MORPHOLOGICAL IDENTIFICATION OF AMACRINE CELLS

IN THE ZEBRAFISH RETINA

By

Jennifer Hsieh

Submitted to the

Faculty of the College of Arts and Sciences

of American University

in Partial Fulfillment of

the Requirements for the Degree of

Master of Science

In

Biology

O r Vint rvr in P P n t i n a i i a h t n n ^-^ Chair:

Dr. Victoria P. Connaughton

Dr.J)avid"B. Carlini

Kathleen L. DeCicco-Skinner icco-Skir

SgfoZ fthi

~yK*+oA&£/ Dean of the College of Arts and Sciences

Date

2008

American University

Washington, D.C. 20016

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Hsieh, Jennifer

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MORPHOLOGICAL IDENTIFICATION OF AMACRINE CELLS

IN THE ZEBRAFISH RETINA

BY

Jennifer Hsieh

ABSTRACT

The zebrafish is a powerful vertebrate model in the study of visual neuroscience.

Amacrine cells in the zebrafish retina were labeled using the DiOlistic technique, in

which a gene gun delivers microcarriers coated with the lipophilic dye Dil (1,1'-

dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) onto retinal slices.

Incorporation of the dye allows for visualization of somata and dendritic processes. The

cells were morphologically classified according to dendritic field widths, dendritic

stratification patterns, and/or soma shape. Eighteen amacrine cell types were identified.

Four A0ff types were monostratified in si, one A0ff type was monostratified in s3, and two

A0ff types ramified simultaneously in sl/s2 and sl/s3. Dendritic fields were 60-130 urn

in both these groups. Six Aon types were identified with dendritic processes innervating

sublamina b of the IPL. Both rounded and pyriform-shaped somata were seen. Dendritic

fields ranged in size from ~0-l 10 urn. Of the five bistratified Abi cell types identified,

one innervated sl/s4, three innervated sl/s5, and one innervated s2/s5. Dendritic fields

were typically 20-140 um. These results are important in determining the function of

each morphological type and how they are affected in visual diseases.

ii

ACKNOWLEDGMENTS

This work would not have been possible without the guidance and unwavering

support of my advisor, Dr. Victoria Connaughton. Her enthusiasm and steadfast

optimism have been crucial in making me a better scientist. I would like to thank Dr.

David Carlini and Dr. Kathleen DeCicco-Skinner for their encouragement and sound

advice. Thank you to Dr. Ralph Nelson for the gene gun and an endless supply of

zebrafish. I would also like to thank my best friend, Daniel Kim, for always believing in

me.

Lastly, and most importantly, I am indebted to my parents for everything.

iii

ABSTRACT

TABLE OF CONTENTS

11

ACKNOWLEDGMENTS iii

LIST OF TABLES vi

LIST OF ILLUSTRATIONS vii

Chapter

1. INTRODUCTION 1

The Vertebrate Retina 1

Amacrine Cells 5

Synaptic Circuitry 8

Morphological Diversity 11

Synaptic Chemistry and Neurotransmitter Distribution 15

Variety of Function 22

All and A17 Amacrine Cell Types 28

The Zebrafish (Danio rerio) 33

Purpose and Objectives 34

iv

2. MATERIALS AND METHODS 36

Retinal Slices 36

DiOlistics 37

Identification of Cell Types and Terminology Used 38

3. RESULTS 40

OFF-type Amacrine Cells 40

ON-type Amacrine Cells 44

Bistratified Amacrine Cells 47

4. DISCUSSION 52

OFF-type Amacrine Cells 54

ON-type Amacrine Cells 56

Bistratified Amacrine Cells 58

5. CONCLUSIONS 60

APPENDIX 63

REFERENCES 64

v

LIST OF TABLES

Table Page

1. Morphological Types of Amacrine Cells Identified in Nine Species of Animals 14

2. Neurochemical Summary of Amacrine Cells 22

3. Morphological Measurements of Amacrine Cell Types in the Zebrafish Retina 51

4. Morphological Types of Amacrine Cells Identified in Nine Species of Animals 61

vi

LIST OF ILLUSTRATIONS

Figure Page

1. 3-D Block of a Portion of the Human Retina 2

2. Simple Diagram of the Organization of the Retina 3

3. Light Micrograph of a Vertical Section of the Human Retina to Show the Inner

Plexiform Layer (IPL) 6

4. Electron Micrograph of a Reciprocal Feedback Synapse of a Rod Amacrine Cell (A17) Upon a Rod Bipolar Axon Terminal 10

5. Morphological Summary Diagram of Seven Amacrine Cell Types Within the

Zebrafish Retina 13

6. Distribution of Choline Acetyltransferase 16

7. Distribution of Glutamic Acid Decarboxylase (GAD) 19

8. Recordings of a Transient Amacrine Cell in Response to a Spot Illumination and to

Annuli of 250 u and 500 u 26 9. Summary Schematic of LY-filled Cells with their Neurotransmitter, Morphology,

and Response Type 27

10. A Golgi-stained Example of an All Amacrine Cell in Cat Retina 29

11. Serotonin-containing Amacrine Cells Stained with Lucifer Yellow in Wholemount Rabbit Retina 31

12. Summary Diagram of the Rod Pathway Neurons and Their Responses 32

vn

13. DiOlistic Stains of OFF-type Amacrine Cells in the Zebrafish Retina 42

14. DiOlistic Stains of ON-type Amacrine Cells in the Zebrafish Retina 46

15. DiOlistic Stains of Bistratified Amacrine Cells in the Zebrafish Retina 48

16. Morphological Summary Diagram of Seven OFF-type Amacrine Cells 56

17. Morphological Summary Diagram of Six ON-type Amacrine Cells 57

18. Morphological Summary Diagram of Five Bistratified Amacrine Cells 59

19. DiOlistic Stains of Amacrine Cells Seen Only Once in the Zebrafish Retina 63

vin

INTRODUCTION

The Vertebrate Retina

The study of the visual system provides an opportunity to explore a unique neural

pathway that is independent from, but connected to, the rest of the central nervous

system. It gains its function primarily from the retina, which is located at the back of the

eye and is largely responsible for the ability of the brain to process incoming information

and impart the capacity to see. In the late nineteenth century, the importance of retinal

research was firmly established by the pioneering Spanish neuroanatomist Santiago

Ramon y Cajal, who recognized that the cellular organization of the retina rendered it an

excellent tool for the understanding of neural activity. Utilizing the Golgi silver method

and light microscopy, Cajal was able to morphologically distinguish between the retinal

cell types and identify their locations, forming the foundation on which retinal research is

based today.

Compared to higher visual centers found in the brain, the vertebrate retina is

easily accessible and possesses a highly organized anatomy composed of seven major

types of cells, each with a specific distribution within the five different layers (Figure 1).

These layers are defined by the aggregation of either all the somata (cell bodies) or all the

dendritic and/or processes of each retinal cell type, creating a system in which all the

somata are dispersed throughout the three nuclear layers and all the synaptic contacts are

made in the two plexiform layers. This basic structure is constant in all vertebrate

1

2

retinas, with variation occurring in terms of complexity, cell size, and extent of dendritic

fields. The cellular structure of the retina has been well-established (reviewed in

Dowling, 1987) and is discussed in detail below.

Figure 1: 3-D Block of a Portion of the Human Retina (Kolb et al, 2003). This drawing shows the five main layers of the retina: outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, and ganglion cell layer.

The retina has adopted an unusual cellular arrangement in which information

processing directionally occurs backwards. Light entering the eye is taken up by the

most distal retinal cells, the photoreceptors, which lie attached to the pigment epithelium

and whose outer segments contain visual pigments (Figure 2). The photoreceptors

3

convert the light into nerve signals in a process called phototransduction. The outer

segments and inner segments of the photoreceptors are visible in distinct layers, followed

by the cell bodies, which make up the outer nuclear layer. Stimulation of the

photoreceptor rods and cones is essential for vision.

Figure 2: Simple Diagram of the Organization of the Retina (Kolb et al, 2003). This drawing shows the neurons that constitute each layer of the retina and their respective synaptic connections. The vertical pathway consists of photoreceptors (rods and cones), which synapse onto bipolar cells, which in turn synapse onto ganglion cells that converge and form the optic nerve. The lateral pathway consists of horizontal cells, which modulate visual information processing in the outer plexiform layer, and amacrine cells, which modulate visual information processing in the inner plexiform layer.

The photoreceptors synapse with the bipolar cells in the outer plexiform layer, but

bipolar cell somata (Figure 2) predominantly occupy the adjacent inner nuclear layer and

4

provide input to the inner plexiform layer. The cell bodies of two additional retinal cells

can also be found in the inner nuclear layer; horizontal cell bodies sit on the distal inner

nuclear layer border and extend their processes into the outer plexiform layer, while

amacrine cell bodies are situated on the proximal inner nuclear layer border and extend

their processes into the inner plexiform layer. Horizontal and amacrine cells (Figure 2)

modulate the vertical visual processing pathway via lateral interactions within the layers

they innervate. This lateral inhibitory activity alters the visual signals carried by the

vertical pathway before it is conveyed to the next step in visual information processing.

The proximal layer of the retina is composed of ganglion cell somata (Figure 2),

which synapse with the bipolar cells in the inner plexiform layer. As the final output

neurons of the retina, ganglion cell axons form the optic fiber layer before converging

and becoming the optic nerve, which relays all visual information perceived by the eye to

the visual processing centers in the brain.

Besides the five retinal cell types that constitute the vertical and lateral visual

pathways, there are two accessory cell types that contribute to the well-defined

framework of the retina. The interplexiform cells (Figure 2) are found amongst amacrine

cells on the proximal inner nuclear layer border and, as their name suggests, these cells

project processes into both the outer plexiform layer and the inner plexiform layer and

transmit information between these two layers. Interplexiform cells make synapses on

bipolar, horizontal, or other interplexiform cells in the outer plexiform layer, but

primarily contact only amacrine cells in the inner plexiform layer.

As the main neuroglia in the retina, the Miiller cells (Figure 2) function as a

physical support network to maintain the well-being of the neurons. Neuroglia are not

5

neurons; instead, they provide nutrition and protection for the surrounding nerve cells.

Miiller cells are associated with proliferating retinal progenitors, which actually become

stem cells that give rise to various types of new retinal neurons following an injury

(Yurco & Cameron, 2005; Raymond et al, 2006). Mtiller cell bodies rest in the middle

of the inner nuclear layer and spread across the entire thickness of the retina, reaching

from the outer limiting membrane (proximal border of outer plexiform layer) to the inner

limiting membrane, which marks the proximal margin of the optic fiber layer.

Amacrine Cells

Amacrine cells have been well-studied and characterized in organisms such as the

cat (Kolb, 1979; Kolb & Nelson, 1981; Kolb et al, 1981; Kolb & Nelson, 1985); the

turtle, Pseudemys scripta elegans (Kolb, 1982); the rhesus monkey, Macaca mulatta

(Mariani, 1990); and the roach, Rutilus rutilus (Wagner & Wagner, 1988; Djamgoz et al.,

1989). The wide range of techniques employed to visualize cells include Golgi

impregnation (Sherry & Yazulla, 1993), light microscopic immunocytochemistry (Sherry

& Yazulla, 1993; Yazulla & Studholme, 2002), dye injection (Yang et al., 1991), and

DiOlistic lipophilic dye staining (Connaughton et al., 2004). Compared to the first three

techniques, however, using lipophilic dyes to label neurons have allowed scientists to

quickly and simultaneously differentiate between types of cells in a complex neuronal

network (Gan et al., 2000). This is incredibly useful in the study of the retina, and will be

applied in this project in order to distinguish amacrine cell bodies and dendritic processes

from the surrounding neurons.

As mentioned above, amacrine cell somata are situated on the proximal inner

6

Figure 3: Light Micrograph of a Vertical Section of the Human Retina to Show the Inner Plexiform Layer (IPL) (Kolb et al, 2003). Amacrine cells sit on the proximal margin of the inner nuclear layer and extend their dendritic processes into the inner plexiform layer.

nuclear layer border and their dendritic processes are distributed throughout the inner

plexiform. Although it is not particularly apparent (Figure 3), the inner plexiform layer

of the mammalian retina (Figure 3) is stratified into five sublayers of equal thickness (~8

um) through which the dendritic processes of bipolar, ganglion, and amacrine cells

extend (Cajal, 1972; Connaughton et al., 2004). Earlier retinal studies determined that

this stratification was related to ON- and OFF-type responses of the ganglion cell and as

a result, the first two sublayers together are called sublamina a, while sublayers 3-5 are

collectively known as sublamina b, in correspondence with the physiology of OFF-and

7

ON-type cells, respectively (Famiglietti & Kolb, 1976). The ON- and OFF-type

responses of ganglion cells are extremely important in vision, as ganglion cells are the

final output neurons of the retina. These signals are relayed through parallel pathways

involving bipolar, amacrine, and ganglion cells. Transmitted by the optic nerve fibers,

ON and OFF electrical signals provide information to the brain about specific

components in the visual field, such as color, movement, and changes in brightness. ON-

type ganglion cell fibers depolarize at the onset of light and maintain a consistent

elevated depolarization rate throughout the length of illumination (Hartline, 1938). These

cells are postsynaptic to ON-type bipolar cell terminals in sublamina b (Nelson et al,

1978). On the other hand, OFF-type fibers respond with depolarization only at the offset

of light, and these cells are postsynaptic to OFF-type bipolar cell terminals in sublamina

a (Hartline, 1938; Nelson et al., 1978). ON-OFF cells respond with depolarization at

both the onset and offset of light, and are bistratified into both sublaminae (Hartline,

1938; Nelson etal., 1978).

Using this stratification criterion, bipolar, ganglion, and amacrine cell types can

be classified according to the sublamina in which their processes are located. Amacrine

cells have also been morphologically categorized based on types of synaptic contacts,

neurochemical content, and physiological responses to light. These categories are

addressed individually in the following sections to provide a comprehensive overview of

the different amacrine cell subtypes. The last section of this introduction summarizes two

of the most widely studied amacrine cells in order to show how these separate criterion

are applied in combination to categorize a given cell type.

8

Synaptic Circuitry

After Cajal first identified amacrine cells as axonless neurons, scientists have

recognized amacrine cell processes as having functions of both axons and dendrites

(Boycott & Dowling, 1969). The processes make synaptic contacts within the inner

plexiform layer and help shape visual information conveyed through the vertical pathway

prior to reaching ganglion cells. In mammals, these cells amount to 32-39% of all

neurons found in the inner nuclear layer; this figure does not include interstitial amacrine

cells or displaced amacrine cells, as their cell bodies are located within the inner

plexiform layer and ganglion cell layer, respectively (Boycott & Dowling, 1969; Perry &

Walker, 1980; Strettoi & Masland, 1995; Jeon et al., 1998). These displaced cells are

typically seen more often in amphibian, avian, and reptilian retinas and less so in

mammals (Boycott & Dowling, 1969).

Since amacrine processes function as both axons and dendrites, these processes

are in fact both presynaptic and postsynaptic along their entire length (Dowling, 1987).

Multiple synaptic connections are made between amacrine cells and interplexiform cell

processes, other amacrine cell processes, occasionally with ganglion cell dendrites and

cell bodies, and most frequently, with bipolar cell terminals (Dowling & Boycott, 1966).

This unique trait provides insight into how other axonless neurons of the brain operate

and suggests that signaling between amacrine cell processes could take place in localized

areas and may not necessarily require the entire neuron. This versatility is crucial to the

amacrine cell's role as an important modulator of the vertical visual pathway.

In both plexiform layers of the retina, there are two types of chemical synaptic

junctions: conventional synapses and ribbon synapses (Dowling and Boycott, 1966).

9

Conventional synapses, involving mostly horizontal, interplexiform, and amacrine cells,

are the typical synapse, characterized by an accumulation of synaptic vesicles in the

presynaptic terminal close to the membrane.

On the other hand, ribbon synapses are unique to the retina. They are commonly

observed between photoreceptors and bipolar cells in the outer plexiform layer, as well as

between bipolar terminals and the postsynaptic ganglion dendrites and amacrine

processes in the inner plexiform layer (Kolb, 1979). They are distinguished by the

presence of an electron-dense ribbon oriented perpendicular to the presynaptic

membrane, and are surrounded by synaptic vesicles (Dubin, 1970). Interestingly, the

ribbons in the outer plexiform layer are consistently longer than those seen in the inner

plexiform layer (Dowling, 1987). The term "dyad" was coined in reference to the two

postsynaptic processes that are contacted by the single presynaptic ribbon terminal; these

are normally found in the inner plexiform layer (Dowling, 1987). Usually, both members

of the postsynaptic dyad are amacrine processes, but in some cases, one element of the

dyad is an amacrine process and the other element is a ganglion dendrite (Dowling &

Boycott, 1966). The ribbon dyad combinations are limited to these possibilities,

however; Dubin (1970) identified several complicated configurations in which one dyad

process is postsynaptic to two ribbons that may be in the same or different bipolar

terminal.

In some dyads (Figure 4), the postsynaptic amacrine process may synapse back

onto the presynaptic bipolar terminal a short distance away from the actual ribbon

synapse, forming a feedback interaction called a reciprocal synapse (Dowling, 1987)

(Figure 4). At the same time, the postsynaptic amacrine process may also synapse onto

10

the other postsynaptic dyad element (Dubin, 1970). Reciprocal synapses consisting of

only amacrine cell processes are called serial synapses, where one amacrine process

synapses onto an adjacent amacrine process, which in turn synapses onto a third

neuron—ganglion dendrites, bipolar terminals, or another amacrine processes are all

possible targets (Dowling, 1987). In some species, a more extensive chain of serial

synapses between amacrine cells has been observed, ones that may include as many as

Figure 4: Electron Micrograph of a Reciprocal Feedback Synapse of a Rod Amacrine Cell (A17) Upon a Rod Bipolar Axon Terminal (Kolb et ah, 2003). Reciprocal synapses consist of a bipolar axon terminal ribbon synapse onto an amacrine cell dendritic process, which in turn reciprocally synapses back onto the same bipolar cell through a conventional, or chemical synapse.

11

four consecutive serial synapses (Dowling, 1987).

Similarly, electrical synapses are also seen in both plexiform layers of the retina.

These synapses are called gap junctions, and they occur mostly between photoreceptor

terminals, between horizontal cells, and between amacrine cells (Naka & Christensen,

1981). Furthermore, gap junctions are also sometimes seen between two amacrine cell

processes or between bipolar cell terminals and amacrine cell processes (Famiglietti &

Kolb, 1975; Dowling, 1987; Mills & Massey, 1995). With their participation in

conventional, ribbon, and electrical synapses, as well as the ability of a single amacrine

cell to be involved in more than one synapse at one time, the number of synapses

mediated by amacrine cell input constitutes approximately 64-87% of all synaptic

contacts in the inner plexiform layer of the mammalian retina (Dubin, 1970; Raviola &

Raviola, 1982; MacNeil et ah, 1999). This impressive percentage arises solely from

synaptic interactions between amacrine, bipolar, and ganglion cells, all of which use an

extensive variety of neurotransmitters as the agent of signal transmission. These

transmitters will be discussed in further detail later.

Morphological Diversity

Morphological characteristics used to distinguish among amacrine cell types

include dendritic field size, dendritic stratification, and cell body shape. In spite of

variations that may occur across different organisms, these morphological parameters

remain relatively fixed across all studies.

Most of the literature that morphologically characterize amacrine cells do so

based on dendritic field diameters, which groups the cells into four main categories:

12

narrow-field (30-150 um), small-field (150-300 urn), medium-field (300-500 urn), and

wide-field (>500 um) (Kolb et al., 1981; Kolb, 1982). These measurements are obtained

by taking the distance between the two most distant ends of the dendritic tree (Wagner &

Wagner, 1988). In cases of a single primary dendritic process descending from the soma

before fanning out into secondary processes, the dendritic field size would be measured

across the bulk of secondary processes. Within these four categories, amacrine cells can

be further morphologically subdivided based on depth of dendritic stratification, dendritic

branching patterns and degree of arborization, as well as somal shape and dimensions.

Dendritic stratification can be determined as the sublayer of the inner plexiform

layer in which the bulk of the dendritic processes lie and the overall depth to which all

processes extend (Connaughton et al., 2004). For example, since the mammalian inner

plexiform layer is comprised of five sublayers, the 20% of the total thickness represented

by each sublayer is often used to define stratification depth (Kolb et al., 1981). Hence, an

amacrine cell with dendritic processes terminating in sublayer 2 of sublamina a would be

designated as s2 and have a percent depth of 20-40%. Although in some organisms a

different number of sublayers would be present—such as in the 6 sublayers seen in

zebrafish (Connaughton et al, 2004) and 7 unequally thick sublayers seen in the roach

(Wagner & Wagner, 1988)—the general equation is still applicable.

Dendritic branching patterns and extent of arborization refer to how many

sublayers the principal dendritic processes innervate and the profusion of branching. The

most regularly used terminology are monostratified, bistratified, and multistratified,

indicating that the dendritic branches innervate one sublayer, two sublayers, or more than

two layers, respectively, and actually form distinct lateral layers themselves within the

13

inner plexiform strata (Wagner & Wagner, 1988). Dendritic processes of diffuse

amacrine cells lack such uniform structure and innervate throughout most or all inner

plexiform sublayers in a profuse arborization (Connaughton et al., 2004). Unlike the

archetypical stratified cell, diffuse amacrine cells have less defined horizontal branching

in each sublayer and have a higher density of branching per unit area (Boycott &

Dowling, 1969). It is important to note that amacrine cells may in fact be stratified and

diffuse at the same time, where dendritic arbor are profuse but aggregate in particular

stratum of the inner plexiform (Boycott & Dowling, 1969).

By applying the aforementioned criteria, amacrine cells have been extensively

studied and morphologically classified in several organisms. The characteristics noted

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Figure 5: Morphogical Summary Diagram of Seven Amacrine Cell Types Within the Zebrafish Retina (Connaughton et al., 2004). Amacrine cells were morphologically classified according to dendritic stratification patterns. OFF-type cells ramified in sublamina a, while ON-type cells ramified in sublamina b. A0frslw is wide-field, monostratified; A0frsln is narrow-field, monostratified. Aon-sl/s5 is bistratified, with dendritic ramifications in both sublaminae. Adifftise-1 is multipolar, with three main varicose dendrites that project throughout the inner plexiform layer.

14

above are used either individually (i.e., a monostratified cell) or in combination (i.e., a

narrow-field, bistratified cell) to describe a given type of amacrine cell. Seven amacrine

cell types were identified in the zebrafish (Danio rerio, Figure 5) based on dendritic

morphology and stratification patterns in the inner plexiform layer, and/or somal shape

(Connaughton et al., 2004). The cells were grouped into ON-type, OFF-type, and diffuse

types; if the primary dendritic branching was located in sublamina b, then the cell was a

designated ON-type, and if the primary dendritic branching was located in sublamina a,

then the cell was a designated OFF-type. Within each category, further distinctions were

made regarding width of dendritic fields, the sublayer in which the majority of the

dendritic arbor was located, and whether the cell body was rounded or mitral-shaped.

Species

Tiger salamander Zebrafish Rat Cat Catfish, Ictalurus punctatus Rhesus monkey, Macaca mulatto Turtle, Pseudemys scripta elegans Rabbit Cyprinid fish, roach

Types

7 7 9 22 22 26 27 28 43

Reference

Yang etal., 1991 Connaughton et al., 2004 Perry & Walker, 1980 Kolb etal, 1981 Chan&Naka, 1976 Mariani, 1990 Kolb, 1982 MacNeil et al, 1999 Wagner & Wagner, 1988; Djamgoz et al., 1989

Table 1: Morphological Types of Amacrine Cells Identified in Nine Species of Animals. The number of morphological types of amacrine cells varies widely between species of animals.

Multiple amacrine cell types have also been identified in a wide range of species,

including other types offish (Chan & Naka, 1976; Djamgoz et al., 1989; Wagner &

15

Wagner, 1988), turtles (Kolb, 1982), and a number of mammals (Kolb et ah, 1981; Perry

& Walker, 1980; MacNeil et ah, 1999; Mariani, 1980). The specific number present,

however, varies among species (Table 1). Amacrine cells have been consistently

classified in these animals according to the aforementioned morphological characteristics,

the most common being dendritic field sizes and stratification patterns.

Synaptic Chemistry and Neurotransmitter Distribution

The large morphological collection of amacrine cells in the retina (Table 1) is

paralleled only by their neurochemical diversity (i.e., the type of neurotransmitter they

release or uptake), which can be described in five major categories: (1) cholinergic, (2)

glycinergic, (3) GABAergic, (4) peptide-immunoreactive, (5) catecholaminergic and

serotonin-accumulating (Vaney, 1990). Using an extensive array of antibodies, Yazulla

and Studholme (2001) performed a comprehensive study on the neurochemical anatomy

of all retinal cells in the zebrafish. These antibodies were able to mark enzymes,

receptors, and transporters that corresponded to these major types of neurotransmitters.

Though the immunocytochemical properties of amacrine cells in many species are

known, only the findings in zebrafish are summarized below, as it is most pertinent to

this project. If possible, however, correlations between the findings in zebrafish and

other species are presented.

Some amacrine cell bodies located in the inner nuclear layer showed intense

labeling for choline acetyltransferase, which is the enzyme responsible for synthesizing

acetylcholine from acetyl-CoA and choline. Therefore, amacrine cells that contain

choline acetyltransferase can be assumed to release acetylcholine as their

16

neurotransmitter. Though labeling also was seen in sublayers 1, 2, 4, and 5, it was most

prominent in sublayers 2 and 4, but appeared discontinuous in sublayers 1 and 5 (Yazulla

& Studholme, 2001) (Figure 6). While cholinergic amacrine cells have been reported as

existing in two mirror-image populations—one in the proximal inner nuclear layer and

one as displaced amacrines found in the ganglion cell layer (Hayden et ah, 1980; Vaney

et ah, 1981)—Yazulla and Studholme (2001) reported weak labeling in displaced

amacrine cells and noted that projection from the inner nuclear layer reached all four

labeled strata. These mirror-image amacrine cell types are also known as the starburst

amacrines, which release acetylcholine directly onto postsynaptic ganglion cells

(Famiglietti, 1983; Vaney, 1984). Acetylcholine has been shown to be strongly

excitatory to many types of ganglion cells (Ames & Pollen, 1969; Negishi et ah, 1978),

Figure 6: Distribution of Choline Acetyltransferase (Yazulla & Studholme, 2001). (A) Amacrine cell bodies located in the inner nuclear layer showed intense labeling for choline acetyltransferase. The labeling was seen in sublayers 1, 2, 4, and 5; it was most prominent in sublayers 2 and 4, but is discontinuous in sublayers 1 and 5. (B) Pyriform amacrine cells with dendritic processes that innervated all four layers (arrow). Scale bar = 20 um.

17

and in the majority of retinas studied, the ON- and OFF-type ganglion cells are

influenced more by acetylcholine than any of the other retinal neurons (Masland & Ames,

1976; Glickman et ah, 1982).

Glycinergic amacrine cells were distinguished by markers that recognize residues

1-10 of the glycine receptor al subunit (Schroder et ah, 1991). Visualization revealed

low levels of receptor immunoreactivity in sublayers 1, 2, 4, and 5; labeling also

traversed intermittently across the inner nuclear and outer plexiform layers, but was not

as intense (Yazulla & Studholme, 2001). These results correspond to in situ

hybridization studies that isolated zebrafish glycine receptor subunits al and p to the

inner plexiform layer, and the a2 subunit to both the outer and inner plexiform layers

(Imboden et ah, 2001). Using antibodies that probed for glycine, Connaughton et al.

(1999) found an intense immunopositive band in sublayer 3 of the inner plexiform layer

that consisted of dendrites originating from amacrine somata in the proximal inner

nuclear or ganglion cell layers.

Probing for the GABAergic system required multiple markers that recognized the

GABA-synthesizing enzyme glutamate decarboxylase (GAD), the GABA-metabolizing

enzyme GABA-transaminase (GABA-T), as well as markers that probed for GABA

transporters and receptor subunits (Yazulla & Studholme, 2001). All four GAD

antibodies produced similar patterns, with intense labeling throughout sublayers 1, 2, 4,

and 5 that were interrupted by a thin and dark band in sublayer 3 of the inner plexiform

layer (Figure 7). Amacrine cell bodies were prominently outlined by GAD-1440 and

GAD-67, but GAD-Wu actually highlighted horizontal cells more and amacrine cells

less. Immunoreactivity of GABA-T, however, showed an absence of the sublayer 3

18

band, and labeling was identified in horizontal cells, random amacrine somata, and

segregated the inner plexiform into three broad immunopositive layers. A colocalization

study performed by Mosinger and Yazulla (1987) demonstrated in the rabbit retina that

all GAD-immunopositive cells are GABA-positive, but only 21% of the GABA-

immunopositive cells are GAD-negative.

When characterizing GABA transporters, three antibodies were used against

transporters that moved GABA across the plasma membrane (GAT-1, GAT-2, GAT-3),

and one antibody was used against a transporter that moved GABA into synaptic vesicles

(VGAT) (Yazulla & Studholme, 2001). GAT-1 labeled amacrine cell bodies and the

inner plexiform layer as a whole, with distinct banding seen in sublayers 1 and 4. GAT-2

was immunopositive in scattered amacrine cells bodies on the proximal inner nuclear

layer border, but did not label any dendrites. GAT-3 fluorescence was not seen in

amacrine cells. VGAT antibodies highlighted the inner plexiform layer in three large

bands. Lastly, amacrine cells were not immunopositive for GABA receptors.

Recognizing the locations of GABA and glycine immunoreactivity is exceptionally

pertinent to the study of amacrine cells, as 40 to 50% of the amacrine cells in the

mammalian retina contain or accumulate these neurotransmitters (Vaney, 1990).

Substance P, cholecystokinin (CCK), enkephalin, neuropeptide Y (NPY), and

somatostatin (SRIF) are all neuropeptides that have been identified in zebrafish amacrine

cells (Yazulla & Studholme, 2001). In many retinas, neuropeptides have been found to

coexist with a number of other neuroactive substances in the same amacrine cell. For

instance, extensive studies of amacrine cells in the bird retina have revealed evidence that

glycine accumulates in about 10 percent of enkephalin-immunopositive cells, as well as

19

those cells that stain positive for somatostatin; some enkephalin-immunopositive cells

also accumulate GABA (Watt et al., 1984). Substance P was seen rarely and only in

pyriform amacrine cells that had a single dendritic process that monostratified in sublayer

3 of the inner plexiform. Vaney (1990) however, reported that amacrine cells

immunoreactive for substance P have cell bodies found both on the proximal inner

nuclear margin and displaced into the ganglion cell layer, with varicose dendrites that

bistratified in sublayers 3 and 4. Amacrine cell bodies that were immunopositive for

Figure 7: Distribution of Glutamic Acid Decarboxylase (GAD) (Yazulla & Studholme, 2001). (A) GAD-1440 labeled the entire inner plexiform layer, with light fluorescence in amacrine cell bodies in the proximal inner nuclear layer. (B and D) GAD-67 strongly labeled the inner plexiform layer, similar to GAD-1440, but more intensely labeled amacrine cell bodies. (C) GAD-Wu highlighted horizontal cells more and amacrine cells less, but intensely labeled the entire thickness of the inner plexiform layer as well. Scale bars = 20 um.

20

cholecystokinin were regularly spaced along the proximal border of the inner nuclear

layer and sent out bistratified dendrites that innervated sublayers 2 and 4, forming two

diffuse bands. Neuropeptide Y was reported in amacrine cell bodies directly on the

proximal nuclear margin that extended processes into sublayers 1 and 5, producing two

dense bands. Somatostatin was localized to three types of amacrine cells: SAa\ and

SAa2 had processes that innervated only sublayer 1, but the former had a larger cell body

and smooth dendrites, while the latter had a smaller cell body with dendritic varicosities;

Sab had a pyriform cell body that sent out a thin process to sublayer 5.

Enkephalins were targeted using Leu-Enk (leucine-enkephalin) and Met-Enk

(methionine-enkephalin) antibodies (Yazulla & Studholme, 2001). Three types of

amacrine cells were identified for Leu-Enk: a spindle-shaped cell in the inner nuclear

layer that ramified in sublayers 1 and 5, a pyriform cell in the inner nuclear layer that

ramified in only sublayer 5, and a displaced cell in the ganglion cell layer that innervated

sublayer 5. Met-Enk immunoreactivity was seen in only a single amacrine cell with

dendritic processes in strata 1 and 5 of the inner plexiform layer.

The last neurochemical category described consisted of the catecholaminergic

amacrine cells, of which serotonin-accumulating cells are a member. In order to identify

the uptake of the neurotransmitter serotonin, antibodies were used against the 5-HT

receptor, which is specific for serotonin (Yazulla & Studholme, 2001). In rabbit, positive

labeling was observed only in the S1 amacrine cell, whose dendritic branching stratifies

mainly in sublayers 1 and 5 and displays varicosities near the terminal ends (Vaney,

1986). Instead of lying directly at the border between the inner nuclear and the inner

plexiform layers, the SI amacrine somata were slightly staggered and seen displaced into

21

both layers (Yazulla & Studholme, 2001). Sandell and Masland (1986) reported an

additional morphological type, designated as the S2 amacrine cell, which also accumulate

serotonin. In zebrafish, positively labeled cells appear to be S1 amacrines, as labeled

processes were restricted to sublayers 1 and 2 (Yazulla & Studholme, 2001).

Phenylethanolamine-N-methyltransferase (PNMT) immunoreactivity was seen

throughout the entire span of the inner plexiform layer, with manifestation in three dense

bands at layers 1, 3, and 4/5 (Yazulla & Studholme, 2001).

While the overlap between amacrine cells described using morphological and

neurochemical techniques (i.e., Figure 5 and Table 2) undoubtedly exists, this correlation

has not yet been established in zebrafish. There are a very limited number of amacrine

cell types in other species that have been classified using all criteria; two well-studied

types, the All and A17 amacrine cells, are described in-depth below. Interestingly, only

7 morphological types of zebrafish amacrine cells have been identified (Connaughton et

al, 2004), but immunocytochemical studies clearly demonstrate that there are more than

14 types in existence (Yazulla & Studholme, 2001; Table 2 below). Marc and Cameron

(2001) also examined the neurochemistry of zebrafish amacrine cells and found 9 types, 7

of which contained GAB A and 2 of which contained glycine. These results imply that a

single morphological type may in fact release or uptake more than one type of

neurotransmitter (colocalization), or that all the morphological types of amacrine cells

have not been identified in zebrafish.

Antibody Comments

Cholecystokinin Few cell bodies; sublayers 2, 4 bistratified Choline acetyltransferase Numerous cell bodies; sublayers 1, 2, 4, 5 of IPL Enkephalin (leucine) Rare, pyriform and stellate Enkephalin (methionine) Rare, pyriform; sublayers 1, 5 bistratified GABA-transaminase Scattered cell bodies; three broad IPL IR layers GABA transporter 1 Numerous cell bodies, all IPL sublayers GABA transporter 2 Scattered cell bodies; no processes Glutamate decarboxylase (all) Numerous cell bodies; sublayers 1, 2, 4, 5 Glycine Sublayers 1, 2, 4, 5; intense sublayer 3 IR Neuropeptide Y Few cell bodies; sublayers 1, 5 bistratified PMNT Numerous cell bodies; three broad IPL IR layers Serotonin S1 cell only; varicosities; sublayers 1, 5 Somatostatin Three cell types; sublayers 1, 5 Substance P Pyriform cell only; sublayer 3 monostratified

Table 2: Neurochemical Summary of Amacrine Cells (Yazulla & Studholme, 2001). Amacrine cells demonstrate a wide neurochemical diversity, which falls into six major categories: cholinergic, serotonin-accumulating, glycinergic, GABAergic, peptide-immunoreactive, and catecholaminergic. Antibodies were used to mark enzymes, receptors, and transporters that corresponded to the major types of neurotransmitters.

Variety of Function

If amacrine cells display such morphological, synaptic, and neurochemical

differences, then it can be assumed that they must perform a wide assortment of functions

in shaping visual information (MacNeil et al., 1999). Furthermore, since amacrine cells

only synapse onto other retinal neurons in the inner plexiform layer, the specialized

functions of each amacrine cell must be carried out chiefly in this region of the retina.

Function is determined by recording the physiological responses of amacrine cells to

different strengths and patterns of light stimuli. These physiological responses are

described by their timecourse (transient or sustained), their response to the onset (ON-

23

cells) or offset (OFF-cells) of light, and/or the presynaptic element (rods or cones).

Morphological correlations to the physiological responses have also been identified.

The stratification of the inner plexiform layer, in particular, is extremely

important in implicating amacrine cell function. The morphological separation of the

inner plexiform layer into five distinct sublayers is related to the physiological ON- and

OFF-center surround responses of the ganglion cells, which indicate that an amacrine cell

with dendritic processes extending into the first two sublayers, or sublamina a,

correspond to an OFF-center cell, while an amacrine cell with dendritic processes

extending into the last three sublayers, or sublamina b, correspond an ON-center cell

(Famiglietti & Kolb, 1976). This has been confirmed physiologically, as cells with

processes in sublamina a are maximally stimulated when the lights are off; whereas, cells

maximally stimulated when the lights are on have processes in sublamina b. It has also

been suggested that sublayer 5 may serve as an intermediate zone between sublamina a

and b, where ON- and OFF-signals converge, as some transient responses generated here

are conveyed to the middle of the inner plexiform layer (Djamgoz et al., 1989).

Although this general scheme describes synaptic connections occurring between

cells of the same polarity, OFF-type cells may in fact receive inputs from ON-type cells,

and vice versa. For example, based on analysis of bipolar axonal stratification patterns,

Connaughton et al. (2004) introduced the possibility that input from multistratified

bipolar cells to OFF-type amacrine cells may actually convey ON-type signals to these

cells. Conversely, ON-type amacrine cells may receive input from multistratified OFF-

type bipolar cells in order to obtain OFF-type signals. Multistratified amacrine cells with

24

dendritic processes ramifying in both sublaminae may assimilate excitatory information

from different categories of bipolar cells before conducting a single output response, or

even individual output responses to postsynaptic processes in each sublamina to facilitate

communication between the sublaminae (Connaughton et ah, 2004).

In addition to ON- vs. OFF-cell types, amacrine cell functions can be classified

according to whether they receive input (1) predominantly from cones, (2) that occurred

by interconnecting the rod and cone pathways, or (3) purely from rods (Kolb & Nelson,

1985). The morphology of amacrine cells in each class may vary widely. For example,

the A4 amacrine cell of the cat cone pathway is small-field, with densely branched

dendritic processes that are confined to the distal inner plexiform layer. These cells are

postsynaptic to cone bipolar cells that innervate sublamina a and are presynaptic to OFF-

center ganglion cells dendrites that also innervate the same sublamina. On the other

hand, the cone-driven the A19 amacrine cell is wide-field, with sparsely branched

dendritic processes that ramify only in sublayer 2. This cell receives input from cone

bipolar cells that terminate in sublamina a, and provides an ON/OFF response to the

onset and offset of light, respectively (Reviewed in Kolb & Nelson, 1985). Further, the

small-field, bistratified A13 cell serves as interneurons between rod bipolar cells and

OFF-center ganglion cells, increasing the ability of these ganglion cells to detect low

levels of light (Nelson & Kolb, 1984; Kolb & Nelson, 1985).

Lastly, amacrine cells can be functionally classified as either sustained or

transient, based on their responses to varied light illuminations that occur in their

receptive fields. Sustained amacrine cells respond to light by either depolarizing or

25

hyperpolarizing; the strength of the response remains constant and does not change in the

face of extreme light differences, forming monophasic receptive fields that determine

orientation specificity (Chan & Naka, 1976; Naka, 1980). In many cases, the

depolarizing phase of the sustained responses is characterized by oscillating, spike-like

potentials that are followed by large hyperpolarizations (Hosokawa & Naka, 1985; Chan

& Naka, 1976). The general morphology of sustained amacrine cells has been identified

as having a mitral-shaped cell body with a single primary dendrite extending into the

inner plexiform layer before spreading out laterally into a profuse arborization (Chan &

Naka, 1976). Variations occur in length and thickness of the primary dendrite, as well as

the symmetry of the dendritic field in relation to the cell body.

Transient amacrine cells display ON- and OFF-depolarizations (Figure 8) in

response to light illumination, and are naturally ideal in mediating motion-sensing

responses; they are also direction-sensitive (Dowling, 1969). While their spindle-shaped

cell bodies are found on the proximal inner nuclear layer margin, their secondary

dendritic processes are nearly always bistratified and connected to the cell body by a

primary dendrite (Chan & Naka, 1976). Though the individual patterns of transient

responses follow a consistent pattern of depolarization at the onset and offset of light,

variations are observed. Some cells have a single peak, while others have multiple peaks

at the onset and offset of light (Chan & Naka, 1976). Some transient amacrine cells have,

for example, a large ON-response and a small OFF-response to spot illumination

(Werblin & Dowling, 1969) (Figure 8). The characteristics of the light response can also

change depending on whether the stimulus is small or large (Werblin & Dowling, 1969)

26

SPOT 250 p

ANNULU5

U L-t - ^ 5 _ ^

i

i

tt_ '̂

A M A C R I N E

r*\ t r _ 1

CELL

500 |i ANNULU5

1

* ^ ^ ^ ^•••r* ^ * i s? *"**

5MV

Figure 8: Recordings of a Transient Amacrine Cell in Response to a Spot Illumination and to Annuli of 250 pi and 500 \i (Werlin & Dowling, 1969). This amacrine cell responded transiently with a large ON-response and a small OFF-response to spot illumination. It then responded with equally large responses at both onset and offset of a small (250 u) annulus, but gave a larger OFF-response with a larger (500 u) annulus.

(Figure 8). There are also transient amacrines that respond with only ON-responses and

some that respond with only OFF-responses, regardless of illumination size and/or

geometry (Dowling, 1969). Transient amacrine cells do not have center-surround

antagonistic receptive fields.

By identifying both the neurotransmitter content and the electrical response to

light stimulation of morphologically different amacrine cells in the tiger salamander

retina, Yang et al. (1991) was able to synthesize these different characteristics to

ascertain the function of these cells (Figure 9). The study concluded that all

multistratified and monostratified glycine-immunoreactive cells displayed typical

transient depolarizations at the onset and offset of light, while most GABA-

immunoreactive cells produced either a sustained ON-response or a sustained OFF-

27

TRANSMITTER MORPHOLOGY RESPONSE

GLYCINE MULTI-

GLYCINE MONO-

6A8A-0N

s r

V V

v V

GABA, Enk

6AIA-OFF

6A8A/ /GLYCINE

V AT

Figure 9: Summary Schematic of LY-filled Cells with their Neurotransmitter, Morphology, and Response Type (Yang et al., 1991). Amacrine cell responses to the onset and offset of light (S) represent excitatory inward currents. Both multistratified and monostratified glycine-IR cells, as well as GABA/glycine cells, were ON-OFF transient. GABA-IR cells were of two types, ON and OFF, which had sustained responses at the onset and offset of light, respectively. GABA/enkephalin cells responded with a sustained ON-response.

28

response to photic stimulation. Amacrine cells that contained both GABA and glycine

showed transient ON/OFF depolarizing responses, but those cells that were

immunopositive for both GABA and enkephalin responded similarly to GABA-

immunoreactive cells (Yang et ah, 1991).

It is extremely important to understand the morphological diversity of amacrine

cells, as this information would be essential in the identification of function. As

exemplified in the Yang et al. (1991) study, bringing together what is known about cell

morphology and the corresponding physiological responses to light can establish the

function of a given amacrine cell type. The following section introduces two of the most

well-studied amacrine cells, the All and A17 cells, in order to illustrate how the different

classification criteria discussed in this proposal can be combined to identify a single cell

type and all of its characteristics.

All and A17 Amacrine Cell Types

In spite of the great morphological diversity of amacrine cells, only 2-3 types,

most notably the All and A17 amacrine cells, have been the primary focus of a number of

studies. Their morphological and physiological properties have been consistently

identified, and their functional roles within retinal circuitry have been widely established.

Before delving into such details, however, it must be understood that mammalian

rod photoreceptors, which are responsible for dark-adapted, or scotopic vision, synapse

onto a collection of bipolar cells that are devoted solely to receiving rod signals (Cajal,

1933; Boycott & Dowling, 1969; Kolb, 1970). Through anatomical work performed on

29

Figure 10: A Golgi-stained Example of an All Amacrine Cell in Cat Retina (Kolb et al, 2003). The All amacrine cell is narrow-field and bistratified, with dendritic ramifications in sublamina a and b of the inner plexiform layer. It possesses a pyriform (conical) arborization.

cats, it was discovered that unlike cone bipolar cells, rod bipolar cells do not directly

synapse onto ganglion cells, but actually go through a population of connecting rod

amacrine cells before reaching the ganglion cell layer (Kolb & Famiglietti, 1974;

Famiglietti & Kolb, 1975; Kolb & Nelson, 1981). In the rabbit, All amacrine cells

connect the rod and cone pathways by linking rod bipolar cells with cone bipolar cells

30

(Figure 12), which ultimately transmit the initial rod signals to the ganglion cells (Strettoi

eta!., 1992).

In both cats and rabbits, All amacrine cells have narrow dendritic fields (Figure

10), which penetrate vertically through all sublayers of the inner plexiform, but possesses

a largely bistratified morphology (Kolb et ah, 1981; Vaney et ah, 1991). A single apical

dendrite descends from the cell body and branches into a conical arborization throughout

both sublaminae a and b (Kolb & Famiglietti, 1974; Famiglietti & Kolb, 1975; Vaney et

ah, 1991). The portion of the cell occupying sublamina a is characterized by short, thin

appendages extending from the primary dendrite that terminate in large varicosities or

lobular appendages; continued descent into sublamina b by the primary dendrite yields

wider ramifications (Kolb & Famiglietti, 1974; Famiglietti & Kolb, 1975; Vaney et ah,

1991).

In the cat, the synaptic terminals of rod bipolar cells directly contact All amacrine

cells, forming one element of the dyad postsynaptic to the bipolar cell ribbon terminals

(Nelson, 1982). In the middle of the inner plexiform layer, these arboreal dendrites

participate in gap junctions with cone bipolar terminals (Kolb, 1979; Strettoi et ah, 1989,

1990). Most importantly, the arboreal dendrites receive significant amounts of

information from rod bipolar terminals (Famiglietti & Kolb, 1975; Strettoi et al, 1990),

and intracellular electrode recordings show that All amacrine and rod bipolar cells share

the same response threshold, causing saturation of both neurons at similar intensities of

light (Nelson, 1982).

The most distal region of the inner plexiform layer is the location of chemical

synaptic output (glycinergic) for All amacrine cells (Nelson, 1982). It is here, in

31

Figure 11: Serotonin-containing Amacrine Cells Stained with Lucifer Yellow in Wholemount Rabbit Retina (Kolb et al, 2003). The A17 amacrine cells are wide-field with diffuse dendritic branching dotted with varicosities. Stratification innervates all sublayers of the inner plexiform layer.

sublamina a, that the lobular dendrites contact cone bipolar terminals in the rabbit

(Strettoi et al., 1989,1990) and with bipolar, ganglion, and other amacrine cells in the cat

(Kolb & Famiglietti, 1974; Famiglietti & Kolb, 1975). It can be said that the All cell is

postsynaptic to rod bipolar cells in sublamina b, whereas sublamina a is reserved for

smaller inputs derived from cone bipolar cells (Strettoi et al., 1992).

A17, on the other hand, is the largest of the rod-driven amacrine cells, with

dendritic fields reaching 500-1,200 um in diameter (Nelson & Kolb, 1984; Nelson &

32

Kolb, 1985) (Figure 11). Extremely thin and straight dendrites spread diffusely

throughout all sublayers of the inner plexiform layer, and run predominantly in sublayers

4 and 5 (Nelson & Kolb, 1985; Sandell & Masland, 1986; Menger & Wassle, 2000). The

dendritic processes are dotted with as many as 1,000 varicosities, or beads, that are either

concentrated near the somal region, or distinctly aggregated near the ganglion cell bodies

around peripheral dendrites (Nelson & Kolb, 1985). Although the cell does not

physically appear so, it can be considered as functionally bistratified—amacrine inputs

occur in sublamina a and bipolar inputs occur in sublamina b (Nelson & Kolb, 1985).

Figure 12: Summary Diagram of the Rod Pathway Neurons and their Responses (Kolb et al, 2003). The All and A17 amacrine cells modulate the rod pathway between the rod bipolar cells and the ON- and OFF-center ganglion cells. Hyperpolarizing rod photoreceptors depolarize rod bipolar cells, which in turn synapse onto the two depolarizing amacrine cells All and Al7.

33

The A17 amacrine cells in the cat and rabbit receive input almost exclusively

from rod bipolar terminal ribbons (Figure 12), and they reciprocally synapse back onto

these same bipolar cells (Kolb & Famiglietti, 1974; Kolb, 1979; Nelson & Kolb, 1985).

In rat, one postsynaptic cell of the dyad usually releases GAB A and reciprocally synapses

back onto the rod bipolar cell (Kim et al., 1998), and it is believed that this corresponds

to the A17 amacrine cell (Menger & Wassle, 2000).

The Zebrafish (Pernio rerio)

Since the early 1990s, the number of scientific studies utilizing zebrafish (Danio

rerio) has increased significantly across a broad range of neuroscience disciplines. The

zebrafish has been recognized as an excellent model in the study of development

neuroscience, particularly with respect to embryology, developmental biology, and

genetics. Furthermore, and most importantly to this study, the similarities of the

zebrafish visual system to visual systems of other vertebrate organisms renders it

extremely valuable for contributions to visual neuroscience. Visual processing pathways

have been well-documented in both adult and developing zebrafish, which has

established an overall understanding of the anatomy, physiology, and behavior of

zebrafish vision. (Reviewed in Bilotta & Saszik, 2001)

Zebrafish do not require special attention or conditions in order to survive in large

numbers; they are easily maintained and reproduce rapidly. A number of techniques have

been developed to introduce genetic mutations into adult zebrafish, which can be detected

in the first generation. These mutations can be sustained for future research by freezing

and storing zebrafish sperm and eggs (Bilotta & Saszik, 2001). Additionally, the fewer

34

number of cells in a zebrafish embryo makes it a suitable model for more complex

vertebrates. The developing embryo is clear, and matures both quickly and externally,

allowing effortless visual access to tissue differentiation events. (Reviewed in Matthews

etah, 2002)

Retinal development in the zebrafish (Schmitt & Dowling, 1999) parallels that of

many other vertebrates, such as mice (Cepko et ah, 1996). In addition, like primates, the

zebrafish retina is duplex (Branchek & Bremiller, 1984), containing both rod and cone

photoreceptors, and exhibits visual behavior as early as 4 days postfertilization (Easter &

Nicola, 1996). Even after hatching, physical structures continue developing (Branchek &

Bremiller, 1984), allowing the simultaneous study of the developing retinal anatomy with

physiology and visual behavior.

In the zebrafish retina, the physiology and/or morphology of photoreceptor

(Branchek & Bremiller, 1984; Raymond et ah, 1993; Robinson et ah, 1993), horizontal

(McMahon, 1994; Connaughton & Dowling, 1998), bipolar (Connaughton & Nelson,

2000), and ganglion cells (Mangrum et ah, 2002) have been reported. A number of

amacrine cell types have been identified and classified both morphologically

(Connaughton et ah, 2004) and physiologically, but neurochemical data (Yazulla &

Studholme, 2001), is strongly indicative of additional types.

Purpose and Objectives

Amacrine cells serve a very critical role in vision. They are lateral modulators of

the vertical information processing pathway and contribute to direction and orientation

specificity, color coding, movement detection, as well as light/dark contrast

35

differentiation. A comprehensive understanding of the neural circuitry of the retina is

dependent upon a thorough knowledge of the morphology, physiology, and

neurochemical diversity of amacrine cells.

The purpose of this study is to identify and morphologically classify amacrine

cells in the zebrafish retina using prepared retinal slices and the DiOlistics lipophilic dye

technique. Although the Dil dye is not specific only for the amacrine cell, it will allow

visualization of individual cells within the layers and subsequent identification of any

present amacrine cells that have incorporated the dye. Previous studies on organisms

such as the cat (Kolb et ah, 1981) and the turtle (Kolb, 1982) have revealed more than 20

morphological types; furthermore, Connaughton et al. (2004) initially identified 7 types

in the zebrafish. Such literature has led us to believe that there is a wide array of

morphological types of amacrine cells in the zebrafish. Most importantly, with the

zebrafish as an excellent model in the study of the vertebrate visual system, data obtained

from this research will be extremely useful and applicable to visual diseases in humans,

such as in Kufs and Tay-Sachs disease where amacrine cells are rendered non-functional.

MATERIALS AND METHODS

Retinal Slices

Adult zebrafish (Danio rerio) were dark-adapted overnight and anesthetized using

0.2% tricaine methanesulfonate. In the light-adapted zebrafish, photoreceptor outer

segments are surrounded by pigment granules. This serves to protect the sensitive rod

outer segments in bright light conditions, and holds the retina to the back of the eye. In

dark adapted conditions, retinomotor movements occur. At this time, the pigment

granules migrate distally and the rod inner segments contract. These movements expose

the rod outer segments to the low light conditions, facilitating vision. Experimentally,

dark-adapted conditions also allow the retinal tissue to be easily separated from the sclera

and prepared for analysis.

Animal care and tissue preparation protocols were approved by the Animal Care

and Use Committee of American University. Retinal slices were prepared according to

methods published by Connaughton (2003). Briefly, after the zebrafish were killed by

decapitation, the eyes were removed, hemisected, and placed vitreal-side down onto the

center of a 0.45-um Millipore filter paper. Following removal of the sclera and isolation

of the retina, the retinal sample was placed into a recording chamber and immersed in

Ringer's solution, which is a salt solution composed of 120 mM NaCl, 2 mM KC1, 1 mM

CaCl2,1 mM MgCl2, 4 mM glucose, and 4 mM HEPES buffer (to pH 7.4-7.5 with

NaOH). This is the standard extracellular solution used in physiological studies of the

36

37

zebrafish retina (Connaughton and Maguire, 1998; Connaughton and Nelson, 2000,

Connaughton, 2003) and it increases the longevity of the excised retina. A tissue sheer

was used to cut the retinal sample into thin (~100 urn) slices, which were rotated 90° and

anchored to Vaseline strips in the recording chamber to view tissue in cross-section and

expose the layers.

DiOlistics

Microcarriers, or tungsten beads, coated with the lipophilic dye Dil (1,1'-

dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes,

Eugene, OR), were used to label retinal tissue. The dye immediately dissolved into the

cellular membrane it contacts and is maintained within the membrane, outlining the cell

in its entirety.

The microcarriers are prepared using published protocols (Gan et al., 2000;

Kettunen et al., 2002). Briefly, 50 mg of tungsten beads (1.3 urn diameter, mesh 20, Bio-

Rad, Hercules, CA) were thinly spread onto a glass plate using a razor blade. The

particles were then mixed with 50 ul of 3% Dil solution (3 mg Dil in 100 ul of methylene

chloride; Sigma, St. Louis, MO) and again, thinly spread across the glass plate surface.

The methylene chloride was allowed to evaporate, which caused the Dil to precipitate

onto the tungsten beads, coating them with the dye. The dye particles were resuspended

in 3 mL of water through sonication. The solution was then drawn into a 15 cm long

length of plastic tubing (Bio-Rad, Hercules, CA, #165-2441) that had an interior pre-

coated with polyvinylpyrrolidone (Bio-Rad, #165-2440, 10 mg/ml in distilled water).

38

The tubing was rotated gently for 45 minutes until the particles completely coated the

interior surface. The tubing was then cut into 13 mm long "bullets".

After removal of the Ringer's solution in the recording chamber, each set of

retinal slices was bombarded with four bullets loaded in a Bio-Rad Helios Gene Gun (190

psi). A 3-urn pore size polycarbonate membrane filter was placed between the sample

and gene gun to prevent clumps and damage to the tissue. Slices were then re-submerged

in Ringer's solution before being viewed under an Olympus compound microscope with

a 40 x water immersion lens and a Dil filter set. Multiple fluorescent images of each

amacrine cell at varying tissue depths were taken with an Olympus Oly-150 camera and

Flashbus software. The images were compiled and edited in Adobe Photoshop (ver. 7.0

or CS3, Adobe Systems, San Jose, CA) to remove peripheral fluorescence emitted by

extraneous dye around the cell. The resulting fluorescent image of each cell was overlaid

onto a Hoffman image of the same field, allowing visualization of the dendritic

stratification patterns of each cell in relation to the retinal layers.

Identification of Cell Types and Terminology Used

A total of 138 amacrine cells were studied and morphologically classified in the

zebrafish retina; from these, 18 different cell types were established. In order to qualify

as a cell type, a specific neuronal morphology had to be seen two or more times, each

time in a different zebrafish eye. Using these criteria, three additional types were

identified, although they are not included in this document because the quality of the

pictures was too poor for them to be definitively characterized. Furthermore, a number of

39

other morphologies (n = 10) were also identified, but will not be reported here because

only one example was observed per type. Six of these are shown in the appendix.

The terminology used to name each cell type follows a previously published

naming scheme (Connaughton et al., 2004). For example, a cell represented by A0ff-sl is

an amacrine cell, designated by "A". The "off subscript indicates that dendritic

branching occurs in sublamina a (OFF layers) of the inner plexiform layer, and "si"

denotes that the dendritic terminals are predominantly found in sublayer 1 of the six IPL

sublayers. It is important to note that in this study, the classification of cells as "on" or

"off types refers solely to the sublamina in which the dendritic branching occurs; in

other words, cells innervating sublamina a are OFF-types and cells innervating sublamina

b are ON-types. Although this terminology is applied here towards morphological

classification, it is also very likely that it corresponds to the cells' physiological responses

to light (Famiglietti & Kolb, 1976; Yang et al, 1991).

Although most literature abide by the four dendritic field diameter ranges

established by Kolb et al. (1981), zebrafish amacrine cells tend to have much smaller

dendritic field widths and would nearly all be characterized as narrow-field (30-150 urn)

under such categories. For the purposes of this study, cells are designated as narrow-field

or wide-field in order to reduce confusion between two cells innervating the same

sublayer, and therefore having the same name. Narrow-field cells are those with

dendritic arbor the same or slightly larger in diameter than the cell body. Dendritic

arbors of wide-field cells, in contrast, are typically more than 3x larger than the diameter

of the cell body. Lastly, cells of similar dendritic width innervating the same sublayer are

differentiated by a hyphenated number, such as " - 1 " .

RESULTS

Amacrine cell types described here were separated based on dendritic field size,

dendritic arborization patterns within the IPL, and/or cell body shape. Of the 18 types of

amacrine cells identified, there were 7 OFF-types, 6 ON-types, and 5 bistratified types.

Although displaced amacrine cells with somata located in the ganglion cell layer certainly

exist (Boycott & Dowling, 1969), only those cells with somata found in the inner nuclear

layer and inner plexiform layer were seen in this study. Additionally, as the lipophilic

Dil dye is not specific for amacrine cells, the random outlining of any retinal cell in the

ganglion cell layer made it difficult to differentiate between displaced amacrine cells and

the ganglion cells themselves.

OFF-type Amacrine Cells

Seven types of A0ff amacrine cells were identified, with dendritic branching

innervating only sublamina a of the inner plexiform layer (Figure 13, Table 3). Four A0ff

cell types exhibited a monostratified layer of primary dendritic processes that were

restricted to sublayer 1. Sublayer 3 was also innervated in a monostratified pattern by

one A0ff cell type. Two A0ff cell types displayed a bistratified dendritic branching pattern,

in which processes laterally ramified in two different OFF-layers (the term "bistratified"

is used here to describe the pattern of dendritic branching, and is not the same as when

applied to cells that innervate both sublaminae). The cell bodies were all round, and

40

41

ranged from very small (5 um) to considerably large (20 um) in diameter. Dendritic

arbors ranged from 50 to 130 um, with an average of-90 urn. There was no direct

correlation between somal diameter and dendritic field width, although cells with larger

somata tended to have larger dendritic fields, and vice versa. All three sublayers of

sublamina a were innervated by at least one cell type.

Four A0ff amacrine cell types were morphological variations on A0ffsl, with

monostratified dendritic branching terminating in sublayer 1 (Figure 13A-C, F). The

Aof^sl-2 cell had a round soma (Figure 13 A), from which a thick apical dendrite

extended proximally before branching horizontally through sublayer 1 in a wide dendritic

field (~110 urn). Few varicosities were seen. The wavy dendrites of the A0frsl-4 cell

were also wide in field (~130 urn), slightly thicker, and extended directly from either side

of the soma (Figure 13C). The cell body was larger compared to other A0ff si cell types

and was situated on the distal border of the inner plexiform layer, rendering the A0ff-sl-4

cell type as interstitial. Extending from either side of the soma, the dendritic processes of

the A0ff sl-3 cell were thin and displayed numerous, small varicosities (Figure 13B). In

most cases, it was difficult to distinguish the cell body, as it was close in size to the

varicosities; furthermore, these varicosities occurred in a continuous, unbroken chain

along the extent of the dendritic breadth. In the fourth A0frsl cell, A0ff-slw, both

dendritic processes projected proximally from the soma before horizontally innervating

sublayer 1 (Figure 13F). Although in the image it appears that the right dendritic process

is shorter than the left, both processes of A0frslw cells are in reality close in length; the

remaining segment of the right dendrite in this example was too blurred by fluorescence

42

A0ff-s1-3 Antf-s1/s3

Figure 13: DiOlistic Stains of OFF-type Amacrine Cells in the Zebrafish Retina. Seven amacrine cell types were identified with dendritic branching innervating only sublamina a of the inner plexiform layer. Four cell types had processes restricted to sublayer 1 (A-C, F), one cell type had processes only sublayer 3 (D), one cell type had processes innervating both sublayers 1 and 2 (G), and one cell type had processes innervating both sublayers 1 and 3 (E). A: A0frsl-2. B: A0frsl-3. C: Aofrsl-4. D: Aoff-s3. E: Aofrsl/s3. F: A0frslw. G: A0ff-sl/s2. Scale bar = 10 urn in G (applies to A-G).

43

to correctly determine its shape.

The dendritic processes of A0ff-s3 cells (Figure 13D) were remarkably varicose,

with small beads prevalent along the primary and secondary dendrites. The primary

dendrite extended proximally from the cell body before ending in a large swelling in

sublayer 3, which was nearly the same size as the soma itself (length x width = 10 x 10

(am). Varicose monostratified dendritic processes spread outwards from this swelling and

ramified laterally throughout sublayer 3. Although the A0frs3 cell in this example

appears to have an asymmetric projection, it is most likely that this asymmetry resulted

from the slicing procedure, which often truncates sections of the dendritic processes and

narrows the actual dendritic field diameter. Of the three total A0ff-s3 cells found in the

zebrafish retina, two demonstrated this asymmetry, while one extended dendritic

processes of equal length.

The first bistratified amacrine OFF-type, A0ff-sl/s2, exhibited primary dendritic

branching that innervated both sublayers 1 and 2 of the IPL (Figure 13G). The dendritic

processes terminating in sublayer 1 extended directly from either side of the soma and

were dotted by large, round varicosities. The dendritic processes branching into sublayer

2 also emerged from either side of the soma, but had smaller varicosities that were less

distinctly spherical in shape.

Bistratification was also identified in A0jrsl/s3 cells (Figure 13E), which

modeled a shape more characteristic of bistratified cells than A0ff-sl/s2 cells. A

monostratified layer of dendritic processes, extending from either side of the cell body,

innervated sublayer 1 of the IPL to a field width of ~60 (am. Another pair of dendritic

processes extended from the cell body proximally and entered the IPL at an angle. One

of these processes simply terminated in a varicosity once arriving in sublayer 3; the other

process, also reaching sublayer 3 in a varicosity, continued laterally in that sublayer. The

sublayer 3 dendritic processes achieved a field diameter of-50 urn, which is slightly

narrower than those of sublayer 1. The dendritic processes of A0ff-sl/s3 cells had

multiple well-defined varicosities.

ON-type Amacrine Cells

Six types of Aon amacrine cells were identified, with dendritic branching

innervating only sublamina b of the inner plexiform layer (Figure 14, Table 3). Because

the dendritic trees of these cells must reach sublamina b, all Aon amacrine cells are

characterized by a long primary dendrite extending proximally from an either round or

pyriform cell body. Varicosities were present on all Aon cell types except for the Aon-s5w

cells. Aon cell types included cells with the smallest dendritic field diameters of 0 to 10

(am; the remaining field widths ranged from 40 to 110 urn, with an average of-70 urn.

All three layers of sublamina b were innervated by at least one Aon cell type.

Two Aon amacrine cell types were primarily characterized by a single apical

dendrite extending proximally from the soma and deep into IPL, passing the first three

sublayers that constitute sublamina a. The primary dendrite always terminated in a large,

bulbous varicosity from which emerged secondary dendrites restricted to monostratified

layer. The Aon-s5/s6 cell (Figure 14A) had a wider dendritic field (-110 |a,m), while the

Aon-s4 cell (Figure 14E) had a narrower dendritic span of-70 \xm.

45

The apical dendrite of Aon-s5/s6 cells extended down into the IPL from the round

or pyriform cell body, ending in a swelling at sublayer 5 before giving rise to laterally

ramifying secondary dendrites. The primary dendrite was sometimes exceptionally thick,

and always extended proximally from a soma 10 urn in diameter. Secondary dendrites

were dotted by many varicosities, which either spread evenly across the entire dendritic

span or clustered mainly to one side of the primary dendrite. In this example, the layers

were compressed during retinal isolation, causing the inner plexiform to appear thinner

than usual. Similarly, Aon-s4 cells had round cell bodies from which a single primary

dendrite extended into the IPL and terminated in a soma-like swelling in sublayer 4.

Varicose secondary dendrites ran horizontally from this swelling through sublayer 4.

Of the remaining four Aon amacrine cell types that were seen in this study, two

types ramified solely in sublayer 5 and two types terminated only in sublayer 6 of the

IPL. The Aon-s5n cells were of narrow dendritic fields averaging a mere 13 um, just

slightly larger than the soma, which were -10 urn in diameter (Figure 14D). These cells

were recognized by a lengthy primary dendrite that innervated sublayer 5, as well as two

very short, varicose secondary dendritic spines that branched into sublayer 3. The

primary dendrite, however, extended somewhat laterally from the cell body, before

making a sharp turn at the INL/IPL border to enter the IPL. At sublayer 5, it curved

slightly and laterally ramified. A large swelling was present at sublayer 4, in addition to

a handful of smaller varicosities that dotted both the primary and secondary dendrites.

Like all ON-type amacrine cell types seen thus far, Aon-s5w cells had an apical

dendrite originating at the cell body, -10 urn in diameter (Figure 14F). This dendritic

process traveled through the IPL until it reached the border between sublayer 3 and

46

••**- fc,r*«-«^»-^"t«jk^, -.*

Figure 14: DiOlistic Stains of ON-type Amacrine Cells in the Zebrafish Retina. Six amacrine cell types were identified with dendritic branching innervating only sublamina b of the inner plexiform layer. One cell type innervated only sublayer 4 (E), three cell types had processes located only in sublayer 5 (A, D, F), and two cell types had processes restricted to sublayer 6 (B, C). A: Aon-s5/s6. B: Aon-s6n. C: Aon-s6w. D: Aon-s5n. E: Aon-s4. F: Aon-s5w. Scale bar = 10 um in F (applies to A-F).

sublayer 4, where it bifurcated into two secondary processes that extended diagonally to

sublayer 5. Here, the secondary processes laterally ramified (dendritic field diameter -40

um). Varicosities were not present on the dendritic processes of this cell type.

47

The Aon-s6n amacrine cell type illustrated one of the simplest possible cell

morphologies—a cell body gave way to a major, apical dendrite (~50 um in length) that

directly penetrated the IPL to sublayer 6 (Figure 14B). Both round and pyriform-shaped

somata were seen in this cell type. Varicosities were prevalent along the dendritic

process.

The last Aon amacrine cell type, Aon-s6w, had a long primary dendrite that

stemmed proximally from the pyriform-shaped soma (Figure 14C). Once it reached

sublayer 5, the dendritic process began curving until it was able to laterally stratify in

sublayer 6, giving a field diameter of approximately 50 um. This cell type was

particularly varicose, with many large, distinct swellings covering the single primary

dendrite.

Bistratified Amacrine Cells

Five types of Abi amacrine cells were identified, with dendritic branching

innervating both sublaminae a and b of the inner plexiform layer (Figure 15, Table 3).

Dendritic arbors ranged from 10 to 140 um, with an average of-60 um. In four

bistratified cells, the field width of dendritic processes that ramified in ON-layers was

significantly smaller than the field width of the OFF-layer processes.

Three Abi amacrine cell types innervated sublayers 1 and 5. The most commonly

seen bistratified amacrine cell type in this study is the Abi-sl/s5-l cell type (Figure 15B).

Varicose dendritic processes extend directly from either side of the cell body and

continued laterally throughout sublayer 1. The dendritic field diameters ranged from 50

um to exceeding 120 um (This variation is most likely because segments of dendritic

48

processes are often lost during retinal slicing, or the cell was located deep in the IPL,

making it difficult to see it in its entirety). Another dendritic process originated at the

base of the soma and traveled down into sublayer 5 of the IPL, ending in a bulbous

swelling. From this process, a short dendritic spine projected into sublayer 4.

Figure 15: DiOlistic Stains of Bistratified Amacrine Cells in the Zebrafish Retina. Five amacrine cell types were identified with dendritic branching innervating both sublamina a and b of the inner plexiform layer. Three cell types had processes restricted to sublayers 1 and 5 (B, C, D), one cell type innervated sublayers 1 and 4 (A), and one cell type had processes located in sublayers 2 and 5 (E). A: Abi-sl/s4. B: Abj-sl/s5-l. C: Abi-sl/s5-2. D: Abi-sl/s5-3. E: Abi-s2/s5. Scale bar = 10 urn in E (applies to A-E).

Abj-sl/s5-2 cells (Figure 15C) had round and pyriform-shaped cell bodies, both of

which were seen equally. In this case, primary dendrites extend from either side of the

pyriform soma and were monostratified in sublayer 1. A single primary dendrite

originated at the proximal end of the soma and descended to the s2/s3 border, where it

bifurcated into two secondary processes that ran diagonally into sublayer 5. In this case,

49

the secondary dendritic processes terminated once reaching sublayer 5 and did not

laterally ramify in this layer. Hence, the field diameters of the two dendritic layers were

very different; the si processes reached up to -70 um in width and the s5 processes

reached only - 20 um. Such disparity in dendritic extent between the ON- and OFF-

layers suggests that these cells process OFF-type inputs gathered from a larger surface

area of the retina than ON-type inputs. In other cells of this type, further lateral

stratification of sublayer 5 was observed, achieving up to ~110 um in dendritic field

width. Although it is not shown here, Abi-sl/s5-2 amacrine cells can also be extremely

varicose.

Abi-sl/s5-3 cells (Figure 15D) had cell bodies located higher in the inner nuclear

layer (-10 um from the INL/IPL border) than most amacrine somata, which are typically

observed hugging the proximal INL border adjacent to the IPL. An apical dendrite

extended proximally from the cell body to sublayer 1 of the IPL, where it diverged into

two secondary dendritic processes. One of the secondary dendritic processes ran laterally

through sublayer 1, while the other continued, eventually curving to terminate in sublayer

5. Varicosities were few to none in amacrine cells of this type. Narrow dendritic field

diameters averaged -20 um.

Abi-sl/s4 cells (Figure 15A) had a single, long primary dendrite that extended

proximally from the round soma and straight down into sublayer 4, where it ended in a

small nodule. In this case, a short (—10 |J.m), varicose secondary dendrite ran laterally in

sublayer 4; in other cells of this type, this secondary dendrite is altogether absent, and the

primary dendrite terminal is not marked by a varicosity. Lastly, in all cells of this type, a

short spine-like secondary dendrite projected from the primary dendrite into sublayer 1.

50

One of the most spectacular amacrine cell types illustrated here is the Abi-s2/s5

cell type, which is characterized by exceptionally thick dendritic processes that span

nearly 14 urn in width (Figure 15E). The soma of this cell is not especially large, as one

would expect from a cell morphology of this size. An apical dendrite extended

proximally from the cell body to sublayer 2, where secondary dendritic processes

laterally stratified to a field diameter of -14 um. A short dendritic spine branched off of

the primary dendrite into sublayer 1. Two tertiary dendritic processes extended from the

secondary processes, where they penetrated the IPL to sublayer 5 before they laterally

ramified in both directions to ~12 um in field width. Large varicosities were apparent on

Abi-s2/s5 cells, although a few were difficult to ascertain due to the thickness of the

dendritic branches.

51

Cell type OFF-type

Aofrslw Aofrsl-2 Aoff-sl-3 AoH-sl-4 A0ff-s3 A0fi-sl/s2 Aofrsl/s3

ON-type A0n"S4

Aon-s5n Aon-s5w Aon-s5/s6 Aon-s6n Aon-s6w

Bistratified Abi-sl/s4

Abi-sl/s5-l

Abi-sl/s5-2

Abi-sl/s5-3

Abi-s2/s5

Dendritic stratification sublayer

si si si si s3

sl-2 si s3

s4 s5 s5

s5-s6 s6 s6

si s4 si s5 si s5 si s5 s2 s5

% depth in IPL

0-17 0-17 0-17 0-17

33-50 0-50 0-17

33-50

50-67 67-83 67-83 67-100 83-100 83-100

0-17 50-67 0-17

67-83 0-17

67-83 0-17

67-83 17-33 67-83

No.

4 2 2 2 3 3 3

4 2 4 6 4 3

2

4

4

2

2

Somal dimensions (length x width)

12.5 x 10.0 10.0 x 10.0 5.0 x 7.5

10.0 x 20.0 10.0 x 10.0 10.0 x 10.0 15.0 x H.7

11.3 x 11.3 10.0 x 10.0 10.0x8.8 12.0 x 12.0 12.5 x 11.3 11.7 x 10.0

12.5 x 12.5

8.8 x 10.0

13.8 x 10.0

10.0 x 7.5

15.0 x 15.0

Dendritic field size

113(90-130) 125(110-140) 120(80-160) 145(130-160) 70(40-90)

117(90-150) 32(20-50) 73(40-120)

90(70-130) 13(10-15) 88(40-130)

122(70-160) —

40(30-50)

12.5(10-15) 12.5(10-15)

88(50-120) 8(0-10)

85(70-100) 25(20-30) 23(20-25)

5(0-10) 140

105(90-120)

Table 3: Morphological Measurements of Amacrine Cell Types in the Zebrafish Retina. Dendritic stratification indicates the sublayer of the IPL in which the bulk of the dendritic processes innervates (sublayer) and the overall vertical depth to which the processes penetrated (% depth). Mean somal dimensions were obtained by measuring along the distal-proximal axes (length) and left to right along the IPL (width). Dendritic field sizes were measured between the two most distant ends of the dendritic tree. Ranges are given in parentheses. All size/dimension values are in urn.

DISCUSSION

In this study, 18 types of amacrine cells were identified in the zebrafish retina.

These types were initially separated into A0ff, Aon, and Abi cells based on depth of

dendritic stratification, but were further described according to dendritic field size,

dendritic stratification patterns within the IPL, and/or cell body shape. Although two

diffuse amacrine cell types were identified by Connaughton et ah (2004), they were not

observed here. This is an apparent indication of their low frequency in the zebrafish, as

was also discussed by Connaughton et ah (2004). Very few diffuse cell types were found

in cat (Famiglietti & Kolb, 1975), turtle (Kolb et ah, 1982), and tiger salamander (Yang

et ah, 1991), but were common in roach (Wagner & Wagner, 1988) and in rabbit

(MacNeil et ah, 1999).

Again, only amacrine cells with somata situated in the inner nuclear layer and

interstitial amacrine cells were identified in this study. Displaced amacrine cells (with

cell bodies in the ganglion cell layer) were not considered, and although they are

undoubtedly present in the zebrafish retina, there was no way to distinguish them from

the ganglion cells. In fact, immunocytochemical studies have revealed that displaced

amacrine cells are extremely rare in the zebrafish retina (Connaughton et al, 1999;

Yazulla & Studholme, 2001). All neurotransmitter markers were detected primarily in

amacrine cells with somata found in the inner nuclear layer (Connaughton et al., 1999;

Yazulla & Studholme, 2001).

52

53

The DiOlistic labeling technique was a rapid and effective method of identifying

amacrine somata and dendritic processes, as any individual neurons contacted by the dye

particles were immediately labeled. Yet because the lipophilic Dil dye is not specific for

amacrine cells, any random combination of the seven retinal neurons was visualized in

each retinal slice. At times, none of the amacrine cells in a single slice were even

labeled; alternatively, numerous bipolar cells, photoreceptors, and ganglion cells could be

brightly fluorescing. Moreover, a successfully labeled set of retinal slices was highly

dependent on several variables. If the retinal slices were too thick, the dye would fail to

incorporate itself into the membrane bilayer; conversely, if the retinal slices were too

thin, the retinal tissue would partially or completely detach from the filter paper. The

amount of dye carried by each bullet also strongly affected the results, since too little dye

generated no labeling and an overabundance of dye caused the outlining of many

interconnected neurons, making it impossible to assign dendrites to a specific cell body.

Each set of slices were "shot" with 2-3 bullets before viewing, which ensured maximal

incorporation of dye without overloading the cells with color.

In the past, a number of techniques were used to visualize retinal cells. These

include Golgi impregnation (Sherry & Yazulla, 1993), light microscopic

immunocytochemistry (Sherry & Yazulla, 1993; Yazulla & Studholme, 2002), dye

injection (Yang et ah, 1991), as well as DiOlistic lipophilic dye staining (Connaughton et

al., 2004). Compared to the first three traditional methods, using lipophilic dyes to

morphologically distinguish amacrine cells is simply faster and easier. Results can be

obtained within minutes of dye-particle delivery and the procedure can be quickly

repeated with the same tissue sample to improve or enhance visualization.

54

Previously, Connaughton et al. (2004) identified seven amacrine cell types in

zebrafish. Three of these types were also identified in this study. In addition, 15 other

cell types are described here.

OFF-type Amacrine Cells

All Aoff amacrine cells had dendritic processes innervating sublayer 1 of the IPL

(Figure 16). Three A0ff types were monostratified in sublayer 1, with dendritic processes

extended either proximally or laterally from a rounded cell body. These cells appear

similar to the monostratified wide-field cells found in rabbit (MacNeil et ah, 1999) and to

one of the stratified wide-field "radiate" cells found in cat (Famiglietti & Kolb, 1975).

Wagner & Wagner (1988) identified an interstitial amacrine cell (A7) in roach that is

very similar to A0frsl-4 cells, with thicker dendritic processes that emerge in both

directions from the cell body. The GAB A immunopositive AM 13 interstitial cell in

lizard (Sherry & Yazulla, 1993) is similar as well. It is likely that the two tyrosine-

hydroxylase immunopositive ON-OFF cells identified in tiger salamander (Yang et ah,

1991) are also similar to the A0ff-sl-4 cells. In A0ff-sl-2 cells, a single apical dendrite

extended proximally from the rounded soma before giving rise to laterally ramifying

secondary dendrites in sublayer 1. These cells appear similar to the monostratified Al

cells found in roach (Wagner & Wagner, 1988), the monostratified "narrow si" cells

found in rabbit (MacNeil et al., 1999), and the A 0 t rs lw cells found also in zebrafish

(Connaughton et al., 2004).

The A0ff-sl/s2 amacrine cell appears most similar in morphology to one of the

stratified wide-field "radiate" cells found in cat (Famiglietti & Kolb, 1975), although

55

these cells are illustrated as having a few more processes terminating in the lower

sublayers of sublamina a. A0frsl/s3 cells are not similar to any previously identified

amacrine cells in other species, although they share a comparable bistratified shape to

A28 cells in roach (Wagner & Wagner, 1988). A28 cells ramify to sublayers 2 and 4,

whereas the A0ff-sl/s3 cells ramify sublayers 1 and 3. However, the fact that zebrafish

have 6 IPL sublayers while roach have 7IPL sublayers may contribute to this difference

in sublayer innervation.

Aoff-slw and A0ff-sl-2 cells appeared similar to the somatostatin-immunopositive

cells described by Yazulla & Studholme (2001). A0ff-sl-2 cells seem to correspond with

the SAal types, whereas A0g-slw cells display similarities to the SAa2 types (Yazulla &

Studholme, 2001). A0frsl/s2 cells may correlate to the serotonin-containing cells also

described by Yazulla & Studholme (2001), although these cells are described to also

ramify in sublayer 5.

A0ff-s3 cells are most similar to the varicose narrow s3 cells identified in rabbit

(MacNeil et al., 1999). Wagner & Wagner (1988) reported the A4 cell in roach as

innervating sublayer 3, but these cells were described as only having varicosities near the

outlying regions of the dendritic processes. Furthermore, double labeling experiments

suggested that the A4 cells may in fact be the OFF-type cholinergic starburst amacrine

cells. Cells of this same starburst morphology were also observed in roach by Djamgoz

et al. (1989), who called them the A4.5 cells. In contrast, immunocytochemical studies

in the zebrafish have revealed that cells immunopositive for the cannabinoid CB1

receptor, calretinin, and the neuropeptide substance P are all characterized by

monostratified dendritic processes in sublayer 3 (Yazulla & Studholme, 2001). In

56

addition, Connaughton et al. (1999) reported an intense band of glycine-immunoreactive

dendritic processes restricted to sublayer 3 in zebrafish. This major difference in

neurochemical content makes it difficult to ascertain whether the A0ff-s3 cells are indeed

starburst amacrine cells.

This study did not find amacrine cells with monostratified dendritic processes

restricted to only sublayer 2, but this is not an indication that these cell types do not exist.

Connaughton et al. (2004) identified A0ff-s2 cells with multiple dendritic branches

terminating in sublayer 2.

A.n-sl-2 Aoff-sl-3 Aofl-s-M Aofpslw

Aon-sl/sS AoB-sl/82

Figure 16: Morphological Summary Diagram of Seven OFF-type Amacrine Cells. A0ff cell types had dendritic trees restricted to sublamina a (sublayers 1-3) of the inner plexiform layer, w, wide-field.

ON-type Amacrine Cells

The dendritic processes of Aon amacrine cells innervated sublamina b of the IPL

and were monostratified (Figure 17). The Aon-s5/s6 cells identified here appear similar to

the sustained depolarizing A4.5 cells in roach (Djamgoz et al, 1989) and monostratified

Aon-s5 cells found by Connaughton et al. (2004) in zebrafish. Aon-s5/s6 cells also

57

resemble A9/A10 amacrine cells in roach (Wagner & Wagner, 1988), although Aon-s5/s6

cells exhibit less branching and more varicosities. Additionally, it is also possible that

the Aon-s5/s6 cells are equivalent to the narrow-field unistratified cells seen in rat (Perry

& Walker, 1980), as these are described to have dendrites that lie in the outer region of

the inner plexiform layer.

A>n-4 A„n-s5n Aon-sSm A„n-s6n A„n-s6w

Figure 17: Morphological Summary Diagram of Six ON-type Amacrine Cells. Aon cell types had dendritic trees restricted to sublamina b (sublayers 4-6) of the inner plexiform layer. Retinal layers of the Aon-s5/s6 cell in this example were compressed during retinal isolation, reducing the IPL thickness, n, narrow-field; w, wide-field.

One of the most frequently seen types, the Aon-s5w cell, shared some physical

similarities with the wide-field, GABA immunopositive AM3.2 cell in lizard (Sherry &

Yazulla, 1993). It is possible that the true dendritic field sizes of the Aon-s5w cells have

been moderately truncated during retinal slicing. The Aon-s5n amacrine cells identified

here did not exhibit definite similarities with previously described cells in any species,

but the closest comparable cell would be the A3 8 cell in roach (Wagner & Wagner,

1988). The A38 cells have profusely branched dendritic processes in sublayers 3 and 4,

whereas the Aon-s5n cell shows a more simplified innervation of sublayer 3.

58

The Aon-s4 cell is morphologically comparable to the A19 cells in roach (Wagner

& Wagner, 1988), which bear highly varicose dendritic processes and few spines. Aon-

s6n cells are most similar to two GABA immunoreactive, sustained ON-type amacrine

cells in tiger salamander (Yang et ah, 1991).

Bistratified Amacrine Cells

Abi amacrine cells have dendritic branching terminating in both sublaminae a and

b (Figure 18). The Abi-s2/s5 cell appears similar to the GABA immunopositive AT2 cell

in lizard (Sherry & Yazulla, 1993) and to the "wavy bistratified" in rabbit (MacNeil &

Masland, 1998).

Like Abj-sl/s5-2 cells, the GABA immunopositive AM2 cell in lizard (Sherry &

Yazulla, 1993) was characterized by a pyriform-shaped soma and two lateral layers of

stratification. The proximal layer of the AM2 cell was extended further laterally than the

distal layer, which agrees with other Abi-sl/s5-2 cells seen in this study. Dendritic

processes were often truncated during slicing, which accounts for the small dendritic

width of the proximal dendrites seen in the figure. The Abi-sl/s5-2 cells also appear to

correspond to the somatostatin immunopositive pyriform cell with dendritic processes

extending laterally in sublayers 1 and 5 of the IPL (Yazulla & Studholme, 2001).

In spite of their high frequency in the zebrafish, Abi-sl/s5-l cells are not similar to

any amacrine cells identified in other species. This result either reflects their rarity in

other species or that these cells are completely exclusive to zebrafish alone.

Yazulla & Studholme (2001) isolated a population of neuropeptide Y

immunoreactive amacrine cells whose dendritic arbors were found exclusively in

59

Abrs1/s4 A,,rs1/s5-1 ^ 1 / 8 5 - 2 ^ 1 / 8 5 - 3 A„rs2/s5

Figure 18: Morphological Summary Diagram of Five Bistratified Amacrine Cells. Abi cell types had dendritic trees innervating both sublamina a and b (sublayers 1-6) of the inner plexiform layer.

sublayers 1 and 5; these cells do not appear morphologically similar to any ones

identified here. Connaughton et al. (2004), however, reported an A0n_sl/s5 bistratified

cell whose morphology corresponds to that described by Yazulla & Studholme.

A study conducted at the University of Texas Health Science Center at Houston

identified a medium-field, bistratified amacrine cell type innervating IPL sublayers 2 and

4 in zebrafish (Miller et al, 2008; #3049: board D645; unpublished data). In light-

adapted retina, the cell displayed strong Lucifer Yellow coupling at its gap junctions.

This cell may correspond to the strong glutamate receptor2/3 immunoreactivity confined

primarily to sublayers 2 and 4 of the zebrafish IPL (Yazulla & Studholme, 2001).

CONCLUSIONS

This study supports previous findings indicating a large morphological diversity

of amacrine cells in zebrafish (Yazulla & Studholme, 2001; Connaughton et al., 2004).

Neurochemical labeling experiments have demonstrated amacrine cells to be

immunopositive for at least 19 various transmitters, voltage-gated channels, and synapse

associated membrane proteins (Yazulla & Studholme, 2001). If each antibody is specific

for an individual cell type, then there are at least 19 different amacrine cell types in

zebrafish. Hence, considering the seven types described by Connaughton et al. (2004)

and the 18 types reported here (three of which were previously identified), it makes sense

to conclude that each cell type must uptake or release more than one neurotransmitter.

This leads us to believe that each cell type may serve more than one retinal function, each

carried out by an individual neurotransmitter.

By determining whether dendritic processes were restricted sublamina a,

sublamina b, or both, each cell type was identified as OFF-type, ON-type, or bistratified.

This classification dictates the nature of the input and output signals corresponding to

each type—for example, OFF-type amacrine cells receive visual information from both

OFF-type bipolar and ganglion cells, but also delivers output signals to these same cells.

In further classifying the cell types according to sublayer innervations, it is possible to

more specifically ascertain the bipolar and ganglion cell types that receive input from and

provide output to each amacrine cell type. The exchange of signals between OFF-type

60

61

and ON-type cells is also highly probable (Connaughton et al., 2004), as well as between

IPL sublaminae as mediated by bistratified amacrine cells (Kolb & Nelson, 1996). In

tiger salamander, bipolar cells exhibit a similar bistratification in the IPL as amacrine

cells (Wu et al., 2000). This bistratification of bipolar axon terminals corresponds with

the segregation of OFF- and ON-type inputs in the IPL, suggesting that in zebrafish,

bistratified amacrine cells may interact with bistratified bipolar cells in the processing of

visual signals. Therefore, a large number of amacrine cell types allows for the complex

signal transmission and processing between bipolar and ganglion cells, which would

otherwise be impossible.

However, when compared to a related cyprinid, the roach, in which 43 amacrine

cell types were reported (Wagner & Wagner, 1988; Djamgoz et al., 1989), the number of

Species

Tiger salamander Zebrafish Rat Zebrafish Cat Catfish, Ictalurus punctatus Rhesus monkey, Macaca mulatta Turtle, Pseudemys scripta elegans Rabbit Cyprinid fish, roach

Types

7 7 9 18 22 22 26 27 28 43

Reference

Yang etal., 1991 Connaughton et al., 2004 Perry & Walker, 1980 This study Kolb et al., 1981 Chan&Naka, 1976 Mariani, 1990 Kolb, 1982 MacNeil et al., 1999 Wagner & Wagner, 1988; Djamgoz et al., 1989

Table 4: Morphological Types of Amacrine Cells Identified in Nine Species of Animals. The number of morphological types of amacrine cells varies widely between species of animals. Seven types of amacrine cells were identified by Connaughton et al. (2004). Three of these types were seen in this study, which identified a total of 18 types.

62

cell types in zebrafish is in fact quite low (Table 4). The roach also has a significant

number of wide-field amacrine cells, with the largest measured being more than 1000 um

in diameter (Wagner & Wagner, 1988). Dendritic field widths of zebrafish are much

smaller, with the largest being -160 um in diameter. It is interesting to see such a high

degree of disparity between two related species, and may be attributed to the fact that

roach, being on average seven times as long as zebrafish, possess a more elaborate visual

system requiring the function of additional amacrine cell types. It may also be possible

that zebrafish amacrine cells co-localize neurotransmitters. At the same time, this

difference may simply indicate the presence of other cell types that remain undocumented

in the zebrafish, which is seen in the 13 morphological types not reported in this study

due to poor image quality and low occurrence. Being able to identify these types more

than once would be crucial in future studies to increase the number of amacrine cell types

established in zebrafish and to identify related cell types between species.

APPENDIX

x

Figure 19: DiOlistic Stains of Amacrine Cells Seen Only Once in the Zebrafish Retina. In the zebrafish, 10 morphological types of amacrine cells were only seen once, and six are shown here. Three cell types had processes restricted to sublayers 1 and 3 (A-C), two cell types innervated sublayers 4 and 6 (E, F), and one cell type had diffuse branching throughout the IPL (D). A: A0frsl/s3. B: A0frsl/s3. C: Aofrsl/s3. D: AdiffUse- E: Aon-s4/s6. F: Aon-s4/s6. Scale bar = 10 urn in F (applies to A-F).

63

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