoustico-lateralis receptor cells (Osborne, 1977; Saito, 1980). The n~orphological similarities among these synapses suggest that they rely on similar synaptic mechanisms.

Electroreceptor cells have been extensively studied in a variety of species with light and electron microscopy (Dotterweich, 1931; Barets & Szabo, 1962; Waltman, 1966; Derbin, 1970; Szabo, 1972). This is the first study of an electroreceptor using the freeze-fracture technique; a preliminary report was presented previously (Sejnowski & Yodlowski, 1978). (See also Akert et a! . , 1980.)

Materials and methods

Nine skates (Rnjn enmcen and Xnjn o~cillnlirf wtth f ~ n spans of 20-30 cm were caught off the shore of Woods Hole, Massachusetts during the iwnmer and kept briefly tn an aquarium with circulating seawater at room temperature Each ai11ma1 was ptthed betore belng transcard~ally perfused wtth hxatlve consssttng of 2% paraformaldehyde and 3'51% glutaraldehyde in elasmobranch Rsnger (200 mh4 urea, 200 mM NaCl, 200 mM sucrose, 20 mM NaHCO;, 5 mh? KC1, 8 mw CaCI,, 0 5 mM Mg12, buffered wtth 30 mM NaHEPES at pH 7 3) One amma1 was anaesthetszed wsth MS-222 before psth~ng After 2-3 11 of ftxatson the hvotd capsules were removed froni the skate and ~ndivtdu~il ampullae were d~siected from the capsules

To prepare the t~ssue for freeze-frncturmg, ampullae were waked tn elasmobranch Rlnger contaming 30% glycerol for 1-2 h, cut wtth a tiisue chopper mto 200 ,urn iltces, mounted on gold discs and frozen in Freon 22 slush The t~ssue was fractured wzth e~ the r kn~ke or 111 a complementary replica holder, and replicated at - 120' C on a Balzeri 360M apparatus fitted w ~ t h an electron beam gun for platinum shadowtng and a quart7 crystal rnonltor for standardizu~g the replica thickne'is. The replicas were cleaned in Purex bleach for l h , rinsed 111 dilute acetic acid, mounted on grids coated with Formvar and carbon, and examined in an AEI 6B electron microscope or a JEOL 100-S electron microscope. The negatives were photographically reversed before being printed so that the platinum deposits appear white and the shadows are black.

To prepare tissue for thin-sectioning, fixed ampullae were thoroughly rlnsed sn elasmobranch

Results

Apicnl surface Only the very apex of the receptor cell faces the lumen of the canal (Fig. 1). When exposed by freeze fracture the lumenal surface shows the supporting cells arranged in a polygonal pattern. The apical surface of each receptor cell protrudes, lke an island, near the edge of a single supporting cell (Fig. 2). The supporting cell wraps around the receptor cell and forms a short border with itself between the receptor cell and the lateral boundary of an adjacent supporting cell. A kinocilium, seen in thin section (Fig. 1) and cross fracture (Fig. 4), arises from each receptor cell. Each supporting cell has a protruding rosette on its P-face, which could be the basal body of a cross-fractured cilium. In addition, the borders of the supporting cells are lined with numerous cross-fractured microvilli.

When the supporting cell is fractured away from the receptor cell, anastamosing tight junctions are se$n to completely encircle the receptor cell near its apical surface (Fig. 3). The P-face of the receptor-cell apical plasmalemma, which faces the lumen, has fewer but larger particles, approximately 10 nm in diameter, than the lateral plasmalemma below the tight junctions (Fig. 3). The P-face of the receptor cell within and immediately around the tight junctions is free of particles.

Bnsnl surfnce Each receptor cell makes several synapses with an afferent nerve fibre; each synapse has a synaptic ribbon which forms the core of a flat process evaginating the postsynaptic nerve fibre (Fig. 5). The ribbon is covered with synaptic vesicles up to a constriction just above the tip of the synaptic evagination. When freeze fractured, the ribbon material can be identified as a double row of large particles adjoining vesicles that appear with either their convex E-faces or concave P-faces (Figs. 6, 11).

Flat processes, Like interposing fingers, extend out from the supporting cells to separate the presynaptic and postsynaptic cells at the insertion of the synaptic evagination (Fig. 5). Dimples are commonly seen on the P-face of the receptor cell in the region overlying the supporting cell, which we call the synaptic arch (Figs. 7, 10). Protrusions are often found on the E-face of the synaptic arch that are complementary to

above this highly organized tip reglon is a second type of particle array composed of somewhat larger particles that are less concentrated and less ordered than at the tip. These particles cleave more often with the P-face (Fig. 8).

Fig. 1. Thm section through the apical surface of a receptor cell (rc) A cil~um ( c ) protrudes Into the lumen of the canal, w h ~ c h is filled with mucous and extracellular material A band of tight junctions (cut transversely here between the arrows) connects the receptor cells to surrourlding supporting cells (cc) x 56 000

Fig. 2. Freeze-fracture plane through the lumeiial aspect of an ampulla revealing the P-faces of receptor cells (arrows) srrt rounded bv supporting cells Cross-fractured rn~crovlllt protrude from the borders of the supporting cells A single protuberarice arises from each supporting cell (circles) w h ~ h may correspoitd w ~ t h the basal bodv of a cilium x 32 000

Fig. 3. Freeze-fractured P-face of a receptor cell Ana.;tomosing tight junctions encircle the protrudtng apical iurface (a) separatmg it from the basal surface (b) of the cell A small portion of the supporting cell E-tace remains adherii~g to the t~gh t lunctxon network (between arrows) The promment strand of the double tlght )unction at the right 1s yresumablv where the iupportlng cell ]oms itself atter ericrrcling rhe receptor cell x 76 000

Fig. 4. The apical P-face of a receptor cell (rc) at high m a g n i f ~ c ~ ~ t ~ o n The klnocillum (c) on the receptor cell has been cross-fractured Coinpare the large particles on the P-face of the receptor cell with the relatlveiy fewer and smaller part~cles on the P-face of the ne~ghbour~ng supporting cell (sc) A piece ot the receptor cell has been fractured away (arrow) revealing a few strands of tight junction on the supporting cell x 100 000

Fig. 5. Thin section through a nbbon svnayse between a receptor cell (rc) and the p n m a v afferent nerve ftbre (no Fingers from the iuypoiting cel!s (sc) are inserted between the receptor cell and the nerve fibre Lwneath the synapttc arch Coated veslcles and prts art found at the top of the synaptic arch (arrowhead) The r~bbon i i covered w ~ t h svnapt~c vesicles except near the tip In this veslcle-tree region aggregates of dense materlal h e the presvnaptlc membrane (black arrows) Postsynaptrc dense rnaterlal is forrnd ~nsicie the nerve fibre hmng the tnvaginatton and 1s particularly dense around the constrlctron above the tip (white arrows) x 167 000

Fig. 6. Freeze-fracture through a ribbon synapse Much of the receptor cell has been fractured away, exposing the evaginatmg process containing the ribbon (r) and spnaptlc vesicles and the E-face of the receptor cell membrane (EF) The postsynaptic nerve fibre (nf) is cross-fractured A portion of P-leaflet of the nerve in the area opposed to the tip of the receptor evaqinatmn contains large particles packed in a regular array (arrows) x 133 000

Fig. 7. Freeze-fracture of a synaptic evagination exposing the P-face (PF) of the receptor cell The ribbon (r) within the invagination has been cross-fractured Numerous large part~cies are packed m a regular array along the tip (arrow) The membrane at the top of the 5vnaptic arch, hke that ot the rest of the basal and lateral receptor-cell surface, is charactenzed bv many small part~cles and several dimples (arrowheads) x 83 000

Fig. 8. Higher magnification of the P-face (PF) of a receptor cell at the t ~ p of an evagmat~ng process Large particles are packed into a t~gh t rect~linear array (arrows) Movmg laterally from the tip the large particles become progresswely less numerous and less ordered x 150 000

Fig. 9. High magnification of the presynaptic reglon showing the E-face (EF) of the receptor cell at the t-~p of the evagmat~on Very few particles are visible but particle imprints and s t r~at~ons mark the tip of the evagination T h ~ s vlew includes a very short segment of cross-fractured cvtoplasm in the evaginatmg process x 150 000

Fig. 10. TWO ribbon wnapses arising from the same receptor cell innervatmg the same cross-fractured nerve ftbrc (nf) The r~bbon or? the right (r) lies iniide a crosi-fractured evagmation A fragment o t the E-face (EF) ot the postsynaptic nerve fibre remains attached to the other synapse to the left A strip of Ixgc particles appedrs along the constriction above the t ~ p of the invag~nat~on (black arrows) and striat~ons are vliible below the conitrrciion Note a l w the dmples (wixte arrowif on the P-face (PF) of the receptor cell Photograph provided b~ Dr john Heuser x 133 000

Fig. 11. Cross-fractured r~bbon and E-face of a receptor cell a t the top of the synaptic. arch after electrically sttmulatmg the skate Numerous protrusion.; (arrow heads) 11e along the synapt~c arch; these may correspond to necks of vewle open~ngs x 83 000

A f f ~ r e n t neme The postsynaptic nerve fibre at the bottom of the invagination has a tight rectilinear

articles on its P-face (Fig. 6) similar to that found on the opposing P-face at the tip of the receptor ce!I (Fig. 7). The corresponding E-face of the nerve fibre at the tip has a complementary striated texture and few particles (Fig. 10). The rows of particles and the striations are about 10 nrn apart and tilted at 45' to the axis along the tip. A narrow, weIi-defined strip of ~rregularly-spaced particles 8-10 nm in diameter is found on the E-face of the nerve fibre adjacent to the constricted neck of the evaginating receptor cell (Fig. 10).

an extensiare

cells confirms the evidence from thm sections that the network of tight lunct~ons is continuous and therefore completely encircles the apical surface (Fig. 3). Complete anastomotic networks of tight junctions are also found between other epithelial cells (Staehelin, 1974), including hair cells in the organ of Corti (Gulley & Reese, 1976).

It has been suggested from physiological experiments on skate ampullae that voltage-sensitive channels on the apical surface of the receptor cell contribute to its high electrical sensitivity (Obara & Bennett, 1972; Clusin et nl . , 1975; Clusin & Bennett, 1977a, b, 1979a, b; Bennett & Clusin, 1979). The tight junctions which electrically isolate the apical from the basal surface of the electroreceptor also separate two regions of membrane with distinctive particle organization. The relatively homogeneous population of large particles found on the apical surface of the receptor may represent the channels that carry the currents observed physiologically, and the tight junctions may contribute to confining these channels to the apical surface; this is regarded as an important function of tight junctions in many epithelial cells (Dragsten et n l . , 1981).

The intramembrane particles that are found in freeze-fractured ribbon synapses of receptor cells in the skate ampulla of Lorenzini are summarized in Fig. 12. Large particles in regular arrays are co-extensive on the P-faces of both the receptor cell and the nerve fibre at the tip of the evagination. Similar arrays of particles are found at the tips of ribbon synapses in vertebrate photoreceptors (Raviola & Gilula, 1975). The synapses in unstimulated receptor cells of both the retina and the ampulla of Lorenzini are tonically active; for example, some nerve fibres from ampullae have resting firing rates of 20-50/sec (Murray, 1962). In cone cell endings of the turtle retina the surface area of the presynaptic membrane is considerably decreased in the light-adapted state when the ribbon synapses are less active (Schaeffer &Raviola, 1978). The symmetry of the particle specializations at the tip of a ribbon synapse suggests that they are junctions between the receptor and the nerve, perhaps serving to anchor the presynaptic and postsynaptic membranes.

Two other types of intramembrane specializations oppose each other across the synaptic cleft immediately lateral to the tip. The large particles that cleave mainly with the presynaptic P-face are similar to the presynaptic P-face particles that are believed to mediate Ca2- entry at other synapses (Pumplinef a1 ., 1981). In a highly restricted region opposite the narrow constriction of the presynaptic cell, ordered aggregates of large particles are found on the E-face of postsynaptic nerve fibres. Neurotransmitter receptor molecules at excitatory synapses often cleave with the E-face of the postsynaptic membrane (Landis et n l . , 1974; Landis & Reese, 1974; Franzini-Armstrong, 1976; Rheuben & Reese, 1978). The locations of the particle specializations in the presynaptic and postsynaptic membranes together with the observation that synaptic vesicles are

Fig. 12. Summary ot principal features and intramembrane particle specializations at a skate electroreceptor ribbon synapse. Particles appearing on the P-face of a membrane are shown as solid circles and E-face pdrtlcles a.; open ctrcles.

found above the constriction but not below indicate that the active zone of the synapse is probably at or above the constricted region.

The large dimples in the P-face and protrusions on the E-face of the receptor cell commonly found near the top of the synaptic arch probably represent sites where vesicles are joined to the membrane. This part of the synapse is unlikely to be part of the

Evldence from freeze-frac

zone lateral to the tip, and rnembra has recently been proposed for ribbon synapses in the turtle retina (Schaeffer et nl , 1982). One difference between the freeze-fracture images of the two ribbon synapses is the E-face protrusions found along the active zones of cone pedicles, which were interpreted as images of exocytosis of synaptic vesicles. We have not found images of vesicle exocytosis at ribbon synapses in skate electroreceptors and have not made measurements to see ~f there is an increase in the number of large particles following stimulation (Heuser & Reese, 1979, 1981). Our identification of the active zone is therefore provisional. The study of membrane dynamics at sensory synapses 1s at a relatively early stage; the homogeneity and high frequency of synapses in the ampulla of Lorenzini make it an attractive preparation for further investigation.

Acknowledgements

This work was initiated during the Neurobiology Course directed by Dr Edward Kravitz at the Marine Biological Laboratory. We are grateful for the help of Dr Thomas Reese in all phases of this study. We thank Dr John Heuser for his help in the beginning of this study, Dr William Huse for introducing us to the dissection, Louise Evans for her technical assistance, Bryan Schroeder and Kathy Cross for photographic help, Dr Bruce Schnapp for reviewing the manuscript, and Trudy Nicholson for her artwork in Fig. 12.

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