Chapter 2 Neural architecture of the cat retina

CHAPTER 2

Neural Architecture of the Cat Retina HELGA KOLB* AND RALPH NELSON**

*University of Utah School of Medicine, Salt Lake City, Utah 84108, USA **The National Eye Institute, Bethesda, Maryland 20205, USA

CONTENTS 1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Light Microscopic A p p e a r a n c e of Retinal Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Golgi Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Reduced Silver Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Retrograde T r a n s p o r t of Horseradish Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Fluorescent Staining for Catecholamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22 22 27 28 28

3. Methods for Understanding Functional Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Electron Microscopy of Golgi Impregnated Retinal Neurons . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Electron Microscopy of Serial Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Intracellular Recording and Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Electron Microscopy of Horseradish Peroxidase Injected and Physiologically Characterized Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. P h a r m a c o l o g i c a l Techniques for Demonstrating the Presence of Neurotransmitters within Neurons of the Retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28 28 29 30

4. The Visual Message is Constructed at Different Levels of the Retina . . . . . . . . . . . . . . . . . . . . . . 4.1. The Outer Plexiform Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. The P h o t o r e c e p t o r Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Bipolar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. H o r i z o n t a l Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The Inner Plexiform Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Cone Pathways to Ganglion Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. B i p o l a r - G a n g l i o n Cell Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Role of the A m a c r i n e Cells in the Cone Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Rod Pathways to Ganglion Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. The Interplexiform Cell of the Cat Retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 39 39 41 43 43 43 44 45 47 53

5. Ganglion Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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H. KOLB and R. NELSON 1. INTRODUCTION

Despite a long tradition of physiological study, surprisingly little was known concerning the structure of the cat retina or the morphology of its neurons until a decade ago. The routing of information between photoreceptors and the then known variety of ganglion cell types was particularly obscure. Functions peformed in this inner retinal region were "black boxes" without attachment to morphologically identified or physiologically studied retinal interneurons. Over the past decade structure has been added to the void. Some known phenomena have been explained and startlingly many unsuspected functional circuits have been uncovered. Beginning with the pioneering work of Granit in the 1940s the retina of the cat was investigated with the electroretinographic and extracellular ganglion cell recordings. There followed a series of classical physiological studies on the ganglion cells (Kuffler, 1953; Barlow et al., 1957; Rodieck, 1965; EnrothCugell and Robson, 1966) which laid the groundwork for the more recent investigations of ever increasing sophistication. On the other hand, on the anatomical front, apart from the few studies that indicated there were probably two types of ganglion cell (Stone, 1965; Brown and Major, 1966) and a single type of horizontal cell (Dowling et al., 1966; Leicester and Stone, 1967), little was known of the morphology of the neurons in the cat retina. Even Ramon y Cajal's (1933) extraordinary Golgi studies of vertebrate retinas did not show many stained examples from cat retina and thus could not be relied on for a detailed morphological understanding. So it is only recently that physiological studies, particularly those using intracellular techniques (Grfisser, 1957; Steinberg, 1969a, b, c; Niemeyer and Gouras, 1973), have stimulated rigorous investigations of the morphology of the different cell types and an understanding of specific synaptic circuitry in the retina of this mammal. Steinberg (1969a, b, c) provided not only intracellular horizontal cell recordings in cat retina but also anatomical investigations that quantitated the ratio of rods and cones to ganglion cells (Steinberg et al., 1973). These studies demonstrated that the cat retina, similar to the retinas of human and subhuman primates, contained a foveal region

of high cone density surrounded by an annulus of high rod density. This confirmed the appropriateness of the cat retina as a model for some aspects of human vison, and, furthermore, since intracellular recordings of the individual neurons could be obtained, confirmed its advantage over human or primate for retinal studies. Response characteristics of cells, as they fitted into functional pathways could now begin to be unravelled in a mammalian retina.

2. L I G H T MICROSCOPIC A P P E A R A N C E OF R E T I N A L NEURONS In order to visualise the overall shape of the neurons of the cat retina four main anatomical techniques have been used; the Golgi technique, the reduced silver staining technique, fluorescence microscopy for catecholamine-containing cells and, more recently, retrograde staining of ganglion cells with the enzyme horseradish peroxidase. 2.1. Golgi Studies

Over the years several attempts have been made to study the morphology of the different retinal neurons in the cat using the method of Golgi (1873) (Dowling et al., 1966; Brown and Major, 1966; Leicester and Stone, 1967; Shkolnick- Yarros, 1971; Gallego, 1971b; Boycott and Kolb, 1973; Boycott, 1974; Boycott and Wfissle, 1974; Kolb and Famiglietti, 1974, 1976; Famiglietti and Kolb, 1975, 1976; Boycott et al., 1978; Wfissle et al., 1978; Wfissle and Rieman, 1978; Kolb et al., 1981). Particularly in the latter studies, examining whole-mounts of retina have given us a comprehensive understanding of the diversity of dendritic field sizes and shapes and complexity of branching patterns of the neurons in this retina. Our presertt knowledge indicates that four of the five basic neural types of the cat retina, the bipolar, horizontal, amacrine and ganglion cells, can be subtyped on morphological criteria that include branching patterns, cell body and dendritic field sizes, and stratification relative to each other. There are two main photoreceptor types in the cat retina, rods and cones. The cones are split into 2 or 3 spectral varieties (Daw and Pearlman, 1970;

23

NEURAL ARCHITECTURE OF THE CAT RETINA

Bat Ahc

Bhc

FIG. 1. Camera lucida drawings of Golgi-impregnated horizontal cell types of the cat retina. The A-type cell (Ahc) is large, stellate and has no axon. The B-type cell (Bhc) is smaller, bushier and emits a long axon that ends at 300 ~m from the cell body in a large multibranched axon terminal (Bat). Scale bar 100/~m. (From Kolb, 1974; Nelson et al., 1975.) Zrenner and Gouras, 1979; Crocker et al., 1980). The rods outnumber the cones in most areas of the retina by 6 3 : 1 except for the area centralis (Plate 1) where the cones are concentrated and packed at a ratio of 1 per 10 rods (Steinberg et al., 1973). At the first synaptic level, the outer plexiform layer (OPL) (Plate 1) rod bipolars and as many as 8 morphologically distinct cone bipolar cells (Fig. 5) connect with the photoreceptors to form the beginnings of the vertical pathways through the retina (Skolnick- Yarros, 1971; Boycott and Kolb, 1973; Kolb et al., 1981). In addition, the O P L is the site of interaction between two types of horizontal cell (Fig. 1), distinguished by the presence (B-type horizontal cell) or absence (A-type horizontal cell) of an axon (Gallego, 1971b; Fisher and Boycott, 1974; Boycott, 1974; Kolb, 1974; Nelson et al., 1975; Boycott et al., 1978), and photoreceptor and bipolar cells. At the second synaptic level, the inner plexiform layer (IPL) (Plate 1), the nine bipolar types have characteristic axonal branching patterns and intermingle with a variety of amacrine and ganglion cells types. It has become evident that the majority of the neurons of the IPL have arrangements of their axonal or dendritic processes in specific strata (Ca-

jal's S1 to $5) or sublaminae (a and b) (Famigletti and Kolb, 1976) so restricting their synaptic interactions (Plate 1). Like most other vertebrates, the majority of the neurons of the cat's IPL are monostratified in one particular stratum, even the axons of the bipolar cells (Fig. 5). In general the neurons with the largest radiate dendritic fields are strictly monostratified (Figs 2, 3 and Plate 3;) while the cells with small, profusely branched dendritic trees tend to be broadly stratified through 2 or 3 strata (Fig. 11) or even diffuse (Plate 2 and Fig. 11). A few cell types have large dendritic fields of extremely fine dendrites that are beaded at regular intervals along their length (Fig. 11). Five cell types of the cat IPL are bistratified and these include a bipolar type, two amacrines and two ganglion cell types (Kolb et al., 1981). The ganglion cells were originally classified into three morphological classes (Boycott and W~issle, 1974) named alpha, beta (Fig. 2) and gamma (Fig. 3) classes which correlated well with three basic physiological classes X, Y and W cells (EnrothCugell and Robson, 1966; Stone and Hoffman, 1972; Cleland and Levick, 1974a, b; Stone and F u k u d a , 1974). M o r e r e c e n t l y , a f u r t h e r physiological subtype with a morphological

24

H . KOLB a n d R. NELSON

lmm

A.C.

,

•i

t

l,

,,/

\

10mm Fl6. 2. Camera lucida drawings of Oolgi-impregnated alpha (a) and beta (/7) type ganglion cells of the cat retina. In the area centralis (A.C.) of the retina the cells have the smallest cell body and dendritic field sizes. Both cell types increase dramatically in size with eccentricity. Scale bars 50/am except for A.C. where it is 15/am. (Modified from Kolb et al., 1981 .)

NEURAL ARCHITECTURE OF THE CAT RETINA

PLATE 1. Light micrograph o f a vertical section of the cat retina at the area centralis. The different layers of the retina are shown. The ganglion cells are small in diameter and densely packed. The inner plexiform layer (IPL) can be subdivided into sublamina a and sublamina b. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer and G, ganglion cell layer. × 600.

25

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H . KOLB a n d R. NELSON

PLATES 2--7. Light micrographs of Golgi-impregnated amacrine and ganglion cells of the cat retina as seen in wholemount views. Plate 2: Small-field, diffusely branched A13 amacrine cell. x 300 Plate 3: Large-field amacrine A19 that stratifies in $2, snblamina a. x 300. Plate 4: Small-field broadly stratified ganglion cell (G9) that branches throughout sublamina a. x 400. Plate 5: Medium-field, delicately branched ganglion cell (G16) that stratifies at the top of sublamina b. x 360. Plate 6: Ganglion cell (GI8) that has dendritic field size and morphology like that of a beta type ganglion cell but it is narrrowly stratified at the top of sublamina a. x 280. P l a t e 7: Beta type ganglion cell occurring close to the G18 ganglion cell for comparison of dendritic morphology, x 280. (Modified from Kolb et al., 1981 ; Kolb and Nelson, 1981.)

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NEURAL ARCHITECTURE OF THE CAT RETINA

/

7b

/

tt

k.

\ \ y

\

~y f /

\

t

:"\\ t \

L \

FIG. 3. Camera lucida drawings of two gamma ganglion cells (ya and yb) (type G3 of Kolb et al., 1981) of the cat retina. Scale bar 100 I~m. (From Kolb et al., 1981.)

counterpart has been described, the epsilon cells (Leventhal et al., 1980; Stone and Clarke, 1980), and the original gamma cells have been subclassified into a further 21 different morphological types (Plates 4 - 6) (Kolb et al., 1981). Amacrine cells of the cat retina appear to come in at least 22 different morphological varieties (Plates 2 and 3; Fig. 11), some of which are now labelling with specific neurotransmitters by autoradiographic or fluorescence techniques (Ehinger and Falck, 1971; Voaden et al., 1977; T6rk and Stone, 1979; Pourcho, 1980a,b, 1982a,b; Nakamura et al., 1978, 1980; Holmgren- Taylor, 1982) indicating chemical as well as morphological differences between these cells.

2.2. Reduced Silver Studies

Two cell types in the cat retina stain well with the neurofibrillar staining methods: A-type horizontal cells and alpha type ganglion cells. (Honrubia and Elliot, 1969; Leicester and Stone, 1967; Gallego, 1971b; Boycott, 1974; W/issle and Rieman, 1978; Peichl and W~issle, 1981; W/issle et al., 1981a). The advantage of this staining technique over the Golgi technique is that throughout the entire retina all cells of these two types are stained, which gives us an understanding of their arrangement in mosaics. However, this technique is primarily useful in a nearest neighbour analysis of cell bodies. The overlapping dendritic fields are difficult to resolve

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H. KOLBand R. NELSON

from each other and the very finest branches and terminals are probably not stained. Even so an approximation of the dendritic field and its overlap with neighbouring fields can be attained. Thus, we now know that the A-type horizontal cells and the alpha ganglion cells are arranged in regular mosaics with increased nearest neighbour distances with eccentricity from the area centralis and concurrent increases in dendritic field sizes. Alpha ganglion cells (Fig. 2) can be distinguished into ON centre and OFF centre varieties each occurring in equal numbers and forming regular, superimposed, independent lattices. (W~ssle et al., 1981a, b). 2.3. Retrograde Transport of Horseradish Peroxidase

Injection of the enzyme horseradish peroxidase (HRP) into various visual nuclei that receive retinal afferents allows the ganglion cells of origin to be retrogradely labelled (Bunt and Minckler, 1977). In the cat visual system this technique has allowed labelling of ganglion cells that project to the lateral geniculate (Stone and Keens, 1980; Illing and W~ssle, 1981; W~.ssle et al., 1981a, b), the superior colliculus (Stone and Keens, 1980; W~ssle and Illing, 1980), the retinal recipient zone (Leventhal et al., 1980) and the accessory optic system (Farmer and Rodieck, 1982). Ganglion cell bodies are particularly well stained with these techniques but the penetration of the reaction product into the dendrites is extremely limited so that complete characterization of a cell's denditic morphology is unattainable for all but the largest diameter dendrites. Only the alpha cells can unequivocally be discerned because they form a unique class of extremely large bodied cells in any event. However, the other ganglion cell types need to be differentiated on the morphology and stratification level of their dendrites, as cell body sizes overlap for at least six different morphological ganglion cell types (Kolb et al., 1981). It is rather clear that the retrograde labelling technique has limitations for morphological descriptions of cells and quantitating specific morphological classes of cells, but immense value for understanding projection sites within the whole visual system.

neurons that contain biogenic amines as their probable neurotransmitter has revealed two different populations of amacrine cells in the cat retina (Ehinger, 1976; Ehinger and Floren, 1976; T6rk and Stone, 1979; H o l m g r e n - T a y l o r , 1982). The fluorescence characteristics of the one type of cell indicates that it is a dopamine-containing neuron. This amacrine typically has a large diameter cell body and a highly stratified dendritic spread close beneath the amacrine cell somata. The flat mount views of these cells (T6rk and Stone, 1979) show that the dendrites are curved around spaces sufficient to house another type of amacrine cell body, suggesting dendro-somatic contacts for this dopaminergic amacrine. Kolb et al. (1981) suggested that a counterpart in Golgi impregnated material was A18. The other amacrine cell type, though not natively fluorescent, can be induced to accumulate indoleamine, and furthermore, the branching pattern is different enough from the dopaminergic cell to distinguish it as a different cell type. It has a small cell body and a bistratified dendritic tree with its major dendritic ramifications in the neuropil proximal to the ganglion cell bodies (Holmgren-Taylor, 1982). The above mentioned anatomical techniques have revealed a profusion of different cell types that can be classified according to morphological criteria in the cat retina. The questions to be asked now are what is the significance of the various morphologies, and how are the different cell types organized into functional circuits? Are we able to understand these circuits in the context of the physiological properties of ganglion cells and the requirements of the visual message? To begin to understand functional circuits we must turn to other finer resolution techniques and combine them with physiological investigations.

3. M E T H O D S

FOR UNDERSTANDING

FUNCTIONAL PATHWAYS 3.1. Electron Microscopy of Golgi Impregnated Retinal Neurons

2.4. Fluorescent Staining for Catecholamines

The Falck Hillarp technique for demonstrating

Using the method first perfected by Stell (1965, 1967) for studying horizontal cell connections with

NEURAL ARCHITECTURE OF THE CAT RETINA

photoreceptors in the goldfish retina it has been possible to elucidate some of the connections of the O P L in the cat retina (Boycott and Kolb, 1973; Kolb, 1974). Thus the two distinct horizontal cell types, A-type and B-type (Fig. 1) can be shown to have connections with photoreceptors. Both the Atype and B-type horizontal cell bodies give rise to a number of fine dendrites bearing clusters of small (0.5 lam diameter) terminals (Plate 8). Each of these clusters or even individual terminals have been seen to contact the cone pedicles and end as lateral elements at the synaptic ribbons (Plates 9 and 10). The axon terminal arborization of the B-type horizontal cell consists of snake-like thick branches (2 - 3 lam diameter) which give rise to thousands of projecting processes (Plate 12) that terminate in the rod spherules, and like the cone contacting terminals, occupy the position of the lateral element (Plate 11). W~issle et al. (1978) consider the axon termina of the B-type horizontal cell to be in contact with 2000 to 3000 rods. The same technique has been applied to Golgi impregnated bipolar cells. Thus, Boycott and Kolb (1973) described the connections of bipolar cells with rods and cones in the cat retina. A rod bipolar was found which contributed dendritic terminals exclusively to approximately 25 rod spherules. Each dendrite occupied a central position below the synaptic ridge and synaptic ribbon in the invaginated group of processes of the rod spherule. Rod bipolar dendrites do not appear to make superficial or basal junctions in the cat, in contrast to the rod-contacting bipolars of some other species where such contacts occur in addition to the ribbon related contacts (Lasansky, 1971, 1973; Stell et al., 1977; West, 1978; Dacheux, 1982). Cone bipolar cells were also found in the cat retina. These were seen to make basal junctions or invaginating ribbon junctions with 4 to 12 cones (Boycott and Kolb, 1973). In that study no cone bipolar cells making both kinds of contact were seen, however. Thus, in the cat rod and cone photoreceptors are connected to independant bipolar cell systems at the OPL, a feature not shared by submammalian species, where many bipolar cell types contact both rods and cones (Lasansky, 1973; Stell et al., 1977; Dacheux, 1982). A further cell type that has been studied by EM of Golgi in the cat retina is the interplexiform cell

29

(Boycott et al., 1975). The dendritic terminals of these cells run in the OPL (Plate 13) but appear not to contact photoreceptors and instead end as projections against bipolar cell bodies or dendrites (Plate 14). The electron dense deposit of the silver chromate stain in Golgi impregnated profiles makes it difficult to discern pre-synaptic specializations within them by electron microscopy. For this reason the EM of Golgi technique has been of value only in looking at stained neurons postsynaptic to photoreceptor cells and has, thus, to the present time been limited to investigation of the OPL in the cat retina. To understand neurons that make dendro- dendritic synapses in the O P L or the IPL it has been necessary to adopt a procedure of examining large areas of the neuropil with EM serial section montages. By this means the synapses of the interplexiform cell upon bipolar cells, for example, could be visualized (Plate 15) (Kolb and West, 1977). 3.2. Electron Microscopy of Serial Sections

Small neurons or portions of large neurons can be reconstructed from serial sections in the electron microscope. The technique has been to trace the outlines of processes from identified cell bodies in the inner nuclear layer (INL) or ganglion cell layer in successive sections and to align and accumulate serial drawings on a sheet of transparent plastic. One then compares reconstructed portions of cells with Golgi-impregnated counterparts. Neurons with small dendritic fields or axonal arborizations can be rather confidently assigned to specific morphological types by this method (Famiglietti and Kolb, 1975; Kolb, 1979). Furthermore, particular neural types often have a distinctive cytological appearance by which their processes can be identified and thus their relationship to new neurons under examination can be established. Plates 1 6 - 19 illustrate an All amacrine cell. Its cytoplasm is characteristically dark and contains large mitochondria. Typically there is a single apical dendrite and cluster of lobular appendages around the neck of the cell body. Serial section analyses and reconstructions show that All cells are postsynaptic to rod bipolar axons at dendritic spines carried on their larger dendrites in the lower portion of the neuropil close to the ganglion cells (Plates 16, 19). In contrast, the

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I--I. KOLB and R. NELSON

lobular appendages (Plates 17, 19) are the presynaptic sites on ganglion cell dendrites, cone bipolar axons and amacrines of the neuropil closer to the amacrine cell bodies. These appendages also serve as sites of input from other amacrine sources, such as the dopaminergic amacrine A18 (Pourcho, 1982a, b). A further characteristic of this amacrine is its long areas of gap junction between its major dendrites and the axon terminals of particular cone bipolars branching in the lower IPL (Plate 18.) Partial reconstruction and valuable information on the synaptic characteristics of the rod bipolar and three cone bipolar types (cbl, cb2 and cbS, see below) have also been achieved by the serial EM technique. An important realization become apparant early in these studies that is fundamental to our understanding of rod and cone pathways. Cone bipolar axons have many direct synapses with ganglion cell dendrites in the IPL but rod bipolars do not. Rod bipolars use instead a number of amacrine cell pathways (the All being one of them) to conduct rod driven information to ganglion cells in the cat retina (Kolb and Famiglietti, 1974). Examination of a group of neighbouring ganglion cells of the area centralis revealed fundamental differences between synaptic input of the proximal and distal neuropil of the IPL to ganglion cell dendrites. Ganglion cells with dendrites close to the amacrine cell bodies received input only from cb 1 and cb2 type cone bipolars, and the apical dendrites of such ganglion cells were not contacted by bipolars such as cb5 or cb6 that branch in the proximal neuropil. Conversely ganglion cells with branching patterns restricted to the proximal neuropil received only the cb5 and cb6 input. In other words cone bipolar input to ganglion cells is restricted to the specific ganglion cells that arborize in the same layer as the bipolar axon terminals (Kolb, 1979). At the same time, intracellular recordings and stainings of ganglion cells to determine stratification of their dendrites, implied that this segregation of cone bipolar innervation patterns onto specific ganglion cell types might be highly significant (Nelson et al., 1978). 3.3. lntraeellular Recording and Staining

Following Steinberg's (1969b, c, d) initial success with intracellular recordings of horizontal cells in vivo in the cat retina, Niemeyer and Gouras (1973)

and ultimately, Nelson et al. (1975) perfected a perfused in vitro preparation (Nelson, 1977) which allowed virtually every cell type of the cat retina to be impaled and recorded with micro-electrodes. Iontophoresis of flourescent dyes such as Procion or Lucifer after recording photoresponses of the cells added the dimension of comparing physiologically characterized cells with Golgi impregnated counterparts (Nelson et al., 1975, 1976), and in some cases with cells serially reconstructed by electron microscopy. The cells of Plate 20 are examples of the two horizontal cells and the axon terminal of one type, the B-type horizontal cells, viewed by f l o u r e s c e n c e m i c r o s c o p y a f t e r intracellular staining. As can be seen these cells match remarkably well the Golgi- impregnated counterparts of Fig. 1 and there is little difficulty in determining which type is which. Their intracellular responses to light are shown to the right of each cell. All three units respond with slow hyperpolarizations to light, typical of horizontal cells, but there are some differences in their waveforms and in the relative amount of rod and cone contributions to the responses (see below). The one drawback of the fluorescent staining techniques has proved to be the inability to look at such stained cells directly in the electron microscope for assessing synaptic circuitry. An alternate staining technique had to be developed. 3.4. Electron Microscopy of Horseradish Peroxidase Injected and Physiologically Characterized Neurons

The use of the enzyme horseradish peroxidase (HRP) instead of the fluorescent dyes for intracellular marking of neurons has opened yet another dimension to the study of functional anatomy. The injected cells, when reacted with diaminobenziadine and H20~ and post-fixed with osmium, contain a fine granular electron dense deposit throughout the cytoplasm spreading into the finest terminals. Stained cells can be viewed by light microscopy and have the appearance of a superior Golgi p r e p a r a t i o n (Plate 21). By electron microscopic examination such stained processes can easily be distinguished and synaptic relationships with surrounding recognisable profiles can be determined. Thus the beta ganglion cell of Plate 21 is

NEURAL ARCHITECTURE OF THE CAT RETINA

PLATES 8-- 12. Light and electron micrographs of Golgi impregnated horizontal cell processes in the cat retina. Plate 8: Cluster or dendritic terminals arising from a major dendrite of an A-type horizontal cell. x 2000. Plate 9: Endings of the clusters of dendritic terminals as lateral, elements in the synaptic ribbon complexes of a cone pedicle (CP). x 24500. Plate 10: A dendritic terminal of a B-type horizontal cell ends as a lateral element at the synaptic ribbon in a cone pedicle (CP). x 28000. Plate 11: A x o n terminal processes of B-type horizontal cells end as lateral elements of the synaptic complex in rod spherules (RS). x 19500, Plate 12: Light micrograph of a vertical section of axon terminal branches (Bat) of a B-type horizontal cell. x 2000. (From Kolb, 1974.)

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H . KOLB a n d R . NELSON

PLATE 13. Light micrograph of a Golgi-impregnated interplexiform cell of the cat retina. The cell body lies in the amacrine cell layer of the INL and the majority of its dendrites branch throughout the IPL. A single process arises from the top o f t h e soma and passes up to the O P L to run for several hundreds of microns, x 900.

PLATE 14. Electron micrograph of a Golgi impregnated profile from an interplexiform cell. The stained process abuts a rod bipolar cell body (RB) at the 1 N L / O P L border, x 12000.

PLATE 15. Interplexiform cell profile as seen in serial section electron microscopy. The process is presynaptic to a rod bipolar cell body (RB) at the I N L / O P L border, x 12000. (From Kolb and West, 1977.)

NEURAL ARCHITECTURE OF THE CAT RETINA

PLATES 1 6 - 19. Electron microscopy and summary drawing of an AII amacrine cell o f the cat retina. Plate 16: AII amacrin~-soma and single apical dendrite (ad) which branches at the sublamina a/sublamina b border to give rise to small spines postsynaptic to rod bipolar axon terminals (RB) (ribbon circled). Portions of the main dendrite form gap junctions (gj) with a cone bipolar type (CB). x 4000. Plate 17: Lobular appendage (la) arises from the apical dendrite (ad) of the AII amacrine cell and is presynaptic to ganglion cells in sublamina a. x 10000. Plate 18: Enlargement of the gap junction between an AII dendrite and a cone bipolar axon terminal (IB). x 88000. Plate 19: Drawing of an AII amacrine cell reconstructed from serial sections. The lobular appendages proximal to the cell body are presynaptic to ganglion cells (G) and cone bipolars (CB). The distal spines are postsynaptic to rod bipolar axons (RB) at chemical synapses and contact cone bipolar axon terminals (cg) and amacrine profiles (ag) at gap junctions. Shaded profiles indicate cut surfaces of the cell going beyond the reconstructed portion. (From Famiglietti and Kolb, 1975; Kolb, 1979.)

33

34

H . KOLB a n d R. NELSON

Ahc

J

Bhc

I

Bat

PLATE 20. Intracellular recordings and stainings of the horizontal cell types of the cat retina. Procion stained, cells on the left and the physiological response of each cell on the right. Superimposed intracellular responses are to rod-saturating 400 n m stimuli of increasing intensity. The largest peak deflection and the longer " r o d after effect" correspond to the highest intensity stimulation. Stimulus duration 520 msec. Calibration bar (left) 50 tam. Calibration bar (right) 10 mV. (From Nelson et al., 1976.)

NEURAL ARCHITECTURE OF THE CAT RETINA

~a

r"

,

III

21

PLATES 21 -- 24. O f f centre beta ganglion cells injected with horseradish peroxidase (HRP) after intracellular recordings. Plate 21: Camera lucida drawing o f the beta cell as seen in vertical section. Scale bar 10/am. Plate 22: H R P stained dendritic varicosity postsynaptic to a cb2 cone bipolar axon terminal, x 22500. Plate 23: H R P stained varicosity postsynaptic to a cbl axon terminal, x 27000. Plate 24: H R P stained apical dendrite postsynaptic to a cb2 axon terminal• x 18000.

35

36

H . KOLB a n d R. NELSON

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PLATE 25. lntracellular recordings and Procion stain of a cat cone. (a) Responses of the cone to rod saturating blue (441 nm) stimuli. Calibration pulse 4.1 mV, flash length, 540 msec. Intensity response characteristics of the cone. 0 , 4 4 1 n m stimuli: II, 647 n m stimuli. (b) Procion stained cone. Calibration bar l0/am. (From Nelson et ak, 1976.)

NEURAL ARCHITECTURE OF THE CAT RETINA

PLATES 26-- 31. Bipolar contacts with photoreceptors. Plate 26: Rod bipolar dendrite (rb) makes a a central invaginating contact at the synaptic ribbon of the rod spherule. B type horizontal cell axon terminals (H) form the lateral elements. x 78000. Plate 27:Cb2 bipolar dendrites end as basal contacts, x 40000. Plate 28:Cb3 bipolars have dendrites ending as central invaginating contacts, x 30000. Plate 29: The dendrites of cb5 end as semi°invaginating contacts flanking the central invaginating bipolar dendrite, x 40000. Cb6 bipolar dendrites make central invaginating contacts but a cap of horizontal cell processes lie between the cb6 dendritic terminal and the synaptic ribbon of the cone pedicle, x 40000. Plate 3 1 : C b 6 dendrites can also end as basal contacts (arrow). x 40000. (From Nelson and Kolb, 1983; Kolb and Nelson, 1983.)

37

NEURAL ARCHITECTURE OF THE CAT RETINA

seen to receive synaptic input from two types of cone bipolar cell, cb 1 and cb2 (Plates 22 - 24) and various amacrines, some of which can be recognised as AII amacrine cells. Photoreceptors, horizontal cells, several bipolar cell types, amacrines and ganglion cells of the cat retina have been studied with the above technique. Much of what will follow in this paper is the data acquired by using the combination of intracellular recordings of physiological responses of the cell, comparisons of HRP stained cells with Golgi counterparts to classify a cell, and an analysis of synaptic circuitry seen with electron microscopy. 3.5. Pharmacological Techniques for Demonstrating the Presence of Neurotransmitters within Neurons of the Retina

Ehinger and Falck (1969) using the F a l c k Hillarp technique for inducing fluorescence in neurons containing catechol and indol derivatives demonstrated amacrine cell types in the cat retina that appeared to contain dopamine. Later, other research has shown (Dowling and Ehinger, 1975; Ehinger and Holmgren, 1979) that catecholamine neurons can be visualised in the electron microscope in order to study synaptic connections, by using a technique of pharmacologic lesioning. Retinas treated with 6-hydroxydopamine or 5,6- or 5,7-dihydroxytryptamine reveal populations of dopaminergic and indolamine containing amacrine cells. Comparisons of their labelling pattern in the IPL as viewed by light microscopy and their synaptic relationships in the IPL as viewed by electron microscopy (Holmgren-Taylor, 1982) makes it possible to draw parallels with the Golgi and intracellular marking techniques and thus identify these amacrine cell types. Thus, the dopaminergic amacrine cell in cat is probably A18 and the amacrine accumulating indoleamines may be A8 (Kolb et al., 1981). Autoradiographic labelling of amacrines with exogenously applied tritiated GABA, glycine, taurine, and dopamine (Pourcho, 1980a, b); 1982a, b; Nakamura et al., 1978; Sterling, 1983; McGuire et aL, 1982) has also been successful in determining the synaptic relationships at the ultrastructural level, of cells that have high affinity uptake for these substances. It is becoming clear that several popula-

39

tions of different morphological types of amacrines are labelling with GABA. Thus, Pourcho (1982a, b) now has evidence for five different amacrine cell types (A3, A13, A10, A17, A19 of Kolb et al., 1981 classification) labelling for GABA. In addition it appears that the interplexiform cell of the cat retina is GABA-ergic (Nakamura et al., 1980; Pourcho, 1981). On the other hand, there is good evidence for the inhibitory transmitter glycine to be contained within a completely different group of amacrine cells, the best characterized of which is the All amacrine (Pourcho, 1980b; Maguire et al., 1982). Evidence from EM autoradiography is also suggesting that the dopaminergic amacrine (probably A18 of Kolb et al., 1981) has specific synaptic connections with the cell body and lobular appendages of the All amacrine (Pourcho, 1982a). We are thus beginning to understand the interrelationships between pharmacologically, morphologically and synaptically distinct neurons of the cat retina. What remains is to bring this information together into an understanding of functional units and pathways, with the aid of data obtained from intracellular recordings.

4. THE VISUAL MESSAGE IS CONSTRUCTED AT D I F F E R E N T LEVELS OF T H E R E T I N A 4.1. The Outer Plexiform Layer

4.1.1. THE PHOTORECEPTORLEVEL Figure 4 summarizes the connections that are presently understood for the outer plexiform layer of the cat retina (Kolb, 1977). The bipolar pathways are segregated into one for rods but several varieties for the cones. Intuitively, one might expect the segregation to remain as strict through channels for rod or cone information with no mixing. However, intracellular recordings of all the purely coneconnected neurons, cone bipolar cells, A-type horizontal cells and B-type horizontal cell bodies, have shown that they all contain a moderate to high percentage of rod input. Plate 25 shows intracellular responses of a cone photoreceptor indentified as such by the iontophoresis of Procion dye [Plate 25(b)]. The intracellular responses [Plate 25(a)] are slow hyperpolarizations with two phases: one due

40

H. KOLB and R. NELSON

FiG. 4. Summary diagram of the neural connections in the outer plexiform layer of the cat retina. At the level of the synaptic terminals, cones (C) have basal processes that make minute gap junctions with rod spherules (R) and larger gap junctions with neighbouring cones. Rod bipolars (RB) connect only to rods while cone bipolars connect only to cones. Each bipolar type is characterized by a particular synaptic arrangement with its photoreceptor type. RB makes invaginating contacts, IB, invaginating contacts and FB, flat contacts. B type horizontal cells (BH) connect with cone pedicles but their axon terminals (AT) contact rod spherules. The axonless horizontal cell (AH) makes exclusiveconnections with cones and is joined to neighbouring A horizontal cells by gap junctions (thick lines). Specialized junctions (shaded) occur between A horizontal cell dendrites and cone bipolar dendrites. The interplexiform cell (I) extends dendrites into the OPL which are presynaptic to bipolar cell somata and dendrites. (From Kolb, 1977.) to cone signals, increasing in a m p l i t u d e with increasing light intensity, a n d the other due to rods b e c o m i n g progressively p r o l o n g e d with brightest stimuli. The intensity response curve to the 441 n m stimulus has a bilobed shape indicative o f a mixture of rod and cone signals. The slow hyperpolarizing after potential seen in the intracellular response is k n o w n to have the spectral characteristics of the rod photoreceptors (Steinberg, 1969c). The intensity response curve for the red 647 n m stimulus has the typical single s m o o t h shape, shifted to higher intensity ranges, of a cone driven response. Thus

the cone p h o t o r e c e p t o r in the cat retina, from the physiological response, appears to carry a high prop o r t i o n o f signals originating in the rod p h o t o receptors (Nelson, 1977). This mixing of rod a n d cone signals in the photoreceptors of the cat retina is carried forward t h r o u g h the n e u r o n s p o s t s y n a p tic to cones which also then c o n d u c t mixed rod/cone information. The answer to the question of how the rod signals are entering the cone p h o t o r e c e p t o r became app a r a n t from the electron microscope analysis of serial sections of the O P L . As Raviola a n d Gilula

NEURAL ARCHITECTURE OF THE CAT RETINA

(1975) described for rabbit OPL, cat cone pedicles contact surrounding rod spherules at small gap junctions (Kolb, 1977) (Fig. 4). There is no direct way of demonstrating whether these gap junctions also pass cone signals into rods because a rod photoreceptor has not been recorded from within the cat retina. However, in most areas of the cat retina rods outnumber cones by as much as 100 to 1 and it seems architecturally unlikely that the scarcer cones could be contacting every one of the surrounding rods in the OPL. In addition, the intracellular recordings of the two neurons that are postsynaptic solely to rods in the OPL, the rod bipolar and the axon terminal of the B-type horizontal cell, show that there is little if any detectable cone signal contributing to their responses. Plate 20 shows the responses of the axon terminal system in comparison with the responses of the Atype and B-type cell bodies to rod saturating blue stimuli. Both the A-type and B-type cells have cone type responses that increase in amplitude with the increased light intensity whereas the axon terminal response is essentially saturated by all stimuli, indicative of a pure rod response. Note the two phase rod-cone responses in the cone driven A- and B-type cell bodies. 4.1.2. BIPOLAR CELLS

The rod bipolar cell of the cat retina is of a single type which contacts rod spherules with an invaginating contact (Plate 26). Its physiological response to light is known to be a hyperpolarization (Nelson et al., 1976; Nelson and Kolb, 1983) similar in all probability to the response of the rod photoreceptor itself (although the responses of rods have yet to be identified in cat retina) (Fig. 6). Cone bipolar cells, on the other hand, make a variety of different contacts with cone pedicles. Basal junctions, invaginating contacts or semiinvaginating contacts have been observed for different bipolar types, so that it might be expected that in cone bipolars different response patterns might reflect the different types of synapses with the cone pedicle. Indeed this is the case. Figure 5 shows the light microscopic appearance of different bipolar types recorded intracellularly and marked with HRP. The cells are identified as to type by comparison with Golgi impregnated ex-

41

amples (Kolb et al., 1981) while, the nature of their dendritic contacts with cone pedicles can be seen by an EM evaluation (Plates 2 6 - 3 1 ) . Bipolar cell types that give hyperpolarizing responses to light include the rod bipolar, cb2 and cb6 (Fig. 6). Their synaptic contact with the photoreceptor can be either invaginating and ribbon related as in RB and cb6, or basal junctions that are not ribbon related as in cb2. Bipolar cells that give a depolarizing response like cb5 (Fig. 6) appear to have semiinvaginating contacts with an area of basal junction along their point of contact with cone pedicle (Plate 29). Thus, there does not seem to be a good correlation between the central invaginating dendritic contact and depolarizing responses, as was originally proposed (Raviola and Gilula, 1975; Famiglietti and Kolb, 1976; Kolb, 1979; Nelson et al., 1981). Although we do not yet fully understand the relationship between the type of synaptic contact and the light response of the cell postsynaptic to the photoreceptors one concept does become clear concerning the organization of the visual message at the OPL. For the cone system the original cone photoresponse of a hyperpolarizing slow potential is split into a two channel pathway consisting of a hyperpolarizing OFF centre and a depolarizing ON centre pathway at the synapse of the bipolar with the photoreceptor. In contrast, for the rod system the bipolar pathway appears to simply reflect the hyperpolarizing or OFF centre response of the rod photoreceptor and transmits it unchanged to the next level of processing. There are, thus, major physiological differences in rod and cone pathways to the IPL. As we have seen, the bipolar signal for the cone system in the cat retina has been divided into ON centre and OFF centre varieties which are handled by different sets of bipolar cells. Similar findings are of course well known now for all vertebrate retinas where bipolar cells have been studied with intracellular recordings (Werblin and Dowling, 1969; Kaneko, 1970; Naka and Ohtsuka, 1975; Yazulla, 1976; Miller and Dacheux, 1976; Lasansky, 1978; Marchiafava and Weiler, 1980). Also, in the other vertebrate species the bipolar cell response is seen to have a concentric organization. The surround reponse, of opposite polarity to the centre response, is thought to come from the

42

H. KOLB and R. NELSON

HRP bipolars /

i

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Flo. 5. Camera lucida drawings of vertically sectioned bipolar cells stained with H R P after intracellular recordings. Rod bipolar (rb) has dendrites reaching above the level of the cone pedicles to rod spherules and a fain:ly stained axon (dotted) that ends in lower sublamina b of the IPL. Cone bipolars cbS, cb6, cb2 and cb3 have clusters of dendritic terminals passing to cone pedicles and are distinguished from each other by the shape and stratification of their axon terminals in the IPL. Cb5 and cb6 have axons in sublamina b, cb2 in sublamina a and cb3 has an axon on the sublamina a/sublamina b border. Scale bar 10/~m. (From Nelson and Kolb, 1983.)

cb2

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FIG. 6. Intracellular responses of the HRP stained bipolar cells of the cat retina. The rod bipolar (rb), the cone bipolars cb2 and cb6 give hyperpolarizing responses to a full field light flash, while the cb5 bipolar gives a depolarizing response. Calibration bars for each cell are 0.5 sec and l mV. (Modified from Nelson and Kolb, 1983.)

horizontal cell, which has a large receptive field and synapses either directly with the bipolar cell (Dowling and Werblin, 1969; Naka, 1976) or indirectly through feedback to the photoreceptor (Baylor et al., 1971; Toyoda and Kujiraoka, 1982; Toyoda and Fujimoto, 1983). Only one of the bipolar types of the cat retina, namely the depolarizing cone bipolar cbS, has a demonstrable antagonistic surround (Nelson et al., 1981). In fact, none of the hyperpolarizing bipolars including the rod bipolar can be induced to show an antagonistic surround-effect. Their responses are very like those of the horizontal cell itself in that they have rather broad receptive fields perhaps ½ to ¼ the size of those horizontal cells. The depolarizing cone bipolar (cbS) has some of the physiological characteristics of a specific type of amacrine cell, the AII. In particular the rod nature of the cb5 responses suggest that, oddly, a component of its receptive field organization is generated in the IPL. An explanation for this will become apparent later when the organization of the IPL is considered. If, then, there is no surround anatagonism built into the bipolar response at the OPL, what is the role of the horizontal cells in the cat retina?

NEURAL ARCHITECTURE OF THE CAT RETINA

4.1.3. HORIZONTALCELLS Feedback shown to be of horizontal cell origin has been demonstrated in the cone photoreceptors of the turtle retina (Baylor et al., 1971) in the perchpike cones (Burkhardt, 1977) and by experimental manipulations of chloride in the Salamander cones (Lasansky, 1981). Feedback has otherwise been rather difficult to demonstrate in vertebrate photoreceptors including cones of the cat retina. The feedback synapse is believed to contribute colour-coding to the horizontal cells in species where there is good colour vision, such as the turtle (Fuortes and Simon, 1974) or carp retina (Toyoda and Fujimoto, 1983). The cat, although it has trichromacy demonstrable at the ganglion cell level (Ringo and Wolbarsht, 1981; Zrenner and Weinrich, 1981) has not yet been shown to have colour-opponency at the horizontal cell level (Nelson, 1977). Thus, feedback for colour-coding at the outer plexiform layer in the cat is not strongly supported. However, the horizontal cells with their greater spatial convergence than the photoreceptors (Nelson, 1977) integrate cone derived signals over a large area, and may adjust the function of the cone synapses in some fashion. The axon terminal system of the cat B-type horizontal cell is thought to function as an independent dendritic system from its cell body (Nelson et al., 1975) and integrates rod information over a field of 3000 rods (W~issle and Rieman, 1978). One possible role of the axon terminal system might be to increase the field size over which a rod bipolar can function by acting as a positive feed-forward system much as the gap junctions between toad rods are thought to do (Fain et al., 1976). Certainly, the receptive field sizes of rod bipolars far exceed their dendritic field sizes or even that expected from rod - rod coupling as seen in other species (Leeper et al., 1978). 4.2. The Inner Plexiform Layer 4.2.1. CONE PATHWAYS TO GANGLION CELLS

In 1969 it was reported that the cone midget bipolar pathway of the monkey retina consisted of a pair of bipolars that made different types of contact with the cone pedicles (Kolb et al., 1969; Kolb, 1970). The two midget bipolars were called invaginating and flat midgets according to their PRR3-B

43

cone contacts but they were also distinguished by the different termination levels of their axons in the IPL. Invaginating midgets end low, close to ganglion cell bodies, and flat midgets end higher, closer to amacrine cell bodies. Kolb (1970) suggested that the two levels of endings of the two midget bipolar cell types were correlated with the two different branching levels of midget ganglion cells (Polyak, 1941). Gouras (1971) further suggested that as ganglion cells of the monkey fovea came in ON centre and OFF centre varieties, the midget bipolar to midget ganglion cell chain was organized to subserve ON and OFF centre pathways. With the finding o f bipolars in cat retina analogous to those of monkey retina (Boycott and Kolb, 1973), although, of course, these were not midget bipolars, the parallel of bipolars subserving ON centre and OFF centre pathways was set for the cat retina too. Intracellular recordings and dye injections of cat ganglion cells revealed a correlation between the level of dendritic branching and receptive field centre response signs (Nelson et al., 1978). Ganglion cells that gave OFF centre responses, irrespective of their being morphologically alpha, beta or g a m m a ( B o y c o t t and W~issle, 1974) or physiologically X, Y or W (Enroth-Cugell and Robson, 1966; Stone and H o f f m a n n , 1972), or brisk sustained or brisk transient (Cleland and Levick, 1974a), had dendrites branching in the neuropil of the IPL closest to the amacrine cells (Fig. 7, top three cells). Conversely, ganglion cells that gave ON centre responses had dendritic branching restricted to the lower IPL nearer the ganglion cell bodies (Fig. 7, lower two cells). With the insight gained f r o m these intracellular recordings, it became clear that the morphological types of ganglion cells known as alpha and beta cells ( B o y c o t t and W~issle, 1974) o c c u r r e d in p a r a m o r p h i c pairs across the entire retina (Famiglietti and Kolb, 1976; W~issle et al., 1981a,b). Both alpha and beta cells occurred as a types that branched preferentially in the IPL close to the amacrines cells (Plate 21) and b types that branched in the IPL close to ganglion cell bodies (Fig. 7, lower two cells). The gamma cells of Boycott and W~issle (1974) have also now been restricted to a single morphological class (G3) that occurs as a and b types (Fig. 3) (Kolb et al., 1981). Famiglietti and Kolb (1976) thought that the ON

44

H. KOLB and R. NELSON

-

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Fic. 7. Intracellular recordings and drawings of Procion stained cat retinal ganglion cells. Cell types that have dendrites ramifying in sublamina a (top three cells) give OFF centre responses to a light flash. The ganglion cells are small bodied and equivalent to a gamma cell (G3), G9 and GI4 respectively (top left, top right and middle, respectively). The lower two ganglion cells are an alpha b type and a beta b type respectively and their intracellular response is ON centre to a flash of light. Both lower cells have their dendrites restricted to sublamina b. Wavelength: 647 nm. Duration 0.5 s. Calibration bars as indicated for each cell's response. Scale bar 100 ~m. (From Nelson et al., 1978.) centre a n d O F F centre b r a n c h i n g p a t t e r n of the g a n g l i o n cells could be extended to other n e u r o n s in the I P L especially to b i p o l a r axon terminals. Thus, the b i s u b l a m i n a r division of the I P L was introduced: s u b l a m i n a a where O F F centre ganglion cells b r a n c h e d was the one third of the neuropil close to the a m a c r i n e cells a n d encompasses C a j a l ' s S1 a n d $2, while s u b l a m i n a b, where O N centre ganglion cells b r a n c h was the two thirds o f the I P L reaching to the g a n g l i o n cell bodies consisting of C a j a l ' s $3, $4 a n d $5 (Plate 1, Fig. 7). Famiglietti e t al. (1977) further extended this conjecture to a m a c r i n e cells. Ironically the early n o t i o n of O N

a n d O F F l a m i n a t i o n of bipolar cell axon terminals, which was so i m p o r t a n t for developing the concept of different stratification for O N and O F F ganglion cells, is now modified. The a x o n terminals o f O N and O F F bipolars m a y be f o u n d at almost any level of the I P L . Nonetheless the different stratification of the dendrites for O N a n d O F F g a n g l i o n cells knows no exception. 4.2.2. B I P O L A R - GANGLION CELL SYNAPSES

A n analysis of E M serial sections of the cat I P L has provided insights into the nature of cone bipolar

NEURAL ARCHITECTURE OF THE CAT RETINA

ON FIG. 8. Wiring diagram of the cone bipolar to ganglion cell connections in the cat retina. OFF centre ganglion cells (Ga) have dendrites branching in sublamina a of the IPL and receive synaptic input from cbl and cb2 cone bipolars only. Cb2 is known to give a centre-hyperpolarizing response to light but the response of cb 1 is not yet known. ON centre ganglion cells branch only in sublamina b of the IPL and receive input from a centredepolarizing bipolar, cb5 and a centre- hyperpolarizing bipolar cb6. Black symbols represent hyperpolarizing, OFF centre units. Open symbols represent depolarizing ON centre units and stippled symbols have, as yet, unknown reponse polarities in this and the following wiring diagrams.

contacts with ganglion cells (Kolb, 1979). Beta type ganglion cells of the area centralis have been reconstructed from serial sections and their major bipolar input studied (Kolb, 1979; Stevens et al., 1980). The beta type a cells receive input only from cone bipolars that have axons restricted to sublamina a which are cbl and cb2. Beta type b cells, on the other hand receive input only from cone bipolars that branch in sublamina b which are cb5 and cb6 (Kolb, 1979; Stevens et al., 1980). The physiological response from intracellular recordings is known for three of these cone bipolar cells (Fig. 6) and the combination of the anatomical and physiological findings results in a wiring diagram (Fig. 8) that summarizes the direct cone bipolar to ganglion cell pathways as we understand them at present in the cat retina. Hyperpolarizing, OFF centre bipolar cells (such as cb2) have synapses with OFF centre ganglion cells and a combination of depolarizing, ON centre (cb5) and hyperpolarizing, OFF centre (cb6) bipolar cells have synaptic input to ON centre ganglion cells. Cb2 and cb5 are

45

thought to have excitatory synapses with the ganglion cells that they contact, however, cb6 might be inhibitory. Thus although the ON centre and OFF centre ganglion cell receptive fields are thought to originate from receptive field characteristics of the bipolar cell driving them (Naka, 1976; Miller, 1980; Marchiafava and Weiler, 1980), the IPL circuitry of cat retina suggests more complex interactions than this. It is quite clear that cb6 a hyperpolarizing, OFF centre bipolar cell has direct synaptic contacts with ON centre ganglion cells of sublamina b (Plate 32) (Nelson and Kolb, 1983). Sterling (1983) and Maguire et al. (1982) have some evidence that a bipolar cell corresponding to our cb6 takes up the inhibitory neurotransmitter glycine. They suggest, therefore, that the cb6 synapses with ON centre ganglion cells will be inhibitory and that cb6 might provide OFF inhibition to these ganglion cells. In the cat retina OFF inhibition in ON centre ganglion cells is sensitive to antagonists of the neurotransmitter GABA (Ikeda and Sheardown, 1983); however, in lower vertebrate retinas such inhibition may be either gabaergic or glycinergic (Frumkes et al., 1981). So far in any vertebrate retina, the cat included, GABA has only been demonstrated in amacrine cells and thus amacrine cells are thought to provide much of the OFF inhibition in ON centre ganglion cells. It would be intriguing if a glycinergic, inhibitory bipolar could be demonstrated to perform this 'amacrine' function as well. 4.2.3. ROLE OF THE AMACRINE CELLS IN THE CONE PATHWAYS

Two amacrine cell types that are thought to play a role in the cone pathways to ganglion cells have been recorded from, marked with dye injection and identified according to the Golgi classification of Kolb et al. (1981). The A4 is a small-field amacrine with a compact tufted dendritic tree that is restricted to the neuropil of sublamina a just above the a - b border. Serial section electron microscope examination (Kolb, 1979) has indicated that A4 receives synaptic input from cone bipolars that branch in sublamina a and synapses upon dendrites of OFF centre ganglion cells. Furthermore, the dendrites of A4 receive large numbers of synapses

46

H. KOLB and R. NELSON

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OFF

Fro. 9. Intracellular responses and wiring diagram of the A4 amacrine cell involved in the cone system. (a) The A4 gives a hyperpolarization to light. The receptive field of the unit has been mapped with a 50 tam wide slit stimulus flashed at different distances from the centre of the receptive field (indicated in Cm to the left of each trace). The limits of the receptive field are approximately 90 tam on either side of the centre. (b) Wiring diagram of connections of A4 as revealed by serial section electron microscopy. The cell is postsynaptic to cb2 bipolar cells in sublamina a of the IPL, makes reciprocal synapses to these bipolars and is presynaptic to OFF centre ganglion cells (Ga). It also receives many synapses from unidentified amacrine cells (A, large arrows). f r o m o t h e r as yet u n i d e n t i f i e d a m a c r i n e cell types. T h e s u m m a r y d i a g r a m (Fig. 9) d e p i c t s its connections a n d intracellular response to light. The cell gives a slow h y p e r p o l a r i z i n g response that is m a i n l y cone d r i v e n a n d has the s a m e sign t h r o u g h o u t its receptive field, so there is no suggestion o f a s u r r o u n d to this unit. Interestingly the receptive field is rather small as indicated by the response a m p l i t u d e to the n a r r o w slit stimulus d i s p l a c e d f r o m the receptive field centre [Fig. 9(a), 0 point]. Because o f this it seems intuitively unlikely that the A 4 is p r o v i d i n g s u r r o u n d i n p u t to the g a n g l i o n cell with which it synapses a n d so is m o r e likely p r o v i d i n g centre i n p u t like that o f the h y p e r p o l a r i z i n g cone b i p o l a r . In a d d i t i o n to the b i s t r a t i f i e d A l l a m a c r i n e cell o f the cat retina, which is k n o w n to be integral to the r o d p a t h w a y s , there is a bistratified a m a c r i n e cell, A8, which appears to be m o r e strongly involved in the c o n e s y s t e m . Serial s e c t i o n e l e c t r o n m i c r o s c o p i c analysis o f an H R P stained A8 shows its s y n a p t i c circuitry [Fig. 10(b)]. The lower s t r a t a o f dendrites are p o s t s y n a p t i c to r o d b i p o l a r a n d

p r e s u m e d cb6 axon terminals in s u b l a m i n a b. There a p p e a r to be reciprocal synapses to the input bipolar in all cases (Plate 35, Fig. 10). The finer d e n d r i t e s b r a n c h i n g in s u b l a m i n a a receive synapses f r o m either or b o t h cb 1 a n d cb2 as well as f r o m a n u m b e r o f u n i d e n t i f i e d a m a c r i n e cells. T h e A8 is also p r e s y n a p t i c in the n e u r o p i l o f s u b l a m i n a a to O F F centre ganglion cell dendrites (Ga) a n d to a m a c r i n e cell d e n d r i t e s o f e x t r e m e l y large d i a m e t e r with a r a d i a t e linear m o r p h o l o g y suggestive o f a m a c r i n e A 1 9 (Kolb et al., 1981). The i n t r a c e l l u l a r r e c o r d i n g s o f A8 show that it is a centre h y p e r p o l a r i z i n g unit with a very p r o n o u n c e d O F F response [Fig. 10(a)]. S t i m u l a t i o n with 647 n m light gave large transient O F F responses which were a b s e n t when the wave length used was the 441 nm light designed to s t i m u l a t e the rods equally. The unit is thus t h o u g h t to be much m o r e s t r o n g l y driven by the cone system. A n analysis o f the receptive field o f A8 [Fig. 10(a)] indicates that a strong depolarizing surround m e c h a n i s m comes into play when the slit stimulus is d i s p l a c e d by 500 to 700/am f r o m the receptive

NEURAL ARCHITECTURE OF THE CAT RETINA

(a)

POSmoN uM or 2~ u, suT

47

(b)

a

b

Ga

OFF

FtG. 10. Intracellular responses and wiring diagram of the A8 amacrine cell, involved in the cone system. (a) Intracellular response is a hyperpolarization to light with a pronounced off depolarization and spikes (0, centred slit). The receptive field indicates an ON-OFF, depolarizing surround response antagonistic to the centre mechanism at 700 pm on either side. (b) Wiring diagram o f A8 as revealed by electron microscopy of the H R P stained unit. This bistratified cell has varicose dendrites in sublamina b which are postsynaptic to cb6 and rod bipolar axon terminals, with reciprocal synapses to the bipolars. The fine dendrites in sublamina a are postsynaptic to cb2 and presynaptic to OFF centre ganglion cell and A19 dendrites. The cell receives many amacrine cell synapses (A, large arrows) on all portions of its dendritic tree, and makes an occasional gap junction (line) with like amacrines.

field centre. In fact the response shape and spatial characteristics of the A8 is very similar to the responses of an OFF centre ganglion cell in cat retina. It is, therefore, reasonable to suppose that the A8 amacrine has direct excitatory synaptic input to the ganglion cells that it is driving. Together with the A4, A8 is another example of an amacrine cell type in the cat retina that may be excitatory to ganglion cells, instead of purely inhibitory as is thought to be the case for amacrine cells in sub-mammalian retinas (Miller, 1980). Recently, however, Hill and associates (1982) have shown that amacrines using neuropeptides as their t r a n s m i t t e r can be e x c i t a t o r y in the vertebrate retina. 4.2.4 ROD PATHWAYSTO GANGLIONCELLS It became clear during the electron microscope analysis of the connections in the I P L of the cat

retina that the rod pathways consist of at least a four neuron chain to ganglion cells as compared to the three neuron chain of the cone pathways (Kolb and Famiglietti, 1974; Kolb, 1979). Rod bipolar axon terminals followed f r o m identified cell bodies in the inner nuclear layer ONE) made ribbon synapses upon two postsynaptic dendrites both of which were amacrine cells. Rod bipolar axon terminals end deep in the I P L proximal to ganglion cells bodies (sublamina b) yet profiles typical of ganglion cells could never be identified postsynaptic to rod bipolars (one cannot rule out the possibility that some small diameter as yet unrecognized ganglion cell dendrite is on rare occasions involved at rod axon terminals). The major output of the rod bipolars in the cat retina appears to be to amacrine cells of one sort or another. The question is then, what are these amacrine cell types and how are they organized to transmit rod bipolar information to all ganglion

48

H. KOLB and R. NELSON

rb

.,.,tS>'- • ~ z•.....•-f

.

A13 ~ .......

A8

,

A6 ~.

........

25klnn FIG. 11. Camera lucida drawing of Golgi or HRP stained cells known to be involved in the rod pathways of the cat retina. r, rod photoreceptor; rb, rod bipolar cell; All, bistratified, small-field amacrine cell; AI7, large-field diffuse amacrine with slender dendrites bearing beads every 10/am along their length. A13, small-field diffusely branched amacrine; A8, bistratified, narrow field amacrine with varicose dendrites in sublamina b and slender dendrites in sublamina b; A6, smallfield, broadly stratified amacrine cell branching primarily in sublamina b. Scale bar: 25 ~m. (From Kolb and Nelson, 1983.)

cell varieties? Another question concerns how rod bipolar signals get from sublamina b to OFF centre ganglion cells that branch in sublamina a. The neurons that are presently known to be involved in the rod system are shown in Fig. 11. The synaptic connections of these amacrines are known as are their physiological responses to light (Fig. 12) and the probable neurotransmitters they use. Most of these cells, through conventional synaptic arrangements transfer rod bipolar signals to OFF and ON centre ganglion cells. In addition, All by

forming gap junctions with axon terminals of cb5, addresses ON centre ganglion cells through the cone bipolar route. A I 7 does not appear to have direct synaptic output to ganglion cells as far as can be determined at present, but synapses back onto rod bipolar terminals. Thus it may act as a local circuit and spatial integrator for the rod system. Amacrines A8 and A6 are both small-field amacrines that appear to be involved with both rod and cone bipolars in the IPL, with A8 more important for the cone system as mentioned

49

NEURAL ARCHITECTURE OF THE CAT RETINA

An

A17

.°

_I

I

j

I

•

~'cI

FIG. 12. Intracellular responses of the cells involved in the rod pathways of the cat retina. The rod bipolar (RB) gives a sustained hyperpolarization to light. Both AI1 and AI7 give depolarizing responses to light flashes with AII the more transient, while A6, A8 and A13 give hyperpolarizing responses. Retinal illumination is with rod-matched 647 n m stimulus at 5 log (quanta/tam2)/(s). (From Kolb and Nelson, 1983.)

Gb ON FIG. 13. Wiring diagram of the rod pathways of the cat retina. Rod bipolar cells contact rods only in the O P L and have output in the IPL to a n u m b e r of amacrine cells. A17 amacrine passes a m o n g s t groups of rod bipolar axons and makes reciprocal synapses with them. AI3 receives synaptic input from rod bipolars, makes reciprocal synapses and is presynaptic to ON centre ganglion cells (Gb). A l l receives synaptic input from rod bipolars in sublamina b and is presynaptic to OFF centre ganglion cells (Ga) in sublamina a. AII also makes gap junctions (g) with the axon terminal of cb5 bipolar cells and has input to ON centre ganglion cells (Gb) through the cb5 axon terminal. A I 8 is the putative dopaminergic amacrine that synapses extensively on AII amacrine cells in sublamina a.

above. Figure 13 summarizes the connections of the rod pathways indicating the major neuronal types involved in processing rod information. The AII amacrine is known to receive synapses from approximately 30 rod bipolar axon terminals in sublamina b and to have synaptic output to ganglion cell dendrites of at least the alpha and beta a type that branch in sublamina a. In addition the AII gets synaptic input in sublamina a from a cone bipolar type, certainly cbl and possibly also cb2, (not shown on Fig. 13) and from amacrine cells (A18). Intracellular recordings of AII amacrines have shown that they have a characteristic short latency depolarizing response to light (Fig. 12) (Nelson, 1982) and the spectral characteristics of the rod system. At higher light intensities some small contribution of their response is due to the cone system. It is thought that the small cone driven signal appears in the AII amacrine via large areas of gap junction between the AII amacrine dendrites and the axon terminals of cb5, depolarizing cone bipolars. Alternatively this cone input could be coming from the cone bipolar synapses in

50

H. KOLB and R. NELSON

sublamina a. The AII amacrine is thought to be glycinergic (Sterling, 1983) and thus to make inhibitory synapses upon the OFF centre ganglion cells it contacts in sublamina a. An interesting finding recently (Pourcho, 1982a) has been that the amacrine cell which labels with tritiated dopamine in the cat retina, A18, makes synaptic contact with the cell body, apical dendrite and lobular appendages of the All amacrine, thus implying that dopamine is a transmitter pertinent to the rod system. The A13 amacrine is a small-to medium-field amacrine with a diffuse dendritic spread (Plate 2 and Fig. 11). The beaded dendrites terminate preferentially in sublamina b. Electron microscopy of an H R P stained A13 indicates that it is postsynaptic to rod bipolar axon terminals primarily with a minor involvement with a cone bipolar type of sublamina b. Reciprocal synapses are made with the rod axon terminal a few microns from the ribbon synapse. In addition the A13 receives input from unidentified amacrine cells in sublamina b and has synaptic output to b type (ON centre) ganglion cells (Plate 34). The intracellular response of the A13 is a hyperpolarization to light (Fig. 12) with the spectral characteristics primarily of the rod system. Its receptive field is considerably larger than its dendritic field (Kolb and Nelson, 1981) suggestive of lateral spatial input perhaps from other amacrine ceils. The A13 has been shown to accumulate tritiated G A B A (Pourcho, 1982b). Its synapses with ON centre ganglion cells are thus t h o u g h t to be i n h i b i t o r y , but synergistic, disinhibiting the centre mechanism. There are, therefore, at least two amacrine cells that are transmitting rod bipolar signals to the ganglion cells: All via a vertical pathway carries rod signals to sublamina a and to OFF centre ganglion cells, while A13 transmits in the local neuropil of sublamina b straight to ON centre ganglion ceils. Both amacrines probably have sign inverting synapses with the ganglion cells and are supplying rod signals for receptive field centre mechanisms. A wide-field amacrine that has been observed to receive synaptic input from rod bipolar axon terminals in sublamina b and make a reciprocal synapse with this bipolar is the A17 (Kolb and Nelson, 1981, 1983). The sites of synaptic exchange are small swellings or beads on the fine radiating

dendrites. When viewed in flat-mounts of retina by light microscopy, as many as 1000 beads can be counted on an H R P or Golgi stained example of an A17. This gives a good indication of the number of rod bipolar axon terminals contacted by a single A17 cell. The cell gives a slow depolarizing response to light stimulation (Fig. 12) with a receptive field size similar to its typical dendritic field size i.e. 8 0 0 - 1 0 0 0 ~m. Ellias and Stevens (1980) have modelled the spread of electrical activity between the synaptic beads for cells with dendrites like the A17 and concluded that without regenerative capacities the signal spread is limited between synaptic sites. The synaptic beads may be essentially electrically isolated and acting in a local circuit m a n n e r at the rod bipolar axon terminal. Physiological measurements, however, indicate a large receptive field, comparable in width to the dendritic span (Kolb and Nelson, 1981). A further route by which we think rod bipolar signals are reaching ON centre ganglion cells is through the depolarizing cone bipolar type, cb5 (Fig. 13). As we have seen the AII amacrine cell makes extensive areas of gap junction with the axon terminals of cb5 bipolars in sublamina b (Plates 18, 19 and 36). Under dark adapted conditions where the rod system is dominant, the response of the cb5 is characterized by a transient component not seen in the cone photoreceptor itself but similar in waveform to that of the All amacrine (compare Figs. 6 cb5, and 12 AII). Under light adapted conditions where the rod signals are eliminated, the waveform of the cb5 becomes sustained while the AII amacrine cell's response is nearly extinguished. These response characteristics are thought to indicate that under the dark adapted conditions the cb5 bipolar cell is strongly driven by the rod system through the All amacrine cell, and in turn transmitted, via direct synapses, to ON centre ganglion cells (Nelson, 1982; Kolb and Nelson, 1983). We consider, therefore, this depolarizing cone bipolar to be acting as another interneuron in the IPL for the rod pathways (Nelson and Kolb, 1983). A similar situation may exist for the amacrine A6 and the hyperpolarizing cone bipolar of sublamina b, the cb6. A6 is a narrow-field amacrine (Fig. 11) that EM of an H R P example indicates to receive its major synaptic input from rod bipolar axon ter-

NEURAL ARCHITECTURE OF THE CAT RETINA

PLATES 3 2 - - 35. Electron micrographs of HRP stained bipolar and amacrine cells in the 1PL. Plate 32: HRP stained axon terminal of a cb6 bipolar cell is presynaptic to amacrines (A) and ON centre ganglion cell types (Gb) (circled). The synaptic ribbons are obscured in the stained terminal but the membrane specialization upon the postsynaptic profiles are evident. Cb6 axon terminals receive many amacrine cell synapses (A arrowhead), x 25000. Plate 33: HRP stained dendrites of A6 amacrines are primarily postsynaptic to rod bipolar axon terminals (rb) in sublamina b of the IPL. x 25000. Plate 34: Dendrite of an A13 amacrine cell is presynaptic to ON centre ganglion cell dendrites (Gb) in sublamina b of the IPL. x 25000. Plate 35: Large varicose dendrite of A8 amacrine is postsynaptic to a cb6 bipolar axon terminal and makes a reciprocal synapse to the bipolar (arrow) in sublamina b of the IPL. x 25000.

51

PLATE. 37. Electron micrograph illustrates the route whereby rod signals may be passing into ON centre ganglion cells via the A6 amacrine cells. Rod bipolars (rb) are presynaptic to A6 dendrites which are then making gap junctions with cb6 axon terminals. Cb6 axon terminals are known to synapses with ON centre ganglion cells (not illustrated, see Plate 32). A: amacrine dendrites postsynaptic to the bipolar cells, x 61000.

PIRATE 36. Electron micrograph illustrates the route whereby rod information passes to ON centre ganglion cell dendrites. The rod bipolar (rb) is presynaptic to All amacrine cell dendrites which, in turn, are forming gap junctions (large open arrow) with cb5 bipolar axon terminals. The cb5 is presynaptic to ON centre ganglion cells (Gb). A: amacrine cells pre- or postsynaptic to the bipolars. Small arrows: amacrine synapses, x 17000.

z

Z

t" t~

O

tji

NEURAL ARCHITECTURE OF THE CAT RETINA

minals (Plate 33). However, A6 also receives both chemical and electrical (gap junctions) synapses from a cone bipolar axon terminal, probably cb6, in sublamina b (Plate 37). The amacrine makes reciprocal synapses with both the rod and the cone bipolar and a further synapse upon the other amacrine cell postsynaptic at the ribbon dyad synapse in both cases. The intracellular response of A6 is a slow hyperpolarization to light (Fig. 12) which follows the sensitivity of the rod system very closely. Its receptive field is large with spatial characteristics of either the rod bipolar or the cb6 bipolar and with no indication of surround antagonism. The gap junctions with cb6 would allow the rod response of A6 to be injected into the cb6 cone bipolar axon terminal and, as with the cb5, influence the ON centre ganglion cells. In conclusion what have we learned concerning the processing of rod information in the cat retina? Rod bipolars appear to transmit only one kind of signal, a hyperpolarization to light, to the IPL. The rod bipolar is essentially an extension of the photoreceptor system which allows convergence and amplification of the rod signal at the O P L but is not further separated into ON centre and OFF centre signals until the IPL. In the IPL the rod signal gets split into ON centre and OFF centre signs and passes through a series of amacrine and cone bipolar interneurons before reaching the ganglion cells of choice: both ON centre and OFF centre ganglion cell types are known to receive rod signals. There appears to be a complicated division of labor involving known and as yet unknown amacrine pathways, which process the rod signal in various ways. Some rod amacrine cells integrate signals over large areas heightening sensitivity (A17) while others (AII) maximize spatial and temporal acuity. GABA and glycine are involved in parallel ON and OFF centre IPL pathways respectively. There is also a feedback loop from the IPL to the O P L which is probably most heavily involved in the rod system of the cat retina. This loop is the interplexiform cell system. 4.2.5. THE INTERPLEXIFORMCELLOF THE CAT RETINA Interplexiform cells were first described in the goldfish retina (Ehinger et al., 1969) and later found to occur in mammalian retinas as well (Gallego, 1971a; Dawson and Perez, 1973: Boycott et ai., PRR3-B*

53

1975). Where in the teleosts and Cebus monkeys these cells are thought to contain catecholamines they do not appear to do so in the cat. Two reports suggest that the interplexiform cell of the cat retina has specific uptake for the neurotransmitter GABA (Nakamura et al., 1978, 1980; Pourcho, 1981). The synaptic connections of the cat interplexiform cell indicates it to be very involved with the rod system. The processes that run in the O P L and through the INL make a multitude of synapses upon the cell bodies and major dendrites of rod bipolar cells (Plates 14, 15). In addition some synapses are also made to cone bipolars (Kolb and West, 1977). The synaptic input of the interplexiform cell is presently only known to be from unidentified amacrine cell types throughout both sublaminae of the IPL. The function of the interplexiform cell is not known for the cat as no intracellular recordings have yet been obtained, but the speculation has been made (Kolb and West, 1977) that this cell is concerned with setting sensitivity levels in the bipolar cell at the input stage i.e. at the photoreceptor level, from integrating units with larger spatial characteristics occuring in the IPL.

5. G A N G L I O N CELLS It was Kuffler (1953) who first described ganglion cells in the cat retina that responded in either an ON or an OFF centre manner. When a small spot, flashed near the recording site, was turned on, some cells were excited (ON centre); others were excited when such a stimulus was turned off (OFF centre). Generally complimentary inhibition was also observed: at stimulus offset in ON centre units, and at stimulus onset in OFF centre units. Furthermore, K u f f l e r (1953) d i s c o v e r e d c e n t r e - s u r r o u n d antagonism. As the stimulus was displaced slightly from the centre of an ON centre unit, responses consisting of excitiation at both onset and offset of the stimulus appeared ( O N - OFF responses). When the stimulus was presented even farther from the centre, the ON response characteristic of the centre disappeared and only OFF responses were evoked. Thus, the notion of an extended receptive field with spatially opponent mechanisms, the concentric cell, was introduced (OFF centre cells were also concentric). These results indicated also that

54

H. KOLB and R. NELSON

O N - O F F responses might simply be due to inappropriately placed stimuli, although subsequent work demonstrated that there are occasional, true O N - O F F receptive fields in cat (Cleland and Levick, 1974b). Nevertheless, the vast majority of ganglion cell receptive fields in cat retina are of the concentric, Kuffler type (Cleland and Levick, 1974a). This is not to say that all concentric units are identical. In addition to the ON and OFF varieties, concentric units may be readily subdivided according to their s p a t i o - t e m p o r a l summation properties. X cells exhibit liner summation and Y cells, nonlinear (Enroth-Cugell and Robson, 1966). Axonal conduction provides another distinguishing characteristic. This amounts to a physiological measure of axonal, and thus likely, cell body diameter, and has introduced a third class, the W cells or small ganglion cell classes (Stone and Hoffman, 1972). S p a t i o - t e m p o r a l summation correlates well with the temporal dynamics of the response: sustained responses are often X, and transient often Y (Ikeda and Wright, 1972; Cleland and Levick, 1974a). "Non-concentric" ganglion cells are sometimes encountered in cat retina and at least 5 classes have been described: local edge detectors, directionally selective cells, colour-coded cells, uniformity detectors, and edge inhibitory OFF centre units (Cleland and Levick, 1974b). It remains a major challenge to correlate most of the physiological classes of cat ganglion cell with the morphological classes as seen in Golgi preparations. Does each physiological receptive field type originate with a particular morphological class of ganglion cell, or alternatively, might a particular morphological class display a variety of receptive field properties? What are the specific connections with amacrine and bipolar cells that underlie the physiological receptive fields and their distinctive properties? Perhaps the clearest c o r r e l a t i o n between a m o r p h o l o g i c a l and physiological class of ganglion cell (Cleland and Levick, 1974a; Peichl and W~ssle, 1981) is between the alpha morphological class (Boycott and W/issle, 1974) and the physiological Y class (or brisktransient class, Cleland and Levick, 1974a). Beta cells (Boycott and W~ssle, 1974) also appear well correlated with the X-type physiological class (or brisk-sustained class, Cleland and Levick, 1974a).

But gamma and delta morphological classes have also been identified (Boycott and W~ssle, 1974) as well as epsilon (Leventhal et al., 1980), and Kolb et al. (1981) have described as many as 23 different morphological types of ganglion cell in the cat retina. Identification of these remaining morphological types with physiological types remains more speculative. Alpha (Y) cells have very large radiate dendritic trees consisting of large calibre dendrites emerging from a very large cell body (approximately 35 ~m diameter) (Fig. 2). Alpha cells come in a and b types branching in sublamina a and b and are equivalent to the OFF centre Y cells and ON centre Y cells respectively (Famiglietti and Kolb, 1976; Nelson et al., 1978; Peichl and W/~ssle, 1981; W~ssle et al., 1981a). Electron microscopy of alpha cells indicate that they receive some input from cone bipolars of the sublamina in which they branch but the greater proportion of their input is from amacrine cells (Kolb, 1979). Concentrically organized Y cells are thought to have receptive field centres made up of discontinuous, non-linear subunits of transient physiological components (Hochstein and Shapley, 1976) which correlates rather well with the anatomical finding of predominant amacrine cell input. It is fairly clear that the receptive field centre of Y cells is not provided solely by bipolar input. In particular we know that some component of the rod input to the receptive field centre comes from the All amacrine cell (see above). Furthermore, Ikeda and Sheardown (1983) have recently demonstrated that iontophoretically applied acetylcholine enhances the centre response of Y cells and that the receptor on the Y cell is nicotininc rather than muscarinic. No bipolar cell of any mammalian retina has yet been suggested to use acetylcholine as a transmitter while at least two varieties of amacrine cell are known to be cholinergic (Masland and Mills, 1979). If the centre of the Y cell receptive field is built by a high proportion of amacrine cell input, there is even more reason to suspect that the surround is also under strong amacrine control. It appears almost certain that no horizontal cell receptive field is large enough to account for surrounds of Y cells (Nelson, 1977) even if it could be demonstrated that the bipolar cells of the cat retina exhibited antagonistic surround behaviour. As noted above,

NEURAL ARCHITECTURE OF THE CAT RETINA

only one bipolar cell type of the cat retina has been shown to have a surround, but this is thought to originate in the IPL rather than the OPL. The X ganglion cells of the cat retina are smallfield, concentric units which also, of course, come in ON centre and OFF centre varieties. The morphological counterparts appear to be the a and b varieties of beta cells (Fig. 2). They have a small profusely branched dendritic tree and a large cell body (approximately 25/am diameter) (Boycott and W~issle, 1974; Famiglietti and Kolb, 1976; Kolb et al., 1981). Both varieties receive the majority of their synaptic input from cone bipolar cells (Kolb, 1979; Stevens et al., 1980; Plates 2 2 - 2 4 ) . The bipolar input is thus thought to form the major proportion of the receptive field centre of such beta cells, although a small field amacrine cell, A4, of the OFF centre system is thought to contribute excitatory input and have small enough spatial characteristics to be invoked as a contributor to this ganglion cell receptive field centre mechanism too (see p. 30). Like Y cells, surrounds from horizontal cell sources have not been elicited in the cone bipolar cells that have direct synaptic input to the receptive field centre of beta ganglion cells. Two amacrine cell types that have been demonstrated morphologically to synapse with OFF centre ganglion cells, namely the AII and the A8, have been shown physiologically to have receptive fields that are already concentrically organized (Nelson, 1982; Fig. 10). So the suggestion that beta cell receptive field surrounds are made up in part by amacrine cell systems has some foundation in both the morphological and physiological evidence. ON-inhibition in OFF centre and OFF-inhibition in ON centre X cells, under cone-driven conditions, has been suggested to be due to glycine- and GABAergic systems respectively (Ikeda and Sheardown, 1983). I n t e r e s t i n g l y , these two i n h i b i t o r y transmitters GABA and glycine have been found specific respectively for the ON and OFF systems of mudpuppy retina too, particularly at the bipolar level (Miller et al., 1981). Several varieties of amacrine cell have now been shown to take up tritiated GABA in the cat retina and striking effects on ganglion cell receptive fields in rabbits as well as cats are seen when antagonists to GABA are used in physiological experiments (Wyatt and Daw, 1976; Ariel and Daw, 1982; Kirby and Enroth-Cugell,

55

1976; Kirby and Schweitzer-Tong, 1981). Concentrically organized alpha and beta ganglion ceils of the cat retina are arranged in regular superimposed bi-level mosaics across the whole retina (W~issle et al., 1981a, b). The cells have the smallest dendritic field sizes at the area centralis and radiate out from the central point with gradually expanding but essentially circular dendritic fields. Each point on the retina is covered by at least one OFF centre cell and one ON centre cell of the alpha and beta type (W/issle et al., 1981a, b). The organization of the cat retina with a concentration of small-field concentrically organized beta cells in the area centralis allows the lowest convergence ratio of cones to both ON centre and OFF centre ganglion cells. This is presumed to be the substrate for the highest acuity pathways. It is of interest to know how many cones are providing input to the smallest beta cells and whether it approaches the one to one ratio of higher primates and humans. The EM serial section analysis of central beta cells (Kolb, 1979) concluded that in the area centralis each ganglion cell received input from a very small number of bipolar cells of a particular type. Considering that the central narrow-field cone bipolar types (cbl, cb5) make connections with 4 to 8 cones (Boycott and Kolb, 1973), it is likely then that the same g r o u p of cones are wired to both ON and OFF beta ganglion cell types of the area centralis through about three cone bipolars per ganglion cell (Kolb, 1983). Among non alpha and beta classes there is one class of W cell that appears to have a concentric organization. This is a sluggish (tonic and phasic) ganglion cell type (Stone and Fukuda, 1974; Cleland and Levick, 1974b) which probably projects to extra-geniculate-striate (Rodieck, 1979), and may be involved in the pupillary reflex pathways. It is possible that the morphological equivalent of this physiological type is the G3 (Kolb et al., 1981) which occurs as a and b subtypes (Fig. 3) and is the gamma cell type of Boycott and W~issle (1974). Nothing is known concerning the functional anatomy of this gamma cell in the cat retina as yet. It has been estimated that between 50 and 60% of all ganglion cells of the cat retina are cells other than alpha and beta types (Fukuda and Stone, 1974; Stone and Fukuda, 1974). Our Golgi study (Kolb et al., 1981) demonstrated that 21 different

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morphological types of ganglion cell in addition to alpha and beta cells are found in the cat retina. The G3 (gamma cell) was proposed to be a concentric type of W cell (Stone and Fukuda, 1974; Cleland and Levick, 1974b), and the remaining G4 to G23 cell types are likely to be either concentric or non concentric W cell types. Some cell types equivalent to G21 and G23 (Kolb et al., 1981) have also been visualized by retrograde H R P filling of visual nuclei. Thus, the epsilon cell type that projects to a part of the pulvinar called the retinal recipient zone (Leventhal et al., 1980) is a large bodied, widefield cell that has some characteristics of the G23 cell type. Unfortunately little is known concerning its response to light, and which of the ubiquitous W cell class it might be. In the same way, Farmer and Rodieck (1982) have successfully stained ganglion cells that correspond to G21, so we now know that this ganglion cell type projects to the accessory optic system, but again we do not know their physiological responses. The speculation is that they might be directionally selective ganglion cells because cells of the medial terminal nucleus (MTN) are reported to be directionally selective (Grasse and Cynader, 1980). A ganglion cell type now identified as G22 of the Kolb et al. classification (1981) was recorded from by intracellular techniques and reported in Nelson et al. (1978) (cell C, 8 of the Table). This ganglion cell type had an OFF centre receptive field which was considerably smaller than its dendritic spread. Furthermore there was no sign of an antagonistic surround mechanism. Interestingly this cell was also found to be driven only by the rod mechanism: intensity response series to rod matched stimuli of 441 nm and 647 nm were indistinguishable for the G22 cell (Nelson et al., 1978). This cell type branches in sublamina a exclusively so its receptive field wiring must be primarily through amacrine cell circuitry of the rod system. Non-concentric ganglion cells of the cat retina may be from a phylogenetically older system. The projections of these cell types to extra-geniculostriate visual centres has been shown in many cases, and substantiates such a conclusion. On this basis it is reasonable to suggest that the circuitry underlying their receptive field organization uses many amacrine cell pathways (Dubin, 1970; Dowling, 1970). In contrast, where high acuity has

developed in mammals, it is served by concentrically organized beta cells (cat) or midget ganglion cells (primates) which project through the lateral geniculate to the visual cortex where more elaborate receptive field properties such as line and edge detection, or directional selectivity are developed. However, it is interesting that a large proportion of non-concentric ganglion cells, with their morphological diversity and elusive physiological characteristics, are still retained in the retina of these mammals. So, even if we come to understand the synaptic circuitry underlying the most simple concentric units of the area centralis, a greater challenge remains in understanding non- concentric complex receptive field type ganglion cells and their contribution to the visual organization of the brain. Acknowledgement- - Supported in part by grant EY 03323 from the National Institutes of Health.

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