Distinct Modulatory Effects of Dopamine on Excitatory

DISTINCT MODULATORY EFFECTS OF DOPAMINE ON EXCITATORY

CHOLINERGIC AND INHIBITORY GABAERGIC SYNAPTIC TRANSMISSION IN

DROSOPHILA

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Ning Yuan

June 2006

This thesis entitled

DISTINCT MODULATORY EFFECTS OF DOPAMINE ON EXCITATORY

CHOLINERGIC AND INHIBITORY GABAERGIC SYNAPTIC TRANSMISSION IN

DROSOPHILA

NING YUAN

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Daewoo Lee

Assistant Professor of Biological Sciences

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract

YUAN, NING, M.S., June 2006, Biological Sciences

DISTINCT MODULATORY EFFECTS OF DOPAMINE ON EXCITATORY CHOLINERGIC

AND INHIBITORY GABAERGIC SYNAPTIC TRANSMISSION IN DROSOPHILA (71 pp.)

Director of Thesis: Daewoo Lee

Dopaminergic neurons and receptors are widely distributed throughout the

Drosophila CNS. However, the role of dopamine in modulating Drosophila central synaptic transmission has not been investigated. By using electrophysiological techniques,

I explored dopamine modulation of cholinergic and GABAergic transmission in primary neuronal cultures. Cholinergic synaptic currents were suppressed by dopamine. This effect was mimicked by the mammalian D1-like receptor agonists SKF38393 and (±)-6- chloro-APB. GABAergic transmission was enhanced by dopamine and the D2-like receptor agonist (-)-quinpirole. These results demonstrated that there are two distinct dopamine signaling pathways modulating cholinergic and GABAergic transmission through D1- and D2-like receptors, respectively. Forskolin modulated cholinergic and

GABAergic transmission exactly opposite to dopamine modulation, suggesting D1- and

D2-like receptor actions are predominantly independent of the increase in intracellular cAMP levels. This study reports dopamine modulation of Drosophila central synaptic transmission for the first time and will help to elucidate mechanisms of dopamine modulation underlying behaviors.

Approved:

Daewoo Lee

Assistant Professor of Biological Sciences 4

Table of Contents

Page

Abstract...... 3

List of Figures...... 7

Introduction...... 9

I. Dopamine as a neurotransmitter...... 9

II. Mammalian dopamine receptors...... 10

III. Dopamine modulation of synaptic transmission in vertebrates...... 11

IV. Dopamine signaling in Drosophila...... 12

V. Drosophila dopamine receptors...... 13

VI. Drosophila primary neuronal cultures as a model to study dopamine modulation in

the CNS ...... 15

Specific Aims...... 17

Aim 1. To examine the modulatory actions of dopamine on Drosophila central synaptic

transmission...... 18

Aim 2. To characterize the pharmacological properties of dopamine receptors which

modulate excitatory cholinergic synaptic transmission...... 18

Aim 3. To characterize the pharmacological properties of dopamine receptors which

modulate inhibitory GABAergic synaptic transmission...... 18

Aim 4. To characterize the molecular properties of dopamine receptors which modulate

Drosophila synaptic transmission ...... 18

Materials and Methods...... 19 5 I. Drosophila stocks...... 19

II. Dopamine receptor deficiency stocks...... 19

III. Drosophila primary neuronal cultures...... 20

a. DAMB deficiency neuronal cultures ...... 21

b. dDA1 deficiency neuronal cultures ...... 21

c. DD2R deficiency neuronal cultures...... 22

IV. Electrophysiology...... 22

V. Pharmacology ...... 23

VI. Data analysis ...... 24

VII. Negative Controls ...... 26

Results...... 27

I. Whole-cell recording in cultured Drosophila primary neurons...... 27

II. Pharmacological characterization of dopamine receptors modulating cholinergic

currents ...... 31

III. Pharmacological characterization of dopamine modulation of GABAergic currents

...... 41

IV. Molecular characterization of dopamine modulation of cholinergic transmission.. 47

a. Dopamine modulation in the DAMB deficiency line...... 47

b. Dopamine modulation in the DD2R deficiency line ...... 50

V. Supplementary data-intracellular signaling pathways...... 51

Discussion...... 54

Pharmacological properties of dopamine modulation of synaptic transmission...... 54

Specificity of pharmacological ligands ...... 55 6 Cellular location of dopamine receptors...... 56

Molecular properties of dopamine modulation of synaptic transmission ...... 57

Potential physiological functions ...... 59

Conclusion...... 60

References...... 61

Appendix...... 69

Intracellular signaling pathways mediated by Drosophila dopamine receptors ...... 69 7 List of Figures

Figure Page

Figure 1. Whole-cell recording of synaptic currents in Drosophila primary neuronal culture...... 29

Figure 2. Spontaneous cholinergic post-synaptic currents (PSCs) recorded in cultured embryonic neurons...... 30

Figure 3. Spontaneous GABAergic PSCs recorded in cultured embryonic neurons...... 30

Figure 4. Spontaneous cholinergic PSCs were down-regulated by the focal application of

500µM dopamine...... 32

Figure 5. Effects of different concentrations of dopamine on the frequency of cholinergic

PSCs...... 33

Figure 6. Effects of 100µM dopamine application on cultured neurons prepared from two

different strains...... 34

Figure 7. Effects of 10µM SKF38393 application on the cholinergic PSCs...... 36

Figure 8. Effects of 10μM (±)-6-chloro-APB application on cholinergic PSCs...... 37

Figure 9. Effects of 10μM (-)-quinpirole application on cholinergic PSCs...... 38

Figure 10. Effects of 20μM SCH23390 on cholinergic PSCs...... 40

Figure 11. Modulation of cholinergic synaptic transmission by dopamine drugs...... 41

Figure 12. Spontaneous GABAergic PSCs were up-regulated by the focal application of

500µM dopamine...... 43

Figure 13. Effects of 10μM (-)-quinpirole on GABAergic PSCs...... 44

Figure 14. Effects of 10μM (-)-quinpirole on GABAergic PSCs in the presence of 10μM

haloperidol...... 45 8 Figure 15. Effects of 10μM SKF38393 on GABAergic PSCs...... 46

Figure 16. Modulation of GABAergic synaptic transmission by various dopamine durgs.

...... 47

Figure 17. Single embryo neuronal cultures prepared from the DAMB deficiency line.. 49

Figure 18. Effects of 10μM SKF38393 on cholinergic PSCs in homozygous DAMB deficiency neurons...... 50

Figure 19. The effects of 20μM forskolin on cholinergic PSCs...... 52

Figure 20. The effects of 20μM forskolin on GABAergic PSCs...... 53

Introduction

I. Dopamine as a neurotransmitter

Dopamine is an important neurotransmitter found mainly in the central nervous

system (CNS) of vertebrates and invertebrates (Carlsson et al, 1958; Monastirioti, 1999;

Benes, 2001; Carlsson, 2001; Prakash and Wurst, 2006). Several dopaminergic neuronal clusters have been found in mammalian brains that project to different areas such as the prefrontal cortex and nucleus accumbens (Bjorklund and Lindvall, 1984; Lewis et al,

1998; Goldman-Rakic, 1998; Yang et al, 1999; Tzschentke, 2001; Durstewitz and

Seamans, 2002; Prakash and Wurst, 2006). Extensive research has shown that dopamine is involved in regulating many brain functions such as emotional state, motor control and

is important in substance abuse (Brozoski et al, 1979; Simon et al, 1980; Le and Mison,

1991). Improper dopamine signaling is associated with psychological disorders such as

schizophrenia or hyperactivity, while lack of dopamine input is the major deficit

underlying Parkinson’s disease, which is the second most common neurodegenerative

disorder worldwide (WHO, 2004).

The first step of dopamine synthesis is the conversion of the amino acid tyrosine

to DOPA by the enzyme tyrosine hydroxylase (TH). DOPA is then quickly converted to

dopamine by the aromatic L-amino acid decarboxylase. The dopamine produced is then

sequestered into synaptic vesicles for storage and released when dopaminergic neurons

are excited. The released dopamine binds to the receptors localized on the postsynaptic

cells to transmit neural signals or to the presynaptic receptors to modulate further release. 10 The effect of dopamine on postsynaptic cells is different from other classical neurotransmitters. For example, classical neurotransmitters such as acetylcholine and

GABA typically generate excitatory post-synaptic potentials (EPSPs) and inhibitory post- synaptic potentials (IPSPs) respectively through the ion channels in their receptors. But no dopamine gated ion channels have been identified. Furthermore, dopamine does not usually directly change the potential of the post-synaptic cell. It either modifies the response of neurons to other neurotransmitters, resulting in amplifying or dampening the effectiveness of ongoing synaptic activity, or influences the synthesis, release, re-uptake or metabolism of other neurotransmitters. Thus, dopamine is classified as a neuromodulator to distinguish it from classical neurotransmitters (Wildmaier et al, 2002).

II. Mammalian dopamine receptors

Although much still needs to be discovered about the neuromodulatory roles of dopamine, a solid understanding of many dopamine receptors in vertebrates has been achieved. Based on their biochemical characteristics, two types of mammalian dopamine receptors, D1- and D2-like receptors, have been found (Neve et al, 2004). Both types of receptors belong to the G-protein coupled receptor (GPCR) superfamily. The major characteristic of D1-like receptors is that they mediate an increase of adenylate cyclase activity via the G protein αs subunit, causing the intracellular cAMP level to increase.

Elevated cAMP activates downstream targets such as cAMP-dependent protein kinases.

These enzymes then phosphorylate substrates which can eventually lead to the modification of cellular functions. On the other hand, activation of D2-like receptors 11 leads to the inhibition of adenylate cyclase via the G protein αi subunit, thus decreasing

the cytoplasmic cAMP level and leading to an inhibitory effect on the cAMP-dependent

signaling pathways (Neve et al, 2004). Other cAMP-independent signaling pathways,

such as those involving direct dopamine receptor cross-talk, have also been revealed for

both types of dopamine receptors (Liu et al, 2000; Gao et al, 2001; Lee et al, 2002; Chen

et al, 2004; Goldman-Rakic et al, 2004; Lee et al, 2005).

Molecular studies have identified several subtypes of vertebrate dopamine

receptors. The D1-like receptor family contains two subtypes, D1 and D5, while the D2-

like receptor family has three subtypes, D2, D3 and D4. They are different in expression

patterns, sequences, and pharmacological characteristics (Jackson and Westlind-

Danielsson, 1994; Lachowicz and Sibley, 1997; Missale et al, 1998; Zhuang et al, 2000;

Neve et al, 2004).

III. Dopamine modulation of synaptic transmission in vertebrates

It has been shown that dopamine modulates both excitatory and inhibitory

synaptic transmission in vertebrates. For example, glutamate synaptic transmission, the

major excitatory synaptic transmission in vertebrates, has been reported to be enhanced

or suppressed by dopamine, depending on the brain area and neuron type studied. Evoked

AMPA-mediated excitatory post synaptic potentials have been reported to be depressed

by dopamine (Law-Tho et al, 1994; Zheng et al, 1999; Urban et al, 2002). Presynaptic

release of glutamate can be inhibited by dopamine in layer V pyramidal neuron circuits

in the prefrontal cortex (Gao et al, 2001). On the other hand, a stimulation of NMDA 12 receptor mediated response by dopamine has been reported in neostriatal, hippocampal and cortical neurons (Umeniya and Raymond, 1997; Neve et al, 2004; Lee et al, 2005).

Similarly, varied effects of dopamine on GABAergic synaptic transmission have also been reported (Starr, 1987; Grobin and Deutch, 1998; Retaux et al, 1991; Neve et al,

2004). In the pars reticulata of the substantia nigra, dopamine released from dendrites

enhanced GABA release, presumably through D1-like receptors located on striatonigral

afferents (Radnikow and Misgeld, 1998). However, in the neostriatum and nucleus

accumbens, dopamine was reported to decrease GABAergic transmission in medium

spiny neurons (Neve et al, 2004). These differences probably result from the differences

in the studied brain areas and neuron types. The extensive and complicated interactions among neurons in the vertebrate CNS make it difficult to fully understand the modulatory

effects of dopamine.

IV. Dopamine signaling in Drosophila

Dopaminergic neurons have been identified in the embryonic, larval and adult

Drosophila CNS by immunochemistry studies. They formed different neuron clusters

which were widely scattered in the Drosophila CNS. The adult Drosophila CNS is

composed of the brain and the thoracic nervous system. Paired and unpaired dopamine

neuron clusters have been found in both parts of the CNS and they innervate major

neuropil areas such as the central complex and the mushroom body (Lundell and Hirsh,

1994; Monastirioti, 1999; Mustard et al, 2005). As in vertebrates, dopamine plays an important role in regulating a variety of behaviors in Drosophila (Feany and Bender, 13 2000; Heisenberg, 2003; Keene and Waddell, 2005; Mustard et al, 2005; Reimensperger et al, 2005). For example, dopaminergic output in the mushroom bodies of the brain is required for the acquisition of shock-reinforced memory (Schwaezel et al, 2003). Flies lacking dopamine in the CNS due to a dopa decarboxylase mutation display learning defects (Tempel et al, 1984). Selective degeneration of dopaminergic neurons causes locomotor dysfunction, similar to those seen in Parkinson’s disease in humans (Feany and Bender, 2000). But compared to its vertebrate counterpart, the Drosophila CNS is much simpler. Genetic manipulations are much easier in this model organism. Thus, given the high degree of conservation between vertebrates and Drosophila in terms of the modulatory functions of dopamine, genetically powered Drosophila can be a good model to study molecular mechanisms underlying dopaminergic signaling. Therefore, the knowledge of dopamine modulatory function in Drosophila will help us understand the functions of dopamine in higher organisms.

V. Drosophila dopamine receptors

By using conserved vertebrate dopamine receptor sequences as a probe, three

Drosophila dopamine receptors (dDA1, DAMB and DD2R) have been identified

(Gotzes, 1994; Sugamori et al, 1995; Feng et al, 1996; Han et al, 1996; Hearn et al, 2002;

Kim et al, 2003). Sequence analysis showed that the receptors all belong to the GPCR superfamily (Mustard et al, 2005). The dopamine receptor dDA1 gene encoded a 385 amino acid protein with high sequence similarity to human dopamine D1 (52.1%) and D5

(51.8%) receptors (Sugamori et al, 1995). In situ hybridization and immunochemistry 14 studies demonstrated that dDA1 is widely expressed in the Drosophila CNS (Gotzes et al, 1994; Kim et al, 2003). Functional expression of the dDA1 gene in HEK, Sf9 and

COS-7 cells caused increased intracellular cAMP level in response to dopamine (Gotzes,

1994; Sugamori et al, 1995). The Drosophila dopamine receptor gene DAMB encoded a protein of 537 amino acids (Han et al, 1996). The predicted hydrophobic core of DAMB showed a high degree of sequence similarity (41% identity) to vertebrate dopamine receptors (Feng et al, 1996). In situ hybridization and immunochemistry analysis revealed that DAMB was highly expressed in the mushroom bodies, a learning and memory center in the fly brain (Han et al, 1996). Stimulation of the DAMB gene expressed in Xenopus oocytes by dopamine increased intracellular cAMP levels (Feng et al, 1996; Reale et al, 1997). Since activation of dDA1 and DAMB both activated adenylate cyclase, they were classified as stimulating D1-like dopamine receptors. The

Drosophila dopamine receptor DD2R gene encoded a receptor ranging in size from 461 to 606 amino acids. Sequence comparison with known vertebrate D2-like dopamine receptors showed a 29%~ 32% identity, higher than the 17%~21% identity to D1-like receptors. Functional expression of the DD2R gene in HEK293 cells indicated that dopamine addition induced a marked decrease in forskolin-induced cAMP level, suggesting DD2R belonged to the inhibitory D2-like dopamine receptor family (Hearn et al, 2002). The expression pattern of DD2R in the Drosophila CNS was not determined.

A common characteristic of these three Drosophila dopamine receptors is that they all showed pharmacological profiles that were different than mammalian dopamine receptors

(Gotzes, 1994; Feng et al, 1996; Han et al, 1996; Hearn et al, 2002; Kim et al, 2003). For example, in Sf9 cells, benzazepine SKF38393, which binds to vertebrate dopamine D1- 15 like receptors with high affinity, were not very efficient in stimulating dDA1 mediated increases of cAMP (Sugamori et al, 1995). In Xenopus oocyte expressing DAMB receptors, vertebrate dopamine D1-like receptor specific agonist R(+)-6-bromo-APB was ineffective in stimulating the increase of cAMP (Reale et al, 1997). In HEK293 cells transfected with DD2R receptor, vertebrate D2-like receptor agonist 7-(OH)-2- dipropylamino-7-hydroxy1, 2, 3, 4-tetrahydronaphthalene hydrobromide (7-OH-DPAT) was unable to stimulate DD2R mediated signaling (Hearn et al, 2002).

VI. Drosophila primary neuronal cultures as a model to study dopamine modulation

in the CNS

Despite the behavior studies of dopamine functions in Drosophila, little is known about the physiological role of dopamine in modulating synaptic transmission in the

Drosophila CNS in vivo. In this respect, the primary Drosophila neuron culture system used in this study provides a good opportunity to examine this question. Synaptic transmission can be directly monitored by recording postsynaptic currents using the whole cell patch clamp technique (Lee and O’Dowd, 1999). Pharmacological studies can be conducted in this culture system through the application of different drugs to neurons by either pressure puffing or bath perfusion. Studying dopamine’s effects on synaptic transmission in this culture system will shed light on dopamine’s potential functions in vivo. In Drosophila, electrophysiological recordings have demonstrated that acetylcholine is the predominant fast excitatory neurotransmitter (Breer and Sattelle, 1987) used by neurons grown in primary cell cultures (Lee and O’Dowd, 1999) while fast inhibitory 16 synaptic transmission is mainly mediated by GABA (Jackson et al, 1990; Mody et al,

1994; Hosie et al, 1997; Lee et al, 2003). Thus, I have focused on studying dopamine modulation on cholinergic and GABAergic synaptic transmissions in this culture system. 17

Specific Aims

Dopamine is an important neuromodulator regulating many brain functions. But,

due to the complexity of the vertebrate nervous system, how dopamine modulates other

neural activity in different brain areas has not been fully understood. Over the past

decades, Drosophila melanogaster has become a useful model animal for studying brain function. Thus, my research has mainly focused on studying the modulatory effects of

dopamine on synaptic transmission in Drosophila primary neuron cultures.

The first step of my research was preparing primary neuronal cultures derived

from Drosophila embryos. After 3 days of incubation (DIV), those cultured neurons were

examined electrophysiologically. Cholinergic and GABAergic post-synaptic currents

(PSCs) were recorded by whole-cell patch recording. Dopamine, its agonists and

antagonists were applied to the patched cells in order to pharmacologically characterize

dopamine modulation of synaptic transmissions. Neuronal cultures from mutant

Drosophila with deficiencies for specific dopamine receptors were also examined to

molecularly characterize the dopamine receptors modulating synaptic transmission. Since

cAMP-dependent intracellular signaling pathways were primary dopamine signaling

pathways in many organisms, the potential involvement of cAMP in dopamine

modulation was studied as the final step of this research.

18 Aim 1. To examine the modulatory actions of dopamine on Drosophila central synaptic transmission

Aim 2. To characterize the pharmacological properties of dopamine receptors which modulate excitatory cholinergic synaptic transmission

Aim 3. To characterize the pharmacological properties of dopamine receptors which modulate inhibitory GABAergic synaptic transmission

Aim 4. To characterize the molecular properties of dopamine receptors which modulate Drosophila synaptic transmission 19

Materials and Methods

I. Drosophila stocks

Two types of fly lines, wild-type fly w118 (a “Cantonized” white stock) and TH-

GFP fly (USA-GFP; TH-GAL4) (Friggi-Grelin et al, 2003) were used in this study. Fly

w118 was obtained from the Bloomington Drosophila stock center. TH-GAL4 and UAS-

GFP fly lines were obtained from Dr. S. Birman (Developmental Biology Institutes of

Marseille, France) and the Bloomington stock center.

The double transgenic line (UAS-GFP; TH-GAL4) was created using a standard

genetic cross method. Tyrosine hydroxylase(TH) is the enzyme that converts tyrosine to

L-Dopa; it is present in dopaminergic neurons.The tyrosine hydroxylase (TH) promoter

drives the expression of the GAL4 protein, which binds to UAS to stimulate the

expression of GFP. Thus the fluorescent marker GFP was only expressed in

dopaminergic neurons. For simplicity, the UAS-GFP; TH-GAL4 fly line has been

designated as TH-GFP in this study. Effects of dopamine and its pharmacological ligands

on synaptic transmission were studied in both wild-type and TH-GAL4 lines to evaluate

potential stock specific differences.

II. Dopamine receptor deficiency stocks

Based on the gene location on the cytogenetic map, the Drosophila dopamine receptor dDA1 deficiency line +/+; +/+; Df(3R)red31/TM6B, Tb[1] (Stock No.1917, Kim et al, 2003), DAMB deficiency line +/+; +/+; Df (3R)L127/TM6, Dp(3;1) B152 (Stock 20 No.3547) and DD2R deficiency line Df(1)mal5, y2ct 1 f 1/Dp(1;Y)y+mal106/C(1)RM,y 1 v

1 f 1mal 2 ; +/+; +/+ (Stock No.5975) were obtained from Bloomington stock center. They

were used to study the contribution of each dopamine receptor to the modulation of

synaptic transmission. FM7i, act-GFP/ C(1) Dx f; +/+; +/+ or +/+;+/+;TM3, Ser, Act-

GFP/Sb fly stock (which are kindly provided by Dr. S Tanda, Ohio University, Athens,

OH) was used as a balancer line to maintain dopamine receptor deficiency chromosome

as these deficiency homozygous lines are embryonic lethal. All fly stocks have been

maintained in the lab at room temperature. The genetic cross used to generate dopamine

receptor deficient neuronal cultures is explained in detail in III a, b, c.

III. Drosophila primary neuronal cultures

To prepare neuronal cultures, the fly stocks were allowed to lay eggs on an agar

plate covered with yeast paste. After four hours, these embryos were collected and

immersed in a 50% bleach solution for four minutes to dechorionate and then rinsed in

sterile water. The dechorinoated embryos were transferred to new Petri dishes and moved

to a laminar flow hood (ThermoForma, Forma Sci Inc, Waltham, MA). A sharp

micropipette was used to harvest neuroblast cells from the midgastrula stage of embryos.

Cells from two embryos (or only one embryo for the neuronal culture of deficiency lines,

see below) were plated on uncoated glass coverslips (Bellco Glass Inc, Vineland, NJ),

flooded with Drosophila culture medium DDM1 and maintained in an incubator supplied

with 5% carbon dioxide at 23℃ to 25℃ for up to 9 days as previously reported

(O’Dowd, 1999). The culture medium DDM1 used in this study was a mixture of high 21 glucose Ham’s F12/DME medium (Irvine Scientific, Santa Ana, CA), L-glutamine

(2.5mM, Irvine Scientific, Santa Ana, CA), HEPES (20mM) and five supplements:

100µM putrescine, 30nM sodium selenite, 20ng/ml progesterone, 100 µg/ml transferrin, and 50 µg/ml insulin (Calbiochem, San Diego, CA).

a. DAMB deficiency neuronal cultures The dopamine receptor DAMB deficiency chromosome Df(3R)L127 was maintained over balancer TM3, Ser, Act-GFP. Since homozygous DAMB receptor deficiency (Df(3R)L127/Df(3R)L127) and homozygous

TM3,Ser, Act-GFP balancer fly lines are lethal, a heterozygous fly line

Df(3R)L127/TM3, Ser, Act-GFP can be maintained for generations. Single embryo neuronal cultures were obtained from this fly line. According to Mendelian genetics, about 25% of the total neuronal cultures prepared should be GFP negative, corresponding to the homozygous DAMB receptor deficiency. The GFP negative phenotype was identified under a fluorescent microscope (Olympus IX71).

b. dDA1 deficiency neuronal cultures The dopamine receptor dDA1 deficiency chromosome Df(3R)red 31 was maintained over a TM3, Ser Act-GFP balancer. For some unknown reason, this Df(3R)red 31/TM3, Ser, Act-GFP line was sterile. Thus, the potential role of dDA1 receptors in dopamine modulation of synaptic transmission was not studied in this research. Different genetic cross strategies will be used in future experiments.

22 c. DD2R deficiency neuronal cultures The dopamine receptor DD2R deficiency chromosome Df(1)mal5,y2ct 1 f 1/Dp(1;Y)y+mal106 was maintained over balancer FM7i,

Act-GFP. Since homozygous DD2R deficiency (Df(1)mal5,y2ct 1 f 1/Dp(1;Y)y+mal106/

Df(1)mal5,y2ct 1 f 1/Dp(1;Y)y+mal106) and homozygous FM7i, Act-GFP balancer fly lines

are embryonic lethal, a heterozygous fly line Df(1)mal5,y2ct 1 f 1/Dp(1;Y)y+mal106/FM7i,

Act-GFP can be maintained for generations. Single embryo neuronal cultures were obtained from this fly line. According to Mendelian genetics, about 25% of the total single neuronal cultures prepared from this fly line should be GFP negative, corresponding to the homozygous DD2R receptor deficiency. The GFP negative phenotype was identified under a fluorescent microscope (Olympus IX71).

IV. Electrophysiology

Each coverslip containing Drosophila neuronal cultures was transferred into a recording chamber containing the following external solution (mM): 140 NaCl, 1 CaCl2,

4 MgCl2, 3 KCl, and 5 HEPES, pH 7.2. Postsynaptic currents were recorded with whole- cell pipettes (tip resistance 3~6 MΩ) filled with internal solutions containing the following ingredients (mM): 120 CsOH, 120 D-gluconic acid, 0.1 CaCl2, 2 MgCl2, 20

NaCl, 1.1 EGTA, and 10 HEPES, pH 7.2. The whole-cell pipettes were prepared by pulling a borosilicated glass pipette with electrode puller PP-830 (Narishige International

USA Inc, East Meadow, NY). The external and internal solutions were prepared fresh every month. An Axopatch 200B amplifier (Axon Instruments Inc, Union City, CA) was used to record currents. For recording cholinergic currents, the voltage was held at -45 23 mV, which is the reversal potential for GABAergic currents (see Figure 3, Lee et al,

2003). For GABAergic current recording, the voltage was held at 0mV, which is the reversal potential for cholinergic currents (see Figure 2, Lee and O’Dowd, 1999).

V. Pharmacology

Several dopamine receptor specific ligands were tested in this study. Dopamine, dopamine D1-like receptor agonist (±)-6-chloro-APB hydrobromide, dopamine D2-like receptor agonist (-)-quinpirole and dopamine D2-like receptor antagonist haloperidol were purchased from Sigma (Sigma-Aldrich Inc, St. Louis, MO). Dopamine D1-like receptor agonist SKF38393 hydrobromide and D1-like receptor antagonist SCH23390 were purchased from Tocris (Tocris Cookson Inc, Ellisville, MO). The solutions containing the desired concentration of the above drugs were prepared fresh from stock solutions daily. The stock solutions were kept at -20degrees Celsius as suggested by the manufacturers.

Dopamine and dopamine agonists were delivered to the patched neurons by pressure ejection (Picospritzer III, Parker Hannifin Corp, Fairfield, NJ) for duration of 30 seconds. Since the amount of pharmacological ligands delivered was very small compared to the large volume of the recording solution in the chamber, the ligands quickly diffused away. After each successful recording, 1 to 2 milliliters of recording solutions were added into the recording chamber to dilute these ligands. Normally, each neuronal culture was discarded after 30 minutes in the chamber or after two successful recordings. The dopamine D1-like receptor antagonist SCH23390 were added to the 24 recording chamber by a perfusion system with vacuum/solution flow control valves FR50

(Warner Instruments Inc, Hamden, CT) at a speed of 1~2 ml/min.

To study the effects of dopamine and dopamine agonists on synaptic transmission, synaptic currents of each patched neuron was continuously recorded for the duration of about 150 seconds. Each recorded trace represented a test conducted on one individual neuron. Each recording can be divided into three experimental phases. The first phase of recording was a 60 seconds recording of the synaptic currents before drug delivery. The second phase of recording was a 30 seconds recording when drug was ejected onto the cell. In order to examine the recovery from a drug effect, the last 60 seconds of recording represented the synaptic currents after the drug application was terminated.

To study the effects of dopamine antagonist SCH23390 alone on synaptic transmission, synaptic currents were recorded for 60 seconds when drug-free external solution was perfused into the recording chamber. Then to each patched neuron, the synaptic currents were recorded for 60 seconds during SCH23390 perfusion and for 60 seconds after wash with drug-free external solution.

VI. Data analysis

Individual postsynaptic currents were analyzed using the Minianalysis detection software (Synaptosoft, Decatur, GA) with amplitude threshold criteria for individual events of 5pA for GABAergic currents and 7.5pA for cholinergic currents (Lee and

O’Dowd, 1999; Lee et al, 2003). Only events with a fast rising and slowly decaying phase were accepted for further analysis. For each acquired trace of data, all events were 25 detected and the frequency was calculated every 5 seconds. If the frequency of postsynaptic currents (PSCs) declined continuously over 20 seconds before drug was applied, a rundown of PSCs was suspected and the data was not further analyzed. For the rest of each recording trace, only PSCs (before drug application) with a stable frequency longer than 20 seconds were used to calculate the control average frequency. This calculated control average frequency was then normalized to 100%. Each 5-second frequency of the entire recording was compared to the control average frequency and their percent frequency was calculated and plotted. Based on the observation, there was a

10 seconds delay time of the effects of dopamine and its agonists on cholinergic transmission and a 20 seconds delay time of the effects of dopamine and its agonists on

GABAergic transmission. So the percent frequency of the last 20 seconds of the 30 seconds in drug application process was used to calculate the average percent frequency during drug application. For GABAergic synaptic transmission experiments, the percent frequency of the last 10 seconds of drug application process and the first 10 seconds after drug application terminated were used to calculate the percent frequency during drug application. For each drug experiment, at least three individual analyzable recordings were required. The percent frequency during drug application from each test was grouped together to calculate the mean percent frequency during drug application. Since few cholinergic PSCs during drug application were seen in this study, the amplitude change of cholinergic PSCs by dopamine and its ligands were not analyzed. For each

GABAergic transmission recording data, however, the amplitude change of GABAergic

PSCs by dopamine or its ligands were analyzed. The control phase and drug application phase were classified the same way as in frequency analysis. The amplitudes of all events 26 in control phase were averaged to calculate the control amplitude. Then the amplitude of all events in drug application phase were averaged and compared to the control to calculate the mean amplitude during drug application. All data reported in this study was shown as mean ±standard error. Number of neurons used in each analysis was represented as n number. Each drug experiment was repeated in neurons from different cultures.

VII. Negative Controls

To investigate whether there were mechanical artifacts caused by either the puffing or perfusion process, it was necessary to have negative controls. Drosophila neurons were clamped as above, but only drug-free external solutions were applied to the cells by pressure puffing or the perfusion system. No changes were seen before and after applying external recording solutions (data not shown). 27

Results

I. Whole-cell recording in cultured Drosophila primary neurons

After 3 days incubation, the prepared Drosophila primary neuronal cultures were

fully differentiated. Some of these cells were well isolated while others were in clusters.

Extensive physical contacts by numerous processes were formed among cells (Figure

1A). Previous immunocytochemistry characterization of these cultures indicated that over

90% of the cells were neurons (O’Dowd, 1995). In the present study, once each culture

was transferred into the recording chamber, they were carefully screened under a phase

contrast microscope. Well-isolated neurons were patched by electrodes as illustrated in

the center of Figure 1A. Another glass pipette filled with pharmacological ligands was

placed near the patched neuron in pharmacological experiments. Those ligands were

ejected by the pressure control system Picospritzer III.

The whole cell patch configuration is illustrated in Figure 1B. The glass pipette

containing electrodes is tightly sealed (gigaohm seal) to the patched neuron. When

neurotransmitters are released from pre-synaptic terminals, any electrophysiological

activities of ion channels located on the post-synaptic neuronal membrane are detected.

As previously reported, the major excitatory post-synaptic currents (PSCs) in this culture system are cholinergic (Lee and O’Dowd, 1999) and the major inhibitory post-synaptic currents are GABAergic (Lee et al, 2003). In order to distinguish between cholinergic

PSCs (Figure 2A) and GABAergic PSCs (Figure 3A), an electrophysiological method was used. Cholinergic synaptic currents are kept very close to zero by holding neurons at 28 0mV, the reversal potential for cholinergic currents (Figure 2B, Lee and O’Dowd, 1999).

GABAergic currents were kept very close to zero by holding neurons at -45mV, the reversal potential of GABAergic PSCs (Figure 3B, Lee et al, 2003). In this study, about

60%~70% of the total neurons successfully patched had spontaneous excitatory postsynaptic cholinergic currents. About 10%~20% of the total patched neurons had inhibitory GABAergic PSCs. Some neurons showed both cholinergic and GABAergic

PSCs. The cultures used in this study were 3 to 9 days old. The incidence and frequency of spontaneous cholinergic and GABAergic PSCs were stable in this stage (Lee and

O’Dowd, 1999). 29 A B

25 μm

Figure 1. Whole-cell recording of synaptic currents in Drosophila primary neuronal culture. (A) Bright field image of Drosophila primary neuronal culture. Extensive physical contacts were formed between neurons in culture. The patch pipette was located on a neuron as shown in the figure and formed a tight seal with one well isolated neuron. Scale bar = 25 μm. (B) Whole-cell patch configuration. The electrode (light yellow) is inserted into a glass pipette (orange), which tightly seals with the membrane of a post- synaptic neuron (green). The neurotransmitters (NT, yellow) are released from pre- synaptic neuron (white). The activity of ligand-gated ion channels localized on the post- synaptic neuron (red) can be detected by the electrode (which is connected to a patch amplifier).

30 A B

Figure 2. Spontaneous cholinergic post-synaptic currents (PSCs) recorded in cultured embryonic neurons. (A) One sample recording of cholinergic PSCs in a neuron held at - 45mV. (B) The amplitude of cholinergic PSCs plotted as a function of the holding potential (adapted from Lee and O’Dowd, 1999). A linear regression fit (orange line) indicates that the reversal potential of acetylcholine receptor is close to 0mV.

A B

Figure 3. Spontaneous GABAergic PSCs recorded in cultured embryonic neurons. (A) One sample recording of GABAergic PSCs in a neuron held at 0mV. (B) The amplitude of GABAergic PSCs plotted as a function of the holding potential (adapted from Lee et al, 2003). A linear regression fit (orange line) indicates that the reversal potential of GABA receptor is close to -45mV.

31 II. Pharmacological characterization of dopamine receptors modulating cholinergic

currents

500µM dopamine application suppressed cholinergic synaptic transmission

(Figure 4A). Further analysis showed that the frequency of detectable cholinergic PSCs was reduced to 6.1%±2.6% (Figure 4B). This reduction was reversible since cholinergic

PSCs quickly recovered after drug application. Higher concentrations of dopamine showed a greater suppression effect (Figure 5). 1μM dopamine had no suppression effect on the frequency of cholinergic PSCs (102.3%±15.2% compared to control). 10μM dopamine suppressed the frequency of cholinergic PSCs down to 51.7%±8.3%. 100μM dopamine decreased the frequency of cholinergic PSCs down to 59.4%±7.5%. 1mM dopamine decreased the frequency down to 3.2%±0.9%. In order to determine whether dopamine efficacy was fly strain-dependent, the effects of 100µM dopamine on cholinergic PSCs were studied in two different Drosophila strains (TH-GFP and wild- type). The results indicated that both types of fly neurons had similar inhibition of cholinergic transmission by dopamine (60.4%±12.7% and 58.6%±9.3% compared to the control, respectively, Figure 6). Thereafter, the data from these two strains were pooled together.

Figure 4. Spontaneous cholinergic PSCs were down-regulated by the focal application of 500µM dopamine. 500µM dopamine was puffed onto a patched neuron for 30 seconds. (A) Spontaneous cholinergic PSCs were suppressed by dopamine. The cholinergic PSCs quickly recovered once drug application was terminated. Cholinergic PSCs on an expanded time scale are shown below the whole recording trace. (B) 500μM dopamine application greatly reduced the frequency of cholinergic PSCs (n=3).

Figure 5. Effects of different concentrations of dopamine on the frequency of cholinergic PSCs.

Figure 6. Effects of 100µM dopamine application on cultured neurons prepared from two different strains. 100µM dopamine was puffed onto a patched neuron for 30 seconds. Three neurons from each fly stock (TH-GFP or wild-type) were tested. The averaged percent frequency during drug application phase was very similar in both strains, demonstrating that the dopamine effect was not variable between different strains.

Both mammalian dopamine D1-like receptor agonists SKF38393 (10µM) and (±)-

6-chloro-APB (10µM) could mimic the dopamine effect (Figures 7 and 8). Once

SKF38393 or 6-chloro-APB was applied, fewer cholinergic PSCs were detected (Figure

7A, 8A). SKF38393 suppressed cholinergic current frequency down to 1.9%±1.2% of the

control (Figure 7B). (±)-6-chloro-APB suppressed cholinergic PSC frequency down to

23.0%±7.0% of the control (Figure 8B). Similar to dopamine, SKF38393 and 6-chloro- 35 APB effects were both reversible. However, the recovery of cholinergic PSCs was much slower in SKF38393 experiments. This phenomenon might due to a longer disassociation time between SKF38393 and its receptors. Or these receptors were much more sensitive to SKF38393 than to dopamine and 6-chloro-APB. Although the local concentration of

SKF38393 declined rapidly after SKF38393 ejection was terminated, the residual

SKF38393 were still able to activate enough receptors to maintain the effect. (-)- quinpirole (10µM), a mammalian dopamine D2-like receptor agonist, caused no obvious changes in cholinergic PSCs (Figure 9). These results indicate that in the Drosophila

CNS, suppression of cholinergic synaptic transmission by dopamine is mediated by receptors with pharmacology similar to vertebrate D1-like receptors.

Figure 7. Effects of 10µM SKF38393 application on the cholinergic PSCs. SKF38393 was puffed onto a patched neuron for 30 seconds. (A) Spontaneous cholinergic PSCs were suppressed by SKF38393. Currents recovered much more slowly compared to after dopamine application. Cholinergic PSCs are illustrated on an expanded time scale below the whole recording trace. (B) SKF38393 application greatly reduced the frequency of cholinergic PSCs (n=3).

Figure 8. Effects of 10μM (±)-6-chloro-APB application on cholinergic PSCs. 6- chloro-APB was puffed onto a patched neuron for 30 seconds. (A) Spontaneous cholinergic PSCs were suppressed by 6-chloro-APB. The current recovery was faster than seen in SKF38393 experiments. Cholinergic PSCs are illustrated on an expanded time scale below the whole recording trace. (B) 6-chloro-APB application greatly reduced the frequency of cholinergic PSCs (n=6).

Figure 9. Effects of 10μM (-)-quinpirole application on cholinergic PSCs. (-)- Quinpirole was puffed onto the patched neuron for 30 seconds. (A) Spontaneous cholinergic PSCs were not affected by (-)-quinpirole. Cholinergic PSCs are illustrated on an expanded time scale below the entire recording. (B) (-)-quinpirole application showed no obvious effect on the frequency of cholinergic PSCs (n=6).

One unexpected result was that the dopamine D1-like receptor antagonist

SCH23390 (1~20µM) showed an agonistic effect. Cholinergic PSCs were recorded for one minute while a drug-free external solution was perfused into the recording chamber.

20 μM SCH23390 was then perfused into the recording chamber. After SCH23390 inside the chamber reached the desired concentration, cholinergic PSCs were recorded for one 39 minute. Then drug-free external recording solution was perfused into the chamber to wash out the SCH23390. Cholinergic PSCs were recorded for one minute after recovery

(Figure 10A). Further analysis showed that the frequency of cholinergic PSCs decreased down to 61.4%±6.8% by SCH23390 perfusion (Figure 10B). After washing, about

84.5%±22.5% of the currents was recovered (Figure 10B). The most likely reason for the partial recovery is that the long process of the test caused a rundown of cholinergic PCSs.

Studies from C. elegans have also reported agonistic effects of SCH23390 (Sanyal et al,

2004; Suo et al, 2004).

The effects of various pharmacological ligands on the frequency of cholinergic synaptic transmission are summarized in figure 11.

Figure 10. Effects of 20μM SCH23390 on cholinergic PSCs. (A) Spontaneous cholinergic PSCs recorded during drug-free external solution perfusion, SCH23390 perfusion and wash stages. The cholinergic currents were suppressed by SCH23390 perfusion. They partially recovered when SCH23390 was washed out. Cholinergic PSCs are illustrated on an expanded time scale for each stage. (B) SCH23390 perfusion reduced the frequency of cholinergic PSCs, which partially recovered after washing with control external solution (n=3).

Figure 11. Modulation of cholinergic synaptic transmission by dopamine drugs. Dopamine: 500μM; SKF38393: 10μM; 6-chloro-APB: 10μM; quinpirole: 10μM. All ligands were puffed onto the neurons for 30 seconds.

III. Pharmacological characterization of dopamine modulation of GABAergic

currents

Since 500μM dopamine application greatly affected cholinergic synaptic

transmission, the same concentration of dopamine was locally applied to neurons with spontaneous GABAergic currents for 30 seconds. Data analysis indicated that both the frequency and amplitude of GABAergic currents were enhanced (148.2%±33.7% and 42 119.9%±10.7%, respectively) by dopamine (Figure 12). A mammalian dopamine D2-like receptor agonist, (-)-quinpirole (10µM), mimicked the dopamine effect (Figure 13). The frequency of GABAergic currents increased up to 368.1%±52.4% and the amplitude increased up to 143.0%±39.1%. This greater increase indicated that (-)-quinpirole is more potent than dopamine in stimulating GABAergic transmission. The enhancement effects of dopamine and quinpirole were reversible. Subsequent experiments showed that the quinpirole effect was abolished in the presence of dopamine D2-like receptor antagonist haloperidol (10µM, Figure 14). In the presence of haloperidol, the frequency of

GABAergic PSCs during (-)-quinpirole application was 88.2%±11.0%. The amplitude of

GABAergic PSCs during (-)-quinpirole application was 101.0%±6.7%. In this experiment, haloperidol was not perfused into the recording chamber. Instead, it was mixed with external solutions directly. Then the mixed solution was added in the recording chamber prior to transferring cultures into it. The effect of haloperidol alone on

GABAergic PSCs was not investigated since the rundown of the GABAergic PSCs over time was severe. Application of mammalian dopamine D1-like receptor agonist

SKF38393 showed no obvious influence on GABAergic currents (Figure 15). These results demonstrate that dopamine enhances GABAergic synaptic transmission in the

Drosophila CNS, most likely through dopamine D2-like receptors.

The effects of pharmacological ligands on the frequency and amplitude of

GABAergic PSCs are summarized in Figure 16.

Figure 12. Spontaneous GABAergic PSCs were up-regulated by the focal application of 500µM dopamine. 500µM dopamine was puffed onto a patched neuron for 30 seconds. (A) Spontaneous GABAergic PSCs were enhanced by dopamine. This effect was reversible. GABAergic PSCs are illustrated as an expanded time scale under the recording. (B) 500μM dopamine application increased the frequency of GABAergic PSCs (n=3).

Figure 13. Effects of 10μM (-)-quinpirole on GABAergic PSCs. (-)-quinpirole was puffed onto a patched neuron for 30 seconds. (A) Spontaneous GABAergic PSCs were enhanced by (-)-quinpirole. GABAergic PSCs are illustrated in an expanded time scale below the entire recording. (B) (-)-quinpirole application greatly increased the frequency of cholinergic PSCs (n=3).

Figure 14. Effects of 10μM (-)-quinpirole on GABAergic PSCs in the presence of 10μM haloperidol. Haloperidol was present in the external recording solutions. Quinpirole was puffed onto a patched neuron for 30 seconds. (A) Spontaneous GABAergic PSCs were not significantly affected by quinpirole in the presence of haloperidol, a D2-like receptor antagonist. GABAergic PSCs are illustrated on an expanded time scale. (B) (-)-quinpirole application did not cause any increase in the frequency of GABAergic PSCs when cultured neurons were pre-incubated with haloperidol (n=4).

Figure 15. Effects of 10μM SKF38393 on GABAergic PSCs. SKF38393 was puffed onto patched neurons for 30 seconds. (A) Spontaneous GABAergic PSCs were not affected by SKF38393. GABAergic PSCs are illustrated on an expanded time scale below the entire recording. (B) SKF38393 application did not change the frequency of cholinergic PSCs (n=4).

Figure 16. Modulation of GABAergic synaptic transmission by various dopamine durgs. Effects of different pharmacological ligands on the frequency of GABAergic PSCs (A) and on the amplitude of GABAergic PSCs (B). Dopamine: 500μM; quinpirole: 10μM; haloperidol: 10μM; SKF38393 10μM. Dopamine, quinpirole and SKF38393 were puffed onto neurons for 30 seconds. Haloperidol was mixed in the solutions in the recording chamber before cultures were transferred into it.

IV. Molecular characterization of dopamine modulation of cholinergic transmission

a. Dopamine modulation in the DAMB deficiency line By using genetic crosses, dopamine receptor DAMB homozygous deficiency neuronal cultures were obtained

(Figure 17). These homozygous deficiency cultures were GFP negative (Figure 17A, B).

Non deficiency neuronal cultures obtained from the same cross were GFP positive

(Figure 17C, D). The GFP negative cultures accounts for 22.9% of the total neuronal cultures prepared for this part of the study. The GFP positive culture accounts for 77.1% of the total cultures prepared. The ratio of GFP negative versus GFP positive cultures were close to the predicted 25% to 75% ratio. 10μM SKF38393 was applied to the homozygous DAMB deficiency neuronal cultures, by the same method as in previous 48 experiments. The results indicated that the effects of SKF38393 on cholinergic PSCs were not affected by the DAMB receptor deficiency (Figure 18A). The frequency of cholinergic PSCs decreased to 6.5%±3.9% (Figure 18B), which was not significantly different from the decrease observed in previous tests in TH-GFP and wild type flies

(1.8%±1.2% compared to the control). This indicated that DAMB is not a likely candidate receptor mediating dopamine modulation of cholinergic synaptic transmission.

Unfortunately, I was unable to obtain dopamine dDA1 receptor homozygous deficiency neuronal culture. Three female virgin Df(3R)red31/TM3, Ser, Act-GFP were crossed with five male Df(3R)red31/TM3, Ser, Act-GFP. But no offspring were obtained from the cross. Repeating the cross did not yield any progeny. Generally, a single pair of flies was sufficient to generate progeny. The reason for the sterility of Df(3R)red31/TM3,

Ser, Act-GFP flies were unknown.

A B

50µm

C D

50μm

Figure 17. Single embryo neuronal cultures prepared from the DAMB deficiency line. (A) Bright field image of a GFP-negative culture. Scale bar = 50μM. (B) Fluorescent image of the same view illustrated in A. The GFP negative culture contained homozygous DAMB deficiency neurons. (C) Bright field image of a GFP positive neuronal culture. Scale bar = 50μM. (D) Fluorescent image of the same field of view illustrated in C. GFP-positive signals were observed.

Figure 18. Effects of 10μM SKF38393 on cholinergic PSCs in homozygous DAMB deficiency neurons. SKF38393 was puffed onto a patched cell for 30 seconds. (A) Spontaneous cholinergic PSCs were greatly decreased by SKF38393. Cholinergic PSCs are illustrated on an expanded time scale under the entire recording trace. (B) SKF38393 application decreased the frequency of cholinergic PSCs (n=4).

b. Dopamine modulation in the DD2R deficiency line Preliminary results from dopamine DD2R receptor homozygous deficiency neuronal cultures indicated that the (-)- quinpirole enhancement effect on GABAergic synaptic transmission was abolished (data not shown), indicating that enhancement of GABAergic currents by dopamine is likely mediated via DD2R receptors. But the DD2R homozygous deficiency neurons were not 51 very healthy based on their morphology, making it difficult to conduct whole-cell patch recordings. Lack genes required for normal neuron development might account for this phenomenon since more than one gene was deleted in DD2R deficiency chromosomes.

V. Supplementary data-intracellular signaling pathways

Studies of Drosophila dopamine receptors in heterologous expression systems indicated that stimulation of dDA1, DAMB receptors by dopamine led to an increase of intracellular cAMP level while stimulation of DD2R receptor by dopamine led to a decrease of intracellular cAMP level (Gotzes, 1994; Sugamori et al, 1995; Feng et al,

1996; Reale et al, 1997; Hearn et al, 2002). In order to study whether dopamine modulation of synaptic transmission was mediated by cAMP-dependent intracellular signaling pathways, the primary dopamine signaling, 20μM forskolin (a membrane permeable adenylate cyclase activator) was puffed onto the patched neurons. The results showed that forskolin application led to an increase in the frequency of cholinergic PSCs

(135.8%±19.8%, Figure 19). In contrast, forskolin application caused a decrease of

GABAergic synaptic transmission (Figure 20). The frequency and amplitude of

GABAergic PSCs was 69.6%±9.6% and 74.1%±9.5% during forskolin application. The forskolin effects on both cholinergic and GABAergic transmission were in contrast to the effects of dopamine. Those results indicated that dopamine modulation of cholinergic and

GABAergic transmission is not simply and directly dependent on the increase of intracellular cAMP level.

Figure 19. The effects of 20μM forskolin on cholinergic PSCs. (A) Forskolin was puffed onto the patched neuron for 60 seconds. Cholinergic transmission was enhanced by forskolin application. (B) Changes of frequencies of cholinergic PSCs after application of 20μM forksolin (n=5) or 500μM dopamine (n=3).

Figure 20. The effects of 20μM forskolin on GABAergic PSCs. Forskolin was puffed onto the patched neuron for 60 seconds. (A) GABAergic transmission was suppressed by forskolin application. (B) Changes of the frequencies and the amplitudes of GABAergic PSCs by the application of 20μM forksolin (n=5) or 500μM dopamine (n=3). 54

Discussion

Pharmacological properties of dopamine modulation of synaptic transmission

The results presented in this study indicated that dopamine played an important

role in modulating synaptic transmission in the Drosophila CNS. Drosophila excitatory

cholinergic synaptic transmission was down-regulated by exogenous dopamine.

Mammalian D1-like receptor specific agonists SKF38393 and (±)-6-chloro-APB also down-regulated cholinergic synaptic transmission. In contrast, a mammalian D2-like receptor specific agonist, (-)-quinpirole, had no significant influence on cholinergic synaptic transmission. These data demonstrated that the effects of dopamine on cholinergic synaptic transmission were mediated by Drosophila D1-like receptors.

Using a similar research strategy, I found that Drosophila inhibitory GABAergic synaptic transmission was up-regulated by exogenous dopamine. This dopamine effect on

GABAergic synaptic transmission was mimicked by (-)-quinpirole, but not by

SKF38393. Subsequent experiments showed that the effect of (-)-quinpirole on

GABAergic synaptic transmission was blocked in the presence of a mammalian D2-like receptor specific antagonist, haloperidol. These results indicated that the up-regulation by dopamine on GABAergic transmission is mediated by Drosophila dopamine D2-like receptors. The different delay time of dopamine effect on cholinergic and GABAergic synaptic transmission also indicate that they were mediated by different signaling pathways. To the best of my knowledge, this is the first report showing that dopamine distinctively modulates excitatory cholinergic and inhibitory GABAergic synaptic 55 transmission in Drosophila. Furthermore, dopamine D1- and D2-like receptors in

Drosophila neurons have been pharmacologically characterized in this study. Given the power of sophisticated Drosophila genetics, primary neuronal culture will be an excellent model to study molecular and cellular mechanisms underlying dopamine modulation and behaviors in the future.

Specificity of pharmacological ligands

The pharmacological ligands used in this study were designed to target specific mammalian dopamine receptor subtypes. Previous studies of Drosophila dopamine receptors using heterologous expression systems indicated quite distinct pharmacological profiles of these receptors compared with their mammalian counterparts (Mustard et al,

2005). For example, (-)-quinpirole had been reported to have no effect in activating

Drosophila dopamine DD2R receptors (Hearn et al, 2002). Mammalian dopamine receptor non-specific agonist 6,7-ADTN was more potent in inducing an intracellular cAMP increase than SKF3893 in dDA1 receptor expressing cells (Sugamori et al, 1995).

However, the results of the present study indicated that pharmacological profiles of

Drosophila dopamine receptors are generally similar to those of mammals, since the receptor specificity of agonists SKF38393, 6-chloro-APB, (-)-quinpirole and antagonist haloperidol appeared to be conserved in Drosophila. One difference found in this study was that SCH23390, a mammalian D1-like receptor antagonist, showed agonist effects.

Similar results were also reported in C. elegans (Sanyal et al, 2004; Suo et al, 2004). The discrepancy between this study and previous reports may result from the different 56 expression systems used. In heterologous expression systems, receptor proteins may function differently from in vivo situations. For example, improper post-translational modification may lead to protein misfolding (Rai and Padh, 2001). Second, the binding affinity of agonists may be altered if there are not sufficient G proteins to couple with the expressed GPCRs (Sarramegana et al, 2003). The primary neuronal culture system used in this study more closely resembled the environment in vivo than do the previously used heterologous expression systems. Thus receptors are expected to have more normal functions in this neuronal culture system.

Cellular location of dopamine receptors

Dopamine modulation of synaptic transmission could be mediated by pre-synaptic receptors, post-synaptic receptors or both. Modulation by pre-synaptic receptors would affect the amount of neurotransmitter released from pre-synaptic terminal, which in turn could cause the change in the frequency of PSCs observed in my study. Modulation by post-synaptic receptors can change either the number of acetylcholine/GABA receptors or receptor sensitivity. These changes would result in changes mainly in the synaptic current amplitude. In the present study, 500µM exogenous dopamine application decreased cholinergic PSC frequency down to 6.1%±2.6%. The mammalian dopamine

D1-like receptor specific agonist SKF38393 suppressed the frequency of cholinergic

PSCs down to 1.9%±1.2%. 6-chloro-APB, another mammalian dopamine D1-like receptor specific agonist, suppressed the frequency of cholinerigic PSCs to 23.0%±

7.0%. However, the number of PSCs detected during drug application was too few to 57 accurately calculate the change in the amplitude. Thus, possible changes in cholinergic

PSCs amplitude were not further analyzed. Whether the suppression of cholinergic transmission by dopamine is due to a pre or post-synaptic effect was unknown.

Determination of the location of dopamine receptors would rely on further pharmacological experiments.

In the study of GABAegic transmission, the application of 500µM dopamine increased the frequency of GABAergic PSCs up to148.2%±33.7% and the amplitude to119.9%±10.7%. A dopamine D2-like receptor agonist (-)-quinpirole increased the frequency of GABAergic PSCs to 368.1%±52.4% and the amplitude to143.0%±39.1%.

I hypothesize that these changes in the frequency and amplitude of GABAergic currents are presynaptic in origin. A change in the frequency of post-synaptic currents generally originates from an altered probability of neurotransmitter release at the pre-synaptic terminal. Although it can be due to changes in the receptor sensitivity, my results favor pre-synaptic alteration because the frequency change (368.1%±52.4% by quinpirole) was much more drastic than that of the amplitude (143.0%±39.1%). However, further pharmacological experiments are needed to determine the location of dopamine receptors since no antibodies to these receptors are currently available.

Molecular properties of dopamine modulation of synaptic transmission

In order to confirm our pharmacological results, I used a genetic approach to study dopamine modulation of synaptic transmission. Because Drosophila dopamine receptor mutants have not yet been reported, I used deficiency stocks to remove the 58 receptor gene. DAMB deficiency neuronal cultures showed SKF38393 effects on the frequency of cholinergic PSCs that were similar to those in non-deficiency flies

(6.5%±3.9% and 1.9%±1.2%), indicating DAMB is not likely to be the receptor mediating dopamine effects on cholinergic transmission. Furthermore, since the patched neurons were randomly chosen and always showed dopamine responsiveness, we hypothesized that the receptors mediating dopamine modulation would be widely expressed in the Drosophila central nervous system. Based on immunostaining results,

DAMB is mainly expressed in mushroom bodies (Han et al, 1996) while dDA1 is more wildly expressed (Kim et al, 2003). Taken together, I hypothesize that the dopamine modulation of cholinergic synaptic transmission is mediated by dDA1 receptors.

Unfortunately, I am currently unable to test this hypothesis in dDA1 deficiency neuronal culture because Df(3R)red31/TM3, Ser, Act-GFP flies are sterile. In future experiments, different dDA1 deficiency and different fluorescent marked balancer stocks will be tested.

As to the DD2R deficiency tests, my initial results showed that the stimulatory effects of (-)-quinpirole on GABAergic transmission was abolished in this deficiency line

(data not shown). It will be very interesting to examine the distribution of DD2R in the

CNS by using DD2R antibodies (when available) or in situ hybridization. If DD2R is exclusively expressed in GABAergic neurons, it would indicate that dopamine modulation of GABAergic transmission is pre-synaptic.

59 Potential physiological functions

The involvement of dopamine in modulating synaptic transmission has been investigated in a variety of animals. For example, dopamine depressed the release of glutamate in the rat nucleus accumbens, causing a decrease of the amplitude of EPSCs

(Harvey and Lacey, 1996; Chergui and Lacey, 1999). In the prefrontal cortex of ferrets, dopamine reduced excitatory neurotransmission in pyramidal cells (Gao et al, 2001). In avian basal ganglia, dopamine also suppressed glutamatergic synaptic transmission (Ding et al, 2003). The enhancement of GABAergic synaptic transmission has been reported previously in vertebrates as well (Wang et al, 2004; Brunig et al, 1999). The physiological roles of dopamine modulation of synaptic transmission include roles in learning and memory formation and involvement in the prefrontal dysfunction of schizophrenia (Brunig et al, 1999; Goldman-Rakic et al, 2004; Wang and Goldman-

Rakic, 2004; Wang et al, 2004). This present study suggests that dopamine modulation of synaptic transmission also exists in the Drosophila central nervous system. The dopamine effects are quite global since randomly patched neurons all showed dopamine responsiveness. Although dopaminergic neurons only count for a small percent of the total neurons in Drosophila CNS, this global effect is not totally unexpected since

Drosophila dopaminergic neurons project to major brain areas such as the central complex and mushroom bodies. In vertebrate, dopamine as a neurohormone can also circulate in bloodstream and regulate different cell functions (Widmaier et al, 2002). In

Drosophila, cholinergic transmission is excitatory and GABAergic transmission is inhibitory, dopamine in Drosophila central nervous system should decrease overall neuronal excitability. This prediction is consistent with a recent report showing 60 hyperexcitable behavior in adult flies when dopamine release was inhibited in vivo

(Friggi-Grelin et al, 2003).

Conclusion

The present study reports that dopamine modulates Drosophila cholinergic and

GABAergic synaptic transmission. The down-regulation of cholinergic transmission was mediated by dopamine D1-like receptors (possibly dDA1 receptors) while the up- regulation of GABAergic transmission was mediated by dopamine D2-like receptors

(possibly DD2R receptors). The overall effect of dopamine in the Drosophila CNS is predicted to be a decrease of neuronal excitability. Previous pharmacological research on invertebrate dopamine receptor ligands was mainly conducted in heterologous systems.

The Drosophila neuronal cultures used in this study are more physiologically intact and thus better represent the dopamine system in vivo. Therefore, Drosophila neuronal cultures expressing functional dopamine receptors will be excellent models to study molecular and cellular mechanisms underlying dopamine modulation of a variety of

Drosophila behaviors. Furthermore, the high degree of conservation between vertebrates and invertebrates in terms of the basic mechanisms important in neuron modulation, suggest that my study in Drosophila is likely to reveal molecular mechanisms involved in human dopamine signaling as well. 61

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Appendix

Intracellular signaling pathways mediated by Drosophila dopamine receptors

The primary intracellular signaling pathways activated by dopamine receptors in

vertebrate and invertebrate are cAMP-dependent (Neve et al, 2004). Thus, in the present

study, I studied the effects of forskolin, a membrane permeable adenylate cyclase

activator, on the cholinergic and GABAergic PSCs. The results showed that application

of 20μM forskolin led to an increase in the frequency of cholinergic PSCs and a decrease

of the GABAergic PSC frequency. These were in sharp contrast with the dopamine

effects, indicating that dopamine modulation of synaptic transmission did not simply and

directly depend on the increase of intracellular cAMP level. cAMP independent signaling pathways have been reported in mammalian dopamine D1- and D2-like receptors. For

example, in the rat nucleus accumbens, dopamine suppression of glutamate excitatory postsynaptic currents (EPSCs) was unaffected by forskolin application (Harvey and

Lacey, 1996). Presynaptic regulation of excitatory neurotransmission in prefrontal circuits by dopamine was cAMP-independent (Gao et al, 2000). Several potential pathways have been proposed as alternatives to cAMP dependent signaling (Neve et al,

2004; Lee et al, 2005). First, protein kinase C or other cAMP-independent protein kinases

could be directly activated by G proteins associated with dopamine receptors. Second,

phospholipase C mediated regulation of intracellular calcium levels might count for some

dopamine D1 receptor modulated behaviors. Third, dopamine D2-like receptor signaling through receptor heteromerization has been reported. Further experiments are needed to identify the intracellular signaling pathways involved in Drosophila. In conclusion, the 70 forskolin experiment results indicated that the dopamine modulation of cholinergic and

GABAergic synaptic transmission observed in this study was not simply mediated by the increase of cAMP level. Secondly, this study also reported for the first time of forskolin effect on Drosophila central synaptic transmission.