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Morphological correlations to an electrical resistance barrier in the eye of the locust Schistocerca… Simpson, William Wilson 1976

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MORPHOLOGICAL CORRELATIONS TO AN ELECTRICAL RESISTANCE BARRIER IN THE EYE OF THE LOCUST SCHISTOCERCA GREGARIA by WILLIAM WILSON SIMPSON B . S c , U n i v e r s i t y of Ottawa, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1976 William Wilson Simpson, 1976 MASTER OF SCIENCE i n In presenting th i s thes is in par t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t i on of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. William W. Simpson, B.Se-. M.Sc. ZOOLOGY Department of _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 June 2/76 Date i ABSTRACT The b a s i c morphology and u l t r a s t r u c t u r e o f the compound eye of the l o c u s t S c h i s t o c e r c a g r e g a r i a , has been i n v e s t i g a t e d . M o r p h o l o g i c a l l y the l o c u s t eye was found to be s i m i l a r to t h a t observed by Horridge (1966) and to those of other i n s e c t s such as the honey-bee drone, Apis m e l l i f e r a ( P e r r e l e t , 1970). U l t r a s t r u c t u r a l l y , t h i s i n v e r t e b r a t e sense organ suggests t h a t there i s l i t t l e e x t r a c e l l u l a r space between the c e l l s o f an ommatidium w i t h i n the r e t i n a , as shown by the presence o f d i f f e r e n t i -ated plasma membranes i n the form of narrow gap j u n c t i o n s . This type o f membrane d i f f e r e n t i a t i o n may be r e l a t e d to the demonstrated e x i s t e n c e of a r e t i n a l r e s i s t a n c e b a r r i e r (Shaw, 1975). This suggests t h a t the p h y s i o l o g i c a l l y demonstrated b a r r i e r may r e q u i r e only minor s t r u c t u r a l a l t e r a t i o n s w i t h i n the r e t i n a . I t i s suggested t h a t the b a r r i e r may be the same i n other i n v e r t e b r a t e s . 11 TABLE OF CONTENTS U l t r a s t r u c t u r e of the eye of the l o c u s t S c h i s t o c e r c a g r e g a r i a (ForskSl) I n t r o d u c t i o n : M a t e r i a l s and Methods: L i g h t and E l e c t r o n Microscopy R e s u l t s : L i g h t Microscopy: General Anatomy of the Locust Eye E l e c t r o n Microscopy: I . General Anatomy and Fine S t r u c t u r e o f the Outer P o r t i o n of the Eye I I . S p e c i a l i z e d Junctions and P o s s i b l e R e s t r i c t i o n s to the E x t r a c e l l u l a r Space i n the R e t i n a D i s c u s s i o n : B a s i c U l t r a s t r u c t u r e i n R e l a t i o n to the Presence o f an E l e c t r i c a l Resistance B a r r i e r References: i i i PLATE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 LIST OF PLATES DESCRIPTION PAGE A schematic diagram o f a l o n g i t u d i n a l g s e c t i o n through an ommatidium of the eye of j3. g r e g a r i a . L i g h t micrograph of the eye and a s s o c i a t e d c e l l types i n S. g r e g a r i a . L i g h t micrographs of the c r y s t a l l i n e 22 c o n e - c e l l area. L i g h t micrographs o f the basement- ^ 4 membrane area. L i g h t micrographs o f the f u s i o n of the c r y s t a l l i n e cones w i t h the rhabdom. Scanning e l e c t r o n micrographs of the l o c u s t eye i l l u s t r a t i n g the surface topography. E l e c t r o n micrographs of the c o n e - c e l l region and an ommatidium. E l e c t r o n micrographs o f the c o n e - c e l l area showing j u n c t i o n a l complexes present. Microdensitometer t r a c i n g of P l a t e 10-D. 18 Low power l i g h t micrographs o f the receptor 20 c e l l s i n l o n g i t u d i n a l s e c t i o n . L i g h t micrographs at the l e v e l o f the 2 2 basement-membrane area. 26 29 31 33 E l e c t r o n micrographs of the same area as 3 5 t h a t of P l a t e 10 showing another j u n c t i o n a l complex. E l e c t r o n micrographs o f ommatidia i n 3 9 c r o s s - s e c t i o n . E l e c t r o n micrographs o f the ommatidia i n 4 3 l o n g i t u d i n a l s e c t i o n showing j u n c t i o n a l complex. iv DESCRIPTION Microdensitometer tracing of Plate 14-C. Electron micrographs of junctional complex in the middle area of the ommatidial region between receptor c e l l s . Microdensitometer tracing of Plate 16-B. Electron micrograph of the junctional complex at the level of the eccentric-cell nuclei in the ommatidial region. Microdensitometer tracing of Plate 19-B. Electron micrographs of receptor-axon bundles at the level of the basement membrane. Electron micrograph of two axon bundles in cross-section at the level of the basement-membrane. Same as that of Plate 21 showing the junctional complex between axons and g l i a l - c e l l processes. Microdensitometer tracing of Plate 22-A. Electron micrographs of the junctional complexes between axons below the basement membrane. Microdensitometer tracing of Plate 24-C. Electron micrographs of gap between adjoining c e l l membranes. Microdensitometer tracing of Plate 26-C. Electron micrographs of another area below the basement- membrane. Microdensitometer tracing of Plate 28-B. Electron micrograph of an axon below the basement-membrane in cross section. V PLATE DESCRIPTION PAGE 31 Same area as that of Plate 30 at higher 7 9 magnification showing extracellular space between axons and g l i a l c e l l s . 32 Microdensitometer tracing of Plate 31-A. 81 33 Electron micrographs at the level below 83 the basement membrane. 34 Same area as that of Plate 33 to show 85 extracellular space present. 35 Diagrams of the morphology and ultrastructure 91 of the eye and associated c e l l types and junctions. 36 Diagram to il l u s t r a t e the barrier in the eye 95 with corresponding morphological landmarks. v i ABBREVIATIONS USED IN PLATES CELL TYPES: c r y s t a l l i n e cone g l i a l c e l l pigment c e l l ... primary pigment c e l l receptor c e l l secondary pigment c e l l ULTRASTRUCTURAL FEATURES: ax axon axb r e c e p t o r - c e l l axon bundle bm basement membrane ch corneal h a i r cor cornea cp c r y s t a l l i n e >cone-cell process ds desmosome en e c c e n t r i c - c e l l nucleus es e x t r a c e l l u l a r space f facet gcp g l i a l - c e l l process Gj gap junction gly glycogen granules gn g l i a l - c e l l nucleus i c intracytoplasmic membrane i s i n t r a c e l l u l a r space Lam lamina lhc large hemolymph channel mi mitochondria mt microtubules mv m i c r o v i l l i nt neurotubules om ommatidia pg pigment granules ppn primary pigment-cell nucleus r c a r e c e p t o r - c e l l axon rcn r e c e p t o r - c e l l nucleus rh rhabdom she small hemolymph channel spn secondary pigment-cell nucleus t r tracheole um unit membrane V v e s i c l e (a pigment granule missing) cc gc pc ppc rc spc v i i ACKNOWLEDGEMENTS I would l i k e t o thank Dr. S. R. Shaw f o r h i s s u p e r v i s i o n o f t h i s research and a s s i s t a n c e i n the pr e p a r a t i o n of t h i s t h e s i s ; and f o r suggesting the germ o f the idea . I would l i k e to thank Dr. A. B. Acton f o r h i s teaching me the theory of E l e c t r o n Microscopy and Mr. L. Veto f o r h i s ear and shoulder to c r y on, not to mention h i s a s s i s t a n c e i n photography and operation o f the E l e c t r o n Microscope, without which t h i s t h e s i s would not be a r e a l i t y . I would a l s o l i k e to thank Dr. T. Bradley f o r h i s h e l p f u l sugges-t i o n s , Mr. J . Spring f o r h i s astounding i n t e r e s t i n t h i s p r o j e c t , and Mrs. J . Ma r t i n f o r her a s s i s t a n c e i n the pr e p a r a t i o n of t h i s t h e s i s . 1 INTRODUCTION One l i n e of i n v e s t i g a t i o n of the mechanisms of nervous i n t e g r a t i o n w i t h i n the arthropod v i s u a l system uses e l e c t r o - p h y s i o l o g i c a l techniques to record and analyze complex neuronal responses. Another important approach i s based on the study of measurable behavioural responses to p a r t i c u l a r s t i m u l i . Such methods r e q u i r e a s o l i d framework of anatomical knowledge upon which to base models of v i s u a l nervous i n t e g r a t i o n . When d e a l i n g w i t h compound eyes, a b a s i c need i s to have a c l e a r p i c t u r e o f the r e t i n a i n the f i r s t p l a c e and of the way i n which i t s b a s i c u n i t s , the ommatidia, are f u n c t i o n a l l y interconnected a t the successive stages i n the v i s u a l pathway. The major organs of l i g h t p e r c e p t i o n i n arthropods are the compound eyes, i n which an ommatidium i s the b a s i c anatomical u n i t or b u i l d i n g b l ock (Smith, 1968; Goldsmith & Bernard, 1974). Each ommatidium con-s i s t s of a polygonal group of u s u a l l y e i g h t receptor or r e t i n u l a c e l l s w i t h rhabdomeres extending toward the centre o f the group forming a rhabdom (Goldsmith & Bernard, 1974). A rhabdomere forms along the sur-face o f the r e t i n u l a c e l l and c o n s i s t s of a c l o s e l y packed array of m i c r o v i l l i which extend from the c e l l ' s surface (Goldsmith & Bernard, 1974). Rhabdomere development has been s t u d i e d i n the f r u i t f l y , D r o s o p h i l a , where the work of Perry (1968) i n d i c a t e s t h a t the rhabdomeres a r i s e by e l a b o r a t i o n and i n f o l d i n g of the plasma membrane. Evidence (Varela & P o r t e r , 1969; Goldsmith & Bernard, 1974) i n d i c a t e s t h a t the rhabdomere contains pigments necessary f o r v i s u a l t r a n s d u c t i o n . The p a r t i c u l a r anatomical arrangement of the receptor c e l l s may suggest an 2 important r o l e i n p o l a r i z e d l i g h t d e t e c t i o n (Shaw, 1966, 1967, 1969b; Kennedy & B a y l o r , 1961), but whatever t h e i r f u n c t i o n , t h e i r axons convey in f o r m a t i o n towards a v i s u a l centre i n the b r a i n . Each compound eye i s composed o f many ommatidia, anat o m i c a l l y i d e n t i -c a l to each other, although they change t h e i r o r i e n t a t i o n w i t h respect to an a r b i t r a r y plane. Surrounding the r e t i n u l a c e l l s i s a sheath of non-nervous pigment c e l l s (Goldsmith & Bernard, 1974) which serves t o i s o l a t e s t r u c t u r a l l y and probably o p t i c a l l y the d i f f e r e n t ommatidia. Each ommatidium has a d i o p t r i c apparatus c o n s i s t i n g of a c u t i c u l a r lens and a c r y s t a l l i n e cone (Roonwall, 1947; Smith, 1968; P e r r e l e t , 1970) ( r e f e r to P l a t e 1 ) , the l a t t e r making d i r e c t contact w i t h the rhabdom. The lenses are modified c u t i c l e and an array of lenses gives a compound eye i t s faceted appearance (Goldsmith & Bernard, 1974). The c r y s t a l l i n e cone i s composed o f four c e l l s , the Semper c e l l s ( P e r r e l e t , 1970; Gold-smith & Bernard, 1974) and provides a medium f o r the tra n s m i s s i o n o f l i g h t from the c u t i c u l a r l ens to the rhabdom. The system i s thought to c o l l e c t l i g h t from i t s v i s u a l f i e l d and convey i t to the c y l i n d r i c a l group o f r e t i n u l a c e l l s or primary sensory neurons of the ommatidium (Smith, 1968). The rhabdomeres of each receptor fuse to form a c i r c u l a r c e n t r a l rhabdom i n many but not a l l i n s e c t s . The d i o p t r i c apparatus o f the l o c u s t , S c h i s t o c e r c a g r e g a r i a (Forsk§l), l i k e t h a t of the honey bee drone, Apis m e l l i f e r a (Higgins) ( P e r r e l e t , 1970), focuses the i n c i d e n t l i g h t on the rhabdom. At the base of the r e t i n a , each r e t i n u l a c e l l of an ommatidium elongates i n t o an axon t h a t p i e r c e s the basement membrane and synapses w i t h second order neurons i n a sy n a p t i c r e g i o n c a l l e d the lamina. 3 Around a c t i v e neural t i s s u e s there are e x t r a c e l l u l a r e l e c t r i c a l f i e l d s which may be b a r r i e r s of high r e s i s t a n c e i n the e x t r a c e l l u l a r space (Burtt & Catton, 1964; Heisenberg, 1971). The r e s i s t a n c e p r o f i l e s of the eye of S_. g r e g a r i a (Shaw, 1975) i n d i c a t e t h a t a b a r r i e r of hi g h e l e c t r i c a l r e s i s t a n c e does e x i s t i n the e x t r a c e l l u l a r space around the photoreceptor axons, and i n order to i n t e r p r e t such p r o f i l e s t h i s space must be a small f r a c t i o n o f the t o t a l eye volume (Ruck, 1964; Krebs et a l . , 1975; Shaw, 1976). In h i s paper, Shaw (1975) discussed the e l e c t r o -p h y s i o l o g i c a l e f f e c t o f such a b a r r i e r upon the r e t i n a l c u r r e n t s , suggesting t h a t the neurons i n the compound eye i n t e r a c t w i t h each other as a consequence of the e x i s t i n g s t r u c t u r e . This b a r r i e r sets up a system of e x t r a c e l l u l a r , p r e s y n a p t i c l a t e r a l i n h i b i t i o n (Shaw, 1975) between receptor c e l l s by d i v e r t i n g receptor current from v i s u a l l y a c t i -vated c e l l s through a r e t u r n c i r c u i t of l e s s a c t i v e neighbouring recep-t o r c e l l s , i n s t e a d of r e t u r n i n g through the e x t r a c e l l u l a r space. Recent evidence (Shaw, 1976) supports the proposed b a r r i e r model i n t h a t the r e t i n a i s i n f a c t sealed o f f from the hemolymph as d i f f u s i o n of substances i n t o the eye i s retarded. I t i s my i n t e n t i o n i n t h i s t h e s i s to examine whether or not such a b a r r i e r i s anatomically present. The approach used i n t h i s study was to i n v e s t i g a t e the morphology and u l t r a s t r u c t u r e of the eye of the l o c u s t S_. g r e g a r i a . I t was hoped t h a t such an i n v e s t i g a t i o n might i n d i c a t e the p a r t i c u l a r m o d i f i c a t i o n s i n s t r u c t u r e o f the plasma membranes which might i n t u r n demonstrate the presence of a r e t i n a l e l e c t r i c a l r e s i s t a n c e b a r r i e r and thereby provide i n f o r m a t i o n and supporting evidence f o r the p r e v i o u s l y proposed physio-l o g i c a l model (Shaw, 1975). Since t h i s organ has not been s t u d i e d 4 u l t r a s t r u c t u r a l l y w i t h regard to the presence of membrane j u n c t i o n a l complexes l e a d i n g to the proposed b a r r i e r , t h i s aspect was examined i n the study. S_. g r e g a r i a was chosen s i n c e the p h y s i o l o g i c a l data previous-l y obtained (Shaw, 1975) was a v a i l a b l e and there are many reviews p e r t a i n i n g to arthropod v i s i o n (Roonwall, 1947; Fernandez-Moran, 1958; Horridge & Barnard, 1965; Horridge, 1966; Northrop & Guignon, 1970). 5 MATERIALS AND METHODS Mature female locusts (Schistocerca gregaria Forskal) which had been reared on a diet of lettuce, bran, powdered milk, yeast and dried grass, were used in this study. These animals were maintained at 28±1°C and at a relative humidity of 50% on a 12 hour dark-light cycle (Phillips & Beaumont, 1971). Light-adapted eyes were removed from the l i v i n g insect by swift decapitation, and transferred immediately to the f i x -ation f l u i d , where further dissection and subdivision of the compound eyes into small segments was carried out. After, two hours at room temp-erature, these segments were then transferred to phosphate buffer for storage prior to dehydration and i n f i l t r a t i o n . The osmotic concentra-tions of a l l solutions used were adjusted so as to approximate that of the eye's f l u i d (370 mOsmol). A Biological Cryostat/Nanoliter Osmometer (Clifton Technical Physics, N. Y., U. S. A.) was used for this procedure. The fixative consisted of 2.5 to 3% glutaraldehyde in 0.34 M phos-phate buffer (354 mOsmol) at room temperature. A one hour period of postfixation at room temperature in 1% OsO^ was followed by a period of dehydration in ethanol, propylene oxide, and a f i n a l stage of i n f i l t r a -tion, with Epon 812 embedding medium. Thick (0.5-1.0 ym) and thin (50-60 nm) sections were cut, using glass knives on a Sorvall (Porter-Blum) MT-2 ultramicrotome. Thick sections were stained with 1% toluidine blue (Horridge & Barnard, 1965) in 1% borax for light microscopic examination. Thin sections were stained in alkaline lead citrate (Reynolds, 1963; Karnovsky, 1961) and additionally treated with uranyl acetate solution in 70% methanol (Watson, 1958; Kellenberger et a l . , 1958) before being 6 examined w i t h H i t a c h i HS-7S and Z e i s s EM-10 e l e c t r o n microscopes. In many cases where j u n c t i o n s between c e l l s were examined, the space between c e l l s i n the micrographs' was very s m a l l . To make objec-t i v e measurement of the gap width some of the negatives were examined w i t h a scanning microdensitometer (Joyce-Loebl type) (Endo, 1964; P e r r e l e t , 1970). Measurements were made w i t h a f a i r l y wide c o l l e c t i n g aperture o f 100 um, but c o n t r o l s i n d i c a t e d t h a t t h i s width d i d not appre c i a b l y downgrade the r e s o l u t i o n of the membrane elements. The . measurements o f the e x t r a c e l l u l a r space from the microdensitometer t r a c i n g s were made from peak t o peak i n each t r a c i n g between two u n i t membranes. 7 RESULTS - LIGHT MICROSCOPY: It i s necessary at this time to familiarize the reader with the general anatomy of the locust eye for the purpose of setting the scene for the detailed ultrastructure which follows in the electron microscopy section. The outermost portion of the ommatidia consists of a cuticular transparent layer, the cornea (Plate 2) . In longitudinal section two layers are seen: an outer, thinner unlaminated layer (6 ym) and an inner, thicker layer (120 ym), irregularly laminated. Lying beneath the cornea is a region of long, slender structures called the crystalline cones (ca. 20 ym wide at the base tapering to 4 ym at the apex, with length 100 ym). The cones vary in dimensions according to the size of the underlying ommatidium (Plate 3-A,B) and extend down into the retina, passing through a region of secondary pig-ment cells and abut directly to the rhabdom. Surrounding the apex of the crystalline cone are the two large oval nuclei of the Semper cells (about 7 ym in diameter). Proximally they abut to the distal ends of the retinula-cell. rhabdomeres while di s t a l l y their outer borders slope inwards to meet the crystalline cones (Plate 3-A,B; 5-A,B). Encircling the crystalline cones and retinulae of the ommatidia are many elongate secondary pigment cells which have long, slender nuclei (ca. 4 ym in diameter). These cells extend from the cornea in the cone-cell region, inward to- the middle of the retina, a distance of 210 ym from the top of the eye (Plate 3-A). Distally near the base of the crystalline cones, 8 A schematic diagram (adapted from Fernandez-Moran, 1958) o f a l o n g i t u d i n a l s e c t i o n through an ommatidium w i t h a p p o s i t i o n type image forma-t i o n showing the rhabdom (rh) medial to the receptor c e l l s ( r c ) ; cc, c r y s t a l l i n e cone; f, f a c e t or corneal l e n s ; pc, pigment c e l l ; r c n , r e c e p t o r - c e l l nucleus; r c a , r e c e p t o r - c e l l axon. 9 10 P l a t e 2 A l i g h t micrograph showing a l o n g i t u d i n a l sec-t i o n through the l o c u s t eye. Note the array of receptor c e l l s , i . e . , the ommatidia (om), as they t r a v e r s e the r e t i n a and p i e r c e the basement membrane (bm) t r a v e l l i n g through the lamina (lam), en route t o the o p t i c lobe i n the b r a i n (cor, cornea; cc, c r y s t a l l i n e cone). 1 cm = 62.5 ym 12 Plate 3-A A low magnification light micrograph of the cone-cell region showing the crystalline cones (cc) where each fuses with the rhabdom of an ommatidium (om). Note also the presence of numerous pigment-cell nuclei (spn, ppn). In this particular section the cornea has been removed. 1 cm = 35 ym Plate 3-B A light micrograph at a higher magnification of the same area as that of Plate 3-A showing a longitudinal section of the crystalline cone (cc) and the pigment-cell nuclei (ppn, spn). Note the distribution of the pigment granules (pg) which are particularly dense near the junction between the cone and rhabdom (arrow). This possibly serves to minimize the lat e r a l scattering of light at the junction. 1 cm = 8.3 ym 14 P l a t e 4-A L i g h t micrograph o f the basement-membrane (bm) region showing the r e c e p t o r - c e l l axon bundles (axb) and the blood channels (he) i n l o n g i -t u d i n a l s e c t i o n . Note the d i s t r i b u t i o n and abundance of pi g m e n t - c e l l granules (pg) con-t a i n e d i n slender g l i a l c e l l s . 1 cm = 10.4 ym P l a t e 4-B L i g h t micrograph under phase c o n t r a s t showing the l a r g e blood channels (lhc) and a s s o c i a t e d g l i a l - c e l l n u c l e i (gn) i n l o n g i t u d i n a l s e c t i o n . Note the axon bundle (axb) p i e r c i n g the base-ment membrane. 1 cm = 24.4 ym 1 5 1 6 the secondary pigment c e l l s are c l o s e l y packed. The r e t i n u l a , which forms the p o s t e r i o r p o r t i o n o f an ommatidium (Roonwall, 1947), l i e s proximal to the c r y s t a l l i n e cone and appears t o be composed o f a c e n t r a l rod or rhabdom (2 ym i n diameter), surrounded by a group o f receptor o r r e t i n u l a c e l l s ( P l a t e 5-B). The rhabdom runs the e n t i r e length of an ommatidium and terminates a short distance above the basement membrane (Plate 7-B). In a d i s t a l s e c t i o n , there appears t o be a polygonal group of c e l l s comprising each ommatidium (Plate 5-B), surrounded by numerous pigment c e l l s c o n t a i n i n g granules (ca. 0.8 ym i n diameter) and the receptor c e l l s surround the rhabdom i n a r o s e t t e - l i k e f a s h i o n ( P l a t e 5-B; 6-B). The ommatidium o f the l o c u s t contains s i x o r seven r e t i n u l a c e l l s which bear rhabdoms (Bennett, T u n s t a l l & Horridge, 1967). P l a t e 5-B, i n oblique cross s e c t i o n , shows the c r y s t a l l i n e cone meeting and f u s i n g w i t h the rhabdom o f an ommatidium. The t i g h t screen o f pigment granules i s c l e a r l y seen and the primary p i g m e n t - c e l l n u c l e i are seen i n t h e i r r e l a t i o n s h i p w i t h the r e t i n u l a . D i s t a l l y the rece p t o r c e l l s converge inwards producing a dome-like appearance, w h i l s t p r o x i m a l l y they reach down to continue across the basement membrane. The length of the receptors t o the basement membrane i s ca. 440 ym (Plate 2). An array of pigment granules covers the outer borders o f the receptor c e l l s as they t r a v e r s e the r e t i n a from top to bottom. The granules appear to be denser at the top of the eye and taper o f f as the basement membrane i s approached ( P l a t e 2; 6-A; 7-A,B). Approximately 160 ym above the basement membrane l o n g , o v a l , s l e n d e r n u c l e i a l i g n the outer borders o f the rece p t o r c e l l s composing an omma-ti d i u m (4 ym i n diameter) (Plate 6-A; 7-A,B). These n u c l e i are thought 17 to be those o f " e c c e n t r i c c e l l s " which extend to the basement membrane and are thought to t r a v e r s e the l a t t e r but whose f u n c t i o n i s not under-stood. P e r r e l e t (1970) s t a t e s t h a t i n the honey-bee drone there i s a s i m i l a r c e l l type c a l l e d the b a s a l r e t i n u l a c e l l . The basement membrane (ca. 10 ym wide) appears t o be very i r r e g u l a r l y s t r i a t e d and non c e l l u -l a r ( P l a t e 2; 4-A,B; 7-A,B), and i s p e r f o r a t e d by the r e c e p t o r - c e l l axons as they pass through i n bundles as w e l l as by a s s o c i a t e d tracheae. As the axons continue down i n t o the o p t i c lobe they decussate below the basement membrane forming a meshwork of nerve f i b e r s . A t t h i s l e v e l below the basement membrane pigment granules (1 ym i n diameter) are a l s o observed i n the g l i a l c e l l s ( Plate 4-A; 6-A; 7-A,B). They are b e l i e v e d to f u n c t i o n i n ca p t u r i n g s c a t t e r e d l i g h t or l i g h t t h a t remains unabsorb-ed by the rhabdom, t h a t would normally r e - e n t e r the r e t i n a and l e a d t o improper v i s u a l i n f o r m a t i o n p r o c e s s i n g (Varela & P o r t e r , 1969). Below t h i s densely pigmented area i s a row of l a r g e o v a l p a r a l l e l hemolymph (blood) channels which cross under the eye about 75-100 ym below the basement membrane i n an a n t e r i o - p o s t e r i o r d i r e c t i o n between the bundles of receptor c e l l axons, but do , not b i f u r c a t e and enter the r e t i n a . • They average 36 ym i n diameter and are surrounded by densely packed g l i a l - c e l l n u c l e i (ca. 7 ym i n diameter) (Plate 4-B), clumped as a r e -s u l t o f the nerve f i b e r s p a s s i n g around each blood channel en route to the o p t i c lobe i n the b r a i n . These blood channels are thought, from the r e s u l t s o f the dye t r a c e r experiments o f Shaw (1976) t o be extensions of the hemolymph. Roonwall (1947) i d e n t i f i e d these channels i n c o r r e c t l y as l a r g e tracheae, w h i l e Northrop and Guignon (1970) named these c o r r e c t -l y as blood channels. The f e n e s t r a t e d appearance below the basement 18 L i g h t micrograph of the cone c e l l r e g i o n i n l o n g i t u d i n a l s e c t i o n showing the r e l a t i o n s h i p of the cone c e l l (cc) w i t h the two primary pigments-cell n u c l e i (ppn) a b u t t i n g the r e t i n -u l a . The rhabdom (rh) i s v i s i b l e at t h i s l e v e l i n some of the ommatidia. 1 cm = 6.9 um L i g h t micrograph o f the c o n e - c e l l r e g i o n merging w i t h the ommatidial r e g i o n i n an oblique cross s e c t i o n . Note the t r a n s i t i o n as the c r y s t a l l i n e cone (cc) meets the rhabdom ( r h ) , and the l a r g e primary pigment-c e l l n u c l e i (ppn) at t h i s l e v e l . Cross s e c t i o n s of the ommatidia (om) show the r e -ceptor c e l l s (rc) and the a s s o c i a t e d pigment granules (pg) i n t h e i r cytoplasm and t o a l e s s e r extent i n the c e l l s surrounding them. 1 cm = 7.5 ym 20 A low power l i g h t micrograph showing the r e -ceptor c e l l s (rc) i n l o n g i t u d i n a l s e c t i o n t r a v e r s i n g the eye and p i e r c i n g the basement membrane (bm). Note the blood channels (he) and the pigment granule (pg) d i s t r i b u t i o n below the basement membrane (en, " e c c e n t r i c -c e l l " n ucleus). 1 cm = 83 ym Same as t h a t of P l a t e 6-A except at a higher m a g n i f i c a t i o n showing more c l e a r l y the p i g -ment granule (pg) d i s t r i b u t i o n above the basement membrane and i n a s s o c i a t i o n w i t h the receptor c e l l s . 1 cm = 33 ym 21 22 P l a t e 7-A Low power l i g h t micrograph showing the recep-t o r c e l l s (rc) i n l o n g i t u d i n a l s e c t i o n p i e r c -i n g the basement membrane (bm). Note the presence o f the " e c c e n t r i c - c e l l " n u c l e i (en) and the blood channels (he) w i t h a s s o c i a t e d t r a c h e a ( t r ) . 1 cm = 27.7 ym P l a t e 7-B Same as P l a t e 7-A except a t higher magnifica-t i o n showing the " e c c e n t r i c - c e l l " n u c l e i (en) bordering the receptor c e l l ( r c ) . Note the d i f f e r e n c e i n the d i s t r i b u t i o n of pigment granules (pg) above and below the basement membrane (bm), and the presence o f the rhabdom (rh). 1 cm = 8.3 ym 23 24 membrane (ca. 8 ym wide) i s thought to be caused by numerous s m a l l blood channels and a s s o c i a t e d tracheoles (Plate 6-A; 7-A). 25 ELECTRON MICROSCOPY: I. General Anatomy and Fine S t r u c t u r e o f the Outer P o r t i o n of the Eye: The f i r s t p a r t o f t h i s s e c t i o n of the r e s u l t s obtained using the e l e c t r o n microscope i s an i n t r o d u c t i o n t o , and a general account o f , the u l t r a s t r u c t u r a l anatomy o f the eye of S_. g r e g a r i a . The d i o p t r i c apparatus of the l o c u s t ommatidium i s formed by a c u t i c u l a r c o r n e a l f a c e t and a c r y s t a l l i n e cone. Examined by the scan-ni n g e l e c t r o n microscope, the compound eye of the l o c u s t , S_. g r e g a r i a , appears egg-shaped i n o u t l i n e ( P l a t e 8). In surface view ( P l a t e 8-B) the corneal lens i s dotted w i t h f i n e h a i r - l i k e extensions (ca. 3 ym long) s i m i l a r to those found i n other i n s e c t s , f o r instance bees ( P e r r e l e t , 1970). In the c o n e - c e l l region, the c r y s t a l l i n e cones are densely g r a n u l a t -ed and bordered by secondary pigment c e l l s (ca. 7 ym i n diameter) dense-l y packed together (Plate 9-A). Again, t h i s s t r u c t u r e resembles t h a t found i n other i n s e c t s , such as the honey-bee drone. The l a c k o f or g a n e l l e s i n cone c e l l s might i n d i c a t e t h a t glycogen p a r t i c l e s cannot be metabolized. Cone glycogen would t h e r e f o r e not have a metabolic f u n c t i o n but an o p t i c a l one, inasmuch as i t could c o n t r i b u t e to the r e f r a c t i v e index ( P e r r e l e t , 1970). The n u c l e i measured from 3 t o 6 ym i n diameter i n the h e a v i l y pigmented c e l l s . Most of the pigment granules had f a l l e n out, which i s a common occurrence w i t h e l e c t r o n microscopy f i x a t i o n (Shaw, personal communication). Some pigment granules were s m a l l , roundish and of uniform e l e c t r o n d e n s i t y which resembled those 26 Scanning e l e c t r o n micrograph of the eye surface of the l o c u s t S_. g r e g a r i a showing the curva-t u r e and egg-shape of the eye. 1 cm = 345 ym Scanning e l e c t r o n micrograph showing the sur-face topography of the eye. Each f a c e t (f) i s hexagonal i n a honeycomb array. Corneal h a i r s (ch) are v i s i b l e . 1 cm = 29.4 ym A micrograph of the same area as t h a t of P l a t e 8-B. The depression i n each f a c e t i s an a r t i f a c t of a i r d r y i n g the specimen. 1 cm = 11.9 ym Same area as' t h a t of P l a t e 8-C at a higher m a g n i f i c a t i o n . 1 cm = 4 um 27 28 present i n the d i s t a l cytoplasm of the r e t i n u l a c e l l s . The d i v i s i o n o f the c r y s t a l l i n e cone i n t o two separate Semper's c e l l s was observed ( P l a t e 9-A). Pigment c e l l s were observed t o be connected t o each other by a j u n c t i o n a l membrane complex, the gap j u n c t i o n (Plate 10-A,B,C,D). The width of the gap or nexus ( G i l u l a , 1974) was not r e s o l v e d at h i g h m a g n i f i c a t i o n . The microdensitometer t r a c i n g of the r e g i o n demonstrated th a t the i n t e r c e l l u l a r . s p a c e or gap measured 1.5 nm wide a t the most (Plate 11). S t r i k i n g features of the pigment c e l l s are t h e i r r i c h n e s s i n m a t e r i a l w i t h the appearance of glycogen (small black dots i n P l a t e 1 0 - A ) t h e presence of few mitochondria and of microtubules i n t h e i r cytoplasm (Baumann, 1975). These fea t u r e s together w i t h t h e i r c l o s e a s s o c i a t i o n w i t h the r e t i n u l a c e l l s and absence of apparent e x c i t a b i l i t y by l i g h t (Baumann, 1975) allow the pigment c e l l s t o be considered as the e q u i v a l e n t of g l i a l c e l l s i n other nervous s t r u c t u r e s , a l b e i t the func-t i o n of g l i a l c e l l s i s nowhere w e l l understood. These gap-junction s p e c i a l i z a t i o n s (Plate 10-B) have a l s o been seen i n the honey-bee drone ( P e r r e l e t , 1970). The pigment c e l l s are probably e l e c t r i c a l l y coupled, which, Baumann (1975) s t a t e s , could e x p l a i n why a l l pigment c e l l s respond s i m i l a r l y to i l l u m i n a t i o n o f the ommatidium even though only a s m a l l number of them i n t e r d i g i t a t e between r e t i n u l a c e l l s . In oblique s e c t i o n s of the j u n c t i o n a p a t t e r n of subunits, the gap-junction l a t t i c e i s seen ( P l a t e 10-D) (van Deurs, 1975). Another type of j u n c t i o n a l membrane complex between two g l i a l c e l l s ( P l a t e 12-A,B) was observed and appeared s i m i l a r t o a macula adhaerens i n t h a t the membranes appeared t o punctate at one p o i n t w i t h no apparent membrane f u s i o n (Goodenough & Revel, 1970). C h a r a c t e r i s t i c a l l y , dense 29 E l e c t r o n micrograph of l o n g i t u d i n a l s e c t i o n showing the c r y s t a l l i n e cone (cc) and pigment (or g l i a l ) c e l l arrangement. Note the l a r g e n u c l e i (spn) and v e s i c l e s formed as a r e s u l t of pigment granules (pg) f a l l i n g out and the r i c h glycogen (gly) content i n the pigment c e l l s (small b l a c k d o t s ) . The arrowhead shows the d i v i s i o n of the cone i n t o one of i t s four Semper 1s c e l l s . 1 cm = 1.4 ym An e l e c t r o n micrograph i l l u s t r a t i n g a cross s e c t i o n of a l o c u s t ommatidium i n the proximal p a r t of the r e t i n a . One sees c l e a r l y t h a t the ommatidium i s formed by e i g h t r e t i n u l a c e l l s (RC 1-8), each of which c o n t r i b u t e s m i c r o v i l l i t o the rhabdom. The ei g h t h receptor c e l l (RC 8) does not c o n t r i b u t e any m i c r o v i l l i to the rhabdom. The r e t i n u l a c e l l s are sur-rounded c l o s e l y by pigment c e l l s , i . e . acces-sory pigment c e l l s which sheath each ommati-dium from the basement membrane. Tracheoles are i n t e r s p e r s e d between the receptor c e l l s . 1 cm = 1.2 ym 30 31 P l a t e 10-A E l e c t r o n micrograph o f the c o n e - c e l l region i n l o n g i t u d i n a l s e c t i o n showing the pigment c e l l s (pc) and t h e i r n u c l e i (spn) i n r e l a t i o n s h i p to the c r y s t a l l i n e cones ( c c ) . Note the many pigment granules (pg) which are i n t a c t and those which have f a l l e n out. 1 cm = 2 ym P l a t e 10-B Same area as t h a t o f P l a t e 10-A. Note the ju n c t i o n s between the g l i a l c e l l s (arrowheads), and the glycogen deposits ( g l y ) . 1 cm = 1 ym P l a t e 10-C P l a t e 10-D Same area as t h a t o f P l a t e 10-B showing the gap j u n c t i o n between two g l i a l c e l l s (arrow-heads) . 1 cm = 100 nm Same as P l a t e 10-C showing a gap of l e s s than 1.5 nm. Note the gap-junction l a t t i c e o f the membrane as i t was cut at an angle (arrowhead) A microdensitometer t r a c i n g between the two dots was made (see P l a t e 11). 1 cm = 50 nm 32 33 P l a t e 11 A microdensitometer t r a c i n g across the two dots of P l a t e 10-D showing a gap width of 1.5 nm. 1 cm = 12 nm 34 35 High m a g n i f i c a t i o n e l e c t r o n micrograph demon-s t r a t i n g the membrane j u n c t i o n a l complex between two pigment c e l l s i n the c o n e - c e l l region of the l o c u s t eye. In t h i s p i c t u r e one sees p a r t s o f the two pigment c e l l s i n l o n g i t u d i n a l s e c t i o n . The cytoplasm o f the pigment c e l l s contains numerous pigment granules (pg). The j u n c t i o n a l complex i s a macula adhaerens (arrowhead). 1 cm = 588 nm Same area as t h a t of P l a t e 12-A showing a t higher m a g n i f i c a t i o n the j u n c t i o n a l complex. Note the presence of dense m a t e r i a l on the cytoplasmic surface of the j u n c t i o n and the microtubules (mt). The macula adhaerens a l s o d i s p l a y s some i n t e r c e l l u l a r m a t e r i a l between the apposed j u n c t i o n a l membranes (arrowhead). 1 cm = 79 nm 36 37 amorphous material was present on the cytoplasmic side of the junction. This macula adhaerens which i s a desmosomal structure and therefore of a supporting nature ( G i l u l a , 1974) also displayed some i n t e r c e l l u l a r material between the apposed ju n c t i o n a l membranes. The d i s c e r n i b l e d i s -tance between the membranes i s approximately 8 nm. Mitochondria appear-ed to vary somewhat i n s i z e (0.5-1 ym i n diameter) and were not numerous, but had the appearance of having been properly f i x e d . The mitochondria (Plate 12-A) are of s i m i l a r structure to those i l l u s t r a t e d by Baumann (1975) i n the honey-bee drone. The cytoplasmic ground substance appear-ed to be r e l a t i v e l y dense and the n u c l e i very large (ca. 5 ym i n diameter). Embedded i n these c e l l s and very concentrated were osmophilic granules (20-24 nm i n diameter) (Plate 12-B). The arrangement of r e t i n u l a c e l l s i s shown i n Plate 9-B. The r e t i n -u l a c e l l s were approximately t r i a n g u l a r i n shape (1-7 ym wide at the base tapering to ca. 100 nm at the apex), the top of the t r i a n g l e being s i t u -ated i n the centre of the ommatidium. Two of the eight c e l l s (Horridge, 1966) d i f f e r e d from the others owing to t h e i r c l e a r cytoplasm which contained only a small number of organelles. The mitochondria (approxi-mately 4 to 5 i n number) varied a l i t t l e i n s i z e (ca. 0.5-0.8 ym i n diameter), and were s i m i l a r to those of the cone-cell region (Plate 11-A). Some appeared improperly f i x e d . The small black dots were thought to be glycogen, s i m i l a r to that found i n Apis m e l l i f e r a (Baumann, 1975). Each receptor c e l l of the ommatidium contained a large mass of electron-dense material measuring ca. 2 ym i n diameter. As i n the cone-cell region there were large v e s i c l e s (ca. 0.8 ym i n diameter) which were believed to have contained pigment granules. V e s i c l e s , as defined by Sjostrand 38 (1956), are rounded o r s p h e r i c a l components bounded by a membrane and appear empty i n f i x e d and embedded specimens. The term " v e s i c l e " r e f e r s to t h e i r appearance and does not exclude the f a c t t h a t they may represent granules t h a t have been d i s s o l v e d during f i x a t i o n and embedding. More-over, numerous pigment granules were present which have been termed accessory pigment ( P e r r e l e t , 1970). They appeared as v e s i c l e s c o n t a i n i n g m a t e r i a l o f v a r i a b l e e l e c t r o n d e n s i t y . S i m i l a r pigment granules to those here and i n the c o n e - c e l l r egion have been described i n Limulus r e t i n u l a c e l l s by Fahrenbach (1969) who i n t e r p r e t e d the dark content as ommochrome, a screening pigment. The v e s i c l e s w i t h c l e a r f i b r i l l a r content (Plate 10-A,B) could represent e a r l y stages i n pigment granule formation as suggested by Shoup (1966) f o r c e r t a i n pigment granules i n the Drosophila eye. E x t r a c e l l u l a r space ( P l a t e 9-B) was abundant, but t h i s was thought to be due to poor i n f i l t r a t i o n and embedding, though a continuous space between r e t i n u l a c e l l and pigment c e l l of Apis m e l l i f e r a has been ob-served by Baumann (1975) as a r e g u l a r gap w i t h a width o f 15 nm, acces-s i b l e to e x t r a c e l l u l a r markers ( P e r r e l e t & Baumann, 1969). In the l i g h t micrographs (Plate 2; 5-B; 7-B) the rhabdom was r e s o l v -ed as a r e f r a c t i l e rod, b e l i e v e d t o be secr e t e d by the sensory c e l l s (Roonwall, 1947) and a c t i n g as a wave guide ensuring t h a t l i g h t e n t e r i n g i t from the d i o p t r i c system would be conducted without l o s s along the c y l i n d e r o f sensory r e c e p t o r s . While the rhabdom may w e l l a c t i n t h i s manner, the e l e c t r o n microscope showed i t to be not a secreted product, but r a t h e r b u i l t up from many short m i c r o v i l l a r processes stemming from the i n n e r surface of a l l the receptor c e l l s ( Plate 9-B; 13-A,B). The rhabdom i n the centre of the ommatidium appeared to be formed mainly by 39 E l e c t r o n micrograph at high m a g n i f i c a t i o n of the rhabdom (rh) i n the same region as P l a t e 9-B showing the m i c r o v i l l i (mv) and the desmosomes (ds) between each r e t i n u l a c e l l (RC 1-7) and the adjacent one. Note the presence of the c r y s t a l l i n e c o n e - c e l l pro-cesses (cp) . 1 cm = 529 nm Same area as t h a t o f P l a t e 13-A showing the m i c r o v i l l a r arrangement and a s s o c i a t i o n w i t h the receptor c e l l s . 1 cm = 444 nm 40 41 the seven r e t i n u l a c e l l s . A j u n c t i o n a l complex resembling a desmosome was formed near the rhabdom between each receptor c e l l and i t s adjacent c e l l (Horridge, 1966), s i m i l a r to that found i n the honey-bee drone (Perrelet, 1970). Behind four of the seven ju n c t i o n a l complexes, a membrane c l e a r l y o u t l i n e d an ovoid structure (ca. 0.3 ym i n diameter) containing microtubules. These structures probably represent extensions of the c r y s t a l l i n e cone, as Waddington and Perry (1960) suggested f o r Drosophila. The m i c r o v i l l i forming the rhabdom were approximately 50\nm\ i n diameter, which i s d i f f e r e n t from that obtained by P e r r e l e t and Baumann (1969)].; for the honey-bee drone (80 nm) , and were seen to touch each other with mere apposition of t h e i r adjoining membranes. In Apis  m e l l i f e r a , the rhabdomeric m i c r o v i l l i are t i g h t l y packed (Perrelet, 1970), and the outer l e a f l e t s of the l i m i t i n g membrane touch each other forming t i g h t junctions. This i s thought to be the same i n the l o c u s t , S_. gregaria. As i n the bee (Perrelet, 1970) , the e x t r a c e l l u l a r space i n the rhabdom appeared to be confined to the narrow t r i a n g u l a r spaces between adjacent m i c r o v i l l i . Throughout the r e t i n a of the locust, i t was observed that the o r i e n t a t i o n of the rhabdomere m i c r o v i l l i was repeated constantly from one ommatidium to the next and within each of these groups of sensory c e l l s , the m i c r o v i l l i of opposite rhabdomeres p a r a l l e l e d each other. 42 I I . S p e c i a l i z e d Junctions and P o s s i b l e R e s t r i c t i o n s to E x t r a c e l l u l a r Space i n the Retina: The search f o r j u n c t i o n a l s p e c i a l i z a t i o n s between the d i f f e r e n t c e l l types of the r e t i n a presented p a r t i c u l a r i n t e r e s t because of the r o l e of c e r t a i n j u n c t i o n s i n r e s t r i c t i n g e x t r a c e l l u l a r f l u i d movement or f o r t h a t matter dyes and r a d i o i s o t o p e t r a c e r s (Shaw, 1976), as w e l l as i n e l e c t r i c a l c o u p l i n g between c e l l s . The f o l l o w i n g r e s u l t s obtained using the e l e c t r o n microscope examine the membrane s p e c i a l i z a t i o n s i n the p a r t i c u l a r p a r t s o f the eye. P r o x i m a l l y towards the basement membrane i n the ommatidial region (ca. 200 ym above the basement membrane), the r e t i n u l a c e l l s were found to d i s p l a y p a r t i c u l a r j u n c t i o n a l s p e c i a l i z a t i o n s . A gap j u n c t i o n o r nexus between r e t i n u l a c e l l s was observed ( P l a t e 14-A,B,C) and was measured from the microdensitometer t r a c i n g ( P l a t e 15) to be of 2 nm width. Numerous holes were observed i n the s e c t i o n but t h i s was b e l i e v e d to be due to poor i n f i l t r a t i o n and embedding of the Epon 812. Another gap was observed more d i s t a l l y from t h i s area i n the ommatidial r e g i o n (Plate 16-A,B) and the microdensitometer t r a c i n g (Plate 17) demonstrated a narrow gap of 1.7 nm. Seven of the e i g h t receptor c e l l s of the ommatidium were d i s t i n c t l y seen i n o b l i q u e - l o n g i t u d i n a l s e c t i o n . The rhabdom was seen i n the centre o f the c e l l s but the rhabdomere m i c r o v i l l i were not as w e l l r e -solved as those of P l a t e 13-A,B. The neurotubules were present (approxi-mately 20 nm wide) and numerous v e s i c l e s were present i n each receptor c e l l resembling those seen i n the c o n e - c e l l r e g i o n . The la r g e space on 43 E l e c t r o n micrograph of an ommatidium approxi-mately 200 ym above the basement membrane i n an ob l i q u e - l o n g i t u d i n a l section showing the rhabdom (rh) and seven of the eight receptor c e l l s (RC 1-7) that comprise i t . The eighth receptor c e l l i s missing due to the f a u l t of t h i n sectioning. Note that there seems to be almost no e x t r a c e l l u l a r space between these c e l l s , but that there seems to be space (s) outside the bundle o f c e l l s . 1 cm = 1.5 ym Same area as that of Plate 14-A (from the c i r c l e d area). Note the lack of any v i s i b l e gap between the c e l l s (arrowheads). 1 cm = 69 nm Same as Plate 14-B except at a higher magnifi-cation i l l u s t r a t i n g the presence of a gap approximately 2 nm between the dots. A micro-densitometer t r a c i n g between the two dots was made (see Plate 15) . 1 cm = 37 nm 44 45 P l a t e 15 • A microdensitometer t r a c i n g across the two dots of P l a t e 14-C showing a gap width of 2 nm. 1 cm = 21 nm 46 e s 47 E l e c t r o n micrograph o f the j u n c t i o n a l membrane complex or nexus i n l o n g i t u d i n a l s e c t i o n be-tween two rece p t o r c e l l s i n the ommatidial r e g i o n approximately 250 microns above the basement membrane. 1 cm = 160 nm Same as t h a t of P l a t e 16-A (from the c i r c l e d area). A gap i s b a r e l y v i s i b l e of 2 nm width approximately (arrowheads). A microdensito-meter t r a c i n g of the area between the two arrowheads was made (see P l a t e 17). 1 cm = 52 nm 48 49 A microdensitometer t r a c i n g across the heads of P l a t e 16-B showing a gap width 1.7 nm. 1 cm = 17 nm 50 51 either side of the receptor c e l l axon bundle (Plate 14-A) is thought to be "space" in the retina between neighbouring ommatidia. As seen in the light micrographs (Plate 6-A; 7-A,B) the ommatidia were distinctly separate from one another and this electron micrograph (Plate 14-A) was of the same section as the one in Plate 6-A. The fact that the ommati-dia are separate may be due to improper fixation, but i t is thought that this space may be present in the intact eye, i.e. an eye not fixed or otherwise prepared for investigation with the electron microscope. Shaw (1976) states that the eye has a 3% volume of extracellular space as determined by the use of dye tracers and this spa.ce noted in the micrograph may account for the space he observed. The receptor cells are probably sealed off from the eye's bathing f l u i d and the junctioning observed here is between the neighbouring receptor cells of one ommati-dium and not between neighbouring ommatidia. Near the basement membrane (Plate 20-A,B) a gap of 7 nm was observed between two receptor c e l l axons from the microdensitometer tracing (Plate 21). At the level of the basement membrane, bundles of axons were observed piercing the structure (Plate 20-A,B; 21). Numerous tracheoles (1-3 ym in diameter) were seen (Plate 20-A) fenestrating the striated basement membrane which was about 15 ym in width. The s t r i a -tions are thought to be collagen fibers (Smith, 1968). Below the base-ment membrane, channels were seen which did not appear to be tracheae, but may be hemolymph channels (Shaw, personal communication). These channels are diagrammed by Northrop and Guignon (1970) but are not labelled. G l i a l cells with large irregularly shaped nuclei were observed E l e c t r o n micrograph o f l o n g i t u d i n a l s e c t i o n showing a l a c k o f e x t r a c e l l u l a r space between two receptor c e l l s near the basement membrane (square). Note the e l e c t r o n dense m a t e r i a l on e i t h e r s i d e o f the j u n c t i o n . 1 cm = 320 nm Same area as P l a t e 18-A (from square). A gap of l e s s than 10 nm i s v i s i b l e . A microdensi-tometer t r a c i n g of the area between the two arrowheads was made (see P l a t e 21). 1 cm = 100 nm 53 54 P l a t e 19 A microdensitometer t r a c i n g across the arrow-heads of P l a t e I8-B showing a gap width o f 7 nm. 1 cm = 35 nm 55 i e s 56 with many pigment granules, both intact and some which had fallen out or had been dissolved during fixation or embedding. In cross section (Plate 20-B) eight receptor c e l l axons were seen to be encompassed by a sheath of basement membrane (ca. 40 nm wide) completely separating i t from other ommatidia and the interommatidial bathing f l u i d . Few pigment granules (ca. 0.3 ym iri diameter) and many mitochondria (ca. 0.2-0.3 ym in diameter) were observed, distally located in the receptor axons (Plate 21). Membranes of the axons and g l i a l - c e l l processes tightly apposed each other and a microdensitometer tracing (Plate 23) at this level revealed a gap of less than 9 nm (Plate 22-A). Mitochondria were ob-served both in the receptor cells and the g l i a l - c e l l process which lay between the axons. Microtubules were cut in cross section and longitud-inal section both in the axons and g l i a l - c e l l process. Below the basement membrane many pigment granules (ca. 0.2 ym in diameter) were evenly distributed around the axon bundle (Plate 20-C) and large oval g l i a l - c e l l nuclei (ca. 6 ym in diameter) were observed. In Plate 20-C a bundle of receptor axons was s t i l l intact and had not fanned out to synapse with second order neurons from the optic lobe, as the lamina or f i r s t neuropile had not yet been reached. Hafner (1973) states that in the crayfish, receptor-cell axons form synaptic links below the basement membrane at different levels in the lamina. Each receptor-cell axon bundle does not forge synapses at the same level or in the same plane. About 35 ym below on the proximal side of the basement membrane (Plate 24-A,B,D) a gap of 9 nm width was observed from the microdensito-meter tracing (Plate 25) between receptor axons as they forged their way 57 P l a t e 20-A E l e c t r o n micrograph showing a bundle o f r e c e p t o r - c e l l axons (axb) i n l o n g i t u d i n a l s e c t i o n p i e r c i n g the basement membrane (bm) w i t h a s s o c i a t e d t r a c h e o l e s ( t r ) . G l i a l - c e l l n u c l e i (gn) are i n c l o s e p r o x i m i t y . 1 cm = 2.6 ym P l a t e 20-B E l e c t r o n micrograph showing a bundle o f r e c e p t o r - c e l l axons i n cross s e c t i o n p i e r c i n g the basement membrane. Note the few pigment granules (pg) and the sheath o f basement mem-brane surrounding the e i g h t receptor axons (large arrowhead). 1 cm = 870 nm P l a t e 20-C An e l e c t r o n micrograph showing a bundle of r e c e p t o r - c e l l s axons (arrowhead) i n l o n g i t u d -i n a l s e c t i o n below the basement membrane as they mesh together as a r e s u l t of decussating w i t h other axon bundles of other ommatidia and i n order to avo i d the hemolymph channels. Other axon p r o f i l e s are a l s o seen but are not numbered. Note the d i s t r i b u t i o n and array of pigment granules (pg). 1 cm = 2.9 ym 5 8 59 E l e c t r o n micrograph of an o b l i q u e - c r o s s sec-t i o n of two receptor-axon bundles p i e r c i n g the basement membrane (bm). Note the numerous mitochondria (mi) and pigment granules(pg) at t h i s l e v e l . 1 cm = 529 nm 60 61 E l e c t r o n micrograph of r e c e p t o r - c e l l axons i n cross s e c t i o n from the same area as t h a t o f P l a t e 21, showing the extent o f the j u n c t i o n -i n g between the c e l l membranes o f the axons and the g l i a l c e l l s . Note the mitochondria (mi) and the numerous neurotubules ( n t ) . A • microdensitometer t r a c i n g between the two dots was made (see P l a t e 23). 1 cm = 111 nm Same area as P l a t e 22-A except a t a higher m a g n i f i c a t i o n showing a gap width o f 5-10 nm. 1 cm = 59 nm 6 2 63 A microdensitometer t r a c i n g across the dots of P l a t e 22-A showing a gap width l e s s than 9 nm. 1 cm = 46 nm 64 e s 65 E l e c t r o n micrograph of receptor axons i n l o n g i -t u d i n a l s e c t i o n showing j u n c t i o n i n g between each and how much e x t r a c e l l u l a r space i s present, a t the l e v e l of the blood channels (he) j u s t below the basement membrane. 1 cm = 540 nm Same as P l a t e 24-A (from c i r c l e d area) except at a higher m a g n i f i c a t i o n to show the pres-ence of a gap of 10 nm width. 1 cm = 108 nm Same as P l a t e 24-A (from square area) showing the presence o f a gap of 9 nm width between two axons (ax). A microdensitometer t r a c i n g between the two arrowheads was made (see P l a t e 25). 1 cm = 108 nm 66 67 P l a t e 25 A microdensitometer t r a c i n g across the two arrowheads of P l a t e 24-C showing a gap width of 9 nm. 1 cm = 35 nm 68 69 E l e c t r o n micrograph of axons (ax) i n l o n g i -t u d i n a l s e c t i o n below the basement membrane Note the numerous neurotubules ( n t ) . 1 cm = 564 nm Same area as t h a t of P l a t e 26-A (from the c i r c l e d area) showing a gap present between g l i a l - c e l l process (gcp) and axon (ax). 1 cm = 173 nm Same as P l a t e 26-B except a t a higher magni f i c a t i o n showing a gap of 8 nm width. A microdensitometer t r a c i n g was made between the two dots (see P l a t e 27). 1 cm = 66 nm 70 71 A microdensitometer t r a c i n g across two dots of P l a t e 26-C showing a gap width of approximate-l y 8 nm. 1 cm = 27 nm 72 e s 73 An e l e c t r o n micrograph showing the presence o a gap between two axons i n l o n g i t u d i n a l sec-t i o n . Note the c o l l a p s e d mitochondria (mi), i n t r a c y t o p l a s m i c membranes (Icm) and neuro-tubules ( n t ) . 1 cm = 132 nm Same as P l a t e 28-A except a t a higher magni-f i c a t i o n showing the presence o f a 10 nm gap. A microdensitometer t r a c i n g was made between the two dots (see P l a t e 29). 1 cm = 50 nm 74 75 P l a t e 29 A microdensitometer t r a c i n g across the two black dots of P l a t e 28-B showing a gap width of 10 nm. 1 cm = 17 nm 76 77 E l e c t r o n micrograph of an axon of one recep-t o r c e l l i n an o b l i q u e - c r o s s s e c t i o n below the basement membrane, showing the amount of e x t r a c e l l u l a r space between g l i a l - c e l l pro-cesses (gcp) and the axons (ax). Note the polygonal l a t t i c e arrangement of the mem-branes (arrowhead) and the c o l l a p s e d mitochondria (mi). 1 cm = 270 nm 78 79 P l a t e 31-A An e l e c t r o n micrograph from the same area as th a t of P l a t e 30 showing the presence o f a gap o f from 11-40 nm. Note the gap-junction l a t t i c e of the u n i t membranes (large arrow-head) . A microdensitometer t r a c i n g between the two arrowheads was made (see P l a t e 32). 1 cm = 142 nm P l a t e 31-B Same as P l a t e 31-A except a t a higher magni-f i c a t i o n showing a gap width of 10 nm approximately. 1 cm = 71 nm 80 81 P l a t e 32 A microdensitometer t r a c i n g across the two arrowheads of P l a t e 31-A showing a gap width of 10 nm. 1 cm = 47 nm 82 83 E l e c t r o n micrograph of the area below the basement membrane i n l o n g i t u d i n a l s e c t i o n . Note the l a r g e amount of space between the axons (ax) which i s thought to be f i l l e d w i t h a g l i a l - c e l l process. 1 cm = 714 nm Same as th a t of P l a t e 33-A (continued from the c i r c l e ) f a r t h e r along from the area l a b e l l e d . 1 cm = 714 nm 84 85 E l e c t r o n micrograph of the same area as t h a t of P l a t e 33 showing the presence of a gap between the axons (ax) and the g l i a l - c e l l process (gcp). Note the areas of high e l e c t r o n d e n s i t y (arrowheads) which may be c e l l contacts between the axon and g l i a l c e l l . 1 cm = 333 nm Same as P l a t e 34-A showing a gap of 13 nm approximately. 1 cm = 133 nm Same as P l a t e 34-A except f a r t h e r along from the arrow, showing a gap of 13 nm. 1 cm = 200 nm 86 87 toward the optic lobe, decussating with other receptor axons of other receptor-axon bundles. At about 70 microns below the basement membrane (Plate 26-A,B,C) the gap width of 8 nm was demonstrated from the micro-densitometer tracing (Plate 27). In approximately the same area as that of Plate 26 a gap of 10 nm was observed .between two axons (Plate 28-B). Two mitochondria were observed but their cristae appeared pushed to one side of the structure, indicating poor fixation of the tissue in this particular section. From the microdensitometer tracing of the area (Plate 29) a gap width of 10 nm was illustrated. Plate 30 shows the g l i a l - c e l l processes interdigitating between the axons at this level below the basement membrane. A gap of 10 nm (Plate 31-B) was observed between the axons. What appeared to be a g l i a l - c e l l process in the upper part of the micrograph (Plate 31-A) was thought to be an artifact due to thin sectioning of the tissue. The microdensitometer tracing (Plate 32) showed a 10 nm gap width. At about 150 microns below the basement membrane (Plate 34-A,B,C) a gap width of 13 nm was observed between two axons and a g l i a l - c e l l process. A substantial amount of electron-dense material was present in the g l i a l - c e l l process. The g l i a l - c e l l process width at one point enlarged to ca. 1.5 ym (Plate 33~A) but remained constant at 60-90 nm between the two axons at this level. 88 DISCUSSION The compound- eye of the l o c u s t i s a t y p i c a l a p p o s i t i o n eye having ommatidia w i t h closed-type rhabdoms. The ommatidium i s formed of e i g h t r e t i n u l a c e l l s and i s a s s o c i a t e d w i t h three types of pigment c e l l s . The number o f r e t i n u l a c e l l s found i n the course of t h i s study i s s i m i l a r to t h a t reported by Horridge (1966) and Bennett jst a l . . (1967) . Small r e t i n -u l a c e l l s have been described i n s e v e r a l o t h e r i n s e c t species as f o r example Dros o p h i l a (Waddington & P e r r y , 1961) , Locusta (Horridge & Barnard, 1965), and Notonecta (Horridge, 1968), but i t i s s t i l l not known whether these c e l l s f u l l f i l a f u n c t i o n d i f f e r e n t to t h a t of the l a r g e c e l l s , ( P e r r e l e t , 1970). I n the d i s t a l e x tremity of the r e t i n u l a c e l l s , few cytoplasmic o r g a n e l l e s were found w h i l e i n the proximal p a r t , the r e t i n u l a c e l l s , s t i l l bearing- rhabdomeres, contained o n l y mitochond-r i a . The d i f f e r e n c e s i n cytoplasmic o r g a n i z a t i o n o f the r e t i n u l a c e l l s , depending on the l e v e l o f the ommatidium, have a l s o been reported i n the honey-bee drone ( P e r r e l e t , 1970) and the worker bee (Varela & P o r t e r , 1969). The pigment c e l l s probably serve as l i g h t s h i e l d s (Goldsmith, 1962; V a r e l a & P o r t e r , 1969), l i m i t i n g the l a t e r a l s c a t t e r i n g o f l i g h t between neighbouring ommatidia, e s p e c i a l l y at the l e v e l o f the d i o p t r i c apparatus, where the l a r g e s t pigment accumulation was found, as w e l l as at the l e v e l below the basement membrane. In a d d i t i o n , the p o s i t i o n o f the pigment c e l l s w i t h respect to the r e t i n u l a c e l l s o r primary sensory neurons, as w e l l as the cytoplasmic feature of microtubules, makes p i g -ment c e l l s resemble g l i a l c e l l s i n the c e n t r a l nervous system of v e r t e b r a t e s and i n v e r t e b r a t e s ( K u f f l e r & N i c h o l l s , 1966; reviewed by 89 P e r r e l e t , 1970; Baumann, 1975). The presence of gap j u n c t i o n s between the outer pigment c e l l s i n d i c a t e s t h a t these c e l l s could be e l e c t r i c a l l y coupled (Baumann, 1975), as g l i a l c e l l s i n i n v e r t e b r a t e and v e r t e b r a t e nervous systems ( K u f f l e r & P o t t e r , 1964). P e r r e l e t (1970) s t a t e s t h a t i n t r a c e l l u l a r r e c o r d i n g i n the outer pigment c e l l s o f the bee during l i g h t s t i m u l a t i o n o f the neighbouring ommatidia revealed no c o u p l i n g between r e t i n u l a c e l l s and outer pigment c e l l s . This he suggests i s due i n f a c t to the continuous e x r a c e l l u l a r space of 10-20 nm between the two c e l l types. However, i n the l o c u s t a gap of 1.5 nm width was recorded between adjacent pigment c e l l s . This width i s c o n s i s t e n t w i t h t h a t c h a r a c t e r i z e d by t h i s j u n c t i o n a l complex as Goodenough and Revel (1970) s t a t e t h a t a gap j u n c t i o n i s c h a r a c t e r i z e d by a 2nm gap between the apposed j u n c t i o n a l membranes (see a l s o Revel & Karnovsky, 1967). In r e c o r d i n g the response to l i g h t of c e l l p a i r s i n the r e t i n a of a honey-bee drone, Shaw (1969a) demonstrated the presence of e l e c t r i c a l c o u p l i n g between c e l l s of the same ommatidium. Guided by e l e c t r o p h y s i o -l o g i c a l data, he hypothesized t h a t the c o u p l i n g occurs p r i n c i p a l l y through the rhabdomeric m i c r o v i l l i , w i t h or without an accessory c o u p l i n g of the r e t i n u l a c e l l s a t the base of the ommatidium. E l e c t r i c a l c o u p l i n g seems p o s s i b l e between rhabdomeric m i c r o v i l l i , s i n c e t i g h t j u n c t i o n s have been found i n the bee ( P e r r e l e t , 1970) between the outer l e a f l e t s o f the m i c r o v i l l i membranes, and i t i s thought t h a t the same occurs i n the l o c u s t , as most other evidence documented p a r a l l e l s t h a t found i n the bee. Further research i n t h i s regard i s needed. Experimentation has pointed out the e x i s t i n g c o r r e l a t i o n between e l e c t r i c a l c o u p l i n g and gap j u n c t i o n s (Payton, Bennett & Pappas, 1969). 90 I t has been found t h a t a gap j u n c t i o n e x i s t s between r e t i n u l a c e l l s , i n t h a t a spacing of 2 run between the j u n c t i o n a l membranes was demonstrat-ed from the microdensitometer t r a c i n g s which agrees w i t h the standards set by Goodenough and Revel (1970). P l a t e 35 i s a diagram i l l u s t r a t i n g the p o s i t i o n i n the eye of S_. g r e g a r i a o f the gap j u n c t i o n complex ob-served i n the cone c e l l , ommatidial, and the basement membrane regions. The receptor c e l l s are separated by an e x t r a c e l l u l a r space measur-i n g ca. 2 nm i n width i n the ommatidial r e g i o n . At the basement membrane though, the membranes of the r e t i n u l a c e l l s come c l o s e to one another, but the microdensitometer t r a c i n g s revealed a 7-10 nm gap between the c e l l s . However, Horridge (1966) s t a t e s t h a t no s t r u c t u r a l feature i s v i s i b l e i n e l e c t r o n micrographs to suggest t h a t any of the l o c u s t r e t i n u l a c e l l s i n t e r a c t , and th a t he has not found any t i g h t j u n c t i o n s or s p e c i a l c e l l c ontacts o t h e r than the elongated desmosomes which l i e between a l l neighbouring c e l l s a t the margin o f the rhabdom. Although the observations of t h i s t h e s i s may be a t t r i b u t e d to f i x a t i o n a r t i -f a c t s , i t was b e l i e v e d to be h i g h l y u n l i k e l y i n t h i s case as f i x a t i o n If ! procedures were un a l t e r e d among specimen p r e p a r a t i o n s . Perhaps the f l u i d i n the c e l l s i s of a much higher o s m o l a r i t y than t h a t measured e x t e r n a l l y . Further s t u d i e s u s i n g a hyperosmotic f i x a t i v e should be undertaken to e s t a b l i s h i f such e f f e c t s as swollen mitochondria s t i l l occur. I t may be noted i n passing t h a t f i x a t i o n procedures have im-proved s i n c e Horridge's p i o n e e r i n g work (1966). This alone may be s u f f i c i e n t to account f o r any d i s c r e p a n c i e s . The number of r e t i n u l a c e l l s observed v a r i e d i n some s e c t i o n s from 7 to 8. I t i s thought t h a t the i n a b i l i t y to s e c t i o n the eye always i n the same plane could be the 91 Plate 35 Diagram to i l l u s t r a t e the r e l a t i v e anatomical p o s i t i o n , extent, and dimensions of the eye of Schistocerca gregaria, and associated junc-tions i n l o n g i t u d i n a l section (cor, cornea; gn, g l i a l - c e l l nucleus; urn, u n i t membrane). 1 cm = 31 ym Plate 35-1 The cone-cell region showing the secondary pigment—cell n u c l e i (spn) and the primary p i g -ment-cell n u c l e i (ppn) and how they associate-with the c r y s t a l l i n e cone (cc) and r e t i n u l a . The i n s e r t i s a schematic diagram of a gap junction which i s found i n t h i s region between pigment c e l l s . The gap measured ca. 2 nm. 1 cm = 25 nm Plate 35-11 The ommatidial region showing the receptor c e l l s (rc) and t h e i r n u c l e i (rcn), the rhabdom (rh), the e c c e n t r i c - c e l l n u c l e i (en) and the sheath of pigment granules (pg) around the receptors. The i n s e r t i s a schematic diagram of a gap junction which.is found i n t h i s region between the receptor c e l l s , measuring from 1.5-2.5 nm. 1 cm = 2 5 nm ..... Plate 35-111 The basement membrane region showing the base-ment membrane (bm) and the r e c e p t o r - c e l l axon bundle (axb) p i e r c i n g i t . The i n s e r t i s a schematic diagram of a gap junction which i s found i n t h i s region between axons and g l i a l -c e l l processes and axons themselves. The gap width measured from 5-15 nm. 1 cm = 50 nm .. . _ •'. 93 cause o f these v a r i e d f i n d i n g s . Horridge (1966) s t a t e s t h a t e i g h t r e -ceptor c e l l s comprise an ommatidium and Bennett e t a l . (1967) s t a t e t h a t 6 or 7 of these r e t i n u l a c e l l s bear rhabdoms. In most cases i t was observed t h a t 7 r e t i n u l a c e l l s of an ommatidium provided rhabdomeres to the c e n t r a l rhabdom. Shaw (1975) suggests the existence o f a b a r r i e r t h a t has an e f f e c t on the r e t i n a l c u r r e n t s by s e t t i n g up a system o f e l e c t r i c a l p r e s y n a p t i c i n h i b i t i o n on c e r t a i n receptor t e r m i n a l s . This system i s thought to a c t through the a v a i l a b l e e x t r a c e l l u l a r space. In h i s paper three b a r r i e r regions were observed: B a r r i e r I l o c a t e d i n the c o n e - c e l l r e g i o n ; B a r r i e r I I i n the ommatidial regio n ; and B a r r i e r I I I i n the basement-mem-brane re g i o n . He s t a t e s t h a t B a r r i e r I represented l o s s of c u r r e n t i n t o the receptor c e l l s and i n f a c t was not a b a r r i e r a t a l l . A t t h i s l e v e l i n the r e t i n a there are many pigment c e l l s present which are coupled by gap j u n c t i o n s (1.5 nm gap width). Baumann (1975) suggests t h a t pigment c e l l s resemble g l i a l c e l l s i n t h e i r cytoplasmic make-up and si n c e g l i a l c e l l s are nervous system i n s u l a t i v e m a t e r i a l , they may absorb the e x t r a -c e l l u l a r c urrent as Shaw observed i n B a r r i e r I . He goes on t o say t h a t B a r r i e r I I was r e a l and e f f e c t i v e l y sealed o f f the e x t r a c e l l u l a r space. To a v o i d the b a r r i e r , current crosses i n t o the r e c e p t o r s , t r a v e l s w i t h i n them and then crosses out again a t the l e v e l of the lamina. The l a s t b a r r i e r , B a r r i e r I I I , was explained as c o n s i s t i n g of t h i s " h i j a c k i n g " c u r r e n t reappearing and flow i n g from the lamina t o the hemolymph chan-n e l s below the basement membrane. Shaw a l s o p o s t u l a t e d t h a t B a r r i e r I I and I I I merge and span approximately the distance between the basement membrane and lamina. This presence of an e x t r a c e l l u l a r b a r r i e r then 94 a f f e c t s the receptors by b l o c k i n g the e x t e r n a l r e t u r n path f o r receptor c u r r e n t t r a v e l l i n g down the axons. In order to complete the c i r c u i t , r e t u r n i n g c u r r e n t s cross i n t o the receptors to avoid the b a r r i e r as do e x t r i n s i c c u r r e n t s . Therefore the non-active axons of neighbouring u n i l l u m i n a t e d receptor c e l l s then c a r r y the r e t u r n c u r r e n t from the e x c i t e d c e l l s (Shaw, 1975). The evidence found i n t h i s study does f i t i n t o Shaw's model o f e l e c t r i c a l p r e s y n a p t i c l a t e r a l i n h i b i t i o n . E x t r i n s i c or e x t r a c e l l u l a r c u r r e n t crosses over to the receptor c e l l s from the e x t r a c e l l u l a r space because there i s a b a r r i e r of high e l e c t r i c a l r e s i s t a n c e present which could i n f a c t be the gap o f e x t r a c e l l u l a r space present i n the ommatidial region . A gap j u n c t i o n of ca. 2 nm width appears to span from the omma-t i d i a l region to the basement membrane re g i o n . Gap j u n c t i o n s , f i r s t demonstrated by Revel and Karnovsky (1967) have been shown to i n c l u d e an i n t e r c e l l u l a r space of 2-4 nm ( S a t i r & G i l u l a , 1973; G i l u l a , 1974). S a t i r and G i l u l a (1973) s t a t e t h a t l a r g e areas or extensive s e r i e s of gap j u n c t i o n s do not o f t e n occur. However i n t h i s study i t i s apparent t h a t t h i s j u n c t i o n a l complex does e x i s t over an extensive area from the ommatidial region of the r e t i n a to a l e v e l c l o s e to the basement membrane. This j u n c t i o n complex appears to be a normal component of i n v e r t e b r a t e t i s s u e (Flower, 1971; G i l u l a & S a t i r , 1971; Lorber & Rayns, 1972). The gap j u n c t i o n t h a t was observed i n t h i s study may be the area of h i g h e l e c t r i c a l r e s i s t a n c e t h a t causes the e x t r i n s i c c u r r e n t as w e l l as the r e t u r n i n g photocurrent to take the path of l e a s t r e s i s t a n c e , t h a t i s , through the receptor c e l l s . One must assume t h a t not a l l the c u r r e n t 95 A diagram showing the approximate l o c a t i o n of the r e s i s t a n c e b a r r i e r proposed by Shaw (1975) (A) and the hypothesized p r o f i l e from the f i n d i n g s of t h i s t h e s i s (B). (bm, basement membrane; cc, c r y s t a l l i n e cone; c or, cornea; Lam, lamina; r c , receptor c e l l ) 96 97 going t o the o p t i c lobe, or r e t u r n i n g , t r a v e l s the same route out of the space. I t must be expected t h a t some of the curr e n t does indeed t r a v e l through the e x t r a c e l l u l a r space even i f t h i s area appears to be of high e l e c t r i c a l r e s i s t a n c e . I t i s the r a t i o of the currents ( I n t r a c e l l u l a r / E x t r a c e l l u l a r ; I i / I e ) i n v o l v e d then t h a t i s i n v e r s e l y p r o p o r t i o n a l t o the r a t i o o f the r e s i s t a n c e s present, i . e . t h a t the path o f l e a s t r e s i s t a n c e has the most cur r e n t . The neighbouring i n a c t i v e receptor c e l l s i n the r e t i n a may then be of l e a s t r e s i s t a n c e and the narrow e x t r a c e l l u l a r spaces or gap j u n c t i o n present of high r e s i s t a n c e . Anatomically there i s a l a r g e gap of 7-10 nm between g l i a l - c e l l processes and axons, or two axons, which extends from twenty microns above the basement membrane ( d i s t a l l y ) to 150 microns below (proximally) the basement membrane. P h y s i o l o g i c a l l y , Shaw (1975) suggests t h a t the curr e n t which crosses over to the neighbouring receptors e x i t s below the basement membrane i n t o the e x t r a c e l l u l a r space i n the lamina. This l a r g e e x t r a c e l l u l a r gap may be the path of l e s s r e s i s t a n c e e l e c t r i c a l l y than a path extending through the r e c e p t o r - c e l l axons t h a t i s taken by the current t r a v e l l i n g towards the v i s u a l centre of the b r a i n - the o p t i c lobe. Assuming t h a t a narrow gap accounts f o r a high r e s i s t a n c e and a wide gap, a low r e s i s t a n c e , i t i s p o s s i b l e t o t r a c e a h y p o t h e t i c a l r e s i s t a n c e diagram from the e l e c t r o n micrographs and the data presented i n t h i s t h e s i s ( r e f e r to P l a t e 36). The r e s i s t a n c e p r o f i l e s are s i m i l a r but s p a t i a l l y d i s p l a c e d . There i s no cu r r e n t explanation f o r the discrepancy i n v e r t i c a l displacement. Perhaps the discrepancy could be re s o l v e d by f u r t h e r study using com-bined e l e c t r o p h y s i o l o g i c a l and u l t r a s t r u c t u r a l techniques. 98 REFERENCES Baumann, F. ( 1 9 7 5 ) , " E l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of the honey-bee retina',' Chapt. 4 In: The Compound Eye and V i s i o n o f I n s e c t s , ed. G. A. Horridge, Oxford Univ. P r e s s , pg. 53-74. Bennett, R. R. , T u n s t a l l , J . & Horridge, G. A. 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