Structure and Function of Gap Junctions in the Photoreceptor Axon

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11 Structure and Function of Gap Junctions in the Photoreceptor Axon Terminals of the Fly Michael J. Wilcox Department of Anatomy, School of Medicine, University of New Mexico, Albuquerque, NM 87131-5211

The structure of the compound eye of dipteran insects provides a unique preparation for investigation of functional coupling by gap junctions in photoreceptor axon terminals in an intact animal. Axon terminals of individual photoreceptors converge and synapse onto a pair of cells that form a visual element. These terminals are electrically coupled by gap junctions. Characterization of the nature of the photoreceptor input at this synapse is a prerequisite to understanding how the stimulus is encoded by a visual element. Control of coupling is imperative to isolate individual photoreceptor cells and preserve their optical disparity. An anatomical substrate that possibly underlies hyperacuity in this animal is shown by optically staining the photoreceptor mosaic in the retina. Although photoreceptor axon terminals are electrically coupled, their gap junctions do not allow dye coupling of the cells. The hypothesis that the gap junctions are voltage sensitive was tested by adaptation of the animal to light or darkness and by injection of depolarizing and hyperpolarizing current directly into the recorded axon terminal after dye injection. Control of the gap junctions by calcium concentration or pH in the terminal was tested by injection of buffers into the terminal. Ongoing experiments will better define these differences and dissect the control mechanism of electrical coupling of photoreceptor axon terminals in a complex but highly structured region of the nervous system.

0065-2393/94/0235-0225$08.00/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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Information Processing in the Retina T h e retina provides a good m o d e l for the study o f information processing i n the nervous system because it is an accessible extension o f the brain, located b e h i n d a clear w i n d o w o f cornea a n d lens; it has a w e l l - d e f i n e d structure; a n d its input elements can be m o d u l a t e d b y an easily controlled s t i m u l u s — l i g h t . T o emphasize the p r o b l e m o f dendritic integration at the first synapse, w e w i l l drastically oversimplify the organization o f the m o r e familiar vertebrate retina. Photoreceptors make synaptic contact w i t h horizontal cells, w h i c h spread lateral interactions a m o n g neighboring synaptic terminals, a n d w i t h bipolar cells, w h i c h i n t u r n contact the ganglion cells, the output o f the retina. Characterization o f interaction at the first synapse is the goal o f o u r research. C o n t r o l o f the gap junctions c o u p l i n g these terminals is essential to that understanding. T h e r e is apparent convergence o f many photoreceptor synapses onto the dendrites o f two types o f bipolar c e l l : " o n c e n t e r " w i t h antagonistic s u r r o u n d and " o f f center" w i t h antagonistic surround ( I ) . T h e dendritic fields o f the two c e l l types have considerable overlap. W h e n several inputs synapse onto a dendrite, the general assumption is that they simply sum i n a linear fashion, as shown i n the classical case o f motor n e u r o n dendrites i n the central nervous system. T h i s assumption may not accurately represent dendritic integration b y the bipolar. H o w e v e r , to test interaction between these convergent synaptic inputs, it is necessary to stimulate i n d i v i d ual photoreceptor cells a n d r e c o r d dendritic integration b y the bipolar c e l l . T h i s procedure is difficult i n the vertebrate retina because physiological stimuli enter the retina through intervening b l o o d vessels, ganglion a n d bipolar c e l l layers, a n d the photoreceptor layer, w h i c h is enshrouded b y p i g m e n t e d e p i t h e l i u m , a n d a clear v i e w o f the receptor matrix is obstructed. T h e usual approach is to remove the retina f r o m the pigment e p i t h e l i u m , w h i c h allows visualization o f the photoreceptors. Unfortunately, this proce d u r e disrupts the matrix that maintains axial orientation o f the outer segment of the photoreceptor that houses the visual pigment. M o r e o v e r , lack o f pigment e p i t h e l i u m increases scatter o f the light stimulus. T h e foregoing problems can be offset b y use o f an intact animal w i t h a s i m p l i f i e d retina. U n l i k e humans, most animals v i e w the w o r l d through c o m p o u n d eyes. H o w e v e r , the cellular architecture i n the retina is parallel i n animals as evolutionarily different as mollusks, insects, a n d vertebrates ( 2 , 3). T h i s commonality suggests that there are only a few good ways to process visual information a n d that far back i n evolution, these processing tricks were adapted a n d adopted. A c o m p o u n d eye consists o f many individual retinas, each o f w h i c h looks at a different point i n space ( F i g u r e 1). Consideration o f only one facet o f the c o m p o u n d eye o f a fly reveals that it is a " s i m p l i f i e d r e t i n a " i n that it has a lens that images a small part o f the w o r l d onto a m i n i r e t i n a that consists of only eight photoreceptor cells, w h i c h are called an o m m a t i d i u m ( F i g u r e 2). A basement m e m b r a n e separates the retina f r o m its



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Gap Junctions in Fly Photoreceptor Axon Terminals

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Figure 1. Scanning electron micrograph of the compound eyes of a female housefly, Musca domestica.

u n d e r l y i n g compartment, w h i c h is called the lamina.

Photoreceptor axons

leave the o m m a t i d i a a n d f o r m bundles that penetrate the basement brane a n d distribute themselves

so that the photoreceptors

mem

that share a

c o m m o n visual axis f o r m synapses w i t h the u n d e r l y i n g structure, w h i c h is called a cartridge

( F i g u r e 3). T h r e e thousand o m m a t i d i a a n d their processing

subunits make u p the c o m p o u n d eye o f a fly a n d provide a nearly panoramic v i e w o f the fly s w o r l d . I n addition to the b r o a d field o f view, this retina enables a male fly to pursue a female i n flight, using relatively few facets, a n d to follow her w i t h amazing accuracy (4) despite his coarse visual acuity, w h i c h is only about 2° ( 5 ) — t h e extent o f the entire h u m a n macula. E v e n w i t h such coarse acuity, the animal can detect small displacements o f a stimulus far better than the resolution dictated b y the spacing o f retinal photoreceptors (6); this is contradictory to the channeling assumptions made i n i n f o r m a t i o n



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Figure 2. Interference contrast micrograph of a frontal section of the compound eye that shows the retina and lamina compartments separated by a basement membrane (BM). Lateral facet lenses are present on the outer edge of the section. Each lens in the eye images light onto a single ommatidium. Peripheral photoreceptors in each diamond-shaped ommatidium project axons across the basement membrane with 1:1 retinotopy onto the lamina cartridges.

processing theory ( 7 ) . S u c h enhanced resolution, b e y o n d the limit i m p o s e d b y the spacing o f receptors

i n the matrix, is called hyperacuity

(8-10).

Presently, machine vision does not have this capability, b u t many animals d o . I f the neural basis f o r hyperacuity c a n b e d e t e r m i n e d , machines c a n b e e n d o w e d w i t h the same ability. H o w hyperacuity works i n any visual system is not understood. F r o m experiments w i t h h u m a n subjects, t i m e delay a n d adaptational constraints suggest that hyperacuity occurs at an early (possibly retinal) level o f visual processing ( 9 ) . W i t h this i n m i n d , h o w the disparity o f i n d i v i d u a l photorecep tor cells can b e maintained w h e n a l l the receptors appear to b e electrically c o u p l e d to one another is even more p u z z l i n g . E l e c t r i c a l c o u p l i n g possibly raises retinal sensitivity to light b y " p o o l i n g " receptor inputs at the first synapse, w h i c h effectively increases the presynaptic surface area o f an i n d i -



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Figure 3. Interference contrast micro graph of a l-μm-thick section of the lamina that shows photoreceptor axon terminals surrounding the paired mono polar neurons in each cartridge.

vidual receptor. T i g h t c o u p l i n g along w i t h a n increase i n synaptic surface w o u l d guarantee transmission o f small signals i n the presence o f noise and, at the same time, reduce the effects o f noise b y shunting current to neighboring terminals. H o w e v e r , this same c o u p l i n g s h o u l d destroy the acuity gained b y the disparity o f individual cells that look at different points i n space. E l e c t r i cal c o u p l i n g o f n e u r a l elements is a n important issue because o f its apparent ubiquity, not only i n the retina ( J , I I ) , b u t also i n the rest o f the nervous system ( 1 2 ) . M o r e o v e r , control mechanisms o f gap junctions offer many possibilities to vary the nature o f functional circuitry. T h e function o f c o u p l i n g is difficult to assess i n a vertebrate retina due to the numbers o f cells c o u p l e d b y gap junctions that f o r m a large syncitium w i t h neighboring cells. Fortunately, a similar arrangement exists i n the fly eye, b u t the arrangement is s i m p l i f i e d b y l i m i t e d c o u p l i n g a n d the optical simplicity o f the c o m p o u n d eye. E l e c t r i c a l c o u p l i n g is l i m i t e d to six photore ceptors that look at the same point i n space ( 1 3 , 14). Optics o f the c o m p o u n d eye aids visualization o f i n d i v i d u a l cells. E a c h photoreceptor

c e l l has a

specialized organelle called a rhabdomere that houses the visual pigment. This highly refractile organelle is an optical waveguide that contains a n d transmits light along its length u n t i l the energy is absorbed b y the visual pigment. T h e distal t i p o f this waveguide is located i n the focal plane o f the facet lens, a n d light reaches the organelle without passing through interven i n g tissue. F o r a n experimenter, this optical arrangement provides a t o o l f o r direct observation o f individual organelles fluorescent

v i a transmitted,

reflected, o r

i l l u m i n a t i o n through the animal's o w n corneal lens. T h u s , the



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rhabdomere becomes a n i n vivo cuvette that is 1 μπι i n diameter a n d 100 μιη long, v i e w e d e n d - o n through a lens w i t h a photographic f n u m b e r o f about 2.0. Alternatively, diffraction o f the lens c a n b e neutralized w i t h water (15) and the organelles c a n be v i e w e d directly b y focusing the microscope o n the tips o f the organelles ( F i g u r e 4). Because the refractive index o f the rhab domere is higher than the s u r r o u n d i n g tissue, light that passes through the cell b o d y is effectively c a p t u r e d a n d p i p e d u p the organelle (16). T h e animal's o w n lens images the light along a w e l l - d e f i n e d axis to b e captured b y the microscope objective. D u e to this e n d - o n geometry, detection o f injected

Figure 4. Micrograph of the rhabdomere tips in a living animal. Because the rhabdomeres act as waveguides, they can trap transmitted, reflected, or fluorescent light. A drop of water placed between the microscope objective and the fly's eye effectively neutralizes diffraction of the cornea and allows the microscope to focus on the distal tips of the rhabdomeres, which are physically and optically isolated from their neighbors. In this animal, the fluorescent dye rhodamine Β was introduced to the retinal extracellular space. Epiillumination was used to excite the dye. Fluorescent light is piped up the organelle into the objective lens. The pattern of organelles shows the ommatidial arrangement, which consists of eight photoreceptor cells: six peripheral cells (R1-R6) that surround two central cells (R7 and R8) whose rhabdomeres are arranged in tandem (R8 below R7, to form a continuous waveguide). Independent stimuli can be selectively imaged onto individual rhabdomeres in this asymmetric pattern of seven optically isolated waveguides.



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dyes at l o w concentration is enhanced. I n all other animals that have c o m p o u n d eyes, the rhabdoms o f the photoreceptors i n each o m m a t i d i u m are fused together into a single waveguide. T h u s , i l l u m i n a t i o n excites all cells i n the o m m a t i d i u m at once. O n l y i n diptera are the rhabdomeres o f each c e l l optically isolated, and, as a consequence, each rhabdomere can be seen a n d independent s t i m u l i can be projected onto each one. T h i s situation allows initiation o f cellular interactions between neighboring cells b y independent stimulation of preselected photoreceptors i n the retinal matrix and simultane ous recording f r o m one of those cells via a microelectrode.

Photoreceptor Matrix in the Retina of Musca domestica E a c h o m m a t i d i u m consists o f six peripheral photoreceptor cells ( R 1 - R 6 ) that surround a pair o f tandemly oriented central cells ( R 7 distal to R8). T h e two central cells bypass the lamina a n d f o r m synapses i n the u n d e r l y i n g n e u r o p i l o f the m e d u l l a . Therefore, the present discussion w i l l ignore effects o f central cell contribution a n d also feedback f r o m the m e d u l l a a n d lateral interactions f r o m neighboring cartridges. Axons o f six peripheral cells, one f r o m each of six adjacent ommatidia, j o i n i n the u n d e r l y i n g layer, w h i c h is called the lamina, to f o r m a structure called a neuroommatidium (17) ( F i g u r e 5). T h e photoreceptors that contribute their axons to a n e u r o o m m a t i d i u m share the same visual axis, that is, they look at the same point i n space (18), as depicted i n F i g u r e 6a. This anatomical and optical relationship is called the p r i n c i p l e of neurosuperposition.

Anatomical Substrate for Hyperacuity Illumination of the animal's head f r o m b e h i n d a n d use of the animal's o w n facet lens as an objective to image the rhabdomeres onto the far-field radiation allowed P i c k (19) to project each rhabdomere i n a single n e u r o o m m a t i d i u m onto the visual w o r l d . P i c k showed that the photoreceptors i n neuroommatidia o f living animals do not share precisely the same visual axis; they are " m i s a l i g n e d " and show divergence w h e n their rhabdomeres are i m a g e d at infinity (effectively 10 m m i n front of the animal). Slight diver gence of the optical axes o f the six photoreceptors results i n an overlapped sampling of a point i n the visual space. C o m p a r e F i g u r e 6a a n d b . S u c h an overlap c o u l d provide a better sampling of the distant point i n space, b u t independent axes c o u l d also f o r m a substrate for hyperacuity. W e p e r f o r m e d an experiment that was the reverse of Pick's a n d used a photosensitization technique to stain photoreceptor cells i l l u m i n a t e d by a h i g h contrast stimulus (20, 21). A spot o f light subtending 8° to the fly's eye i l l u m i n a t e d a small portion of the retinal far field (depicted b y the large circle i n F i g u r e 6a a n d b). Photoreceptors oriented o n axis to the stimulus incorporated a fluorescent



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Figure 5. Light micrograph of a few facets of a fly's compound eye. Dark spots that represent the rhabdomeres of the photoreceptors have been superimposed onto each corneal lens to demonstrate the principle of neuro-superposition. The central corneal facet has been removed from the photograph to depict the underlying lamina cartridge. Anatomically, six peripheral photoreceptor axon terminals (R1-R6) synapse with two second-order cells (LI and L2) in the underlying neuropil that is called the lamina. Each of these six photoreceptors is illuminated by a different lens, but optically they share the same visual axis; that is, they look at the same point in space. This lamina subunit is known as neuroommatidium. Axons of the central receptor cells (R7 and R8) from the overlying ommatidium pass close to this cartridge, but simply bypass the lamina and do not contribute synapses at this neural level.

dye. I f all the receptors i n a single n e u r o o m m a t i d i u m l o o k e d at precisely the same point i n space a n d that point was i l l u m i n a t e d , the receptors w o u l d a l l take u p the dye a n d all the photoreceptor axon terminals i n a single cartridge w o u l d b e stained as depicted i n F i g u r e 6 a a n d c. H o w e v e r , i f the axes o f the receptors were

different,

only the i l l u m i n a t e d photoreceptors

would be

stained, w h i l e the n o n i l l u m i n a t e d photoreceptors i n the same n e u r o o m m a t i d i u m w o u l d not b e stained; the pattern shown i n F i g u r e 6 b a n d d w o u l d b e p r o d u c e d . C o m p a r e d w i t h the i n situ v i e w o f F i g u r e 4, the asymmetric array o f rhabdomeres i n F i g u r e 6 b appears upside d o w n . H o w e v e r , recalling that the

animal's o w n facet lens is b e i n g u s e d to project a n image

rhabdomeres

o f the

at infinity, this inversion is expected. E a c h rhabdomere was

m a p p e d f r o m the far-field radiation ( F i g u r e 6b) onto its target i n the l a m i n a



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Figure 6. The staining pattern of photoreceptor somas (a and b) and axon terminals in the lamina (c and d) predicted by the principle of neurosuperposition (c) and the pattern predicted using the "misaligned" optical axes of the photoreceptors discovered by Pick (d). If every photoreceptor in the neuroommatidium looked at the same point in space (depicted as a dot in a), illumination of that point in space by a spot of light (as depicted by the circle in a) should induce all of those cells to incorporate dye and stain all photoreceptor axons in a cartridge. Uniform staining of each neuroommatidium would occur (as depicted in cj. However, if the optical axes of the photoreceptors in a neuroommatidium are different, some of the cells would look at the bright light while others would not (b). Photoreceptors stained in b would produce a pattern like the one shown in d. The pattern in e was produced by staining a portion of the retina with an 8° spot of light. This pattern closely resembles the predicted pattern shown in d.


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( F i g u r e 6d). Results show that the axon terminals i n the n e u r o n o m m a t i d i a were not u n i f o r m l y stained ( F i g u r e 6e), contrary to the p r i n c i p l e o f neurosu perposition, where photoreceptor images are perfectly superimposed. This means that the disparate visual axis o f each receptor i n a single n e u r o o m m a t i d i u m is maintained a n d that those cells can be selectively stained b y use o f a h i g h contrast stimulus i m a g e d i n the animal's field o f view. W e think that the animal w o u l d not maintain such precise alignment o f individual

receptors


w i t h i n a n e u r o o m m a t i d i u m i f the eye c o u l d not make use o f the advantage gained b y disparate photoreceptor axes. M o r e o v e r , i f the disparate i n p u t is not maintained a n d e n c o d e d b y the monopolar cells, it is difficult to imagine h o w hyperacuity c o u l d be recovered by a downstream circuit.

Electrical Coupling of Axon Terminals in a Single Neuroommatidium W e hypothesized that optically disparate photoreceptor inputs that f o r m an overlapped sampling o f a distant point c o u l d f o r m the substrate for hyperacu ity i n the fly eye. H o w l a m i n a cartridges use this disparity to increase acuity without p r o p e r n e u r a l circuitry is h a r d to imagine. R e c a l l that gap junctions are present between the six photoreceptor axon terminals i n each l a m i n a cartridge (13,

14).

E l e c t r i c a l c o u p l i n g s h o u l d p o o l the information o f all six

terminals and smear the i n d i v i d u a l inputs. Scholes (22) first suggested that tight c o u p l i n g b y the gap junctions was functional. Shaw (23)

refined the

measurement by use o f a fiber optic to stimulate cells i n a single o m m a t i d i u m w h i l e recording f r o m photoreceptors. Because he stimulated all cells u n d e r the lens, equivalent to seven sampling points i n space, many lateral interac tions were i n d u c e d , w h i c h complicated the

analysis. V a n H a t e r e n

(24)

avoided this p r o b l e m b y stimulating only the cells that contributed axons to a cartridge, via the technique o f corneal neutralization (25), a n d recorded f r o m photoreceptors.

V a n H a t e r e n showed that the

six terminals i n a single

n e u r o o m m a t i d i u m are electrically c o u p l e d . T h e soma is separated f r o m the axon t e r m i n a l b y a long, narrow axon. Signals that originate i n a fellow neuroommatidial photoreceptor must traverse its o w n axon, cross f r o m t e r m i nal to t e r m i n a l t h r o u g h the gap junctions, a n d invade the soma o f the recorded c e l l t h r o u g h its o w n narrow axon. T h e signal is attenuated w h e n it invades the larger soma. Therefore, significant c o u p l i n g measured at the distant soma suggests that the terminals are i n d e e d tightly c o u p l e d . G a t i n g o f these gap junctions c o u l d be pivotal for information processing at the

first

synapse, between photoreceptors a n d interneurons. O p e n i n g a n d closing gap junctions, as a function o f either adaptation or neural feedback, w o u l d be one way to effectively couple a n d decouple i n d i v i d u a l cells i n the n e u r o o m m a t i d -


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G a p junctions can be o p e n e d either b y l o w e r i n g the intracellular free calcium concentration or b y raising the cytoplasmic p H (26). L i g h t - a d a p t e d photoreceptors have a higher intracellular c a l c i u m concentration than darkadapted photoreceptors. Therefore, a simple solution to the p r o b l e m w o u l d be that the junctions c o u l d o p e n a n d close w i t h respect to the state o f adaptation of the receptor c e l l . T h u s , sensitivity c o u l d be elevated b y p o o l i n g receptor inputs w h e n the c e l l is dark-adapted a n d acuity c o u l d be elevated b y isolating disparate inputs w h e n the c e l l is light-adapted. H o w e v e r , this does not appear to be the case i n the excised vertebrate retina, w h e r e c e l l c o u p l i n g is resistant to extracellular perfusion ( I J) that w o u l d decouple epithelial cells. I n that study, the c e l l b o d y was recorded. C y t o p l a s m i c c a l c i u m concentration was not controlled f r o m inside the cell, near the sight o f the gap junctions. T h e axon terminal, v i e w e d as a structured process w i t h all the necessary machinery for intercellular c o m m u n i c a t i o n f i r m l y i n place, becomes another i n vivo cuvette, isolated f r o m the soma o f the c e l l b y a narrow axon that crosses the basement m e m b r a n e between retina and lamina. A l o n g axon effectively compartmentalizes the soma o f the terminal, a n d impairs d i f f u sion. M o r e o v e r , each cartridge is electrically insulated f r o m its neighbors b y a sheath o f glial cells ( 2 7 ) . Therefore, the t e r m i n a l is appropriately h a n d l e d as an independent entity. T h e intracellular c a l c i u m concentration of the soma, k n o w n to be affected b y light adaptation, may not be controlled i n the same way i n the t e r m i n a l . T h e basement m e m b r a n e also provides an effective barrier for the diffusion o f dyes a d d e d to the retina. T h u s , two separate extracellular compartments are accessible for perfusion o f the soma a n d the axon t e r m i n a l . F u r t h e r m o r e , we can pharmacologically manipulate this iso lated corner o f the c e l l b y a c o m b i n a t i o n o f pressure a n d electrophoretic injection into the terminal near the site o f the gap junctions, w h i l e continuing electrical recording, and then recover the t e r m i n a l for histological examina tion ( F i g u r e 7). T h u s , control o f c o u p l i n g i n the receptor axon terminals seems pivotal, not only for mechanisms o f hyperacuity but also for characteri zation o f gap junctions i n this highly developed nervous system.

Dye Coupling between Photoreceptors Classical approaches to verification o f functional gap junctions i n c l u d e c o n ductance measurement, pharmacological manipulation, a n d demonstration o f dye c o u p l i n g (28). D y e c o u p l i n g is a straightforward m e t h o d for demonstrat i n g gap junctional function. Injection experiments are simple and results are easily interpreted. T h e outcome is assessed immediately w i t h an i n vivo preparation. A lower dye concentration is necessary for detection because dye lost d u r i n g fixation a n d tissue processing is avoided. I n the fly, w h e n the recording electrode crosses the basement m e m brane, the polarity o f the extracellular potential i n response to light, w h i c h is



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Figure 7. Fluorescence micrograph of a l-pum-thick section of the lamina of a male fly that shows stained photoreceptor axon terminals cut parallel to their long axis. Gap junctions between the six peripheral cells occur in the outer 10 pirn of the axon terminal, just below the somas of the monopolar neurons. Because recorded cells are located in reference to the corneal facet matrix, the location and orientation of that cell in the underlying neuropil is predetermined, so it can be sectioned in any given plane and recovered for histological and histochemical examination.

called the electroretinogram, inverts. T h u s , the electrode position i n the eye can be verified. O n c e a n axon t e r m i n a l is penetrated, the c e l l is brought o n axis to the stimulus b y m o v i n g the animal through orthogonal arcs u n t i l the cell responds to stimulation w i t h a localized m a x i m u m . F u r t h e r m o r e , the facet lens that illuminates the c e l l can be localized b y imaging a

field

diaphragm onto the corneal surface. F i g u r e 8 shows the result o f a n experi ment i n w h i c h the axon t e r m i n a l o f a photoreceptor was coinjected w i t h 6-carboxyfluorescein a n d the c a l c i u m chelator, ethyleneglycoltetraacetic a c i d ( E G T A ) . T h e steady-state response o f the receptor potential was augmented after injection, whereas the transient response r e m a i n e d unchanged, w h i c h



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Figure 8. Fluorescence micrograph of the surface of the fly eye with a single facet lit. Although only one photoreceptor under that lens is stained (confirmed by examination of the ommatidium via corneal neutralization), the whole facet is illuminated like the lens of an oncoming car. If dye had spread to its neuroommatidial neighbors, adjacent facets would light up in response to fluorescent excitation.

indicates that E G T A h a d l o w e r e d the free c a l c i u m i o n concentration w i t h i n the t e r m i n a l (29). E G T A chelation o f cytosolic calcium near the site o f c o u p l i n g s h o u l d have o p e n e d gap junctions a n d p e r m i t t e d dye c o u p l i n g . T h e same experiment has b e e n p e r f o r m e d w i t h L u c i f e r yellow, fluorescein, 6carboxyfluorescein, fluorescein isothiocynanate, c o u m a r i n 175, Lissamine rhodamine B , a n d rhodamine 123, all o f w h i c h have l o w molecular weight, either neutral, positive, or negative charge, a n d d i f f e r i n g partial solubilities i n l i p i d . Attempts also have b e e n made to raise the cytoplasmic p H i n the t e r m i n a l b y coinjection o f alkaline dye solutions and buffers w i t h dye. E a c h experiment has failed to show dye c o u p l i n g , even though there was clear evidence o f dye filling the injected c e l l a n d we used the e n d - o n enhanced sensitivity of illumination, w h i c h was sensitive to dye filling o f the i m p a l e d c e l l b y diffusion alone. A l t h o u g h the results were negative, these experiments suggest either that control o f gap junctional permeability is not a simple function o f the cytosolic free c a l c i u m concentration or p H or that some other process interferes w i t h dye c o u p l i n g through this protein. A l t h o u g h Shaw a n d Stowe (14) reported that L u c i f e r y e l l o w d i d not cross gap junctions o f these


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cells, they neither injected dye directly into the t e r m i n a l n o r t r i e d to manipulate the permeability o f the j u n c t i o n to i n d u c e c o u p l i n g . Because w e used dyes of various l i p i d solubility a n d charge, i f the channel was open, dye solubility, resident charge i n the channel, or charge o f the molecule d i d not exclude passage o f the tracer. T h e simplest explanation o f o u r negative result is either that the channel i n the gap j u n c t i o n is too small to accommodate the dyes used or that some other molecule obstructs the entrance to the channel. M a n y insect tissues are c o u p l e d b y gap junctions. I n salivary glands (30), cuticular epithelia (31, 32), a n d the segmental partition o f imaginai discs (33), dye c o u p l i n g v i a gap junctions occurs readily. H o w e v e r , d u r i n g develop ment, the extent o f dye c o u p l i n g diminishes as compartmentalization o f the tissue proceeds, whereas electrical c o u p l i n g remains. T h e process i n v o l v e d i n sealing off these compartments may be similar to the process seen i n photoreceptor axon terminals. I n o u r future research, dissection o f the control mechanism w i l l focus o n manipulation o f the m e m b r a n e a n d gap j u n c t i o n proteins themselves i n the intact preparation. C o u p l i n g effects o n synaptic transmission can be assessed b y r e c o r d i n g f r o m these interneurons. T h e c o m b i n e d approach o f pharmacology, electrophysiology, a n d histochem istry o f the same tissue w i l l lead to a better understanding o f not only function o f neuronal gap junctions i n their native membranes, but also information processing at the first synaptic layer o f the retina. C o n d u c t a n c e o f gap junctions between salivary gland cells o f another dipteran, Drosophila, is voltage sensitive b o t h to the intracellular voltage as w e l l as the transjunctional voltage (34). If gap junctions i n the photoreceptor terminals o f o u r preparation w e r e o p e n , dye s h o u l d have entered the c o u p l e d cell. I f gating the junctions were a function o f adaptation, light or dark adaptation of the cells s h o u l d have o p e n e d the junctions a n d allowed dye coupling. E v e n though w e obtained stable recordings f r o m the terminals, w e saw no dye c o u p l i n g , perhaps because the i m p a l e d t e r m i n a l was damaged b y the electrode a n d the resting potential was no longer physiological. B a s e d o n this premise, we injected current f r o m 0.5 to 30 n A o f b o t h polarities to modulate transjunctional as w e l l as intracellular voltage o f the t e r m i n a l . Still, no dye c o u p l i n g was seen.

Voltage Coupling across Gap Junctions in Axon Terminals T h e strength o f electrical c o u p l i n g between adjacent terminals was measured b y intracellular recording directly f r o m the axon t e r m i n a l . A spatially filtered and expanded laser b e a m i l l u m i n a t e d two diaphragms that were i m a g e d at the back focal plane o f a 2 5 - m m achromat lens used as an objective. T h i s apparatus p r o d u c e d a small luminous spot that subtended a solid angle o f 3.4 a r c m i n to the fly eye. T w o other, moveable diaphragms were p l a c e d i n the image plane of the same objective to f o r m field diaphragms that allowed



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illumination o f any two facet lenses i n the field. O n c e a photoreceptor axon t e r m i n a l was recorded, the c e l l was brought o n axis to the stimulus b y m o v i n g the animal i n orthogonal arcs u n t i l a local m a x i m u m response was recorded. T h e animal was m o v e d i n planar coordinates u n t i l the facet that contained the photoreceptor entered the i l l u m i n a t e d field. B o t h field diaphragms were i m a g e d onto the same facet. B o t h b e a m intensities were adjusted to give a criterion response i n the r e c o r d e d photoreceptor axon terminal. T h e n the field diaphragms were used to selectively illuminate photoreceptors whose c o u p l e d axon terminals f o r m e d synapses i n the same cartridge as the r e c o r d e d cell. Experiments show that neighboring terminals are tightly c o u p l e d at all intensities f r o m the p h o t o n range to near saturation. T h e c o u p l i n g ratio can be defined as the voltage r e c o r d e d w h e n the adjacent t e r m i n a l is stimulated d i v i d e d b y the voltage recorded i n response to stimulation o f the c e l l whose t e r m i n a l is i m p a l e d b y the electrode. I n this case, the ratio is between 0.7 a n d 0.9, w h i c h indicates tight c o u p l i n g ( F i g u r e 9a). Imaging stimuli onto cells that do not contribute to the same cartridge resulted i n either an unrecordable response or a response f r o m the i m p a l e d t e r m i n a l w i t h less than 1 0 % o f the amplitude o f a response f r o m c o u p l e d cells. Therefore, 1 0 % is considered the m a x i m u m contribution due to light scatter i n o u r imaging system. T h i s c o u p l i n g factor compares w i t h 0.5 for V a n H a t e r e n s measurements v i a corneal neutralization. B o t h stimuli were brought closer together u n t i l they overlapped i n t i m e . T h e two signals s u m m e d unexpectedly ( F i g u r e 9b). Because stimulus inten sity was the same for b o t h photoreceptors, voltage i n each t e r m i n a l s h o u l d have b e e n the same, w h i c h w o u l d cancel any c o u p l i n g current. T h i s s u m m a tion suggested that o u r original assumption—that all inputs to the monopolar dendrites are e q u a l — m a y not be correct. I f so, the nonlinear interaction should be integrated by the monopolar nonlinearly. T o verify this, w e p e r f o r m e d the same experiment w h i l e r e c o r d i n g f r o m a monopolar c e l l . W h e n b o t h receptors were i l l u m i n a t e d together, the amplitude o f the monopolar response was actually less than the amplitude w h e n either recep tor was i l l u m i n a t e d ( F i g u r e 9c). S u c h an interaction suggests an inhibitory process. Perhaps the e n c o d i n g o f the monopolar dendrite is a differential process rather than a summatory process. T h e observed nonlinear behavior may be due to feedback synapses or other lateral interactions, w h i c h for this discussion we have ignored. O p t i c a l isolation o f the stimulus to photorecep tors that contribute synapses only to the cartridge that contains the r e c o r d e d monopolar c e l l suggests that lateral interactions are m i n i m i z e d . I f lateral interactions are present at all, they must be due to light scatter o f the stimulus to neighboring ommatidia, w h i c h w e measured at less than 1 0 % o f the response to stimulation o f cells directly synapsing w i t h the recorded monopolar c e l l . A n inhibitory process is consistent w i t h lateral current shunting through the gap junctions to neighboring terminals. A mechanism o f electrotonic




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Figure 9. Traces of intracellular recordings from photoreceptor axon terminals (a and b) while the soma of the cell and the soma of a coupled cell were stimulated by a laser and from a monopolar cell (c) while two coupled photoreceptors were stimulated. Half-second stimuli were used in all three cases. In a and b the photoreceptor that contributes its axon terminal to the same cartridge was stimulated by a laser with increasing intensity, and the voltage was recorded from the impaled terminal. The recordings show that coupling is strong between adjacent axon terminals and also that disparate information is retained and encoded by the monopolar cell.


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inhibition, w h i c h involves voltage i n a restricted extracellular space, was first proposed for the w e l l - k n o w n M a u t h n e r c e l l (35, 36). A modification o f that m o d e l can be used to explain o u r experimental results. G l i a l isolation of each cartridge ( 2 7 )

together w i t h h i g h o h m i c resistance across the basement

m e m b r a n e imparts h i g h input i m p e d e n c e for the photoreceptor i n p u t to the lamina. H i g h i m p e d e n c e prevents voltage degradation a n d improves Somas o f nonstimulated photoreceptors

fidelity.

w i t h i n the same cartridge w o u l d


provide a virtual g r o u n d for current that w o u l d be d r a w n through the gap j u n c t i o n a n d back to nonstimulated somas. T h e distal position o f gap j u n c tions (13) prevents passage o f current through the m a i n b o d y o f a nonstimu lated photoreceptor terminal; however, current is directed antidromically through its axon. Therefore,

current w o u l d not contribute to

transmitter

release f r o m that t e r m i n a l a n d input w o u l d be i n h i b i t e d functionally. M a i n t e nance of extracellular voltage c o u l d explain the apparent voltage summation i n the terminals. I n this case, depolarization o f the extracellular space w o u l d i n d u c e current flow back toward the retina through the unstimulated p h o toreceptors. A t l o w luminance, extracellular voltage does not saturate a n d shunting of small currents c o u l d actually s u m i n the electrically c o u p l e d terminals. A similar m o d e l was proposed b y Shaw (23) as a nonsynaptic basis for lateral i n h i b i t i o n w i t h neighboring cartridges. T h i s mechanism, w h i c h operates between cartridges, may be responsible for an antagonistic surround. If a similar mechanism operates w i t h i n a single n e u r o o m m a t i d i u m , it c o u l d f o r m a substrate for hyperacuity i n the c o m p o u n d eye a n d functional units i n the vertebrate retina as w e l l . B y use o f a high-contrast stimulus to differentially stain the cells i n the n e u r o o m m a t i d i u m , w e have shown that optical disparity o f individual p h o toreceptors

that f o r m the input to l a m i n a cartridges i n the fly eye

is

maintained. E l e c t r i c a l c o u p l i n g o f these same axon terminals is h i g h at l o w and moderate

luminance. E v e n though evidence shows that c o u p l i n g is

structurally mediated b y gap junctions, these junctions do not allow dye c o u p l i n g a n d dye c o u p l i n g is insensitive to treatment that w o u l d o p e n closed junctions i n other cells. Quantification o f electrical c o u p l i n g remains to be d e t e r m i n e d b y c o n ductance measurement. These experiments are i n progress. H o w e v e r , at l o w a n d moderate luminance, it is sufficient that the gap junctions r e m a i n i n a conducting state to accomplish b o t h goals o f this system: elevated sensitivity at l o w luminance b y allowing lateral spread o f current to c o u p l e d photorecep tor terminals a n d contrast enhancement o f individual inputs f r o m optically disparate receptors; b o t h functions use current shunted through gap j u n c tions. Therefore, the original postulate that gap junctions o p e n a n d close w i t h respect to light adaptation is not even necessary.

M o r e o v e r , the use

of

constitutively o p e n gap junctions that enables the transition f r o m summation at l o w intensity to i n h i b i t i o n at moderate intensity c o u l d be a continuous function o f stimulus contrast. T h e morphological a n d physiological evidence


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presented here does not prove that the proposed mechanism underlies hyperacuity. H o w e v e r , it is a plausible explanation that functions w e l l i n a computer simulation (37) a n d shows that, at least at the level o f the monopolar dendrites, disparate information is maintained a n d e n c o d e d b y the monopolar cells. T h e advantage o f this system is that information about the absolute position o f a point source o f light is preserved i n an analog signal and yields h i g h resolution that is not l i m i t e d b y spacing o f retinal detectors.

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RECEIVED for review January 2 9 , 1991. ACCEPTED revised manuscript January 26,

1993.


Structure and Function of Gap Junctions in the Photoreceptor Axon

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