Astrocytes Role in Lipid Mediated Synaptic Activity By Nathan Anthony Smith Submitted in Partial ...

 

Astrocytes Role in Lipid Mediated Synaptic Activity

By Nathan Anthony Smith Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

Supervised by Professor Maiken Nedergaard Neuroscience School of Medicine and Dentistry University of Rochester Rochester, New York 2013

ii  

I dedicate this work to my family, especially my mother and grandmother because I would not be where I am today if it were not for their love and support.

iii  

Biographical Sketch The author was born in Baton Rouge, Louisiana. He attended Xavier University of Louisiana, and graduated with a Bachelor of Science Degree in Biology Pre-Med. He began his doctoral studies in the Neuroscience at the University of Rochester in 2005. He was award a National Institutes of Health Training Grant in 2007 and a National Institutes of Health Training Grant in 2011. He was awarded Masters of Science degree from the University of Rochester in 2010. He pursed his research in cellular neuroscience under the direction of Professor Maiken Nedergaard. The following publications were a result of work conducted during doctoral study: Smith NA, Wang F, Xu Q, Fujita T, Baba A, Matsuda T, Takano T, Bekar L, Nedergaard M (2012) Astrocytes modulate neural network activity by Ca(2)(+)-dependent uptake of extracellular K(+). Science signaling 5:ra26. Lovatt D, Xu Q, Liu W, Takano T, Smith NA, Schnermann J, Tieu K, Nedergaard M (2012) Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity. Proc Natl Acad Sci U S A 109:6265-6270. Fujita T, Williams EK, Jensen TK, Smith NA, Takano T, Tieu K, Nedergaard M (2012) Cultured astrocytes do not release adenosine during hypoxic conditions. J. Cereb. Blood Flow Metab 32:e1-7. Kaback A, Soung DY, Naik A, Smith N, Schwarz EM, O’Keefe RJ, Drissi H (2008) Osterix/Sp7 Regulates mesenchymal stem cell mediated endochodral ossification. Journal of Cellular Physiology 214:173-82. Wang YJ, Zhou CJ, Shi O, Smith N, Li TF (2007) Aging delays the regeneration process following sciatic nerve injury in rats. Journal of Neurotrauma. 24(5):885-94. Smith N, Dong Y, Lian JB, Prata J, Kingsley PD, Wijnen AJ, Stein JL, Schwatz EM, O’Keefe RJ, Stein GS, Drissi H (2005) Overlapping Expression of Runx 1 (Cbfa2) and Runx2 (Cbfa1) transcription factors support cooperative induction of skeletal development. Journal of Cellular Physiology 203: 133-143.

iv  

Acknowledgments I would like to thank my advisor, Dr. Maiken Nedergaard, for her unwavering support and constant guidance. I would also like to thank my committee members, Drs. Kerry O’Banion, Jian Kang, and John Olschowka. An additional thanks to Drs. Kim Tieu,

Edward Vates, and Hicham Drissi for their strong letters of support. I would also like to give a special thanks to Drs. Takahiro Takano and Lane Bekar for their constant support and training throughout my graduate career. I also extend my sincerest appreciation to all of laboratory colleagues in the Center for Translational Neuromedicine. My sincerest gratitude goes out to my funding sources from the National Institutes of Health for National Service Research Award and Neuroinflammation and Glial Cell Biology Training Grant. I am very thankful for all the love and support I received from my family. Most importantly, I am extremely grateful to both my mother and my grandmother who have been a driving force in my life for years and for teaching me that education is the key to achieve all of my dreams.

v  

Abstract Astrocytes are a major cell type in both the human and rodent central nervous systems. Due to their inability to be electrically excited, astrocytes have been viewed as supportive cells providing a suitable environment for neuronal signaling without participating in information processing. This simplistic view has been overturned in the past few decades by evidence showing that astrocytes can respond directly to local neuronal activity with subsequent modification. Neurotransmitters such as glutamate can initiate intracellular Ca2+ signaling in astrocytes that leads to astrocyte release of gliotransmitters, including glutamate, D-serine, ATP or endocannabinoids, which can modulate nearby synaptic strength. Moreover, astrocytes release Arachidonic Acid metabolites, such as PGE2, modulating local blood flow to meet the energy demands of increased neuronal activity. Observations in our lab have demonstrated that vasodilation is seemingly dissociable from astrocytic calcium dynamics in many cases, thus prompting us to test whether astrocytes, through lipid release, can signal on a faster signaling scale in a calciumindependent manner. Thus far, virtually all studies looking at astrocytic signaling mechanisms focus on intracellular Ca2+, which is on a signaling time scale of seconds. However, astrocytes are capable of Ca2+ independent signaling that is potentially on a time scale one to two orders of magnitude faster (msec). In line with this, astrocytes possess Ca2+-independent PLA2 that can lead to the production of AA in the absence of calcium. However, further investigation is needed to reveal the existence of Ca2+independent signaling from astrocytes and whether this can influence physiological function. We report that upon Ca2+ chelation and receptor stimulation astrocytes can

vi  

release lipid in a Ca2+ independent manner and that these lipids can indeed affect neuronal signaling. Furthermore, using a model of transient Heterosynaptic Depression (tHSD) in cortical slices, we report that astrocytic modulation of synaptic transmission occurs independent of traditional gliotransmitters, such as ATP and glutamate, but through astrocytic release of endocannabinoids. Taken together, these results provide novel insight into the controversial role of astrocytes in synaptic regulation and thereby in higher information processing.

vii  

Contributors and Funding Sources This work was supervised by a dissertation consisting of Professors Maiken Nedergaard (advisor), Kerry O’Banion of the Department of Neurobiology and Anatomy, Jian Kang of the Department of Cell Biology and Anatomy at New York Medical, and John Olschowka of the Department of Neurobiology and Anatomy. The data analyzed in the last figure of chapter 2 was provided by Dr. Fushun Wang. All other work conducted for the dissertation was completed by the student independently. Graduate study was supported by a National Institutes of Health Training Grant for Neuroinflammation and Glial Cell Biology and a National Institutes of Health National Research Service Award.

viii  

Table of Contents

Chapter 1: Introduction

1

1.1: Astrocytes are multifunctional cells

2

1.2: Astrocytic Gliotransmitters

2

1.3: Astrocytes role in Blood Flow

7

1.4: Astrocytic modulation of Synaptic Activity

10

1.5: Astrocytic Calcium Signaling

15

1.6: Astrocytic Receptor Mediated Lipid Release

16

1.7: Significance

18

Chapter 2: Calcium Independent Astrocytic Release of Lipid Modulation 20 Abstract

21

2.1: Introduction

22

2.2: Results

23

2.2.1: GPCR-mediated Ca2+-independent release of 3H-AA and/or its metabolites from astrocytic cultures

23

2.2.2: iPLA2 activity is essential for Ca2+-independent liberation

25

2.2.3: GPCR-mediated Ca2+-independent release of PGE2 from astrocytic cultures

31

2.2.4: Connexin 43 does not mediate PGE2 release

35

2.2.5: Ca2+-independent astrocytic lipid release enhances mEPSCs via Kv channels blockade

39

2.2.6: Discussion

44

ix  

Chapter 3: Astrocytic Endocannabinoids Mediate Transient Heterosynaptic Depression

48

Abstract

49

3.1: Introduction

50

3.2: Results

52

3.2.1: Astrocytes are necessary for tHSD

52

3.2.2: Group II mGluR are necessary for tHSD

56

3.2.3: Astrocytic vesicular release is not involved in

60

Group II mGluR-mediated tHSD 3.2.4: CB1R antagonists block Group II mGluR-mediated tHSD

66

3.2.5: Discussion

68

Chapter 4: General Discussion

71

Chapter 5: Experimental Procedures

82

5.1: Culture, Ca2+ imaging of cultured cells, and small interfering RNA

83

5.2: Isolation of human fetal astrocytes

84

5.3: Immunocytochemistry

85

5.4: Radiolabelling and Assessment of AA Release

85

5.5: PGE2 Release Assessment

86

5.6: Slice preparation and electrophysiology/Patch clamp

86

5.7: Animals

88

5.8: Slice preparation for tHSD

88

5.9: Electrophysiology Recording and Analysis for tHSD

88

6.0: Statistical Analysis

89

x  

References

89

List of Figures Figure

Title

Page

Figure 1.1.

Mechanisms of Gliotransmitter Release

5

Figure 1.2.

Astrocytic modulation of blood flow

8

Figure 1.3.

Astrocytic modulation of synaptic activity

13

Figure 2.1.

Agonist mediated Calcium Rises in Astrocytic Cultures

27

Figure 2.2.

GPCR-mediated Ca2+ independent release of 3H-AA and/or its metabolites from astrocytic cultures

29

Figure 2.3.

GPCR-mediated Ca2+ independent release of PGE2 from astrocytic cultures

33

Figure 2.4.

Connexin 43 does not mediate PGE2 release

37

Figure 2.5.

Ca2+-independent astrocytic lipid release enhances mEPSCs via Kv channel blockade

42

Figure 3.1.

Astrocytes are necessary for tHSD

54

Figure 3.2.

Group II mGluR are necessary for tHSD

58

Figure 3.3.

Astrocytic vesicular release is not involved in Group II mGluR mediated tHSD

62

Figure 3.4.

CB1R antagonist blocks Group II mediated MGluR-mediated tHSD

66

Figure 4.1

Astrocytic Processes

73

Figure 4.2.

Astrocytes role in lipid mediated synaptic activity

77

xi  

List of Abbreviations Ach

Acetylcholine

2AG

2-Arachidonoylglycerol

AA

Arachidonic Acid

AEA

Anandamide

Bel

Bromoenol Lactone

CMZ

Calmidazolium

CPA

Cyclopiazonic Acid

cPLA2

Ca2+ dependent Phospholipase 2

DPCPX

Dipropylxanthine

DHA

Docosahexaenoic Acid

DOX

Doxycycline

EPSPs

Excitatory Postsynaptic Potentials

fEPSPs

Excitatory Postsynaptic Potentials (field)

mEPSCs

Excitatory Postsynaptic Currents (miniature)

HSD

Heterosynaptic Depression

iPLA2

Ca2+ independent Phospholipase 2

NE

Norepinephrine

mGluR

Metabotropic Glutamate Receptors

PGE2

Prostaglandin E2

PUFA

Polyunsaturated fatty acids

tACPD

trans-1-amino-cyclopentane-1,3-dicarboxylic acid

xii  

tHSD

Transient Heterosynaptic Depression

1  

Chapter 1 Introduction

2  

1.1 Astrocytes are multifunctional cells Astrocytes are one of the major glial cells in the CNS and have long been ignored because of their inability to be electrically excitable. However, these cells are remarkably multifunctional (Ransom et al., 2003). Traditionally, astrocytes have been regarded as the supportive cells of the nervous system and their main function was to optimize the environment for synaptic transmission. Some of their supportive tasks include the control of ion and water homeostasis, and the maintenance of blood brain barrier integrity (Ballabh et al., 2004). Astrocytes are also involved in the production, removal, and breakdown of several neurotransmitters, such as glutamate and GABA (Anderson and Swanson, 2000). They have also been shown to play an important role in sequestration and/or redistribution of K+ during neural activity as well as ammonium detoxification. Intense research has shown that astrocytes respond to neuronal activity with increases in cytosolic calcium and subsequent release of gliotransmitters that modulate the activity of surrounding neurons (Haydon, 2001; Nedergaard et al., 2003; Newman, 2003). Recently, a new study revealed that astrocytes also modulate synaptic activity with the uptake of K+ due to increases in cytosolic calcium (Wang et al., 2012). More and more studies are providing evidence of astrocytes being multifunctional cells as well as being active partners in synaptic activity. 1.2 Astrocytic Gliotransmitters It is well known that astrocytes possess a wide variety of functional neurotransmitter receptors with most of these being metabotropic G-protein coupled receptors, which are positively linked to phospholipase C. It is also known that activation of theses receptors

3  

leads to increases in intracellular Ca2+ in astrocytes, which in turn causes the release of gliotransmitters, such as eicosanoids (Xu et al., 2003; Zonta et al., 2003; Takano et al., 2006), glutamate (Parpura et al., 1994; Bezzi et al., 1998; Kang et al., 1998a), D-Serine (Schell et al., 1995; Mothet et al., 2000; Yang et al., 2003), ATP (Coco et al., 2003; Hamilton and Attwell, 2010; Parpura and Zorec, 2010), TNFα (Chung and Benveniste, 1990; Beattie et al., 2002), and GABA (Angulo et al., 2008; Velez-Fort et al., 2012). Upon activation these gliotransmitters can leave astrocytes by two mechanisms (Figure 1.1). The first is a non-vesicular pathway, which consists of volume-activated anion channels (Takano et al., 2005), hemi-channels (Cotrina et al., 1998b), and P2X7 receptors (Virginio et al., 1999). The second mechanism of vesicular release has been extensively studied. The general mechanism behind vesicular release from astrocytes is as follows: vesicles are formed and released from the Golgi complex; they accumulate transmitters via transporters and go on to dock on the plasma member; intracellular rises in Ca2+ triggers exocytosis. Astrocytes were shown to possess vesicles as well as all required proteins for vesicular release of gliotransmitters. For example, astrocytic cultures provided direct evidence of Ca2+ dependent vesicular glutamate release (Bezzi et al., 2004). Later studies provided further evidence that astrocytes also express the capability for vesicular release in situ (Zhang et al., 2004a; Zhang et al., 2004b). Unlike neurons, this astrocytic form of vesicular release is very slow and depends on internal stores of Ca2+. After release, these gliotransmitters are responsible for paracrine actions on astrocytes (Guthrie et al., 1999) as well as affecting many other cell types in the CNS. Most importantly, they have also been shown to modulate blood flow (Zonta et al., 2003;

4  

Mulligan and MacVicar, 2004; Takano et al., 2006) and synaptic activity (Kang et al., 1998a; Fiacco and McCarthy, 2004; Mothet et al., 2005).

5  

Figure 1.1: Mechanisms of Gliotransmitters Release Astrocytes can release gliotransmitters through two distinct mechanisms. The first is through hemichannels, P2X7 receptors, and volume sensitive chloride channels. The second is through exocytosis of vesicles. In brief, vesicles are released from the Golgi; the released vesicles accumulate transmitter; Ca2+ increase regulates vesicles docking and exocytosis by triggering conformational changes of syntaxin, synaptobrevin, and synaptotagmin. (from Verkhratsky and Butt, 2007)

6  

Nonvesicular Gliotransmitter Release

Vesicular Gliotransmitter Release

7  

1.3 Astrocytes role in Blood Flow Because of their unique position between neurons and cerebrovascular surfaces, astrocytes have the exceptional ability to sense neuronal activity and modulate cerebral blood flow (Figure 1.2). Evidence of this phenomenon was first seen in brain slice preparations. In 2003, Zonta et al. demonstrated that electrical stimulation of cortical slices resulted in activation of astrocytic mGluR, thus triggering astrocytic release of the vasodilator AA metabolite, PGE2 (Zonta et al., 2003). However, another study showed that photolysis of Ca2+ in astrocytic processes caused astrocytes to release the vasoconstrictive metabolite, 20-HETE, leading to cerebrovasoconstriction (Mulligan and MacVicar, 2004). These conflicting results caused much debate about the exact mechanism of astrocytic modulation of blood flow. In 2006, a subsequent study shed new light on the subject by looking at astrocytic modulation of local microcirculation in vivo. This study showed that photolysis of Ca2+ in astrocytic endfeet processes led to vasodilation in the intact brain (Takano et al., 2006). Furthermore, this study also demonstrated that the activation of mGluR in turn prompts astrocytes to release vasodilator metabolites leading to increased blood flow in the barrel cortex, thus giving insight in the role of astrocytes in functional hyperemia (Takano et al., 2006). Nonetheless, there is still much work to be done to understand the exact physiological conditions in which astrocytes modulate cerebral blood flow.

8  

Figure 1.2: Astrocytic modulation of blood flow. Upon mGluR receptor activation, astrocytes can release a number of vasoactive mediators that can cause vasodilation (PGE2) or vasoconstriction (20-HETE). (from Gordon, 2011)

9  

10  

1.4 Astrocytic Modulation of Synaptic Activity Many studies have implicated astrocytes role in modulating synaptic activity through several mechanisms. They first were shown to regulate synaptic strength by controlling the concentration of neurotransmitters in the synaptic cleft via glial transporter (Iversen and Neal, 1968; Tong and Jahr, 1994). Recently, a new study revealed a novel way for astrocytes to modulate synaptic activity. It demonstrated that upon receptor stimulation and intracellular Ca2+ increases, astrocytes buffer extracellular K+ via the Na-K ATPase (Wang et al., 2012). However, the majority of studies focusing on astrocytic modulation of synaptic activity have been viewed through the release of gliotransmitter (Figure 1.3). The two gliotransmitters responsible for characterizing most of astrocytic involvement in synaptic activity are glutamate and ATP (Haydon and Carmignoto, 2006). Although most of the studies have focused on astrocytic effects at the local synapse, attention has now turned to astrocytic modulation of neural networks. Over the last decade, several studies have demonstrated that astrocytes modulate neural networks though both Heterosynaptic (HSD) and Transient Heterosynaptic Depression (tHSD). HSD is a form of intersynaptic communication in which active synapses decrease the efficacy of neighboring inactive synapses. The phenomenon of HSD is induced with tetanic or high frequency stimulation (10-100 pulses at 10-100 Hz) of one pathway, which results in the depression of another pathway. The duration of this inhibition can be seen on a scale of 5 to 10 minutes (Lynch et al., 1977; Manzoni et al., 1994). Furthermore, HSD is believed to increase contrast between potentiated and nonpotentiated inputs in the CA1 region of the hippocampus. In 1977, Gribkoff’s group

11  

was the first to show that HSD is a correlate of long-term potentiation in the hippocampus (Lynch et al., 1977). However, the exact mechanism behind HSD remained elusive for years. In 1994, HSD was first proposed to be mediated by activation of NMDA receptors on interneurons, which led to the release of adenosine inhibiting presynaptic neurotransmitter release (Manzoni et al., 1994). As research on HSD intensified, astrocytes became another important player in uncovering the mystery of HSD. Astrocytes have been shown to be closely associated with neuronal synapses (Ventura and Harris, 1999) with one rodent astrocyte domain encompassing approximately 90,000 synapses (Bushong et al., 2002; Oberheim et al., 2009). Of equal importance, synaptic activity has been shown to increase calcium signaling in astrocytes (Porter and McCarthy, 1996; Pasti et al., 1997). This rise in internal calcium has been demonstrated to lead to the release of gliotransmitters such as ATP/Adenosine, which have been linked to HSD (Zhang et al., 2003; Pascual et al., 2005; Serrano et al., 2006). A recently study from our lab showed that HSD could be due to neuronal release of adenosine rather than astrocytic release of ATP, which is later converted to adenosine (Lovatt et al., 2012). However, there is still much work to be done to understand the exact mechanism behind astrocytic modulation of HSD. On the other hand, tHSD is a rapid form of HSD. Unlike HSD, tHSD can be seen on a millisecond time scale and is only induced with mild stimulation. In addition, tHSD was also shown to be independent of NMDA or adenosine A1 receptors activation. Astrocytic modulation of tHSD was based on several lines of experimental data: 1) tHSD was absent in animals with immature astrocytes 2) tHSD was blocked by fluoroacetate, a

12  

chemical that blocks astrocytic metabolism (Fonnum et al., 1997), 3) tHSD was blocked by the connexin hemichannel antagonists, carbenoxolone and endothelin-1, and 4) tHSD was blocked by group II mGluR antagonist (Andersson et al., 2007). However, the exact mechanism of how astrocytes mediate tHSD is still largely unknown. It was postulated that tHSD is due to astrocytic glutamate release following mGluR receptor activation, and this release leads to the suppression of transmitter release from the presynaptic terminal. Gliotransmitter release, however, is a slow and unpredictable event. For example, glutamate release from astrocytes is inconsistent, triggered only by selective astrocytic stimulation and occurs with a delay of 4-20 seconds (Parpura and Haydon, 2000), which makes it very difficult to explain how astrocytes could regulate tHSD through the release of glutamate. Based on the findings of Andersson’s study reporting astrocytic modulation of fast synaptic activity and that glutamate release from astrocytes is know to be Ca2+ dependent and thus occurs on the second time scale, we surmise that a second, faster and Ca2+-independent astrocytic signaling pathway may potentially underlie tHSD.

13  

Figure 1.3: Astrocytic modulation of synaptic activity Astrocytes modulate synaptic activity through the release of gliotransmitters such as glutamate, ATP, and D-serine. A. and B.) mGluR mediated astrocytic released glutamate can increase excitability and potentate neurons by action on the pre or post synaptic membrane NR2B containing NMDA receptors. C.) GABAβ mediated astrocytic released glutamate can inhibit neuronal activity by acting on presynaptic GABA receptors on interneurons. D.) GABAβ mediated astrocytic released glutamate modulate Heterosynaptic Depression by inhibiting presynaptic glutamate receptors. E.) mGluR mediated astrocytic release D-serine modulate synaptic activity by acting on postsynaptic NMDA receptors. F.) GABAβ mediated astrocytic released ATP, which is converted into adenosine modulate Heterosynaptic Depression by inhibiting transmitter release on presynaptic membrane adenosine receptors. (from Hamilton and Attwell, 2010)

14  

15  

1.5 Astrocytic Calcium Signaling It is well established that astrocytes are nonexcitable cells because they are unable to generate action potential even though they express voltage gated ion channels. It was not until the early 90s that astrocytes were shown to exhibit a different form of excitability through receptor mediated rises in intracellular Ca2+ (Cornell-Bell et al., 1990). Later research revealed that astrocytes express a wide variety of functional neurotransmitter receptors with most of these being metabotropic G-protein couple receptors (GPCR) (Verkhratsky and Kettenmann, 1996). Of these receptors, metabotropic glutamate (mGluR 3/5) and purinergic P2Y receptors are the two major types responsible for rises in intracellular Ca2+. These receptors are positively coupled to phospholipase C (PLC). Upon activation of these GPCR, PLC leads to the hydrolysis and activation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Volterra and Meldolesi, 2005). This increase in IP3 goes on to activated the IP3R2, which is specially found in astrocytes on the endoplasmic reticulum (ER), thus increasing intracellular astrocytic Ca2+ (Volterra and Meldolesi, 2005). Once Ca2+ rises in astrocytes, it can be propagated to neighboring astrocytes by direct intracellular diffusion of IP3 via gap junctions (Charles et al., 1992), the release of ATP, which can activate P2Y receptors on neighboring astrocytes (Cotrina et al., 2000; Arcuino et al., 2002), or the release of ATP from a single astrocyte that can diffuse over a long distance (Arcuino et al., 2002; Nedergaard et al., 2003). After the ER stores are depleted of Ca2+, astrocytic store-operated Ca2+ channels (SOCC) are activated to replenish the depleted stores(Parekh and Putney, 2005).

16  

Furthermore, research in astrocytic Ca2+ signaling has intensified. More groups have turned their attention to characterizing astrocytic Ca2+ signal in the soma verses astrocytic processes. Over all, astrocytic Ca2+ signaling is considered slow and has been shown to be on a time scale of 3 to 6 seconds (Cornell-Bell et al., 1990; Wang et al., 2006). In contrast, a few studies have now demonstrated that astrocytic Ca2+ signaling can occur on a faster time scale within the processes that is within the range of hundreds of milliseconds (Winship et al., 2007; Santello et al., 2011). There is still much work to be done to figure out the spatial temporal domain of astrocytic Ca2+ signaling and what it means for astrocytic modulation of fast synaptic activity, but in the meantime, we also should investigate Ca2+ independent signaling pathways, which have the potential to modulate rapid forms of synaptic activity. 1.6 Astrocytic Receptor Mediated Lipid Release Astrocytes are capable of releasing more than traditional gliotransmitters upon receptor stimulation and increases in intracellular Ca2+. These other transmitters are lipids. Astrocytes are able to release AA in culture upon activation of muscarinic cholinergic receptors (Felder et al., 1989), adrenoceptors (Kanterman et al., 1990), thrombinactivated receptors (Sergeeva et al., 2002), metabotropic glutamate receptors (Stella et al., 1994), and ATP (Bruner and Murphy, 1990; Stella et al., 1997b; Chen and Chen, 1998). The mechanism of activation includes the formation of 1,4,5-inositol triphosphate (IP3) and diacylglycerol production, which leads to increased intracellular Ca2+ and the activation of protein kinase C (PKC), thus leading to the activation and phosphorylation of cPLA2 to go on to release AA (Dennis, 1994; Boarder et al., 1995). AA release from

17  

membrane phospholipids is regulated by the activity of phospholipase A2 (PLA2). Once Arachidonic Acid is released from the Sn2 position of the membrane by phospholipase A2, it can be metabolized into one of its many metabolites by cyclooxygenase, lipoxoygenase, or cytochrome P450 (Ackermann and Dennis, 1995). Arachidonic Acid and its numerous metabolites play a major role in both functional hyperemia and synaptic modulation. Until now all studies looking at receptor mediated lipid release from astrocytes have only focused on the Ca2+ dependent release thru cPLA2 activity, but astrocytes also express Ca2+ -independent PLA2 (Sun et al., 2005), which can cause the release of AA and/or its metabolites in the absence of Ca2+ and has the potential to initiate signaling on a faster time scale. Of equal importance, another group of lipid molecules that are released upon receptor stimulation from astrocytes are endocannabinoids (Walter and Stella, 2003; Stella, 2004; Walter et al., 2004). Endocannabinoids are derived from AA through conjugation with ethanolamine or glycerol (Rodriguez de Fonseca et al., 2005). The two major endocannabinoids found in the CNS are Anandamide (AEA) and 2-Arachidonoyl glycerol (2AG). AEA is produced by phospholipase D through a Ca2+ dependent process (Cravatt et al., 2001; Okamoto et al., 2004). 2-AG, however, is unique because it can be produced through a Ca2+ dependent process by diacylglycerol lipase or a Ca2+ independent process by lysophospholipase C (Dinh et al., 2002b; Sugiura et al., 2002; Bisogno et al., 2003). Like AA metabolites, endocannabinoids play a major role in synaptic modulation; one in particular, is their ability to inhibit neurotransmitter release. Thus, AA and its metabolites, as well as endocannabinoids, provide a novel solution to

18  

the apparent disparity in the temporal dynamics of astrocytic modulation of tHSD and astrocytic vesicular release of glutamate. 1.7 Significance Paramount to uncovering the possible mechanism through which astrocytes affect neuronal processes is the identification of different chemical messengers used by astrocytes. For example, new roles have been uncovered for lipids in modulating neuronal signaling. In the brain, the conduction of electrical impulse, synaptic function, and complex signaling pathways depends on temporally and spatially coordinated interaction of lipids (Gross et al., 2005). Since astrocytes release the majority of AA, DHA, and other lipids into the extracellular fluid (Moore, 1993), it is important to clarify the role of astrocyte lipid signaling specifically on neuronal transmission; yet these processes are poorly understood. Although studies have looked at the effects of lipid signaling on neurons, none have looked at the role of astrocytic lipid signaling specifically on neuronal transmission. Regarding lipid signaling, astrocytes have been shown in culture to release AA and/or its metabolites through receptor stimulation; however, receptor-mediated release of lipids involves rises in intracellular Ca2+. Equally important, astrocytes are also capable of Ca2+ independent lipid release. However, the physiological importance of a Ca2+ independent lipid pathway in astrocytes has yet to be explored and the role that it may potentially play in synaptic activity still remains a mystery. The lack of understanding pertaining to astrocyte and neuronal transmission warrants further investigation, which will be addressed through examining the role that astrocytes play in lipid mediated modulation of synaptic activity. In the first part of my

19  

thesis, I will demonstrate that upon Ca2+ chelation and receptor stimulation, astrocytes release lipids in a Ca2+ independent manner and that these lipids can indeed affect neuronal activity. In the second part of my thesis, I will show that astrocytes are essential for tHSD and that group II mGluR plays a very important role in astrocytic modulation of tHSD. Furthermore, I will also demonstrate that astrocytic modulation of this rapid synaptic phenomenon is not through vesicular released gliotransmitters but receptor mediated astrocytic lipids, more specifically the endocannabinoid 2AG. This study has the potential to redefine the role(s) of astrocytes in higher brain function. Moreover, this pathway will be the first described mechanism by which astrocytes regulate synaptic transmission in a Ca2+ independent manner. Since astrocytes have been implicated in a number of CNS functions, including synaptic depression, long term potentiation (LTP), and sleep, it is of utmost importance to define how astrocytes impact neural networks. This knowledge may be a key for development of novel therapeutics for treatment of diseases such as epilepsy. Targeting astrocytes may be advantageous because it may be possible to reduce side effects such as sedation and impairment of memory, compared with the traditional therapeutic pathways that directly target synaptic function.

     

20  

Chapter 2: Calcium Independent Astrocytic Release of Lipid Modulators

21  

ABSTRACT Astrocytes have in recent years been shown to actively participate in higher neural processes in the central nervous system (CNS). Most of these studies have pointed to a key role of Ca2+-dependent gliotransmitter release. However, the astrocytic fine perisynaptic processes are essentially devoid of organelles. Since mobilization of intracellular Ca2+ stores is not possible in processes lacking endoplasmic reticulum (ER), we here asked whether astrocytes could release lipid modulators through Ca2+-independent phospholipase A2 (iPLA2) activity. Our analysis showed that in response to agonist exposure, cultured astrocytes released Arachidonic acid (AA) and its metabolites, including PGE2 and that buffering of cytosolic Ca2+, which is a key element in astrocytic excitation, was linked to a sharp potentiation of lipid release. In acute brain slices, astrocytic release of PGE2 enhanced mEPSCs by inhibiting the opening of Kv channels, thus modulating neuronal excitability. This study provides the first evidence for the existence of a Ca2+-independent pathway for AA and PGE2 release in astrocytes and furthermore demonstrates a functional role for astrocytic lipid release in modulation of synaptic activity.

22  

2.1 INTRODUCTION Comprising the majority weight of the adult brain, lipids are an essential component of the phospholipid bilayer and, of that, a large percent are in the form of long chain polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA) and Docosahexaenoic Acid (DHA) (Sinclair, 1975). The liver is the major organ in the body for synthesis of AA, but the brain is also capable of producing AA and DHA from their precursor fatty acids, linoleic and linolenic acids (Dhopeshwarkar and Subramanian, 1976). Astrocytes play a pivotal role in this process. By possessing a key anatomical location between the vasculature and neurons, astrocytes receive all incoming fatty acid precursors and serve as the major site for processing essential fatty acids in the CNS (Moore, 1993). Lipids have gained much attention for their role as bioactive mediators in the CNS. Numerous studies have focused on lipids in both functional hyperemia and synaptic activity. For instance, PGE2 has been shown to be a potent vasodilator and vasoconstrictor in CNS blood flow regulation (Zonta et al., 2003; Takano et al., 2006; Dabertrand et al., 2013) as well as a regulator of membrane excitability in CA1 pyramidal neurons during synaptic activity (Chen and Bazan, 2005). Furthermore, AA and its metabolic products are important secondary messengers that can modulate the activity of a variety of ion channels (Piomelli, 1993; Meves, 1994; Horimoto et al., 1997). For example, PGE2 has been shown to suppress K+ current in sensory neurons (Nicol et al., 1997; Evans et al., 1999), and AA has also been shown to suppress Kv channels in the soma or dendrites, and therefore enhances EPSPs (Ramakers and Storm, 2002). Other lipids such as endocannabinoids can suppress neurotransmitter release from the

23  

presynaptic membrane through the activation of cannabinoid receptor 1 (CB1) (Fride, 2002; Hashimotodani et al., 2007). However, these studies focused on neuronal release of lipids and paid little or no attention to the fact that astrocytes can release lipid mediators via a receptor-mediated pathway. Since the majority of AA, DHA, and other lipids present in the extracellular fluid are produced by astrocytes (Moore et al., 1991), it is crucial to establish whether astrocytic lipid release actively modulates synaptic activity. In culture, astrocytes can release AA in a Ca2+-dependent pathway upon activation of metabotropic glutamate and P2Y purine receptors (Bruner and Murphy, 1990; Stella et al., 1994; Stella et al., 1997b; Chen and Chen, 1998). However, astrocytes also express the Ca2+-independent PLA2 (iPLA2)(Sun et al., 2005). This PLA2 isoform, like cPLA2, requires the G-protein βγ subunit for activation (Jelsema and Axelrod, 1987; Murayama et al., 1990; van Tol-Steye et al., 1999), but it does not require Ca2+ or PKC phosphorylation for further activation. iPLA2 has also been shown to release AA and DHA upon receptor stimulation in numerous cell types (Gross et al., 1993; Akiba et al., 1998; Seegers et al., 2002; Tay and Melendez, 2004). Because astrocytic Ca2+ signaling is regarded as a key step in neuroglia signaling, a possible role of iPLA2 in receptormediated astrocytic lipid release has not yet been considered to our knowledge. 2.2 RESULTS 2.2.1 GPCR-mediated Ca2+-independent release of 3H-AA and/or its metabolites from astrocytic cultures To test whether astrocytes could mediate the release of AA in the absence of intracellular calcium, we first needed to establish a method by which we could evaluate the effect of calcium independent signaling. To define and control for the effect of receptor mediated

24  

intracellular Ca2+ elevations, cultured rat astrocytes were loaded with the Ca2+ indicator Fluo 4-AM (4.5µM) for 30 minutes and then stimulated with the purine agonist ATP. ATP (100µM, n= 5) induced a prompt increase in cytosolic Ca2+ in astrocytes that was completely blocked by the specific inhibitor of the ER Ca2+ pump cyclopiazonic acid (CPA) (20µM, n= 5) or the cytosolic Ca2+ chelator 1, 2 Bus (2-aminophenoxy) ethaneN,N,N.N-tetra-acetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) (20µM, n= 5) (20µM, n= 5) (Figure 2.1). Thus, CPA and BAPTA efficiently blocked ATP mediated rises in intracellular Ca2+ in astrocytes. We next assessed Ca2+ independent release of AA and/or its metabolites using a 3H-AA assay (Figure 2.2A). Cultured rat astrocytes were pre-incubated for 24hrs with 3H-AA, followed by exposure to agonists. ATP (n= 12) on its own failed to induce the release of AA and/or its metabolites (Figure 2.2B); however, in cultures pretreated for 10 to 12 minutes with CPA (n= 12) or BAPTA-AM (n= 12), ATP evoked a robust increase in the release of AA and/or its metabolites, whereas CPA (n= 7) alone had no effect (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.2B). Similarly, we found that stimulation of metabotropic glutamate receptors (mGluR) and AMPA receptors elicited calcium independent release of AA and/or its metabolites from cultured astrocytes (Figure 2.2C). Upon stimulation with a combination of tACPD (100µM), an mGluR agonist, and AMPA (100µM), an ionotropic GluR agonist (n= 7), we observe little to no release of AA and/or its metabolites (n= 7), however, when cytosolic calcium was blocked with CPA, tACPD and AMPD (n= 7) evoked a significant increase in 3H-AA and/or its metabolites (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.2C). These observations suggest that

25  

astrocytes can release AA, a key precursor from bioactive eicosanoids, but that AA release is surprisingly inhibited by increases in cytosolic Ca2+. 2.2.2 iPLA2 activity is essential for Ca2+ independent liberation of AA. As demonstrated above (Figure 2.2B), ATP triggered little to no release of AA and/or its metabolites in astrocytic cultures. However, if cytosolic Ca2+ increases were inhibited by CPA or BAPTA-AM significant lipid release was consistently induced by receptor stimulation. To explore the mechanism of Ca2+-independent release of 3H-AA and/or its metabolites, we next evaluated whether inhibition of the Ca2+-sensitive cPLA2 or alternatively of the Ca2+-insensitive iPLA2 enzymes would reduce 3H-AA release. Astrocytes were pretreated with 10µM of Methylarachidonyl Fluorophosphates (MAFP), a nonspecific inhibitor of both cPLA2 and iPLA2 or 10µM of Bromoenol Lactone (Bel), a specific inhibitor of iPLA2. MAFP (n= 12) significantly decreased the amount of 3H-AA and/or its metabolites released in response to ATP in the presence of CPA (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.2D). The same decrease was observed in the presence of Bel (n= 12), thus affirming a role for iPLA2 in Ca2+-independent lipid release (Figure 2.2D). 3H-AA release was only observed when Ca2+ was blocked (Figure 2.2B and C), suggesting that intracellular Ca2+ must inhibit the Ca2+-independent iPLA2. Calmodulin has previously been shown to be a potent Ca2+-dependent inhibitor of iPLA2 (Wolf and Gross, 1996). To assess the interaction between calmodulin and iPLA2, the cells were treated with Calmidazolium (CMZ), an inhibitor of Ca2+/calmodulin interaction, which has been shown to remove the calmodulin block of iPLA2 (Wolf and Gross, 1996). In the presence of CMZ (2µM, n= 10), ATP led to a significant release of

26  

3

H-AA and/or its metabolites, which was comparable to blocking increases in cytosolic

Ca2+ by preloading with either CPA or BAPTA (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.2D). This observation suggests that Ca2+ acts primarily as a brake, through calmodulin, that effectively inhibits iPLA2 activity (Wolf and Gross, 1996; Wolf et al., 1997). Furthermore, these findings are also reflective of previous studies that have shown iPLA2 involvement in receptor mediated AA release in pancreatic islet cells (Gross et al., 1993), smooth muscle cells (Lehman et al., 1993), and endothelium cells (Seegers et al., 2002). Taken together, this data provides evidence of a new signaling mechanism in astrocytes through Ca2+ independent iPLA2, which has been shown to be the major isoform in the brain that accounts for 70% of PLA2 activity (Yang et al., 1999).

27  

Figure 2.1: Agonist mediated Calcium Rises in Astrocytic Cultures Cultured astrocytes were labeled with Fluo-4 AM (4.6µM) and the fluorescent changes associated with intracellular calcium measured using confocal microscopy. BAPTA-AM (20µM) and CPA (20µM) eliminated the ATP (100µM) evoked Ca2+ increases in fluorescent. MAFP (10µM), Bel (10µM), and CMZ (2µM) did not inhibit ATP (100µM) induced Ca2+rises in Astrocytes. *ANOVA followed by Tukey post-test; p < 0.05 All   bar  graphs  show  means  ±  s.e.m.

28  

Control

+ ATP

1600

F/F

1400 1200 ATP CPA

1000 800 600 400

0

20 40 60

80 100 120 140 160

time

150

+ ATP

Z CM

Be l

FP MA

pta

-A M

*

Ba

CP A

ol

* ntr

100 50 0 -50

Co

F/F

250 200

29  

Figure 2.2: GPCR-mediated Ca2+ independent release of 3H-AA and/ or its metabolites from astrocytic cultures. A.) Schematic of the 3H-AA Radioactive Assay. B.)The effects of P2YR agonist ATP (100uM) on Ca2+ independent 3H-AA and/or its metabolites release in astrocytic cultures compared to control. C.) The effects of mGluR agonists, tACPD (100µM) and AMPA (100µM), on Ca2+ independent 3H-AA and/or its metabolites release in astrocytic cultures compared to control. (D) Effects of the iPLA2 inhibitor, BEL (10µM), cPLA2 inhibitor, MAFP (10µM), or Calmodulin/Ca2+ complex inhibitor, CMZ (2µM) on Ca2+ independent 3H-AA and/or its metabolites release in astrocytic cultures compared to control. *ANOVA followed by Tukey post-test; p < 0.05. All  bar  graphs  show  means   ±  s.e.m.

30  

B.

A.

250

*

200 A

Astrocytes

*

150 100

recover.

50

PA A

A

+ Agonist/CPA or BAPTA

PA

AT P

0 + Agonist

D.

250

250

150

50

0

0

*

P AT

A

A

P

A

P

PA

*

BE L

50

PA

100

PA

100

PA /

AA

150

200

*

*

A

A

200

31  

2.2.3 GPCR-mediated Ca2+ independent release of PGE2 from astrocytic cultures. Due to the nonspecific nature of the 3H-AA radioactive assay, it was not possible to assess which specific metabolites were released upon receptor stimulation. Given that we have previously demonstrated astrocyte PGE2 release following photolysis-induced calcium uncaging (Takano et al., 2006), we here evaluated whether PGE2 could also be released via a Ca2+-independent mechanism. Using a PGE2 ELISA assay, we found that in the presence of CPA, ATP (n= 12) evoked a significant increase in the release of PGE2, whereas little to no release was observed in the presence of ATP (n=12) alone (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.3A). Using the same experimental protocol, rat astrocytic cultures were stimulated with tACPD and AMPA in the presence and absence of CPA. In the absence of CPA (n= 8), little to no release of PGE2 was observed (Figure 2.3A). However, when CPA blocked internal stores of Ca2+, tACPD and AMPA (n= 8) significantly increased PGE2 release (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.3A). To extend the observation to other species, we preformed the same experiments using human astrocytes. In the presence of CPA, ATP (n= 12) evoked a significant increase in the release of PGE2 from human astrocytes (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.3B), whereas little to no release was observed with ATP (n= 12) alone. Likewise, co-application of tACPD and AMPA showed the same effect (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.3B). Additional analysis showed that cultured mouse astrocytes similar to rat and human astrocytes, also exhibited PGE2 release upon Ca2+ chelation and ATP stimulation (n= 12)

32  

(Figure 2.3C). However, in contrast to human and rat astrocytes, mouse astrocytes also released a significant amount of PGE2 when stimulated with ATP (n= 12) in the absence of CPA or BABTA AM preloading (Figure 2.3C), as already demonstrated previously (Dennis, 1994; Stella et al., 1994; Boarder et al., 1995). These findings show that cultured mouse astrocytes express Ca2+ dependent PLA2 as opposed to cultured human or rat astrocytes.

33  

Figure 2.3: GPCR-mediated Ca2+ independent release of PGE2 from astrocytic cultures. A.) The effects of GPCR agonists ATP (100uM) and tACPD (100µM) and AMPA (100µM) on Ca2+ independent PGE2 release in rat astrocytic cultures compared to control. B.) The effects of P2YR agonist ATP (100uM) on Ca2+ independent PGE2 release in human astrocytic cultures compared to control. C.) The effects of P2YR agonist ATP (100uM) on Ca2+ independent PGE2 release in mouse astrocytic cultures compared to control. *ANOVA followed by Tukey post-test; p < 0.05. All  bar  graphs  show  means  ±   s.e.m.

C. 200 180 160 140 120 100 80 60 40 20 0

/C PA

l

tA C

200

100

0

Mouse

* *

AT P

l

nt ro

Co

* * % of control of PGE2 Release

AT P/ CP A

/A M PA /C PA

/A M PA

B.

tA CP

PD

PD

AT P

Rat 400

300

200

AT P/ CP A

D/ AM PA PD /A M PA /C PA

tA C

tA C

l

nt ro

300

AT P

nt ro

Co

400

AT P

Co

% of control of PGE2 Release

A.

% of control PGE2 Release

34  

Human

* *

100 0

35  

2.2.4 Connexin 43 does not mediate PGE2 release Hemichannels formed by Connexin 43 (Cx43) have been shown to mediate, at least in part, PGE2 release in bone osteocytes (Jiang and Cheng, 2001; Jiang and Cherian, 2003). To test if astrocytes release PGE2 using a similar mechanism, we used pharmacology, as well as siRNA antisense to Cx43. In the presence of the connexin hemichannel blocker carbenoxolone (100µM), we observed little to no PGE2 release when stimulated with ATP (n= 8) (Figure 2.4A). However, when Ca2+ was blocked with CPA (n= 8), significant release of PGE2 occurred in the presence of carbenoxolone suggesting that carbenoxolone did not block PGE2 release (One Way ANOVA, Tukey Post Hoc) (Figure 2.4A). To further assess whether PGE2 release requires functional Cx43 hemichannel or gap junctions, we utilized Cx43 siRNA antisense and its scramble sequence. The Cx43 siRNA efficacy was confirmed by staining astrocytic cultures with an antibody for Cx43. The immunostaining revealed that Cx43 was indeed reduced in Cx43 siRNA treated cells compared with cells treated with the scrambled sequence (Figure 2.4B). Despite this reduction, ATP (n= 8) still evoked a significant increase in the release of PGE2 in cells loaded with CPA (One Way ANOVA, Tukey’s Post Hoc Test) (Figure 2.4A). It is worth noting that the actual amount of PGE2 release between siRNA and carbenoxolone treated cultures showed no significant difference when comparing controls (range: 25 - 28 pg/ml) and ATP stimulated (range: 25 - 28 pg/ml) samples, respectively (One Way ANOVA, Tukey’s Post Hoc Test). Combined, these observations indicate that ATP induced PGE2 release from astrocytes does not involve connexin 43 gap junctions or hemichannels.

36  

This finding is not surprising since lipids are lipophilic and can readily cross the membrane (Figure 2.4C).

37  

Figure 2.4: Connexin 43 does not mediate PGE2 release. (A) Effects of the Connexin 43 inhibitors, Carbenoxolone (100µM) and siRNA antisense on Ca2+ independent PGE2 release in astrocytic cultures compared to control. B.) Immunohistochemical analysis of Connexin 43 (green) expression in siRNA scrambled (upper panel) and antisense (lower panel) treated rat astrocytic cultures. Scale bars are 100µm. (C) Schematic of receptor mediated Ca2+ independent lipid release across the astrocytic membrane. *ANOVA followed by Tukey post-test; p< 0.05. All bar graphs show means ± s.e.m.

38  

B. *

500 400

*

300 200 100 siRNA

CBX

siRNA

CBX

siRNA

CBX

Cx43-siRNA Cx43 Dapi AT P

nt ro

AT P/ CP A

0

Co

C.

Scramble siRNA Cx43 Dapi

600

l

% of control of PGE2 Release

A.

39  

2.2.5 Ca2+-independent astrocytic lipid release enhances mEPSCs via Kv channel blockade Experiments thus far have demonstrated that astrocytes can indeed release lipids in a Ca2+-independent manner. Because previous studies have shown that AA and PGE2 can modulate neuronal potassium currents (Kv) (Horimoto et al., 1997; Nicol et al., 1997; Evans et al., 1999) and enhance synaptic activity (Chen and Bazan, 2005; Sang et al., 2005), we next evaluated whether astrocytic derived lipids can do the same in acute brain slices. To our knowledge, no studies have directly assessed the functional significance of a potential Ca2+-independent astrocyte-neuron lipid signaling mechanism in situ. Here we performed dual patch-clamp of neighboring neurons and astrocytes in acute hippocampal slices from 12-18 day old mice (Figure 2.5A). In order to test the effects of this proposed pathway on neuronal synaptic activity, we employed the use of MrgA1 transgenic animals (MrgA1+/-), which specifically express the exogenous Gq-coupled MRG receptor (MrgA1) under control of the astrocyte selective GFAP promoter. Use of these animals enabled the selective assessment of the astrocytic influence on neighboring neuronal Kv currents. As K+ currents are sensitive to AA and/or its metabolites, we isolated Kv currents in neighboring patch-clamped pyramidal neurons with 1 mM QX314 in the patch pipette to block sodium channels and imposed a voltage ramp (from -100 mV to 50 mV) every 5 seconds to enable using Kv channel current as a metric for detecting agonist-induced astrocytic lipid release (Figure 2.5B). FMRF (15 µM), a MrgA1 agonist that induces potent increases in astrocytic cytosolic Ca2+ in hippocampal slices (Fiacco et al., 2007; Agulhon et al., 2010; Wang et al., 2012), did not induce detectable changes in neuronal Kv current when the neighboring patched

40  

astrocyte did not contain BAPTA in the pipette solution (Figure 2.5B). However, when BAPTA (5 mM) was included in the astrocyte patch pipette, a marked decrease in neuronal Kv current was evoked by FMRF exposure (Figure 2.5B). A similar transient decrease in Kv current was observed when TFLLR-NH2 (30 µM), an agonist of proteaseactivated receptor-1 (PAR1) that is primarily expressed by astrocytes (Shigetomi et al., 2008), was added. Consistent with the action of FMRF, neuronal Kv currents were unaffected when TFLLR was applied without BAPTA in the astrocytic pipette solution (Figure 2.5E). Stimulation with ATP (100µM) in the presence or absence of BAPTA in astrocytes produced a comparable change to neuronal Kv (Figure 2.5C). Importantly, direct addition of PGE2 and AA induce similar changes in Kv current in the absence of buffering astrocytic Ca2+ increases (Figure 2.5C). Thus, these observations suggest that astrocytes modulate neuronal Kv current via previously undocumented Ca2+-independent lipid release. In order to test whether a decrease in neuronal Kv current was a consequence of Ca2+independent astrocytic lipid release, we next employed specific lipid receptor antagonists for PGE2 and endocannabinoids. In the presence of AH6809 (10 µM), a PGE2 EP1/2 antagonist, and GW627368X (3 µM), a PGE2 EP4 antagonist, in the perfusion solution, the TFLLR, FMRF, and ATP Ca2+-independent induced decrease in neuronal Kv current was abolished (Figure 2.5C); In contrast, the CB1 antagonist AM251 (5µM) failed to abolish the decrease in Kv current (Figure 2.5C). Taken together, this data suggests that the observed decrease in neuronal Kv current was due to astrocytic Ca2+-independent release of PGE2.

41  

Interestingly, the presence or absence of BAPTA in the astrocytic pipette solution also affected agonist-induced changes in neuronal membrane potential (Figure 2.5D). Without BAPTA in astrocytes, TFLLR induced hyperpolarization (1.4 ± 0.26 mV, n = 6), as has been shown in a previous study to be due to a decrease in extracellular potassium (Wang et al., 2012). However, with BAPTA present in astrocytes, TFLLR induced depolarization (2.1 ± 0.25 mV, n = 5), an effect we suggest might be attributable to the blockage of potassium current. As PGE2 was previously shown to enhance neuronal mEPSCs (Chen and Bazan, 2005), we next tested the effect of astrocytic calcium independent lipid release on synaptic activity. Following stimulation with FMRF in the presence of astrocyte BAPTA, we observed an increase in the amplitude and frequency of mEPSCs (Figure 2.5E). This is consistent with a previous report showing a PGE2-mediated enhancement of synaptic transmission (Sang et al., 2005) and supports the notion that astrocytic Ca2+-independent lipid release may function as a signaling mechanism capable of modulating synaptic activity.

42  

Figure 2.5: Ca2+-independent astrocytic lipid release enhances mEPSCs via Kv channel blockade A.) Schematic of Patching astrocyte and neuron. (B.) Typical traces show application of FMRF (15µM) in the MrgA1+/- mice with BAPTA decreased Kv current, but not in the absence of BAPTA. TFLLR (30µM) with BAPTA also decreased Kv current, as can be seen in the peak currents; and increased amplitude and frequency of mEPSCs can also be seen. Left traces are an extension of the left traces. (C.) Quantification of effects of different drugs on the holding voltage ramp induced voltage gated K+ currents (*, P < 0.05, **, p < 0.01, ANOVA followed by Tukey post test, n =5-7). (D.) Typical traces show the membrane potentials changes with the agonists, and quantification was shown in inlet (**, p < 0.01, t-test, n = 5). (E) Changes of frequency and amplitude of mEPSCs from recording A, 20s traces before and after application of Par1 were analyzed below. Inset shows membrane potential changes by agonists were different with BAPTA present or absent. Inset shows the statistics. All  bar  graphs  show  means  ±  s.e.m.

43  

44  

2.2.6 DISCUSSION In the present study, we demonstrated that upon Ca2+ chelation and glutamatergic or purinergic receptor stimulation astrocytes release AA and/or its metabolites (Figure 2.2). We also demonstrated that this receptor mediated Ca2+ independent lipid release is iPLA2 dependent and that this release is blocked in the presence of Bel (Figure 2.2). Furthermore, we found that by blocking the Ca2+ calmodulin complex we were able to induce Ca2+ independent Lipid release in the absence of Ca2+ chelation, thus further confirming the importance of iPLA2 activity. Using the PGE2 ELISA, we found PGE2 was one of the major metabolites released upon receptor stimulation and Ca2+ chelation. In addition, we demonstrated that receptor mediated Ca2+ independent PGE2 was preserved across mouse, rat and human astrocytes (Figure 2.3). We also demonstrated that Connexin 43 hemi-channels are not involved in Ca2+ independent PGE2 release (Figure 2.4). Lastly, we demonstrate that astrocytic Ca2+ independent PGE2 release in brain slices reduces neuronal Kv current, which results in enhanced synaptic activity as observed by increases in mEPSCs (Figure 2.5). The observations reported here represent, to our knowledge, the first demonstration of Ca2+- independent release of gliotransmitters in the form of lipid mediator. To date, many studies have shown the multifaceted functions of PLA2 in the CNS, but iPLA2 is the major PLA2 isoform, and accounts for 70% of the PLA2 activity in the brain (Yang et al., 1999). Although cPLA2 has been demonstrated to play several roles in CNS activity (Malaplate-Armand et al., 2006; Schaeffer and Gattaz, 2007; Kim et al., 2008), iPLA2 has been shown to modulate phospholipid remodeling (Sun et al., 2004), regulate

45  

hippocampal AMPA receptors and learning and memory (Menard et al., 2005), regulate store-operated Ca2+ entry in cerebellar astrocytes (Singaravelu et al., 2006), and provide neuroprotection against oxygen glucose deprivation (Strokin et al., 2006). Astrocytes express both isoforms of PLA2 (Sun et al., 2005). However, little attention has been given to astrocytic iPLA2 activity as the hallmark of astrocytic signaling centers around agonistinduced rises in intracellular Ca2+. Instead, numerous excellent studies have evaluated Ca2+ dependent cPLA2 involvement in receptor mediated AA and/or its metabolites release from astrocytes (Bruner and Murphy, 1990; Stella et al., 1994; Stella et al., 1997b; Chen and Chen, 1998). Unlike the previous studies, we explored iPLA2 lipid pathway in astrocytes by chelating intracellular Ca2+. This pathway has most likely been overlooked because Ca2+ is the hallmark of astrocytic excitation. Like previous studies, we observed Ca2+ dependent release of PGE2 upon receptor stimulation in mouse astrocytes as well (Figure 2.3). Furthermore, we believe these observed difference seen in our study when compared to previous studies could be reflected through species differences. Mouse astrocytes have been shown in culture to express high levels of cPLA2 compared to iPLA2 (Balboa et al., 2002). On the other hand, rat and human astrocytes have been shown to express low levels of cPLA2, and the expression is increased in the setting of inflammation (Yoshihara et al., 1992; Clemens et al., 1996; Stephenson et al., 1996; Stephenson et al., 1999). For example, rat astrocytic cultures were shown to increase lipid release upon receptor stimulation in a Ca2+ dependent manner, after being pretreated with a proinflammatory cytokine (Katsuura et al., 1989)

46  

Of equal importance, this is the first study to demonstrate that astrocytic released lipids modulate neuronal activity through voltage gated K+ channels. Voltage gated potassium channels are located on the dendrites of hippocampal pyramidal neurons (Johnston et al., 2000), and these channels play a major role in controlling dendritic excitability potently altering the shape and amplitude of EPSCs. A morphological study found that the density of K+ channel in the dendrites of pyramidal neurons (Hoffman et al., 1997) increased 5fold from the soma to the most distal point measured in the apical dendrites. Closure of voltage gated K+ currents will enhance EPSPs, possible explaining why PGE2 enhance the synaptic transmission and LTP (Sang et al., 2005). This observation provides new evidence of how astrocytes can modulate neuronal activity in the absence of traditional gliotransmitters, such as glutamate or ATP. Furthermore, agonist-induced astrocytic Ca2+ increases occur on a time scale of seconds (Cornell-Bell et al., 1990; Wang et al., 2006) and have been shown to be linked to the release of gliotransmitters, such as glutamate (Parpura et al., 1994; Bezzi et al., 1998; Kang et al., 1998b), D-Serine (Mothet et al., 2000; Yang et al., 2003), and ATP (Coco et al., 2003). However, despite many suggestive studies, the concept of gliotransmission is still under considerable debate (Nedergaard et al., 2003; Agulhon et al., 2010; Hamilton and Attwell, 2010; Nedergaard and Verkhratsky, 2012a). Recently, we showed that agonist-induced astrocytic Ca2+ signaling can modulate synaptic activity by active K+ uptake resulting in a transient lowering of extracellular K+ (Wang et al., 2012) and depression of synaptic activity. Thus, astrocytes may regulate synaptic transmission by Ca2+-dependent K+ uptake, in addition to release of ‘so-called’ gliotransmitters. However,

47  

the agonist-induced K+ uptake is also driven by Ca2+ signaling, this neuron-glia signaling pathway is, therefore, slow. However, agonist-induced Ca2+-independent iPLA2 lipid release, does not require mobilization of intracellular Ca2+ stores, has the potential to signal on a much faster time scale (possibly milliseconds). It is possible that iPLA2 mediated lipid release acts as a feedback system to modulate fast synaptic transmission confined to a single synapse level, whereas Ca2+ is more suited toward the slow and widespread modulation of brain activity that occurs in the setting of, for example, activation of locus coeruleus and associated norepinephrine release (Guthrie et al., 1999; Cotrina et al., 2000). In conclusion, Ca2+indendent astrocytic lipid release constitutes a largely unexplored participant in complex neuro-glial signaling interactions. Taken together, the analysis presented here adds a new dimension to agonist-induced Ca2+ signaling by demonstrating that several Gq-linked receptors can mediate release of lipid modulators and that increases in cytosolic Ca2+ acts as a brake that prevent lipid release. Our study also highlights that important species differences exist, and that observations in rodent astrocytes must be confirmed in their human equivalents whenever possible.

48  

        Chapter 3 Astrocytic Endocannabinoids Mediate Transient Heterosynaptic Depression                                        

49  

Abstract Astrocytes are highly dynamic cells that have been shown to modulate many forms of synaptic transmission ranging from Long Term Potentiation (LTP) to Heterosynaptic Depression (HSD), but these forms of modulation occurs within a temporal domain of seconds to minutes. However, recent studies have revealed that astrocytes can also modulate a faster form of synaptic activity known as Transient Heterosynaptic Depression (tHSD), which occurs in a temporal domain of milliseconds to seconds. The mechanism behind astrocytic modulation of tHSD is unclear and it is debated whether this modulation is through traditional gliotransmitters such as ATP or Glutamate or through some yet unexplored new gliotransmitter such as lipids. Using fluoroacetate and IP3R2 -/- mice, we further confirmed astrocytes are critical for tHSD. We also confirmed the importance of group II mGluR in astrocytic modulation of tHSD. Next, using dominant negative SNARE mice, we found that vesicular release gliotransmitters were not required for tHSD. Since astrocytes have been shown to release lipids upon receptor stimulation, we next asked the question whether astrocytic released lipids, more specially endocannabinoids, could modulate this process. Using a CB1R antagonist, we found that tHSD was completely blocked. Taken together, this study provides the first evidence for group II mGluR-mediated astrocytic endocannabinoid release, which in turn blocks presynaptic neurotransmitter release thus inducing tHSD.

50  

3.1 Introduction Over the past decade, astrocytes have gained notoriety for being active participants in synaptic activity rather than merely supportive bystanders. Recent studies show that astrocytes experience rises in intracellular Ca2+ upon receptor stimulation that can lead to the release of gliotransmitters such as glutamate (Parpura et al., 1994; Bezzi et al., 1998; Kang et al., 1998a), D-serine (Mothet et al., 2000; Yang et al., 2003), or ATP (Coco et al., 2003). Gliotransmission is thought to be critical in calcium wave propagation by astrocytic paracrine signaling (ATP) (Cotrina et al., 1998a; Guthrie et al., 1999) and modulating synaptic activity by signaling to neurons (Kang et al., 1998a; Mothet et al., 2005). However, the question of whether astrocytes modulate synaptic activity through the release of gliotransmitters is still under debate (Agulhon et al., 2010; Hamilton and Attwell, 2010; Nedergaard and Verkhratsky, 2012b). Nonetheless, astrocytes have been shown to play a unique role in information processing by modulating a very rapid form of intersynaptic communication known as transient Heterosynaptic Depression (tHSD) (Andersson et al., 2007). Transient HSD is a faster form of HSD that can act as early as 100 ms after stimuli and lasts only a few seconds (Andersson et al., 2007). It is induced via modest synaptic stimulation (2-5 pulses at 50 Hz), reflecting persistently occurring activity seen in the hippocampus (Lisman, 1997; Buhl and Buzsaki, 2005; Andersson et al., 2007). Furthermore, as a result of its low threshold stimuli, tHSD is not dependent on activation of NMDA or Adenosine A1 receptors (Gustafsson et al., 1989; Isaacson et al., 1993). Most importantly, astrocytes have also been shown to mediate this faster form of

51  

intersynaptic communication (Andersson et al., 2007). The study postulated that tHSD occurs due to astrocytic vesicular release of gliotransmitters following mGluR receptor activation, leading to suppression of transmitter release from presynaptic terminals. Gliotransmitter release, however, is a slow and unpredictable event. Furthermore, gliotransmitter release from astrocytes is inconsistent, triggered only by selective astrocytic stimulation and occurs with a delay of 4-20 seconds (Parpura and Haydon, 2000). It is difficult to explain how astrocytes could regulate tHSD through the release of glutamate when such a release is inconsistent with the temporal domain of tHSD. So we begin to explore if alternative astrocytic transmitters, such as lipids, which are well suited to modulate neuronal activity, could be responsible for tHSD. One particular lipid candidate is the endocannabinoids since they are known to inhibit neurotransmitter release (Szabo et al., 2006), making them ideal candidates’ for astrocyte-mediated induction of tHSD. Now the question remains if astrocytes modulation of tHSD is through a Ca2+ dependent or a Ca2+ independent release of endocannabinoids. Although astrocytes have been shown to release endocannabinoids in a calcium dependent manner (Walter and Stella, 2003; Stella, 2004; Walter et al., 2004), there is some evidence that endocannabinoids can also undergo calcium independent release (Dinh et al., 2002b; Sugiura et al., 2002; Bisogno et al., 2003). Here we show that when astrocytic intracellular Ca2+ is disrupted using the IP3R2 -/- mice, tHSD was blocked. Furthermore, we confirmed that astrocytic group II mGluR activation is important for the induction of tHSD, and that astrocytic vesicular release of gliotransmitter, using the dnSNARE mice, was not important for tHSD. Next we were

52  

able to block the astrocytic group II mGluR mediated tHSD in the presence of CB1 receptor antagonist. Combined, these findings show that astrocytes modulation of tHSD is Ca2+ dependent and requires astrocytic released endocannabinoids. 3.2 RESULTS 3.2.1 Astrocytes are necessary for tHSD It has been shown that astrocytes modulate tHSD; however, the exact mechanism of how they modulate this depression remains incomplete (Andersson et al., 2007). Using a distinct stimulation protocol to induce tHSD (Figure 3.1A), we first confirmed previous finding that astrocytes are essential by employing fluoroacetate, which inhibits astrocytic metabolism (Fonnum et al., 1997). In the presence of fluoroacetate (10uM) tHSD was eliminated (Figure 3.1B, t-test, n= 4), thus confirming the critical role of astrocytes in this synaptic phenomenon. To further test the role of astrocytes in tHSD, we employed the use of connexin 43/30, which are gap juctions specifically found in astrocytes, double knockout mice (Wallraff et al., 2006) and the IP3R2 -/- mice. First, we employed the use of the Connexin 43/30-/- mice to determine whether disrupting intracellular astrocytic communications via gap junctions affects tHSD. We found a partial inhibition of tHSD in Connexin 43/30-/- mice (Figure 3.1C, t-test, n= 8). Previous studies that used pharmacological methods found that tHSD is completely inhibited in the presence of the gap junction blocker carbenoxolone. However, this inhibitor is non-specific and these results were not confirmed using the Connexin 43/30-/- used in our study. Next, we took advantage of the IP3R2-/- mice. In these animals, the astrocytic IP3R2, an isoform only found in astrocytes, is knocked out (Sharp et al., 1999; Holtzclaw et al.,

53  

2002; Hertle and Yeckel, 2007). Astrocytes are thus unable to signal with rises in intracellular Ca2+ upon receptor stimulation. Upon application of ATP, we observered increases in intracellular Ca2+ in slices from wildtype (IP3R2+/+) mice but failed to elicit Ca2+ responses in the IP3R2-/- mice (Figure 3.1D, upper panel), consistent with previous studies (Li et al., 2005; Petravicz et al., 2008; Wang et al., 2012). Interestingly, tHSD was completely inhibited in the IP3R2-/- mice, but remained intact in the wildtype (Figure 3.1D, t-test, n= 4 to 6). This confirms that astrocytic calcium signaling plays an essential role in tHSD. Taken together, these data provides evidence that astrocytes are necessary for tHSD.

54  

Figure 3.1: Astrocytes are necessary for tHSD A.) A schematic illustration that shows the placement of condition stimulating electrode and test stimulating electrode in the stratum radiatum of the CA1 region. tHSD is induced by stimulating one group of synapses with a short burst (3 pulses at 50Hz: conditional stimulation), which elicits a depression of fEPSPs at another synapse (test stimulation). Lower Panel illustrates a depression of fEPSPs during tHSD. B.) Bar graph comparing tHSD before and after exposure to Fluoroacetate (10 µM), an astrocytic toxin, in wildtyped mice hippocampal slices. C.) Bar graph comparing tHSD in Connexin 43/30+/+ and Connexin 43/30-/- mice hippocampal slices. D.) Image of ATP (100 µM) failed to increase Ca2+ in slices prepared from IP3R2-/- mice loaded with Fluo-4AM (4.5 µM) in left panel, whereas right panel show ATP (100µM) induced Ca2+ increase in a slice prepared from IP3R2+/+ mice. Lower panel, Bar graph comparing tHSD in IP3R2+/+ and IP3R2-/- mice hippocampal slices. *P < 0.05, paired and unpaired t-test. All  bar  graphs   show  means  ±  s.e.m.

55  

B. %Change of Baseline

A.

0 5 -10 -15 -20 -25 -30 -35 -40 -45 -50

*

Control

Fluoroacetate

tHSD

D.

0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

IP3R2

Cx43/30+/+

Cx43/30-/-

%Change of Baseline

%Change of Baseline

C.

IP3R2

-/-

0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

+/+

*

IP3R2

+/+

IP3R2

-/-

56  

3.2.2 Group II mGluR are necessary for tHSD After confirming that astrocytes are essential for the induction tHSD, our next step was to explore the mechanism by which astrocytes induce tHSD. It was previously shown that tHSD was dependent on group II metabotropic glutamate receptor (mGluR) activation (Andersson et al., 2007). This group is made up of mGluR2 and mGluR3, which are Gi coupled receptors that negatively regulate adenylate cyclase (Pin and Duvoisin, 1995). The most prominent metabotropic glutamate receptors found in astrocytes are mGluR 3 and mGluR 5 (Testa et al., 1994; Petralia et al., 1996; Balazs et al., 1997; Sun et al., 2013). However, the majority of focus has been devoted to the action of astrocytic mGluR5 since it is a Gq coupled receptor that is positively linked to polyphosphoinositide (PI) hydrolysis, which stimulates intracellular Ca2+ increases (Nakahara et al., 1997), but Gi coupled receptors can also signal with rises in intracellular Ca2+ by crossing talking with Gq receptors (Werry et al., 2003) or through its βγ subunit (Taussig and Zimmermann, 1998). Recently, a new study revealed that these receptor types are developmentally regulated in astrocytes with mGluR 5 being down regulated with age and mGluR 3 persisting throughout adulthood (Sun et al., 2013). In addition, anatomical studies have also revealed that the majority of mGluR 3 expression seen in the CA1 region of the hippocampus is localized in astrocytic processes (Petralia et al., 1996; Tamaru et al., 2001). Lastly, hippocampal astrocytes associated with synaptic terminals in the CA1 region have been shown to respond to neuronal release of glutamate (Porter and McCarthy, 1996).

57  

First, we assessed direct activation of astrocytic group II mGluR on synaptic activity in the striatum radium of the CA1 region by picospritzing trans-1-amino cylopentane-1, 3dicarboxylic acid (tACPD), a specific group II mGluR agonist (Figure 3.2A). In the presence of 50µM tACPD, we observed a significant depression of synaptic activity (Figure 3.2B, t-test, n= 6). Because local application of tACPD could potentially activate neuronal mGluR (Pacelli and Kelso, 1991), we next investigated electrically induced activation of astrocytic group II mGluR using the stimulation protocol (Figure 3.1A). In the presence of 20µM LY341495, a specific antagonist for group II mGluR, we observed tHSD was significantly blocked (Figure 3.2C, t-test, n= 6), thus confirming previous findings and further establishing a link between astrocytic group II mGluR and tHSD. It is worth mentioning that we observed no effect on the baseline field potentials, thus indicating that blockage of astrocytic group II mGluR does not inhibit basic synaptic transmission. Taken together, these data demonstrate that astrocytic group II mGluR activation is necessary for the induction of tHSD. This is a significant finding since the majority of astrocytic modulation of neuronal activity has been seen through the activation of astrocytic group I mGluR rather than group II.

58  

Figure 3.2: Group II mGluR are necessary for tHSD A.) A schematic illustration that shows the placement of picospritzer and test stimulating electrode in the stratum radiatum of the CA1 region. This model induced depression of fEPSPs at a local synapse (test stimulation) by picospritzing tACPD. B.) Bar graph comparing the effects of group II mGluR agonist mediated depression before and after AM251 (4µM), CB1 antagonist, exposure in wild-typed mice hippocampal slices. C.) Bar graph comparing tHSD before and after exposure to LY341495 (20 µM), mGluR group II antagonist in wild-typed mice hippocampal slices. *P < 0.05, paired t-test. All  bar   graphs  show  means  ±  s.e.m.

59  

A.

% Change of Baseline

B. 0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

*

aCSF/tACPD

%Change of Baseline

C.

0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

*

Control

LY341495

aCSF/tACPD/CB1R antagonist

aCSF/tACPD

60  

3.2.3 Astrocytic vesicular release is not involved in Group II mGluR-mediated tHSD Next, we investigated the possible mechanism behind group II mGluR mediated tHSD. It was proposed that tHSD was due to glutamate induced vesicular glutamate release from astrocytes that acted on presynaptic terminals and inhibited neurotransmitter release (Andersson et al. 2007). Since astrocytes have been shown to respond to neuronal release of glutamate with Ca2+ rises (Cornell-Bell et al., 1990; Porter and McCarthy, 1996; Wang et al., 2006) and subsequent gliotransmitter release (Parpura et al., 1994; Mothet et al., 2000; Coco et al., 2003), we therefore investigated the role of vesicular release of gliotransmitters in tHSD. Using the previously described stimulation protocol to induced tHSD, we employed the transgenic, dominant-negative SNARE mice (Figure 3.1A). It has been shown that core proteins of the snare complex, synaptobrevin 2 and synataxin, are expressed in astrocytes (Parpura et al., 1995). The dominant negative SNARE mice are conditional knockouts that express a disrupted synaptobrevin 2 protein in astrocytes when animals are taken off doxycycline (DOX), thus blocking astrocytic vesicular transmitter release (Pascual et al., 2005). We performed a beta-galactosidase (β-Gal) expression assay to confirm the appropriate expression profile of the dominant negative SNARE protein. In the presence of DOX, no β-Gal expression was observed indicating the slices had functional snare proteins (Figure 3.3A, right panel). In the absence of DOX, β-Gal expression was observed in astrocytes (Figure 3.3A, right panel). In the dominant negative mice with disrupted vesicular trafficking, tHSD was still present suggesting that vesicular release of gliotransmitters does not play a role in tHSD (Figure 3.3A, t-test, n= 6). Furthermore, we induced tHSD in the presence of 8-cyclopentyl-1, 3,

61  

dipropylxanthine (DPCPX), which is an adenosine A1 receptor antagonist. In the presence of DPCPX (1µM), tHSD was not suppressed (Figure 3.3B, t-test, n= 4), thus providing more evidence that traditional gliotransmitters are not required for tHSD.

62  

Figure 3.3: Astrocytic vesicular release is not involved in Group II mediated tHSD A.) Bar graph comparing tHSD in dnSNARE mice treated with ±DOX as well as before and after AM251 (4µM) exposure in hippocampal slices. Right panel, image of X-Gal staining of brain tissue from dnSNARE mice treated with ±DOX. B.) Bar graph comparing tHSD before and after exposure to DCPCX (1µM), Adenosine A1 Receptor antagonist, in wild-type mice hippocampal slices. *P < 0.05, paired and unpaired t-test. All  bar  graphs  show  means  ±  s.e.m.

63  

A.

(+) DOX

%Change of Baseline

0

%Change of Baseline

B.

-5 -10 -15 -20 -25 -30 -35 -40 -45 -50

*

*

(-) DOX (+)Dox

CB1R antagonist

(-)Dox

CB1R antagonist

0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

Control

DCPCX

64  

3.2.4 CB1R antagonists block Group II mGluR-mediated tHSD After determining that vesicular gliotransmitter release was not involved in tHSD, we next sought to investigate what specific astrocytic transmitter is responsible for tHSD. However, upon closer evaluation, we observed that the tACPD activation of astrocytic mGluR3 induced depression of synaptic transmission was blocked in the presence of AM251 (4µM), a CB1R antagonist (Figure 3.2B, t-test, n= 6). Just like glutamate, endocannabinoids are also known to inhibit neurotransmitter release by activating CB1R on presynaptic terminals. A few studies have shown that endocannabinoids are released from the post synaptic membranes in response to high frequency stimulation through the activation of NMDA receptors (Ohno-Shosaku et al., 2007), but astrocytes are also capable of releasing endocannabinoids. Astrocytes have been shown to release endocannabinoids upon receptor stimulation in both a Ca2+ dependent (Walter and Stella, 2003; Stella, 2004; Walter et al., 2004) and Ca2+ independent fashion (Dinh et al., 2002b; Sugiura et al., 2002; Bisogno et al., 2003). To further evaluate astrocytic endocannabinoids in tHSD, we next induced tHSD in the presence of CB1R antagonist. In the presence of AM251 (4µM), tHSD was significantly depressed in burst-induced suppression of the test synapse (Figure 3.4A, t-test, n= 6). These findings suggest that CB1 endocannabinoid receptors are crucial for tHSD. We next wanted to explore which astrocytic endocannabinoid could be responsible for tHSD. Using URB754 (1µM), a specific inhibitor of monoacyl glycerol Lipase (MGL), which inactivates of 2AG (Dinh et al., 2002b; Dinh et al., 2002a), we observed a significant enhancement in tHSD (Figure

65  

3.4B, t-test, n= 6), further endorsing the role of endocannabinoids, more specifically 2AG, in tHSD (Figure 3.4C).

66  

Figure 3.4: CB1R antagonist blocks Group II mGluR mediated tHSD A.) Bar graph comparing tHSD before and after exposure to AM251 (4µM) in wild-type mice hippocampal slices. B.) Bar graph comparing tHSD before and after exposure to JZL184 (1µM), an MGL inhibitor. C.) Schematic illustration of one active synapse (glutamatergic) and one inactive synapse (glutamatergic) with and astrocyte endfeet inbetween. The astrocyte is equipped with Group II mGluR, whose activation can increase Ca2+ and lead to the production of 2AG. 2AG can activate the CB1R on the presynaptic terminal and inhibit release probability at glutamatergic terminals. *P < 0.05, paired and unpaired t-test. All  bar  graphs  show  means  ±  s.e.m.

67  

A.

B. 0

0 % Change of Baseline

-5 % Change of Baseline

-10 -15

*

-20 -25 -30 -35

-10 -20 -30 -40

-40

-50

-45

-60

-50

Control

CB1R Antagonist

* Control

C.

Endocannabinoids (2AG / AEA) Ca2+

CB1R

DAG VSCC

Glu AMPAR

mGluR NMDAR

Ca2+ ER

Ca2+

Active synapse

Astrocyte

Inactive synapse

JZL184

68  

3.2.5 DISCUSSION In the present study, we show that astrocytic endocannabinoids are essential for tHSD. Using the IP3R2-/- mice, we demonstrated that tHSD was blocked in the absence of astrocytic Ca2+ (Figure 3.1). We also confirmed the importance of astrocytic group II mGluR through a specific group II antagonist (Figure 3.2). Next, using the dnSNARE mice, we demonstrated that vesicular release of traditional gliotransmitters is not required for tHSD (Figure 3.3). Finally, we show that tHSD is dependent on astrocytic endocannabinoids, more specifically 2AG (Figure 3.4). A previous study suggested that activation of group II mGluR causes vesicular released gliotransmitters (glutamate) from astrocytes, which in turn activates presynaptic neuronal group II mGluR, thus inducing tHSD (Andersson et al., 2007). This was an attractive hypothesis since glutamate have been shown to inhibit neurotransmitter release from presynaptic terminals by activating group II mGluR in different areas of the CNS (Bushell et al., 1996; Glitsch et al., 1996; Macek et al., 1996; Yokoi et al., 1996; Scanziani et al., 1997; Dube and Marshall, 2000; Kew et al., 2001). However, based on autoradiography and histological studies, the majority of group II mGluR expression was localized in astrocytic processes rather than presynaptic neuronal terminals within the striatum radium in the CA1 region of the hippocampus (Poncer et al., 1995; Petralia et al., 1996; Tamaru et al., 2001), thus limiting the possibility for neuronal group II mGluR participation. Furthermore, the possibility of vesicular release of gliotransmitters was also reduced with the use of the dnSNARE mice. Most importantly, we were able to

69  

inhibit the group II mGluR mediated tHSD in the presence of CB1 antagonist, thus suggesting a role for endocannabinoids in tHSD. Endocannabinoids are unique lipid modulators that suppress neuronal activity by acting on their respective receptors located on presynaptic terminals (Mechoulam et al., 1996; Stella et al., 1997a; Di Marzo et al., 1998; Freund et al., 2003). Furthermore, endocannabinoid modulation of synaptic activity has only been shown through retrograde signaling of endocannabinoids from the postsynaptic neuronal membrane, which is only inducible by high frequency depolarization or direct activation of NMDA or mGluR 1 receptors on the postsynaptic neuronal membrane (Maejima et al., 2001; Ohno-Shosaku et al., 2001; Varma et al., 2001; Zhu, 2006). Be that as it may, the possibility of retrograde signaling of endocannabinoids in the current study is very slim for several reasons. First, tHSD was shown to be independent of NMDA receptor activation but dependent on astrocytic group II mGluR (Andersson et al., 2007). Finally, tHSD is not dependent on high frequency stimulation (10 pulses at 100 Hz) but physiological stimulation (3 pulses at 50 Hz). Equally important, astrocytes can also release endocannabinoids upon receptor stimulation (Walter and Stella, 2003; Stella, 2004; Walter et al., 2004), but no one, until now, has evaluated their role in modulating synaptic activity. We initially thought that because tHSD occurred on a millisecond time scale that it could be due to astrocytic Ca2+ independent release of endocannabinoids because a number of studies have demonstrated that Ca2+ increases in astrocytes occur between 3 to 6 seconds (Cornell-Bell et al., 1990; Wang et al., 2006; Hartline and Colman, 2007). However, the

70  

IP3R2-/- transgenic mice proved that tHSD was in fact Ca2+ dependent, thus making it the first study to demonstrate a sub-second astrocytic mediated synaptic effect. Recently, several studies have shown that astrocytes can in fact exhibit fast Ca2+ signaling (Winship et al., 2007; Nimmerjahn et al., 2009). One in particular demonstrated the correlation of sub-second blood flow responses with rapid sub-second Ca2+ signaling in astrocytes, which is consistent with the time frame we observed (Winship et al., 2007). These findings are significant because they provide first hand evidence of sub-second Ca2+ signaling in astrocytes that can affect many CNS functions. In conclusion, the current study has huge potential in advancing our understanding of astrocytic modulation of neuronal activity beyond traditional gliotransmitter in the form of lipids. It is well known that during development astrocytes provide all of the lipids of the brain (Moore et al., 1991) but now we provide evidence for a possible role of astrocytic lipid modulation of fast synaptic activity. Furthermore, this study puts astrocytes at the center of information processing and could have implications in helping us understand and possibly treat diseases such as epilepsy and attention-deficit disorder (ADD), being that endocannabinoids have been shown to block epilepsy (Alger, 2004) and play a beneficial role in ADHD 2003 (Adriani et al., 2003).

71  

Chapter 4 General Discussion

72  

In my thesis work, I have demonstrated for the first time that Ca2+ chelation and glutamatergic or purinergic receptor stimulation potentiate release of AA and/or its metabolites, especially PGE2, from astrocytic cultures prepared from human and rat brains. I have also demonstrated for the first time that astrocytes in the hippocampal slice upon Ca2+ chelation and receptor stimulation release lipids, which in turn modulate neuronal excitability by acting on voltage gated K+ channels. Furthermore, using a model of transient Heterosynaptic Depression (tHSD), I demonstrated that astrocytes modulate rapid intersynaptic communication through the release of the endocannabinoid 2AG. This pathway of glia-neuronal signaling was dependent on astrocytic mobilization of intracellular Ca2+ stores and is thereby Ca2+ dependent. However, this does not preclude a role for Ca2+ independent lipid release in modulating synaptic activity and other CNS functions. Most likely, the Ca2+ independent response occurs in the distal thin processes known as leaflets that lack organelles for Ca2+ signaling (Bushong et al., 2002; Bergersen et al., 2012), and Ca2+ is propagated from the soma and thick processes known as branches and branchlets, where organelles for Ca2+ signaling reside (Di Castro et al., 2011; Reeves et al., 2011) (Figure 4.1). Once Ca2+ reaches the distal processes, it shuts down the Ca2+ independent lipid-signaling pathway, which potentiates synaptic activity, and activates the Ca2+ dependent lipid-signaling pathway, which depresses synaptic activity. Combined, these findings would expand our understanding of astrocytic modulation of information processing on two levels of lipid signaling in synaptic potentiation and synaptic depression.

73  

Figure 4.1: Astrocytic Processes Two-photon confocal imaging of an enhanced green fluorescent protein (eGFP)expressing astrocyte in a cortical slice, illustration the dense array of processes from a single cell. Inlet: Zoomed in image of the thin and thick astrocytic processes that engulf neuronal synapses. Take from Nedergaard, Ransom, and Goldman, 2003.

74  

eGFP-expressing astrocyte

Vessel

75  

Until now, the majority of studies implicating astrocytes in modulating synaptic activity have been viewed through Ca2+ dependent release of traditional gliotransmitters, such as D-serine, ATP, and glutamate (Hamilton and Attwell, 2010). For example, in the hippocampus, astrocytes are known to respond to neuronal activity with increases in intracellular Ca2+ (Porter and McCarthy, 1996; Haydon, 2001) and then release traditional gliotransmitters that modulate both excitatory and inhibitory synaptic activity (Kang et al., 1998b; Fiacco and McCarthy, 2004; Mothet et al., 2005). However, several studies have questioned the concept of traditional gliotransmitters and their roles in modulating synaptic activity (Petravicz et al., 2008; Agulhon et al., 2010). For example, McCarthy’s group used the MrgA1 and IP3R2-/- transgenic animals to demonstrate that in the presence or absence of receptor mediated intracellular Ca2+ rises astrocytes had no effect on short or long term plasticity (Petravicz et al., 2008; Agulhon et al., 2010). This controversy has throw the glial field into a stalemate and prompted other groups to look for alternative mechanisms by which astrocytes can actively modulate neuronal activity. One such approach involved receptor mediated intracellular Ca2+ rises that directly enhanced the activity of the Na+/K+ ATPase pump, thus modulating neuronal activity through K+ uptake (Wang et al., 2012). However, both receptor mediated gliotransmitter release and receptor mediated K+ uptake are forms of modulation that are slow and occur within seconds to minutes, which makes it difficult to explain how astrocytes could modulate rapid forms of synaptic activity. So the question remains whether astrocytes could modulate rapid forms of synaptic activity and by what possible mechanism.

76  

This newly discovered Ca2+-independent lipid signaling pathway could potentially occur within a time frame of msec. This observation is important because it suggests that astrocytes can actively participate in fast synaptic events. One possible hypothesis based on my work is that astrocytes, by an orchestrated release of lipid, support the precision and termination of synaptic activity by transiently switching between Ca2+ independent and Ca2+ dependent lipid release mechanisms. The first and faster astrocytic response is through receptor mediated Ca2+ independent PGE2 release. As shown in chapter 2, PGE2 will facilitate synaptic transmission by blocking voltage gated K+ channels. In the second and somewhat delayed phase, astrocytes will release 2AG in a Ca2+ dependent process, which depresses activity of inactive synapses. In this proposed model, astrocytes aid fast and precise transmission of synaptic inputs, but also facilitate termination of synaptic activity, thus enabling the neural network to quickly adapt to the ever-changing pattern of novel sensory input (Figure 4.2).

77  

Figure 4.2: Astrocytes role in lipid mediated synaptic activity Schematic illustrating astrocytes modulation synaptic potentiation and synaptic depression through Ca2+ independent PGE2 release and Ca2+ dependent 2AG release.

78  

PGE2 iPLA2

Endocannabinoids (2AG / AEA)

Ca2+

CB1R

DAG VSCC Ca2+

Glu AMPAR

NMDAR

ER

Ca2+

Active synapse

Astrocyte

Inactive synapse

79  

Equally important, this study could have major implications in advancing our understanding of astrocytes’ role in learning and memory consolidation. For example, by strengthening the firing of the active synapse and preventing the unwanted firing of inactive synapses, astrocytes become the key mediator that facilitate the passage of important brain signals that are cleaner and crisper, thus putting astrocytes at the center of information processing. In addition, it could also provide novel insight from an astrocytic viewpoint for a better understanding of neurological diseases, such as epilepsy and attention-deficit disorder (ADD), that disrupt the passage of important and concise neuronal signaling. Nonetheless, the regulation of synaptic transmission in both physiological and pathophysiological conditions can have multiple mechanisms that coexist at the same synapse to regulate the strength or efficacy of neuronal firing. Astrocytes are uniquely positioned to provide such regulation through lipid signaling. Future studies are needed to fully elucidate the role of astrocytic released lipids in both normal and disease CNS function. Although my research aims were reached, the current study has several limitations that warrant further investigation. First, by using mature acute brain slices in the tHSD model, I was unable to bulk load slices with membrane permeable organic Ca2+ indicator dyes to evaluate astrocytic Ca2+ rises within the astrocytic processes in response to the tHSD stimulation paradigm. However this limitation can be overcome with the use of GCaMP animals. GCaMP is a genetically encoded Ca2+ indicator (GECI) that is created from a fusion of green fluorescent protein (GFP), calmodulin, and M13 (Shigetomi et al., 2013; Tong et al., 2013). This genetically encoded indicator can be expressed as a

80  

cytosolic indicator or a membrane bound indicator specifically in astrocytes (Shigetomi et al., 2013; Tong et al., 2013). Unlike the membrane permeable organic Ca2+ indicators, GECI can be expressed throughout the entire astrocyte including, and most importantly, astrocytic processes that engulf neuronal synapses. As of now, the new forms of GCaMP such as GCaMP 3 and GCaMP 5 have higher signal to noise ratio and faster kinetics. Even though the IP3R2-/- animals suggest Ca2+ is important for tHSD, it is essential to further confirm what role, if any, Ca2+ is playing in tHSD as well as other neurological processes. A second potential caveat to my work is the use of fluoroacetate to confirm the role of astrocytes in tHSD. Fluoroacetate is a very toxic drug that is exclusively taken up by astrocytes. It inhibits glycolysis in astrocytes, which in turn affects metabolic support that astrocytes provide to neurons, thus making it a very poor candidate for studying astrocytic modulation of neuronal activity especially in tHSD. To over come this particular limitation, I would employ certain transgenic animal models in which astrocytes can be specially activated. For example, I would employ the MrgA1 transgenic animals to specially activate astrocytes to try to induce tHSD in the absence of the conditional input. Finally, tACPD was used to characterize mGluR 3 activation in the tHSD model. To further characterize mGluR 3’s role in tHSD, more specific mGluR 3 agonists, such as NAAG, should be used. In addition, I would also evaluate several other signaling molecules, especially neuromodulators such as Norepinephrine (NE) and Acetylcholine (Ach). Each of them has been shown to play a role in numerous brain functions especially affecting neural networks by modulating signal to noise ratios (Hirata et al., 2006; Benarroch, 2010). It is very likely that astrocytes serve as effector

81  

cells for these neuromodulators and upon activating their respective receptors on astrocytes they could potentially control signal to noise ratio by modulating tHSD through release of endocannabinoids. Taken together these studies would solidify astrocytes’ role as an active participant in synaptic activity and open the door to new possibilities for neuron-glia interactions. In conclusion, by moving beyond the idea of traditional gliotransmitters, this thesis work identifies a new astrocytic signaling mechanism that has the potential to modulate synaptic activity in real time. In particular, astrocytic lipid release might improve the temporal precision of synaptic transmission. My analyses suggest that astrocytic receptor activation initially potentiate local synaptic transmission by Ca2+ independent release of PGE2 followed by sub-second delayed Ca2+ dependent release of 2AG, which ultimately terminates synaptic activity.

82  

Chapter 5 Experimental Procedures

83  

5.1 Culture, Ca2+ imaging of cultured cells, and small interfering RNA Cultured neocortical astrocytes were prepared from 1 to 2 day old Wistar rat pups (Taconic Farms, Inc.) as previously described (Lin et al., 1998). Briefly, cerebral cortices were dissected and meninges were removed. The tissue was washed 3 times in Hank’s balanced salt solution without Ca2+. Once washed the tissue was triturated, filtered through 70µm nylon mesh and then centrifuged. The pellet was resuspended in 10 % FBS in DMEM/F12 containing 100 IU ml1 penicillin and 100 µg ml-1 streptomycin, and transferred to culture flasks. Cells were maintained at 37 °C in an incubator containing humidified air and 5% CO2. Medium was changed after 24 h and twice a week thereafter. More than 95% of the cells stained positive for GFAP. When the cells became confluent, they were rinsed two times in Hanks without Ca2+, suspended in 0.05 % trypsincontaining solution for 1 min, resuspended in DMEM/F12, centrifuged to remove trypsin, and then plated in 24 well plates. Experiments were performed when the cells were 95% confluent. For calcium imaging, cells were loaded with Fluo-4 acetomethoxy ester (Fluo4AM) 4.6µM and incubated with various pharmacological agents for 30 minutes at 37˚C. Using confocal microscopy (Olympus FV500), calcium wave activity was evoked by adding an equal volume of medium containing 100µM of ATP to each well. Relative increases in fluorescence signal evoked by agonist exposure over baseline fluoresce (ΔF/F) were calculated as previously described (Nedergaard, 1994). Small interference RNA (siRNA) targeting rat Cx43 (GenBank accession number NM_012567) was designed using a web based program (http://www.dharmacon.com/sidesign) and manufactured by Dharmacon (Chicago, IL). The Sequence

84  

ACAUCAUUGAGCUCUUCUA and it complimentary strand were chosen for the current study. The transfection mixture containing double stranded annealed Cx43 siRNA (42 pmol/24 well and Lipofectamine 2000 (.6µl/24-well) was added directly to rat astrocytic cultures once they are about 70 to 80% confluent. Sister cultures were transfected with stealth RNAi low GC duplex (Invitrogen) as negative control (Lin et al., 2008). The experiment using the siRNA treated cultures was evaluated 5 to 6 days post transfection with 80 to 90% inhibition of Cx43. 5.2 Isolation of Human Fetal Astrocytes Human fetal forebrain tissues were obtained from second-trimester aborted fetuses of 20 weeks gestational age. Tissues were obtained from aborted fetuses, with informed consent and tissue donation approval from the mothers, under protocols approved by the Research Subjects Review Board of the University of Rochester Medical Center. No patient identifiers were made available to or known by the investigators; no known karyotypic abnormalities were included. The forebrain tissue samples were collected and washed 2-3 times with sterile Hank’s balanced salt solution with Ca2+/Mg2+ (HBSS+/+). The cortical plate region (CTX) of fetal forebrain was dissected and separated from the ventricular zone/subventricular zone (VZ/SVZ) portion. The CTX was then dissociated with papain as previously described (Keyoung et al., 2001). The cells were resuspended at 2-4 x 106 cells/ml in DMEM/F12 supplemented with N2, 0.5 % FBS and 10 ng/ml bFGF and plated in suspension culture dishes. The day after dissociation, cortical cells were recovered and subjected to magnetic activated cell sorting (MACS) for purification of the astrocyte progenitor population. Briefly the recovered cells were incubated with

85  

CD44 microbeads as per the manufacturer’s recommendations (Miltenyi Biotech). The cells were then washed, resuspended in Miltenyi Washing buffer and bound to a magnetic column (Miltenyi Biotech). The bound CD44+ astrocyte progenitor cells were eluted, collected and then washed with DMEM/F12. The purified human fetal astrocyte progenitors were cultured in DMEM/F12 supplemented with N2 and 5% characterized FBS to further differentiate them. To prepare culture dishes for ATP release assays or immunocytochemistry, the fetal cortical astrocytes were dissociated with TrypLE (Invitrogen) into single cells and then plated onto poly-L-ornithine/laminin coated 24 well plates (50,000 cells per well). 5.3 Immunocytochemistry   Cultures were stained for the astrocytic progenitor marker, CD44 as described in the previous section. CD44 was localized on live cells that were then fixed with 4% paraformaldehyde. CD44 supernatant was used at a dilution of 1:200 (DSHB) in DMEM/F12/N1 and was applied for 40 minutes at 4°C. Post-fixation, cultures were stained for astrocyte markers, GFAP (1:800, Abcam) and AQP4 (1:200, Chemicon), and for the neuronal marker, HuC/D (clone 16A11, Invitrogen). To determine if any oligodendrocytes were remaining in the culture, oligodendrocyte lineage cells were labeled using Olig2 (1:200, R&D). Secondary antibodies, Alexa-488, 594 and 647 conjugated goat-anti mouse IgM, rabbit and chicken antibodies respectively were used at a dilution of 1:500 (Invitrogen). Fixed cultures were counterstained with DAPI (10 ng/ml; Invitrogen). 5.4 Radiolabeling and Assessment of AA Release

86  

Confluent rat astrocytic cultures were loaded with 100nCi 3H-AA(Perkin Elmer) 24 hrs prior to experiments. The cells were washed three times with serum free medium. After washes, the cells were allowed to recover for twenty minutes. Before stimulation with 100µM of ATP, the cells were incubated with appropriate inhibitors for fifteen minutes. Aliquots of medium were taken before and after stimulation and 3H-AA and/or its metabolites were measured by liquid scintillation counting. 5.5 PGE2 Release Assessment Confluent rat astrocytic cultures were washed three times with serum free medium. After washes, the cells were allowed to recover for twenty minutes. Before stimulation with 100µM of ATP, the cells were incubated with appropriate inhibitors for fifteen minutes. Aliquots of medium were taken after stimulation and measured using a PGE2 Enzyme Immunoassay Kit from Cayman Chemicals following the manufacturer’s manual. 5.6 Slice preparation and electrophysiology Unless otherwise noted, 15-21 day old C57BL/6 (Charles River, Wilmington, MA), MrgA1+/-, MrgA1-/- pups were used for preparation of hippocampal slices as previously described (Wang et al., 2012). The pups were anesthetized in a closed chamber with isoflurane (1.5%), and decapitated. The brains were rapidly removed and immersed in ice-cold cutting solution that contained (in mM): 230 sucrose, 2.5 KCl, 0.5 CaCl2, 10 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, pH=7.2-7.4. Coronal slices (400µm) were cut using a vibratome (Vibratome Company, St. Louis) and transferred to oxygenated artificial cerebrospinal fluid (aCSF) that contained (in mM): 126 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, pH = 7.2-7.4,

87  

osmolarity = 310 mOsm. Slices were incubated in aCSF for 1-5 hours at room temperature before recording. Experiments were performed at room temperature (21-23 ºC). During the recordings, the slices were placed in a perfusion chamber and super fused with aCSF gassed with 5% CO2 and 95% O2 at room temperature. Cells were visualized with a 40X water-immersion objective and differential inference contrast (DIC) optics (BX51 upright microscope, Olympus Optical, New York, NY). Patch electrodes were fabricated from filament thin-wall glass (World Precision Instruments) on a vertical puller; resistance of the pipette is around 6-9 mΩ with intracellular pipette solution added. I-V curves of voltage-gated potassium currents were recorded under voltage-clamp using an AxoPatch MultiClamp 700B amplifier (Axon Instruments, Forster City, CA). The pipette solution contained (in mM) 140 K-gluconate, 5 Naphosphocreatine, 2 MgCl2, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP (pH adjusted to 7.2 with KOH). QX314 0.5 mM was also added in the pipette solution, except those experiments for Na currents. With these solutions, the IPSCs reversed at -70 mV, and EPSCs reversed at about 0 mV. Voltage clamp was used to record miniature EPSCs, which were isolated by adding 0.5 µM TTX and 10 µM bicuculline to the bath solution and maintaining a holding potential of -70 mV. The junction potential between the patch pipette and bath solution was zeroed before forming a giga-seal. Patches with seal resistances of less than 1 GΩ were rejected. Data were low-pass filtered at 2 kHz and digitized at 10 kHz with a Digidata 1440 interface controlled by pClamp Software (Molecular Devices, Union City, CA). All results are reported as mean ± s.e.m. and significance was determined by paired or unpaired t-tests or Tukey-Kramer post hoc multiple comparison tests.

88  

5.7 Animals Connexin 43/30 -/- mice were bred as described previously (Wallraff et al., 2006), and both knockout and their wildtypes littermates from 18 to 30 day old were used. Dominant negative SNARE mice were bred as described previously (Pascual et al., 2005). In brief, doxycycline (Sigma-Aldrich, St. Louis, MO, 100µg/ml) was administered and continued on (+) DOX and off (-) DOX for 4 weeks prior to electrophysiological recording. Animals between the ages of 18 and 30 day old were used. IP3R2-/- mice were generated as described previously (Sharp et al., 1999; Holtzclaw et al., 2002; Hertle and Yeckel, 2007), and the animals from 18 to 30 days old were used. C57BL/6J mice were obtained from Charles River Laboratories. The Animal Care and Use Committee of the University of Rochester approved all animal experiments. 5.8 Slice preparation for tHSD Experiments were performed on hippocampal slices from 18 to 30 day old C57BL/6J mice. The mice were anaesthetized with isoflurane and decapitated, and the brain was quickly removed in ice cold cutting solution containing (in mM) 124 NaCl, 1 KCl, 1.25 NaH2PO4, .5 CaCl2, 10 MgSO4, .04 ascorbic acid, 1 Kynurenic acid, 26 NaHCO3, 10 glucose. In the same solution, transverse hippocampal slices (400µM thick) were cut with a vibratome. Slices were kept at 30-32 °C in ACSF saturated with 95% O2, 5% CO2. Slices were studied while submerged and under continuous super fusion (~2 ml/min-1) in ACSF saturated with 95% O2, 5% CO2 at 30-32 °C. Picrotoxin (100um) was always present in the perfusion fluid to block GABAA receptor mediated activity. 5.9 Electrophysiological Recording and Analysis for tHSD

89  

Electrical Stimulation of Schaffer collateral-commissural axons and recording of synaptic responses were carried out in the stratum radiatum of the CA1 region. Stimuli consisted of biphasic constant current pulses (Master 8, AMPI, Israel) delivered through tungsten wires (~0.1MΩ). Two stimulation electrodes were positioned in the stratum radiatum with a distance of about 500µm from each other. They were activated every 10s. Field EPSPs were recorded using a glass micropipette filled with 1M NaCl (resistance 1-2MΩ) placed between the two stimulation electrodes. Fields EPSPs were sampled at 10 kHz with an AxoPatch 200B amplifier (Axon Instrument) and filtered at 1kHz. Evoked responses were analyzed off line using pClamp 10. Data analyzed were the peak amplitude of the baseline fEPSPs and of fEPSPs evoked after application of the conditioning pulses. fEPSPs amplitudes were expressed as percentage of baseline to control for differences in absolute magnitude across slices and conditions. 6.0 Statistical Analysis Data are expressed as means ± s.e.m. Statistical significance (*P< 0.05) for paired and unpaired samples were evaluated using Student’s t-test and one-way analysis of variance (Tukey Post Hoc Test).

Reference Ackermann EJ, Dennis EA (1995) Mammalian calcium-independent phospholipase A2. Biochim Biophys Acta 1259:125-136. Adriani W, Caprioli A, Granstrem O, Carli M, Laviola G (2003) The spontaneously hypertensive-rat as an animal model of ADHD: evidence for impulsive and nonimpulsive subpopulations. Neurosci Biobehav Rev 27:639-651.

90  

Agulhon C, Fiacco TA, McCarthy KD (2010) Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling. Science 327:1250-1254. Akiba S, Hayama M, Sato T (1998) Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1 beta and dibutyryl cAMP in rat mesangial cells. FEBS Lett 437:225-228. Alger BE (2004) Endocannabinoids and their implications for epilepsy. Epilepsy currents / American Epilepsy Society 4:169-173. Anderson CM, Swanson RA (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32:1-14. Andersson M, Blomstrand F, Hanse E (2007) Astrocytes play a critical role in transient heterosynaptic depression in the rat hippocampal CA1 region. J Physiol 585:843852. Angulo MC, Le Meur K, Kozlov AS, Charpak S, Audinat E (2008) GABA, a forgotten gliotransmitter. Prog Neurobiol 86:297-303. Arcuino G, Lin JH, Takano T, Liu C, Jiang L, Gao Q, Kang J, Nedergaard M (2002) Intercellular calcium signaling mediated by point-source burst release of ATP. Proc Natl Acad Sci U S A 99:9840-9845. Balazs R, Miller S, Romano C, de Vries A, Chun Y, Cotman CW (1997) Metabotropic glutamate receptor mGluR5 in astrocytes: pharmacological properties and agonist regulation. J Neurochem 69:151-163. Balboa MA, Varela-Nieto I, Killermann Lucas K, Dennis EA (2002) Expression and function of phospholipase A(2) in brain. FEBS Lett 531:12-17. Ballabh P, Braun A, Nedergaard M (2004) The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 16:1-13. Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC (2002) Control of synaptic strength by glial TNFalpha. Science 295:2282-2285. Benarroch EE (2010) Acetylcholine in the cerebral cortex: effects and clinical implications. Neurology 75:659-665. Bergersen LH, Morland C, Ormel L, Rinholm JE, Larsson M, Wold JF, Roe AT, Stranna A, Santello M, Bouvier D, Ottersen OP, Volterra A, Gundersen V (2012) Immunogold detection of L-glutamate and D-serine in small synaptic-like

91  

microvesicles in adult hippocampal astrocytes. Cereb Cortex 22:1690-1697. Bezzi P, Gundersen V, Galbete JL, Seifert G, Steinhauser C, Pilati E, Volterra A (2004) Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nat Neurosci 7:613-620. Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T, Volterra A (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391:281-285. Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, Matias I, SchianoMoriello A, Paul P, Williams EJ, Gangadharan U, Hobbs C, Di Marzo V, Doherty P (2003) Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J Cell Biol 163:463-468. Boarder MR, Weisman GA, Turner JT, Wilkinson GF (1995) G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci 16:133-139. Bruner G, Murphy S (1990) ATP-evoked arachidonic acid mobilization in astrocytes is via a P2Y-purinergic receptor. J Neurochem 55:1569-1575. Buhl DL, Buzsaki G (2005) Developmental emergence of hippocampal fast-field "ripple" oscillations in the behaving rat pups. Neuroscience 134:1423-1430. Bushell TJ, Jane DE, Tse HW, Watkins JC, Garthwaite J, Collingridge GL (1996) Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices. Br J Pharmacol 117:14571462. Bushong EA, Martone ME, Jones YZ, Ellisman MH (2002) Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J Neurosci 22:183192. Charles AC, Naus CC, Zhu D, Kidder GM, Dirksen ER, Sanderson MJ (1992) Intercellular calcium signaling via gap junctions in glioma cells. J Cell Biol 118:195-201. Chen C, Bazan NG (2005) Endogenous PGE2 regulates membrane excitability and synaptic transmission in hippocampal CA1 pyramidal neurons. J Neurophysiol 93:929-941. Chen WC, Chen CC (1998) ATP-induced arachidonic acid release in cultured astrocytes is mediated by Gi protein coupled P2Y1 and P2Y2 receptors. Glia 22:360-370.

92  

Chung IY, Benveniste EN (1990) Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta. J Immunol 144:2999-3007. Clemens JA, Stephenson DT, Smalstig EB, Roberts EF, Johnstone EM, Sharp JD, Little SP, Kramer RM (1996) Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat. Stroke 27:527-535. Coco S, Calegari F, Pravettoni E, Pozzi D, Taverna E, Rosa P, Matteoli M, Verderio C (2003) Storage and release of ATP from astrocytes in culture. J Biol Chem 278:1354-1362. Cornell-Bell AH, Finkbeiner SM, Cooper MS, Smith SJ (1990) Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247:470473. Cotrina ML, Lin JH, Lopez-Garcia JC, Naus CC, Nedergaard M (2000) ATP-mediated glia signaling. J Neurosci 20:2835-2844. Cotrina ML, Kang J, Lin JH, Bueno E, Hansen TW, He L, Liu Y, Nedergaard M (1998a) Astrocytic gap junctions remain open during ischemic conditions. J Neurosci 18:2520-2537. Cotrina ML, Lin JH, Alves-Rodrigues A, Liu S, Li J, Azmi-Ghadimi H, Kang J, Naus CC, Nedergaard M (1998b) Connexins regulate calcium signaling by controlling ATP release. Proc Natl Acad Sci U S A 95:15735-15740. Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, Lichtman AH (2001) Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc Natl Acad Sci U S A 98:9371-9376. Dabertrand F, Hannah RM, Pearson JM, Hill-Eubanks DC, Brayden JE, Nelson MT (2013) Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles. J Cereb Blood Flow Metab 33:479-482. Dennis EA (1994) Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem 269:13057-13060. Dhopeshwarkar GA, Subramanian C (1976) Biosynthesis of polyunsaturated fatty acids in the developing brain: I. Metabolic transformations of intracranially administered 1-14C linolenic acid. Lipids 11:67-71.

93  

Di Castro MA, Chuquet J, Liaudet N, Bhaukaurally K, Santello M, Bouvier D, Tiret P, Volterra A (2011) Local Ca2+ detection and modulation of synaptic release by astrocytes. Nat Neurosci 14:1276-1284. Di Marzo V, Melck D, Bisogno T, De Petrocellis L (1998) Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci 21:521-528. Dinh TP, Freund TF, Piomelli D (2002a) A role for monoglyceride lipase in 2arachidonoylglycerol inactivation. Chemistry and physics of lipids 121:149-158. Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL, Kathuria S, Piomelli D (2002b) Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci U S A 99:10819-10824. Dube GR, Marshall KC (2000) Activity-dependent activation of presynaptic metabotropic glutamate receptors in locus coeruleus. J Neurophysiol 83:11411149. Evans AR, Vasko MR, Nicol GD (1999) The cAMP transduction cascade mediates the PGE2-induced inhibition of potassium currents in rat sensory neurones. J Physiol 516 ( Pt 1):163-178. Felder CC, Kanterman RY, Ma AL, Axelrod J (1989) A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis. J Biol Chem 264:20356-20362. Fiacco TA, McCarthy KD (2004) Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons. J Neurosci 24:722-732. Fiacco TA, Agulhon C, Taves SR, Petravicz J, Casper KB, Dong X, Chen J, McCarthy KD (2007) Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity. Neuron 54:611-626. Fonnum F, Johnsen A, Hassel B (1997) Use of fluorocitrate and fluoroacetate in the study of brain metabolism. Glia 21:106-113. Freund TF, Katona I, Piomelli D (2003) Role of endogenous cannabinoids in synaptic signaling. Physiol Rev 83:1017-1066. Fride E (2002) Endocannabinoids in the central nervous system--an overview. Prostaglandins, leukotrienes, and essential fatty acids 66:221-233.

94  

Glitsch M, Llano I, Marty A (1996) Glutamate as a candidate retrograde messenger at interneurone-Purkinje cell synapses of rat cerebellum. J Physiol 497 ( Pt 2):531537. Gross RW, Ramanadham S, Kruszka KK, Han X, Turk J (1993) Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells. Biochemistry 32:327336. Gross RW, Jenkins CM, Yang J, Mancuso DJ, Han X (2005) Functional lipidomics: the roles of specialized lipids and lipid-protein interactions in modulating neuronal function. Prostaglandins & other lipid mediators 77:52-64. Gustafsson B, Asztely F, Hanse E, Wigstrom H (1989) Onset Characteristics of LongTerm Potentiation in the Guinea-Pig Hippocampal CA1 Region in Vitro. Eur J Neurosci 1:382-394. Guthrie PB, Knappenberger J, Segal M, Bennett MV, Charles AC, Kater SB (1999) ATP released from astrocytes mediates glial calcium waves. J Neurosci 19:520-528. Hamilton NB, Attwell D (2010) Do astrocytes really exocytose neurotransmitters? Nat Rev Neurosci 11:227-238. Hartline DK, Colman DR (2007) Rapid conduction and the evolution of giant axons and myelinated fibers. Curr Biol 17:R29-35. Hashimotodani Y, Ohno-Shosaku T, Kano M (2007) Endocannabinoids and synaptic function in the CNS. Neuroscientist 13:127-137. Haydon PG (2001) GLIA: listening and talking to the synapse. Nat Rev Neurosci 2:185193. Haydon PG, Carmignoto G (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 86:1009-1031. Hertle DN, Yeckel MF (2007) Distribution of inositol-1,4,5-trisphosphate receptor isotypes and ryanodine receptor isotypes during maturation of the rat hippocampus. Neuroscience 150:625-638. Hirata A, Aguilar J, Castro-Alamancos MA (2006) Noradrenergic activation amplifies bottom-up and top-down signal-to-noise ratios in sensory thalamus. J Neurosci 26:4426-4436.

95  

Hoffman DA, Magee JC, Colbert CM, Johnston D (1997) K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature 387:869-875. Holtzclaw LA, Pandhit S, Bare DJ, Mignery GA, Russell JT (2002) Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors. Glia 39:69-84. Horimoto N, Nabekura J, Ogawa T (1997) Arachidonic acid activation of potassium channels in rat visual cortex neurons. Neuroscience 77:661-671. Isaacson JS, Solis JM, Nicoll RA (1993) Local and diffuse synaptic actions of GABA in the hippocampus. Neuron 10:165-175. Iversen LL, Neal MJ (1968) The uptake of [3H]GABA by slices of rat cerebral cortex. J Neurochem 15:1141-1149. Jelsema CL, Axelrod J (1987) Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci U S A 84:3623-3627. Jiang JX, Cheng B (2001) Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2. Cell communication & adhesion 8:283-288. Jiang JX, Cherian PP (2003) Hemichannels formed by connexin 43 play an important role in the release of prostaglandin E(2) by osteocytes in response to mechanical strain. Cell communication & adhesion 10:259-264. Johnston D, Hoffman DA, Magee JC, Poolos NP, Watanabe S, Colbert CM, Migliore M (2000) Dendritic potassium channels in hippocampal pyramidal neurons. J Physiol 525 Pt 1:75-81. Kang J, Jiang L, Goldman S, Nedergaard M (1998a) Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nat Neurosci 1:683-692. Kang J, Jiang L, Goldman SA, Nedergaard M (1998b) Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nat Neurosci 1:683-692. Kanterman RY, Axelrod J, Felder CC (1990) Alpha-1 adrenergic receptor mediates the release of arachidonic acid in spinal cord neurons independent of phosphatidylinositol-specific phospholipase C. Advances in second messenger and phosphoprotein research 24:158-163. Katsuura G, Gottschall PE, Dahl RR, Arimura A (1989) Interleukin-1 beta increases prostaglandin E2 in rat astrocyte cultures: modulatory effect of neuropeptides.

96  

Endocrinology 124:3125-3127. Kew JN, Ducarre JM, Pflimlin MC, Mutel V, Kemp JA (2001) Activity-dependent presynaptic autoinhibition by group II metabotropic glutamate receptors at the perforant path inputs to the dentate gyrus and CA1. Neuropharmacology 40:2027. Kim DH, Fitzsimmons B, Hefferan MP, Svensson CI, Wancewicz E, Monia BP, Hung G, Butler M, Marsala M, Hua XY, Yaksh TL (2008) Inhibition of spinal cytosolic phospholipase A(2) expression by an antisense oligonucleotide attenuates tissue injury-induced hyperalgesia. Neuroscience 154:1077-1087. Lehman JJ, Brown KA, Ramanadham S, Turk J, Gross RW (1993) Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent phospholipase A2. J Biol Chem 268:2071320716. Li X, Zima AV, Sheikh F, Blatter LA, Chen J (2005) Endothelin-1-induced arrhythmogenic Ca2+ signaling is abolished in atrial myocytes of inositol-1,4,5trisphosphate(IP3)-receptor type 2-deficient mice. Circ Res 96:1274-1281. Lin JH, Weigel H, Cotrina ML, Liu S, Bueno E, Hansen AJ, Hansen TW, Goldman S, Nedergaard M (1998) Gap-junction-mediated propagation and amplification of cell injury. Nat Neurosci 1:494-500. Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K, Yang J, Kang J, Nedergaard M (2008) A central role of connexin 43 in hypoxic preconditioning. J Neurosci 28:681-695. Lisman JE (1997) Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci 20:38-43. Lovatt D, Xu Q, Liu W, Takano T, Smith NA, Schnermann J, Tieu K, Nedergaard M (2012) Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity. Proc Natl Acad Sci U S A 109:62656270. Lynch GS, Dunwiddie T, Gribkoff V (1977) Heterosynaptic depression: a postsynaptic correlate of long-term potentiation. Nature 266:737-739. Macek TA, Winder DG, Gereau RWt, Ladd CO, Conn PJ (1996) Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses. J Neurophysiol 76:3798-3806.

97  

Maejima T, Ohno-Shosaku T, Kano M (2001) Endogenous cannabinoid as a retrograde messenger from depolarized postsynaptic neurons to presynaptic terminals. Neurosci Res 40:205-210. Malaplate-Armand C, Florent-Bechard S, Youssef I, Koziel V, Sponne I, Kriem B, Leininger-Muller B, Olivier JL, Oster T, Pillot T (2006) Soluble oligomers of amyloid-beta peptide induce neuronal apoptosis by activating a cPLA2-dependent sphingomyelinase-ceramide pathway. Neurobiol Dis 23:178-189. Manzoni OJ, Manabe T, Nicoll RA (1994) Release of adenosine by activation of NMDA receptors in the hippocampus. Science 265:2098-2101. Mechoulam R, Ben Shabat S, Hanus L, Fride E, Vogel Z, Bayewitch M, Sulcova AE (1996) Endogenous cannabinoid ligands--chemical and biological studies. Journal of lipid mediators and cell signalling 14:45-49. Menard C, Valastro B, Martel MA, Chartier E, Marineau A, Baudry M, Massicotte G (2005) AMPA receptor phosphorylation is selectively regulated by constitutive phospholipase A(2) and 5-lipoxygenase activities. Hippocampus 15:370-380. Meves H (1994) Modulation of ion channels by arachidonic acid. Prog Neurobiol 43:175186. Moore SA (1993) Cerebral endothelium and astrocytes cooperate in supplying docosahexaenoic acid to neurons. Adv Exp Med Biol 331:229-233. Moore SA, Yoder E, Murphy S, Dutton GR, Spector AA (1991) Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega6). J Neurochem 56:518-524. Mothet J, Parent A, Wolosker H, Jr RB, Linden D, Ferris C, Rogawski M, Synder S (2000) D-serine is an endogenous ligand for the site of N-methyl D-aspartate receptor. Proc Natl Acad Sci U S A 97:4926-4931. Mothet JP, Pollegioni L, Ouanounou G, Martineau M, Fossier P, Baux G (2005) Glutamate receptor activation triggers a calcium-dependent and SNARE proteindependent release of the gliotransmitter D-serine. Proc Natl Acad Sci U S A 102:5606-5611. Mulligan SJ, MacVicar BA (2004) Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature 431:195-199. Murayama T, Kajiyama Y, Nomura Y (1990) Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets. J Biol Chem

98  

265:4290-4295. Nakahara K, Okada M, Nakanishi S (1997) The metabotropic glutamate receptor mGluR5 induces calcium oscillations in cultured astrocytes via protein kinase C phosphorylation. J Neurochem 69:1467-1475. Nedergaard M (1994) Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263:1768-1771. Nedergaard M, Verkhratsky A (2012a) Artifact versus reality-How astrocytes contribute to synaptic events. Glia 60:1013-1023. Nedergaard M, Verkhratsky A (2012b) Artifact versus reality-How astrocytes contribute to synaptic events? Glia. Nedergaard M, Ransom B, Goldman SA (2003) New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci 26:523-530. Newman EA (2003) Glial cell inhibition of neurons by release of ATP. J Neurosci 23:1659-1666. Nicol GD, Vasko MR, Evans AR (1997) Prostaglandins suppress an outward potassium current in embryonic rat sensory neurons. J Neurophysiol 77:167-176. Nimmerjahn A, Mukamel EA, Schnitzer MJ (2009) Motor behavior activates Bergmann glial networks. Neuron 62:400-412. Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, Xu Q, Wyatt JD, Pilcher W, Ojemann JG, Ransom BR, Goldman SA, Nedergaard M (2009) Uniquely hominid features of adult human astrocytes. J Neurosci 29:3276-3287. Ohno-Shosaku T, Maejima T, Kano M (2001) Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals. Neuron 29:729-738. Ohno-Shosaku T, Hashimotodani Y, Ano M, Takeda S, Tsubokawa H, Kano M (2007) Endocannabinoid signalling triggered by NMDA receptor-mediated calcium entry into rat hippocampal neurons. J Physiol 584:407-418. Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N (2004) Molecular characterization of a phospholipase D generating anandamide and its congeners. J Biol Chem 279:5298-5305. Pacelli GJ, Kelso SR (1991) Trans-ACPD reduces multiple components of synaptic

99  

transmission in the rat hippocampus. Neurosci Lett 132:267-269. Parekh AB, Putney JW, Jr. (2005) Store-operated calcium channels. Physiol Rev 85:757810. Parpura V, Haydon PG (2000) Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc Natl Acad Sci U S A 97:8629-8634. Parpura V, Zorec R (2010) Gliotransmission: Exocytotic release from astrocytes. Brain Res Rev 63:83-92. Parpura V, Fang Y, Basarsky T, Jahn R, Haydon PG (1995) Expression of synaptobrevin II, cellubrevin and syntaxin but not SNAP-25 in cultured astrocytes. FEBS Lett 377:489-492. Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG (1994) Glutamatemediated astrocyte-neuron signalling. Nature 369:744-747. Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY, Takano H, Moss SJ, McCarthy K, Haydon PG (2005) Astrocytic purinergic signaling coordinates synaptic networks. Science 310:113-116. Pasti L, Volterra A, Pozzan T, Carmignoto G (1997) Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. J Neurosci 17:7817-7830. Petralia RS, Wang YX, Niedzielski AS, Wenthold RJ (1996) The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Neuroscience 71:949-976. Petravicz J, Fiacco TA, McCarthy KD (2008) Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. J Neurosci 28:4967-4973. Pin JP, Duvoisin R (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34:1-26. Piomelli D (1993) Arachidonic acid in cell signaling. Current opinion in cell biology 5:274-280. Poncer JC, Shinozaki H, Miles R (1995) Dual modulation of synaptic inhibition by distinct metabotropic glutamate receptors in the rat hippocampus. J Physiol 485 ( Pt 1):121-134.

100  

Porter JT, McCarthy KD (1996) Hippocampal astrocytes in situ respond to glutamate released from synaptic terminals. J Neurosci 16:5073-5081. Ramakers GM, Storm JF (2002) A postsynaptic transient K(+) current modulated by arachidonic acid regulates synaptic integration and threshold for LTP induction in hippocampal pyramidal cells. Proc Natl Acad Sci U S A 99:10144-10149. Ransom B, Behar T, Nedergaard M (2003) New roles for astrocytes (stars at last). Trends Neurosci 26:520-522. Reeves AM, Shigetomi E, Khakh BS (2011) Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps. J Neurosci 31:9353-9358. Rodriguez de Fonseca F, Del Arco I, Bermudez-Silva FJ, Bilbao A, Cippitelli A, Navarro M (2005) The endocannabinoid system: physiology and pharmacology. Alcohol Alcohol 40:2-14. Sang N, Zhang J, Marcheselli V, Bazan NG, Chen C (2005) Postsynaptically synthesized prostaglandin E2 (PGE2) modulates hippocampal synaptic transmission via a presynaptic PGE2 EP2 receptor. J Neurosci 25:9858-9870. Santello M, Bezzi P, Volterra A (2011) TNFalpha controls glutamatergic gliotransmission in the hippocampal dentate gyrus. Neuron 69:988-1001. Scanziani M, Salin PA, Vogt KE, Malenka RC, Nicoll RA (1997) Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature 385:630-634. Schaeffer EL, Gattaz WF (2007) Requirement of hippocampal phospholipase A2 activity for long-term memory retrieval in rats. J Neural Transm 114:379-385. Schell MJ, Molliver ME, Snyder SH (1995) D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proc Natl Acad Sci U S A 92:3948-3952. Seegers HC, Gross RW, Boyle WA (2002) Calcium-independent phospholipase A(2)derived arachidonic acid is essential for endothelium-dependent relaxation by acetylcholine. J Pharmacol Exp Ther 302:918-923. Sergeeva M, Strokin M, Wang H, Ubl JJ, Reiser G (2002) Arachidonic acid and docosahexaenoic acid suppress thrombin-evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid liberation. J Neurochem 82:1252-1261.

101  

Serrano A, Haddjeri N, Lacaille JC, Robitaille R (2006) GABAergic network activation of glial cells underlies hippocampal heterosynaptic depression. J Neurosci 26:5370-5382. Sharp AH, Nucifora FC, Jr., Blondel O, Sheppard CA, Zhang C, Snyder SH, Russell JT, Ryugo DK, Ross CA (1999) Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain. J Comp Neurol 406:207-220. Shigetomi E, Bowser DN, Sofroniew MV, Khakh BS (2008) Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. J Neurosci 28:6659-6663. Shigetomi E, Bushong EA, Haustein MD, Tong X, Jackson-Weaver O, Kracun S, Xu J, Sofroniew MV, Ellisman MH, Khakh BS (2013) Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J Gen Physiol 141:633-647. Sinclair AJ (1975) Long-chain polyunsaturated fatty acids in the mammalian brain. The Proceedings of the Nutrition Society 34:287-291. Singaravelu K, Lohr C, Deitmer JW (2006) Regulation of store-operated calcium entry by calcium-independent phospholipase A2 in rat cerebellar astrocytes. J Neurosci 26:9579-9592. Stella N (2004) Cannabinoid signaling in glial cells. Glia 48:267-277. Stella N, Schweitzer P, Piomelli D (1997a) A second endogenous cannabinoid that modulates long-term potentiation. Nature 388:773-778. Stella N, Tence M, Glowinski J, Premont J (1994) Glutamate-evoked release of arachidonic acid from mouse brain astrocytes. J Neurosci 14:568-575. Stella N, Estelles A, Siciliano J, Tence M, Desagher S, Piomelli D, Glowinski J, Premont J (1997b) Interleukin-1 enhances the ATP-evoked release of arachidonic acid from mouse astrocytes. J Neurosci 17:2939-2946. Stephenson D, Rash K, Smalstig B, Roberts E, Johnstone E, Sharp J, Panetta J, Little S, Kramer R, Clemens J (1999) Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration. Glia 27:110-128. Stephenson DT, Lemere CA, Selkoe DJ, Clemens JA (1996) Cytosolic phospholipase A2 (cPLA2) immunoreactivity is elevated in Alzheimer's disease brain. Neurobiol

102  

Dis 3:51-63. Strokin M, Chechneva O, Reymann KG, Reiser G (2006) Neuroprotection of rat hippocampal slices exposed to oxygen-glucose deprivation by enrichment with docosahexaenoic acid and by inhibition of hydrolysis of docosahexaenoic acidcontaining phospholipids by calcium independent phospholipase A2. Neuroscience 140:547-553. Sugiura T, Kobayashi Y, Oka S, Waku K (2002) Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance. Prostaglandins, leukotrienes, and essential fatty acids 66:173-192. Sun GY, Xu J, Jensen MD, Simonyi A (2004) Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases. Journal of lipid research 45:205-213. Sun GY, Xu J, Jensen MD, Yu S, Wood WG, Gonzalez FA, Simonyi A, Sun AY, Weisman GA (2005) Phospholipase A2 in astrocytes: responses to oxidative stress, inflammation, and G protein-coupled receptor agonists. Mol Neurobiol 31:27-41. Sun W, McConnell E, Pare JF, Xu Q, Chen M, Peng W, Lovatt D, Han X, Smith Y, Nedergaard M (2013) Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 339:197-200. Szabo B, Urbanski MJ, Bisogno T, Di Marzo V, Mendiguren A, Baer WU, Freiman I (2006) Depolarization-induced retrograde synaptic inhibition in the mouse cerebellar cortex is mediated by 2-arachidonoylglycerol. J Physiol 577:263-280. Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, Nedergaard M (2006) Astrocyte-mediated control of cerebral blood flow. Nat Neurosci 9:260-267. Takano T, Kang J, Jaiswal JK, Simon SM, Lin JH, Yu Y, Li Y, Yang J, Dienel G, Zielke HR, Nedergaard M (2005) Receptor-mediated glutamate release from volume sensitive channels in astrocytes. Proc Natl Acad Sci U S A 102:16466-16471. Tamaru Y, Nomura S, Mizuno N, Shigemoto R (2001) Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites. Neuroscience 106:481-503. Taussig R, Zimmermann G (1998) Type-specific regulation of mammalian adenylyl cyclases by G protein pathways. Advances in second messenger and phosphoprotein research 32:81-98.

103  

Tay HK, Melendez AJ (2004) Fcgamma RI-triggered generation of arachidonic acid and eicosanoids requires iPLA2 but not cPLA2 in human monocytic cells. J Biol Chem 279:22505-22513. Testa CM, Standaert DG, Young AB, Penney JB, Jr. (1994) Metabotropic glutamate receptor mRNA expression in the basal ganglia of the rat. J Neurosci 14:30053018. Tong G, Jahr CE (1994) Block of glutamate transporters potentiates postsynaptic excitation. Neuron 13:1195-1203. Tong X, Shigetomi E, Looger LL, Khakh BS (2013) Genetically encoded calcium indicators and astrocyte calcium microdomains. Neuroscientist 19:274-291. van Tol-Steye H, Lodder JC, Mansvelder HD, Planta RJ, van Heerikhuizen H, Kits KS (1999) Roles of G-protein beta gamma, arachidonic acid, and phosphorylation inconvergent activation of an S-like potassium conductance by dopamine, AlaPro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2. J Neurosci 19:3739-3751. Varma N, Carlson GC, Ledent C, Alger BE (2001) Metabotropic glutamate receptors drive the endocannabinoid system in hippocampus. J Neurosci 21:RC188. Velez-Fort M, Audinat E, Angulo MC (2012) Central role of GABA in neuron-glia interactions. Neuroscientist 18:237-250. Ventura R, Harris KM (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J Neurosci 19:6897-6906. Verkhratsky A, Kettenmann H (1996) Calcium signalling in glial cells. Trends Neurosci 19:346-352. Virginio C, MacKenzie A, Rassendren FA, North RA, Surprenant A (1999) Pore dilation of neuronal P2X receptor channels. Nat Neurosci 2:315-321. Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci 6:626-640. Wallraff A, Kohling R, Heinemann U, Theis M, Willecke K, Steinhauser C (2006) The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus. J Neurosci 26:5438-5447. Walter L, Stella N (2003) Endothelin-1 increases 2-arachidonoyl glycerol (2-AG) production in astrocytes. Glia 44:85-90.

104  

Walter L, Dinh T, Stella N (2004) ATP induces a rapid and pronounced increase in 2arachidonoylglycerol production by astrocytes, a response limited by monoacylglycerol lipase. J Neurosci 24:8068-8074. Wang F, Smith NA, Xu Q, Fujita T, Baba A, Matsuda T, Takano T, Bekar L, Nedergaard M (2012) Astrocytes modulate neural network activity by Ca(2)(+)-dependent uptake of extracellular K(+). Science signaling 5:ra26. Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9:816-823. Werry TD, Wilkinson GF, Willars GB (2003) Mechanisms of cross-talk between Gprotein-coupled receptors resulting in enhanced release of intracellular Ca2+. Biochem J 374:281-296. Winship IR, Plaa N, Murphy TH (2007) Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo. J Neurosci 27:6268-6272. Wolf MJ, Gross RW (1996) The calcium-dependent association and functional coupling of calmodulin with myocardial phospholipase A2. Implications for cardiac cycledependent alterations in phospholipolysis. J Biol Chem 271:20989-20992. Wolf MJ, Wang J, Turk J, Gross RW (1997) Depletion of intracellular calcium stores activates smooth muscle cell calcium-independent phospholipase A2. A novel mechanism underlying arachidonic acid mobilization. J Biol Chem 272:15221526. Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, Gonzalez FA, Weisman GA, Wood WG, Sun GY (2003) Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents. Prostaglandins, leukotrienes, and essential fatty acids 69:437-448. Yang HC, Mosior M, Ni B, Dennis EA (1999) Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A2 from rat brain. J Neurochem 73:1278-1287. Yang Y, Ge W, Chen Y, Zhang Z, Shen W, Wu C, Poo M, Duan S (2003) Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine. Proc Natl Acad Sci U S A 100:15194-15199. Yokoi M, Kobayashi K, Manabe T, Takahashi T, Sakaguchi I, Katsuura G, Shigemoto R, Ohishi H, Nomura S, Nakamura K, Nakao K, Katsuki M, Nakanishi S (1996)

105  

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science 273:645-647. Yoshihara Y, Yamaji M, Kawasaki M, Watanabe Y (1992) Ontogeny of cytosolic phospholipase A2 activity in rat brain. Biochem Biophys Res Commun 185:350355. Zhang JM, Wang HK, Ye CQ, Ge W, Chen Y, Jiang ZL, Wu CP, Poo MM, Duan S (2003) ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression. Neuron 40:971-982. Zhang Q, Fukuda M, Van Bockstaele E, Pascual O, Haydon PG (2004a) Synaptotagmin IV regulates glial glutamate release. Proc Natl Acad Sci U S A 101:9441-9446. Zhang Q, Pangrsic T, Kreft M, Krzan M, Li N, Sul JY, Halassa M, Van Bockstaele E, Zorec R, Haydon PG (2004b) Fusion-related release of glutamate from astrocytes. J Biol Chem 279:12724-12733. Zhu PJ (2006) Endocannabinoid signaling and synaptic plasticity in the brain. Crit Rev Neurobiol 18:113-124. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, Carmignoto G (2003) Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6:43-50.