Adenosinergic and Gabaergic Modulation of Neuronal Activity in The

Home , Bicuculline

Adenosinergic and GABAergic modulation of neuronal activity in the

hypoxia-tolerant pond snail Lymnaea stagnalis

Aqsa Malik

A thesis submitted in conformity with the requirements

For the degree of Master of Science

Graduate Department of Cell and Systems Biology

University of Toronto

Adenosinergic and GABAergic modulation of neuronal activity in the hypoxia-tolerant

pond snail Lymnaea stagnalis

Aqsa Malik Master of Science (2010) Department of Cell and Systems Biology University of Toronto

ABSTRACT

The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABAA receptor because of sensitivity to GABAA receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity.

ii ACKNOWLEDGEMENTS

This thesis is the result of the wonderful company, guidance, and inspiration that I received from my mentors, collaborators, and friends during the last two years of my graduate education. I would like to extend my gratitude to the following people, without whom the completion of my degree would not have been possible.

Firstly, I am indebted to my mentor and supervisor, Dr. Leslie Buck. His enthusiastic disposition and passion for science shaped the development of my positive perspective towards scientific research. His experimental and academic support was instrumental in my success as a graduate student. I am also grateful for his approachable and friendly mentoring approach.

I would also like to thank my committee advisors Dr. Melanie Woodin and Dr. Zhong-

Ping Feng for their invaluable technical and conceptual help throughout the course of my thesis project. I am thankful for their time, criticism, and attention to my work.

I am also very appreciative for having the opportunity to work amongst such bright and welcoming colleagues and peers. I am grateful to Dave Hogg and Matthew Pamenter for their engaging discussions and for their assistance in data analysis. I am thankful to George Zivkovic for his unrelenting technical support and encouragement. I owe special gratitude and recognition to Brooke Acton for being a source of friendship, wisdom, and laughter. I would also like to thank Ian Buglass for helping me navigate through the administrative aspects of graduate life.

I am thankful to my brothers and sister for their generosity and kindness and for inspiring me to live a well-balanced fulfilling life. From the three of you I learned the meaning of “If you want to go fast, go alone. If you want to go far, go together.” Finally, and most importantly, I am deeply grateful to my parents for teaching me the value and power of education, encouraging me to have confidence in my abilities, and supporting my academic pursuits and career goals.

iii TABLE OF CONTENTS ABSTRACT ...... ii ACNOWLEDGEMENTS...... iii TABLE OF CONTENTS ...... iv LIST OF TABLES AND FIGURES ...... vii ABBREVIATIONS ...... viii

CHAPTER 1: INTRODUCTION ...... 1

1.1. Anaerobic metabolism and evolution of O2 ...... 1

1.2. Mammalian neurons—hypoxia-sensitive...... 2

1.3. Mechanisms of hypoxia tolerance...... 3

1.3.1. Metabolic Arrest ...... 3

1.3.2. Channel Arrest ...... 4

1.4. Adenosine-mediated neuroprotection ...... 5

1.4.1. Adenosine structure, receptors & function ...... 5

1.4.2. Adenosine and ischemic preconditioning ...... 7

1.4.3. Role of adenosine in hypoxia tolerance—vertebrates ...... 8

1.4.4. Role of adenosine in hypoxia tolerance—invertebrates ...... 11

1.5. GABA-mediated neuroprotection ...... 14

1.5.1. GABA—primitive developmental signal ...... 14

1.5.2. GABA receptors ...... 15

1.5.3. Polarity of GABA transmission and cation-chloride cotransporters ...... 16

1.5.4. GABA and ischemic preconditioning ...... 18

1.5.5. Role of GABA in hypoxia tolerance ...... 19

1.6. Lymnaea stagnalis as an invertebrate model of hypoxia tolerance ...... 20

1.7. Rationale and Hypotheses ...... 22

CHAPTER 2: METHODS ...... 24

2.1. Animals ...... 24

2.2. Anoxia tolerance ...... 24

2.3. Solutions and dissection ...... 24

2.4. Electrophysiology ...... 26

2.5. Fluo-4 intracellular Ca2+ imaging ...... 26

2.6. Chemicals ...... 27

2.7. Statistical Analysis ...... 29

CHAPTER 3: RESULTS ...... 30 3.1. Anoxia tolerance ...... 30

3.2. Adenosinergic modulation of neuronal activity ...... 31

3.3. GABAergic modulation of neuronal activity ...... 39

CHAPTER 4: DISCUSSION ...... 46 4.1. Anoxia tolerance ...... 46

4.2. Modulation of neuronal activity by adenosine ...... 46

4.2.1. Summary of findings...... 46

4.2.2. Mechanisms of adenosine-mediated depression ...... 47

4.2.3. Seasonal differences in adenosinergic transmission ...... 48

4.3. Modulation of neuronal activity by GABA ...... 49

4.3.1. Summary of findings...... 49

4.3.2. Regulation of cation-chloride cotransporter function ...... 52

4.3.3. Seasonal differences in GABAergic neurotransmission ...... 54

4.3.4. Physiological significance of excitatory GABA ...... 55

4.3.5. Conclusions and future directions ...... 56

CHAPTER 5: REFERENCES ...... 58

vi LIST OF TABLES AND FIGURES CHAPTER 1: INTRODUCTION Table 1-1: Concentration and effects of adenosine on various invertebrate preparations 11

CHAPTER 2: MATERIALS AND METHODS Table 2-1: Cluster F neurons of L. stagnalis pedal ganglia ...... 25

Figure 2-2: Effects of DMSO on AP frequency in cluster F neurons ...... 28

CHAPTER 3: RESULTS Table 3-1: Anoxic tolerance of L. stagnalis ...... 30

Table 3-2: Effect of adenosine on AP frequency measured in summer animals ...... 33

Figure 3-1: AP frequency remains stable under control conditions ...... 34

Figure 3-2: Effects of A1 receptor activation or antagonism on AP frequency ...... 35

Figure 3-3: A1 receptor-mediated decrease in AP frequency ...... 36

Table 3-3: Effects of adenosine, CPA and DPCPX on membrane potential of neurons from animals obtained during the winter months ...... 36

Figure 3-4: Concentration-response curve for adenosine effect on AP frequency ...... 37

Figure 3-5: Effects of adenosine on intracellular calcium concentrations in cluster F neurons ...... 38

Figure 3-6: Effects of GABAA receptor activation or antagonism on AP frequency in neurons obtained during the fall months ...... 41

Figure 3-7: Effects of GABAA receptor activation or antagonism on AP frequency in neurons obtained during the winter months ...... 42

Figure 3-8: GABAA receptor-mediated decrease and increase in spike frequency in fall (F) and winter (W) groups, respectively ...... 43

Table 3-4: Effects of GABAA receptor antagonism on membrane potential in fall (F) and winter (W) groups ...... 44

Figure 3-9: Seasonal changes induce an inhibition to excitation trend in neurons obtained during the fall and winter months, respectively ...... 44

Figure 3-10: Pharmacological inhibition of cation-chloride cotransporter activity blocks GABAA receptor-mediated changes in spike frequency ...... 45

vii ABBREVIATIONS

µmol micromolar mmol millimolar

MΩ megaohm

2+ [Ca ]i intracellular calcium concentration

APf action potential frequency

A1R adenosine receptor type I subclass

ANOVA Analysis of Variance

Bic bicuculline methiodide

BMT bumetanide

CCC cation-chloride cotransporter

CPA N6-cyclopentyladenosine

DPCPX 8-cyclopentyl-1,3-dipropylxanthine

GABA gamma-aminobutyric acid

ECD excitotoxic cell death

FRS furosemide

Hz hertz

IPC ischemic preconditioning

KCC2 K-Cl cotransporter isoform 2

NKCC1 Na-K-2Cl cotransporter isoform 1

O2 oxygen

PeDG pedal dorsal ganglia

Vm membrane potential

viii Chapter 1: INTRODUCTION

1.1. Anaerobic metabolism and evolution of O2

Before the accumulation of oxygen in the atmosphere that led to the profound changes in

the surface chemistry of Earth, organisms were dependent on a variety of anaerobic metabolic

pathways. Energy sources for early anaerobic metabolism including H2, H2S, and S°, likely

originated from deep-sea and hydrothermal systems. The rapid cooling of volcanic gases

containing H2S and SO2 could have been the source of S° (Grinenko and Thode, 1970), while

H2S would have originated directly from hydrothermal systems in order to be utilized by

autotrophs in terrestrial hydrothermal environments (Canfield, 2005). Elemental sulfur reduction

to hydrogen sulfide using both H2 and organic compounds as the electron donor is widespread

within both the Bacteria and Archaea Domains (Canfield, 2005). Similarly, the capacity to carry

out anoxygenic photosynthesis by Green sulfur bacteria indicates that sulfur metabolism was

prevalent in Precambrian history (Canfield and Raiswell, 1999). Fe2+ could also have fueled

anoxygenic photosynthesis as it can dissolve in marine waters to significant concentrations

(Canfield, 2004). The evolution of oxygen-producing cyanobacteria, however, posed a great

threat to anaerobic organisms that possessed few, if any, antioxidants. Thus, cyanobacterial

evolution played a central role in the oxygenation of Earth’s surface and was a notable event in

the history of life itself (Canfield, 2005).

Increasing levels of oxygen in the atmosphere some 2.3 billion years ago substantially

augmented the level of primary production leading to a multitude of biological consequences

including the emergence of large animals (Canfield and Raiswell, 1999). Because oxygen is an

electron acceptor, it is used during cellular respiration by both aquatic and terrestrial organisms

that have a range of different respiratory structures involved in gas exchange. Cellular

1 respiration accounts for the production of virtually all of the adenosine triphosphate (ATP) that is necessary for homeostatic cellular processes and survival. ATP is the molecular energy currency of the cell as it is produced and metabolized in different subcellular organelles in order to be utilized for ion pumping, biosynthetic reactions, protein synthesis, and cell division. ATP production is tightly coupled to ATP demand for the maintenance of transmembrane ion gradients among other essential cellular functions. Considering the ubiquitous importance of

ATP, it becomes clear that oxygen is critical to almost all life on earth because it makes mitochondrial oxidative phosphorylation possible. Furthermore, mammals have a significantly higher standard metabolic rate than other vertebrates and the mammalian brain with its limited scope for metabolic activity makes oxygen an essential requirement for mammalian brain (Lutz,

1992).

1.2. Mammalian neurons—hypoxia sensitive

Oxygen limitation (hypoxia), even temporarily can lead to irreversible cellular damage.

Local hypoxia, as observed during stroke and cardiac infarction can be deleterious within minutes, and is one of the major causes of mortality in humans. Acute hypoxia or cerebral ischemia leads to neuronal death due to failure of ATP-driven ion transporters (i.e. Na+/K+

ATPase) that follows diminished ATP production. Breakdown of transmembrane ion gradients and the subsequent membrane potential depolarization (termed anoxic depolarization) causes release of excitatory amino acids including aspartate and glutamate. Calcium influx upon glutamate binding to postsynaptic receptors activates enzymes such as proteases, lipases, and endonucleases that ultimately compromise neuronal integrity. The toxic accumulation of cytosolic calcium through glutamate receptors is a hallmark of excitotoxic cell death (ECD), a term used to describe the cascade of events that lead to cell death upon oxygen deprivation.

2 Beyond ECD, cellular damage is further exacerbated by oxygen reperfusion injury that

constitutes the synthesis of nitric oxide, release of oxygen radicals, inflammation, and

uncoordinated firing of inhibitory and excitatory neuronal populations (Berger et al., 2002). This

hypoxia/ischemia-induced neuronal death is a common phenomenon among a range of vertebrate

species including fish and mammals, even after temperature effects are accounted for (Lutz et

al., 2003).

1.3. Mechanisms of hypoxia-tolerance

1.3.1. Metabolic Arrest

Many organisms, however, tolerate prolonged periods of hypoxia without the

susceptibility to neuronal damage that is characteristic of the mammalian brain. Freshwater

turtles of the genera Trachemys and Chrysemys, crucian carp (Carassius carassius) and goldfish

(Carassius auratus) are four of the most anoxia-tolerant vertebrates known, as they are capable of tolerating weeks without oxygen at low temperatures (Bickler and Buck, 2007; Lutz and

Nilsson, 1997). Trachemys scripta and Chrysemys scripta have been used extensively in

research to elucidate the mechanisms that allow neuronal survival during exposure to hypoxia.

A true facultative anaerobe, C. scripta can survive months in the total absence of oxygen by

depressing its metabolic rate such that energy utilization is equal to energy production by means

of anaerobic pathways. This phenomenon, termed metabolic arrest, refers to lowering of ATP

turnover via decreases in glycolysis and oxidative phosphorylation in order to match depressed

energy consumption through a decline in protein synthesis and membrane permeability

(Hochachka et al., 1996; Hochachka and Lutz, 2001). Manifestations of this mechanism include

the ability of C. scripta to survive months solely on anaerobic metabolism, decrease heart rate

from 10 to 0.4 beats per minute, and greatly reduced muscle activity (Jackson et al., 1984). By

3 bypassing an energy deficit in such a way, the catastrophic drop in ATP levels observed in

mammalian cells is prevented in this anoxia-tolerant species.

1.3.2. Channel Arrest

Along with metabolic arrest, there is a coordinated regulation of ionic conductances and

pumps, referred to as “channel arrest” (Hochachka, 1986). The theory posits that the plasma

membrane of anoxia-tolerant organisms has an inherently low permeability due to reductions in

channel density and/or activity and that membrane permeability decreases further during periods

of reduced oxygen supply. Indeed, comparisons of membrane permeability to Na+ and K+ in

mammals and reptiles of comparable size and body temperature show that reptilian membranes

are fivefold less leaky than mammalian membranes (Else and Hulbert, 1987). Ion leakage is also

further reduced during anoxia in the turtle brain (Chih et al., 1989). Indirect evidence of ion

channel arrest includes maintenance of membrane potential despite a 75% decrease in Na+/K+

ATPase activity (Buck and Hochachka, 1993) and an anoxia-induced 42% decrease in voltage- gated Na+ channel activity in turtle cerebellum (Perez-Pinzon et al., 1992). Robust decreases in

whole-cell glutamate receptor currents (Shin and Buck, 2003; Pamenter et al., 2008) and single- channel open time (Buck and Bickler, 1998) provide direct indications of ion channel arrest.

Further decreases in neuronal energy requirements are achieved by inhibition of excitatory neurotransmitter release such as dopamine (Milton and Lutz, 1998) and glutamate (Milton et al.,

2002), along with an elevation in the release of inhibitory neurotransmitter GABA (Nilsson and

Lutz, 1991) and neuromodulator adenosine (Nilsson and Lutz, 1991). Thus, by employing metabolic and channel arrest, anoxia-tolerant species avoid the energy deficit that induces cell death in anoxia-sensitive species.

4 1.4. Adenosine-mediated neuroprotection

1.4.1. Adenosine structure, receptors & function

Adenosine is an important neuromodulator with neuroprotective roles. Chemically, adenosine consists of a purine base adenine linked to a sugar ribose, thus it is classified as a nucleoside. It is one of the most ubiquitous metabolic intermediates in the cell needed for nucleic acid synthesis, formation of ATP and is also implicated as a neurotransmitter (Snyder,

1985). Actions of adenosine either decrease the activity of excitable tissues, such as bradycardia observed in heart, or increase in the delivery of metabolic substrates such as vasodilation observed in the cerebrovasculature, thus it helps to couple the rate of energy expenditure to that of energy supply (Dunwiddie and Masino, 2001). Under conditions of increased demand and reduced supply of energy, such as hypoxia, there is an increase in adenosine turnover and adenosine receptor stimulation (Roman et al., 2008). Adenosine receptors are G protein-coupled receptors that are evolutionarily well conserved (Fredholm et al., 2001a). A1 and A3 receptors

couple to G proteins of the Gi family, while A2A and A2B receptors couple to proteins of the Gs

family (Fredholm et al., 2000). Adenosine as an agonist is equipotent at A1, A2A, and A3

receptors, although the potency of endogenous adenosine is simultaneously dependent on the

receptor number and on the type of response measured (Fredholm et al., 2001b). This arises

from the difficulty in determining the affinity of adenosine to its receptors by direct binding

studies since adenosine is quickly metabolized and also rapidly formed in membrane

preparations along with other types of biological assays (Fredholm, 2010). Adenosine plays

various different roles as an intercellular messenger, especially in the brain, which expresses

high concentrations of adenosine receptors and where its role is implicated in both normal and

pathophysiological processes. The neurophysiological functions of adenosine are primarily

5 inhibitory, and mostly involve inhibition of excitatory neurotransmitter release achieved by the blockade of calcium influx into nerve terminals which results in inhibition of glutamate release and reduction of excitatory effects at a postsynaptic level (Snyder, 1985; Roman et al., 2008).

The important neuroprotective role of endogenous adenosine in the central nervous system (CNS) has been well characterized. Physiologically, there is a very low concentration of adenosine in the extracellular fluid (30-200 nM); however, it rises dramatically up to 30 µM during increased nerve activity, hypoxia or ischemia by the breakdown of high-energy phosphates (Oldenburg et al., 2003; Fredholm, 2010). During these conditions, degradation or transport of adenosine is inhibited by adenosinergic transmission-potentiating agents (adenosine deaminase and kinase inhibitors), and elevated adenosine levels offer protection against ischemic or excitotoxic neuronal damage (Wardas, 2002). The neuroprotective actions of adenosine are mediated predominantly through the A1 receptor. The cellular mechanisms that could potentially be involved include: inhibition of neurotransmitter release (glutamate in particular), hyperpolarization of neurons, and direct inhibition of certain kinds of Ca2+ channels (Dunwiddie and Masino, 2001). All of these mechanisms could reduce excitotoxicity by inhibiting Ca2+ entry, and by decreasing metabolic demand, which would help to conserve ATP stores that would otherwise be required for pumping Ca2+ out of the cell. Experiments with neuronal tissue suggest that the number of A1 receptors expressed might serve as a limiting factor in acute protection because enhanced A1 receptor binding increases neuroprotection (Halle et al., 1997).

Some of the protective effects could be conferred through the A3 receptor; however, the A2A receptor contributes to tissue damage because in experiments with knockout mice lacking A2A receptors, reduced brain damage was shown following focal ischemia (Chen et al., 1999).

6 1.4.2. Adenosine and ischemic preconditioning

Adenosine also confers neuroprotection during ischemic preconditioning (IPC). IPC is

an endogenous defense mechanism where a brief sublethal ischemic episode leads to robust and

sustained neuroprotection against a subsequent ischemic insult of increased severity several

hours or even days later. It is established that any subthreshold stressor of a short duration is

capable of conferring significant neuroprotection (Dirnagl et al., 2003). The neuroprotective

effects of adenosine during IPC in neuronal tissue appear to be mediated through A3 as well as

A1 receptors. In the brain, global ischemic tolerance involves a cascade of events that includes

+ release of adenosine, stimulation of adenosine A1 receptors, and opening of ATP-sensitive K

(KATP) channels via the A1 receptor. The role of adenosine A1 receptors during ischemic

preconditioning and mitochondrial KATP (mKATP) channels during subsequent severe ischemia

has also been implicated in the development of focal cerebral ischemic tolerance. Yoshida et al.

(2004) found that the neuroprotective effect of preconditioning was attenuated by application of

an adenosine A1 receptor antagonist before conditioning ischemia, and by the administration of

mKATP channel blocker before test ischemia. Opening of mKATP channels or mitochondrial depolarization causes release of Ca2+, elevating intracellular Ca2+ levels. Adenosine receptor

2+ activation may also cause an increase in cytosolic Ca via an inositol-3-phosphate (IP3)-

mediated pathway, as all but adenosine A2A receptors activate phospholipase C (PLC), an

enzyme converting phospholipids into diacylglycerol and IP3 (Dunwiddie and Masino, 2001).

Thus, adenosine appears to be a good candidate as an intercellular messenger that could be

generated rapidly and locally from the breakdown of ATP (Buck, 2004).

7 1.4.3. Role of adenosine in hypoxia tolerance—vertebrates

The neuroprotective role of adenosine during decreased oxygen availability has also been

explored in anoxia-tolerant vertebrates. The main strategy employed by freshwater turtles to

survive anoxic episodes appears to be a decrease in energy utilization, in order for ATP demand

to be met by glycolytic production alone. Nilsson et al. (1990) found that in response to anoxia,

levels of inhibitory compounds such as GABA, glycine and taurine increased in the brain, with a

simultaneous decrease in the excitatory neurotransmitter glutamate. Further, they measured

increases in the extracellular concentrations of GABA, glycine and taurine, but the increase was

observed after 100 minutes of anoxia (Nilsson and Lutz, 1991). Thus, they hypothesized that

although inhibitory amino acids are important mediators of suppressed energy usage in the long- term; their role in the initial decrease in brain activity needed to meet a diminished rate of energy production is not likely. Consequently, they measured the effects of anoxia on the extracellular level of adenosine in the Trachemys scripta striatum. An increase in extracellular adenosine was observed in response to N2 exposure; however, this increase was only temporary and began to

decrease after 90-120 minutes. The decrease in adenosine concentration coincided temporally

with the increase in levels of inhibitory amino acids (Nilsson and Lutz, 1992). This leads to the

assertion that the initial energetic shift observed in the turtle brain is due to adenosine signaling,

while the subsequent release of inhibitory neurotransmitters plays a pivotal role in maintaining

the remarkably depressed metabolic rate that allows prolonged anoxia survival.

The general mechanisms through which adenosine confers neuroprotection during

oxygen deprivation in anoxia-tolerant species has also been studied. The enhanced ability of the

turtle brain to survive anoxia is possible by decreasing energy expenditure in order to meet

reduced energy production. Among other things, reduction in K+ flux or ion leakage is one way

8 that the anoxic turtle brain conserves energy. Following inhibition of the Na+/K+-ATPase with ouabain, Pek and Lutz (1997) measured reduction in ionic leakage by approximately 70% and a

3-fold increase in the time to reach full depolarization in the anoxic turtle brain when compared with normoxic controls. Application of the general adenosine receptor blocker theophylline, or the specific adenosine A1 receptor blocker, 8-cyclopentyltheophylline to the ouabain-treated brain before and during anoxia significantly reduced the time to full depolarization along with increasing the rate of K+ efflux. These experiments suggest that expression of anoxia-induced ion channel arrest in the turtle brain is mediated by adenosine acting through its A1 receptor.

Similar experiments were done to determine the effects of adenosine on cerebral blood flow (CBF) in the crucian carp, C. carassius. Because C. carassius has large liver glycogen stores, increased cerebral blood flow in order to maintain glycolytic flux during anoxia would be expected. Nilsson et al. (1994) found that normoxic adenosine application increased CBF to the same degree as anoxic perfusion. Further, application of the adenosine receptor blocker aminophylline to the brain inhibited the effect of anoxia on CBF. These data suggest that adenosine also mediates the anoxia-induced increase in CBF of the crucian carp.

Although adenosine appears to have a clear role in mediating anoxia tolerance in turtles and the crucian carp, its effects are variable across other vertebrate species. The role of adenosine and anoxia in modulating CBF has been examined in the leopard frog, Rana pipiens

(Soderstrom-Lauritzsen et al., 2001). Exposure of the telencephalon to anoxia caused an initial

225% increase in CBF that was reversible after another 20 minutes of anoxia. Application of 50

µM adenosine during normoxia caused a 52% increase in CBF, a higher concentration resulted in no further increase and the effects of all doses were completely inhibited by the adenosine receptor blocker aminophylline. However, application of aminophylline to the brain during

9 anoxia did not block the anoxia-induced increase in CBF, thus CBF can be increased by

adenosine but it is not likely that adenosine is the primary mediator of the anoxia-induced

increase in CBF in R. pipiens. Similar effects of adenosine were observed in the hypoxia-

tolerant shark Hemiscyllium ocellatum, where adenosine caused an increase in CBF during

normoxia and this effect could be diminished by aminophylline, but the adenosine receptor

antagonist had no effect on the preservation of CBF, nor did hypoxia (Soderstrom et al., 1999).

Therefore, unlike most other hypoxia-tolerant vertebrates, in R. pipiens and H. ocellatum, the hypoxia-induced increase in CBF is not mediated through adenosine.

10 1.4.4. Role of adenosine in hypoxia tolerance—invertebrates

Although invertebrates comprise the major number of anoxia-tolerant species, few studies have utilized invertebrate models to examine a possible neuromodulatory role of adenosine (Table 1-1).

Table 1-1. Concentration and effects of adenosine on various invertebrate preparations

Species Concentration Physiological Actions

Helix aspersa 0.6 μM Depression of acetylcholine-mediated depolarization in identified neurons of the

isolated subesophageal ganglionic massa Mytilus edulis 1-10 μM Inhibition of neurotransmitter (monoamines) release from the pedal gangliab

Sipunculus nudus 30 µM Decrease in oxygen consumption rate of isolated body wall musculaturec

Panulirus argus 100 μM Inhibitory effect on the spontaneous activity of interneurons; certain cells exhibit excitationd Calliphora vicina 10-500 µM Decrease in amplitude of evoked EPSCs from the neuromuscular junction 200-500 μM Decrease in frequency of mEPSCse

aCox and Walker 1987; bBarraco and Stefano 1990; cReipschlager et al 1997; dDerby et al 1987; eMagazanik and Federova 2003

Several lines of evidence indicate physiological changes mediated by adenosine in invertebrates. Lazou (1988) studied the presence of adenylate-metabolizing enzymes in bivalves, gastropods, echinoderms, crustaceans, cephalopods, and polychaetes and found that adenosine is metabolized by all the invertebrate tissues studied. In support of the theory that adenosine modulates blood flow, it was demonstrated that aerobic muscles had higher levels of adenosine kinase than anaerobic muscles, allowing rapid regulation of adenosine concentrations.

11 It also indicates that adenosine plays a regulatory role in muscles with very high adenosine

kinase activity (Lazou, 1988).

Studies have also been done on individual organisms to determine the effects of

adenosine on neurophysiology. Adenosine was shown to modify the acetylcholine-induced

depolarization of a specific neuron in the isolated suboesophageal ganglionic mass of Helix

aspersa (Cox and Walker, 1986). The magnitude of the response was dependent on the final

bath concentration of adenosine; lower concentrations (0.6-6 nM) increased whereas higher

concentrations (60 nM-0.6 µM) reduced the depolarization caused by acetylcholine. In the pedal

ganglia of the marine bivalve Mytilus edulis, modulation of neurotransmitter release by

adenosine and its agonists was examined (Barraco and Stefano, 1990). The adenosine agonist,

5’-N-ethylcarboxamidoadenosine (NECA; 10 nM), inhibited the release of serotonin, dopamine, and to a lesser degree norepinephrine, while theophylline blocked the inhibitory effects of NECA on neurotransmitter release. Adenosine itself also inhibited neurotransmitter release, although

100-fold higher concentrations were required to achieve levels of inhibition similar to NECA.

Slightly different results were found using interneurons in the circumesophageal ganglia of the spiny lobster, Panulirus argus. Both AMP and adenosine were found to have an inhibitory effect on the spontaneous activity and responsiveness of certain interneurons to chemical or electrical stimuli, while the response of others was enhanced rather than depressed by AMP or adenosine (Derby et al., 1987). The modulatory actions of adenosine in P. argus were receptor-

mediated because the adenosine receptor antagonist, 1,3-dipropyl-8-p-sulfophenylxanthine

(DSPX) inhibited the effects of adenosine, although theophylline was not an effective antagonist of adenosine receptors in this study. Derby et al. (1987) account for this discrepancy by suggesting that DSPX is known to be more potent than theophylline in certain mammalian CNS

12 preparations. Similarly, the excitable characteristics of adenosine appear contrary to the overall

trend of its depressive effects, but this finding also has parallels in vertebrates where purinergic

modulators occasionally cause excitation rather than inhibition.

There have been few studies examining the role of adenosine during decreased oxygen

availability in invertebrates. Michaelidis et al. (2002) measured adenosine levels in the brain,

heart and haemolymph of the land snail Helix lucorum and in the brain, heart and blood of the

lizard Agama stellio stellio during long-term hibernation. Adenosine levels in the brain of hibernating H. lucorum significantly decreased after two months in hibernation and remained depressed for the following two months. In A. stellio stellio, adenosine levels were maintained in the brain during the first five days, but decreased significantly during prolonged hibernation.

These findings are in accordance with the suggestion that the initial energetic shift observed in anoxia-tolerant organisms is due to the effects of adenosine signaling.

Adenosinergic modulation has also been examined in the marine invertebrate Sipunculus nudus. Reipschlager et al. (1997) measured concentrations of various neurotransmitters in nervous tissue of S. nudus after exposure of anoxia, hypercapnia, or anoxic hypercapnia. S. nudus depresses its metabolic rate by 70% during anoxia, and by 20-35% during hypercapnia.

Among all the compounds studied (monoamines and amino acids), adenosine levels changed concomitantly in a manner that is consistent with its role in metabolic depression under all conditions studied. Adenosine levels increased in the nervous tissue during anoxia, hypercapnia, and to an even greater degree during anoxic hypercapnia. Adenosine applied under control conditions, when basal adenosine levels are minimal, resulted in depressed oxygen consumption for more than 90 minutes. During hypercapnia, when adenosine levels are high and aerobic metabolic rate is decreased, application of theophylline increased the oxygen consumption rate

13 after 30 minutes of infusion. Thus, adenosine is involved in anoxia-mediated metabolic depression since increased levels reduce oxygen consumption.

Considering data from vertebrates and invertebrates, it appears that adenosine has an important neuroprotective role in animals that routinely experience severe hypoxia. ATP is rapidly broken down to adenosine in response to hypoxic stress and this rapid increase in adenosine concentration serves as a potent signal of metabolic stress. Adenosine release and its effects mediated by A1 receptors are considered essential to hypoxia tolerance in many

vertebrates, although it remains to be determined whether a similar role exists in hypoxia-tolerant

invertebrates.

1.5. GABA-mediated neuroprotection

1.5.1. GABA—primitive developmental signal

γ-amino butyric acid (GABA) operates as a highly conserved developmental signal in a

wide variety of species, including insects at early developmental stages. For example, post-

embryonic brain development in the beetle Tenebrio molitor involves GABA expression

(Wegerhoff, 1999). In Drosophila, GABA postsynaptic currents are observed at the mid- gastrula stage (Lee and O’Dowd, 1999), and inhibition of GABA uptake increases the amplitude of endplate junction potentials (Delgado et al., 1989). Several GABAergic-modulated behaviours have been identified in Drosophila by utilizing a pharmacological approach that disrupts GABA transporter function. Adult female flies systemically treated with GABA transporter inhibitors exhibit diminished locomotor activity, convulsive behaviours, aberrant geotaxis, and a secondary loss of the righting reflex (Leal and Neckameyer, 2002). Further, alterations in neural development (e.g. increase in latency of evoked quantal release) occur if

GABAergic neurotransmission is potentiated by a generalized treatment of GABA uptake

14 inhibitors in Drosophila (Neckmeyer and Cooper, 1998). Mushroom bodies, key features of the insect brain circuitry involved in associative learning, exhibit synchronous calcium oscillations

that are modulated by GABA receptors, and GABA also has important roles in integration of

insect olfactory cues (Rosay et al., 2001). Thus, GABA is a primitive communicative signal

involved in early development in various invertebrate species. Despite the present lack of

evidence, it is probable that mechanisms that regulate the development of enzymes and

transporters involved in GABAergic neurotransmission are evolutionarily conserved in

invertebrates like worms and insects (Ben-Ari, 2002).

1.5.2. GABA receptors

The details of GABA synthesis and breakdown were discovered shortly after its

identification as a neurotransmitter in mammalian brain tissue in the 1950s (Purves et al., 2001).

GABA is the principal inhibitory neurotransmitter in the mature CNS, which expresses several

types of GABA receptors (GABAA and GABAB) and where its role has been implicated in both

normal and pathological processes (Krnjevic, 1997). Unlike glutamate, GABA is not an

essential precursor in any metabolic process, nor is it involved in protein synthesis. As such, the

presence of GABA alone is a reliable indication that the neuron being studied uses GABA as a

neurotransmitter (Purves et al., 2001). The fast response of neurons to GABA is due to direct

activation of GABAA receptors that are heteropentameric chloride-sensitive ligand-gated ion

channels (Moult, 2009). This receptor belongs to a superfamily of Cys-loop ligand-gated ion

channels that possess a characteristic loop formed by a disulfide bond between two cysteine

residues (Sieghart and Sperk, 2002). GABAA receptors have a large N-terminal extracellular

domain, four transmembrane regions (T1-4), and an intracellular loop between TM3 and TM4

segments (Nayeem et al., 1994). The diversity of GABAA receptor subunits is the largest of any

15 mammalian ion channel receptor (D’Hulst et al., 2009). The subunits are divided into eight classes based on sequence similarity: α (1-6), β (1-3), γ (1-3), δ, ε, ρ (1-3), θ, and π (Olsen and

Sieghart, 2008). This extensive diversity is further complicated by alternative splicing that

generates multiple forms of the α, β, and γ subunits, these subunits also comprise the majority of

native receptors in the CNS (Simon et al., 2004). GABAA receptors can be allosterically

modulated by benzodiazepenes, barbiturates, steroids, anaesthetics, and convulsants along with

many other drugs (Sieghart, 1995).

1.5.3. Polarity of GABA transmission and cation-chloride cotransporters

Inhibitory actions of GABA are due to the expression of the K-Cl cotransporter isoform

2 (KCC2), a member of the larger family of cation-chloride cotransporters (CCCs). Decreased intracellular chloride concentrations maintained by KCC2 result in chloride influx when GABAA

receptors are activated (Blaesse et al., 2009). Influx of negatively charged chloride ions hyperpolarizes the membrane of the postsynaptic neuron, resulting in an inhibitory postsynaptic potential that functions to decrease the probability of spiking. Excitatory actions of GABA have been reported during development of the nervous system (Taketo and Yoshioka, 2000) or in certain cell populations (Lamsa and Taira, 2003). This phenomenon is due to increased intracellular chloride concentrations maintained by the Na-K-2Cl cotransporter isoform 1

(NKCC1), also a member of the CCC protein family, leading to chloride extrusion upon GABAA

receptor activation.

Transporter proteins such as CCCs are thought to have evolved by duplication of genes

encoding channel-type protein subunits consisting of a small number of membrane-spanning segments (Saier, 2003). The CCC family has origins in early evolution, as there are putative

CCC homologs in a cyanobacterium and in C. elegans (Xu et al., 1994). In accordance with this

16 suggestion, homologs of CCCs exist in a wide range of species. Diverse plant species have a

CCC gene that encodes for a Na-K-Cl cotransporter that is both functional and bumetanide-

sensitive (Colmenero-Flores et al., 2007). Moreover, expression of the only KCC gene present

in Drosophila is neuron-specific (Hekmat-Scafe et al., 2006), as such it is possible that the

evolution of the first KCC in animals was for the proper functioning of inhibitory

neurotransmission (Blaesse et al., 2009).

The CCC family, of which both NKCC1 and KCC2 are members of, consists of nine

transporters encoded by the genes Slc12a1-9 in mammals (Blaesse et al., 2009). The CCC

proteins are glycoproteins with molecular weights of 120-200 kDa (Kahle et al., 2008). Out of the nine CCCs studied thus far, seven are plasmalemmal ion transporters (Gamba, 2005) that functionally fall under three categories: two members are Na-K-2Cl cotransporters (NKCCs; isoforms NKCC1 and NKCC2), one is a Na-Cl cotransporter (NCC), and four are K-Cl cotransporters (KCCs; isoforms KCC1-4). Of all the CCCs, the predicted secondary structure has been validated for only NKCC1, consisting of 12 transmembrane segments flanked by intracellular termini that compose nearly half of the entire protein (Gerelsaikhan and Turner,

2000). Although information about how oligomerization affects CCC function is lacking at present, hetero- and homo-oligomers have been confirmed for all the CCCs (Blaesse et al.,

2006).

All CCCs are expressed in neurons or glia at some stage of CNS development, with the exception of NKCC2 and NCC that are mostly found in the kidney (Blaesse et al., 2009). KCC1 performs homeostatic functions in various cell types including glial cells; however, present evidence suggests minimal expression in central neurons (Payne et al., 1996). The KCC2 isoform of KCCs is exceptional, as it is exclusively expressed in CNS neurons (Karadsheh and

17 Delpire, 2001). NKCC1 has an essential role in neuronal proliferation as it is highly expressed

in embryonic ventricular zones (Li et al., 2002); it is also expressed in dorsal root ganglion cells

(Rocha-Gonzales et al., 2008) and in olfactory receptor neurons (Reisert et al., 2005). CCCs do not directly contribute to neuronal membrane potential as their ion transport stoichiometry renders them electrically neutral (Blaesse et al., 2009). However, several reports suggest that subtle changes in plasmalemmal ion-transport mechanisms have a profound effect on the output of neuronal networks in the adult CNS (Coull et al., 2003; Laviolette et al., 2004). One recent finding provides the first demonstration of ionic plasticity (downregulation of KCC2) in response to physiological stimuli, namely acute restraint stress, leading to increased activity of neuroendocrine cells (Hewitt et al., 2009). GABAergic transmission is tightly coupled to intraneuronal chloride gradients, thus spatially distinct expression patterns of KCC2 and/or

NKCC1 can lead to localized compartmentalization of steady-state chloride gradients having functional implications at an individual neuronal and the network level (Szabadics et al., 2006).

1.5.4. GABA and ischemic preconditioning

Since GABA has important functional roles in maintaining existing neuronal circuits, it is conceivable that aberrant GABAergic transmission might also contribute to neuronal death during excitotoxicity. Indeed, the disruption of excitatory and inhibitory equilibrium induced by ischemia leads to neuronal damage by reducing the expression of GABAA receptors (Wang et al,

2007; Hall et al., 1997; Schiene et al., 1996). In animal models of ischemia, both GABA uptake

inhibitors and GABAA receptor agonists have proven to be neuroprotective (Green et al., 2000;

Gilby et al., 2005). Oxygen-glucose deprivation also causes a significant reduction in cell surface GABAA receptors, which suggests that decreased density of surface GABAA receptors

may enhance neuronal death during ischemia (Mielke and Wang, 2005). As with adenosine, the

18 role of GABA in conferring neuroprotection during IPC has also been examined. One of the many neuronal changes that underlie tolerance associated with IPC involves elevated GABA release (Grabb et al., 2002) along with upregulation of GABAA receptors (Sommer et al., 2002) during ischemia after IPC induction. Moreover, application of GABAA receptor antagonist bicuculline during IPC blocks IPC-mediated neuroprotection, suggesting that GABAA receptor activation at least partially contributes to ischemic tolerance (Lange-Asschenfeldt et al., 2005).

1.5.5. Role of GABA in hypoxia tolerance

Modulation of GABAergic synaptic transmission in response to decreased oxygen levels has been observed in a wide variety of hypoxia-tolerant species; extracellular levels of GABA rise in the anoxic brain of the shore crab Carcinus naenus (Nilsson and Winberg, 1993), Crucian carp (Hylland and Nilsson, 1999), leopard frog Rana pipiens (Milton et al., 2003), and the red- eared turtle Trachemys scripta (Nilsson and Lutz, 1991). GABA concentrations in the turtle brain increase 45-60% over the first 2-4 hours of anoxia, and a further 127% after 13 hours

(Nilsson et al., 1990). The breakdown of GABA to succinate is dependent on oxygen availability since the enzymes involved in its degradation, GABA aminotransferase and succinic semialdehyde dehydrogenase, are both mitochondrial enzymes (Purves et al., 2001). Contrarily, robust increases in GABA concentration are possible during anoxic conditions because the conversion of glutamate to GABA by glutamate acid decarboxylase (GAD) is an anaerobic process (Milton and Prentice, 2007). In addition to the accrual of extracellular and tissue GABA levels, the effectiveness of GABAergic inhibition is enhanced during anoxia by an increase in the density of GABAA receptors that continues to rise for at least 24 hours of anoxia (Lutz and

Leone-Kabler, 1995). Thus, it appears that pharmacological interventions, such as, preventing

GABA reuptake and enhancing GABAA receptor activity with agonists, that allow prolonged

19 neuronal survival during ischemia in the mammalian brain are endogenously employed by

several anoxia-tolerant species (Milton and Prentice, 2007).

1.6. Lymnaea stagnalis as an invertebrate model of hypoxia tolerance

Lymnaea stagnalis is a bimodal breather, utilizing skin transpiration under water along

with periodic lung exchange with air via its pneumostome (Jones, 1961). The anoxia tolerance

of L. stagnalis is about 40 hours in a N2-bubbled environment at 20°C, with a mortality rate of

10% on day 7 of normoxic recovery (Wijsman et al., 1985). 48 hours of anoxia resulted in a

30% decrease in carbohydrate levels, while haemolymph and tissue anaerobic end products

(succinate and D-lactate) increased significantly after 24 hours of anoxia (Wijsman et al., 1985).

Further, a linear increase in calcium was observed during the anoxic episode due to buffering of acid end products by dissolution of calcium carbonate (Wijsman et al., 1985). In addition to data on anaerobic metabolism, L. stagnalis has been used extensively in neurophysiological studies due to its well-characterized respiratory system and neuronal morphology (Inoue et al., 2001;

Syed et al., 1990; Taylor and Lukowiak, 2000; Kyriakides et al., 1989). Recently, its ability to tolerate hypoxia has been utilized to examine hypoxia-induced modulation of different protein profiles (Fei and Feng, 2008; Fei et al., 2007; Silverman-Gavrila et al., 2009).

GABAergic transmission has been studied extensively in L. stagnalis. Two types of

GABAA receptor subunits with 30-50% identity to vertebrate GABAA receptors have been

cloned in L. stagnalis (Harvey et al., 1991; Hutton et al., 1993). Studies of GABA-like

immunoreactive neurons in the CNS of L. stagnalis have been done using a combination of

immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations.

GABA-like immunoreactivity was detected in all ganglia leading to detailed maps of the central

GABA-like immunoreactive neurons in juveniles and adults of L. stagnalis (Hatakeyama and Ito,

20 2000). In systemic studies, Romanova et al. (1996) have shown that GABA injected into the haemolymph of intact L. stagnalis immediately evoked rhythmic movements of its radula including: protraction, rasping, and swallowing. These results indicate that GABA receptors in the CNS of L. stagnalis can modulate behaviours, such as feeding activity. Given the importance of inhibitory compounds such as adenosine and GABA in regulating hypoxia-induced inhibition of neuronal activity that is necessary for preservation of ATP stores, it is important to examine their role in regulating excitability of neurons in L. stagnalis central ring ganglia.

21 1.7. Rationale and Hypotheses:

The apparent dearth of knowledge regarding effects of adenosine in CNS of invertebrates

led me to examine the neuromodulatory role of adenosine in L. stagnalis. The effects of

adenosine perfusion on neuronal activity as assessed by changes in action potential (AP)

frequency and membrane potential (Vm) will be studied.

Hypothesis 1a: Consistent with its actions in mammalian neurons, adenosine will hyperpolarize

Vm and decrease AP frequency.

Hypothesis 1b: Since adenosine signaling has effects on intraneuronal calcium homeostasis,

adenosine perfusion will also cause changes in intracellular calcium levels of cluster F neurons.

The effects of GABA on neuronal activity of L. stagnalis remain inconclusive as different

studies report contradictory results. Cheung et al., (2006) reported that hypoxia-induced

depression in neuronal activity is mediated by excitatory actions of GABA; these results are

consistent with those of Rubakhin et al. (1996), who also reported excitatory effects of GABA in

the adult L. stagnalis CNS. In contrast, Moccia et al. (2009) and Molnar et al. (2004) report

inhibitory actions of GABA in the mature L. stagnalis CNS. Given the lack of consistency found

in these reports, I will examine the effects of GABA in adult L. stagnalis CNS. As with the

experimental paradigm proposed in the adenosine study, effects of GABA on neuronal activity of

cluster F neurons will be monitored.

Hypothesis 2a: GABA perfusion onto central ring ganglia will depolarize Vm and increase AP frequency. Any observed changes in neuronal activity will likely be mediated by the GABAA

receptor, thus pharmacological inhibition of GABAA receptors will block the effects of GABA.

22 Hypothesis 2b: The contribution of NKCC1 and KCC2 to the maintenance of intraneuronal chloride concentrations is well documented; thus, pharmacological inhibition of these CCCs will also have an effect on any observed changes in neuronal activity induced by GABA.

23 Chapter 2: MATERIALS AND METHODS

2.1. Animals

Laboratory-raised stocks of the freshwater snail L. stagnalis were maintained on a natural photoperiod for Toronto, ON, Canada. All animals were raised in aquaria filled with well-

aerated filtered tap water at 22°C. L. stagnalis were fed lettuce ad libitum and experiments were

performed on snails with a shell length of 10-20 mm (~ 2 months old).

2.2. Anoxia tolerance

Because food consumption stops during anoxia, snails were deprived of lettuce one day

prior to the start of anoxic incubation. Groups of snails were incubated at 22°C in nitrogen- bubbled water for various periods. The anoxic aquarium was built with a mesh cover that was submerged in the water column to prevent gas exchange at the air-water interface and bubbled with >99% nitrogen. After incubation, snails were transferred to glass beakers filled with well- aerated filtered tap water to monitor their food consumption, locomotor activity and mortality rate.

2.3. Solutions and dissection

All experiments were conducted at room temperature of 22°C. L. stagnalis saline

-1 solution included (in mmol l ): 10 glucose, 51.3 NaCl, 1.7 KCl, 4 CaCl2, 1.5 MgCl2, 10 HEPES, buffered to pH 7.9 using 12 mol l-1 HCl; 144 mOsm. In experiments aiming at inhibition of

polysynaptic transmission, a high divalent cation (Hi-Di) solution (6x Ca2+/6x Mg2+ in mmol l-1):

10 glucose; 45.0 NaCl; 1.7 KCl; 10 HEPES; 24.0 CaCl2; and 9.0 MgCl2) was superfused onto the

ganglion. Snails were anesthetized briefly in L. stagnalis saline solution containing 30%

Listerine, then de-shelled with forceps. Snails were pinned dorsal surface up to the bottom of a

Sylgard-filled dissection dish, covered with L. stagnalis saline solution, and a medial incision

24 was made from the base of the mantle to the head. Central ring ganglia were removed with a

pair of fine surgical scissors, and placed in a modified flow-through perfusion chamber (RC-26 with a P1 platform, Warner Instruments, CT, USA) before being pinned out. The bottom of an

RC-26 chamber was modified by replacing the 22mmX40mm glass coverslip with

22mmX40mm plexiglass plates and a 4 mm layer of Sylgard (total thickness 5 mm). The connective tissue sheath was removed from cluster F neurons within the LPeDG and RPeDG with a pair of fine forceps (Fig. 2-1). After being transferred to the microscope stage, the chamber was perfused at a rate of 2-3 ml min-1 by gravity flow.

Neurons were perfused with L. stagnalis saline solution until AP frequency and Vm

stabilized (10-20 min). and Vm were recorded for 20 min before exposing the preparations to

treatment saline for up to 40 min. The preparation was then reperfused Following this

stabilization period, baseline AP frequency with control saline solution to wash out the treatment

saline. Each preparation was exposed to only one treatment protocol.

Figure 2-1. Cluster F neurons of the L. stagnalis pedal ganglia. (A) The central ring ganglia isolated from L. stagnalis. Recordings were made from neurons in cluster F (white region) on the dorsal surface of either the left or right pedal ganglion. (B) Schematic of the L. stagnalis central ring ganglia. Recordings were not obtained from the identified neurons L- or RPeD1. Adapted from Cheung et al. (2006).

25 2.4. Electrophysiology

Whole-cell recordings were performed using pipettes with resistance in the range of 2-3

MΩ. Pipettes were pulled from borosilicate glass capillaries (Fisher Scientific, Napean, ON,

Canada) and filled with a filtered intracellular solution (pH 7.4) composed of the following (in

-1 mmol l ): 8 NaCl, 0.0001 CaCl2, 10 NaHEPES, 110 potassium gluconate, 1 MgCl2, 0.3 NaGTP

and 2 NaATP (280-290 mOsm). An Axopatch-1D amplifier (Axon Instruments, CA, USA) was

used for recordings. The series resistance was monitored throughout each recording and if it

varied by >10%, the recording was rejected. No electronic compensation for series resistance

was used. After obtaining a gigaohm-seal onto neurons, whole-cell patches were obtained under

voltage-clamp mode at a holding membrane potential of -60 mV by applying a brief suction.

Spontaneous neuronal activity was recorded after switching to current clamp mode. Recordings

were low-pass filtered at 2 KHz using a CV-4 headstage, and a Digidata 1200 (Axon

Instruments, CA, USA) and then digitized and stored on a computer using Clampex 7 software

(Axon Instruments, CA, USA).

Intracellular recordings using sharp electrodes were made with glass microelectrodes

(Harvard Apparatus, Edenbridge, UK) filled with a saturated solution of K2SO4 (tip resistance

50-60 MΩ). Intracellular recordings were amplified with a Multiclamp 700B amplifier

(Molecular Devices, CA, USA) and recorded using Clampex 10 software. Electrophysiological experiments were analyzed using Clampfit 10 software (Molecular Devices, CA, USA).

2.5. Fluo-4 intracellular Ca2+ imaging

The Ca2+-sensitive, membrane-impermeable pentapotassium salt of fluo-4 (Molecular

2+ 2+ Probes Inc.) was used to determine intracellular Ca [Ca ]i levels. Fluo-4 was used because of

its low background absorbance and increased brightness at lower concentrations (Paredes et al.,

26 2008). Fluo-4 was dissolved in the intracellular recording solution at a final concentration of 100

µmol l-1. Fluo-4 was excited at 488 nm for 0.1 s using a DeltaRam X high-speed random-access monochromator and a LPS220B light source (PTI, NJ, USA). Fluorescent emissions above 510 nm were isolated using an Olympus DM510 dichroic mirror and fluorescence images were acquired (526 nm) at 10-s intervals to limit photobleaching, using an Olympus BX51W1 microscope and a QImaging Rolera MGi EMCCD camera (Roper Scientific Inc., IL, USA). The obtained images were quantitatively analyzed for changes in fluorescence intensities within cells using EasyRatioPro software (PTI). Values of fluo-4 measurements are reported as means ± standard error (s.e.m.), with N being the number of cell bodies tested from different animals.

The amplitude of the fluo-4 responses was analyzed from the soma and the data expressed as relative fluorescence intensity.

2.6. Chemicals

All chemicals were obtained from Sigma Chemical Co. (Oakville, ON, Canada).

Adenosine was dissolved in L. stagnalis saline solution at a final concentration of 200 µmol l-1.

N6-cyclopentyladenosine (CPA) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were initially dissolved in 100% dimethylsulfonic acid (DMSO); and then diluted in L. stagnalis saline solution to a final concentration of about 0.3% v/v. Vehicle application alone did not affect the stability of patch-clamp recordings (Fig. 2-2).A final concentration of 100 µmol l-1 was used for

both CPA and DPCPX. GABA was dissolved in L. stagnalis saline solution and applied to the

pedal ganglia using a VC-6 perfusion valve control system (Warner Instruments, CT, USA) for

duration of 10 seconds. VC-6 perfusion valve was connected to a plastic capillary with an

orifice of 0.2 mm positioned between the water immersion lens used to visualize neurons and the

ganglion under study. A 100 mmol l-1 stock solution of bicuculline methiodide was prepared in

27 DMSO and then dissolved to a final concentration of 100 µmol l-1. A 100 mmol l-1 stock solution of bumetanide and furosemide were prepared in ethanol and DMSO, respectively; then diluted to a final concentration of 100 µmol l-1 in L. stagnalis saline solution.

20 mV 30 s B

1.4

1.2

1.0

0.8

0.6

(Hz)APFrequency 0.4

0.2

0.0 Control DMSO Recovery

Figure 2-2. Effects of DMSO on AP frequency in cluster F neurons of the L. stagnalis pedal ganglia. (A) Raw trace of a recording from a cluster F neuron maintained under control conditions. A switch from control to saline containing 100 µL DMSO occurred at the arrow. (B) Mean group data (N=4) showing that addition of DMSO alone did not cause significant changes in AP frequency (from 1.01±0.09 to 1.00±0.07 spikes s-1).

28 2.7. Statistical analysis

For adenosine-related experiments AP frequency and Vm were monitored for a 5 min interval in

the last 5 min of control (immediately before the switch to treatment perfusion), last 5 min of

treatment, and last 5 min of each wash-out perfusion at a sampling rate of 5 KHz. There was significant variation in AP frequency between neurons (minimum number of APs: 0.02 spikes s-

1; maximum number of spikes: 3.2 spikes s-1). Therefore, in order to compare AP frequency between neurons and show the extent of variance within the control values, 5 min of control recordings were used as a baseline to normalize data during a single experiment. To assess if neuronal activity changes as a function of time under control conditions without any pharmacological intervention, AP frequency and Vm were determined by analyzing a continuous

trace for a 5 min interval at the end of every 10 min, for up to 60 min. For concomitant AP

frequency and fluorescence measurements, AP frequency and fluo-4 signals were analyzed for a

1 min interval every 5 min of treatment perfusion for 20 min. Adenosine dose-response curve was fitted to the Four-Parameter Logistic (4PL) equation. For GABA-related experiments AP frequency and Vm were analyzed in manner similar to that described above, however; interval

duration was set to 1 min rather than 5 min. All results are reported as means ± s.e.m. Statistical

analysis was performed using SigmaStat software (Point Richmond, CA, USA). Results were

analyzed using repeated measures one-way ANOVA or a paired t-test, and significance was

determined at P<0.05 unless otherwise indicated. Values expressed as percentage change were

calculated as measured value minus baseline value divided by baseline value multiplied by

100%.

29 Chapter 3: RESULTS

3.1. Anoxia tolerance

Survival studies were done during the winter months. At room temperature, adult L. stagnalis survived a maximal anoxic period of 4 hours (Table 3-1). Snails were completely motionless after being in anoxic water for 1 hour, and heart rate had decreased to 10 beats/minute (data not shown). After being transferred to aerated water, snails recovered within

3 hours and began food consumption shortly thereafter. Mortality increased as length of the anoxic period increased, the majority of snails beyond 4 hours of incubation in anoxic water did not survive even after being transferred to aerobic conditions.

Table 3-1. Anoxic tolerance of L. stagnalis.

Anoxic Incubation % Mortality (Hours) Day 1 Day 3 Day 5

1 0 0 0 2 0 10 0 3 0 0 20 4 0 10 20 6 20 80 - 8 100 - -

For each time period 10 animals were incubated in anoxic conditions at 22ºC.

30 3.2. Adenosinergic modulation of neuronal activity

I used whole-cell patch-clamp techniques and fluorescence microscopy to measure

changes in neuronal electrical properties and intracellular calcium levels in response to adenosine

receptor modulation (vertebrate A1R subclass-like) using neurons from cluster F of the left or

right pedal dorsal ganglion (LPeDG or RPeDG) within the central ring ganglia of L. stagnalis.

Recordings were restricted to cluster F of the PeDG because they have similar

electrophysiological properties and their action potential shape has been well characterized

(Kyriakides et al., 1989). The majority of neurons tested were spontaneously active, showing

APs along with excitatory and inhibitory postsynaptic potentials at their resting membrane potential. Modulation of neuronal activity was determined by changes in AP frequency and resting membrane potential (Vm). In cells obtained during the summer (May to August),

adenosine-mediated decreases in AP frequency were minute and were not statistically significant at any given concentration (Table 3-2). Significant changes in neuronal activity in response to adenosine treatment were observed during winter months (December to March), and the following results represent data collected during the wintertime.

Under control conditions, there was no significant change in AP frequency for up to 60 min. AP frequency remained stable: 1.77±0.56 spikes s-1 at 30 min and 1.72±0.39 spikes s-1 at

60 min (Fig. 3-1A,B; N=8). Similarly, no significant change in Vm occurred during the first (-

64.9±3.1 mV) and second (-64.2±3.1 mV) halves of the recording period (Table 3-3; N=8).

Application of 200 µmol l-1 adenosine caused a significant decrease in AP frequency from

1.08±0.22 to 0.57±0.14 spikes s-1, a 47% reduction (Fig. 3-2A and Fig. 3-3; N=14). AP

frequency during the recovery period increased to 0.78±0.16 spikes s-1 and was not statistically different from the control recording prior to the adenosine perfusion (Fig. 3-2B and Fig. 3-3;

31 N=14). Adenosine application also hyperpolarized Vm from -63.8±3.6 to -70.5±4.6 mV (Table

3-3; N=14). An adenosine concentration of 200 µmol l-1 was chosen because this was the lowest

concentration that resulted in changes in Vm and AP frequency. Cells did not respond to 50 or

100 µmol l-1 adenosine, but did respond to 200, 500 µmol l-1 and 1 mmol l-1 (Fig. 3-4).

To determine whether changes in neuronal activity caused by adenosine were A1R-

mediated, AP frequency and Vm were analyzed in the presence of the A1R agonist CPA.

Addition of 100 µmol l-1 CPA to the bulk perfusion resulted in a 43% reduction of AP frequency,

from 1.11±0.27 to 0.63±0.11 spikes s-1 (Fig. 3-2C and Fig. 3-3; N=7). As with adenosine

application, AP recovered to 0.71±0.11 spikes s-1 after CPA addition, and was not significantly

different from the control value prior to treatment onset (Fig. 3-2C and Fig. 3-3; N=7). However, an all pair-wise post-hoc test revealed that only recovery from the adenosine treatment was significantly different from adenosine treatment alone. This may be the result of tighter binding of the pharmacological modulators and a requirement for a longer washout period. CPA application also hyperpolarized Vm, from -72.4±2.6 to -76.9±2.1 mV (Table 3-3; N=7). The

A1R-mediated change was further tested by incubation of the preparation with the A1R

antagonist DPCPX prior to and during adenosine application. Addition of adenosine to DPCPX-

treated neurons caused no significant change in AP frequency (from 1.09±0.06 to 0.81±0.03

-1 spikes s ; Fig. 3-2D and Fig. 3-3; N=6) or Vm (from -56.4±2.8 to -60.8±3.9 mV; Table 3-3;

N=6).

The effect of adenosine on Ca2+ homeostasis of cluster F neurons was examined using the

single wavelength Ca2+ indicator fluo-4. Since neurons were not incubated with fluo-4 and the

dye was applied through a patch pipette, at least 15 min of baseline recording was required prior

to any treatment application to allow maximal loading of cells with the dye and for the

32 fluorescence trace to reach a plateau. The spontaneous activity (both AP frequency and Vm) of

these neurons did not change under control conditions similar to that observed in previous

recordings lacking fluo-4 (data not shown). Thus inclusion of the dye to the intracellular

recordings solution did not adversely affect electrical properties. Application of adenosine

provoked a significant 12.5±1.5% increase in the fluo-4 fluorescence; this response had a slow onset and it reached a maxima at ~20 min (Fig. 3-5A,B; N=4). AP frequency and fluo-4

fluorescence were then analyzed at 5 min intervals after treatment onset for 20 min to determine

whether the two measurement variables covaried. In response to adenosine perfusion,

correlation analysis revealed a linear correlation between the two variables; as Ca2+

concentration increased, AP frequency decreased. The correlation between intracellular Ca2+

concentration and AP frequency was significant (r2=0.91, P=0.0125) and a linear regression

analysis of the data resulted in a slope of -0.11 (Fig. 3-5C; N=4). Conversely, application of

adenosine to DPCPX-treated neurons caused no significant change in intracellular calcium levels

(Fig. 3-5A,B; N=4).

Table 3-2. Effects of adenosine on AP frequency measured in summer animals

AP frequency (Hz)

No. of Cells Control Control 100 µM 200 µM 500 μM 1 mM

8 0.84±0.09 0.85±0.17

7 1.36±0.18 1.12±0.23

13 1.38±0.35 1.36±0.37 10 2.22±0.74 1.95±0.64

11 0.95±0.25 0.72±0.21

Data are presented as means ± SEM. Repeated measures one-way ANOVA with a Tukey (all pairwise) post-hoc test was used to determine significance between treatments (recovery data not shown).

33 A

20 mV 5 min

2 AP Frequency (Hz) Frequency AP

0 0 10 20 30 40 50 60 70

Time (min)

Figure 3-1. AP frequency remains stable under control conditions. (A) Raw continuous trace of a recording from a cluster F neuron with a relatively low APf maintained under control conditions. A switch from control to control saline occurred at the arrow, 20 min into the recording. (B) Mean group data (n=8) showing that APf did not change significantly throughout the recording period. APf was assessed every 10 min over a 5 min interval (see Methods). APf: AP frequency.

34 A

20 mV 1 min

20 mV 20 s Adenosine

20 mV 20 s CPA

20 mV 20 s DPCPX + Adenosine

Figure 3-2. Effects of A1 receptor activation or antagonism on APf. (A) Raw continuous trace from an experiment undergoing control to adenosine transition. APf during last 5 min of control and 15 min of adenosine perfusion is shown, adenosine application began at the arrow. Raw discontinuous traces from individual experiments during control, treatment perfusion (B) adenosine; (C) CPA; (D) DPCPX + adenosine and subsequent washout. Discontinuous traces for treatment and recovery parameters are shown for the purposes of clarity and brevity because individual APs were difficult to distinguish in a highly compressed 60 min trace.

35 Figure 3-3. A1 receptor-mediated decrease in APf. Summary of changes in APf during control, treatment with adenosine and/or adenosine receptor modulators, and recovery following treatment. Variance within control values was determined by comparison with a five minute recording period preceding the control interval. Data represent 6 to 14 replicate experiments and are expressed as means ± SEM. Asterisks indicate data significantly different from control values (P<0.05; one-way RM ANOVA). APf: AP frequency. Table 3-3. Effects of adenosine, CPA and DPCPX on membrane potential of neurons from winter animals

Membrane Potential Vm (mV)

No. of Control Control Adenosine DPCPX + CPA Cells Adenosine

8 -64.9±3.1 -64.2±3.1 14 -63.8±3.3 -70.5±4.6*

6 -56.4±3.8 -60.8±3.9 7 -72.4±2.6 -76.9±2.1*

Values are means ± SEM. Asterisk indicates significant difference from control value (P<0.05; paired t-test).

-20

-40 (% of baseline activity) of baseline (% AP f -60

-80 0 200 400 600 800 1000 1200 [Adenosine] uM

Figure 3-4. Concentration-response curve for adenosine effect on APf (normalized values in percent of control). 5 min of control recording immediately prior to treatment onset was used to determine changes in APf with adenosine perfusion. Data are presented as means ± SEM. Each point represents results of 4 to 14 separate experiments.

2+ Figure 3-5. Effects of adenosine on [Ca ]i in cluster F neurons. (A) Raw data traces of fluo-4 fluorescence changes from neurons undergoing treatments as specified by the solid bars under the individual traces. Each trace represents the change in a single neuron. Note that the Ca2+ signal does not increase indefinitely, but reaches a plateau towards the end of the recording. (B) Summary graph of normalized changes in fluo-4 fluorescence from neurons undergoing A1R antagonism with DPCPX (n=4) or a control to adenosine transition (n=4). Data are expressed as means ± SEM. The fluo-4- 2+ induced Ca signals were quantified as∆F/F o multiplied by 100%. Asterisk indicates significant change from baseline following treatment onset (P<0.05; paired t-test). (C) 2+ Upon adenosine perfusion, AP frequency varies linearly with [Ca ]i (n=4). Error bars are obscured by symbol in some cases (Y=2.45).

38 3.3. GABAergic modulation of neuronal activity

To examine the effect of GABA on neuronal activity, intracellular recordings using sharp

electrodes were made from non-identified cluster F neurons on the dorsal surface of either the L-

or RPeDG due to their close proximity to GABAergic neurons (Hatakeyama and Ito, 2000). In

the majority of neurons obtained during the fall (October and November), GABA decreased

neuronal activity by hyperpolarizing Vm and inhibiting electrical firing. Application of 500 µmol l-1 GABA caused a significant decrease in AP frequency from 1.15±0.16 to 0.17±0.08 spikes s-1

(Fig. 3-6A and Fig. 3-8; N=21). AP frequency during the recovery period increased to 1.18±0.18

spikes s-1 and was not statistically different from the control recording prior to GABA perfusion

(Fig. 3-8; N=21). GABA application also led to a significant hyperpolarization of Vm from -

63.8±2.4 to -76.8±2.6 mV, while Vm after GABA washout was not significantly different from

the control period (Table 3-4). To determine whether the changes in neuronal activity caused by

GABA were GABAA receptor-mediated, neurons were incubated with the GABAA receptor

antagonist bicuculline prior to and during GABA application. Addition of GABA to bicuculline-

treated neurons caused no significant change in AP frequency (from 1.71±0.24 to 1.64±0.41

-1 spikes s ; Fig. 3-6B and Fig. 3-8; N=5) or Vm (from -55.4±3.6 to 53.7±3.1 mV; Table 3-4). The

depressive effects of GABA were maintained when polysynaptic transmission was inhibited with

the use of high divalent cation saline (data not shown), indicating that the observed changes in

neuronal activity were not due to non-specific effects of GABA on other neurons in the CNS

(Nesic et al., 1996).

In contrast, GABA perfusion onto the majority of neurons obtained during the winter

months (December-March) potentiated neuronal activity. Application of 500 µmol l-1 GABA

caused a significant increase in AP frequency from 1.74±0.25 to 2.81±0.40 spikes s-1 (Fig. 3-7A

39 and Fig. 3-8; N=22) and depolarization of Vm from -62.5±2.4 to -54.7±2.6 mV (Table 3-4). AP

frequency and Vm during the recovery period were not statistically different from the control

recording prior to GABA application (Fig. 3-8 and Table 3-4). GABA also initiated spiking in

electrically quiescent neurons that exhibited EPSPs but failed to reach AP threshold at the resting

membrane potential (data not shown). The excitatory actions of GABA were also mediated

through the GABAA receptor as perfusion of GABA onto bicuculline-treated neurons failed to elicit an excitatory response (from 1.34±0.32 to 1.31±0.31 spikes s-1; Fig. 3-7B, Fig. 3-8, and

Table 3-4; N=5). Thus, a GABA-mediated inhibition to excitation trend was observed in neurons obtained in the fall compared to winter months (Fig. 3-9) and both excitation and inhibition were mediated through the GABAA receptor (Fig. 3-8).

Since both excitatory and inhibitory actions of GABA were mediated through the same

receptor, I tested the contribution of NKCC1 and KCC2 in rendering GABA excitatory and

inhibitory, respectively. To perturb intracellular chloride concentrations, neurons obtained

during the winter months were incubated with the NKCC1 inhibitor bumetanide. AP frequency

was not statistically different during 100 μmol l-1GABA application in bumetanide-treated neurons when compared to control conditions (from 1.62±0.58 to 1.26±0.41 spikes s-1; Fig. 3-10;

N=6). To determine the contribution of KCC2 to GABAA-receptor mediated inhibition in the

fall, neurons were perfused with furosemide during and prior to GABA application. GABA (100

µmol l-1) perfusion onto neurons incubated with furosemide failed to elicit an inhibitory response

as AP frequency was not statistically different from the control period (from 2.11±1.15 to

1.45±0.59 spikes s-1; Fig. 3-10; N=5). Since the excitatory effects of GABA were blocked by

both bicuculline and bumetanide perfusion and the GABA-mediated depression of neuronal activity was blocked by furosemide and bicuculline treatment, it is likely that the relative activity

40 and/or expression levels of KCC2 and NKCC1 underlie the inhibition to excitation trend observed in fall and winter neurons, respectively.

A Control

20 mV 10 s GABA B 20 min Bicuculline treatment

20 mV 10 s GABA

Figure 3-6. Effects of GABAA receptor activation or antagonism on AP frequency in neurons obtained during the fall months. (A) Raw trace demonstrating the change in spike frequency in response to application of GABA (at bar) onto the central ring ganglia. An ~ 10 mV hyperpolarization of Vm was observed. Dotted line represents the resting membrane potential recorded 1 min prior to GABA perfusion. (B) Raw trace of a neuron incubated with the GABAA receptor antagonist bicuculline followed by GABA application (at bar).

Figure 3-7. Effects of GABAA receptor activation or antagonism on AP frequency in neurons obtained during the winter months. (A) Raw trace demonstrating the change in spike frequency in response to application of GABA (at bar) onto a cluster F neuron. Vm depolarized by ~ 7 mV. Dotted line represents the resting membrane potential observed 1 min prior to GABA perfusion. (B) Raw trace of a neuron incubated with the GABAA receptor antagonist bicuculline followed by GABA application (at bar).

Figure 3-8. GABAA receptor-mediated decrease and increase in spike frequency in fall (F) and winter (W) groups, respectively. Summary of changes in AP frequency during control, treatment with GABA and/or GABAA receptor antagonist bicuculline (Bic), and recovery following treatment. Variance within control values was determined by comparison with a 1 min recording period preceding the control interval. Data represent 6-22 replicate experiments and are expressed as means ± s.e.m. Asterisks indicate data significantly different from control values (P<0.05; one- way RM ANOVA).

43 Table 3-4. Effects of GABA and GABAA receptor antagonism on membrane potential in fall (F) and winter (W) groups

Control GABA (F) GABA+Bic (F) GABA (W) GABA+Bic (W)

-63.8±2.4 -76.8±2.6* -55.4±3.6 -53.7±3.1 -62.5±2.4 -54.7±2.6* -64.2±3.8 -65.7±3.8 Values are means ± s.e.m. Asterisk indicates significant difference from control value (P<0.05). Repeated measures one-way ANOVA with a Holm-Sidak post-hoc test was used to determine significance between treatments (recovery data not shown). Membrane potential is given in mV.

Figure 3-9. Seasonal changes induce an inhibition to excitation trend in neurons obtained during the fall and winter months, respectively. GABA application in the majority of neurons (>65%) obtained in winter (Dec-Mar) elicited an excitatory response while depression of electrical activity was observed in neurons tested in the fall (Oct and Nov).

44 Figure 3-10. Pharmacological inhibition of cation-chloride co-transporter activity blocks GABAA receptor-mediated changes in spike frequency. Addition of GABA in the presence of NKCC1 antagonist bumetanide (BMT) failed to increase AP frequency in neurons that initially responded with elevated spike frequency to GABA application alone. Perfusion of KCC2 inhibitor furosemide (FRS) blocked the GABA-mediated depression in AP frequency observed in neurons obtained during the fall months. Data represent 5-6 replicate experiments and are expressed as means ± s.e.m. RM one-way ANOVA was used to determine significance between treatment groups.

45 Chapter 4: DISCUSSION

4.1. Anoxia tolerance

I found that snails only survived 4 hours of complete anoxia at room temperature.

Contrary to the findings of Wijsman et al. (1985), who reported the anoxic tolerance of L.

stagnalis at 20°C to be 40 hours. I observed similar behavioural changes after anoxic incubation

as Wijsman et al. (1985); exploratory behaviour stopped and snails became completely

motionless within an hour. Anoxic incubation of longer than 4 hours led to significant mortality

assessed 24 hours after being transferred to normoxic conditions. I attribute the disparity of

these findings to the inability of Wijsman et al. (1985) to achieve complete anoxia in the aquaria.

It is unlikely that the large discrepancy between the results of this study and that of Wijsman et

al. (1985) is due to differences in the age of the animals because both studies used 2-3 month old

snails. I conclude that L. stagnalis is not anoxia-tolerant and it would be more accurate to

categorize L. stagnalis as hypoxia-tolerant.

4.2. Modulation of neuronal activity by adenosine

4.2.1. Summary of findings

I examined the neuromodulatory effects of adenosine in the central ring ganglia of the

pond snail L. stagnalis. In response to application of adenosine, cluster F neurons exhibited an

11% hyperpolarization of resting membrane potential along with a 47% decrease in action

potential firing frequency. To further explore this possibility, I used a pharmacological A1R

agonist and antagonist to determine changes in neuronal electrical properties. CPA and DPCPX

were selected because of their relative A1R specificity and because the latter has previously

proved effective as an A1R antagonist in invertebrate preparations (Magazanik and Federova,

2003). When A1-receptor mediated neurotransmission was antagonized with DPCPX, adenosine

46 failed to elicit a robust inhibitory response. We further demonstrated that the inhibitory effects

of adenosine are mediated via the A1 receptor, by showing that the response is CPA sensitive.

CPA caused a reduction in AP frequency and hyperpolarization of Vm that were comparable to

those elicited by adenosine. The inhibitory actions of adenosine are consistent with a previous

investigation (Barraco and Stefano, 1990), which showed that adenosine inhibited the release of

excitatory neurotransmitters including serotonin and dopamine from the pedal ganglia of the

marine bivalve Mytilus edulis, whereas an adenosine antagonist blocked the inhibitory effects on

neurotransmitter release. The agonist and antagonist specificity as well as the reversibility of the

depression in neuronal activity upon reperfusion with control saline suggest that this

phenomenon is receptor-mediated.

4.2.2. Mechanisms of adenosine-mediated depression

Depression of neurotransmitter release (glutamate in particular) by activation of

adenosine receptors is achieved through G-protein-coupled inhibition of Ca2+ influx in nerve

endings (Fredholm and Dunwiddie, 1988; Wu and Saggau, 1997) along with enhancement of K+

and Cl- conductance (Trussel and Jackson, 1985; Mager et al., 1990). Adenosine receptor

activation may also cause an increase in cytosolic Ca2+ levels via an inositol 3-phosphate

(Ins(3)P)-mediated pathway. All adenosine receptors except the A2A subtype activate phospholipase C (PLC), an enzyme converting phospholipids into diacylglycerol and Ins(3)P

(Bickler and Buck, 2007; Dunwiddie and Masino, 2001). Ins(3)P receptor activation is

consistent with the elevation of intracellular Ca2+ observed in cluster F neurons upon perfusion

with adenosine. Furthermore, a strong correlation was observed between elevated cytosolic Ca2+

and decreasing AP frequency. The reversibility of this phenomenon could not be tested because

of technical limitations (whole cell patches were difficult to maintain beyond 60 min). Despite

47 this shortcoming, it is clear the Ca2+ increase was adenosine dependent because incubation with

DPCPX failed to elicit a response in the presence of adenosine.

Unlike the deleterious accumulation of Ca2+ that occurs during ECD in response to lack of oxygen in hypoxia-sensitive species (Choi, 1994; 1992), minute increases in cytosolic Ca2+

levels can be neuroprotective in certain anoxia-tolerant species (Bickler et al., 2000; Shin et al.,

2005). One of the outcomes of elevated Ca2+ is inactivation of N-methyl-D-aspartate receptors

(Ehlers et al., 1996) that are Ca2+ permeable glutamate-gated ion channels regulated by

intracellular Ca2+ and required for fast excitatory neurotransmission in the central nervous

system (Albensi, 2007). It has been well established that through its actions on presynaptic

receptors, adenosine depresses the activation of NMDA receptors due to reduced

neurotransmitter release (Poli et al., 1991). Signaling through the A1 receptor also results in

inhibition of NMDA receptor-mediated excitatory postsynaptic potentials and currents by a

postsynaptic mechanism (De Mendoca and Ribiero, 1992; De Mendoca et al., 1995). Moreover,

adenosine A1 receptor agonism also attenuates NMDA receptor-mediated excitotoxicity (Finn et al., 1991). Thus, the adenosine-mediated decrease in AP frequency and hyperpolarization of Vm

along with a simultaneous increase in Ca2+ levels suggest a possible synergistic mechanism resulting in global depression of neuronal activity.

4.2.3. Seasonal differences in adenosinergic neurotransmission

L. stagnalis is a pulmonate that hibernates during the winter (Jones, 1961). Tolerance of variable oxygen concentration is often associated with inactivity and hypometabolism induced by cold temperatures. There is a “hemispheric asymmetry” in cold tolerance amongst species that frequently undergo freeze-thaw events (Sinclair et al., 2003; Chown et al., 2004). Because these events occur year-round in high latitudes of the Southern Hemisphere, endemic freeze-

48 tolerant species retain their ability to survive cold temperatures throughout the year without

energetically demanding metabolic shifts. By contrast, temperatures in high-latitude areas of the

Northern Hemisphere remain sub-zero for several months during the winter and rise well above

freezing for long periods during the summer. Consequently, native species lose their cold

tolerance in the summer, but show exceptional freeze tolerance in winter that requires costly

physiological adaptations. These differing strategies amongst freeze-tolerant species in the

Southern and Northern hemispheres raise the possibility that hypoxia tolerance of L. stagnalis

may have a seasonal dependence. Furthermore, it might explain our observation of diminished

responsiveness to adenosine modulation in neurons obtained during the summer months (May to

August). Sensitivity of L. stagnalis neurons to seasonal changes has been reported previously.

Zapara et al. (2004) found that neurons obtained during summertime exhibited a depolarized resting membrane potential (anoxic depolarization) in response to ischemia and/or hypoxia, whereas those obtained during the winter showed resistance to anoxic depolarization. More research is required to elucidate the mechanisms that render neurons more sensitive to adenosine regulation during winter (December to March) than during summer.

4.3. Modulation of neuronal activity by GABA

4.3.1. Summary of findings

In the mammalian central nervous system, GABA is the principal inhibitory

neurotransmitter, although in certain cases its actions can also be excitatory. GABAA receptor activation gives rise to fast postsynaptic responses lasting up to 4 milliseconds by the transmembrane flow of chloride ions through its channel pore. Electrophysiological studies utilizing Xenopus laevis oocytes injected with cloned homo-oligomeric molluscan GABA receptor from cDNA showed that the receptor is permeable to chloride and reversibly blocked by

49 both bicuculline and picrotoxin (Bhandal et al., 1995). Immunohistochemistry studies found a widespread distribution of GABAergic cell bodies in all ganglia within the adult CNS of L. stagnalis (Hatakeyama and Ito, 2000).

The work presented here examined the effects of GABA on electrical activity of cluster F neurons found on the dorsal surface of the right and left pedal ganglia. Clusters of GABA-like immunoreactive cell bodies that make extensive connections to adjacent ganglia were largely found on the dorsal edge of the right or left pedal ganglion (Hatakeyama and Ito, 2000), thus we restricted our experiments to specifically this area (although identified neurons L- and RPeD1 were not used in this study). We found that GABA hyperpolarized the resting membrane potential and inhibited action potential firing in the majority of neurons obtained in the fall. This inhibitory effect of GABA on neuronal activity failed to occur when GABAA receptor neurotransmission was blocked by a pharmacological antagonist. These results are consistent with those previously reported by Molnar et al. (2004) and Moccia et al. (2009). Molnar et al.

(2004) performed experiments on RPeD1 using the isolated suboesophageal ring, similar to the preparation used in our study. However, they collected their animals during the spring and summer months and used 2.5 mmol l-1 KCl electrode solution to record neuronal activity.

Moccia et al. (2009) used 2-3 month old snails and performed experiments on RPeD1 utilizing an isolated brain preparation. Similar to our conditions, they carried out experiments at 22°C using an electrode solution of saturated K2SO4. Both studies found inhibitory effects of GABA on neuronal activity that could be blocked by antagonism of GABAA receptor with a pharmacological antagonist.

In contrast, neurons obtained during the winter months exhibited increased action potential firing and depolarization of resting membrane potential upon GABA perfusion that was

50 reversible upon washout of the treatment saline with control saline. As with inhibitory

neurotransmission, excitatory effects were also mediated via the GABAA receptor since

potentiated neuronal activity in response to GABA failed to persist after bicuculline perfusion.

The data presented here are in accordance with those obtained by Rubakhin et al. (1996) and

Cheung et al. (2006). Rubakhin et al. (1996) performed experiments on adult animals collected

during the months of May-October. Unlike this study, they studied single neurons that were

isolated mechanically using 3 mol l-1 KCl as their electrode solution. In their conditions, GABA

depolarized the membrane of the neuron RPeD1 isolated from L. stagnalis CNS. They found

that in the majority of neurons tested, GABA-induced depolarization was associated with fluctuations in membrane conductance related to changes in chloride permeability. Similarly,

Cheung et al. (2006) found depolarizing actions of GABA using cluster F neurons under the same conditions employed in this study. Importantly, they found that the excitatory GABAergic drive was decreased via regulation of NKCC1 when neurons were treated with hypoxic saline such that depressed neuronal activity would lead to a significant reduction in energetic requirements of the brain under conditions of limited ATP stores.

Given that both the excitatory and inhibitory effects of GABA observed in this study were mediated by the bicuculline-sensitive GABAA receptor, we tested the contribution of

NKCC1 and KCC2 to GABA-mediated neuromodulation. To perturb intracellular concentrations, neurons obtained during the winter months were incubated with the NKCC1 inhibitor bumetanide. Bumetanide, a diuretic compound, has an up to 500-fold higher affinity for NKCC1 than KCC2 (Payne et al., 2003). Similar to GABAA receptor antagonism with bicuculline, bumetanide blocked the excitatory effects of GABA in neurons obtained during the winter months. This phenomenon is similar to that observed by Cheung et al. (2006), these

51 authors found that bumetanide perfusion alone was sufficient to cause a significant decrease in action potential firing, consistent with bumetanide being a potent blocker of NKCC1 in pulmonate neurons.

Next, we tested whether pharmacological inhibition of KCC2 transporter activity would have an effect on GABA-mediated depression of neuronal activity observed in the fall. To this end, we used furosemide, another diuretic compound frequently employed as a KCC2 cotransporter antagonist. Furosemide is used at concentrations of 100 µmol l-1 or higher to inhibit KCC2 since it blocks both NKCC1 and KCC2 with equal potency (Blaesse et al., 2009).

As with NKCC1 inhibition, GABA perfusion onto neurons incubated with furosemide failed to effect neuronal activity in a manner similar to GABA perfusion alone. A confounding effect of furosemide is the simultaneous inhibition of NKCC1, such that even 100 µmol l-1 concentrations do not provide a complete block of KCC2. Furthermore, results obtained with furosemide use alone cannot be considered conclusive, as it is not a pharmacologically specific compound. For example, it has inhibitory effects on both NMDA and GABAA receptors (Staley, 2002), thus the observed changes may be due to non-specific effects of furosemide on other ionotropic receptors.

4.3.2. Regulation of cation-chloride cotransporter function

Since the excitatory effects of GABA were blocked by both bicuculline and bumetanide perfusion and the GABA-mediated depression of neuronal activity was blocked by furosemide and bicuculline treatment, it is likely that the relative activity and/or expression levels of KCC2 and NKCC1 underlie the inhibition to excitation trend in a subset of cluster F neurons observed during fall and winter neurons, respectively. Kinetic activity of CCCs is predominantly regulated by phosphorylation mechanisms. Studies of chloride transport using shark rectal glands demonstrate that phosphorylation activates NKCC1 (Lytle and Forbush, 1992), such that

52 decreases in intracellular chloride concentrations below a homeostatic “set point” lead to direct

phosphorylation and activation of NKCC1, resulting in increased intracellular chloride

concentrations (Blaesse et al., 2009; Russell, 2000).

Data on KCC2 remains inconclusive since several studies contradict the established perspective that phosphorylation inhibits KCC2 function. Vale et al. (2005) suggest that KCC2 is anatomically present but functionally inactive during development, only becoming active when inhibitory synaptogenesis begins to take place. This suggestion finds support in their finding that the phosphorylation dynamics of KCC2 change from early to postnatal development, with the phosphorylated state of the protein being the active form later in development, coinciding with the emergence of inhibitory transmission. Similarly, Wake et al. (2007) found that tyrosine phosphorylation plays a pivotal role in the maintenance of KCC2 activity and kinetic regulation. Using rat hippocampal neurons, they found dephosphorylation of KCC2 correlated with decreases in its transport activity and surface expression, along with reduced intraneuronal chloride concentrations. Contrarily, in situ hybridization shows that transporter mRNA is present and Western blot analysis show that phosphorylated KCC2 is also present early in development before the GABA switch from excitation to inhibition has occurred, suggesting the preponderance of transcriptional upregulation of KCC2 over endogenous phosphorylation by tyrosine kinases (Stein et al., 2004). There is consensus, however; that direct phosphorylation by protein kinase C regulates KCC2 by strengthening the surface stability of the phosphorylated protein (Lee et al., 2007; Banke and Gegelashvili, 2008). These studies do not provide confirmation of whether changes in activation or inactivation of KCC2/NKCC1 demonstrate alterations in intracellular trafficking of proteins to the plasma membrane or their rate of ion movement (Blaesse et al., 2009). Further, it is difficult to speculate whether mechanisms

53 regulating cotransporter activity and/or expression in mammalian neurons also apply to

invertebrate neurons.

4.3.3. Seasonal differences in GABAergic neurotransmission

Other considerations in our study beyond the regulation of CCC activity are the environmental stimuli associated with seasonal changes that would lead to such an effect. These can include many variables, of which temperature, ambient oxygen tension, and photoperiod appear to be the most important. Temperature-dependent variations in mechanisms that provide protection from hypoxia and ischemia have been reported in hibernating ground squirrels

(Frerichs and Hallenbeck, 1998). Dynamics of antioxidant regulation in anoxia-tolerant turtles also have a temperature and seasonal dependence (Perez-Pinzon and Rice, 1995). Specifically, it was found that cerebral antioxidants such as ascorbic acid and glutathione, were lower in turtles exposed to colder temperatures reflecting the depressed state of metabolism during winter hibernation. Similarly, histochemical changes in serotonin and bioactive peptides including

Substance P and endothelin have also been reported in the CNS of hibernating Helix aspersa

(Bernocchi et al., 1998). Because temperature and ambient oxygen are kept constant in our laboratory conditions, it is likely that variation in photoperiod associated with seasonal changes is leading to the plasticity observed in GABAergic transmission.

This polarity in GABAergic transmission accompanies seasonal changes in the behaviour of L. stagnalis, as these molluscs hibernate during the winter under ice-covered lakes (Jones,

1961). It is well known that animals are deprived of several external sensory stimuli during hibernation and do not engage in important behaviours such as, feeding, locomotion, and copulation. Exposure of L. stagnalis to hypoxia results in near cessation of heart rate, depressed food consumption (Wijsman et al., 1985), decreased ventilation (Taylor et al., 2003), suppressed

54 motor behaviour (Silverman-Gavrila et al., 2009), delayed response to light stimuli (Fei and

Feng, 2008), and reduced righting movement (Fei et al., 2007). With regard to these behaviours and the general hypometabolic state characteristic of organisms that routinely experience hypoxia, excitatory actions of GABA, especially during hypoxic exposure, might appear contradictory. However, from an energetic standpoint, extruding large amounts of chloride to allow for hyperpolarizing GABA responses in concert with exporting sodium is more expensive than transporting it into the cell (Ben-Ari, 2002). In fact, low intracellular chloride concentrations are considered atypical in comparative cellular physiology as the evolutionary trade-offs for this mechanism include, but are not restricted to, a reduced capacity for the control of intracellular pH and cellular volume (Rivera et al., 2004).

4.3.4. Physiological significance of excitatory GABA

Excitatory actions of GABA that mainly function to generate giant depolarizing potentials (GDPs) are considered primitive patterns of electrical activity that are conserved throughout developmental evolution. It has been suggested that depolarizing effects of GABA underlying GDPs carry limited informational content that is later replaced with behaviourally relevant patterns of activity (Ben-Ari, 2001). Depolarizing actions of GABA are observed in peripheral neurons, and higher intracellular chloride concentrations are also found in cardiac cells, along with muscular and digestive systems (Hume et al., 2000; Liu et al., 1987;

Baumgarten and Fozzard, 1981). These findings have led to the suggestion that high intracellular chloride levels might serve an evolutionary conserved feature for purposes other than GABA excitation (Ben-Ari, 2002). For example, immature neurons that exhibit depolarizing GABA actions are also very tolerant of anoxia (Krnjevic et al., 1989). Furthermore, inhibitors of oxidative phosphorylation induce an increase in intracellular chloride concentrations

55 reflecting the ability of mitochondria and cell metabolism to regulate chloride homeostasis

(Garcia et al., 1997). Oxidative stress and hyperexcitability also lead to dephosphorylation of

KCC2, resulting in increased intraneuronal chloride levels due to decreased activity, mRNA and

surface expression of the cotransporter (Wake et al., 2007). Similar mechanisms indicating

variations in ambient oxygen levels might cause changes in cotransporter activity leading to

excitatory GABAergic transmission observed in L. stagnalis during the winter months.

4.3.5. Conclusions and future directions

I have demonstrated that adenosine depresses neuronal activity in cluster F neurons of L. stagnalis via the A1R (type 1 subclass) and this inhibition correlates with changes

in intracellular Ca2+. Additional experiments are required to ascertain if the adenosine response is mediated entirely through the A1R, by testing the effects of type 2 receptor modulators.

Moreover, it remains to be determined whether adenosine confers protection during severe hypoxia and whether this is also mediated via changes in cytosolic Ca2+ levels.

While examining the effect of GABA on neuronal activity, I found that ionic plasticity of

GABAergic neurotransmission in adult L. stagnalis CNS is seasonally dependent. Inhibitory and

excitatory effects of GABA in the fall and winter months, respectively, are mediated via changes

in the relative expression and/or activity of NKCC1 and KCC2. Perturbing intracellular chloride

concentrations with bumetanide blocked the GABAA receptor-mediated excitation in neuronal

activity, consistent with GABA being an excitatory neurotransmitter in L. stagnalis during the

winter months. Similarly, reducing the activity of KCC2 with furosemide occluded GABAA

receptor-mediated depression of neuronal activity, suggesting that KCC2 activity underlies the

inhibitory response of cluster F neurons to GABA in the fall. It remains to be determined

whether manipulation of environmental stimuli (e.g. photoperiod) can induce changes in CCC

56 activity. Finally, use of voltage-clamp and/or perforated patch techniques to measure GABA- mediated currents or fluorescent chloride imaging would provide for direct measurement of intracellular chloride concentrations and confirm the seasonal plasticity of the response.

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