CHROMATIC ORGANIZATION AND SE

From: PHOTORECEPTORS Edited by A. Borsellino and L. Cervetto (Plenum Publishing Corporation, 1984)

CHROMATIC ORGANIZATION AND SEXUAL DIMORPHISM OF THE FLY RETINAL MOSAIC Nicolas Franceschini Institut de Neurophysiologie et Psychophysiologie CNRS, 31, Chemin Joseph-Aiguier 13277 Marseille Cedex 9, France ·INTRODUCTION Whether in vertebrates or in invertebrates, mapping the spectral organization of a retinal mosaic with single cell resolution often remains an insuperable task, for which microspectrophotometry and intracellular recordings appear as cumbersome tools •. The need for methods capable of revealing at a glimpse the mosaic pattern of the indjvidual spectral types across a large receptor array has led to many ingenious techniques, some of which are listed in Table 1. This account presents the knowledge we have gained over the last few years about the spectral properties of individual receptor cells and their topographie distribution across the fly retinal mosaic. Several methods used for analyzing the properties of single cells (microspectrophotometry, intracellular recordings, intracellular dye injections and electron microscopy) have been associated with a technique of "ommatidial fundus fluoroscopy". This technique reveals individual spectral types of receptors in the live insect from their characteristic autofluorescence colour (Fig. 2). Like many vertebrates, flies possess a duplex retina. The chromatic organization of the retinal mosaic now appears amazingly complex with a number of spectral types that is reminiscent of the highly differentiated avian retina (Bowmaker and Knowles, 1977; Mariani and Leurre du Pree, 1978).

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Table 1.

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Sorne methods which have been used to reveal the topographie distribution of the various spectral types of retinal receptors with single cell resolution. The methods of group (I) merely provides a way of labelling various receptor types. The possible presence of screening or sensitizing pigments in some photoreceptor cells calls for caution in inferring the spectral sensitivity from transmission measurements. The methods of group (II) offer a more direct insight into the spectral sensitivity of the revealed photoreceptor cells.

Method

Authors

Spectral absorbance

Denton and Wyllie Scholes Kirschfeld et al.

(1955) (1975) (1978)

Receptor autofluorescence

Liebman and Leigh Franceschini et al.

(1969) (1981)

Use of oil droplets

Brown Granda and Haden Meyer and May Bowmaker and Knoles Mariani and Leure Du Pree Kolb and Jones

(1969) (1981 a, b) (1969) (1970)

Slective uptake of dyes

De Monastrio et al.

(1981)

Activity staining

Gribakin Marc and Sperling Levine et al. Basinger et al. Fernald

(1969) (1976) (1979) (1979) (1981)

Butler Menzel

(1971) (1972)

Selective induction of pigment migration

I

(1978) (1982)

Il

SEXUAL DIMORPHISM OF THE FLY RETINAL MOSAIC

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As regards the neural processing of visual information, an interesting aspect of insects and in particular flies, is the possibility to correlate physiological and behavioural data. Hence the two last chapters concern a characteristic sexual dimorphism of the receptor mosaic which is likely to play a role in the sexual pursuit of the female by the male. The emerging message is of ethoneurological nature, which states that looking at the behaviour of an animal may help understand even the post peripheral part of a neurosensory circuitry. STRUCTURE AND AXONAL PROJECTIONS OF THE PHOTORECEPTOR CELLS Two compound eyes containing a total of ~ 50 000 receptor cells confer upon the housefly a panoramic vision (Fig. 9a). In each of the 3000 ornmatidia of an eye, the receptor cells are arranged in groups of 8, according to a characteristic, asyrnmetrical pattern (Fig. la, b, c). The light-sensitive part of a cell is called a rhabdomere (hatched in Fig. la, b, c), which is a slender rod (diameter ~ 1 µm) acting as an absorbing waveguide. Six peripheral cells (R 1-6) encircle two smaller cells (R 7-8) whose rhabdomeres curiously lie on top of each other and build a continuous light-guide (Fig. le). As a consequence, R7 inevitably acts as a screen for the underlying R8. Bath peripheral (Rl-6) and central (R7-8) cells respond to light with a depolarizing receptor potential (J~rcilehto, 1971; Smala and Meffert, 1975; Eckert et al., 1976; Hardie, 1977) having a peak level of~ 50 mV and a time-to-peak of ~10-50 ms (Scholes, 1969; Hardie, 1977) . Due to the neat separation of the 7 rhabdomere endings in the focal plane of the corneal lenslet, each ornmatidium samples the few degrees of its visual field along seven eigen-direction (Kirschfeld, 1967; Kirschfeld and Franceschini, 1968). But the projection of the receptor axons onto the first optic ganglion (the lamina or outer plexiform layer) takes advantage of this situation not for improving the angular resolution of the eye but rather for improving its quantum catch. Each cartridge of the lamina receives axons from six receptor cells (of type 1-6) which belong each to a different ornmatidium but which look all in the same direction of space. This is the remarkable connectivity principle of the "neural superposition eye" of flies (Vigier, 1909; Braitenberg, 1967; Kirschfeld, 1967; Reviews in Kirschfeld, 1972; Braitenberg and Strausfeld, 1973; Shaw, 1981). In view of the photon noise affecting each receptor channel, a ben-

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Fig. 1.

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(a-c) Sensory outfit of a housefly ommatidium (after Trujillo-Cenoz and Melamed, 1966; Boschek, 1971). (a) six receptor cells Rl-6 whose longitudinal section is shown in (b) surround two smaller, central cells R7 and RB which lie on top of each other as shown in (c). The hatched part of each cell is the rhabdomere, which houses the visual pigment(s). (d) method used for visualizing the rhabdomeres in vivo in a large number of ornmatidia (see Fig. 2). "Onnnatidial fundus fluoroscopy" is achieved by optically neutralizing the corneal surface with a drop of water and observing the distal receptor endings with epi-fluorescence microscopy (blue excitation). (from Franceshini et al., 1981 b).

eficial effect of the signal summation which takes place within a cartridge (Scholes, 1969) appears to be an improvement (b y factor 16) in the signal-ta-noise ratio, a parameter which sets limits to bath absolute and contrast sensitivity. A slight variant of this scheme is encountered at the equator of the eye, on each side of which the rhabdomere patterns exhibit a mirror-image symmetry (Dietrich, 1909). Here 7 to 8 receptor cells of type 1-6 looking in the same direction of space (Kirschfeld , 1967) and it has been shown that the lamina cartridges correspondingly receive 7 to 8 axons instead of the usual 6 (Horridge and Meinertzhagen, 1970; Boschek, 1971). This provides a kind of "visual streak" with improved quantum catch that could help the fly navigate in low light level environments (Franceschini, 1975). For each sampling direction, vision is brought about by two visual subsystems having co-axial receptive fields. The one is mediated by the second order neurons leaving each cartridge and projecting to


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various sublaminae of the medulla, the other is mediated by the two central cells R7 and R8 whose axons bypass the underlying cartridge and project directly to the medulla (Cajal and Sanchez, 1915; Trujillo-Cenoz and Melamed, 1966; Campos-Ortega and Strausfeld, 1972). An optical phenomenon which we have called the "reduced corneal pseudopupil" provides direct evidence for the strict coaxiality of the elementary receptive fields of these two integrated visual subsystems (Franceschini and Kirschfeld, 197lb; and Franceschini, 1975). The present review summarizes our knowledge about the spectral properties of these two visual subsystems and about a conspicuous departure from the general connectivity pattern outlined above which affects a strategic region of the male eye. NATURAL FLUORESCENT LABEL ON EACH SINGLE CELL Early intracellular recordings made by Burkhardt (1962) have revealed that the compound eye of flies is equipped with receptor cells of various spectral sensitivities. The recent discovery of rhabdomere autofluorescence has shed a new light upon the precise chromatic organization of the retinal mosaic. The convex corneal lenslets of the fly's eye can be optically neutralized if one applies a clear medium of appropriate refractive index ( ~ 1.5) onto the corneal surface (Franceschini and Kirschfeld, 197la). Under such conditions (Fig . ld) each lenslet (diameter ~ 30µ m, see Fig. la) becomes a kind of tiny flat window through which one can discover the intimacy of the retinula. Recent association of this technique of "optical neutralization of the cornea" with epifluorescence microscopy has revealed that most rhabdomeres of the eye of flies are fluorescent under various excitations (Franceschini, 1977; Franceschini et al., 198lb). The most colourful palette is observed under blue excitation (e.g. 436 nm Hg-line) which simultaneously reveals a homogeneous population of red-emitting R 1-6 rhabdomeres and a mixed population of green- and non-fluorescing R7's (Fig. 2). These fluorescence phenomena are interesting in three respects. Firstly, they allow visual pigment properties to be studied in individual, living cells by using in vivo microspectrofluorimetry (review in Franceschini, 1982). Secondly, they confer upon each individual cell a characteristic color label which allows detailed mapping of the retinal mosaic to be carried out with single cell resolution (sections 7-8). Thirdly, they can be used for attributing to a given cell the results of an intracellular electrical recording (section 5).

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Fig. 2.

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In vivo epifluorescence observation of the individual photoreceptor cells in the eye of a f emale housefly (mutant white). The whole retina is more than 100 times larger than the retinal patch viewed here. Calibration bar: 10 µm. The method of "ommatidial fundus fluoroscopy" schematically described in Fig. ld was used here (neutralizing agent: water; objective: 25x water, numerical aperture 0,65; blue excitation: mercury arc 100 Watts with interference filter 436 nm; barrier filter: 510 nm; aperture filtering as described in Franceschini, 1982). On this black-and-white print of the original colour slide (Ektachrome 400 Asa), the colour-code is the following: grey spots = red-fluorescing Rl-6 rhabdomeres; bright central spots = green-fluorescing R7 rhabdomeres (R7y); dark central spots= non-fluorescing R7 rhabdomeres (R7p). The two bright and large spots reveal the yellow f luorescence of the two cells which have been impaled by thf microelectrode and stained by iontophoretic injection of procion yellow (see Section 5). (From Franceschini and Hardie, in preparation).

So far our analysis of the fluorescence colours observed under blue excitation can be summarized as follows. The red emission stems from a related form, M' of metarhodopsin, which is created under intense blue irradiation (Franceschini et al., 198lb; Stavenga et al., in prep.). The green emission belongs to a peculiar class of central rhabdomeres (R7y) which contain, in addition to their rhodop-


325

sin, a blue-absorbing accessory-pigment (probably S-carotene according to the three-fingered absorption spectrum measured with microspectrophotometry: Kirschfeld et al., 1978; Mac Intyre and Kirschfeld, 1981). Evidence that the green emission stems from this accessory pigment (rather than from the specific rhodopsin of this cell: Kirschfeld, 1979) is provided by recent fluorescence observations done under polarized blue excitation (Franceschini, unpublished). By contrast to the red emission from R 1-6 which is maximal when the E-vector of the exciting light is parallel to their microvilli (hatched in Fig. la) the green emission is maximal when the E-vector is perpendicular to the R7 microvilli. This is precisely what is expected if the ernission stems from the accessory pigment because the latter conf ers upon the cell an unusual dichroisrn such that maximal absorption in the blue occurs when the E-vector is perpendicular to the microvilli (Kirschfeld and Franceschini, 1977). However, assignment of the green emission to S-carotene is sornehow provocative in view of the reputedly non-fluorescing property of this molecule even at low temperature (Tric and Lejeune, 1970; Song and Moore, 1974). We are left with the hypothesis that the microenvironment of this molecule in the rnicrovillar membrane would be such as to allow some radiative deexcitation, whether the carotene remains as such or suffers a "retro" isornerism (Wallcave and Zechrneister, 1953). The fluorescence intensity of all cell types remains stable over hours under rnoderate blue excitation, thus allowing scrutiny of the retinal rnosaic (see section 6-8). Before we report the spectral sensitivity of these various receptor cells we first present some methods we now have on hand that allow rapid measurernent of the spectral sensitivity and rapid recovery of an impaled cell. FAST MEASUREMENT OF THE SPECTRAL SENSITIVITY OF SINGLE CELLS WITH A VOLTAGE-CLAMP METHOD The spectral sensitivity of a receptor cell can be defined ~s the reciprocal of the photon flux required at each wavelength to induce a criterion response (depolarizing or hyperpolarizing receptor potential). The voltage-clamp method we have devised (Franceschini, 1979) is a straightforward application of this definition. Its principle is to have the light flux impinging upon the cell autornatically adjusted at each wavelength in such a way that the receptor potential rernains clarnped at a given reference value V ref· This is achieved by a neutral density wedge whose position is co ntinuously controlled by the errorsignal between the reference voltage and the actual voltage delivered by the cell (Fig. 3).

326

N. FRANCESCH INI

MOT·

1 1

'11

1

1 1

t.,-.

Fig. 3.

Voltage-clamp technique for fast measurement of the spectral sensitivity of a receptor cell (a fly photoreceptor cell of type Rl-6 is here .schematically depicted, hit by a microelectrode). The purpose of the electromechanical feedback is to have the cell deliver a constant output voltage (equal to a reference voltage V ref.) and to record the grey wedge settings (POT . = Potentiometer) which realize this condition for each colour. The error signal between the actual receptor potential and the reference voltage V is amplified by a high-gain differential amplifier wh~~~· output controls the rotation of a miniature DC-motor (MOT.) and in turn the rotation of a quartz neutral density wedge (optical density 0-4).


327

In the "open loop mode" (Fig. 4a) the grey wedge has a fixed setting and stepping of the interference filter wheel induces a jump of receptor potential which depends upon many parameters (spectral sensitivity and characteristic curve of the cell, spectral emission of the lamp and transmission of each fil ter). In the "closed loop mode" on the other hand (Fig. 4b) any deviation of the cell's output from the reference voltage (here Vref = 8mV) is automatically nulled out. The grey wedge (Fig. 4c) continuously searches for the setting which yields a constant receptor potential (Fig. 4b). Under these conditions, the wedge setting D (À), which is translated by a potentiometer (Fig. 3), is a useful output from which a genuine spectral sensitivity of the cell can be determined subsequently. For this purpose each pair À, D is set again after the electrophysiological experiment and a radiometer reads the corresponding light flux, whose inverse is plotted versus wavelength as the spectral sensitivi ty of the cell (Fig. 4d). The main advantage of this method lies in the rapid uptake of the essential information required to (subsequently) determine the spectral sensitivity. In Fig. 4, it took only 20 seconds to explore a one-octave wavelength range in 20 discrete points. But the relatively rapid settling time (T~lOO ms) of the electromechanical servo-system allows spectral sensitivity measurements to be done within only three seconds provided the spectrum in continuously scanned rather than discretely stepped (Franceschini, in preparation). FAST RECOVERY OF DYE-INJECTED CELLS IN THE LIVING ANIMAL The blindly impaled receptor cell whose spectral sensitivity is being determined can subsequently be recovered in vivo provided a fluorescent dye be injected into the cell at the end of the electrophysiological recording (Franceschini and Hardie, 1980). In Fig. 2, two such cells were recovered in this way. Under blue excitation the stained receptor cells were identified imrnedia tely after the injections by the bright (yellow) fluorescence of their rhabdomeres (and to a lesser extent of their cell bodies) standing out from the f ainter (red or green) autofluorescence of most rhabdomeres. The two cells were penetrated one after the other with a micropipette filled with procion yellow. Following physiological measurements, iontophoretic injection of the dye was done in each case by passing a negative d.c. current (2 nA for 1 min.; i.e. ~ 0 . 1 µ C) across the "preparation". The two spotlighted rhabdorneres

328

N. FRANCESCHINI

..

CLAMPEO

i-...f 1

.

d ·l

•J JOO

40 0

500

WA.V ELENGTH

Fig. 4.

6 00

(nm]

To illustrate the operation of the servo-system schematized in Fig. 3 (a) "open-loop mode"; the feedback is disconnected and the receptor potential of the cell is free to jump from one value to another as the interference filter wheel is stepped at 1 Hz. Following a steady illumination at 620 nm, the filter wheel successively presents its 20 interference filters (halfwidth