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Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.)
Exp, Eye Res. (1979) 29, 45-60
Fine Structure of the Lamprey Photoreceptors and Retinal Pigment Epithelium (Petromyzon marinus L.) D.
HOWARD
(Receivrd
Drcrtsox
AND
11 Jwnuccry
DEBRA
_1. GRAVES
1979, London)
The retinal photoreceptor cells and pigment epithelium of the lamprey, Petrornyzon mrinus have been examined with the electron microscope. The pigment epithelial cells in this avascular retina are characterized by surface specializations in the form of apical microvilli and basal infoldings. These cells contain myriad myeloid bodies, some phagosomes and residual bodies, as well as sparse. apically positioned melanin granules. Two types of photoreceptor cells are described in the retina of this animal. The long receptors have conical outer segments, slender elongated myoid regions and their nuclei are positioned in the outer portion of the outer nuclear layer near t,he external limit,@ membrane. The pyramid-shaped synaptic terminals of these cells are found deep in the outer plexiform layer, are less electron dense than those of the short rereptors and their synaptic I-esicles appear to be less densely packed than in the long receptor terminals. The short receptors have slender, elongated outer segments, their nuclei are positioned deeper in the outer nuclear layer, while the spherical synaptir terminals are located in the scleral region of the outer plexiform layer. The outer segments of both cell types however, show similar cone-like characteristics at the ultrastructural level, being composed of membranebound stacks of double-membrane discs, some of which are in continuity with the extracellular space. The outer segment of both t’ypes of receptors is joined to the inner segment through a slender ciliary stalk and is surrounded by calycal processes. Autoradiographic analysis shows that protein renewal in the photoreceptor outer segments of both long and short rereptors takes place in a similar diffuse manner. giving rise are to the suggestion that both photoreceptor types in t’he retina of Petromyzon n~~inu.s cones. Key zc*ordx: lamprey; cyclostomes; retina: photorecaeptors: pigment, epithelium, autoradiography; outer segment renewal.
1. Introduction The lamprey is of special interest to the biologist becausethese animals are living relat,ives of the now extinct ostracoderms. As such. the nature of the retinal photoreceptor cells in these vertebrate. q has engendered much interest and has been discussedand speculated on by numero~lsauthors in this century and the last,. as outlined by Walls (1935), Duke-Elder (195X) and Kleerekoper (1972). Only in the last deca.de,through electrophysiological and electron microscopical studies on the river lamprey (Lmnpetm JEuviatilis) by Ohman and Holmberg, has progress been made t,oward their identification (Ohman, 1971; Holmherg a)nd Ohma,n, 1956; Ohman, 1976; Holmberg, Ohman and Dreyfert, 1977). Even so: there remains a certain degree of uncertainty with regard to t,he classification of the allparent two types of photoreceptor cells in the lamprey retina. This paper attempts to clarify the situation in the CcJlllnlOn North American wadromous lamprey, Petromyzoa ~snri~us, through electron microscopic studies of the retina and pigment epithelium, and through autoradiographic analysis of the lq’take cf labelled precursors of the phot,orecept,or out’er segment proteins. ~10144835/79/070045+
16 $01.00/O
Q 45
1979 Academic
Press Inc.
(London)
Limited
2. Materials
and Methods
,\dult speciniens of the anadromoussea l;mipre~. ~&‘OW~/,OJ~ /i/f/i’l’lll/S \Vt?l’t’~X~l~~lf’~‘~~ in early spring. Young, recently transforlued, parasitic:adults were ot)&ne(l prior to their’ migration to the sea,from Gaspreauby fishermenon Washadenloak1,:&e i 1, Sew lhw~~wick: sexually rnat.ureadults iserecaptured in the Ln Have River in SOY;~Scot&t. 2~s t,lltx\. migrated upstreamto spawn.All animalswere maintained in the labmttory in fresh wtrr at 21°Cunder cycled light conditions. Electfm microscopy Light adapted animalswere anaesthetizedin a solution of tricaine ~nethanesulplloll;~tc: (MS-222); the eyes were removed, opened at the ora serrata with a sharp blade am1 immersedfor six hours in a fixative (860+1( 1 mosmol) containing 27; glut~wr;~ldeh~tlt: (Ladd ResearchInc.), lo/b paraformaldehyde (Fisher Chemcial), and CaCl, in :I, conceutration of 2.5 to 3.0 mu, buffered with 0.02 M sodium cacodylate at, pH 7.4. A ‘L-hr post, fixation in 176 osmium tetroxide was followed by staining e,zbloc with saturated uranyl acetatefor 1.5 hr. All specimenswereembeddedin Taab resin(Taab Laboratories England) ~(1 sectionsprepared for electron microscopy were st#ainedfor one minute iu Reynolcls lead citrate (Reynolds, 1963), and photographed in il Zeiss 108 transmissionelect,rorr microscope. adult animals mere given an intraocular injec+ion of a 50 PuCimixture of I,-leucinr [4,VH(N)] (specific.activity-60.0 Ci/mmol) and L-phenylalanine[alanine-“H(N)J (specific activityPm22.(! Ci/mmol), containing equal amounts of radioactivity due to each cornpound. The experiment was terminated 24 ht following the injection, and the eyes were removed and processedas describedabove. Half micron sectionswere cut, mounted on glassslidesand dipped ill Kod:d~, NTH;Z liquid emulsion. Following an exposure of 3 weeksat 4”C., the slideswere developed in Kodak D-19 developer for 2 mins at 19°C. The emulsionwas fixed and wadled, and the sectionsu;erestained through the emulsionwith cresyl violet. 3. Results The retinas of adult, upstream migrants and parasitic lampreys were examined and no significant differences were detected in either the pigment epithelial or ljhotoreceptor cells in samplestaken from these two extremes in the adult life cycle.
The pigment epithelium is composedof a single layer of cuboidal cells which rests on a basal lamina that forms part of Bruch’s membrane (Figs 1, 2 and 3). Bruch’s membrane in the lamprey is for the most part a bilaminar structure being composed of the basal lamina of the pigment epithelial cells (0.12 ~111thick) together with a O-7 pm thick zone of loosely woven collagen fibers (Figs 3 and 4). A thin and intermittent basal lamina of the choriocapillaris may also be seen and in these areas the memhra.neis tripartite (Fig. 3). The pigment epithelial cells are specialized at both their apices and bases.The apices are covered with irregular, elongate microvilli which project into the optic ventricle and surround the tips of the photoreceptor outer segments. The basesof these cells are characterized by a high degree of irregular infoldings (Figs 2 and 3). Although there are no identified attachments between pigment epithelial cells and the photoreceptors, adjacent epithelial cells are joined near their apices by a junc-
RETINA
OF
THE
LAMPREY
17
F11:. I. J,iyht mirrograph of a half micron, plastic section of parasitic lamprey retina. piymrnt (*l)ilhrtiun~ and choriocapiilaris. The choroid capillsrks (CC’) are separated from t.he pigment, epithclliunl i I’IC) by Krnch’s Jlembrane (BM). The short apical (inner) processes of the pigment epithrlium envelol~ “III> I he outer ae~ments of the long receptors (LR). Outer segments of the short receptors (SR) Irarc4~ ~;r.~h darkly stained ellipsoids (E) IW~II I,~I I hc pigment tlpithelium. Both receptor types show prominent, scler;rl to the external limiting membrane (ELM -arrows). The photorecept,or cell nuclei a~‘<’ l;)vr~~l within the ontcr nuclear layer (ONL) and the synaptic terminals of both receptor cell typvs a~‘<’ I~~~;~i~c~rl 111the outer region of the outer plexiform layer (OPL - arrows). lVote: The large black WI’OW on 21.CII,CIII;~L i\-bite backgronnd indicates the direction of the outside of the cyr.
-tional complex which consists of an inner zonulrr. occlz4rbns and all out,er ant\ Iw:: taxtensive zora~lnccdherms(Fig. 5). The nucleus is found within the basal (outer) region of the pigment epithelial cell, Ijut in light adapted animals, the melanin granules are found exclusively in apical cytoplnsmic extensions of the eelI. The majority of t,he cytoplasm of these ~11s is filled with profiles of smooth endoplasmic reticulunl. Small quantities of rough twinplasmic reticulum are present as well, as are free ril)osomes and some small n&ochondria (Fig. 3). Xyeloid bodies too are present. and are a common feature of the cytoplasm of these cells. They consist of lenticular stacks of flattened. discoid saccules, and are in direct continuity with the smooth endopluwnic reticulum (Figs 3 and 4).
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FII c:. 5. Low magnitiwt~or~ c4rctron micropr;~ph of iw 2Irlnlt 1amprl.y wtina shou il of th e choroid cspilhwirs (Cl), Rruch’s Mrmlwanr (19X). pigment cpithcliurn (I’E) within t hp pigsrnrnt, ~~pithelium WC the ftatt,enel inner . and outer segments. Pwmincnt m~4anosom~~. I’hs~oaomrs ( Py or 111peloid bodies (My) as well as epically positiwcd The, mter segments of the long receptor (LR) arc short and conical in shspc, and art’ i with the pigment epithelial cell apical processes. wherras those of the short recrpto and (elongate, and barely reach the pigmcnb epithelimn. Roth the outer segment and filled ellipsoid (E) of the long receptor extend beyond the shorter rrcrptor ~11s. ‘I’hc segm lents are joined to the cell body by a slender myoirl region (Mtl).
t,he cndot heli um nd photorrcec kor membrane sta CliR tre also obser\ Yxl. intimate coot act (SR) are slcn der he mitochondr ,ialrceptor ccl1 01Aer
RETIXA
OF
FIG. ::. Electron micrograph of the retinal long receptor cells. The end&helium (En) of an ilncomplete basal lamina. Bruch’s membrane infoldings of the pigment epithelium. The somcs (11) and phagosomes (Pg) apically, centrally, while the basal regions of the cell, 1”~:. 4. High magnification ment, epithelial basal lamina lamina of the choriocapillaris
electron (arrows), endothelial
THE
LAMPREY
pigment epithelium (PE) and the outer segments (OS) of the choroid capillaries is seen to he fen&rated and to h:,\ c (BJI) separates the endothelium from the basal (ollt(.r) cytoplasm of the pigment rpithelial cells contains m&11(,myeloid bodies (My) and smooth mdoplasmic rcticulunl contain secondary lysosomes and rcsitlual bodies.
micrograph of Bruch’s JIembrane which is composed loosely arranged collagen fibers and where present, cells (not shown here-see Fig. 3).
of thr ljict,ht% lt;~s;tl
Fro. 5. High magnification micrograph of the apical (inner) margin of two adjacent pigment epithc4ial cells. Large stacks of membranes (myeloid bodies) fill t,he cytoplasm and the apical margins of the cells are joined by a junctional complex consisting of an inner zonuta ocetudens [a) and an oator, less extensive zonula adherens (b). FIG. 6. High magnification micrograph large residual body or secondary lysosome sm~loth endoplasmic reticulum.
of the central cytoplasm (arrow), my&id body
of a pigment epithelial (&My). free pol~ysomes
cell, showing and nhundaut
50
II.
H. l~I(‘I~SOS
.ANl,
I). 11. c:l:AVF:6
Mlpeloid hodics mnge in size from small t&its slightly larger t8hall IIX~~II~IJ pwlul~~,-c to large st’ructures that measure 1.5 iJy 7.0 ,um. and are found throughout, thrb ~11. exclusive of the smaller apical cytoplasmic ext)ensions. Phagosomes are a,lso fount1 within thr pigment epithelial cells in a variet.\- of li)rtt 14. These tnembrane hJluld inclusion bodies. when &erred ntl:~r the apex of t.ht WI\. appear as loose, distorted niemhrane whorls; in the hasal region of the cell tlJ(LL present. as compact and more electron tlellse structures wit,11 t II(a t,ypical alq~ara& of secondary lysosomes or residual bodies (Figs 3 antI 6). Photoreceptor cells In Petrompon., two types of 1Jhotorece~~tur cells are identified on the tJa,siS of light l~~icroscopieal differenees (Fig. 1). They are classified as long arltI short receptors a.rd are present wit)hin the retina in a ratio of one to three respectively.
The most obvious structural di#erences between the two receptor types art: observed in the region of the inner and outer segments. The myoid is that portion ;)f the inner segment which extends between the ellipsoid end the nuclear region of the cell. In the long receptor it is slender and elongate, measuring up to 40 pm in length; in the shorter receptor it measures approximately 12 pm in length. The niyoiti region of both receptor cell types contains some rough endoplasmic: reticulum and polysomes, as well as abundant profiles of smooth endoplasmic reticulum (Fig. 2). In both ccl1 types, the myoid has a broad outer region and a slender. elongate inner region which contains bundles of longitudinally oriented microtubules (Figs 1. 2 and 3). Both cell types possess large mitochondrial-filled ellipsoids. The ellipsoidal region of the l(u~g receptor is more plump and the mitochondria more electron tlerlse. than those of t.ht: short receptor (Fig. 2). Outer seqmen.ts The outer segments of the short receptors are slender: elongate structures measuring approximately 25 pm in length; long receptor outer segme& are on the other hantl much shorter (approximately 7 pm in length). The long receptor outer segments are closely associated with apical pigment epithelial cell processes, whereas only the t,ips of the short receptor outer segments are in intimate association with the pigment epithelium (Fig. 2). In longitudinal section. it can he seen that the outer segmcnt)s of these two receptor types are quite similar in structure. Each is COJWJIOSCX~ of R membrane-bound stack of double-membrane discs or lamellae. Occasionally. t,he outer limiting niemhrane of both receptor types is seen to invaginate. giving rise to a single lamella which is in direct continuity with the extracellular space (Figs 7 ant1 8). The intradisc and interdisc spacing in both receptor types was found to he the same. The outer segments of both recept,or types are joined to the inner segments through a slender ciliary stalk which originates from a centriole and rootlet complex within the scleral end of the inner segment. Also originating from the scleral end of each inner segment are 11 to 15 slender calycal processes which extend in a scleral direction (Fig. 9). In cross section they can be seen to surround the outer segment in a palisadelike arrangement (Fig. 10).
RETINA
OF
THE
LAMPREY
5
FIC:S. 7, 8. High magnification micrographs of the outer segment of a long receptor (Fig. 7) and shot,t recept,or (Fig. 8). Note the areas (arrows) where the outer segment lamellae are in continuity with tbt, rxt~rarellular space. Outer segment interlamellar and intralamellar spaces are the same for both rrcvpt~~r types. FJC:. 9. Elect,ron micrograph of short receptors showing the mitochondrial-filled ellipsoid (E) joinul through a connecting ciliary stalk (insert) to the outer segment (OS). C’alycal processes (arrows) SILO originate from the inner segment and these filamrnt-fiiled processes extend along the length of the outr.1 s~wnmts. h F”xc:. 10. Elect,ron micrograph of the lamprey outer retina taken at right angles to Fig. 9. (Iross sectional profiles of long receptor inner segment (IS) and short, receptor outer segments (Oti) are ohaervc~t!. ‘I’hr outer segments are Buyrounded by calycal processes (small arrows) while the ciliary process (tarpr arrow) is ohsrrrrd on onp side only.
The external limiting membrane (EL;\l) is composed simply of a series of atllwritlg zonules found between adjacent Killer cells and also t wtween Jliillrr cells and phot,oreceptor myoid regions. ;\lliiller cell apical nlicrovilli extend hevontl the ELM ilrt’cb t II(J optic ventricle (Figs 11, 12 and 13).
The oval photoreceptor nuclei form the outer nuclear (OKL) layer of the retimt. In general, nuclei of the short receptor type are positioned deeper within the ONL. while those nuclei identified as belonging to the long receptors are found near the ELXand often protrude beyond. into the optic ventricle (Figs 12 and 13). Large buntllrs of microtubules are found in the perinuclear cytoplasm of the long receptors and these extendecl in a scleral and vitreal direction from the cell nucleus (Fig. 12). Receptor cell cytoplasm becomes attenuated in the region of the nucleus but expands again near the outer plexifornl layer to give rise to the synaptic terrnina.1 of the cell (Figs
FIG. 11. Micrograph of the extend limiting membrane region of the lamprey ret’ina. Junctions (arrows) in the form of ,zoudn ndherms are ubscrvctl between adjacent Miillw cells (Mu) and between Miiller cells and the myoitl regions of adjacent receptor cells (R).
R
OF
THE
.XI
LAkMPRE\
FIG. 1% Electron micrograph mont.age of lamprey photoreceptor shown in its entirety from the slender inner segment mvoid (Md), microtubule-filled axon, to the pyramid-shaped synaptic terminal +kveral short receptors (XR) are also shown. The external limiting HPI’OW htaads.
cells. A long receptor (LR) cell is through cell body with nucleus and within the outer plexiform layer. membrane is indicated with small
Fro. 13. Electron micrograph montage of lamprey rct,ina showing a complatr longitudinal t,hruugh a short receptor (SR) extending from the inner segment ellipsoitls (E) and my&l (Md) the cell body, to t.he synaptic terminal.
section through
FIG:. 11. Elcctwn micrograph nxcptor tvtn~innla aw generally they ;we lwgcr, pvt,nmitl-shaped insert shams at high rrsolution
of qwaptic ttl~minalx of long (I,li) anal stlol’t ($I:) 1’wY.[~l1~1’ (.t.li,s. I,‘>112 less elcctt~on &IW tlw in par’t to Itw (l(wsdy I)wlwl ,*I niiptic \i,ui(.lw: synaptic wcqdow, ;urtl rrmtain tnainl,v i~il~b~~rl s,vnnl,~~~s (ilw1~w4i. ‘1’11,. receptor fwminals of both thcl shod (SR) atvl Irma (IAl:) typtx
12 and 13). 8iilCe reCcptOr terminals Cc~ldd IJe positiv(?ly itht&?d whhtlrr CWlti~lllit~ was established up to the inner segment. some differences were ol~srrvttl twtww wceptc)r synaptic terminals of the two cell types. In general. ~~erminals of the hlg cells are less electron dense hut contain synaptic vesicles of the same size :ls tlIe short receptors; however, the vesicles are less densely packed in the long receptor tcrttliw~ls (Fig. 14). Long receptor terminals were always found deeper in the outer plcxiforn~ layer than those of the short receptors (Fig. 13). The short receptor synaptic teminals appear as dilated, oval terminations of the axon (spherules), while those of the long receptors present a pyramidal or pedicle-like shape (Figs Id and 14). -_~~-~~__-FIG. 15. Electron (black arrows).
micrograph
of a short
recept,or
(SR)
synaptic
terminel
showing
ribbon
syvl~apses
FIG. 16. Composite light micrograph of lamprey retina. At the left a small strip of normally stained retina shows pigment epithelium (PE), long (LR) and short (SR) photoreceptors. At the right an nlctoradiograph of a half micron plastic section of lamprey retina. The animal was injeotcrl int~rsrwulady with a 50 &i mixture of tritiated leucine ant1 phrnylalanine 24 hr prior to fix&on of the tissue. Heavy random l&belling is observed over the outer segments of the short receptors: the long receptor outer segments are also labelled randomly but, the label is less dcnsr than that o\w tho short receptors.
RETINA
OF
THE
(f&e czptions
LAMPREY
opposite.)
55
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I).
H. l~I(‘li6OS
ANI)
1). A. (iltAvE:.s
Both terminal types are filled with electron-lucent synaptic vesicles \\.llictl tll(‘;lsurt’ 54&A nm in diameter. and they make synaptic contact with inner retinal tleuroll(Figs 14 and 15). Kibhon synapties arc characterized hy the presence of iIt1 cl(>ctrc,ll dense complex composed of a sickle-shaped synaptic ribbon which is ~urroutlclfld iJ\synaptic vesicles and capped wit.h an arciform tlensit,y (Figs 1-l antI 1r)). ‘l’hc~c~ synapses also show post synaptic membrane densit.ies.
Autoradiographic sections of lamprey retina. fixed 24 hr subsequent to an intraocular injection of a 50 $2 mixture of t,ritiated leucine and phenylalanine show dense random labelling over the whole of the outer segment regions of the short. receptors. The entire outer segment regions of the long receptors show random labelling as well, but the density of silver grains over these regions was less than that observed over the shorter receptors (Fig. 16). 4. Discussion The pigment
epitheliurrl
Considering the primitive phylogenetic position of this species of vertebrate, the retinal pigment epithelium is remarkably similar in most respects to that of avascular retinas of other lower vertebrates, such as the newt (Dickson and Hollenberg, 1971). frog* (Porter and Yamada, 1960), and Laripetm jluwitrtilis (Ohman, 1974). Myeloid bodies are a common feature of this and some other avascular retinas. They have been described in the pigment epithelial cells of the frog (Leuenherger et nl., 1978; Porter and Yamada, 1960), newt (Dickson and Hollenherg, 1971), lizard (Anh, 1971) and pigeon (Marshall and Ansell, 1971). Considerable controversy &ill exists with regard to the origin and functional significance of these elaborate tncmbranous structures. Initially, myeIoic1 bodies were thought to play a role in regulating the movenlent of pigments in some species (Porter and Yamada, 1960): in the reconstitution of bleached visual pigments (Bliss, 1951; Porter and Yamada., 1960; Peterson, 1971), and in the metabolism of visual pigments (Nguyen-Legros, 1975). Leuenberger et nl. (1978) suggested myeloid bodies as a possible source of acid hydrolases and so they would he important to the pigment epithelial cell in digesting phagocytosed outer segment tips. In addition. recent work I)y Dickson and C!ollard (1978) on the developing retina of the larval lamprey, has demonstrated the likelihood of a functional relationship between myeloid bodies and differentiating photoreceptor cells. The lamprey pigment epithelium also contains many phagosomes and secondar) lysosomes or residual bodies. Since recent studies of vertebrate retinas indicate t’hat not only are rod outer segment discs phagocytosed by the pigment epithelium. but those of cones as well (Young, 1976; Hogan and Wood, 1974), lamprey photoreceptors could then be either rods or cones.
The external limiting membral:e in the vertebrate retina generclly consists of bhe estahlished junctional complexes between adjacent photoreceptor and glial elements. * The anur’an 1973).
frog
rye has a rascular
~nemhranr
which
lies on the surface
of tht
rctina
(Up+
anil
Bmds~,
RETINA
OF
THE
LAMPREY
57
These attachments have been described in a wide variety of vertebrates and were found to include in most instances, a gap junctional component (Miller and Dowling, 1970; Uga and Smelser, 1973). Recently, an extensive investigation (Tonus and Dickson, 1978) has revealed that the external limiting membrane in the avascular newt, retina is formed in part by a very elaborate gap-junctional network between t#he-retinal glial elements (Miiller cells). It is speculated that this junctional network link:-: the retinal glia, forming a large metabolite reservoir for the retinal neurons. In view of the similar avascular nature of the lamprey retina, it is surprising that, 110 gap junctions were observed in the region of t)hc external limiting memhrant>. The photoreceptor
cells
Although there have been many attempts to reveal the nat,ure of the photoreceptor cells in the lamprey retina, the controversy still rages, concerning the classification of the long and short receptors (seeWalls, 1935 and Kleerekoper, 1973 for review). Attempts have been made to classify the two cell t)ypes as rods and/or cones, hut these have to date been both inconclusive and conflicting. The most recent electron microscope studies of Ohman and Holmberg, in the river lamprey (Lanrpetrn jhcintilis), suggest that both types of photoreceptors are rod-like (Ohman, 1971, 1976: Holmberg and Ohman, 1976), mrhereas,the tracer studies with Pro&on Yellow (Oh.man, 1971) and ERG-recordings (Hohnberg, Ohman and Dreyfert, 1977)indicat,c the presenceof both rods and cones. In examining the fine structure of the two varieties of photoreceptor cells in tshc retina of P. m~wrinus, features common to both rods and cones of other \~c~rtrbrato retinas were revealed. By both light microscopy and low magnification electrorl microscopy, the outer segmentsof the short receptors were revealed to J)e rod-like. whilr those of t,he long receptors were distinctly conical in profile. It is of course Ilo\\ a \vell known fact that the general shape of the outer segment is not necessarily indicative of t’he receptor type, sincethe hunlan macula contains coneswith tIistinctI\ rotl-like morphological characteristics. More cxt,ensi\-e ult,rastructural study of the 1amJ)reynuter segment,has revealetl both here. in P~trorr~yaor~ and in Lcmpetrcc (Ohman, 1971) that the outer segmentsof both receptor types are surrounded by a menlbranc which is charactcrizetl I)y nunferolts infoldings or invagina,tions that, extend hctween the outer scgmc~nt(I~scs. rnlike the appearance of cone outer segmentsin other lower vertct)ratesz where tticl discs are formed through a continual infolding of the outer segment nlenlt)r.;me (( 2)tl(~n.1970; Nilsson. 1965), the outer seglllents(lrscrit,cd for ljoth l;r.mJ)rc:yrc>cel)tol tylws rt,semlIle closely the model ~~ropwc~ i hy Young (1976) for n~anln~aliancorl(:s. \rhcre the outer segment is enclosed by a membrane which makes nunlwo~ls slrl>rll infoldings t#hat remain connected t,o the ext,racellular space. I’reviously reported descriptions of photoreceptors of other lowt>r verteI,~~;ltt~
5s
I).
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in both and since neither ljossessrs an electron (lenso intr:llan~c~llar I~aricl. thcb\- r(~s~~1111)l~~ the cone outer segmenbs of other lower vertelmltes. Anot)her difference het,ween rod a.nd cone outrr segments is secn wh(att t,fris rvgio~~ is examined in cross-section. Rod outer segment discs in amphi hiam (Hrow~. (4 1)l)ons and Wald. 1963; Dickson and Hollenbcrg. 1971) and fish (Fint,ran and Xicol. 1973: Borwein and Hollenherg, 1973) 1Iresent a distinctly lobulated appear;:nc~‘. (Iuc to (IP(JI~ incisures of the disc perimeters. In man and prinlates. the incisures are tnort’ Sll~‘t’l‘fieial, while in other mammals, they may be single deep fissures ((!ohcn, 1972). On thr other hand, cone outer segments never appear to have incisurex. and alt~hough the significance of the incisures in rod discs is not clear, it does seem to 1w char;:ctwistic: of this receptor cell type. hterestin~ly, the outtcr segments of tleither the http ttoJ to the short receptors in t,he Lamprey have incisures. a fact wflicf1 further at,t.cBsts their cone-like nat,ure. As outlined by Young (1969), t,lle basic morpholog,v of phot’oreceptor synapt,ic terminals is similar in most mammals, with rod terminals being ovoid (sphcrulcs) and cone terminals being conical in shape (pedicles). Studies on the newt retina (Dickson and Hollenberg, 1971) and mudpuppy (Dowling and Werl)lin. 1969) harr shown that rod terminals are pedicular and extend laterally some dist*ancebefore synapses are established; these are in contrast with the cone terminals which are more spherical. Differences in the density of rod and cone synaptic ternlinals hsvr also been reported for a number of classesof lower vertebrates. which include atnphiIlia (Evans, 1966; Dickson and Hollenherg, 1951; Keefe 19il ; Lasanskp. 19’i3) and fish (Borwein and Hollenberg, 1973; Villegas. 1960). In the lamprey. Petrovrh!/zu/r.\vc’ have noted that tM-o types of receptor terminals are present and that they differ in shapeand density of staining. The terminals of the short, receptors resemblespherules and are more electron densethan the slightly pedicular terminals of the long reccpt)ors. Ohman (1976) on the other hand, reports the presence of only spherical receptor terminals in the Lamprey, Lmrvpetm, while ate11(1972) has demonstrated that froth receptors in the American brook lamprey. Et~~tosp1wm.s are pedicles. In recent years, much interest has been focused on the concefd of photoreceptor outer segment renewal, since the initial confirmation of the process1)yYoung in 196i. Thesestudieshave involved the useof radioactively labelled precursorsof outer segment’ membranesand have clearly established that the eater segn’ents of rods and cones are renewed in different ways. When proteins reach the outer segments of rocls, thck majority is incorporated into the membranes at, the very baseof t,hr outer segments. Subsequent to the administration of radioactively labellcd amino acids, a I)antl of newlv formed protein is always identified in rods but has never heen ohserved in cones, of any of the speciesof amphibia. reptiles, fish, birds or mammals stutlied (Young, 1976). Th e renewal process differs in cones regardless of their shape. and labelling with membrane precursors always gives rise to a diffuse scaMering of newI> incorporated molecules throughout the length of the outer segment. Since even the rod-like fovea1 COIVX of the primate ret,& consistently label in this same diffLtuse manner (Young, 1971), we suggestthis t)echniqueas being a useful method of differentiating rods from cones. The majority of the morphological evidence present.ed here suggest,sthat t.11~ two types of receptors in the Lamprey are cone-like; the diRering features of the two receptor cell synaptic terminals are the only exceptions. However, it is our belief, based on the autoradiographic evidence of similar diffuse patterns of probein renewal in the outer segments of both the long and the short receptors, that these
RETIKA
OF
THE
two photoreceptor cells in Petromyzon mnriwus are in fact both cones.
:i!$
LAMPREY
are of the same t,ype, and that they
ACBXOWLEDGMESTS
The authors would like to express their appreciation to Dr John Youson of the Departof Zoology, University of Toronto, for providing the vounp, parasitic adult, lamprevs used in t,his study. This resea.rch was supported by grants from the Xedical Research Council of Cauad;l and the Canadian National Institute for the Blind, out of the E.A. Baker Foundation +‘()I the Prevention of Blindness. ment,
REFERENCE8 ;\nh,
J. N. H.
morphologie Bliss.
(1971).
Les corps
myeloides
de 1’Cpithelium
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r&inien. 1. Rhpartition. 115, 508-23. in the regeneration of rodopsin. J. Hiol.
et rapports avec les organites cellulaires. %. Zellforsch.
A. F. (1951). Properties of pigment layer factor r’hem. 193 5”5-31. Borwein. R. an)d Gollenberg, M. J. (1973). The photoreceptors of the ” E’ our-eyed” fish, dscchl~ps nncrbleps L. J. Xorphol. 140, 40542. Brown, I’. R., Gibbons, I. R. and Wald, G. (1963). The visual cells and visual pigments of the mudpuppy, Secturus. J. Cell Biol. 19, 79-106. Hunt. A. H. (1978). Fine structure and radioautography of rabbit photorecept,or cells. ZIL~.~SI. Ophthdmol. 17, Y&104. (lohen. A. (1970). Further studies on the question of the patency of saccr~les in outer seemerits of vertebrate photoreceptors. Fis. Res. 10, 445-53. (‘ohen. A. 1. (1972). Rods and cones, In Handbook of Sensory Physiology l-01. r-IZ/?, Phjysiology of f>hotoreceptor Organs (Ed. Fuortes. $1. G. F.) Springer-Verlag. Kety York. 1)ickson. D. H. and Hollenberg, M. J. (1971). The fine strncture of the pigment epithelium and photoreceptor (tells of the newt, Triturus airirfescens dorsnlia (Rafinesque). b. Morph,ol. 135, 389-43’~ . 1. Dickson. D. H. and Collard, T. R. (1979) Retinal development in the lamprey (Pdrow!lm,c morinus L.): premetamorphic ammocoete eye. An/. J. &at. 154, 321-36. Dow-ling. ,J. E. and Werblin, F. S. (1969). Organization of retina of the mlcdpuppy, Nechrus ~~nncuZo.sus. 1. Synaptic structure. J. Neurophysiol. 32, 315-38. Duke-Elder, S. (1958). The eyes of cyclostomes. In Systetn oj” Ophthm~rno~ogy. 1.01. 1. 3Cy iu &*olution. Pp. 259-72. Nosby Co.. St. Louis. Evalls. E. M. (1966). On the ultrastructure of the synaptic region of the visual receptors in wrhiu vcrtebmtes. 2. ZelZforsch. 71, 499p51fi. Fineran. K. A. and Sicol, J. A. C. (1974). Studies on the ryes of Sew Zealand parrot-fishes (Labridae). t’roc. El. Sot. Land B. 186, 21747. Hog;Ln. M. 1. and \l’ood, I. (1974). Phagocyt’osis by pigment) epithelium of human retinal (‘ones. il’ntwre, (Lo&on) 252, 305-6. Holrnberg, K. and Ohman, P. (1976). Fine structure of retinal synaptic organelles in lamprey and hagfish photoreceptors. Vis. Res. 16, 237- 9. Holmbng, K., Ohman P. and Dreyfert’, T. (1977). ERG-recording from the retina of the river lamprey (Lnmpetm ,j7wintilis). I’is. Res. 17, 715-17. Keefi!. J. R. (1971). The fine structure of the ret,ina in the Newt. Trifurus r,iridescrns. .I. Exp. ZOOI. 177, m-93. Kletsrekoper, H. (1972). The sense organs. In Th.e Hiology sf Lnnlpreys (Eds Hard&y. 11. iv. and Pot,ter. I. (1.). Academic Press, London. Lasansky, A. (1973). Organization of the outer synaptic layer in the retina of the larval tiger salamander. Phil. Tsans. Roy. Sot. (Lond.) Ser. B. 265, 417-89. Leuanberger. $2.. Demolis, Ph. and Keller, G. (1978). Cytoplasmic variations in pigment, epithelium (PE) during cyclic lighting. Invest. Ophthal. Suppl. 1978, p. 135, no. 6 (Abst.) Marshall, J. and Ansell, I?. L. (1971). Membranous inclusions in the retinal pigment epithelium: pha.goxometi and myeloid bodies. J. Amt. 110, 91-104.
cm Miller.
TJ. tl.
IJI~'IiSON
ziXT)
I). ;I. (:It,.A\'E:5
K. F. and Dowling. .1. E. (1970). Intrwrllulur responses of the i\ldler ((klial) cvlls 01’ I hr. mudpuppy retina: their relat,ion to h-warr of t hc rlectrol~tinoRlan1. -1. Se~oph!yskd. 33, :1:3- 41. Nilsson, S.-E. G. (1%X). The ultrastructure of the rweptor outer scgmcwts in the: ret,ina of’ I IN’ leopard frog, Hnru~ p@iens. J. I%rcrst. Res. 12, 207-Z< 1. Nguyen-T,egros, J. (1975). .A propos ties Corps My&loides de l,‘~pit.h~lium Pignwntairca tL(* Ia R&tine des Vert6brks. J. Ciltmst. 12esch. 53, Is?-63. Ohman. P. (1971). The photoreceptor outer segments of the river lamprey (Lnmpetm ,jfzwkrtili.s). An &&on-. fluorescenceand light, microscopic study. Actrc Zoologicrc 52, %7- 97. Ohmun, L’. (1976). Fine structure of photorecept,ors and associated neurons in the retina of Lnncpetrtc Jluviutilis (Cyclostomi). Via. Res. 16, 659-ti. Pet,erson. P. 4. (1971). Studies on the intern&w between prealbumin, retinolhinding protein ant1 vitamin A. J. Binl. Chem. 246, 449. Port,er, K. R. and Ynmada, E. (1960). Studies on endoplssmic reticulum (ER). V. 1 ts fornl ant1 differenti:Aon in pigment epithelial cells of the frog ret.ina. J. Hiophys. Biochenj. C’ytol. 8, 181--‘05. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 20%ItJ. Stell. W. K. (197%). The morphological organization of t,he vertebrate retina. In IIa~adbool~ of Memory Physiology Vol. VII/Z, Physiology of Photoreceptor 0rqnm.s (Ed. Fuort,es, M. (:. I’.). Springer-Verlag. New York. Tonus, J. (:. and Dickson, D. H. (1979). Neuro-glial relationships at, the external limit,ing mww branc of t,he newt retina. Exp. Eye RN. 28, 93-100. Uga, S. and Smelser, (:. K. (1973). Comparative study of the fine structure of retinal Miiller r,rllx in various vertebrates. Iwest. O~hthnlmol. 12, 434-48. Villeges, C:. JI. (1960). Electron microscopic study of the vertebrate retina. J. Cen. Physiol. 43, 15-43. Walls, G. I,. (1935). The visual cells of lampreys. Br. .J. Ophthnlmd. 19, 129-48. Yacob, A., Wise, C. and Kung, Y. W. (1977). The accessory outer segment of rods and cones in the retina of the guppy, Poe& reticulntrr P. (Teleostei). Cell Tiss. Rrs. 177, 181-93. Young, R. 1%‘. (1967). The renewal of photoreceptor outer segments. J. Cell Viol. 33, 61-71. Young. R. W. (1969). The organization of vertebrate phot,oreceptor cells. In The Ret&au: dlorphology, F’unction, rrnrl C’linicnl Chnrncteristics (Eds Straatsma, 1%. It.. Hall, R1. 0.. Alleu, R. 4. and Crescitelli. F.). Univ. of CaLfornia Press, Berkeley and Los Angeles. Young, R. 12:. (1971). The renewal of rod and cone outer segments in the rhesus monkey. .J. ( ‘rll Bid. 49, 303-18. Young. R. IV. (1976). Visual cells and the concept of renewal. Imtsf. Ophthnlwol. 15, 7OO-m2fi.
Fine Structure of the Lamprey Photoreceptors and Retinal Pigment Epithelium (Petromyzon marinus L.) D.
HOWARD
(Receivrd
Drcrtsox
AND
11 Jwnuccry
DEBRA
_1. GRAVES
1979, London)
The retinal photoreceptor cells and pigment epithelium of the lamprey, Petrornyzon mrinus have been examined with the electron microscope. The pigment epithelial cells in this avascular retina are characterized by surface specializations in the form of apical microvilli and basal infoldings. These cells contain myriad myeloid bodies, some phagosomes and residual bodies, as well as sparse. apically positioned melanin granules. Two types of photoreceptor cells are described in the retina of this animal. The long receptors have conical outer segments, slender elongated myoid regions and their nuclei are positioned in the outer portion of the outer nuclear layer near t,he external limit,@ membrane. The pyramid-shaped synaptic terminals of these cells are found deep in the outer plexiform layer, are less electron dense than those of the short rereptors and their synaptic I-esicles appear to be less densely packed than in the long receptor terminals. The short receptors have slender, elongated outer segments, their nuclei are positioned deeper in the outer nuclear layer, while the spherical synaptir terminals are located in the scleral region of the outer plexiform layer. The outer segments of both cell types however, show similar cone-like characteristics at the ultrastructural level, being composed of membranebound stacks of double-membrane discs, some of which are in continuity with the extracellular space. The outer segment of both t’ypes of receptors is joined to the inner segment through a slender ciliary stalk and is surrounded by calycal processes. Autoradiographic analysis shows that protein renewal in the photoreceptor outer segments of both long and short rereptors takes place in a similar diffuse manner. giving rise are to the suggestion that both photoreceptor types in t’he retina of Petromyzon n~~inu.s cones. Key zc*ordx: lamprey; cyclostomes; retina: photorecaeptors: pigment, epithelium, autoradiography; outer segment renewal.
1. Introduction The lamprey is of special interest to the biologist becausethese animals are living relat,ives of the now extinct ostracoderms. As such. the nature of the retinal photoreceptor cells in these vertebrate. q has engendered much interest and has been discussedand speculated on by numero~lsauthors in this century and the last,. as outlined by Walls (1935), Duke-Elder (195X) and Kleerekoper (1972). Only in the last deca.de,through electrophysiological and electron microscopical studies on the river lamprey (Lmnpetm JEuviatilis) by Ohman and Holmberg, has progress been made t,oward their identification (Ohman, 1971; Holmherg a)nd Ohma,n, 1956; Ohman, 1976; Holmberg, Ohman and Dreyfert, 1977). Even so: there remains a certain degree of uncertainty with regard to t,he classification of the allparent two types of photoreceptor cells in the lamprey retina. This paper attempts to clarify the situation in the CcJlllnlOn North American wadromous lamprey, Petromyzoa ~snri~us, through electron microscopic studies of the retina and pigment epithelium, and through autoradiographic analysis of the lq’take cf labelled precursors of the phot,orecept,or out’er segment proteins. ~10144835/79/070045+
16 $01.00/O
Q 45
1979 Academic
Press Inc.
(London)
Limited
2. Materials
and Methods
,\dult speciniens of the anadromoussea l;mipre~. ~&‘OW~/,OJ~ /i/f/i’l’lll/S \Vt?l’t’~X~l~~lf’~‘~~ in early spring. Young, recently transforlued, parasitic:adults were ot)&ne(l prior to their’ migration to the sea,from Gaspreauby fishermenon Washadenloak1,:&e i 1, Sew lhw~~wick: sexually rnat.ureadults iserecaptured in the Ln Have River in SOY;~Scot&t. 2~s t,lltx\. migrated upstreamto spawn.All animalswere maintained in the labmttory in fresh wtrr at 21°Cunder cycled light conditions. Electfm microscopy Light adapted animalswere anaesthetizedin a solution of tricaine ~nethanesulplloll;~tc: (MS-222); the eyes were removed, opened at the ora serrata with a sharp blade am1 immersedfor six hours in a fixative (860+1( 1 mosmol) containing 27; glut~wr;~ldeh~tlt: (Ladd ResearchInc.), lo/b paraformaldehyde (Fisher Chemcial), and CaCl, in :I, conceutration of 2.5 to 3.0 mu, buffered with 0.02 M sodium cacodylate at, pH 7.4. A ‘L-hr post, fixation in 176 osmium tetroxide was followed by staining e,zbloc with saturated uranyl acetatefor 1.5 hr. All specimenswereembeddedin Taab resin(Taab Laboratories England) ~(1 sectionsprepared for electron microscopy were st#ainedfor one minute iu Reynolcls lead citrate (Reynolds, 1963), and photographed in il Zeiss 108 transmissionelect,rorr microscope. adult animals mere given an intraocular injec+ion of a 50 PuCimixture of I,-leucinr [4,VH(N)] (specific.activity-60.0 Ci/mmol) and L-phenylalanine[alanine-“H(N)J (specific activityPm22.(! Ci/mmol), containing equal amounts of radioactivity due to each cornpound. The experiment was terminated 24 ht following the injection, and the eyes were removed and processedas describedabove. Half micron sectionswere cut, mounted on glassslidesand dipped ill Kod:d~, NTH;Z liquid emulsion. Following an exposure of 3 weeksat 4”C., the slideswere developed in Kodak D-19 developer for 2 mins at 19°C. The emulsionwas fixed and wadled, and the sectionsu;erestained through the emulsionwith cresyl violet. 3. Results The retinas of adult, upstream migrants and parasitic lampreys were examined and no significant differences were detected in either the pigment epithelial or ljhotoreceptor cells in samplestaken from these two extremes in the adult life cycle.
The pigment epithelium is composedof a single layer of cuboidal cells which rests on a basal lamina that forms part of Bruch’s membrane (Figs 1, 2 and 3). Bruch’s membrane in the lamprey is for the most part a bilaminar structure being composed of the basal lamina of the pigment epithelial cells (0.12 ~111thick) together with a O-7 pm thick zone of loosely woven collagen fibers (Figs 3 and 4). A thin and intermittent basal lamina of the choriocapillaris may also be seen and in these areas the memhra.neis tripartite (Fig. 3). The pigment epithelial cells are specialized at both their apices and bases.The apices are covered with irregular, elongate microvilli which project into the optic ventricle and surround the tips of the photoreceptor outer segments. The basesof these cells are characterized by a high degree of irregular infoldings (Figs 2 and 3). Although there are no identified attachments between pigment epithelial cells and the photoreceptors, adjacent epithelial cells are joined near their apices by a junc-
RETINA
OF
THE
LAMPREY
17
F11:. I. J,iyht mirrograph of a half micron, plastic section of parasitic lamprey retina. piymrnt (*l)ilhrtiun~ and choriocapiilaris. The choroid capillsrks (CC’) are separated from t.he pigment, epithclliunl i I’IC) by Krnch’s Jlembrane (BM). The short apical (inner) processes of the pigment epithrlium envelol~ “III> I he outer ae~ments of the long receptors (LR). Outer segments of the short receptors (SR) Irarc4~ ~;r.~h darkly stained ellipsoids (E) IW~II I,~I I hc pigment tlpithelium. Both receptor types show prominent, scler;rl to the external limiting membrane (ELM -arrows). The photorecept,or cell nuclei a~‘<’ l;)vr~~l within the ontcr nuclear layer (ONL) and the synaptic terminals of both receptor cell typvs a~‘<’ I~~~;~i~c~rl 111the outer region of the outer plexiform layer (OPL - arrows). lVote: The large black WI’OW on 21.CII,CIII;~L i\-bite backgronnd indicates the direction of the outside of the cyr.
-tional complex which consists of an inner zonulrr. occlz4rbns and all out,er ant\ Iw:: taxtensive zora~lnccdherms(Fig. 5). The nucleus is found within the basal (outer) region of the pigment epithelial cell, Ijut in light adapted animals, the melanin granules are found exclusively in apical cytoplnsmic extensions of the eelI. The majority of t,he cytoplasm of these ~11s is filled with profiles of smooth endoplasmic reticulunl. Small quantities of rough twinplasmic reticulum are present as well, as are free ril)osomes and some small n&ochondria (Fig. 3). Xyeloid bodies too are present. and are a common feature of the cytoplasm of these cells. They consist of lenticular stacks of flattened. discoid saccules, and are in direct continuity with the smooth endopluwnic reticulum (Figs 3 and 4).
Il.
H. lJI(‘KSON
ASI)
I). -1. (:I:.\\‘IC$
FII c:. 5. Low magnitiwt~or~ c4rctron micropr;~ph of iw 2Irlnlt 1amprl.y wtina shou il of th e choroid cspilhwirs (Cl), Rruch’s Mrmlwanr (19X). pigment cpithcliurn (I’E) within t hp pigsrnrnt, ~~pithelium WC the ftatt,enel inner . and outer segments. Pwmincnt m~4anosom~~. I’hs~oaomrs ( Py or 111peloid bodies (My) as well as epically positiwcd The, mter segments of the long receptor (LR) arc short and conical in shspc, and art’ i with the pigment epithelial cell apical processes. wherras those of the short recrpto and (elongate, and barely reach the pigmcnb epithelimn. Roth the outer segment and filled ellipsoid (E) of the long receptor extend beyond the shorter rrcrptor ~11s. ‘I’hc segm lents are joined to the cell body by a slender myoirl region (Mtl).
t,he cndot heli um nd photorrcec kor membrane sta CliR tre also obser\ Yxl. intimate coot act (SR) are slcn der he mitochondr ,ialrceptor ccl1 01Aer
RETIXA
OF
FIG. ::. Electron micrograph of the retinal long receptor cells. The end&helium (En) of an ilncomplete basal lamina. Bruch’s membrane infoldings of the pigment epithelium. The somcs (11) and phagosomes (Pg) apically, centrally, while the basal regions of the cell, 1”~:. 4. High magnification ment, epithelial basal lamina lamina of the choriocapillaris
electron (arrows), endothelial
THE
LAMPREY
pigment epithelium (PE) and the outer segments (OS) of the choroid capillaries is seen to he fen&rated and to h:,\ c (BJI) separates the endothelium from the basal (ollt(.r) cytoplasm of the pigment rpithelial cells contains m&11(,myeloid bodies (My) and smooth mdoplasmic rcticulunl contain secondary lysosomes and rcsitlual bodies.
micrograph of Bruch’s JIembrane which is composed loosely arranged collagen fibers and where present, cells (not shown here-see Fig. 3).
of thr ljict,ht% lt;~s;tl
Fro. 5. High magnification micrograph of the apical (inner) margin of two adjacent pigment epithc4ial cells. Large stacks of membranes (myeloid bodies) fill t,he cytoplasm and the apical margins of the cells are joined by a junctional complex consisting of an inner zonuta ocetudens [a) and an oator, less extensive zonula adherens (b). FIG. 6. High magnification micrograph large residual body or secondary lysosome sm~loth endoplasmic reticulum.
of the central cytoplasm (arrow), my&id body
of a pigment epithelial (&My). free pol~ysomes
cell, showing and nhundaut
50
II.
H. l~I(‘I~SOS
.ANl,
I). 11. c:l:AVF:6
Mlpeloid hodics mnge in size from small t&its slightly larger t8hall IIX~~II~IJ pwlul~~,-c to large st’ructures that measure 1.5 iJy 7.0 ,um. and are found throughout, thrb ~11. exclusive of the smaller apical cytoplasmic ext)ensions. Phagosomes are a,lso fount1 within thr pigment epithelial cells in a variet.\- of li)rtt 14. These tnembrane hJluld inclusion bodies. when &erred ntl:~r the apex of t.ht WI\. appear as loose, distorted niemhrane whorls; in the hasal region of the cell tlJ(LL present. as compact and more electron tlellse structures wit,11 t II(a t,ypical alq~ara& of secondary lysosomes or residual bodies (Figs 3 antI 6). Photoreceptor cells In Petrompon., two types of 1Jhotorece~~tur cells are identified on the tJa,siS of light l~~icroscopieal differenees (Fig. 1). They are classified as long arltI short receptors a.rd are present wit)hin the retina in a ratio of one to three respectively.
The most obvious structural di#erences between the two receptor types art: observed in the region of the inner and outer segments. The myoid is that portion ;)f the inner segment which extends between the ellipsoid end the nuclear region of the cell. In the long receptor it is slender and elongate, measuring up to 40 pm in length; in the shorter receptor it measures approximately 12 pm in length. The niyoiti region of both receptor cell types contains some rough endoplasmic: reticulum and polysomes, as well as abundant profiles of smooth endoplasmic reticulum (Fig. 2). In both ccl1 types, the myoid has a broad outer region and a slender. elongate inner region which contains bundles of longitudinally oriented microtubules (Figs 1. 2 and 3). Both cell types possess large mitochondrial-filled ellipsoids. The ellipsoidal region of the l(u~g receptor is more plump and the mitochondria more electron tlerlse. than those of t.ht: short receptor (Fig. 2). Outer seqmen.ts The outer segments of the short receptors are slender: elongate structures measuring approximately 25 pm in length; long receptor outer segme& are on the other hantl much shorter (approximately 7 pm in length). The long receptor outer segments are closely associated with apical pigment epithelial cell processes, whereas only the t,ips of the short receptor outer segments are in intimate association with the pigment epithelium (Fig. 2). In longitudinal section. it can he seen that the outer segmcnt)s of these two receptor types are quite similar in structure. Each is COJWJIOSCX~ of R membrane-bound stack of double-membrane discs or lamellae. Occasionally. t,he outer limiting niemhrane of both receptor types is seen to invaginate. giving rise to a single lamella which is in direct continuity with the extracellular space (Figs 7 ant1 8). The intradisc and interdisc spacing in both receptor types was found to he the same. The outer segments of both recept,or types are joined to the inner segments through a slender ciliary stalk which originates from a centriole and rootlet complex within the scleral end of the inner segment. Also originating from the scleral end of each inner segment are 11 to 15 slender calycal processes which extend in a scleral direction (Fig. 9). In cross section they can be seen to surround the outer segment in a palisadelike arrangement (Fig. 10).
RETINA
OF
THE
LAMPREY
5
FIC:S. 7, 8. High magnification micrographs of the outer segment of a long receptor (Fig. 7) and shot,t recept,or (Fig. 8). Note the areas (arrows) where the outer segment lamellae are in continuity with tbt, rxt~rarellular space. Outer segment interlamellar and intralamellar spaces are the same for both rrcvpt~~r types. FJC:. 9. Elect,ron micrograph of short receptors showing the mitochondrial-filled ellipsoid (E) joinul through a connecting ciliary stalk (insert) to the outer segment (OS). C’alycal processes (arrows) SILO originate from the inner segment and these filamrnt-fiiled processes extend along the length of the outr.1 s~wnmts. h F”xc:. 10. Elect,ron micrograph of the lamprey outer retina taken at right angles to Fig. 9. (Iross sectional profiles of long receptor inner segment (IS) and short, receptor outer segments (Oti) are ohaervc~t!. ‘I’hr outer segments are Buyrounded by calycal processes (small arrows) while the ciliary process (tarpr arrow) is ohsrrrrd on onp side only.
The external limiting membrane (EL;\l) is composed simply of a series of atllwritlg zonules found between adjacent Killer cells and also t wtween Jliillrr cells and phot,oreceptor myoid regions. ;\lliiller cell apical nlicrovilli extend hevontl the ELM ilrt’cb t II(J optic ventricle (Figs 11, 12 and 13).
The oval photoreceptor nuclei form the outer nuclear (OKL) layer of the retimt. In general, nuclei of the short receptor type are positioned deeper within the ONL. while those nuclei identified as belonging to the long receptors are found near the ELXand often protrude beyond. into the optic ventricle (Figs 12 and 13). Large buntllrs of microtubules are found in the perinuclear cytoplasm of the long receptors and these extendecl in a scleral and vitreal direction from the cell nucleus (Fig. 12). Receptor cell cytoplasm becomes attenuated in the region of the nucleus but expands again near the outer plexifornl layer to give rise to the synaptic terrnina.1 of the cell (Figs
FIG. 11. Micrograph of the extend limiting membrane region of the lamprey ret’ina. Junctions (arrows) in the form of ,zoudn ndherms are ubscrvctl between adjacent Miillw cells (Mu) and between Miiller cells and the myoitl regions of adjacent receptor cells (R).
R
OF
THE
.XI
LAkMPRE\
FIG. 1% Electron micrograph mont.age of lamprey photoreceptor shown in its entirety from the slender inner segment mvoid (Md), microtubule-filled axon, to the pyramid-shaped synaptic terminal +kveral short receptors (XR) are also shown. The external limiting HPI’OW htaads.
cells. A long receptor (LR) cell is through cell body with nucleus and within the outer plexiform layer. membrane is indicated with small
Fro. 13. Electron micrograph montage of lamprey rct,ina showing a complatr longitudinal t,hruugh a short receptor (SR) extending from the inner segment ellipsoitls (E) and my&l (Md) the cell body, to t.he synaptic terminal.
section through
FIG:. 11. Elcctwn micrograph nxcptor tvtn~innla aw generally they ;we lwgcr, pvt,nmitl-shaped insert shams at high rrsolution
of qwaptic ttl~minalx of long (I,li) anal stlol’t ($I:) 1’wY.[~l1~1’ (.t.li,s. I,‘>112 less elcctt~on &IW tlw in par’t to Itw (l(wsdy I)wlwl ,*I niiptic \i,ui(.lw: synaptic wcqdow, ;urtl rrmtain tnainl,v i~il~b~~rl s,vnnl,~~~s (ilw1~w4i. ‘1’11,. receptor fwminals of both thcl shod (SR) atvl Irma (IAl:) typtx
12 and 13). 8iilCe reCcptOr terminals Cc~ldd IJe positiv(?ly itht&?d whhtlrr CWlti~lllit~ was established up to the inner segment. some differences were ol~srrvttl twtww wceptc)r synaptic terminals of the two cell types. In general. ~~erminals of the hlg cells are less electron dense hut contain synaptic vesicles of the same size :ls tlIe short receptors; however, the vesicles are less densely packed in the long receptor tcrttliw~ls (Fig. 14). Long receptor terminals were always found deeper in the outer plcxiforn~ layer than those of the short receptors (Fig. 13). The short receptor synaptic teminals appear as dilated, oval terminations of the axon (spherules), while those of the long receptors present a pyramidal or pedicle-like shape (Figs Id and 14). -_~~-~~__-FIG. 15. Electron (black arrows).
micrograph
of a short
recept,or
(SR)
synaptic
terminel
showing
ribbon
syvl~apses
FIG. 16. Composite light micrograph of lamprey retina. At the left a small strip of normally stained retina shows pigment epithelium (PE), long (LR) and short (SR) photoreceptors. At the right an nlctoradiograph of a half micron plastic section of lamprey retina. The animal was injeotcrl int~rsrwulady with a 50 &i mixture of tritiated leucine ant1 phrnylalanine 24 hr prior to fix&on of the tissue. Heavy random l&belling is observed over the outer segments of the short receptors: the long receptor outer segments are also labelled randomly but, the label is less dcnsr than that o\w tho short receptors.
RETINA
OF
THE
(f&e czptions
LAMPREY
opposite.)
55
.x
I).
H. l~I(‘li6OS
ANI)
1). A. (iltAvE:.s
Both terminal types are filled with electron-lucent synaptic vesicles \\.llictl tll(‘;lsurt’ 54&A nm in diameter. and they make synaptic contact with inner retinal tleuroll(Figs 14 and 15). Kibhon synapties arc characterized hy the presence of iIt1 cl(>ctrc,ll dense complex composed of a sickle-shaped synaptic ribbon which is ~urroutlclfld iJ\synaptic vesicles and capped wit.h an arciform tlensit,y (Figs 1-l antI 1r)). ‘l’hc~c~ synapses also show post synaptic membrane densit.ies.
Autoradiographic sections of lamprey retina. fixed 24 hr subsequent to an intraocular injection of a 50 $2 mixture of t,ritiated leucine and phenylalanine show dense random labelling over the whole of the outer segment regions of the short. receptors. The entire outer segment regions of the long receptors show random labelling as well, but the density of silver grains over these regions was less than that observed over the shorter receptors (Fig. 16). 4. Discussion The pigment
epitheliurrl
Considering the primitive phylogenetic position of this species of vertebrate, the retinal pigment epithelium is remarkably similar in most respects to that of avascular retinas of other lower vertebrates, such as the newt (Dickson and Hollenberg, 1971). frog* (Porter and Yamada, 1960), and Laripetm jluwitrtilis (Ohman, 1974). Myeloid bodies are a common feature of this and some other avascular retinas. They have been described in the pigment epithelial cells of the frog (Leuenherger et nl., 1978; Porter and Yamada, 1960), newt (Dickson and Hollenherg, 1971), lizard (Anh, 1971) and pigeon (Marshall and Ansell, 1971). Considerable controversy &ill exists with regard to the origin and functional significance of these elaborate tncmbranous structures. Initially, myeIoic1 bodies were thought to play a role in regulating the movenlent of pigments in some species (Porter and Yamada, 1960): in the reconstitution of bleached visual pigments (Bliss, 1951; Porter and Yamada., 1960; Peterson, 1971), and in the metabolism of visual pigments (Nguyen-Legros, 1975). Leuenberger et nl. (1978) suggested myeloid bodies as a possible source of acid hydrolases and so they would he important to the pigment epithelial cell in digesting phagocytosed outer segment tips. In addition. recent work I)y Dickson and C!ollard (1978) on the developing retina of the larval lamprey, has demonstrated the likelihood of a functional relationship between myeloid bodies and differentiating photoreceptor cells. The lamprey pigment epithelium also contains many phagosomes and secondar) lysosomes or residual bodies. Since recent studies of vertebrate retinas indicate t’hat not only are rod outer segment discs phagocytosed by the pigment epithelium. but those of cones as well (Young, 1976; Hogan and Wood, 1974), lamprey photoreceptors could then be either rods or cones.
The external limiting membral:e in the vertebrate retina generclly consists of bhe estahlished junctional complexes between adjacent photoreceptor and glial elements. * The anur’an 1973).
frog
rye has a rascular
~nemhranr
which
lies on the surface
of tht
rctina
(Up+
anil
Bmds~,
RETINA
OF
THE
LAMPREY
57
These attachments have been described in a wide variety of vertebrates and were found to include in most instances, a gap junctional component (Miller and Dowling, 1970; Uga and Smelser, 1973). Recently, an extensive investigation (Tonus and Dickson, 1978) has revealed that the external limiting membrane in the avascular newt, retina is formed in part by a very elaborate gap-junctional network between t#he-retinal glial elements (Miiller cells). It is speculated that this junctional network link:-: the retinal glia, forming a large metabolite reservoir for the retinal neurons. In view of the similar avascular nature of the lamprey retina, it is surprising that, 110 gap junctions were observed in the region of t)hc external limiting memhrant>. The photoreceptor
cells
Although there have been many attempts to reveal the nat,ure of the photoreceptor cells in the lamprey retina, the controversy still rages, concerning the classification of the long and short receptors (seeWalls, 1935 and Kleerekoper, 1973 for review). Attempts have been made to classify the two cell t)ypes as rods and/or cones, hut these have to date been both inconclusive and conflicting. The most recent electron microscope studies of Ohman and Holmberg, in the river lamprey (Lanrpetrn jhcintilis), suggest that both types of photoreceptors are rod-like (Ohman, 1971, 1976: Holmberg and Ohman, 1976), mrhereas,the tracer studies with Pro&on Yellow (Oh.man, 1971) and ERG-recordings (Hohnberg, Ohman and Dreyfert, 1977)indicat,c the presenceof both rods and cones. In examining the fine structure of the two varieties of photoreceptor cells in tshc retina of P. m~wrinus, features common to both rods and cones of other \~c~rtrbrato retinas were revealed. By both light microscopy and low magnification electrorl microscopy, the outer segmentsof the short receptors were revealed to J)e rod-like. whilr those of t,he long receptors were distinctly conical in profile. It is of course Ilo\\ a \vell known fact that the general shape of the outer segment is not necessarily indicative of t’he receptor type, sincethe hunlan macula contains coneswith tIistinctI\ rotl-like morphological characteristics. More cxt,ensi\-e ult,rastructural study of the 1amJ)reynuter segment,has revealetl both here. in P~trorr~yaor~ and in Lcmpetrcc (Ohman, 1971) that the outer segmentsof both receptor types are surrounded by a menlbranc which is charactcrizetl I)y nunferolts infoldings or invagina,tions that, extend hctween the outer scgmc~nt(I~scs. rnlike the appearance of cone outer segmentsin other lower vertct)ratesz where tticl discs are formed through a continual infolding of the outer segment nlenlt)r.;me (( 2)tl(~n.1970; Nilsson. 1965), the outer seglllents(lrscrit,cd for ljoth l;r.mJ)rc:yrc>cel)tol tylws rt,semlIle closely the model ~~ropwc~ i hy Young (1976) for n~anln~aliancorl(:s. \rhcre the outer segment is enclosed by a membrane which makes nunlwo~ls slrl>rll infoldings t#hat remain connected t,o the ext,racellular space. I’reviously reported descriptions of photoreceptors of other lowt>r verteI,~~;ltt~
5s
I).
H.
l)I('I\SOS
XX
I)
I).
.A. (:It.%vb:s
in both and since neither ljossessrs an electron (lenso intr:llan~c~llar I~aricl. thcb\- r(~s~~1111)l~~ the cone outer segmenbs of other lower vertelmltes. Anot)her difference het,ween rod a.nd cone outrr segments is secn wh(att t,fris rvgio~~ is examined in cross-section. Rod outer segment discs in amphi hiam (Hrow~. (4 1)l)ons and Wald. 1963; Dickson and Hollenbcrg. 1971) and fish (Fint,ran and Xicol. 1973: Borwein and Hollenherg, 1973) 1Iresent a distinctly lobulated appear;:nc~‘. (Iuc to (IP(JI~ incisures of the disc perimeters. In man and prinlates. the incisures are tnort’ Sll~‘t’l‘fieial, while in other mammals, they may be single deep fissures ((!ohcn, 1972). On thr other hand, cone outer segments never appear to have incisurex. and alt~hough the significance of the incisures in rod discs is not clear, it does seem to 1w char;:ctwistic: of this receptor cell type. hterestin~ly, the outtcr segments of tleither the http ttoJ to the short receptors in t,he Lamprey have incisures. a fact wflicf1 further at,t.cBsts their cone-like nat,ure. As outlined by Young (1969), t,lle basic morpholog,v of phot’oreceptor synapt,ic terminals is similar in most mammals, with rod terminals being ovoid (sphcrulcs) and cone terminals being conical in shape (pedicles). Studies on the newt retina (Dickson and Hollenberg, 1971) and mudpuppy (Dowling and Werl)lin. 1969) harr shown that rod terminals are pedicular and extend laterally some dist*ancebefore synapses are established; these are in contrast with the cone terminals which are more spherical. Differences in the density of rod and cone synaptic ternlinals hsvr also been reported for a number of classesof lower vertebrates. which include atnphiIlia (Evans, 1966; Dickson and Hollenherg, 1951; Keefe 19il ; Lasanskp. 19’i3) and fish (Borwein and Hollenberg, 1973; Villegas. 1960). In the lamprey. Petrovrh!/zu/r.\vc’ have noted that tM-o types of receptor terminals are present and that they differ in shapeand density of staining. The terminals of the short, receptors resemblespherules and are more electron densethan the slightly pedicular terminals of the long reccpt)ors. Ohman (1976) on the other hand, reports the presence of only spherical receptor terminals in the Lamprey, Lmrvpetm, while ate11(1972) has demonstrated that froth receptors in the American brook lamprey. Et~~tosp1wm.s are pedicles. In recent years, much interest has been focused on the concefd of photoreceptor outer segment renewal, since the initial confirmation of the process1)yYoung in 196i. Thesestudieshave involved the useof radioactively labelled precursorsof outer segment’ membranesand have clearly established that the eater segn’ents of rods and cones are renewed in different ways. When proteins reach the outer segments of rocls, thck majority is incorporated into the membranes at, the very baseof t,hr outer segments. Subsequent to the administration of radioactively labellcd amino acids, a I)antl of newlv formed protein is always identified in rods but has never heen ohserved in cones, of any of the speciesof amphibia. reptiles, fish, birds or mammals stutlied (Young, 1976). Th e renewal process differs in cones regardless of their shape. and labelling with membrane precursors always gives rise to a diffuse scaMering of newI> incorporated molecules throughout the length of the outer segment. Since even the rod-like fovea1 COIVX of the primate ret,& consistently label in this same diffLtuse manner (Young, 1971), we suggestthis t)echniqueas being a useful method of differentiating rods from cones. The majority of the morphological evidence present.ed here suggest,sthat t.11~ two types of receptors in the Lamprey are cone-like; the diRering features of the two receptor cell synaptic terminals are the only exceptions. However, it is our belief, based on the autoradiographic evidence of similar diffuse patterns of probein renewal in the outer segments of both the long and the short receptors, that these
RETIKA
OF
THE
two photoreceptor cells in Petromyzon mnriwus are in fact both cones.
:i!$
LAMPREY
are of the same t,ype, and that they
ACBXOWLEDGMESTS
The authors would like to express their appreciation to Dr John Youson of the Departof Zoology, University of Toronto, for providing the vounp, parasitic adult, lamprevs used in t,his study. This resea.rch was supported by grants from the Xedical Research Council of Cauad;l and the Canadian National Institute for the Blind, out of the E.A. Baker Foundation +‘()I the Prevention of Blindness. ment,
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A. F. (1951). Properties of pigment layer factor r’hem. 193 5”5-31. Borwein. R. an)d Gollenberg, M. J. (1973). The photoreceptors of the ” E’ our-eyed” fish, dscchl~ps nncrbleps L. J. Xorphol. 140, 40542. Brown, I’. R., Gibbons, I. R. and Wald, G. (1963). The visual cells and visual pigments of the mudpuppy, Secturus. J. Cell Biol. 19, 79-106. Hunt. A. H. (1978). Fine structure and radioautography of rabbit photorecept,or cells. ZIL~.~SI. Ophthdmol. 17, Y&104. (lohen. A. (1970). Further studies on the question of the patency of saccr~les in outer seemerits of vertebrate photoreceptors. Fis. Res. 10, 445-53. (‘ohen. A. 1. (1972). Rods and cones, In Handbook of Sensory Physiology l-01. r-IZ/?, Phjysiology of f>hotoreceptor Organs (Ed. Fuortes. $1. G. F.) Springer-Verlag. Kety York. 1)ickson. D. H. and Hollenberg, M. J. (1971). The fine strncture of the pigment epithelium and photoreceptor (tells of the newt, Triturus airirfescens dorsnlia (Rafinesque). b. Morph,ol. 135, 389-43’~ . 1. Dickson. D. H. and Collard, T. R. (1979) Retinal development in the lamprey (Pdrow!lm,c morinus L.): premetamorphic ammocoete eye. An/. J. &at. 154, 321-36. Dow-ling. ,J. E. and Werblin, F. S. (1969). Organization of retina of the mlcdpuppy, Nechrus ~~nncuZo.sus. 1. Synaptic structure. J. Neurophysiol. 32, 315-38. Duke-Elder, S. (1958). The eyes of cyclostomes. In Systetn oj” Ophthm~rno~ogy. 1.01. 1. 3Cy iu &*olution. Pp. 259-72. Nosby Co.. St. Louis. Evalls. E. M. (1966). On the ultrastructure of the synaptic region of the visual receptors in wrhiu vcrtebmtes. 2. ZelZforsch. 71, 499p51fi. Fineran. K. A. and Sicol, J. A. C. (1974). Studies on the ryes of Sew Zealand parrot-fishes (Labridae). t’roc. El. Sot. Land B. 186, 21747. Hog;Ln. M. 1. and \l’ood, I. (1974). Phagocyt’osis by pigment) epithelium of human retinal (‘ones. il’ntwre, (Lo&on) 252, 305-6. Holrnberg, K. and Ohman, P. (1976). Fine structure of retinal synaptic organelles in lamprey and hagfish photoreceptors. Vis. Res. 16, 237- 9. Holmbng, K., Ohman P. and Dreyfert’, T. (1977). ERG-recording from the retina of the river lamprey (Lnmpetm ,j7wintilis). I’is. Res. 17, 715-17. Keefi!. J. R. (1971). The fine structure of the ret,ina in the Newt. Trifurus r,iridescrns. .I. Exp. ZOOI. 177, m-93. Kletsrekoper, H. (1972). The sense organs. In Th.e Hiology sf Lnnlpreys (Eds Hard&y. 11. iv. and Pot,ter. I. (1.). Academic Press, London. Lasansky, A. (1973). Organization of the outer synaptic layer in the retina of the larval tiger salamander. Phil. Tsans. Roy. Sot. (Lond.) Ser. B. 265, 417-89. Leuanberger. $2.. Demolis, Ph. and Keller, G. (1978). Cytoplasmic variations in pigment, epithelium (PE) during cyclic lighting. Invest. Ophthal. Suppl. 1978, p. 135, no. 6 (Abst.) Marshall, J. and Ansell, I?. L. (1971). Membranous inclusions in the retinal pigment epithelium: pha.goxometi and myeloid bodies. J. Amt. 110, 91-104.
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K. F. and Dowling. .1. E. (1970). Intrwrllulur responses of the i\ldler ((klial) cvlls 01’ I hr. mudpuppy retina: their relat,ion to h-warr of t hc rlectrol~tinoRlan1. -1. Se~oph!yskd. 33, :1:3- 41. Nilsson, S.-E. G. (1%X). The ultrastructure of the rweptor outer scgmcwts in the: ret,ina of’ I IN’ leopard frog, Hnru~ p@iens. J. I%rcrst. Res. 12, 207-Z< 1. Nguyen-T,egros, J. (1975). .A propos ties Corps My&loides de l,‘~pit.h~lium Pignwntairca tL(* Ia R&tine des Vert6brks. J. Ciltmst. 12esch. 53, Is?-63. Ohman. P. (1971). The photoreceptor outer segments of the river lamprey (Lnmpetm ,jfzwkrtili.s). An &&on-. fluorescenceand light, microscopic study. Actrc Zoologicrc 52, %7- 97. Ohmun, L’. (1976). Fine structure of photorecept,ors and associated neurons in the retina of Lnncpetrtc Jluviutilis (Cyclostomi). Via. Res. 16, 659-ti. Pet,erson. P. 4. (1971). Studies on the intern&w between prealbumin, retinolhinding protein ant1 vitamin A. J. Binl. Chem. 246, 449. Port,er, K. R. and Ynmada, E. (1960). Studies on endoplssmic reticulum (ER). V. 1 ts fornl ant1 differenti:Aon in pigment epithelial cells of the frog ret.ina. J. Hiophys. Biochenj. C’ytol. 8, 181--‘05. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 20%ItJ. Stell. W. K. (197%). The morphological organization of t,he vertebrate retina. In IIa~adbool~ of Memory Physiology Vol. VII/Z, Physiology of Photoreceptor 0rqnm.s (Ed. Fuort,es, M. (:. I’.). Springer-Verlag. New York. Tonus, J. (:. and Dickson, D. H. (1979). Neuro-glial relationships at, the external limit,ing mww branc of t,he newt retina. Exp. Eye RN. 28, 93-100. Uga, S. and Smelser, (:. K. (1973). Comparative study of the fine structure of retinal Miiller r,rllx in various vertebrates. Iwest. O~hthnlmol. 12, 434-48. Villeges, C:. JI. (1960). Electron microscopic study of the vertebrate retina. J. Cen. Physiol. 43, 15-43. Walls, G. I,. (1935). The visual cells of lampreys. Br. .J. Ophthnlmd. 19, 129-48. Yacob, A., Wise, C. and Kung, Y. W. (1977). The accessory outer segment of rods and cones in the retina of the guppy, Poe& reticulntrr P. (Teleostei). Cell Tiss. Rrs. 177, 181-93. Young, R. 1%‘. (1967). The renewal of photoreceptor outer segments. J. Cell Viol. 33, 61-71. Young. R. W. (1969). The organization of vertebrate phot,oreceptor cells. In The Ret&au: dlorphology, F’unction, rrnrl C’linicnl Chnrncteristics (Eds Straatsma, 1%. It.. Hall, R1. 0.. Alleu, R. 4. and Crescitelli. F.). Univ. of CaLfornia Press, Berkeley and Los Angeles. Young, R. 12:. (1971). The renewal of rod and cone outer segments in the rhesus monkey. .J. ( ‘rll Bid. 49, 303-18. Young. R. IV. (1976). Visual cells and the concept of renewal. Imtsf. Ophthnlwol. 15, 7OO-m2fi.