shua Nathaniel hng

MODULATION OF OFF RESPONSE OUTPUT FROM MOUSE RETINAL GANGLION

CELLS BY MGLUR6, CB1, AND GABAc RECEPTORS

AN ABSTRACT

SUBMITTED ON THE SECOND DAY OF APRIL 2013

TO THE NEUROSCIENCE PROGRAM

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

OF THE SCHOOL OF SCIENCE AND ENGINEERING

OF TULANE UNIVERSITY

FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

BY

APPROVED: 1,6

Guoyo .D., Ph.D Director

/7

Jeffrey G. Taster, Ph.D.

aura A. Schra , .D.

FionaM. Inglis, Ph.D.

Beamin J. Hall, Ph.D.

ABSTRACT

The retina is a sensory tissue that converts optical images into neural signals

known as light responses. Light responses are transmitted from photoreceptors to

bipolar cells to retinal ganglion cells (RGCs) in parallel pathways specific for either

light increments or light decrements. This improves vision by doubling the retina’s

dynamic range and increasing contrast sensitivity. Research has shown that Off

pathways, which are sensitive to light decrements, are likely modulated by the

activity of metabotropic glutamate receptor 6 (mGluR6) receptors, cannabinoid 1

receptors (CB1Rs), and -aminobutyric acid C (GABAC) receptors. In this

dissertation, I investigate how these neurotransmitter receptors modulate Off

responses in the retina by performing whole-cell recordings of mouse RGCs.

On bipolar cells express mGluR6 receptors, a type of glutamate receptor that

hyperpolarizes bipolar cells when bound to glutamate. Previous research has shown

that these receptors modulate Off responses under dark adaptation, but effects

under light adaptation were unclear. My research has shown that mGluR6 receptor

agonist DL-2-amino-4-phosphonobutyric acid (APB) decreases light-evoked Off

responses under light adaptation by disrupting dopaminergic transmission between

amacrine cells and Off bipolar cells.

CB1Rs are localized to many cell types including cone and bipolar cell axon

terminals, each of which release glutamate. Research primarily in brain has shown

that cannabinoid receptor activation prevents neurotransmitter release from the

presynapse. This has led to the hypothesis that CB1R activation would decrease

glutamate release in Off pathways and attenuate Off responses. My research shows

that CB1R agonists differentially modulate Off responses. Based on my results, I

suggest that CB1R agonists increase light-evoked Off responses in one population of

RGCs by reducing GABA transmission between GABAergic amacrine cells and Off

bipolar cells.

GABAergic amacrine cells feed back onto bipolar cell axon terminals that

express GABAC receptors. Previous research has shown that GABAC receptor

antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) alters

On responses, but effects on Off responses are unclear. I show that TPMPA

modulates kinetics of both On and Off responses recorded from On-Off RGCs. All

together, the results in this dissertation indicate that mGluR6 receptors, CB1Rs, and

GABAC receptors modulate Off responses, and therefore vision.

oshua Nathaniel P ng

APPROVED: Guoy • ng W ng, M.D., Director

MODULATION OF OFF RESPONSE OUTPUT FROM MOUSE RETINAL GANGLION

CELLS BY MGLUR6, CB1, AND GABAc RECEPTORS

A DISSERTATION

SUBMITTED ON THE SECOND DAY OF APRIL 2013

TO THE NEUROSCIENCE PROGRAM

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

OF THE SCHOOL OF SCIENCE AND ENGINEERING

OF TULANE UNIVERSITY

FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

BY

Jeffrey„p. Tasker, Ph.D.

Schrader, Ph.D.

61, ( S

Fion . Inglis, Phi.

Benj in J. Hall, Ph.D.

aura

ii

ACKNOWLEDGMENTS

Completing this dissertation would not have been possible without the

support of many people. First, I thank my parents for instilling a life-long interest in

science and for nurturing a curious mind. Especially, I want to thank my mom,

Colleen Marvin, who has made many personal sacrifices for my education and

happiness. I am exceedingly grateful for my fiancée, Amanda Hill, for her love and

support. She has often been a sounding board for the ideas and experiments in this

dissertation. I also want to express my deep appreciation for my advisor, Dr.

Guoyong Wang. Dr. Wang has been a true mentor for me the past five years.

Whenever I needed assistance with experiments, understanding scientific concepts,

or even life advice, Dr. Wang was available and eager to help. Under Dr. Wang’s

direction, I have grown both as a scientist and as a young man. In addition, I want to

thank my committee: Dr. Jeffrey Tasker, Dr. Laura Schrader, Dr. Fiona Inglis, and Dr.

Ben Hall. I am very grateful for their guidance and mentorship over the years.

I deeply appreciate all of my fellow Wang Lab members. Patch clamp

electrophysiology is an extremely challenging technique. I attribute much of my

success with the technique to Dr. Joey Nemargut who trained me on the patch clamp

rig. Also, I am very appreciative of Joey’s support and friendship outside of the lab.

In addition, I want to thank our post-doctoral fellow Dr. Jinnan Yang and our lab

technician Wei Huang for support in the lab. Finally, I would like to extend my

iii

thanks to our lab undergraduates Nathan Pham and Carter Kelly for all their

assistance inside and outside of lab.

Finally, I want to thank my many friends in the Neuroscience Program at

Tulane. I especially want to thank Sherrie Calogero for guiding me through the

program. To all those I have mentioned here and countless others I have shared

experiences with at Tulane, thank you for helping to make the last five years so

enjoyable.

iv

FOREWARD

Joshua Nathaniel Pahng has conducted many experiments to examine the

roles of mGluR6, CB1, and GABAC receptors in shaping light-evoked Off responses in

the mouse retina. His research is primarily focused on synaptic circuitry in the light-

adapted retina. Over the course of his graduate career, he has co-authored a peer-

reviewed journal article and published two abstracts at the Society for Neuroscience

conference. Much of the research presented in this dissertation is also presented in

the following:

Yang J, Pahng J, Wang GY (2012) Dopamine modulates the Off pathway in light-adapted mouse retina. J Neurosci Res. 91 (1), 138-150.

Pahng J, Yang J, Wang GY. Cannabinoid receptor 1 agonist 2-arachidonoylglycerol

differentially affected light-evoked responses in mouse retinal ganglion cells under light adaptation. Program No. 775.10. Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2010. Online.

Pahng J, Yang J, Wang GY. Cannabinoid antagonist differentially modulated light-

evoked responses of mouse retinal ganglion cells under light adaptation. Program No. 557.3. Neuroscience Meeting Planner. Chicago, IL: Society for Neuroscience, 2009. Online.

v

TABLE OF CONTENTS

ACKNOWLEDGMENTS............................................................................................................................ii FOREWARD………………………………………………………………………………………………………..iv LIST OF FIGURES………………………………………………………………………………………………vii INTRODUCTION………………………………………………………………………………………………….1 Retina structure and organization……………………………………………………………...3 On and Off pathways………………………………………………………………………………….6 mGluR6 receptors in the retina………………………………………………………...………10 CB1 receptors in the retina………………………………………………………………………11 GABAC receptors in the retina…………………………………………………………………..14 Hypothesis………………………………………………………………………………………………15 Statement of specific aims………………………………………………………………………..16 CHAPTER 1: Dopamine modulates the Off pathway in light-adapted mouse

retina Introduction……...…………………………………………………………………………………….19 Materials and Methods…………………………………………………………………………….21 Results……………………………………………………………………………………………………27

vi

Discussion………………………………………………………………………………………………33 CHAPTER 2: CB1Rs modulate Off responses in light-adapted mouse retina Introduction……...…………………………………………………………………………………….54

Materials and Methods…………………………………………………………………………….56 Results……………………………………………………………………………………………………58 Discussion…………………………………………………………………………………….…………63 CHAPTER 3: GABAC receptor antagonist TPMPA modulates light-evoked Off

responses in light-adapted mouse retina Introduction……...…………………………………………………………………………………….82 Materials and Methods…………………………………………………………………………….85 Results……………………………………………………………………………………………………87 Discussion………………………………………………………………………………………………90 CONCLUSION…………………………………………………………………………………………………..102 LIST OF ABBREVIATIONS……………………………………………………………………………….107 LIST OF REFERENCES……………………………………………………………………………………..109

vii

LIST OF FIGURES 1. Diagram of rod and cone signaling pathways………………………………………………9 2. The effects of APB on RGC Off responses under different ambient light

conditions……………………………………………………………………………………………….39 3. The effects of L-AP4 and DCPG on RGC Off responses under different ambient

light conditions………………………………………………………………………………………..41 4. Antagonists of glycine, GABAA, and GABAC receptors did not eliminate the

APB-induced reductions of Off responses of RGCs in light-adapted mouse retinas…………………………………………………………………………………………………….43

5. Effects of dopamine receptor antagonists on the APB-induced reductions of

Off responses of RGCs in light-adapted mouse retinas……………………………….45 6. Effects of APB on light-evoked Off responses when D1 receptors and

inhibitory receptors are blocked………………………………………………………………47 7. HCN channel antagonist ZD 7288 prevented the APB-induced reduction in Off

responses under light adaptation……………………………………………………………..49 8. A depolarized holding potential of -40 mV, which inactivates HCN channels in

the recorded RGCs, did not prevent the APB-induced reduction of Off responses under light adaptation……………………………………………………………..51

9. A diagram showing a functional circuit from On pathways to Off pathways

under light adaptation……………………………………………………………………………..53 10. DSE of light-evoked Off EPSCs recorded from RGCs…………………………………..71 11. Effects of CB1R antagonist O-2050 on light-evoked Off responses of RGCs…73 12. Effects of CB1R agonist WIN on light-evoked Off responses of RGCs…………..75 13. Effects of CB1R agonist 2-AG when crossover inhibition is blocked with

mGluR6 agonist APB………………………………………………………………………………..77

viii

14. Increases in Off response amplitudes are prevented when GABAC receptors are blocked by TPMPA……………………………………………………………………………..79

15. Diagram showing a functional circuit for CB1R-mediated modulation of light-

evoked Off response amplitudes………………………………………………………………81 16. GABAC antagonist TPMPA increases the decay time of On and Off responses

in On-Off RGCs under low photopic conditions…………………………………………95 17. GABAC antagonist TPMPA increases the decay time of On and Off responses

in On-Off RGCs under high photopic conditions………………………………………..97 18. Blocking crossover inhibition with mGluR6 agonist APB does not prevent the

TPMPA-induced increase in Off response decay time under high photopic conditions………………………………………………………………………………………………99

19. Diagram showing a functional circuit for GABAC receptor-mediated

modulation of Off responses…………………………………………………………………..101

1

INTRODUCTION

The retina is a thin piece of neural tissue that senses photons and is crucial

for vision (1). It develops from the maturing diencephalon and, as such, is

considered part of the brain. Optical images are initially transformed into neural

signals by retinal photoreceptors that are sensitive to photons. The photoreceptors

can be subdivided into rods and cones that are active in dim and bright ambient

light intensities, respectively. The neural signal, also known as a light response, is

then transferred to interneurons known as bipolar cells, and ultimately to ganglion

cells whose axons terminate in the lateral geniculate nucleus of the thalamus. This

simple circuit uses glutamate as the primary neurotransmitter, and thus is known as

the glutamatergic through-pathway. Signal processing occurs within the retina prior

to the information reaching the lateral geniculate nucleus, both along the through-

pathways and by lateral transmission from amacrine and horizontal cells.

One way that the retina processes images is by dividing its neural signals

between parallel circuits (2). The circuit that is sensitive to light increments, that is

light with intensity above the mean ambient light intensity, is known as the On

pathway. On the other hand, the circuit that is sensitive to light decrements, or light

with intensity below the mean ambient light intensity, is known as the Off pathway.

This division into parallel circuits has important

2

consequences for vision. Separate channels are used to encode distinct parts of an

image. The specialization of one channel exclusively for light intensities above a

mean and another channel exclusively for light intensities below a mean results in

robust signal transfer for both light increments and light decrements; this is because

RGCs respond to both light increments and light decrements with high frequency

action potentials that are sent along the optic nerve to the lateral geniculate nucleus

(1). In addition, the division into parallel On and Off pathways is critical because it

results in the doubling of the retina’s dynamic range, or the range of light intensities

that can be effectively encoded into neural signals. The effects of halving the retinal

dynamic range are dramatic (3). In the rhesus monkey, the On channel was

reversibly blocked by intraocular injection of mGluR6 agonist APB. With only active

Off channels, the monkeys were unable to detect light increments. Moreover, the

monkeys experienced a severe loss in contrast sensitivity. Therefore, the parallel On

and Off pathways are critical for normal contrast sensitivity, which in turn is crucial

for resolving detail in everyday visual scenes. Since these parallel pathways are so

important for normal vision, it is vital that both the functional mechanisms of each

pathway and the differences between them are deeply understood. This thesis

focuses on the Off pathways and Off response modulation for two important

reasons. First, Off pathways form one half of the parallel circuits that are so

important for contrast sensitivity, and therefore should be studied in their own

right. Second, Off pathways have a relatively simple glutamatergic through-pathway

unlike On pathways that have an inverted synapse between the cones and bipolar

3

cells (4). Thus, for practical experimental reasons, we focus on modulation of Off

responses.

In this thesis, I examine the modulation of Off responses in the light-

adapted mouse retina. APB, a potent agonist of mGluR6 receptors has been shown to

increase Off response amplitudes under dark adaptation by disrupting crossover

inhibition from On pathways to Off pathways (5, 6, 7, 8). Therefore, I investigated

the effects of APB on Off response amplitudes under light adaptation and attempted

to determine the mechanism of APB’s effects, as detailed in Chapter 1. Investigators

have found that CB1Rs are localized to numerous sites in the mouse retina, but the

effects of this endocannabinoid (eCB) system on light responses are not understood

(9). I used CB1R agonists and antagonists to study the role the retinal eCB system

plays in modulating Off responses, as discussed in Chapter 2. Finally, previous

research has shown that GABAC receptors are localized to many bipolar cell axon

terminals, and these receptors modulate On response kinetics (10). I used GABAC

receptor-specific antagonist TPMPA to study the role of GABAC receptors in shaping

Off responses.

1. Retina structure and organization

The retina lines the posterior portion of the eye and is around 200 microns

thick in both humans and mice (11). Three main stages of processing occur within

the retina before the visual signals are sent to the lateral geniculate nucleus of the

thalamus (1). First, the photoreceptors transform the optical image made up of

photons into a neural signal in a process known as transduction. Second, the neural

4

signal is transmitted to interneurons including bipolar cells. Third, the neural signal

is transmitted to the RGCs that fire action potentials that travel to the thalamus.

Other retinal neurons include the horizontal cells with somas in the outer plexiform

layer (OPL) and amacrine cells with somas in the inner plexiform layer (IPL).

Photoreceptors are localized to the most posterior layer of the retina known

as the photoreceptor layer (12). In the absence of photons, photoreceptors are

depolarized due to a constant influx of sodium ions known as the dark current.

When photons are present, however, photoreceptors hyperpolarize. Rods are the

photoreceptor subtype that are active in dark adaptation and as such are involved in

night vision. They are extremely sensitive and can transduce single photons (13,

14). Cones, on the other hand, are active in light adaptation and are commonly

associated with visual acuity and color vision. They are not as sensitive as rods and

need the relatively abundant amount of photons present in daylight to function.

Both rods and cones release glutamate from their axon terminals.

Bipolar cells and horizontal cells receive direct glutamatergic input from the

photoreceptors (1). Their cell bodies are localized to the inner nuclear layer (INL)

and their dendrites stratify in the OPL. Rod bipolar cells receive inputs from rods,

cone bipolar cells receive inputs from cones, and one type of mouse bipolar cell

receives inputs from both rods and cones. The bipolar cell axon terminals stratify in

the IPL and release glutamate. Horizontal cells mediate lateral transmission in the

retina (15). They release GABA from their axon terminals that are localized to the

OPL. Depending on subtype, horizontal cells may feed back onto photoreceptors

5

and/or feed forward onto bipolar cells. Functionally, the horizontal cells average

photoreceptor signals (1).

The amacrine cells receive direct glutamatergic inputs from bipolar cells (16,

17). Most amacrine cell bodies are localized to the INL, but so-called displaced

amacrine cell bodies can be found in the ganglion cell layer (GCL). There are

approximately 40 subtypes of amacrine cell. The AII amacrine cell mediates forward

transmission and is crucial under dark adaptation. It receives inputs exclusively

from rod bipolar cells and connects to either On-cone bipolar cells via gap junctions

or Off-cone bipolar cells via glycinergic synapses. Other amacrine cell subtypes

mediate lateral transmission. Depending on subtype, amacrine cells can release

neurotransmitters including but not limited to glycine, GABA, dopamine, or

acetylcholine (18).

RGCs are the final output neurons of the retina. Their cell bodies are localized

to the GCL, the innermost layer of the retina, and they receive direct inputs from

bipolar cells and amacrine cells (1). Ganglion cells have diverse morphology and

physiology and can be subdivided into at least 20 subtypes. The On subtype has

dendrites that stratify in sublamina b, the innermost part of the IPL whereas the Off

subtype has dendrites that stratify in sublamina a, the outermost part of the IPL (19,

20). On RGCs respond with action potentials when there are light increments in the

center of their receptive field and Off RGCs respond with action potentials when

there are light decrements in the center of their receptive field.

6

2. On and Off pathways

The retina divides its neural signals into parallel circuits for light increments

and light decrements (1). In the light-adapted retina, also known as photopic

conditions, there is one circuit for light increments known as the On-cone signaling

pathway and one circuit for light decrements known as the Off-cone signaling

pathway. The On-cone signaling pathway proceeds as follows: 1) cones

hyperpolarize in response to photons, 2) glutamate release from cones onto On-

cone bipolar cells ceases, 3) mGluR6 receptors localized to On-cone bipolar cells are

inactive resulting in depolarization of the On-cone bipolar cells, 4) glutamate

released from On-cone bipolar cells depolarizes On ganglion cells. For light

decrements, the Off-cone signaling pathway proceeds as follows: 1) cones

depolarize in response to a decrease of photons, 2) cones release glutamate onto

Off-cone bipolar cells, 3) ionotropic glutamate receptors localized to Off-cone

bipolar cells are activated resulting in depolarization of the Off-cone bipolar cells, 4)

glutamate released from Off-cone bipolar cells depolarizes Off ganglion cells.

Under scotopic conditions of the dark-adapted retina, there are two circuits

for light increments (21). The main pathway for light increments in scotopic

conditions is known as the primary rod On pathway, and it proceeds as follows: 1)

rods hyperpolarize in response to photons, 2) glutamate release from rods onto rod

bipolar cells ceases, 3) mGluR6 receptors localized to rod bipolar cells are inactive

resulting in depolarization of the rod bipolar cells, 4) glutamate released from rod

bipolar cells depolarizes AII amacrine cell, 5) On-cone bipolar cells also depolarize

because of their gap junctions with AII amacrine cells, 6) glutamate released from

7

On-cone bipolar cells depolarizes On ganglion cells. The other pathway for light

increments in scotopic conditions is known as the secondary rod On pathway. In

this pathway, the rods form gap junctions with cones so when the rods

hyperpolarize the cones do as well. The neural signal then travels down the On-cone

signaling pathway just as it would under light adaptation.

There are three circuits for light decrements under scotopic conditions (22).

The primary rod Off pathway is as follows: 1) rods depolarize in response to

absence of photons, 2) rods release glutamate onto rod bipolar cells, 3) rod bipolar

cells expressing mGluR6 receptors hyperpolarize in response to glutamate, 4)

glutamate release from rod bipolar cells to AII amacrine cells ceases, 5) AII amacrine

cells hyperpolarize and cease release of glycine onto Off-cone bipolar cells, 6) Off-

cone bipolar cells depolarize and release glutamate onto Off ganglion cells. In the

secondary rod Off pathway, rods make glutamatergic synapses directly onto Off-

cone bipolar cells. Therefore, when the rods depolarize, they release glutamate onto

Off-cone bipolar cells that release glutamate onto Off RGCs. Finally, in the tertiary

rod Off pathway, rods form gap junctions with cones (23). Thus, when the rods

depolarize, the cones do as well resulting in a release of glutamate from cones that

depolarizes Off-cone bipolar cells, and ultimately depolarizes Off ganglion cells. For

both light increments and light decrements, the pathways are ranked (i.e. primary,

secondary, etc.) in order from most to least sensitive. The On and Off signaling

pathways are shown in Figure 1.

8

Figure 1. Diagram of rod and cone signaling pathways. A: One cone pathway and

two rod pathways for transmitting On responses. On cone signaling pathway: cones

> On-cone bipolar cells > On RGCs. Primary rod On pathway: rods > rod bipolar cells

> AII amacrine cells > gap junction > On-cone bipolar cells > On RGCs. Secondary rod

On pathway: rods > gap junction > cones > On-cone bipolar cells > On RGCs. B: One

cone pathway and three rod pathways for transmitting Off responses. Off cone

signaling pathway: cones > Off-cone bipolar cells > Off RGCs. Primary rod Off

pathway: rods > rod bipolar cells > AII amacrine cells > Off-cone bipolar cells > Off

RGCs. Secondary rod Off pathway: rods > Off-cone bipolar cells > Off RGCs. Tertiary

rod Off pathway: rods > gap junction > cones > Off-cone bipolar cells > Off RGCs. R=

rod, C= cone, RB= rod bipolar cell, On CB= On-cone bipolar cell, Off CB= Off-cone

bipolar cell, AII= AII amacrine cell, On RGC= On retinal ganglion cell, Off RGC= Off

retinal ganglion cell. Dotted lines represent gap junctions. Bolded cell types express

mGluR6 receptors at the dendrites.

9

Figure 1

10

In the lab, we are capable of experimentally determining whether a ganglion

cell receives inputs from On pathways, Off pathways, or both On and Off pathways.

RGCs are voltage-clamped in the whole-cell mode and a spot of light, centered on the

ganglion cell, is shined onto the retina. On ganglion cells respond with an inward

current at light onset whereas Off ganglion cells respond with an inward current at

light offset. On-Off ganglion cells, with bistratified dendrites that collect inputs from

both On and Off pathways, respond with an inward current both at light onset and at

light offset. These inward currents, provided they are large enough to reach

threshold, would trigger action potentials in vivo that would be transmitted via the

optic nerve (24).

3. mGluR6 receptors in the retina

In the retina, mGluR6 receptors have been localized to rod bipolar cell and

On-cone bipolar cell dendrites (25, 26). These receptors are G-protein coupled and

close a cation channel when bound to glutamate. Research has shown that this

cation channel may be TRPM1 (27). As a result of the cation channel closure in the

rod bipolar cell or On-cone bipolar cell dendrites, the bipolar cell hyperpolarizes.

However, Off-cone bipolar cells do not express mGluR6 receptors (28, 29, 30).

Instead, they express a combination of AMPA and Kainate receptors that depolarize

the Off-cone bipolar cell when bound to glutamate. It is this difference in glutamate

receptor expression at the bipolar cell that creates the distinct On and Off pathways.

While the On and Off pathways carry distinct neural signals representing

different parts of an optical scene, these pathways also crosstalk. One way that On

11

and Off pathways crosstalk is through crossover inhibition (31, 32, 33, 34).

Activation of On pathways inhibits Off pathways and vice versa via glycinergic

amacrine cells that are involved in lateral transmission (35, 36, 37). DL-2-Amino-4-

phosphonobutyric acid (APB) is a potent mGluR6 agonist (38). When it binds to

mGluR6 receptors on On-cone bipolar cell dendrites, the cell hyperpolarizes and

glutamate release from the On-cone bipolar cell axon terminal ceases. Since Off-cone

bipolar cells do not express mGluR6 receptors, APB does not directly affect them.

Thus, APB has been used experimentally to specifically block On pathways.

Previous studies have shown that in the dark-adapted retina, APB blocks On

responses but increases the amplitude of Off responses (5, 6, 7, 8). In this scenario,

APB inactivates the On pathway preventing crossover inhibition of Off pathways

and resulting in potentiation of Off responses. Since these previous studies have

been performed under dark adaptation, the rod pathways were active. During

application of APB, rod bipolar cells and On-cone bipolar cells are hyperpolarized

resulting in the total block of On pathways, and partial block of Off pathways. As the

primary rod Off pathway is blocked by APB, Off responses are carried by the

secondary and possibly by the rare mouse tertiary rod Off pathways. The effects of

APB on Off responses in the light-adapted retina are unknown, however.

4. CB1 receptors in the retina

Previous investigations of the effects of Cannabis use on vision provided the

first evidence of a retinal eCB system (9). Anecdotal evidence was provided by

Jamaican fishermen who claimed that smoking marijuana improved their night

12

vision and helped them catch fish at night (39). Cannabis sativa or

tetrahydrocannabinol administration was found to improve night vision in a field

study in Morocco (40). The field researcher hypothesized that these effects were

mediated at the retinal level. Marijuana smoking has also been shown to have other

visual effects such as a decrease in visual acuity during testing with a Snellen chart

and an increase in photosensitivity (41). Only after the eCB 2-AG was isolated in the

retina was the existence of a retinal eCB system confirmed (42).

The expression of CB1Rs in the retina has been investigated in human,

mouse, rat, chick, tiger salamander, goldfish, and rhesus monkey (9, 42, 43, 44). In

one study, CB1Rs were found to be expressed in rod synaptic terminals, cone

synaptic terminals, amacrine cells, RGCs, and sporadically in the IPL (42). Distinct

bands of expression were observed in the IPL near the INL and GCL. In another

study and using a different antibody, CB1R expression was detected in rat rod

bipolar cells, PKC-immunoreactive GABAergic amacrine cells, the outer plexiform

layer, and the IPL but not the GCL (43). In goldfish, CB1R immunoreactivity was

detected in cone pedicles, Müller’s cells, bipolar cell bodies, bipolar cell axon

terminals, and in the IPL, but CB1Rs were not detected in rod spherules or in

horizontal cells (44). Within the IPL, CB1R expression was strongest in the On

sublamina.

Various experiments have been performed to determine the functions of the

retinal eCB system. The majority of these studies have focused on the role this

system plays in modulating ion channel currents. Brain researchers have shown that

CB1Rs are activated by endogenous retrograde neurotransmitters (45, 46). In the

13

retina, KCl or mGluR1 agonist DHPG puff onto bipolar cell bodies elicited release of a

retrograde neurotransmitter that reversibly inhibited cone IK(V) currents by

approximately 25% (47). This effect was blocked by CB1R antagonist SR141716A

indicating that the retrograde transmitter was an eCB acting at CB1 receptors. Bath

application of CB1R agonist modulates several voltage-dependent ion channel

currents including HVA Ca2+ currents, ICa currents, and IK(V) currents in

photoreceptors, bipolar cells, and cultured RGCs (42, 44, 47, 48, 49, 50, 51, 52).

CB1R agonist concentrations as high as 10M were used in several studies because

biphasic effects emerge at lower concentrations like 1M, and because researchers

wanted to ensure that the lipophilic drug would penetrate all retinal synaptic layers

(48, 53, 54, 55, 56).

Several studies have investigated the role of the retinal eCB system in

regulating neurotransmitter release. A variety of CB1R agonists have been shown to

decrease generalized norepinephrine, dopamine, and glutamate release in the retina

(57, 58, 59). This is consistent with findings in other studies demonstrating that

CB1Rs activate Gi/o to reduce neurotransmitter release (60). CB1R agonists also

affect miniature post-synaptic current (mini) frequency. In cultured chick amacrine

cells with a low initial mini frequency, CB1R agonists increased the mini frequency

via a CB1R-mediated mechanism (61). CB1R agonists also decreased spontaneous

excitatory post-synaptic current (EPSC) and inhibitory post-synaptic current (IPSC)

frequencies recorded from mouse RGCs (62). Only one study has investigated CB1R

effects on light-evoked responses (63). In that study, researchers found that cone

responses to light offset returned to baseline faster following CB1R agonist

14

application. CB1R antagonist SR141716A did not block this phenomenon, however,

possibly indicating a non-CB1R effect.

5. GABAC receptors in the retina

GABAC receptors, like GABAA receptors, are ionotropic GABA receptors that

flux chloride (64). TPMPA is an antagonist that specifically blocks GABAC receptors,

but GABAA receptors can be blocked specifically by bicuculline and picrotoxin (PTX)

blocks both GABAA and GABAC receptors (65, 66). While GABAA receptor expression

is predominant in other parts of the central nervous system, GABAC receptors are

highly expressed in the retina. In mammals, GABAC receptors are localized to rod

and cone bipolar cell axon terminals (67). Bipolar cell axon terminals receive GABA

inputs from GABAergic amacrine cells (1). Light GABAA expression has also been

observed in bipolar cell axon terminals, but the majority of GABAA receptors are

expressed in cones, bipolar cell dendrites, and ganglion cells (68). In ferret, the ratio

of GABAC to GABAA (GABAC: GABAA) expression is higher in On bipolar cells than Off

bipolar cells indicating differences in GABA receptor expression between On and Off

pathways (69). Further, GABAC: GABAA expression is higher in rod bipolar cells than

cone bipolar cells. Following with the morphological data, GABAC receptors mediate

the majority of chloride current induced by GABA puff onto bipolar cell axon

terminals (70). In the study, the ratio of GABAC to GABAA-mediated current differed

between bipolar cells in tiger salamander retina, but overall, 80% of the current

induced by GABA puff was mediated by GABAC receptors.

15

GABAC receptors modulate bipolar cell excitability and help shape light-

evoked responses. It has been shown that GABAC-mediated currents in bipolar cells

exhibit a longer latency and time course than GABAA-mediated currents (71, 72, 73).

Light-evoked inhibitory currents recorded from bipolar cells were blocked by PTX,

the GABAA and GABAC antagonist, but not by the GABAA-specific antagonist

bicuculline (74). This research indicates that GABAC receptors mediate inhibition

onto bipolar cells, modulate excitability of bipolar cells, and ultimately modulate

neurotransmission from bipolar cells to RGCs. Indeed, under one level of light

adaptation, block of GABAC receptors with 50M TPMPA increased total charge

transfer and decay time of On EPSCs recorded from mouse RGCs (10). In contrast,

TPMPA did not affect Off responses recorded from Off RGCs. GABAC receptor

knockout decreased On RGC but not Off RGC dynamic range, or the range of light

intensities that elicit between 5% and 95% of the maximum response, compared to

wild type.

6. Hypothesis

The retina uses parallel circuits to effectively transmit neural signals for both

light increments and light decrements (1). Off pathways form one half of these

parallel circuits and are crucial for proper contrast sensitivity and therefore, vision.

Previous investigations, including receptor localization studies, indicate that

mGluR6 receptors, CB1 receptors, and GABAC receptors may modulate Off

responses. I performed whole-cell voltage clamping of mouse RGCs and record light-

evoked Off responses primarily under light adaptation. To determine the role of

16

mGluR6 receptors in shaping Off responses under light adaptation, I applied

mGluR6-specific agonist APB. To examine the role of CB1 receptors in shaping Off

responses, I used CB1R agonists 2-arachidonoyl glycerol (2-AG) and WIN 55,212-2

(WIN) and various CB1R antagonists. To study how GABAC receptors modulate Off

responses under different levels of light adaptation, I applied the GABAC- specific

antagonist TPMPA. In these studies, I tested the hypotheses that: APB increases Off

response amplitudes under light adaptation as it does under dark adaptation by

reducing crossover inhibition from On pathways to Off pathways; CB1R agonists

reduce glutamate release along the Off pathway resulting in a decrease in Off response

amplitudes; and TPMPA increases the decay time of Off responses as has been

previously shown in On responses.

7. Statement of specific aims

Specific Aim 1: investigated whether mGluR6 agonist APB increases RGC

light-evoked Off amplitudes by reducing crossover inhibition of Off pathways under

light adaptation. Retinal On pathways exhibit crossover inhibition of Off pathways

and vice versa via glycinergic amacrine cells (31, 32, 33, 34). The mGluR6 agonist

APB is commonly used to block On pathways and has been shown to prevent

crossover inhibition of Off pathways under dark adaptation resulting in an increase

in Off response amplitudes (5, 6, 7, 8). We hypothesized that APB would also

potentiate Off responses under light adaptation by reducing crossover inhibition of

Off pathways. For experiments in this specific aim, I performed whole-cell voltage

clamp on mouse RGCs under different adaptation levels and used APB to determine

17

if Off responses were uniformly potentiated. In addition, I applied different mGluR6

agonists to ensure that APB effects were mediated by mGluR6 receptors. Further, I

applied dopamine receptor antagonists to determine if the APB effects were

dopamine transmission-dependent. The experiments for this specific aim are

explained in detail in Chapter 1.

Specific Aim 2: investigated whether cannabinoids acting at CB1Rs

attenuate RGC light-evoked Off responses under light adaptation. Classically,

presynaptic CB1Rs are activated by eCBs released by postsynaptic cells to decrease

neurotransmitter release from the presynapse (75, 76, 77, 78, 79). In the retina,

CB1Rs are localized at presynaptic sites at glutamatergic synapses along the Off

pathway (9, 42, 43, 44). Therefore, we hypothesized that bath application of CB1R

agonists would reduce glutamatergic transmission and decrease Off response

amplitudes. For experiments in this specific aim, I performed whole-cell patch

clamping of mouse RGCs under light adaptation. I applied either CB1R antagonists,

such as O-2050 or AM251, or CB1R agonists, such as 2-AG and WIN, and recorded

light-evoked responses to determine the role of CB1 receptors in modulating Off

responses. Also, I co-perfused APB during CB1R agonist treatment to eliminate

crossover inhibition. Further, I applied TPMPA both prior to and during CB1R

agonist perfusion to determine if CB1R agonists increased Off amplitudes in some

RGCs via disinhibition. The experiments for this specific aim are explained in detail

in Chapter 2.

Specific Aim 3: investigated whether GABAC receptor antagonist TPMPA

modulates Off responses. GABAC receptors have been localized to the axon terminals

18

of bipolar cells (67). Under one level of light adaptation, block of GABAC receptors

with TPMPA has been shown to selectively potentiate On EPSCs recorded from On

RGCs (10). TPMPA releases bipolar cells from inhibition leading to greater release of

glutamate from bipolar cells onto RGC dendrites. For experiments in this specific

aim, I performed whole-cell patch clamping of mouse On-Off RGCs under different

levels of light adaptation. I applied TPMPA to determine if both On and Off response

decay times increase with GABAC receptors blocked. Additionally, I applied APB

both prior to and during application of TPMPA to investigate whether TPMPA

increases Off responses via a decrease in crossover inhibition. Further, experiments

were performed at different ambient light intensities to determine if GABAC-

mediated modulation of Off responses is consistent or dynamic. The experiments for

this specific aim are explained in detail in Chapter 3.

19

CHAPTER 1

Dopamine modulates the Off pathway in light-adapted mouse retina

Introduction

In the retina, light increment information is transmitted by On pathways and

light decrement information is transmitted by Off pathways. Regardless of

adaptation level, On responses require transmission by On-cone bipolar cells (1).

The On-cone bipolar cells express mGluR6 receptors at their dendrites. These

mGluR6 receptors differ from the ionotropic glutamate receptors expressed by Off-

cone bipolar cells in two crucial ways. First, mGluR6 receptors are metabotropic G-

protein coupled receptors. Second, mGluR6 receptor activation closes a cation

channel resulting in the hyperpolarization of the On-cone bipolar cell (27, 80). Thus,

mGluR6 agonist APB can be applied to hyperpolarize On-cone bipolar cells and

block On pathways.

Off-bipolar cells do not express mGluR6 receptors and therefore cannot be

directly affected by APB. Nevertheless, under dark adaptation, Off response

amplitudes have been shown to increase following application of APB (5, 6, 7, 8). It

has been shown that APB hyperpolarizes On-cone bipolar cells resulting in a

disruption of glutamate release onto glycinergic amacrine cells (34, 35, 36, 37). The

amacrine cells cease

20

releasing glycine onto Off-cone bipolar cells resulting in disinhibition and larger Off

responses. These studies have revealed that On and Off pathways are not completely

discrete. Instead, the pathways communicate through crossover inhibition.

However, the studies have not demonstrated that the effects of APB are consistent

across adaptation levels.

In the current study, patch clamp recordings were made from RGCs in mouse

retinas under different adaptation levels. We found that APB decreases Off response

amplitudes under light adaptation by inhibiting dopaminergic amacrine cells that

synapse onto Off-cone bipolar cells resulting, ultimately, in inhibition of Off-cone

bipolar cell hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The

results presented in this study suggest that crosstalk between On and Off pathways

occurs under light adaptation via dopaminergic amacrine cells.

21

Materials and Methods

The basic methods used in this study were similar to those used previously

(81, 82, 83, 84). All procedures were in compliance with National Institutes of

Health guidelines and were approved by the campus animal use committees of

Tulane University. Animals were dark-adapted overnight prior to the experiments.

All procedures, including animal surgery, dissection of retinas, and recordings from

cells, were performed in complete darkness. Infrared goggles were used to visualize

the tissue on the dissecting and recording microscopes and to maneuver in the

recording room. LEDs (850nm) were used to provide light to the dissecting

microscope while the illumination from the recording microscope was passed

through a 850-nm cutoff filter.

Retinal preparation. Retinas were obtained from 3-4-month-old mice (C57BL/6

from Charles River). After a lethal dose of barbiturate (Beuthanasia-D; 360 mg/kg

i.p.), the eyes were removed and placed in oxygenated L15 (Sigma, St. Louis, MO;

L1518) at 37C for 12 min. The retinas were then carefully peeled from the eyecup

and stored at room temperature in minimum essential medium Eagle (MEME;

Mediatech, Herndon, VA; catalog No. 51-010-PC), and continuously bubbled with

95% O2+ and 5% CO2. There was no cesium in the bath solution. A small piece of

retina was placed GCL upward in the recording chamber and stabilized with an

overlying piece of filter paper. A 2-mm hole in the filter paper provided access for

the recording electrode. Cells were visualized through a 40X objective mounted on

an upright epifluorescence microscope (Nikon).

22

During recordings, the retina was perfused continuously with MEME (1.5

ml/min) through a gravity-fed line, heated with a dual channel temperature

controller (Warner Instruments, Hamden, CT), and continuously bubbled with 95%

O2 and 5% CO2. A calibrated thermocouple monitored the temperature in the

recording chamber, which was maintained at 35C. Recordings from each individual

cell usually lasted 30-120 min., and retinal segments from which recordings were

made typically remained viable for 8-12 hr. Patch electrodes were filled with a

solution containing (in mM) cesium methanesulfonate 118, CsCl 12, CaCl2 0.5, MgCl2

0.5, HEPES 10, EGTA 5, 0.5% Lucifer yellow, QX-314 3, pH 7.4, osmolarity 290

mOsm. The chloride equilibrium potential (ECl) with this internal solution was

approximately -58.0 mV. QX-314 (3 mM) was included in the electrode solution to

eliminate sodium currents in the recorded cell. All recordings were made with the

whole-cell configuration. By the end of the experiment, the soma and the dendritic

arborizations of the recorded cell were usually completely filled with Lucifer yellow.

Once complete filling had been achieved, the retina was removed and fixed in 4%

paraformaldehyde for 6-8 hr at 4C.

Subsequently, by using a Leica TCS SP2 confocal microscope (Leica

Microsystems, Heidelberg, Germany), high-resolution and three-dimensional images

were made of each cell. Scans were taken at 0.25-0.7-m intervals along the z-axis

depending on the objective used. The dendritic stratifications of RGCs in the IPL

were determined by rotating the confocal stack image 90. DAPI was used to label

the nuclei of the GCL and the INL. The depth of the IPL was defined as the area

between the GCL and the inner border of the INL. The outer two-fifths of the IPL is

23

sublamina a, the Off sublamina; the inner three-fifths of the IPL is sublamina b, the

On sublamina. Ganglion cells with their dendrites ramify in sublamina a, sublamina

b, or both are Off, On, or On-Off RGCs, respectively. Only morphologically identified

Off and On-Off RGCs were included in this study. Because we focused mainly on the

effects of APB on the Off responses under dark and light adaptation, and for

statistical analyses, we grouped Off and On-Off cells together. Images of

morphological Off RGCs and On-Off RGCs are similar to those from our previous

studies (83, 84, 85).

Electrophysiology. Whole-cell patch clamp recordings were made from RGCs in dark-

and light-adapted retinas. Patch pipettes with a tip resistance between 3 and 7 M

were pulled from thick-walled 1.5-mm-ODS borosilicate glass on a Sutter

Instruments (Novato, CA) puller (P-97). Whole-cell patch clamp recordings were

made with a Multiclamp 700B patch clamp amplifier (Axon instruments,

Burlingame, CA). The data were low-pass filtered at rates between 1 and 2 kHz and

digitized at rates of 5 kHz before storage on a computer for subsequent offline

analysis. To attain whole-cell access, the vitreous and the limiting membrane

overlying the recording area were removed by gently brushing the retinal surface

with the tip of a glass pipette. Recordings were made from ganglion cells located

around a region about midway between the geometric center and the peripheral rim

of the retina and obtained by patching onto cells with clear, nongranular cytoplasm.

High-resistance seals were obtained by moving the patch electrode onto the cell

membrane and applying gentle suction. After formation of a high-resistance seal

24

between the electrode and the cell membrane, transient currents caused by pipette

capacitance were electronically compensated by the circuit of the MultiClamp 700B

patch clamp amplifier. Recordings from cells with a seal resistance 1 G were

discarded. After forming the whole-cell configuration by gentle suction, the series

resistance was measured, and it was 7-16 M. Recordings were terminated

whenever significant increases (20%) in series resistance occurred. Light-evoked

EPSCs were recorded at the membrane potential of -58 mV, the chloride reversal

potential of the RGCs. For light-evoked EPSCs, the averaged amplitudes of each cell

before and after drug application were obtained from five trials. The results were

expressed as mean SE.

Light stimulus. Light-evoked responses were obtained by delivering square-wave

spots of light to the retina from a 1-in.-diameter computer monitor, with a green

(P43, 545 nm light) phosphor (Lucivid MR1-103 MicroBrightField, Colchester, VT),

through the camera port of the microscope (86). Light responses to a 600 m spot of

light were recorded for each cell. Then, the size of the spot of light was adjusted to

evoke the maximal current recorded under voltage clamp before the effects of APB

were tested. For each cell, different-sized spots, from 200 to 600 m were used. The

spots of light were always centered on the soma. The stimuli were programmed in

Matlab (Mathworks, Natick, MA), using the Psychophysics Toolbox extensions (87,

88). The intensity of the spot of light was calibrated with a spectroradiometer/

photometer (UDT instruments, S350/268R) and expressed in terms of the time-

averaged rate of photoisomerizations per rod per second (Rh/rod/sec), the only

25

unit that the retina understands (89). The instrument was calibrated relative to

standards of the National Institute of Standards and Technology.

The method of recording from light-adapted retinas is essentially the same as

in our previous studies (83, 84). Initially, the whole-cell patch clamp recordings

were made from a ganglion cell under the dark-adapted condition; then, a

background light was delivered to the retina to induce light adaptation, and the

recording was continued from the same cell. For these studies, a background light of

constant brightness at 150, 1500, or 15000 Rh/rod/sec was provided full-field by

the computer-controlled 1-in.-diameter monitor (Lucivid) for 10 min to allow the

transition from dark adaptation to adaptation under the background light. The

background light with intensity at 150 Rh/rod/sec is in the mesopic range (21, 90).

With this background light, both rods and cones can be activated. In contrast, the

background light with intensity at 1500 or 15000 Rh/rod/sec is in the photopic

range. Rods are completely inactivated by 1500 or 15000 Rh/rod/sec background

light (83, 84).

Light stimuli with intensity of 350 Rh/rod/sec and light stimuli with

intensities greater than that of the background light were used in the dark- and

light-adapted retina, respectively to evoke light responses from ganglion cells. For

each cell, different intensities were used to evoke light responses under light

adaptation. Then, the lowest intensity required to evoke an optimal response was

determined and used in the experiments to test the effects of APB on the ganglion

cell. An optimal response was characterized as the largest synaptic current

amplitude (in voltage clamp mode) of the light-evoked responses of each cell. Under

26

dark or different background light conditions, light stimuli were delivered once

every 20 sec to limit alteration of the adaptation level.

Drug application. APB (20 M; Sigma), L-2-amino-4-phosphonobutyric acid (L-AP4;

4 M; Sigma), (S)-3,4-dicarboxyphenylglycine (DCPG; 3 M; Tocris, Ellisville, MO),

strychnine (STR; 2 M; Sigma), picrotoxin (PTX; 100 M; Sigma), (1,2,5,6-

tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA; 50 M; Sigma), SCH 23390

hydrochloride (20 or 10 M; Tocris), spiperone hydrochloride (20 or 10 M;

Tocris), and ZD 7288 (100 or 30 M; Tocris) were freshly dissolved in MEME on the

day of the experiment and administered through a gravity-fed line. The pH was

adjusted to 7.4. All drugs were prepared and stored in accordance with the

manufacturer’s recommendations. The solutions were heated with a dual-channel

temperature controller (Warner Instruments) and continuously bubbled with 95%

O2 and 5% CO2. A six-position rotary valve (Western Analytical Products, Wildomar,

CA) was used to switch between bath and drug solutions.

27

Results

Effects of APB and L-AP4 on the light-evoked Off responses in Off and On-Off ganglion

cells under dark adaptation and different ambient light conditions

To determine the effects of APB on Off responses, morphologically identified

Off and On-Off mouse RGCs were voltage-clamped and light-evoked Off responses

were recorded before and after bath application of APB. In addition, these cells were

either dark-adapted or adapted to another ambient light intensity. Researchers have

identified three Off pathways that function under dark adaptation: 1) the primary

pathway: rod > rod bipolar cell > AII amacrine cell > glycinergic synapse > Off-cone

bipolar cell > Off ganglion cell; 2) the secondary pathway: rod > gap junction > cone

> Off-cone bipolar cell > Off ganglion cell; and 3) the tertiary pathway: rod > Off-

cone bipolar cell > Off ganglion cell (2, 4, 21, 91). APB blocks the primary pathway

by hyperpolarizing rod bipolar cells and the tertiary pathway is rarely seen in the

mouse retina (23). Thus, following APB application in the dark-adapted retina, Off

responses are mainly mediated by the secondary Off pathway. However, the light

stimulus intensity of 350 Rh/rod/sec that was used to elicit responses in dark-

adapted cells can also activate M cones (92). Therefore, some of the Off responses

under dark adaptation were likely mediated by the Off cone pathway.

Bath applied APB (20 M) completely blocked On responses in On-Off RGCs

and under all tested ambient light intensities (Figure 2). The effects of APB on Off

responses varied depending on adaptation, however. Under dark adaption, APB

significantly increased the amplitude of Off responses recorded from Off and On-Off

RGCs (Figure 2A,B, n= 15). After washout, the potentiated Off amplitudes returned

28

to baseline. At a background light intensity of 150 Rh/rod/sec, however, APB

effects were inconsistent and no significant increase or decrease in Off amplitude

was observed (Figure 2C,D, n= 7). The effects of APB were also tested under a

background light intensity of 1500 Rh/rod/sec (Figure 2E,F, n=20) or 15000

Rh/rod/sec (Figure 2G,H, n= 8). In both conditions, APB significantly reduced the

amplitude of Off responses. These attenuated Off responses recovered following a

washout.

In order to confirm that the observed APB effects are mediated by mGluR6

receptors, we repeated the above experiment with the more specific mGluR6

agonist, L-AP4 (Figure 3A-D, n= 8). Effects of L-AP4 were similar to those of APB.

Following bath application of L-AP4, On responses were blocked in all recorded On-

Off RGCs. In addition, L-AP4 increased the amplitude of Off responses under dark

adaptation (Figure 3A,B, n= 8). Further, L-AP4 decreased the amplitude of Off

responses under a background of 1500 Rh/rod/sec (Figure 3C,D, n=8). These

results confirm that the APB effects under dark and light adaptation are due to

activation of mGluR6 receptors.

Previous researchers have shown that L-AP4 can activate mGluR8 (93, 94).

To ensure that L-AP4 effects are not in fact mediated by mGluR8 receptors, we

repeated the above experiment with mGluR8 agonist DCPG (Figure 3E-G, n= 7). The

effects of DCPG were not similar to those of APB and L-AP4. Under a background of

1500 Rh/rod/sec, DCPG increased On response amplitude (Figure 3F, n= 7) but did

not consistently affect Off response amplitudes (Figure 3G, n= 7). These data suggest

29

that APB and L-AP4 are not acting at mGluR8 receptors to affect Off responses under

light adaptation.

Effects of strychnine, picrotoxin, and TPMPA on the APB-induced reduction of Off

responses in the light-adapted mouse retina

Previous research has shown that crossover inhibition between On and Off

pathways occurs via glycinergic neurotransmission. To determine if inhibitory

mechanisms are involved when APB reduces Off responses under light adaptation,

glycine receptor antagonist STR, GABAA/C receptor antagonist PTX, and GABAC

receptor antagonist TPMPA were bath applied before and during application of APB

(Figure 4). When STR was applied to block glycine receptors, APB still reduced Off

response amplitudes under light adaptation (Figure 4A,B, n= 16). Similar results

were seen when both GABAA and GABAC receptors were blocked by PTX (Figure

4C,D, n= 16) and also when only GABAC receptors were blocked by TPMPA (Figure

4E,F, n= 16). Further, simultaneous application of STR, PTX, and TPMPA did not

prevent the reduction of Off response amplitudes by APB (Figure 4G,H, n= 7). These

results indicate that the underlying mechanism of APB-induced reduction of Off

responses under light adaptation does not involve glycinergic or GABAergic

transmission.

Effects of dopamine receptor antagonists on the APB-induced reduction of Off

responses of RGCs

30

As the retina transitions from dark adaptation to light adaptation, dopamine

begins to be released by dopaminergic amacrine cells (95, 96, 97). Dopamine has

been shown to increase the amplitude of light-evoked responses in RGCs (98). Thus,

it is possible that a disruption in dopamine transmission may reduce Off response

amplitudes. Indeed previous studies have shown that APB inhibits dopaminergic

amacrine cells (99, 100). To determine if APB reduces Off responses in the light-

adapted mouse retina by disrupting dopamine receptor activation, D1 and D2

antagonists (SCH 23390 and spiperone, respectively; 20 M) were bath applied

before and during APB application (Figure 5). Our results show that Off responses

decreased with D1 receptors blocked (Figure 5A,B, n= 10) and Off responses

increased with D2 receptors blocked (Figure 5C,D, n= 8). When APB was applied

after D1 block, Off amplitudes no longer decreased. Rather, APB increased Off

amplitudes following D1 block. However, when APB was applied after D2 block, Off

amplitudes still decreased. These results indicate that dopaminergic transmission

involving D1 receptors is necessary for the APB-induced reduction of Off responses

under light adaptation. Similar results were seen when experiments were repeated

with L-AP4 (Figure 5G,H, n= 5).

To further study the role of D1 and D2 receptors in APB-induced reduction in

Off amplitudes, both SCH 23390 and spiperone were co applied (Figure 5E,F, n= 9).

Off amplitudes increased with both D1 and D2 receptors blocked. APB did not

significantly change the Off amplitudes when D1 and D2 receptors were blocked.

Moreover APB did not significantly change Off amplitudes under light adaptation

when SCH 23390, STR, PTX, and TPMPA were applied (Figure 6A,B, n= 7). Under a

31

background light of 150 Rh/rod/sec, APB did decrease Off amplitudes when STR,

PTX, and TPMPA were applied (Figure 6C,D, n= 7). These results indicate that under

mesopic conditions, blocking inhibitory neurotransmission allows APB to reduce Off

amplitudes as was also seen under light adaptation.

HCN channel blocker (ZD 7288) prevented the APB-induced reduction of Off responses

in RGCs in the light-adapted mouse retina

Dopamine has been shown to modulate HCN channels in the retina (101,

102). Additionally, APB inhibits a subgroup of dopaminergic amacrine cells (99,

100). Thus, APB may affect HCN function resulting in the observed reduction in Off

responses under light adaptation. To determine if HCN channels are involved in

APB-induced reduction of Off responses under light adaptation, HCN antagonist ZD

7288 (100 M) was applied before and during bath application of APB (Figure 7).

Under dark adaptation, ZD 7288 did not significantly change On responses (Figure

7A,B, n= 12), but it did significantly decrease Off responses (Figure 7A,C, n= 15). On

the contrary, ZD 7288 decreased both On (Figure 7D,E, n= 11) and Off (Figure 7D,F,

n= 15) responses under light adaptation. The modulatory effects of ZD 7288 were

more pronounced under light adaptation. These data follow a previous study

showing that HCN channels are activated in bright light conditions (103).

When ZD 7288 blocked HCN channels under dark adaptation, APB increased

Off amplitudes similarly to how Off amplitudes increased without HCN channel

block (Figure 7C, n= 15). In contrast, under light adaptation, APB no longer

decreased Off amplitudes when HCN channels were blocked by ZD 7288 (Figure 7F,

32

n= 15). These results indicate that HCN channels are indeed involved in the

mechanism underlying the APB-induced reduction of Off responses under light

adaptation.

APB-induced reduction of Off responses did not depend on the membrane potentials of

RGCs in the light-adapted mouse retina

HCN channels are expressed by Off-cone bipolar cells, but also by RGCs (103,

104). Thus, it is possible that ZD 7288 blocked RGC-expressed HCN channels to

prevent the APB-induced reduction of Off responses. HCN channels are active when

the neuron is hyperpolarized but inactive in depolarized conditions like -40 mV

(102, 105). Therefore, if RGC-localized HCN channels were the major site involved in

the APB-induced reduction of Off responses, then the APB-induced reductions of Off

responses should be eliminated by RGC depolarization. To test that possibility, RGCs

were voltage-clamped at depolarized, resting, and hyperpolarized membrane

potentials under light adaptation and APB was applied (Figure 8). In all three

conditions, APB still decreased Off amplitudes indicating that HCN channels

localized to RGCs do not underlie the APB-induced reduction of Off responses

(Figure 8B, n= 10).

33

Discussion

Previous studies have shown that On and Off pathways cross talk via

crossover inhibition mediated by glycinergic amacrine cells (34, 35, 36, 37). Other

researchers have shown that under dark adaptation, APB activates mGluR6

receptors causing a disruption in crossover inhibition and ultimately increasing Off

response amplitudes (5, 6, 7, 8). The results of the present study demonstrate that

APB does indeed increase Off response amplitudes under dark adaptation, but

effects on Off and On-Off RGCs differ under different adaptations. In the light-

adapted retina, APB significantly decreased Off response amplitudes. When D1

receptors or HCN channels were blocked, APB-induced reduction of Off responses

under light adaptation was prevented. These findings provide evidence that under

light adaptation, On and Off pathways cross talk via dopaminergic amacrine cells

that modulate HCN channel activity at the Off-cone bipolar cell. Moreover, these

findings demonstrate that mGluR6 receptor activity modulates this dopaminergic

mechanism of crosstalk. Taking together the findings in this study and previous

studies, I identified that mGluR6 receptors play an important role in modulating Off

response amplitudes.

APB differentially modulated Off responses of RGCs in dark- and light-adapted mouse

retinas

APB is an mGluR6 agonist that is commonly used in retinal research to block

On pathways. Off responses are also affected by APB, however. Under dark

adaptation, it has been shown that APB blocks APB-sensitive Off responses in the

34

primary rod pathway (81, 106). The remaining Off responses, mainly from the

secondary Off pathway, increase following APB application. Most likely, APB reduces

crossover inhibition via glycinergic amacrine cells under dark adaptation to

increase the Off amplitudes. However, the effects of APB at different adaptation

levels are unclear. The current study used voltage clamp recordings to show that

APB differentially affects Off responses of mouse RGCs depending on adaptation

level. Similar to previous studies, APB increased Off amplitudes under dark

adaptation. Under light adaptation, however, APB decreased Off amplitudes.

We simulated mesopic conditions by adapting the retina to a background

light intensity of 150 Rh/rod/sec. Under these conditions, with both rod and cone

pathways active, APB neither increased nor decreased Off response amplitudes

(107). Therefore, the effect of APB to increase Off amplitudes under dark adaptation

and the effect of APB to decrease Off amplitudes under light adaptation may have

cancelled out. Indeed, when crossover inhibition was blocked with GABA and

glycine antagonists under mesopic conditions, APB decreased Off amplitudes. Taken

together, these results indicate that under mesopic conditions, Off potentiation

requiring crossover inhibition and Off attenuation requiring dopamine occlude each

other.

Glycinergic and/ or GABAergic inhibition and other members of group III mGluRs were

not involved in the APB-induced reduction of Off responses of RGCs in the light-adapted

mouse retina

35

Block of inhibitory neurotransmission with STR, PTX, TPMPA or a

combination of these drugs did not prevent APB-induced reduction of Off responses.

These results indicate that while inhibitory neurotransmission is involved in the

APB effects under dark adaptation, it is not involved in the mechanism under light

adaptation.

In previous studies, APB and L-AP4 have been shown to activate group III

mGluRs including mGluR4, mGluR6, mGluR7, and mGluR8 (108, 109, 110, 111). The

effects of activation of group III mGluRs other than mGluR6 on Off responses in the

mouse retina is currently unclear. In the present study, we applied the mGluR8

agonist DCPG and found that it did not significantly change Off amplitudes. The

results indicate that APB and L-AP4 effects on Off responses occur primarily due to

activation of mGluR6 receptors.

Dopamine receptors were involved in the APB-induced reduction of Off responses of

RGCs in the light-adapted mouse retina

Dopamine release in the retina is stimulated by light adaptation (95, 96, 97).

APB has been previously shown to inhibit dopaminergic amacrine cells, resulting in

a decrease in dopamine release (99, 100). The results from the present study show

that the D1 antagonist SCH 23390 prevents APB from decreasing Off responses

under light adaptation. This indicates that dopaminergic transmission is involved in

the APB-induced reduction of Off responses.

D1 receptor activation has been shown to activate HCN channels (102). When

HCN channels were blocked in the present study, Off response amplitudes

36

decreased. Therefore, it is possible that under light adaptation, APB disrupts D1

activation resulting in HCN inhibition and Off response reduction. When D1

receptors were blocked with SCH 23390, APB actually increased the Off response

amplitudes. These results suggest that under light adaptation, APB has competing

effects. The first effect is the one described above that results in Off response

amplitude reduction. The second is that APB increases Off responses by removing

crossover inhibition, as occurs under dark adaptation. Thus, the mechanism

underlying APB-induced increases of Off responses observed under dark adaptation

is still active under light adaptation. However, under light adaptation, the

mechanism involving disruption of dopamine transmission has more robust effects,

resulting in a net decrease in Off response amplitudes. These findings are supported

by our results showing that APB did not significantly change Off amplitudes when

both the glycinergic and dopaminergic mechanisms were blocked.

While blocking D1 receptors prevented APB-induced reduction of Off

responses under light adaptation, blocking D2 receptors did not. D1 receptor block

decreased Off responses and D2 receptor block increased Off responses. Thus, D1

and D2 receptors may play opposite roles in the retina to modulate light-evoked

responses under light adaptation. It remains unclear why D2 receptor block

significantly increased Off response amplitudes. Future studies should identify the

functional role of D2 receptors in shaping light responses in the retina.

HCN channels were involved in the mechanisms that underlie the APB-induced

reduction of Off responses of RGCs in the light-adapted mouse retina

37

HCN channel block by ZD 7288 prevented the APB-induced reduction of Off

responses. It has been shown that dopamine activates HCN channels, so both

dopamine and HCN may be involved in the APB-induced reduction of Off responses

under light adaptation (102). In the present study, we demonstrated that HCN

channels upstream of RGCs, and not localized to RGCs themselves, are involved in

the APB effects. Previous studies showed that HCN channels are also localized to Off-

cone bipolar cells, cones, and amacrine cells and it is activation of these channels

that is likely modulated by APB application (101, 103, 112, 113, 114).

The results presented here, along with previous studies, indicate that under

light adaptation in the mouse retina, APB inhibits dopaminergic amacrine cells

which inhibits HCN channels resulting in a decrease in Off response amplitudes. The

hypothesized functional circuit illustrated in Figure 9 is as follows: On-cone bipolar

cells activate dopaminergic amacrine cells, which then activate D1 receptors on Off-

cone bipolar cells; after that, HCN channels are activated and glutamate release from

the Off-cone bipolar cell is elevated. APB disrupts this circuit by inhibiting the On-

cone bipolar cells, preventing the activation of dopaminergic amacrine cells. This

circuit demonstrates another way in which On and Off pathways are capable of

crosstalk.

38

Figure 2. The effects of APB on RGC Off responses under different ambient light

conditions. A: Recordings from an On-Off RGC under dark adaptation. APB increased

the amplitude of light-evoked Off responses of this cell, indicated by arrows, and the

response recovered after wash-out. The stimulus light intensity was 350 Rh*/rod/s.

The light onset and offset are indicated above the recording traces. B: Bath

application of APB significantly increased the remaining Off responses of recorded

RGCs under dark adaptation (n= 15, 4 Off cells, and 11 On-Off cells; ** p= 0.001,

paired t-test). C, D: Under a background light of 150 Rh*/rod/s, APB had little effect

on Off responses of RGCs (n= 7, 1 Off cell, 6 On-Off cells; N.S. p= 0.111, paired t-test).

The stimulus light intensity was 6000 Rh*/rod/s. E, F: Under a background light of

1500 Rh*/rod/s, APB significantly decreased the averaged amplitude of Off

responses of the recorded cells (n= 20, 6 Off cells, 14 On-Off cells, ** p= 0.007, paired

t-test). The stimulus light intensity was 15000 Rh*/rod/s. G, H: Under a background

light of 15000 Rh*/rod/s, APB significantly decreased the averaged amplitude of Off

responses of the recorded cells (n= 8, 2 Off cells, 6 On-Off cells; ** p= 0.004, paired t-

test). The stimulus light intensity was 25000 Rh*/rod/s.

39

Figure 2

40

Figure 3. The effects of L-AP4 and DCPG on RGC Off responses under different

ambient light conditions. A, B: Under dark adaptation, mGluR6 agonist L-AP4

significantly increased the amplitudes of Off responses of recorded RGCs (n= 8, 2 Off

cells, 6 On-Off cells; ** p= 0.001, paired t-test). The stimulus light intensity was 350

Rh*/rod/s. C, D: Under the background light of 1500 Rh*/rod/s, L-AP4 significantly

decreased the amplitudes of Off responses of recorded RGCs (n= 8, 1 Off cell, 7 On-

Off cells; ** p= 0.009, paired t-test). The stimulus light intensity was 15000

Rh*/rod/s. E, F, and G: Under a background light of 1500 Rh*/rod/s and a stimulus

light intensity of 15000 Rh*/rod/s, mGluR8 agonist DCPG significantly increased the

averaged amplitude of On responses of recorded RGCs (n= 7, 7 On-Off cells; * p=

0.040, paired t-test). However, DCPG had little effect on the averaged amplitude of

Off responses of recorded RGCs (n= 7, 7 On-Off cells; N.S. p= 0.263, paired t-test).

41

Figure 3

42

Figure 4. Antagonists of glycine, GABAA, and GABAC receptors did not eliminate the

APB-induced reductions of Off responses of RGCs in light-adapted mouse retinas.

Recordings were made under the background light of 1500 Rh*/rod/s from three

On-Off RGCs as shown in A, C, and E, respectively. A, C, and E: APB still decreased

the Off responses of the three cells following perfusion with STR, PTX, and TPMPA,

respectively. B, D, and F: The APB-induced reductions of Off responses were not

eliminated by either STR, or PTX, or TPMPA, respectively. G and H: STR, PTX and

TPMPA together did not prevent the APB-induced reductions of Off responses of

RGCs in light-adapted mouse retinas (** p= 0.006, *p= 0.014, ** p=0.005, and ** p=

0.001 in B, D, F and H, respectively, paired t-test).

43

Figure 4

44

Figure 5. Effects of dopamine receptor antagonists on the APB-induced reductions

of Off responses of RGCs in light-adapted mouse retinas. All recordings were made

from RGCs with a background light of 1500 Rh*/rod/s and a stimulus light intensity

of 15000 Rh*/rod/s. A, B: A D1 blocker, SCH 23390, significantly decreased the light-

evoked Off responses of the recorded cells, but it prevented and reversed the APB-

induced reduction of the Off responses in these cells (n= 10, 2 Off cells, 8 On-Off

cells; * p= 0.027 for SCH 23390 alone and ** p= 0.007 for APB with SCH 23390,

paired t-test). C, D: A D2 blocker, spiperone, significantly increased the light-evoked

Off responses of the recorded cells, but did not prevent the APB-induced reduction

of the Off responses in these cells (n= 8, 3 Off cells, 5 On-Off cells; ** p= 0.008 for

spiperone alone and * p= 0.015 for APB with spiperone, paired t-test). E, F: Bath

application of D1 and D2 blockers together significantly increased the light-evoked

Off responses in the recorded cells. APB, when applied with SCH 23390 and

Spiperone, had little effect on Off responses of RGCs (n= 9, ** p=0.006, N.S. p= 0.966,

paired t-test). G, H: SCH 23390 significantly decreased the light-evoked Off

responses of the recorded cells, but it prevented and reversed the L-AP4-induced

reduction of the Off responses in these cells (n= 5, 1 Off cell, 4 On-Off cells; * p=

0.022 for SCH 23390 alone and * p= 0.013 for L-AP4 with SCH 23390, paired t-test).

45

Figure 5

46

Figure 6. Effects of APB on light-evoked Off responses when D1 receptors and

inhibitory receptors are blocked. A, B: Under a background light of 1500 Rh*/rod/s,

APB had little effect on the averaged amplitude of Off responses of the recorded cells

after D1, GABA, and glycine receptors blocked together (n= 7, N.S. p= 0.101, paired t-

test). The stimulus light intensity was 15000 Rh*/rod/s. C, D: Under a background

light of 150 Rh*/rod/s, following GABA and glycine receptor block by PTX, TPMPA,

and STR, bath application of APB decreased the averaged amplitude of Off responses

of the recorded cells (n= 7, * p= 0.026, paired t-test).

47

Figure 6

48

Figure 7. HCN channel antagonist ZD 7288 prevented the APB-induced reduction in

Off responses under light adaptation. A: Recordings from an On-Off RGC in a dark-

adapted retina. The stimulus light intensity was 350 Rh*/rod/s. B: ZD 7288 did not

significantly decrease the light-evoked On responses of the recorded On-Off cells

under dark adaptation (n= 12, N.S. p= 0.170, paired t-test). C: ZD 7288 significantly

decreased the amplitudes of light-evoked Off responses of the recorded cells under

dark adaptation (n= 15, 3 Of cells, 12 On-Off cells, * p= 0.041, paired t-test). The

average percent reduction was (9.2 ± 1.3) %. After blocking HCN channels with ZD

7288, APB still significantly increased the amplitude of light-evoked Off responses of

the recorded cells under dark adaptation (n= 15, ** p= 0.001, paired t-test). D:

Recordings were made from an On-Off ganglion cell under a background light of

1500 Rh*/rod/s. The stimulus light intensity was 15000 Rh*/rod/s. E: ZD 7288

significantly decreased the light-evoked On responses of the recorded On-Off cells

under the background light of 1500 Rh*/rod/s (n= 11, * p= 0.047, paired t-test). F:

ZD 7288 significantly decreased the amplitudes of light-evoked Off responses of the

recorded cells under a background light of 1500 Rh*/rod/s (n= 15, 4 Off cells, 11

On-Off cells, * p= 0.015, paired t-test). The average percent reduction was (47.6 ±

7.5) %. After blocking the HCN channels with ZD 7288, APB did not further decrease

the averaged amplitude of light-evoked Off responses in the same recorded cells (n=

15, N.S. p= 0.131, paired t-test).

49

Figure 7

50

Figure 8. A depolarized holding potential of -40 mV, which inactivates HCN

channels in the recorded RGCs, did not prevent the APB-induced reduction of Off

responses under light adaptation. A: Under a background light of 1500 Rh*/rod/s,

recordings were made from an On-Off ganglion cell with holding potentials at -40

mV (left panel), -60 mV (middle panel), and -90 mV (right panel). The stimulus light

intensity was 15000 Rh*/rod/s. Bath application of APB decreased the Off

responses of these cells at all three holding potentials. B: Under the background

light of 1500 Rh*/rod/s, the averaged Off response amplitudes of the recorded cells

were significantly reduced by APB at the holding potentials of -40 mV, -60 mV, and -

90 mV (n= 10, 2 Off cells; 8 On-Off cells, * p= 0.032 at -40 mV, * p= 0.015 at -60 mV,

and ** p= 0.004 at -90 mV, paired t-test).

51

Figure 8

52

Figure 9. A diagram showing a functional circuit from On pathways to Off pathways

under light adaptation: On-cone bipolar cells activate dopaminergic amacrine cells

that release dopamine, which in turn to activates D1 receptors on Off-cone bipolar

cells. Then, D1 receptors activate HCN channels on Off-cone bipolar cells. C: cone; ON

CB: On-cone bipolar cell; OFF CB: Off-cone bipolar cell; ON RGC: On retinal ganglion

cell; OFF RGC: Off retinal ganglion cell; DA: dopaminergic amacrine cell; D1:

dopaminergic type 1 receptor; HCN: hyperpolarization-activated cyclic nucleotide-

gated channel.

53

Figure 9

54

CHAPTER 2

CB1Rs modulate Off responses in light-adapted mouse retina Introduction

CB1Rs are metabotropic G-protein coupled receptors that are involved in

regulating neurotransmitter release (115). When post-synaptic neurons are excited

and depolarize, they release eCBs that travel retrograde to the presynaptic neuron

and bind to CB1Rs (79). Following signal transduction, neurotransmitter release

from the presynapse is reduced (116). Activity-dependent eCB suppression of

excitatory neurotransmitter release is known as depolarization-induced

suppression of excitation (DSE), and activity-dependent eCB suppression of

inhibitory neurotransmitter release is known as depolarization-induced

suppression of inhibition (DSI) (75, 117, 118). Thus far, the majority of CB1R

research has focused on functions in the brain, but CB1Rs have also been discovered

in the retina.

CB1Rs are expressed widely in the retina of various species including mouse.

One study has shown that CB1Rs are localized to rod synaptic terminals, cone

synaptic terminals, amacrine cells, RGCs, and sporadically throughout the IPL (43).

Another study used a different antibody and found CB1R expression in rod bipolar

cells, PKC-immunoreactive GABAergic amacrine cells, in the OPL, in the IPL, but not

in the GCL (42). While the localization studies do not completely agree, it is clear

55

that CB1Rs are positioned to affect neurotransmission at glutamatergic and possibly

GABAergic synapses.

Previous studies investigating the functions of CB1Rs have mainly focused on

shaping ion channel currents such as the HVA Ca2+ currents in photoreceptors,

bipolar cells, and cultured RGCs (9). Application of CB1R agonists has been shown to

decrease the release of neurotransmitters like dopamine and glutamate, which

follows with previous studies in the brain showing that CB1R activation prevents

neurotransmitter release (57, 59). Only one study investigated CB1R and its role in

shaping light-evoked responses (63). The researchers found that the cone response

to light offset returned to baseline faster following application of CB1R agonist.

However, previous studies have not demonstrated the role of the eCB system in

shaping light responses in RGCs, the final output neurons of the retina.

In the current study, patch clamp recordings were made from RGCs in mouse

retinas under light adaptation. I found that a phenomenon similar to DSE occurs in

some RGCs, but it is unclear if CB1Rs are involved in the underlying mechanism.

Further, I found that there are two functional populations of RGCs: one where CB1R

agonists increase Off responses, and one where CB1R agonists decrease Off

responses. The results presented in this study suggest that CB1R activation

increases Off response amplitudes by disinhibiting Off-cone bipolar cells.

56

Materials and Methods

The basic methods used in this study were similar to those used previously in

this laboratory (81, 82, 83, 84). The materials and methods used for the experiments

outlined in this Chapter are also similar to those described in Chapter 1. All

procedures were performed under light adaptation with a background light

intensity of 2.9x104 photons/m2/s.

Retinal preparation. Retinal preparations performed in these experiments are

similar to those described in Chapter 1. For current-clamp experiments, patch

electrodes were filled with a solution containing (in mM) potassium gluconate 120,

NaCl 10, MgCl2 0.5, CaCl2 0.5, HEPES 10, EGTA 5, 0.5% Lucifer yellow, pH 7.4,

osmolarity 280 mOsm. The ECl with this internal solution was approximately -58

mV.

Electrophysiology. The basic electrophysiology methods were similar to those

described in Chapter 1, except that all experiments were performed under light

adaptation. For DSE experiments, RGCs were first maintained at -58 mV, the

chloride reversal potential. Three light responses at least 10 seconds apart were

recorded (pre-depolarization) and then the membrane potential was depolarized to

0 mV for 10 seconds before returning to -58 mV for the rest of the recording.

Immediately following membrane depolarization, light responses were recorded

every ten seconds for 3 minutes. The first three light responses after depolarization

are henceforth referred to as “post-depolarization” and the first three light

57

responses at least two minutes after depolarization are referred to as “recovery.”

For current clamp experiments, current was applied so that the baseline membrane

voltage was approximately -58 mV.

Light stimulus. The light stimuli were presented to the light-adapted retina using the

Lucivid, as described in Chapter 1. For these studies, a background light of constant

brightness, 2.9x104 photons/m2/s, was provided full-field by lights in the

recording room. At this background light intensity, rods are completely inactivated

and only cone-mediated responses are recorded (83).

The method of recording from light-adapted retinas is similar to those in our

previous studies. Throughout the procedure, including during retinal preparation,

dissection, and recordings, the retina was light-adapted. Light stimuli with intensity

greater than 2.9x104 Rh/rod/sec were used to evoke light responses from ganglion

cells.

Drug application. 2-arachidonoyl glycerol (2-AG; 5 M; Tocris), AM251 (2 M;

Tocris), APB (20 M; Sigma), O-2050 (2-10 M; Tocris), (1,2,5,6-tetrahydropyridin-

4-yl)methylphosphinic acid (TPMPA; 50 M; Sigma), WIN 55,212-2 (WIN; 10 M;

Sigma) were freshly dissolved in MEME on the day of the experiment and

administered through a gravity-fed line.

58

Results

DSE in RGC light-evoked Off responses

In certain preparations, when postsynaptic neurons are transiently

depolarized, glutamate release from presynaptic neurons is reduced resulting in

smaller EPSCs in the postsynaptic cell (118). It is believed that the depolarized

postsynaptic neuron releases eCBs that travel retrogradely to activate

presynaptically localized CB1Rs that reduce neurotransmitter release. To determine

if DSE occurs in the retina, specifically at the synapses between Off-cone bipolar

cells and RGCs, morphologically identified Off and On-Off mouse RGCs were voltage-

clamped and light-evoked Off responses were recorded before and after transient

membrane depolarization of RGCs (Figure 10).

Light responses were recorded both before and immediately after a 10

second RGC-membrane depolarization to 0 mV that was designed to evoke DSE.

Responses were again recorded 2 minutes after membrane depolarization to

determine if response amplitudes recovered from DSE. Experiments were first

carried out in MEME without drugs applied to the bath solution (Figure 10A-C, n=

12). The averaged response amplitude after depolarization did not significantly

decrease in the 12 total recorded RGCs (Figure 10B, n= 12). However, in one

population of RGCs, Off response amplitudes significantly decreased following

depolarization, which is consistent with DSE (Figure 10A,C, n= 7). The decrease in

amplitude following depolarization in these RGCs was 23%, similar to EPSC

amplitude reductions reported in other studies (45, 79). In these cells where DSE

occurred, Off responses returned to pre-depolarization amplitudes by 2 minutes

59

post-depolarization, similar to the recovery time reported in the other studies.

These results suggest that DSE occurs at some, but not all synapses between Off-

cone bipolar cells and RGCs.

The protocol used to evoke DSE in control conditions was repeated following

bath application of 2M CB1R antagonist O-2050 (Figure 10D-F, n= 10). Again, the

averaged response amplitude after depolarization did not significantly decrease in

the 10 total recorded RGCs (Figure 10E, n= 10). However, in the same 7 RGCs where

DSE occurred in control conditions, DSE also occurred following bath application of

O-2050 (Figure 10F, n= 7). The amplitude reduction due to depolarization in the

presence of O-2050 was 11%. These results indicate that blocking CB1Rs may

reduce, but not totally prevent DSE in this preparation. Moreover, the observed DSE

may be mediated by mechanisms that do not involve CB1Rs. Previous researchers

using other preparations have also found that CB1R antagonists could not block DSE

(118, 119).

Effects of CB1R antagonist O-2050 on light-evoked Off responses of RGCs

CB1Rs are localized to glutamatergic synapses in the retina and CB1R

antagonists have been shown to increase neurotransmitter release in other

preparations (120). Therefore we hypothesized that bath application of CB1R

antagonist O-2050 would increase glutamate release in Off pathways and increase

Off response amplitudes. To further our understanding of eCB functions in the retina

and determine if CB1R antagonists increase light-evoked Off responses in RGCs, Off

responses were recorded before and after bath application of 2M CB1R antagonist

60

O-2050 (Figure 11). Consistent with our hypothesis, O-2050 significantly increased

Off response amplitudes in one population of Off and On-Off RGCs (Figure 11A-C, n=

8). However in another population of Off and On-Off RGCs, O-2050 significantly

decreased Off response amplitudes (Figure 11D-F, n= 28). Experiments were

repeated with CB1R antagonist AM251 (2 M) and similar results were found (data

not shown). These data suggest that there are two functional populations of RGCs:

blocking CB1Rs in one population of RGCs increases Off responses, and blocking

CB1Rs in another population of RGCs decreases Off responses.

Effects of CB1R agonist WIN on light-evoked Off responses of RGCs

We originally operated under the hypothesis that activating CB1Rs with WIN

would reduce glutamate release and reduce Off responses (9). To determine if CB1R

agonists decrease Off response amplitudes, Off responses were recorded before and

after bath application of 10 M CB1R agonist WIN (Figure 12). In one population of

Off and On-Off RGCs, WIN significantly decreased Off response amplitudes, as we

hypothesized (Figure 12D-F, n= 9). However, in another population of Off and On-

Off RGCs, WIN significantly increased Off response amplitudes (Figure 12A-C, n= 7).

These results are consistent with our previous findings of two functional

populations of RGCs after O-2050 application. Experiments were repeated with the

eCB and CB1R agonist 2-AG (5 M) and similar results were found (data not shown).

Washout of WIN or 2-AG for between 20 and 30 minutes did not reliably return Off

amplitudes to control levels.

61

Effects of CB1R agonist 2-AG when crossover inhibition is blocked with mGluR6 agonist

APB

On pathways inhibit Off pathways via crossover inhibition (31, 32, 33, 34).

The circuit for crossover inhibition is as follows: cone > On-cone bipolar cell >

glycinergic amacrine cell > Off-cone bipolar cell > RGC. Applying mGluR6 agonist

APB blocks crossover inhibition. Activation of mGluR6 receptors, which are

localized to On-cone bipolar cell dendrites, results in hyperpolarization of On-cone

bipolar cells. This prevents glycinergic amacrine cells from being activated,

disinhibits Off-cone bipolar cells, and increases Off responses (5, 6, 7, 8). As

explained previously, in one population of Off and On-Off RGCs, application of CB1R

agonists increased the amplitude of Off responses, contrary to our original

hypothesis. To determine if CB1R agonists increase Off responses by reducing

crossover inhibition of Off pathways, we applied APB (20 M) to block crossover

inhibition and then recorded Off responses before and after bath application of 5 M

CB1R agonist 2-AG (Figure 13). With crossover inhibition blocked by APB, 2-AG still

increased the amplitude of Off responses in one population of Off and On-Off RGCs

(Figure 13A,B, n= 15) and decreased the amplitude of Off responses in another

population of Off and On-Off RGCs (Figure 13C,D, n= 10). APB eliminated On

responses in every recorded On-Off cell, indicating that On pathways were blocked,

On-cone bipolar cells were hyperpolarized, and crossover inhibition was indeed

blocked. These results indicate that CB1R agonists increase Off response amplitudes

in one population of Off and On-Off RGCs, but the underlying mechanism does not

involve modulation of crossover inhibition.

62

Off response amplitude enhancement by CB1R agonists is prevented when GABAC

receptors are blocked by TPMPA

Much of the CB1R immunoreactivity in the retina is localized to

glutamatergic synapses (9). However, in one study, CB1R immunoreactivity was

discovered at the axon terminals of a subtype of PKC-immunoreactive GABAergic

amacrine cell (42). Previous researchers studying eCBs in the brain have found that

eCBs can reduce GABA release from presynaptic neurons (75, 121). Activation of

CB1Rs localized to GABAergic amacrine cells could prevent GABA release and

disinhibit cells that receive inputs from GABAergic amacrine cells. Off-cone bipolar

cells that preferentially express GABAC receptors, as opposed to GABAA receptors,

receive inputs from GABAergic amacrine cells (73, 74, 122). On-cone bipolar cells

also express GABAC receptors (123). To determine if CB1R agonists increase Off

amplitudes in one population of RGCs by reducing GABAergic inhibition onto Off-

cone bipolar cells, we applied APB (20 M) to block crossover inhibition, we applied

TPMPA (50 M) to block GABAC receptors, and we recorded Off responses before

and after application of 5 M 2-AG (Figure 14). Bath application of CB1R agonist 2-

AG significantly reduced Off response amplitudes in all recorded Off and On-Off

RGCs (Figure 14B, n= 5). Thus, increases in Off response amplitudes by a CB1R

agonist were prevented when GABAC receptors were blocked. These results suggest

that CB1R agonists may disinhibit Off-cone bipolar cells that mostly express GABAC

receptors resulting in greater Off amplitudes in one population of Off and On-Off

RGCs. We suspect that CB1R agonists reduce GABA release from GABAergic

amacrine cells that both express CB1Rs and synapse onto Off-cone bipolar cells.

63

Discussion

Studies of the retinal eCB system have focused primarily on CB1R

localization and CB1R-mediated modulation of isolated channel currents (9). The

available CB1R localization studies do not completely agree (42, 43). However, each

study has reported CB1R expression at the glutamatergic synapses in the OPL and

IPL as well as at amacrine cells. In particular importance to the present study, CB1R

expression has also been reported at the axon terminals of a subtype of PKC-

immunoreactive GABAergic amacrine cell. CB1Rs have been found to modulate

voltage-dependent potassium, chloride, and calcium currents in various retinal

neurons (42, 44, 47, 48, 49, 50, 51, 52). In addition, CB1R agonists decrease

generalized dopamine and glutamate release in the retina, consistent with brain

studies showing that CB1R activation decreases neurotransmitter release (58, 59).

In the only study that investigated the role of CB1Rs in shaping light-evoked

responses, researchers found that CB1R agonist WIN decreased the latency for cone

responses to return to baseline following light offset (63). However, the role of

CB1Rs in shaping light-evoked response output has not yet been investigated. The

results of the present study demonstrate that DSE, a phenomenon involving CB1Rs

that has been studied extensively in the brain, occurs in some but not all RGCs.

There are functionally two populations of Off and On-Off RGCs: 1) in the first

population, CB1R activation increases Off amplitudes; 2) in the second population,

CB1R activation decreases Off amplitudes. Reduction of crossover inhibition is not

the mechanism underlying CB1R agonist-mediated increases in Off response

amplitude. Rather, our results indicate that CB1R activation can reduce GABAergic

64

inhibition onto Off-cone bipolar cells resulting in the observed increase in Off

response amplitudes. Taken together, these findings provide evidence that under

light adaptation, CB1Rs participate in shaping light-evoked Off responses.

DSE occurs in some RGCs but CB1Rs may not be involved

DSE occurs when a postsynaptic neuron depolarizes and subsequently

synthesizes eCBs that activate CB1Rs localized to presynaptic glutamatergic

neurons (118, 124). The activation of CB1Rs leads to a reduction of glutamate

release from the presynaptic neuron and a subsequent reduction in excitation of the

postsynaptic neuron. DSE, along with its counterpart DSI, which occurs at inhibitory

synapses, is thought be a key mechanism of eCB functions in the brain. However,

even though CB1Rs have been discovered in the retina, DSE has not yet been studied

in the retina. The current study used voltage clamp recordings to show that a

phenomenon similar to DSE occurs at the synapse between Off-cone bipolar cells

and RGCs.

Under light adaptation, we recorded light-evoked Off responses in Off and

On-Off RGCs before (pre-depolarization) and after (post-depolarization) a 10 second

RGC membrane depolarization to 0 mV. In previous studies, the excitatory current-

reducing effect of DSE weakened over several minutes until current amplitudes

returned to pre-depolarization levels (118, 124). Therefore, we also recorded Off

responses 2 minutes after depolarization to see if Off amplitudes returned to pre-

depolarization levels (recovery). Off response amplitudes decreased following

depolarization in some but not all recorded RGCs. This may be due to different

65

expression levels of CB1Rs at different synapses. In cells where DSE did occur, CB1R

antagonist O-2050 reduced but did not eliminate the DSE. This data may indicate

that at least some of the DSE we observed in RGCs is not mediated by CB1R

activation. It is possible that the DSE that is not blocked following O-2050

application is mediated by an undiscovered cannabinoid receptor. Indeed, in a

previous study, cannabinoid receptor agonist WIN was able to reduce EPSCs

recorded from hippocampal neurons in a CB1R knockout mouse (119). The

researchers in that study hypothesize that cannabinoids may be acting at

presynaptic glutamatergic neurons expressing non-CB1R cannabinoid receptors.

Taken together, these results indicate that DSE occurs in some RGCs but may only be

partially mediated by CB1Rs.

Effects of manipulating retinal CB1R activity on light-evoked Off responses in RGCs

CB1Rs are expressed at glutamatergic synapses in the retina (9). In a 2008

review, Dr. Yazulla hypothesized that application of CB1R agonists would decrease

glutamate release at these synapses and reduce light-evoked responses. Thus, we

hypothesized that blocking CB1Rs with specific antagonists would increase light-

evoked responses. The current study used voltage clamp recordings to show that O-

2050 does indeed increase Off responses in one population of RGCs but decreases

Off responses in another population of RGCs.

Consistent with our hypothesis, O-2050 increased the amplitude of light-

evoked Off responses in some RGCs. Taking into account CB1R localization studies,

it is likely that O-2050 is blocking CB1Rs and preventing eCBs from reducing

66

glutamate release. Contrary to our hypothesis, however, O-2050 reduced the

amplitude of light-evoked Off responses in other RGCs. To test if O-2050 similarly

modulated RGC action potentials, a major way that the retina communicates with

the brain, we repeated the experiment in current clamp (data not shown). In some

cells, Off action potential frequency increased following O-2050 application as we

hypothesized, but in other cells Off action potential decreased. This data follows

with the voltage clamp data above. In addition, voltage clamp experiments were

repeated with another CB1R antagonist, AM251. Similar results to those seen with

O-2050 were also seen with AM251, indicating that the effect was mediated by

CB1Rs.

We had hypothesized that CB1R agonists would decrease the amplitude of

Off responses. In one population of RGCs, CB1R agonist WIN decreased Off

responses as we hypothesized. Contrary to our hypothesis, however, WIN increased

Off responses in another population of RGCs. Experiments were repeated with the

CB1R agonist 2-AG yielding similar results. Washout of CB1R agonists was carried

out but effect was non-reversible. This was not surprising since difficulty in washing

out the lipophilic CB1R agonists has been reported in the past (50, 116, 125, 126).

These data follow with the O-2050 data indicating that there are functionally two

populations of RGCs in terms of CB1R effects on Off responses. We believe that in

cells where CB1R antagonists increased Off responses, CB1R agonists would have

decreased Off responses. Further, we believe that in cells where CB1R antagonists

decreased Off responses, CB1R agonists would have increased Off responses. Due to

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difficulties with washouts, however, carrying out the above experiment would have

proved problematic.

Crossover inhibition does not underlie CB1R agonist-mediated increases in Off

response amplitude

Given the expression of CB1Rs at retinal glutamatergic synapses, we

hypothesized that CB1R agonists would reduce glutamate release and decrease Off

responses. In many RGCs, however, CB1R agonists increased Off responses.

Therefore, we began to investigate alternative ways in which CB1R agonists may

increase Off responses. On pathways inhibit Off pathways via crossover inhibition. It

is possible that CB1R agonists increased Off responses in some RGCs by reducing the

amount of crossover inhibition of Off pathways. For instance, if CB1R agonists

decreased glutamate release in On pathways, glycinergic amacrine cells could be

inhibited and release of glycine onto Off pathways could be reduced. This crossover

inhibition can be reliably blocked with mGluR6 agonist APB. In the present study,

we showed that even with crossover inhibition blocked by APB, CB1R agonist still

increased Off response amplitudes in one population of RGCs and decreased Off

response amplitudes in another population of RGCs. These results suggest that a

reduction in crossover inhibition is not the mechanism underlying the CB1R

agonist-mediated increases in Off amplitudes in one population of RGCs.

GABAC-mediated inhibition is necessary for the CB1R agonist-mediated increases in Off

response amplitude

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Since we found that crossover inhibition did not underlie CB1R agonist-

mediated Off response amplitude increases, we explored another possible

explanation. Studies have shown that CB1Rs are not only localized to glutamatergic

synapses (9). Rather, studies agree that CB1Rs are expressed at the axon terminals

of amacrine cells. In one study, CB1R immunoreactivity was found at the axon

terminals of a subtype of PKC-immunoreactive GABAergic amacrine cell indicating

that CB1Rs may regulate retinal GABA release (43). These GABAergic amacrine cells

often feed forward onto the axon terminals of cone bipolar cells (127). Therefore,

CB1R agonists may reduce GABA release from these amacrine cells and disinhibit

Off-cone bipolar cells upstream of our recorded Off and On-Off RGCs. In the present

study, we found that with crossover inhibition blocked by APB and with GABAC

receptors blocked by TPMPA, 2-AG was no longer able to increase Off response

amplitudes recorded from RGCs. In the retina, GABAC receptors are primarily

expressed by On and Off-cone bipolar cells (128). Therefore, we believe that a

reduction in GABAergic input at the synapse between amacrine cells and Off-cone

bipolar cells underlies the CB1R agonist-mediated increases in Off responses in one

population of RGCs.

Taken together, the results of this study and results from previous studies

indicate that Off responses can be differentially modulated by CB1R agonists and

antagonists. Our hypothesized circuit for the CB1R agonist-mediated increases in Off

response, as illustrated in Figure 15, is as follows: 1) CB1R agonist activates CB1Rs

at GABAergic amacrine cells that provide inputs to Off-cone bipolar cells, 2) GABA

release is reduced and GABAC receptors on Off-cone bipolar cells are not activated

69

resulting in disinhibition, and 3) Off-cone bipolar cells release more glutamate onto

Off and On-Off RGCs. For the population of RGCs where CB1R agonists decreased Off

responses we hypothesize the following circuit: 1) CB1R agonist activates CB1Rs at

cone and Off-cone bipolar cell synaptic terminals, 2) Glutamate release along the Off

pathway is reduced, and 3) RGC Off responses are reduced. We believe that both

circuits may be involved in modulating individual Off responses. However, the

relative strength of one circuit compared to the other due to differences in CB1R

expression, number of synapses, or another variable may result in the two

populations of RGCs we have observed in this study.

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Figure 10. DSE of light-evoked Off EPSCs recorded from RGCs. Recordings were

made from RGCs with a background light of 1.6x104 photons/m2/s and a stimulus

light intensity of 6.7x106 photons/m2/s. A: Averaged Off responses before RGC

depolarization (black trace), shortly after depolarization (dotted trace), and two

minutes after depolarization (gray trace) in one On-Off RGC perfused with normal

bath solution. In this cell, 10 second RGC membrane depolarization temporarily

reduced the averaged Off response amplitude. The averaged Off response amplitude

recovered two minutes after depolarization. B: RGC depolarization did not

significantly reduce Off response amplitudes in all recorded RGCs (n= 12, 2 Off cells,

10 On-Off cells; N.S. p= 0.158, paired t-test). C: RGC depolarization significantly

reduced Off response amplitudes in one population of recorded RGCs (n= 7, 7 On-Off

cells, * p= 0.041, paired t-test). D: Averaged Off responses before RGC depolarization

(black trace), shortly after depolarization (dotted trace), and two minutes after

depolarization (gray trace) in one On-Off RGC with bath applied CB1R antagonist O-

2050. In this cell, 10 second RGC membrane depolarization temporarily reduced the

averaged Off response amplitude. The averaged Off response amplitude recovered

two minutes after depolarization. E: RGC depolarization did not reduce Off response

amplitudes in all recorded cells with CB1Rs blocked by O-2050 (n= 10, 2 Off cells, 8

On-Off cells, N.S. p= 0.801, paired t-test). F: RGC depolarization significantly reduced

Off response amplitudes in the same cells depicted in panel C even though O-2050

was included in the bath solution (n= 7, 7 On-Off cells, ** p= 0.009, paired t-test).

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Figure 10

72

Figure 11. Effects of CB1R antagonist O-2050 on light-evoked Off responses of

RGCs. O-2050 increased OFF responses in one population of recorded RGCs (A, B,

and C). A: Recording traces from an OFF RGC. O-2050 increased the amplitude of

OFF responses in this cell. B: Recording traces from an ON-OFF RGC. O-2050

increased the amplitude of OFF responses in this cell. C: In one population of RGCs,

O-2050 significantly increased the amplitude of OFF responses (n= 8, 1 Off cell, 7

On-Off cells; ** p= 0.004, paired t-test). O-2050 decreased OFF responses another

population of recorded RGCs (D, E, and F). D: Recording traces from an OFF RGC. O-

2050 decreased the amplitude of Off responses in this cell. E: Recording traces from

an On-Off RGC. O-2050 decreased the amplitude of Off responses in this cell. F: In

one population of RGCs, O-2050 significantly decreased the amplitude of Off

responses (n= 28, 9 Off cells, 19 On-Off cells; *** p0.001, paired t-test).

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Figure 11

74

Figure 12. Effects of CB1R agonist WIN on light-evoked Off responses of RGCs. WIN

increased Off responses in some RGCs (A, B, and C). A: Recording traces from an Off

RGC. WIN increased the amplitude of Off responses in this cell. B: Recording traces

from an On-Off RGC. WIN increased the amplitude of Off responses in this cell. C: In

one population of recorded RGCs, WIN significantly increased the amplitude of Off

responses (n= 7, 2 Off cells, 5 On-Off cells; * p= 0.028, paired t-test). WIN decreased

Off responses in some RGCs (D, E, and F). D: Recording traces from an Off RGC. WIN

decreased the amplitude of Off responses in this cell. E: Recordings from an On-Off

RGC. WIN decreased the amplitude of Off responses in this cell. F: In one population

of recorded RGCs, WIN significantly decreased the amplitude of Off responses (n= 9,

1 Off cell, 8 On-Off cells; * p= 0.013, paired t-test).

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Figure 12

76

Figure 13. Effects of CB1R agonist 2-AG when crossover inhibition is blocked with

mGluR6 agonist APB. 2-AG increased Off responses in some RGCs even with

crossover inhibition blocked by APB (A and B). A: Recording traces from an On-Off

RGC. APB blocked the On responses and decreased the Off response amplitudes in

this cell. 2-AG increased the amplitude of Off responses in this cell. B: In one

population of recorded RGCs, 2-AG in the presence of APB increased the amplitude

of Off responses (n= 15, 9 Off cells, 6 On-Off cells; *** p0.001, paired t-test). 2-AG

decreased Off responses in some RGCs even with crossover inhibition blocked by

APB (C and D). C: Recording traces from an On-Off RGC. APB blocked the On

responses and decreased the Off response amplitudes in this cell. 2-AG decreased

the amplitude of OFF responses in this cell. D: In one population of recorded RGCs,

2-AG in the presence of APB decreased the amplitude of Off responses (n= 10, 1 Off

cell, 9 On-Off cells; * p= 0.012, paired t-test).

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Figure 13

78

Figure 14. Increases in Off response amplitudes are prevented when GABAC

receptors are blocked by TPMPA. 2-AG decreased Off responses in all recorded RGCs

with crossover inhibition blocked by APB and GABAC transmission blocked by

TPMPA (A and B). A: Recording traces from an On-Off RGC. APB blocked the On

responses and decreased the Off response amplitudes in this cell. TPMPA, co-

perfused with APB, increased Off response amplitudes indicating GABAC block and

subsequent disinhibition. 2-AG, co-perfused with APB and TPMPA, decreased Off

response amplitudes in this cell. B: After application of APB and TPMPA, 2-AG

significantly decreased the amplitude of Off responses in recorded RGCs (n= 5, 2 Off

cells, 3 On-Off cells, * p= 0.015, paired t-test).

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Figure 14

80

Figure 15. Diagram showing a functional circuit for CB1R-mediated modulation of

light-evoked Off response amplitudes: CB1R agonists activate CB1Rs at cone

terminals and/or Off-cone bipolar cell terminals to decrease glutamate release and

decrease Off amplitudes. Alternatively, CB1R agonists activate CB1Rs at GABAergic

amacrine cell terminals to decrease GABA release onto Off-cone bipolar cell

terminals that express GABAC receptors resulting in an increase in OFF amplitudes.

C: cone; OFF BC: Off-cone bipolar cell; OFF RGC: Off retinal ganglion cell; A:

GABAergic amacrine cell; GABAC: GABAC receptor.

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Figure 15

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CHAPTER 3 GABAC receptor antagonist TPMPA modulates light-evoked Off responses in light-adapted mouse retina Introduction One way that Off pathways are modulated is by lateral inhibitory inputs from

amacrine cells (129, 130). Many GABAergic amacrine cells make synapses with the

axon terminals of Off-cone bipolar cells to provide this lateral inhibition. In the

mammalian retina, both On and Off-cone bipolar cells express GABAA and GABAC

receptors, but they do not express the metabotropic GABAB receptors (128, 131).

GABAC receptor expression is much greater than GABAA expression, however, in

both On and Off bipolar cell terminals (67, 132). Indeed, almost all GABA-evoked

current in bipolar cells is mediated by GABAC receptors (69, 122 133). The GABA-

evoked current can be parsed out experimentally using receptor-specific

antagonists. GABAA receptors can be specifically blocked with bicuculline, GABAC

receptors can be specifically blocked with TPMPA, and both GABAA and GABAC

receptors are blocked by PTX (134).

Several experiments have investigated the functional roles of GABAC

receptors. Blocking GABAC receptors with TPMPA alters bipolar cell responses to

GABA puff (135, 136). GABA-induced responses

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became shorter following GABAC block leading researchers to hypothesize that

GABAC receptors may primarily modulate sustained glutamate release from bipolar

cells. Experimenters have also shown that GABAA versus GABAC expression differs

by bipolar cell type with important functional implications (69). In ferret, rod

bipolar cell responses were primarily mediated by GABAC receptors and were

slowest. On bipolar cell responses were slightly faster and were mediated by both

GABAA and GABAC receptors. Off bipolar cells had the fastest responses and these

responses were mostly mediated by GABAA receptors. These results indicated that

On pathways may be modulated more strongly by GABAC receptors than Off

pathways. Indeed, in the light-adapted GABAC receptor knockout mouse retina, On

responses evoked by electrical stimulation of bipolar cells were significantly

different from wild-type (10). However, Off response kinetics in GABAC-null mice

were not significantly different from wild-type. The researchers in the above study

concluded that GABAC receptors more effectively limit signaling from On than Off-

cone bipolar cells. However, the previous retinal GABAC studies have not

demonstrated that GABAC receptors modulate light-evoked responses consistently

under different ambient light conditions.

In the current study, patch clamp recordings were made from RGCs in mouse

retinas under two ambient light conditions. Under low photopic conditions, I found

that GABAC block by TPMPA increased both On and Off response decay times

recorded from On-Off RGCs. Further, I found that under high photopic conditions,

TPMPA had similar effects to those observed in dim light. Blocking crossover

inhibition with APB did not prevent TPMPA-induced increases in Off decay time.

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The results presented in this study suggest that GABAC receptors shape both On and

Off responses in the light-adapted mouse retina under both low and high photopic

ambient light conditions.

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Materials and Methods

The basic methods used in this study were similar to those used previously in

the laboratory (81, 82, 83, 84). The materials and methods used for the experiments

outlined in this Chapter are also similar to those described in Chapters 1 and 2.

Retinal preparation. Retinal preparations performed in these experiments are

similar to those described in Chapters 1 and 2.

Electrophysiology. Whole-cell patch clamp recordings were made from RGCs in light-

adapted retinas, as described in Chapters 1 and 2. Measurements for light-evoked

EPSCs are detailed in Chapter 1. Three light-evoked responses were averaged under

control conditions and also under each different drug condition.

Light stimulus. The light stimuli were presented to the light-adapted retina using the

Lucivid, as described in Chapter 1. For these studies, each RGC was dissected and

prepared with a background light intensity of 3000 photons/m2/s (low photopic).

Control responses were recorded at the above background light intensity. After

control responses were recorded, drugs were bath applied and the retina was

adapted to a background light intensity of 2.9x104 photons/m2/s (high-photopic).

At both of these background light intensities, rods are completely inactivated and

only cone-mediated responses are recorded.

Light stimuli with intensities greater than that of the background light were

used to evoke light responses from RGCs. For each cell, different intensities, ranging

from 3.2x103 photons/m2/s to 1.6x104 photons/m2/s under the low photopic

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conditions and between 6.25x104 photons/m2/s and 6.7x106 photons/m2/s

under the high photopic conditions were used to evoke light responses.

In this study, we investigated the role of TPMPA in modulating light-evoked

response kinetics. The time for light-evoked EPSCs to decay to 10% of the peak

amplitude was measured with Minianalysis (Synaptosoft, Inc.). EPSC traces were

visualized using Clampfit 9 (Molecular Devices, Inc.) and averaged within drug

conditions using Origin 6.0 (OriginLab Corporation). Averaged traces before and

after TPMPA application were normalized by amplitude and overlaid to illustrate

differences in light response kinetics.

Drug application. APB (20 M; Sigma) and (1,2,5,6-tetrahydropyridin-4-

yl)methylphosphinic acid (TPMPA; 50 M; Sigma) were freshly dissolved in MEME

on the day of the experiment and administered through a gravity-fed line.

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Results

TPMPA increased the decay time of both On and Off responses under low photopic

background illumination

In a previous study performed under one background light intensity, GABAC

receptor antagonist TPMPA significantly increased On but not Off response decay

time (10). Moreover, On responses in GABAC knockout mice strongly differed from

On responses in wild-type, but Off responses in GABAC knockout mice were no

different from Off responses in wild-type. Responses were stimulated by electrical

tetanus localized to the OPL where bipolar cell dendrites stratify. To determine if

TPMPA selectively increases light-evoked On response decay time under dim light

conditions, we recorded light-evoked responses from On-Off RGCs under a low

photopic ambient light intensity of 3000 photons/m2/s before and after bath

application of 50 M TPMPA (Figure 16). With GABAC receptors blocked by TPMPA,

light-evoked On response average time to decay to 10% of peak amplitude

significantly increased (Figure 16A,B, n= 12). The average On decay time increased

62%. However, Off response decay times also significantly increased (Figure 16C,D,

n= 12). The average Off decay time increased 82%. These results indicate that under

low photopic conditions, TPMPA alters both light-evoked On and light-evoked Off

kinetics.

TPMPA increased the decay time of both On and Off responses under high photopic

background illumination

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As was discussed in Chapter 1, lateral inhibition of Off pathways changes

depending upon the intensity of background illumination. In the previous study

demonstrating the effects of TPMPA in RGC responses, experiments were only

performed at one background light intensity (10). To determine if TPMPA increases

light-evoked On and Off response decay time consistently at different ambient light

intensities, we increased the background light intensity from 3000 photons/m2/s

to a high photopic intensity of 2.9x104 photons/m2/s and recorded light-evoked

responses before and after bath application of 50 M TPMPA (Figure 17). The same

cells were used for both the low photopic and high photopic conditions. After the

effects of TPMPA were recorded in the low photopic condition, washout of TPMPA

proceeded for 10 minutes. At the same time, the retina was illuminated with and

adapted to the high photopic ambient light intensity. Under high photopic

conditions, GABAC receptor block by TPMPA significantly increased On response

average time to decay to 10% of peak amplitude (Figure 17A,B, n= 11). The On

response decay time increased 67%. Moreover, Off response average time to decay

to 10% of peak amplitude under photopic conditions also significantly increased

(Figure 17C,D, n= 11). TPMPA increased the Off response decay time 85%. These

results indicate that under high photopic conditions, TPMPA alters both On and Off

response kinetics.

Blocking crossover inhibition with mGluR6 agonist APB does not prevent the TPMPA-

induced increase in Off response decay time

89

Researchers found that only On response kinetics were significantly altered

in GABAC knockout mice or after application of TPMPA (10). However, we found that

both On and Off decay times significantly increased following bath application of

TPMPA. In our experiment, we used light to evoked RGC responses. As we

demonstrated in Chapter 1, light-evoked Off responses can be modulated by

crossover inhibition from On pathways. To determine if TPMPA modulates

crossover inhibition to increase Off response decay times, we bath applied APB (20

M) to block crossover inhibition and then recorded light-evoked responses from

On-Off RGCs before and after application of 50 M TPMPA (Figure 18). Even with

crossover inhibition blocked by APB, Off response average time to decay to 10% of

peak amplitude significantly increased in TPMPA. Indeed, Off response decay times

increased 64%. These results indicate that a reduction in crossover inhibition does

not underlie the TPMPA-induced increases in Off response decay time.

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Discussion

Both On and Off-cone bipolar cells express GABAC receptors at their axon

terminals (137, 138, 139, 140). GABAergic amacrine cells release GABA that

activates the chloride-fluxing GABAC receptors at bipolar cell terminals (128). This

results in inhibition of the bipolar cell and reduces subsequent release of glutamate

from bipolar cells onto RGCs. GABAergic transmission in the IPL, like that described

above, plays an important role in shaping the surround inhibition and temporal

responses in RGCs (135, 141). Thus, GABAC mediated transmission is crucial to

important visual properties like edge detection, motion sensitivity, and direction

sensitivity.

TPMPA can be used to specifically block GABAC receptors (65). When GABAC

receptors are knocked out or blocked by TPMPA, GABAR-mediated chloride

currents become more transient, with a fast rise and decay time, because of the

elimination of a longer-latency chloride current (10). This alters neurotransmitter

release from bipolar cells and alters kinetics of EPSCs recorded from RGCs. While

GABAC receptors are expressed at both On and Off-cone bipolar cell axon terminals,

a previous study using a ferret model demonstrated that GABA-induced currents in

bipolar cells differed depending on cell type (69). GABA-induced currents in ferret

On bipolar cells were mostly mediated by GABAC receptors, whereas GABA-induced

currents in ferret Off bipolar cells were mostly mediated by GABAA receptors and

were transient. Moreover, in GABAC knockout mice, On responses recorded from

RGCs were significantly different from wild-type, but Off responses were not

significantly different from wild-type (10). Thus, the previous studies indicate that

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GABAC-mediated inhibition plays a larger role in modulating On responses than Off

responses. However, whether these previous findings are consistent under different

background light intensities is not known. Further, it is not yet clear whether the

results from previous studies will be consistent with results of experiments where

TPMPA is used to block GABAC receptors and light is used to evoke responses. The

results of the present study demonstrate that TPMPA increases both On and Off

response decay times under dim ambient light conditions. Similar results were

found when the background light intensity was increased. Crossover inhibition does

not underlie the TPMPA-induced increase in Off decay times. Taken together, these

findings provide evidence that under two background light intensities, GABAC block

by TPMPA alters both On and Off response kinetics.

TPMPA increases both On and Off response decay time under low photopic background illumination Under a low photopic background light intensity of 3000 photons/m2/s,

TPMPA altered both On and Off response amplitude and kinetics. By blocking GABAC

receptors with TPMPA, both On and Off pathways were disinhibited. This facilitated

increased glutamate release from bipolar cells onto the recorded RGCs. In addition,

the temporal properties of both On and Off responses changed. On EPSCs had a

much longer time to decay. Surprisingly, Off EPSC decay times also increased,

contrary to the findings in previous studies (10). Indeed, TPMPA increased Off decay

times to a greater extent than On decay times. In the earlier study, RGC responses

before and after application of TPMPA were evoked with an electrical stimulus. It is

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possible that the differences between the previous study and the current study are

due to differences in stimulation methods. However, light-evoked responses were

also used in the aforementioned previous study to determine the differences in both

On and Off kinetics between wild-type and GABAC knockout mice. In that study,

GABAC knockout mice had significantly different On but not Off responses compared

to wild-type. Currently it is unclear why Off responses were affected by TPMPA in

the present study, but not in the previous study.

TPMPA increases both On and Off response decay time under high photopic background illumination Given the surprising result of TPMPA-induced differences in Off response

kinetics, we repeated the above experiment at the higher background light intensity

of 2.9x104 photons/m2/s. From our results in Chapter 1, in addition to results from

other studies, we hypothesized that the relative strength of inhibition may change at

different ambient light intensities. Therefore, we adapted the retina to a high

photopic, but still light-adapted, ambient illumination. In the present study, we

showed that under high photopic background illumination, TPMPA still increased

both On and Off response amplitude and decay times. In fact, the factors by which

TPMPA increased both On and Off decay times were consistent across the two

background illumination levels. These results suggest that our surprising finding

that TPMPA altered Off response kinetics was not due to differences in ambient light

intensity compared to the previous study.

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Blocking crossover inhibition with APB does not prevent TPMPA-induced increases in Off response decay time In the previous study, TPMPA only increased On response decay times (10).

These On responses were evoked electrically with the stimulus localized to the OPL

where bipolar cell dendrites stratify. Given the results from the previous study, we

had not expected that TPMPA would increase Off response decay times. However,

we used light to evoke the stimulus rather than an electric tetanus. It is possible that

differences in the stimulus methods resulted in different circuits being activated.

For instance, light-evoked Off responses are known to be modulated by crossover

inhibition from On pathways. Crossover inhibition of Off pathways could potentially

be inactive when using an electrical stimulus, and this could underlie the differences

found between the previous and the current study. Therefore, we blocked crossover

inhibition with APB and recorded light-evoked responses before and after

application of TPMPA. Even with crossover inhibition blocked by APB, TPMPA still

increased Off response decay times. These results indicate that modulation of

crossover inhibition does not underlie the TPMPA-induced increases in Off response

decay times.

Taken together, the results of this study and those of previous studies

indicate that GABAC receptors play a crucial role in shaping the kinetics of light

responses in RGCs. Studies showing GABAC expression at Off-cone bipolar cell

terminals, as well as our present study, suggest that both On and Off responses are

modulated by GABAC receptors (Figure 19). It is currently unclear why the results of

our study differed from the results in 10. Future experiments should be conducted

to determine the basis for the inconsistency.

94

Figure 16. GABAC antagonist TPMPA increases the decay time of On and Off

responses in On-Off RGCs under low photopic conditions. Recordings were made

from RGCs with a background light of 3000 photons/m2/s and a stimulus light

intensity of 1.6x104 photons/m2/s. TPMPA increased the On decay time in On-Off

RGCs (A and B). A: Overlaid On responses normalized by amplitude from a single

On-Off RGC before (black trace) and after bath application of TPMPA (gray trace). B:

TPMPA significantly increased the average time for the On response to decay to

10% of the peak On amplitude (n= 12, 12 On-Off cells, * p= 0.010, paired t-test).

TPMPA increased the Off decay time in On-Off RGCs (C and D). C: Overlaid Off

responses normalized by amplitude from the same On-Off RGC as A before (black

trace) and after bath application of TPMPA (gray trace). D: TPMPA significantly

increased the average time for the Off response to decay to 10% of the peak Off

amplitude (n= 12, 12 On-Off cells, *** p< .001, paired t-test).

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Figure 16

96

Figure 17. GABAC antagonist TPMPA increases the decay time of On and Off

responses in On-Off RGCs under high photopic conditions. Recordings were made

from RGCs with a background light of 2.9x104 photons/m2/s and a stimulus light

intensity of 6.7x106 photons/m2/s. TPMPA increased the On decay time in On-Off

RGCs (A and B). A: Overlaid On responses normalized by amplitude from a single

On-Off RGC before (black trace) and after bath application of TPMPA (gray trace). B:

TPMPA significantly increased the average time for the On response to decay to

10% of the peak On amplitude (n= 11, 11 On-Off cells, * p= 0.014, paired t-test).

TPMPA increased the Off decay time in On-Off RGCs (C and D). C: Overlaid Off

responses normalized by amplitude from the same On-Off RGC as A before (black

trace) and after bath application of TPMPA (gray trace). D: TPMPA significantly

increased the average time for the Off response to decay to 10% of the peak Off

amplitude (n= 11, 11 On-Off cells, *** p< .001, paired t-test).

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Figure 17

98

Figure 18. Blocking crossover inhibition with mGluR6 agonist APB does not prevent

the TPMPA-induced increase in Off response decay time under high photopic

conditions. TPMPA increases Off response decay time recorded from On-Off RGCs

with crossover inhibition blocked by APB (A and B). A: Overlaid Off responses

normalized by amplitude from an On-Off RGC during bath application of APB (black

trace) and bath application of APB plus TPMPA (gray trace). B: TPMPA significantly

increased the average time for the Off response to decay to 10% of peak Off

amplitude even with crossover inhibition blocked by APB (n= 11, 11 On-Off cells, p=

0.015, paired t-test).

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Figure 18

100

Figure 19. Diagram showing a functional circuit for GABAC receptor-mediated

modulation of Off responses. GABAergic amacrine cells release GABA onto Off-cone

bipolar cells. The Off-cone bipolar cells, which express GABAC receptors at the axon

terminals become inhibited and subsequent glutamate release from the Off-cone

bipolar cell is reduced. Ultimately, Off responses recorded from Off RGCs are

reduced. TPMPA blocks this circuit by blocking GABAC receptors and preventing

GABA from inhibiting the Off-cone bipolar cells. C: cone; OFF BC: Off-cone bipolar

cell; OFF RGC: Off retinal ganglion cell; A: GABAergic amacrine cell; GABAC: GABAC

receptor.

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Figure 19

102

CONCLUSION

Neural signals in the retina are carried by parallel pathways dedicated to

light increments and light decrements (1). Having parallel circuits doubles the

retina’s dynamic range, increases signal transfer efficiency, and facilitates contrast

sensitivity. Loss of function of either pathway severely impairs vision (3). Therefore,

it is crucial to deeply understand both the circuit for light increments, known as the

On pathway, and the circuit for light decrements, known as the Off pathway. In this

thesis, modulation of Off responses by mGluR6, CB1, and GABAC receptors were

studied using patch clamp electrophysiology of RGCs and pharmacological

manipulations. The experiments I have described in this thesis demonstrate how

several neurotransmitter receptors shape the responses recorded from RGCs, the

final output neurons of the retina.

Even though On and Off pathways are parallel and have opposite functions,

they are not completely isolated from one other. In previous experiments performed

in the dark-adapted retina, it has been shown that On pathways can inhibit Off

pathways via crossover inhibition (31, 32, 33, 34). When On-cone bipolar cells

become excitable they release glutamate that activates glycinergic amacrine cells.

These amacrine cells then release glycine onto Off-cone bipolar cells and inhibit Off

responses. Blocking

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crossover inhibition with mGluR6 agonist APB disinhibits the Off-cone bipolar cells

and increases the amplitude of RGC Off responses recorded under dark adaptation

(5, 6, 7, 8). The results from the experiments detailed in Chapter 1 confirm that APB

increases Off response amplitudes under dark adaptation. However, we found that

APB decreases Off response amplitudes under light adaptation. This suggests that

the impact of crossover inhibition is dynamic and depends on the ambient light

intensity. APB-induced increases in Off response amplitude under dark adaptation

required glycinergic transmission. We found that the APB-induced decreases in Off

response amplitude under light adaptation, however, did not involve inhibitory

neurotransmission because the effect persisted even with STR, PTX, or TPMPA

included in the bath solution. This indicated that On pathways may modulate Off

pathways using a novel mechanism that does not involve crossover inhibition. When

we blocked D1 receptors, APB-induced decreases in Off responses under light

adaptation were blocked. This suggested that dopaminergic transmission involving

D1 receptors is necessary for the observed effects of APB under light adaptation.

Finally, blocking HCN channels also prevented APB-induced decreases in Off

responses under light adaptation. Taken together these results indicate a novel way

in which On pathways can modulate Off responses. Under dark adaptation, On

pathways inhibit Off pathways via glycinergic amacrine cells. Under light adaptation,

this crossover inhibition pathway is still active but is occluded by a stronger and

opposite crosstalk mechanism: 1) On-cone bipolar cells excite dopaminergic

amacrine cells that are only active during light adaptation, 2) dopamine is released

from these amacrine cells and binds to D1 receptors expressed by Off-cone bipolar

104

cells, 3) activation of the metabotropic D1 receptors results in activation of HCN

channels , and 4) HCN channel activation increases glutamate release from Off-cone

bipolar cells resulting in greater Off EPSCs in RGCs. APB blocks the above

mechanism by hyperpolarizing On-cone bipolar cells resulting in a decrease in Off

response amplitudes. Our findings in dark and light adaptation suggest that mGluR6

receptors, despite being localized to On pathways, are capable of modulating Off

responses. Moreover, mGluR6-mediated modulation of Off responses is dynamic

and the mechanisms involved are dependent on the retinal adaptation.

The retinal eCB system is a relatively new field of study. CB1R localization

and functions in the retina have been investigated, however, the role of CB1Rs in

shaping light-evoked output from the retina is not yet clear (9). The results

presented in Chapter 2 support our hypothesis that the retinal eCB system does

modulate Off response output from the light-adapted retina. We found that DSE, an

extensively studied mechanism of eCB function in the brain, also occurs at some

synapses between Off-cone bipolar cells and RGCs. The DSE may not be completely

mediated by CB1Rs, however. In addition, we found that there are functionally two

populations of RGCs. In the first population, CB1R antagonists increase Off

responses and CB1R agonists decrease Off responses. We suggest that the

antagonists increase glutamate release from cones and bipolar cells resulting in a

larger Off response. The agonists decrease glutamate release at the same cells

resulting in a smaller Off response. In the second population, CB1R antagonists

decrease Off responses and CB1R agonists increase Off responses. Our results

indicate that the antagonists decrease Off responses by increasing GABA release

105

from GABAergic amacrine cells that provide inputs to Off-cone bipolar cells.

Consistent with this, our results suggest that agonists increase Off responses by

decreasing GABA release from GABAergic amacrine cells that provide inputs to Off-

cone bipolar cells. Taken together, the results from this study indicate that the

retinal eCB system does modulate signal output from the retina and likely has an

impact on vision.

As previous studies have shown, including our own in Chapter 1, lateral

inhibition modulates Off pathways (129, 130). Lateral inhibition from GABAergic

amacrine cells to bipolar cells is mainly mediated by GABAC receptors (134).

However, in a previous study, even though both On and Off-cone bipolar cells

express GABAC receptors, knocking out or blocking GABAC receptors only affected

On but not Off responses in RGCs under light adaptation (10). The results from the

experiments detailed in Chapter 3 indicate that both On and Off responses are

affected by TPMPA, a GABAC specific antagonist. Contrary to the results in the

previous study, TPMPA increased both On and Off response decay times as would be

expected if GABAC receptors, with their long-latency and sustained effects, were

blocked. These results were consistent at two levels of ambient light intensity and

with crossover inhibition blocked by APB. It remains unclear why our results differ

from those of the previous study. Taken together, the results presented in this study

indicate that GABAC receptors play an important role in modulating the strength and

temporal properties of Off responses.

In conclusion, the data contained in this thesis indicate that mGluR6, CB1,

and GABAC receptors are all capable of modulating Off response output from the

106

retina. Collectively, the results in this thesis suggest several methods in which Off

pathways can be modulated. First, we showed that crosstalk between On and Off

pathways modulates Off responses under dark and light adaptation. Second, we

demonstrated that retrograde neurotransmitter systems affect Off response

strength. Finally, we illustrated how lateral inhibition shapes Off responses. The

mechanisms described in this thesis illustrate both the complexity and the

importance of the retina with regard to visual signal processing.

107

LIST OF ABBREVIATIONS

2-AG Endocannabinoid and CB1R agonist 2-arachidonoyl glycerol APB mGluR6 receptor agonist DL-2-amino-4-phosphonobutyric acid CB1R Cannabinoid 1 receptor DCPG mGluR8 agonist (S)-3,4-dicarboxyphenylglycine DSE Depolarization-induced suppression of excitation DSI Depolarization-induced suppression of inhibition ECl Chloride equilibrium potential eCB Endocannabinoid, an endogenous cannabinoid EPSC Excitatory post-synaptic current GABA Inhibitory neurotransmitter -aminobutyric acid GCL Ganglion cell layer HCN Hyperpolarization-activated cyclic nucleotide-gated channel INL Inner nuclear layer IPL Inner plexiform layer IPSC Inhibitory post-synaptic current MEME Bath solution used in experiments, minimum essential medium Eagle mGluR6 Metabotropic glutamate receptor 6 ONL Outer nuclear layer OPL Outer plexiform layer

108

PTX GABAA/C receptor antagonist picrotoxin RGC Retinal ganglion cell SE Standard error STR Glycine receptor antagonist strychnine TPMPA GABAC receptor specific antagonist (1,2,5,6-tetrahydropyridin-4-

yl)methylphosphinic acid WIN Synthetic CB1R agonist WIN 55, 212-2

109

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BIOGRAPHY

Joshua Nathaniel Pahng was born to Sung-Cue Pahng and Colleen Marvin at

Camp Lejune Naval Hospital in Jacksonville, North Carolina on November 24th, 1985.

In 2000, he and his family moved to Manassas, Virginia where he attended

Stonewall Jackson High School. He earned his Bachelor of Science degree at the

College of William and Mary in Williamsburg, Virginia in 2008. In the fall of 2008,

Joshua entered the Ph.D. Program in Neuroscience at Tulane University in New

Orleans, Louisiana. There, he conducted his research under the mentorship of his

advisor, Dr. Guoyong Wang.