Interactions Between Brain-Derived Neurotrophic Factor And

Interactions between Brain-Derived Neurotrophic Factor and

Endocannabinoids at Neocortical Inhibitory Synapses

Liangfang Zhao, Ph.D.

University of Connecticut, 2014

Endogenous cannabinoids (endocannabinoids; eCBs) play important roles

in synaptic transmission. Known as retrograde messengers, eCBs are released

on demand from postsynaptic sites and suppress presynaptic neurotransmitter

release in several brain regions where the primary receptor, the type 1

cannabinoid receptor (CB1R) is expressed. Similarly, neurotrophins, particularly

brain-derived neurotrophic factor (BDNF), act as potent modulators of synaptic

transmission in many brain regions, most notably the neocortex and

hippocampus. The effect of BDNF in synaptic transmission is mainly mediated by

activation of tropomyosin receptor kinase B (trkB) receptors.

There is growing evidence for cross-talk between BDNF and eCB signaling.

Recently it has been shown that BDNF can induce eCB release at cortical inhibitory synapses, but little is known about the underlying signaling pathways or

functional relevance of this interaction. In the present studies, we examined the

intracellular signaling pathways that underlie BDNF-induced eCB release, as well

as their interactions in regulating activity-dependent long-term depression at

Liangfang Zhao – University of Connecticut, 2014

inhibitory synapses (iLTD). Using pharmacological approaches and whole-cell

patch clamp recordings from layer 2/3 pyramidal neurons in mouse somatosensory cortical slices, we found that PLCγ underlies BDNF-induced eCB release, as it was blocked by PLC inhibitors. Other downstream signaling pathways of trkB, namely protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, are not involved.

This effect is also independent of mGluR activation. Furthermore, we found that

theta burst stimulation (TBS) induces a form of eCB-mediated LTD at inhibitory

synapses (eCB-iLTD) that is independent of mGluR activation but requires

endogenous BDNF as well as downstream PLCγ signaling. Endocannabinoid-

mediated iLTD can also be induced by combining a subthreshold concentration of exogenous BDNF with weak TBS that is insufficient to induce iLTD alone.

Taken together, these results identified the signaling pathway underlying BDNF- induced eCB release, and identified a novel form of eCB-iLTD that requires endogenous BDNF signaling. Our studies may contribute to the understanding of functional interactions between BDNF and eCB signaling, as well as provide

insights in understanding activity-dependent modulation of cortical circuits.

Interactions between Brain-Derived Neurotrophic Factor and

Endocannabinoids at Neocortical Inhibitory Synapses

Liangfang Zhao

B.S., Capital Normal University, Beijing, China, 2009

A Dissertation

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

at the

University of Connecticut

2014

Liangfang Zhao

2014 APPROVAL PAGE

Doctor of Philosophy Dissertation

Interactions between Brain-Derived Neurotrophic Factor and

Endocannabinoids at Neocortical Inhibitory Synapses

Presented by

Liangfang Zhao, B.S.

Major Advisor ______Eric S. Levine, Ph. D.

Associate Advisor ______Richard E. Mains, Ph. D.

Associate Advisor ______Xin-Ming Ma, Ph. D.

Associate Advisor ______Douglas L. Oliver, Ph. D.

University of Connecticut

2014

Dedications

To my beloved grandparents Qiang-Sheng Sun (1930 - ) and Kuang-Yi Liang

(1927 - 2014), for everything they have given me.

Acknowledgements

This dissertation could not have been written without the help and guidance

of my major advisor, Dr. Eric Levine, who has provided me tremendous

inspiration, wisdom and challenge throughout my graduate career. I am forever

grateful for his strict but supportive training in the purpose of teaching me to fish,

as well as the intellectual freedom and friendship he has provided me. I would also like to thank my thesis advisory committee, Drs. Douglas Oliver, Richard

Mains, and Xin-Ming Ma, for their persistent guidance and constructive criticism to make me a better scientist. I also deeply appreciate the past and current members of the Levine Lab: Dr. Lawrence Hsieh, Dr. Fouad Lemtiri-Chlieh,

Tiwanna Robinson, James Fink, Kia Bolduc, Dr. Mason Yeh, for their support, companionship and scientific insights.

I am also greatly indebted to all my lovely friends, in particular, Xi Bie, Yi-

Zhou Zhu, Yi-He Ma, Qi-Yu Liang, Tong Chen, Zi-Yan Zhang, Ying-Hui Zhang, as well as my family, for giving me the emotional support and encouragement any-time when I needed them. Even if there are always significant time differences and distances, they always feel so close to me. Most of all I would like to thank my loving boyfriend Daniel Sheehy, for his undying love and support that are beyond the power of language during this academic endeavor.

iii

Table of Contents

Chapter 1. Introduction, Background, and Rationale ...... 1

1.1 Overview ...... 1 1.2 The endogenous cannabinoid system ...... 2 1.2.1 Cannabinoid receptors ...... 2 1.2.2 Endogenous ligands in the brain and their synthesis ...... 6 1.2.3 Endocannabinoid mobilization and metabolism ...... 8 1.3 Endocannabinoid-mediated synaptic plasticity ...... 12 1.3.1 Endocannabinoid-mediated short-term plasticity ...... 12 1.3.2 Endocannabinoid-mediated long-term plasticity ...... 14 1.4 The brain-derived neurotrophic factor (BDNF) system ...... 18 1.4.1 BDNF, trkB receptor and BDNF-trkB signaling pathways .... 18 1.4.2 Distribution of trkB receptors ...... 20 1.4.3 Distribution and release of endogenous BDNF ...... 21 1.5 Functional role of BDNF in synaptic plasticity ...... 23 1.5.1 Acute effect of BDNF on synaptic transmission ...... 23 1.5.2 BDNF and long-term synaptic plasticity ...... 26 1.6 Interactions between BDNF and endocannabinoid systems ...... 28 1.7 Rationale and hypothesis...... 31

Chapter 2. Materials and Methods ...... 35

2.1 Animal handling and slice preparation ...... 35 2.2 Electrophysiology ...... 36 2.3 Chemicals ...... 37 2.4 Data analysis ...... 38

Chapter 3. BDNF-endocannabinoid interactions at neocortical inhibitory synapses require phospholipase C signaling ...... 39

3.1 The effect of BDNF on IPSC requires postsynaptic phospholipase-C signaling ...... 42 3.2 The effect of BDNF is independent of PKC, MAPK and PI3K signaling ...... 46 3.3 The effect of BDNF is independent of mGluR signaling ...... 49

3.4 Summary and discussion ...... 52

Chapter 4. Role for Endogenous BDNF in Endocannabinoid-mediated Long-Term Depression at Neocortical Inhibitory Synapses ...... 53

4.1 Strong theta frequency burst stimulation induces eCB-dependent iLTD ...... 55 4.2 iLTD in layer 2/3 of somatosensory cortex is independent of mGluR signaling ...... 57 4.3 iLTD in layer 2/3 of somatosensory cortex requires endogenous BDNF and phospholipase-C signaling ...... 59 4.4 Exogenous BDNF facilitates iLTD induction ...... 61 4.5 Summary and discussion ...... 65

Chapter 5. Discussion and conclusions ...... 66

5.1 Summary and interpretation of findings ...... 66 5.2 Control experiments for pharmacological experiments ...... 72 5.2.1 Controls for negative drug effects ...... 73 5.2.2 Controls for positive drug effects ...... 75 5.3 Limitations of the present studies ...... 77 5.4 Functional impact of BDNF-eCB interactions on cortical synaptic plasticity ...... 80 5.5 Conclusions and future directions for BDNF-eCB interactions ...... 83

References ...... 87

Chapter 1

Introduction, Background, and Rationale

This chapter is a modified version of a review article in preparation: Zhao L and Levine ES, It takes two to tango: Interactions between brain-derived neurotrophic factor and endogenous cannabinoid.

1.1. Overview

Synaptic plasticity is a fundamental property of the brain, which allows us to learn and remember, adapt to new situations, refine movements, predict and obtain reward, and recover function after injury. For over a century, neuroscientists have endeavored to understand the mechanisms of synaptic plasticity, in other words, how transient neuronal activity causes enduring synaptic changes, which ultimately leads to modification of brain function (Citri and Malenka 2008; Hebb 1949; James

1890). The neocortex, composed of six distinct layers of orderly connected synapses both within- and between-layers (Bannister 2005; Cajal 1911), is a major brain region for synaptic plasticity. It controls sensory and motor perceptions, as well as advanced cognitive tasks such as problem solving and decision making, all of which are heavily influenced by prior experience and usage. Acquisition and processing of information within the neocortex involve a sequence of synaptic relays across different layers. Local microcircuits extensively integrate all the excitatory and inhibitory information received in each layer and thus exert an important role in shaping synaptic plasticity.

The activity-dependent modulation of synaptic plasticity is a complex process involving multiple synaptic and cellular mechanisms that occurs during development and throughout life. Brain-derived neurotrophic factor (BDNF) and endogenous cannabinoids (endocannabinoids, or eCB) are two of the most important classes of molecules that modulate cortical synaptic plasticity (Alger 2002; Aloyz et al. 1999;

Bracken and Turrigiano 2009; Jiang et al. 2010; Johnston 2009; Kullmann et al.

2012; Lu et al. 2009). There is also growing evidence for functional interactions between BDNF and endocannabinoid signaling, and it has been suggested that they may work in concert in modulating synaptic plasticity, yet our knowledge about their interactions is still limited. The proposed studies will examine the functional significance of BDNF-eCB interaction in the cortex by 1) identifying the intracellular signaling pathway underlying BDNF-induced eCB release; 2) examining the role of

BDNF in eCB-mediated synaptic plasticity.

1.2. The endogenous cannabinoid system

The endocannabinoid system includes cannabinoid receptors, endogenous ligands, and enzymes for ligand biosynthesis, mobilization and metabolism. Below, I will describe these components in detail. The functional role of the endocannabinoid system in synaptic plasticity will be discussed in Chapter 1.3.

1.2.1. Cannabinoid receptors

Two types of cannabinoid receptors have been identified, type-1 and type-2 cannabinoid receptors (CB1R and CB2R), both of which are Gi/o-linked G-protein 2 coupled receptors (GPCRs). CB1Rs are the predominant cannabinoid receptors found in the brain (Howlett et al. 2002). CB2Rs are expressed in the immune system, although recent studies have also shown that they are expressed in both glial and neuronal processes in a number of brain regions, including (but not limited to) the cerebral cortex, striatum, thalamic nuclei, hippocampus and amygdala (Gong et al. 2006; Van Sickle et al. 2005). In the following paragraphs I will focus on

CB1Rs, as their function in synaptic plasticity is well characterized compared with

CB2Rs.

As a Gi/o-protein coupled receptor, activation of CB1R negatively regulates voltage-gated calcium channels (VGCCs), in particular N-type and P/Q- type

(Mackie et al. 1995; Twitchell et al. 1997) . Activation of CB1R also leads to suppression of adenylyl cyclase and cAMP production, as well as modulation of various potassium channels, including suppressing D- (Mu et al. 1999) and M- type

K+ channels (Schweitzer 2000) and activation of A-type (Hampson et al. 1995) and

G protein-coupled inwardly-rectifying (GIRK, Mackie et al. 1995) potassium channels.

The CB1R is one of the most highly expressed GPCRs in the brain (Howlett

1998), with highest levels in the cerebellum, basal ganglia, hippocampus, cerebral cortex, amygdala, and nucleus accumbens (Egertova et al. 2003; Herkenham et al.

1990; Matsuda et al. 1993). CB1Rs are found predominantly at presynaptic axonal terminals of both GABA and glutamate axons (Katona et al. 1999; Katona et al.

2006). Evidence for CB1R expression on neuronal somata (Freund et al. 2003), astrocytes (Navarrete and Araque 2008; 2010), and microglia (Molina-Holgado et al.

2002; Ramirez et al. 2005) has also been found, although at a much lower density than that on presynaptic neuronal terminals. The major physiological effect of CB1R activation is to suppress presynaptic neurotransmitter release (Kreitzer and Regehr

2001; Wilson and Nicoll 2001). Together with the postsynaptic site release (which will be further described in the following sections), the presynaptic location of

CB1Rs allows endocannabinoids to act as retrograde synaptic signals (for detailed reviews please see Alger 2002; Freund et al. 2003).

In the neocortex, CB1R expression exhibits a laminar-specific pattern, and the distribution varies in different cortical regions as well as in different species. For example, in rodents, CB1R is highly expressed in layers 2/3, 5a and 6 of the somatosensory cortex (Deshmukh et al. 2007; Egertova et al. 2003; Egertova and

Elphick 2000; Marsicano and Lutz 1999; Tsou et al. 1998). In the entorhinal cortex,

CB1R is most intense in layers 2 and 6 (Tsou et al. 1998). On the other hand, the expression of CB1R in prefrontal and cingulate cortices is concentrated in layer 4 in monkeys (Eggan and Lewis 2007), but layer 4 in these cortical areas has the lowest

CB1R expression in rat (Egertova and Elphick 2000). The layer- and species- specific distribution of CB1R suggested that eCBs might modulate cortical functions differently in different cortical regions and species.

CB1R expression also displays a cell-type specific distribution pattern. High levels of CB1R expression have been found in terminals and preterminal axons of large cholecystokinin (CCK)-containing interneurons and calbindin-positive interneurons that surround pyramidal neurons (Bodor et al. 2005; Eggan and Lewis

2007; Harkany et al. 2005; Marsicano and Lutz 1999). CCK-containing interneurons

4 are basket cells that mainly innervate the perisomatic area of pyramidal neurons

(Neu et al. 2007), while calbindin-positive interneurons resemble dendrite targeting double bouquet or bitufted interneurons that target distal dendrites of pyramidal neurons in the cortex (DeFelipe et al. 1989; Wedzony and Chocyk 2009). However,

CB1Rs are rarely detected in parvalbumin-, calretinin-, somatostatin- or vasoactive intestinal peptide (VIP)-containing interneurons in the cortex (Bodor et al. 2005;

Marsicano and Lutz 1999), although single-cell PCR have detected CB1 mRNA in the latter two types of interneurons (Hill et al. 2007). Significant amount of CB1Rs are also found at excitatory terminals, but much lower than the concentration at inhibitory ones (Lafourcade et al. 2007).

Some other cannabinoid-related receptors include transient receptor potential vanilloid type-1 ion channel (TRPV1) and GPR55 receptor. TRPV1 belongs to the transient receptor potential family, and is a Ca2+ permeable, nonselective cation channel (De Petrocellis et al. 2000). TRPV1 is not activated by 2- arachidonoylglycerol (2-AG), one of the major endogenous ligands of CB1R, or synthetic cannabinoids and is thus not categorized as a cannabinoid receptor.

However, the other major endogenous ligand, anandamide (AEA), appears to be a full agonist of TRPV1 (thus AEA is also called endovanilloid), implicating a potential cross-talk between the eCB and endovanilloid systems (Maccarrone et al. 2008).

GPR55 is an orphan GPCR that has recently been recognized as a novel cannabinoid receptor. It can be activated by a number of cannabinoid ligands but has distinct pharmacological properties compared to CB1R and CB2R. GPR55 can be activated by Δ9-THC, CP55,940, and endocannabinoids including AEA, 2-AG,

5 noladin ether, and virodhamine, but not by the synthetic compound WIN55,212-2

(WIN). GPR55 is also known to be activated by some non-CB1R or CB2R fatty acid ligands such as palmitoylethanolamide and oleoylethanolamide (Ryberg et al. 2007).

Although GPR55 mRNA is detected in the brain, it is not evident that functional

GPR55 proteins are actually expressed, and the function of this novel cannabinoid receptor is currently unknown.

1.2.2. Endogenous ligands in the brain and their synthesis

Several endogenous brain lipids are found to bind to and activate CB1R receptors and are thus called endogenous cannabinoids (endocannabinoids, or eCBs). The two best characterized endogenous CB1R ligands are 2- achidonoylglycerol (2-AG) and arachidonoylethanolamide (anandamide, or AEA).

There are also other potential endogenous ligands, such as noladin ether (Hanus et al. 2001), virodhamine (Porter et al. 2002), N-arachidonoyl dopamine (NADA,

Huang et al. 2002), dihomo-γ-linolenoyl ethanolamine (Hanus et al. 1993), and oleamide (Leggett et al. 2004). Interestingly, these lipids do not share structural similarities to Δ9-tetrahydrocannabinol (THC), the active component of marijuana. In the paragraphs below, I will focus on the synthesis, mobilization and metabolism of

AEA and 2-AG.

Anandamide (AEA) was the first identified endocannabinoid, originally isolated from pig brain (Devane et al. 1992). It is named after the Sanskrit word for bliss, ananda, based on the euphoria associated with marijuana use. AEA acts as a

6 partial agonist for both CB1 and CB2 receptors, and also acts as a full agonist for the vanilloid receptor TRPV1 (Ross et al. 2001; Starowicz et al. 2007).

Three pathways have been identified for anandamide synthesis; the first and major synthetic route of AEA involves a two-step process. First, the precursor N- arachidonoyl phosphatidylethanolamine (NArPE) is synthesized in a Ca2+- dependent manner by the enzyme N-acyltransferase (NAT) (Cadas et al. 1997).

Second, NArPE is cleaved to AEA and phosphatidic acid through the action of a phosphodiesterase (NArPE-specific phospholipase D, or NArPE-specific PLD, Di

Marzo et al. 1994). AEA can be alternatively synthesized by direct condensation of free arachidonic acids and ethanolamine (Deutsch and Chin 1993), although the physiological significance of this pathway is questioned (Sugiura et al. 1996; Ueda et al. 1995). A third synthesis pathway, which was found recently, is independent of

NArPE-specific PLD. In this pathway, NArPE is first converted into phospho-AEA and then to AEA via phospholipase C (PLC) signaling and the following action of various phosphatases (Liu et al. 2008).

The other well studied endocannabinoid, 2-AG, is a full agonist of CB1R and

CB2R, and its concentration in brain tissue is about 200-fold higher than that of AEA

(Stella et al. 1997; Sugiura et al. 1995), suggesting that 2-AG may be the main eCB in the brain. 2-AG and AEA are independent endocannabinoids that are synthesized via different lipid metabolism pathways, and are believed to mediate different functions upon activating CB1R and CB2R receptors (Piomelli et al. 1998). The main synthesis pathway of 2-AG in brain involves hydrolysis of phosphatidylinositol

(PI) by phospholipase C (PLC) and subsequent hydrolysis of 1,2-diacylglycerol

(DAG) by a DAG lipase (DGL) (Stella et al. 1997). There are two isoforms of DGL,

DGLα and DGLβ (Bisogno et al. 2003). DGLα is distributed in the spine head and neck, and is thus believed to be related to activity-dependent 2-AG release (Kano et al. 2009). DGLβ has been suggested to contribute to non-retrograde signaling- related 2-AG (Alger and Kim 2011), although its role in modulating synaptic activity cannot be excluded (Min et al. 2010). Alternative pathways of 2-AG synthesis include hydrolysis of PI via phospholipase A1 (PLA1) and lysophosphatidylinositol- specific PLC (Tsutsumi et al. 1994), as well as conversion of 2-arachidonoyl lysophosphatidic acid (LPA) to 2-AG via an unidentified phosphatase (Nakane et al.

2002).

1.2.3. Endocannabinoid mobilization and metabolism

Unlike classical hydrophilic neurotransmitters that are packed in presynaptic vesicles and travel anterogradely to activate postsynaptic receptors, eCBs are best known as retrograde signaling molecules. Typically, eCBs are synthesized and released from postsynaptic membrane lipid precursors in a non-vesicular manner, and travel retrogradely to activate presynaptic CB1Rs. The synthesis and release of eCBs are usually considered as one integrated process, as it is hard to distinguish these two processes in most experiments due to the on-demand mobilization of eCBs.

Several known mechanisms have been found to induce eCB release. The first form is calcium-induced eCB release (Ca2+-ER), which is driven by strong depolarization to the postsynaptic neurons and large increase of intracellular

8 concentration of Ca2+ via VGCCs (Pitler and Alger 1992) and NMDA receptors

(Ohno-Shosaku et al. 2007). Ca2+-ER is independent of PLC signaling, because inhibitor of PLC failed to block depolarization-induced eCB-release in hippocampus and cerebellum (Chevaleyre and Castillo, 2003; Edwards et al., 2006; Szabo et al.,

2006). In addition, genetic ablation of various isoforms of PLC did not change eCB- mediated short-term depression (Hashimotodani et al. 2008; Hashimotodani et al.

2005; Maejima et al. 2005).

Activation of Gq/11-coupled receptor also drives eCB release (receptor-driven eCB release, RER). RER requires activation of PLCβ, which is downstream of Gq- protein coupled receptor (GqPCR) activation such as group I metabotropic glutamate receptors (I-mGluRs, Maejima et al. 2001), M1/M3 muscarinic acetylcholine receptors (Kim et al. 2002; Straiker and Mackie 2007), or orexin receptors (Haj-Dahmane and Shen 2005). Importantly, RER is independent of postsynaptic Ca2+ elevation (Hashimotodani et al. 2007a; Maejima et al. 2001).

Therefore, mechanisms of the mGluR1-driven endocannabinoid release were assumed to be distinct from those driven by calcium.

It has also been found that manipulations that elevate intracellular Ca2+ level can greatly enhance the effect of Gq-driven RER. This form of eCB release is termed Ca2+-assisted RER. Calcium entry through postsynaptic depolarization in combination with mGluR or muscarinic agonists such as DHPG or carbamylcholine

(CCh) enhances eCB-release (Martin and Alger 1999; Ohno-Shosaku et al. 2002;

Straiker and Mackie 2007).

A fourth mechanism of eCB release is induced by BDNF. This form of eCB release is triggered by activation of trkB receptors upon binding of BDNF, and PLCγ and elevated intracellular Ca2+ level are both required (Lemtiri-Chlieh and Levine

2010; Zhao and Levine 2014). This form of eCB release shares some similarities with the Ca2+-assisted RER but also has distinct differences that it is not simply the summation of Ca2+-ER and RER, but requires both factors.

After release from postsynaptic cell and activates presynaptic CB1Rs, AEA and 2-AG are rapidly deactivated by specific serine hydrolyzing enzymes. AEA is degraded into arachidonic acid and ethanolamine by fatty acid amide hydrolase

(FAAH) that exists mostly in postsynaptic membranes (Cravatt et al. 1996; Hillard et al. 1995; Schmid et al. 1985). 2-AG is degraded into fatty acid and glycerol by monoacylglycerol lipase (MGL) located in presynaptic cytosol (Dinh et al. 2002).

FAAH (Goparaju et al. 1998) and α/β hydrolase 6 and 12 (ABHD6 and ABHD 12,

Blankman et al. 2007) have also been found to target 2-AG degradation as well, although the majority (85%) is hydrolyzed by MGL (Blankman et al. 2007).

Quite a few pharmacological tools have been developed to study the reuptake and metabolism of eCBs, including FAAH and MGL inhibitors as well as eCB transport inhibitors. However, the selectivity of these drugs is far from satisfying. For example, N-(4-hydroxyphenyl)-arachidonylamide (AM404), has been shown to block anandamide uptake with IC50 ranges from 1-11 μM (Beltramo et al. 1997;

Piomelli et al. 1999). However, AM404 also targets CB1R (IC50 = 1.76 μM) (Pertwee

2000), TRPV1 (pEC50 = 7.4 μM) (Zygmunt et al. 2000) and is susceptible to FAAH

(De Petrocellis et al. 2000; Schmid et al. 1985). Similar issues of low selectivity also exist among most FAAH and MGL inhibitors.

It is not clear however, how hydrophobic fatty acids like eCBs are transported in and out of the lipid membrane for release and reuptake processes. In fact, not only can eCBs cross the lipid membrane, they seem to be able to cross the membrane very rapidly (Bojesen and Hansen 2005). Several models have been proposed to explain this puzzle. The most influential model suggested a facilitated transport (Di Marzo et al. 1994; Hillard et al. 1997). However, different results both in favor and against this model have been reported. For example, studies have found that the intracellular accumulation of AEA is saturable, and time- and temperature-dependent, but is not ATP-dependent or coupled to any ion gradient

(Di Marzo et al. 1994; Hillard et al. 1997; Hillard and Jarrahian 2000), thus indicating an active mechanism. However, Thors and Fowler (Thors and Fowler 2006) suggested that the time and temperature dependence is a reflection of the concentration of AEA available for uptake, rather than the uptake process itself. In addition, (Deutsch et al. 2001) suggested that FAAH might act as an intracellular sequestration mechanism of AEA which keeps the free [AEA]i low and thus allow further passive diffusion, further questioning the facilitated transport mechanism model. It has been suggested that eCBs utilize more than one mechanism at the same time (Ehehalt et al. 2006). However, the identification and/or cloning of the transporter protein is still lacking. Taken together, without better pharmacological tools, it is still difficult to fully characterize the eCB system at the current stage.

1.3. Endocannabinoid-mediated synaptic plasticity

Endocannabinoids acutely suppress inhibitory and excitatory transmission throughout the forebrain, as well as mediate several different forms of short- and long-term depression at both excitatory and inhibitory synapses.

1.3.1. Endocannabinoid-mediated short-term plasticity

Endocannabinoid-mediated short-term depression (eCB-STD) includes depolarization-induced suppression of inhibition (DSI) and excitation (DSE). They were first found in hippocampus and cerebellum almost at the same time in 2001

(Kreitzer and Regehr 2001; Wilson and Nicoll 2001). In neocortex, DSI was first reported by our group in layer 2/3 mouse auditory and visual cortices (Trettel and

Levine 2003). Later studies have also reported DSI/DSE in basal lateral amygdala

(Zhu and Lovinger 2005), basal ganglia (Hashimotodani et al. 2013; Narushima et al.

2006), ventral tegmental area (Melis et al. 2004) and hypothalamus (Jo et al. 2005), suggested a widespread function of eCB signaling throughout the brain. During DSI and DSE, eCBs are released from postsynaptic sites in response to depolarization- induced calcium influx and act retrogradely via CB1Rs to suppress transmitter release (Wilson and Nicoll 2001). The transient suppression under DSE/DSI is believed to be mediated by direct βγ-subunit of Gi/o protein interacting with N-type and P/Q-type voltage-gated calcium channels (VGCCs, Wilson et al. 2001), leading to a rapid suppression of presynaptic Ca2+ influx. Activation of CB1Rs also leads to an increase in K+ channel conductances, although their role in DSI lacks compelling evidence (for review see Chevaleyre et al. 2006). Interestingly, our group has found

12 that only pyramidal neurons (PNs), but not interneurons (INs), express DSI (Lemtiri-

Chlieh and Levine 2007). In this study, we found that in layer 2/3 of somatosensory cortex, the CB1R agonist WIN suppresses GABA release onto regular spiking nonpyramidal, irregular spiking, and fast spiking INs, suggesting that all three classes of INs receive cannabinoid-sensitive inhibitory inputs. However, none of these classes of INs were able to express DSI even with prolonged high-frequency spike trains that cause increases in intracellular calcium similar to that seen in pyramidal neurons. Nor did eCB reuptake inhibitors unmask any DSI in these interneurons. It is not clear whether these interneurons lack the enzymes for endocannabinoid synthesis and/or release, or the amount released by depolarization alone is below the detection threshold using electrophysiological methods.

DSI also exhibits a laminar-specific expression pattern in the somatosensory cortex, which has been investigated in details by Bodor and colleagues (Bodor et al.

2005). They found that CB1R immunoreactivity is most dense in layers 2/3, 5a and

6 of somatosensory cortex. In correlation with the laminar distribution pattern of

CB1R immunoreactivity, most layer 2/3 and a small population of layer 5a pyramidal neurons displayed eCB-dependent DSI (Bodor et al. 2005).

Although a role for eCBs in mediating DSI/DSE is clear, attempts to identify the specific endogenous ligand involved have yielded conflicting results. Inhibition of

MGL lengthens DSI, thus 2-AG is considered as the eCB that mediates DSI

(Hashimotodani et al. 2007b; Makara et al. 2005). However, many pharmacological studies using various DGL inhibitors, namely RHC-80267, tetrahydrolipstatin (THL)

13 or the novel compound OMDM-188, have reported no inhibition of DSI/DSE, even though these inhibitors are able to block other forms eCB signaling (for detailed review see Min et al. 2010). Meanwhile, two recent studies took a transgenic approach and found that in mice lacking DGLα, DSI and DSE are abolished, suggested the involvement of DGL. Notably, such eCB-mediated signaling is intact in animals lacking DGLβ (Gao et al. 2010; Tanimura et al. 2010).

1.3.2. Endocannabinoid-mediated long-term plasticity

Endocannabinoids mediate several forms of long-term depression at both excitatory (eCB-LTD) and inhibitory synapses (eCB-iLTD). eCB-LTD and/or eCB- iLTD has been reported in many brain regions including dorsal striatum (Gerdeman et al. 2002; Kreitzer and Malenka 2005), nucleus accumbens (NAc, Mato et al. 2008;

Robbe et al. 2002), sensory cortex (Bender et al. 2006; Crozier et al. 2007; Huang et al. 2008; Jiang et al. 2010; Lefort et al. 2013; Min and Nevian 2012; Nevian and

Sakmann 2006; Sjostrom et al. 2004; 2003), prefrontal cortex (Chiu et al. 2010;

Lafourcade et al. 2007), cerebellum, (Safo and Regehr 2005), basal lateral amygdala (BLA, Azad et al. 2004; Marsicano et al. 2002) and hippocampus

(Chevaleyre and Castillo 2004; 2003; Chevaleyre et al. 2007; Yasuda et al. 2008).

Induction of eCB-LTD/iLTD requires activation of CB1R for several minutes, but once induced, becomes independent of CB1R activation (Chevaleyre and

Castillo 2003). The relatively long induction period (compared with eCB-mediated short-term plasticity) suggests that CB1 receptors may engage a different signaling pathway than which mediates transient suppression of transmitter release as in

14 eCB-STD. The cyclic AMP/protein kinase A (cAMP/PKA) pathway might be a good candidate for the downstream signaling cascade mediating eCB-iLTD (Heifets and

Castillo 2009). Gi/o-coupled receptors have a well-documented inhibitory effect on adenylyl cyclase and PKA activity via actions of the α-subunit (Childers and

Deadwyler 1996; Howlett et al. 1986), which has a slower onset of action compared with β/γ subunit-mediated actions. Indeed, Chevaleyre and colleagues found that in the hippocampus and the amygdala, TBS-induced eCB-iLTD is prevented by blocking presynaptic cAMP/PKA signaling. Furthermore, they also showed that the active zone protein RIM1α, which can be phosphorylated by PKA, is required for eCB-iLTD, indicating that activation of CB1R may lead to regulation of the release machinery for GABA (Chevaleyre et al. 2007). However, whether this mechanism applies to eCB-LTD/iLTD induced in other brain regions or by other induction protocols is not known.

Several forms of eCB-mediated long-term depression at glutamatergic synapses have been reported in neocortex and hippocampus. Endocannabinoid- mediated timing-dependent plasticity (tLTD) has been reported at different excitatory synapses in the cortex. For example, Sjostrom and colleagues reported that tLTD at excitatory synapses between synaptically connected pairs of layer 5 pyramidal neurons in visual cortex requires eCB release (Sjostrom et al. 2003).

They also postulated that tLTD induction requires a coincident activation of CB1R and presynaptic NMDA receptor activation. However, mGluRs are not involved. An eCB-dependent tLTD induced by similar induction protocol has been reported at layer 4 to 2/3 excitatory synapses of the somatosensory cortex (Bender et al. 2006).

A different induction protocol that combined pairing of presynaptic firing with subthreshold postsynaptic depolarization (depolarization-induced LTD, or dLTD) was also found to be dependent on eCB release (Sjostrom et al. 2004).

Endocannabinoid-mediated LTD can be also induced by presynaptic spikes alone.

In prefrontal cortex, continuous stimulation at 10 Hz (10 min) of layer 2/3 afferents has been reported to induce a robust eCB-LTD in layer 5/6 pyramidal neurons, which requires activation of postsynaptic mGluR5 and PLC signaling, as well as rise in postsynaptic Ca2+ (Lafourcade et al. 2007). The authors also suggested that 2-

AG is the eCB that mediates this LTD, because blocking the degradation of 2-AG

(URB602) but not AEA (URB597) lowered the threshold of LTD induction. In addition, blocking the synthesis of 2-AG with the DGL inhibitor tetrahydrolipstatin

(THL) blocked LTD. High frequency stimulation (HFS, 100 Hz/1s, 2 trains with 20 s inter-train interval) has also been found to elicit mGluR-dependent eCB-iLTD in hippocampal CA1 neurons (Chevaleyre and Castillo 2003).

The eCB-iLTD can be also induced by a combination of pharmacological and electrical stimulation. For example, pairing subthreshold TBS stimulation with a low dose of the mGluR agonist DHPG induced iLTD comparable to strong TBS alone in hippocampal CA1 neurons (Younts et al. 2013). In addition, in layer 5 of prefrontal cortex, coactivation of CB1R and dopamine type 2 receptors (D2Rs), which are both highly expressed in the region and colocalize to presynaptic terminals of GABAergic synapses, triggers eCB-iLTD. In addition to D2R coactivation, this iLTD also requires activation of group I mGluR for induction (Chiu et al. 2010).

The eCB-mediated LTD and iLTD are developmentally regulated, suggesting a role in shaping cortical circuit of sensory cortexes during critical period of postnatal development. In layer 2/3 pyramidal neurons of visual cortex, TBS of layer 4 axons induces eCB-LTD in a mGluR5-dependent, but postsynaptic calcium- and NMDA receptor-independent, manner. Interestingly, this LTD does not occur in animals that has passed their critical period (P 35-41, Huang et al. 2008). Similarly, Jiang and colleagues found that long-term depression of inhibitory synaptic transmission

(iLTD) at the same synapses is eCB-dependent, as it is mimicked by CB1R agonists, blocked by CB1R antagonists and is absent in CB1R knockout mice. This eCB-iLTD was rapidly lost by the fifth postnatal week, which correlates with changes in CB1R sensitivity that is regulated by age and visual experience (Jiang et al. 2010). In summary, the studies reviewed above cover various forms of LTD/iLTD in neocortex and hippocampus, suggesting that eCBs may have multiple roles in regulating long-term plasticity and network circuitry. However, further studies are needed to distinguish the roles of eCBs in the various types of LTD/iLTD.

1.4. The brain-derived neurotrophic factor (BDNF) system

1.4.1. BDNF, trkB receptor and BDNF-trkB signaling pathways

BDNF belongs to the neurotrophin gene family which has long been known to be important for neuronal development and differentiation. It is also a potent modulator of synaptic plasticity. BDNF acutely enhances excitatory synaptic activity via both pre- and postsynaptic mechanisms, suppresses inhibitory synaptic activity, and has been found to be essential for long-term potentiation (LTP, see Gottmann et al. 2009 for review).

Several types of receptors have been found to be responsible for BDNF

-11 signaling: the high affinity full-length tropomyosin receptor kinase B (trkB, Kd ~ 10

M) which mediates the majority of BDNF signaling and its role in synaptic plasticity; the truncated form of trkB (trkB-T1) which lacks protein-tyrosine kinase activity, and

-9 the lower affinity, pan-neurotrophin receptor p75NTR (Kd ~ 10 M), which is a member of the tumor necrosis factor superfamily (Berg et al. 1991; Kaplan et al.

1991; Rodriguez-Tebar and Barde 1988). TrkB-T1 has a widespread distribution in the brain (Fryer et al. 1996; Ohira et al. 1999), and its level of expression increases as the brain ages (Ohira et al. 1999). It is best known as a dominant-negative inhibitor of full-length trkB function (Eide et al. 1996; Saarelainen et al. 2000), although some recent studies also reported its role in regulating cytoskeletal changes in neurons and glia (Ohira et al. 2006; Yacoubian and Lo 2000). The underlying signaling pathways of trkB-T1 are largely unknown. P75NTRs primarily mediate actions that oppose the effects of trkB receptor activation, such as inhibition of axonal outgrowth (Kohn et al. 1999; Walsh et al. 1999; Yamashita et al.

1999) and apoptosis (Barrett 2000; Bunone et al. 1997; Frade and Barde 1999;

Majdan et al. 1997; Rabizadeh et al. 1993). It has also been found that the precursor of BDNF (proBDNF) preferentially binds to p75NTR (Lee et al. 2001; also see Lu et al. 2005 for review). I will focus on trkB receptors in the following paragraphs.

The trkB receptor is a single-transmembrane domain receptor tyrosine kinase.

Upon binding of BDNF, trkB receptors dimerize and phosphorylate each other to enhance catalytic activity of the kinase. Activation results in stimulation of at least three downstream intracellular signaling pathways via tyrosine phosphorylation:

Ras/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinases

(PI3K)/Akt pathways, and the phospholipase Cγ (PLCγ) pathway. Activation of the

Ras/MAPK and PI3K/Akt pathways is critical for neuronal survival and differentiation

(reviewed in Huang and Reichardt 2003; Patapoutian and Reichardt 2001; Segal and Greenberg 1996). Activation of PLCγ leads to cleavage of phosphatidylinositol

4, 5-bisphosphate (PIP2) into second messengers inositol trisphosphate (IP3) and

2+ diacylglycerol (DAG). IP3 induces Ca release from intracellular calcium stores upon binding to its receptor and thus increases intracellular calcium concentration

2+ ([Ca ]i). DAG is involved in several intracellular signaling pathways, one of which is stimulation of protein kinase C (PKC)-δ, which is required for neurite outgrowth

(reviewed in Huang and Reichardt 2003), and has been found to underlie the role of

BDNF in modulating inhibitory synaptic transmission (Henneberger et al. 2002;

Jovanovic et al. 2004).

1.4.2. Distribution of trkB receptors

TrkB mRNA and protein are widely distributed across the brain, with highest densities in the neocortex, hippocampus, cerebellum, amygdala and dorsal root ganglia (Ernfors et al. 1990; Gorba and Wahle 1999; Hofer et al. 1990). The mRNA and protein levels of trkB are also developmentally regulated. The mRNA levels for trkB reaches its peak by birth and remains stable or undergoes a slight decrease throughout adulthood in most regions of rat forebrain (Fryer et al. 1996), while trkB protein reaches peak density as well as stable layer distribution around postnatal day 10, with highest density in neocortical layers 2/3 and 5 (Cabelli et al. 1996).

At the synaptic level, trkB is expressed in both presynaptic terminals and postsynaptic dendrites in the hippocampus and cortex (Aoki et al. 2000; Drake et al.

1999). Presynaptic immunoreactivity for trkB was found at both asymmetric synapses (indicating glutamatergic presynaptic terminals) as well as symmetric synapses (indicating GABAergic or neuromodulatory synapses) using electron microscopy (Drake et al. 1999). Inagaki and colleagues reported that at layer 5 inhibitory synapses in visual cortex, HFS-induced LTP was blocked by bath application of a trk inhibitor, but loading the postsynaptic cell with the inhibitor had no effect on HFS-induced LTP (Inagaki et al. 2008). This physiological evidence, although indirect, supports the existence of presynaptic trkB receptors at inhibitory synapses. The existence of trkB at both pre- and postsynaptic components suggests that BDNF may modulate activity-dependent synaptic plasticity at both sides of the synapse.

1.4.3. Distribution and release of endogenous BDNF

Similar to trkB, BDNF is widely distributed throughout the brain, suggesting an essential role at a variety of synapses. In rodents, BDNF mRNA and protein levels peak shortly after birth (~ 2-3 weeks) and remain stable (Friedman et al. 1991;

Katoh-Semba et al. 1997; Maisonpierre et al. 1990; Patz and Wahle 2006). BDNF mRNA has been found in both neurons (Conner et al. 1997; Wetmore et al. 1990) and astrocytes (Wu et al. 2004), although its level in astrocytes is much lower than in neurons (Zafra et al. 1990). At the subcellular level, BDNF mRNA is distributed not only in somatic structures but also in dendritic compartments, (An et al. 2008;

Capsoni et al. 1999; Tongiorgi et al. 1997), suggesting local BDNF protein synthesis and activity-dependent release of endogenous BDNF (Tongiorgi et al. 2004;

Tongiorgi et al. 2006).

BDNF-containing vesicles have been found at postsynaptic sites (Aoki et al.

2000; Matsuda et al. 2009; Tongiorgi et al. 1997; Wetmore et al. 1991) and presynaptic terminals (Aloyz et al. 1999; Conner et al. 1997; Danzer and McNamara

2004; Kohara et al. 2001; Matsuda et al. 2009). Specifically, Danzer and McNamara reported presynaptic localization of BDNF at glutamatergic mossy fiber-CA3 synapses. Most of the studies that have been done, however, used mixed neuronal cultures, thus it is not clear whether BDNF-containing vesicles only exist at excitatory axonal terminals, or exist at inhibitory terminals as well.

In accordance with the distribution of BDNF-containing vesicles, activity- dependent release of BDNF has been found from both postsynaptic dendrites

(Dean et al. 2012; Hartmann et al. 2001; Matsuda et al. 2009) and presynaptic terminals (Aloyz et al. 1999; Canossa et al. 2001; Dean et al. 2012; Kohara et al.

2001; Matsuda et al. 2009). BDNF release can be triggered by 1) Ca2+ influx through ionotropic glutamate receptors or voltage-gated channels (VGCCs)

(Hartmann et al. 2001); 2) Activation of Group I mGluR receptors, which

2+ subsequently triggers IP3-mediated Ca release from intracellular calcium store

(Canossa et al. 2001); 3) Ca2+-dependent Ca2+ release from intracellular stores by ryanodine receptors (RyR, Balkowiec and Katz 2002), although specific mechanisms for pre- or postsynaptic release have not been identified.

Interestingly, it has been shown that both pre- and postsynaptic sites can release BDNF simultaneously but exert distinct functions in modulating synaptic transmission. Dean and colleagues suggested that postsynaptically released BDNF acts as a retrograde messenger and modulates presynaptic transmitter release frequency, while presynaptically released BDNF might act as an anterograde signal and modulate the amplitude of mEPSCs (Dean et al. 2012). In addition, Matsuda and colleagues reported that brief spiking activity (1 min TBS) is able to induce substantial BDNF secretion at the dendrite, while the same stimulation resulted in very little BDNF secretion at the axon. On the other hand, prolonged high-frequency activity (50 Hz or TBS for 3 min), a condition neurons may encounter during epileptic discharge, induces full vesicular fusion with BDNF secretion at the axon

(Matsuda et al. 2009). This study suggested that activity-dependent axonal secretion of BDNF might represent extreme discharge conditions that neurons may encounter during an epileptic seizure, and BDNF secretion from postsynaptic

22 dendrites might be responsible for normal neural activities. Astrocytes can also release BDNF after stimulation with glutamate, which requires mGluR signaling

(Jean et al. 2008). In addition, BDNF can also be released constitutively in neuronal cell lines (Goodman et al. 1996), although its function is not clear.

1.5. Functional role of BDNF in synaptic plasticity

1.5.1. Acute effect of BDNF on synaptic transmission

BDNF acutely modulates synaptic transmission at both excitatory and inhibitory synapses. BDNF has been found to acutely enhance glutamatergic transmission in various preparations. For example, BDNF enhances glutamate transmission in hippocampal and cortical cultures (Lessmann and Heumann 1998;

Levine et al. 1995; Li et al. 1998), in different regions of hippocampus in acute slices

(CA1, Madara and Levine 2008; dentate gyrus, Messaoudi et al. 1998) and in layer

5 (Carmignoto et al. 1997; Madara and Levine 2008), layer 2/3 (Akaneya et al. 1997) of cortical slices from juvenile rodents, in accordance with trkB expression pattern in the cortex.

BDNF enhances excitatory synaptic transmission both presynaptically, by enhancing glutamate release (Lessmann et al. 1994; Lohof et al. 1993; Madara and

Levine 2008), as well as postsynaptically, by enhancing the response of NMDA glutamate receptors (Crozier et al. 1999; Levine et al. 1998). Importantly, BDNF has been found to enhance both pre- and postsynaptic mechanisms in the same preparation (Madara and Levine 2008). In this study, BDNF rapidly enhanced

23 miniature excitatory postsynaptic current (mEPSC; i.e. action potential- independent currents) decay time and frequency, suggesting simultaneous post- and presynaptic effects. Moreover, the authors found that the pre- and postsynaptic effects of BDNF are independent of each other. Selectively blocking postsynaptic trkB receptors or postsynaptic NMDA receptors by intracellular application of antagonists blocked the postsynaptic effect on mEPSC decay time but did not block the presynaptic effect on mEPSC frequency. The presynaptic effect, on the other hand, was dependent on presynaptic trkB and NMDA receptors.

While many studies have examined the mechanisms underlying BDNF modulation of excitatory synaptic transmission, current knowledge of the role and underlying mechanisms of BDNF in modulating inhibitory synaptic transmission is much less clear. The acute effect of BDNF at inhibitory synapses is quite varied, depending on the age of the animal used, tissue preparation (slice or cell culture) and brain region being studied. The majority of studies find that BDNF acutely suppresses GABAergic synaptic transmission in many different brain regions, with the exception in Purkinje cells, where BDNF enhances responsiveness of postsynaptic GABA receptors (Cheng and Yeh 2005). BDNF has been found to suppress inhibitory synaptic transmission via both presynaptic (Frerking et al. 1998) and postsynaptic mechanisms (Henneberger et al. 2002; Tanaka et al. 1997). The major evidence for a postsynaptic mechanism in these studies is the involvement of postsynaptic trkB receptors, because the suppressive effect of BDNF is blocked by a trk inhibitor loaded into the recording pipette. While these results affirmed the involvement of postsynaptic trkB receptors, they did not exclude the possibility of

24 concurrent pre- and postsynaptic effects, as these early studies did not examine presynaptic effects in their preparations, or take retrograde signaling into consideration. Indeed, BDNF has been found to simultaneously decrease amplitude and frequency of miniature IPSCs (mIPSCs) in the same preparation (Cheng and

Yeh 2003; Hewitt and Bains 2006), suggesting simultaneous pre- and postsynaptic effects. A recent study from our lab showed that at inhibitory synapses onto layer

2/3 cortical pyramidal neurons of juvenile mice, acute application of BDNF rapidly suppresses evoked and spontaneous IPSCs. This effect requires activation of postsynaptic trkB receptor, but was expressed as a suppression of presynaptic

GABA release probability. We further showed that eCBs are the retrograde signaling mediating the effect of BDNF, as the effect is blocked in the presence of

CB1R antagonist as well as when endocannabinoid synthesis or release is blocked

(Lemtiri-Chlieh and Levine 2010). However, the signaling pathway linking BDNF- trkB activation to endocannabinoid mobilization is not known. Some studies have shown that activation of PLC (Tanaka et al. 1997) and/or downstream activation of

PKC (Henneberger et al. 2002; Jovanovic et al. 2004) are required for the effect of

BDNF upon activation of postsynaptic trkB receptors. It is not clear, however, whether PLC and/or PKC underlie all of the BDNF effects reported so far, nor is it clear whether other downstream signaling pathways of trkB activation, such as the

MAPK and PI3K pathways, are involved in the effect of BDNF at inhibitory synapses.

Taken together, these results suggested that BDNF utilizes multiple mechanisms in modulating inhibitory synaptic transmission and may have diverse outcomes, which

25 further complicates our understanding of the effect of BDNF on synaptic transmission.

1.5.2. BDNF and long-term synaptic plasticity

The activity-dependent secretion of endogenous BDNF, along with its potent effects on synaptic transmission, makes it a compelling candidate for activity- dependent plasticity, such as long-term potentiation (LTP). A functional role for endogenous BDNF-TrkB receptor signaling in LTP is well established in the hippocampus and neocortex. In heterozygous BDNF knockout mice, LTP is dramatically reduced at hippocampal Schaffer collateral-CA1 synapses (Korte et al.

1995). Similar results have been found in animals that lack trkB receptors in CA1

(Xu et al. 2000) or forebrain (Minichiello et al. 1999). The deficits in LTP of BDNF knockout mice are not due to developmental consequences since LTP can be rescued by acute BDNF treatment (Patterson et al. 1996) or by re-expression of the

BDNF gene through virus-mediated transfection (Korte et al. 1996). Similarly, application of exogenous BDNF enhances the magnitude of LTP in rat visual cortex

(Akaneya et al. 1997). Furthermore, several studies have indicated an essential role for endogenous BDNF in the induction of LTP in hippocampus and cortex (Abidin et al. 2006; Aicardi et al. 2004; Chen et al. 1999; Inagaki et al. 2008; Lu et al. 2010), while applying function-blocking antibodies against trkB (trkB-Fc) or BDNF scavenger, trkB-IgG fusion protein, prevented LTP in hippocampus and cortical slices (Akaneya et al. 1997; Chen et al. 1999; Kang et al. 1997) .

It has been found that high frequency stimulation during LTP induction releases endogenous BDNF (Aicardi et al. 2004; Chen et al. 1999; Gartner and

Staiger 2002). In particular, using specific antibodies for BDNF, NT-3 and NT-4/5,

Chen and colleagues have found that BDNF, but not other trkB ligands, is necessary for hippocampal LTP. Furthermore, they showed that LTP was attenuated if BDNF was blocked before and during TBS stimulation, but not after stimulation (Chen et al. 1999), suggesting the release of endogenous BDNF during high frequency stimulation is an important mechanism of LTP induction.

Besides its direct effect in inducing and maintaining long-term potentiation of excitatory synapses, BDNF has also been found to facilitate LTP by suppressing inhibitory transmission at GABAergic synapses. In a recent study from Lu and colleagues, the authors showed that in cocaine withdrawal animals, BDNF facilitates LTP induction at layer 2/3 to layer 5 synapses in medial prefrontal cortex

(mPFC) by suppressing GABAergic inhibition and enhancing pyramidal neuron excitability (Lu et al. 2010), suggesting that BDNF plays a role in circuit sensitization during relapse.

BDNF can also contribute to LTP by protecting activated synapses from LTD.

Tsumoto and colleagues showed that low frequency stimulation (1 Hz, 15 min) induces LTD at layer 4 to layer 2/3 excitatory synapses in visual cortex, while application of BDNF prevented this LTD (Akaneya et al. 1996). Notably, 12 years after this finding, the same group refined the mechanism of the anti-LTD effect of

BDNF by adding eCB signaling into the equation. They found that BDNF prevents

LTD at these synapses by counteracting the action of eCBs, which are released by

TBS stimulation and induces LTD in the absence of BDNF (Huang et al. 2008). This advanced the understanding of synaptic plasticity from solely recognizing a BDNF effect to highlighting the interactions between BDNF and eCB. This progress has been reflected throughout the entire field over the past decade, as the importance of

BDNF-eCB interaction has been brought to light. I will discuss BDNF-eCB interaction in more detail in the following section.

1.6. Interactions between BDNF and endocannabinoid systems

BDNF is traditionally known for its trophic role in neurodevelopment, such as axonal guidance and neuronal migration (Lykissas et al. 2007) as well as neuronal survival and neurogenesis (Barde 1994; Newton and Duman 2004). More recent findings have highlighted the importance of BDNF signaling in synaptic transmission and plasticity (see Chapter 1.5). Endocannabinoid signaling was initially studied in the context of synaptic transmission (see Chapter 1.3), and recently gained attention due to its involvement in axonal pathfinding and neurogenesis (Oudin et al.

2011). In addition, the expression of trkB receptors and CB1Rs in the brain shows a strong overlapping pattern. Both receptors have high expression levels in brain regions such as cerebral cortex, hippocampus, amygdala, striatum and cerebellum

(Egertova et al. 2003; Fryer et al. 1996; Gomes et al. 2006; Hofer et al. 1990;

Masana et al. 1993; Matsuda et al. 1993; Moldrich and Wenger 2000; Tsou et al.

1998). Within the neocortex, trkB is predominantly localized to layers 2/3 and 5

(Cabelli et al., 1996; Fryer et al., 1996; Miller and Pitts, 2000), and the highest levels

28 of CB1R in the neocortex are also found in layers 2/3 and 5 (Matsuda et al., 1993;

Tsou et al., 1998; Marsicano and Lutz, 1999; Egertova et al., 2003). Furthermore, unpublished studies from our lab also suggested that at cellular level, trkB and

CB1R colocalize to the same neurons (Jeff Forte and Dr. Mason Yeh, unpublished observations), which provides an anatomical basis for potential functional and physiological interactions between BDNF and eCB signaling in the neocortex.

The earliest evidence of BDNF-eCB interactions comes from animal models of disease, in which activation of one system leads to transcriptional changes of the other. For example, BDNF and eCB are both known for their neuroprotective effects against depression, and abnormal function of either BDNF or eCB signaling has been found to be related to depression (for detailed reviews see Ashton and Moore

2011; Autry and Monteggia 2012). Studies have suggested that the antidepressant effects of endocannabinoids in depression are mediated by BDNF. CB1R activation leads to increased BDNF expression (Aso et al. 2008; Vinod et al. 2012). In addition, chronic exposure to Δ9-THC, the primary psychoactive constituent of marijuana, leads to an up-regulation of BDNF in vivo in brain areas that are implicated in reward and addiction processes (Butovsky et al. 2005). Furthermore, BDNF levels are decreased in CB1R-null mice, while administration of BDNF in the hippocampus reversed the enhanced stress response in CB1R knockouts (Aso et al. 2008).

Taken together, these studies suggest that the antidepressant effects of cannabinoids are mediated by downstream upregulation of BDNF.

Activation of CB1R also governs BDNF signaling during corticogenesis. During cortical interneuron development, AEA regulates the migration of CCK-expressing

29 interneurons by transactivating TrkB receptors. Meanwhile, it inhibits BDNF-induced neurite outgrowth and differentiation, suggesting that eCBs can regulate the broad spectrum of BDNF-mediated responses to ensure proper order of spatiotemporal activation (Berghuis et al. 2005).

In turn, BDNF also regulates eCB signaling. In cultured cerebellar granule neuron cultures, BDNF has been found to increase the expression of CB1Rs, decrease MGL expression and promote neuronal sensitivity to eCBs (Maison et al.

2009). A few studies have reported BDNF-eCB interactions at the synaptic level.

Our lab recently found that in layer 2/3 of neocortex, the suppressive effect of BDNF on inhibitory postsynaptic currents (IPSCs) is mediated by endocannabinoids

(Lemtiri-Chlieh and Levine 2010). As briefly mentioned in Chapter 1.5.2, Huang and colleagues found that TBS simultaneously induces LTP and LTD at Layer 4 to layer

2/3 excitatory synapses in visual cortex, however the net effect is a potentiation of synaptic transmission at these synapses. The LTP is mediated by TBS-induced release of endogenous BDNF, since it is blocked by trkB-IgG. The LTD, on the other hand, is dependent of TBS-induced eCB, because it is expressed as a decrease in presynaptic release and can be blocked by CB1R antagonists.

Interestingly, application of BDNF blocked the eCB-LTD. The authors thus suggested that BDNF induces LTP by competing against eCB-mediated LTD induction, presumably via antagonizing downstream signaling cascade of CB1R activation (Huang et al. 2008). In striatum, activation of CB1R suppresses both excitatory and inhibitory transmission. However, BDNF selectively antagonizes

CB1R receptor function at inhibitory synapses, while leaving the CB1R-mediated

30 suppression of excitatory transmission intact. The effect of BDNF at inhibitory synapses was found to be mediated by increasing cholesterol content in lipid rafts, as depletion of cholesterol or inhibiting its synthesis rescued CB1R activation- induced suppression of IPSCs (De Chiara et al. 2010; De Chiara et al. 2013). In hippocampus, hours of BDNF incubation suppresses mGluR-induced eCB signaling, while enhances depolarization-induced eCB signaling at hippocampal excitatory synapses, suggesting that BDNF-eCB interactions may involve multiple mechanisms (Roloff et al. 2010). Taken together, these studies suggest that the physiological and functional levels of BDNF-eCB interactions may have diverse impact on synaptic transmission at different synapses and in different brain regions, although much is still unknown at this stage.

1.7. Rationale and hypothesis

The studies carried out in this dissertation examine the functional relevance of

BDNF-eCB interactions at cortical inhibitory synapses. We recently found that in layer 2/3 of somatosensory cortex, the effect of BDNF on inhibitory postsynaptic currents (IPSCs) is mediated by a retrograde effect on presynaptic terminals

(Lemtiri-Chlieh and Levine 2010). Specifically, we found that BDNF causes a rapid suppression of IPSCs, which is blocked by the trk inhibitor K252a or the calcium chelator BAPTA applied selectively to the postsynaptic cell, indicating involvement of postsynaptic trkB activation and subsequent increase in intracellular Ca2+.

Surprisingly, rather than a change in postsynaptic responsiveness, the suppression

31 of IPSCs is due to a decrease in presynaptic GABA release. BDNF caused an increase in the paired-pulse ratio and coefficient of variation of evoked responses, as well as a decrease in the frequency of action potential–independent miniature

IPSCs (mIPSCs), all of which indicate decreased presynaptic release. In contrast,

BDNF had no effect on the amplitude of mIPSCs, nor did BDNF alter the postsynaptic response to locally applied GABA.

A postsynaptic induction and a presynaptic expression suggested the involvement of retrograde signals. Indeed, we also showed that the BDNF effect is blocked by CB1R antagonists or eCB release inhibitor. In addition, the effect of

BDNF is occluded by CB1R agonist WIN, which further suggested that BDNF causes eCB release and subsequent activation of CB1R to suppress IPSCs. The

DGL inhibitor RHC-80267 also blocked the effect of BDNF, indicating that BDNF induces the synthesis of 2-AG. Taken together, these results provide strong evidence that BDNF induces eCB release at layer 2/3 inhibitory synapses. The signaling pathway that links BDNF/trkB activation to eCB, however, is not known, nor is it clear how such interactions will influence synaptic plasticity.

We hypothesize that BDNF induces eCB release via phospholipase Cγ (PLCγ) signaling based on the following reasons. First, the β isoform of PLC (PLCβ), which is activated downstream of Gq protein-coupled receptors, is known to be involved in mobilizing endocannabinoids during receptor-driven eCB release (RER, see

Chapter 1.3) (Galante and Diana 2004; Hashimotodani et al. 2005; Varma et al.

2001). Second, PLCγ signaling has been implicated in the effect of BDNF at inhibitory synapses where postsynaptic trkB activity is required (Cheng and Yeh

2005; Tanaka et al. 1997). Third, in the previous study, the effect of BDNF depends on elevated intracellular calcium level in the postsynaptic cell. Release from intracellular calcium store is most likely the source of Ca2+, as neurons were voltage-clamped at -70 mV throughout the experiments, and activation of PLCγ is known to induce Ca2+ release from intracellular store (Blum and Konnerth 2005).

Fourth, the results from DGL inhibitor suggested involvement of 2-AG, which also points to PLC signaling, as activation of PLCγ results in increased levels of DAG, the direct precursor of 2-AG (discussed in Chapter 1.2).

We use eCB-mediated iLTD as a model to examine the functional revelance of

BDNF-eCB interaction. Theta-frequency burst stimulation is known to induce various forms of eCB-mediated long-term depression at both excitatory (eCB-LTD) and inhibitory (eCB-iLTD) synapses (see Chapter 1.3). Interestingly, the same pattern of high frequency stimulation that induces eCB release during eCB-iLTD can also induce release of endogenous BDNF (Aicardi et al. 2004; Akaneya et al. 1997;

Balkowiec and Katz 2002; Li et al. 2010). It is tempting to speculate that BDNF can induce eCB release in the context of iLTD. We hypothesize that TBS stimulation induces release of endogenous BDNF. Release of endogenous BDNF activates postsynaptic trkB receptors, which then triggers eCB release and further leads to eCB-dependent iLTD. We also hypothesize that BDNF will facilitate weak induction protocols that are insufficient to induce iLTD.

The studies presented in this dissertation examined 1) the signaling pathways that underlie BDNF-induced eCB release; and 2) the role of endogenous BDNF in eCB-mediated long-term plasticity at cortical inhibitory synapses. Understanding the

33 physiological relevance of such interactions will provide knowledge to better understand modulation of cortical inhibitory microcircuits. In addition, because

BDNF and eCBs are implicated in an overlapping set of neurological and psychiatric disorders (for detailed reviews see Ashton and Moore 2011; Autry and Monteggia

2012), better understanding of BDNF-eCB interactions may ultimately lead to novel therapeutic strategies for disease treatment. The results of the present studies will be presented in Chapters 3 and 4 and will be discussed in Chapter 5.

Chapter 2

Materials and Methods

2.1. Animal handling and slice preparation

All animal procedures were approved by the University of Connecticut Health

Center Animal Care and Use Committee. Postnatal day 15-27 Swiss CD-1 mice of either sex (Charles River, Wilmington, MA) were anesthetized by 3.5% isoflurane inhalation, followed by decapitation. Whole brains were removed and immersed in ice-cold slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 1.25

NaH2PO4∙H2O, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2∙6H2O, 25 dextrose, 11.6 sodium ascorbate, 3.1 sodium pyruvate, equilibrated with 95% O2-5% CO2 (pH 7.3, 310 ± 5 mOsm/kg). Transverse slices (350 µm) containing somatosensory cortex were cut with a Dosaka EM DTK-1000 vibratome (Kyoto, Japan) and transferred to an incubating chamber. Slices were then incubated for 15 min at 33-35 °C in carboxygenated incubating solution containing (in mM) 125 NaCl, 2.5 KCl, 1.25

NaH2PO4∙H2O, 25 NaHCO3, 0.5 CaCl2, 3.5 MgCl2∙6H2O, 4 sodium lactate, 2 sodium pyruvate, 25 dextrose, and 0.4 ascorbic acid (pH 7.3, 310 ± 5 mOsm/kg) before being transferred to room temperature. Slices were then individually transferred to a recording chamber at (room temperature) fixed to the stage of an Olympus BX51WI upright microscope fitted with a 40X water-immersion objective lens (0.8 NA). The recording chamber was continuously perfused at 1-1.5 ml/min with carboxygenated artificial cerebrospinal fluid (aCSF) consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25

NaH2PO4∙H2O, 25 NaHCO3, 2 CaCl2, 2 MgCl2∙6H2O, 25 dextrose (pH 7.3, 305 ± 5 mOsm/kg).

2.2. Electrophysiology

Whole-cell recordings were obtained from layer 2/3 somatosensory cortex pyramidal neurons. Pyramidal neurons were identified by their morphology and position under infrared differential interference contrast (IR-DIC) video microscopy.

Patch electrodes (2–3 MΩ) were pulled from borosilicate glass capillaries using a

Flaming/Brown P-97 micropipette puller (Sutter Instrument, Novato, CA). Pipette internal solution contained (in mM): 130 CsCl, 10 HEPES, 1 EGTA, 0.1 CaCl2, 1.5

MgCl2, 4 Na2-ATP, 0.3 Na-GTP, 10 di-tris-phosphocreatine and 5 QX-314 (pH 7.3,

290 ± 5 mOsm/kg). A bipolar tungsten electrode (1 MΩ; WPI, Sarasota, FL)was positioned 70-150 µm lateral to the patched pyramidal neuron to elicit electrically- evoked inhibitory postsynaptic currents (eIPSCs). Extracellular stimuli consisted of individual square-wave current pulses (170 µs; 4-30 µA) and were delivered every

20 seconds through a stimulus isolator (ISO-Flex, A.M.P.I, Jerusalem, Israel).

Stimulation strength was set to a level that evoked 30-70% of maximal response for each individual cell.

The chloride equilibrium potential (ECl) using the above internal and external solutions was close to 0 mV; thus, IPSCs were recorded as inward currents.

Ionotropic glutamate receptor antagonists 6, 7-dinitroquinoxaline-2, 3-dione (DNQX;

10 µM) and 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP; 3 µM)

36 were used to isolate inhibitory activity through all experiments. Cells were voltage clamped at -70 mV during recording. All electrical events were filtered at 2.9 kHz and digitized at >6 kHz using a HEKA EPC9 amplifier and ITC-16 digitizer (HEKA

Elektronik, Darmstadt, Germany). Series resistance (Rs) was compensated up to 40% at 100 µs lag. Input resistance (Ri) was monitored with 10 mV (50 ms) hyperpolarizing voltage steps at the end of each sweep. Cells were rejected from analyses if Ri fell below 50 MΩ or holding current dropped by 150 pA during the course of an experiment.

2.3. Chemicals

Unless otherwise stated, all drugs were from Tocris Biosciences (Bristol, UK) and were delivered by bath perfusion. Drugs were first prepared as concentrated stock solution in solvents and stored at -20 °C. Drug stock solutions were diluted into aCSF on the day of recording to the final concentrations. The stock solutions of

DNQX, AM251, U-73122, U-73343, GF 109203X, PD 98059, LY 294002, (S)-3, 5-

DHPG, ANA-12, K252a and SR 141716A were dissolved in 100% dimethyl sulfoxide (DMSO). ET-18-OCH3 was dissolved in anhydrous ethanol. The final concentration of DMSO did not exceed 0.1%, and the final ethanol concentration was 0.04%, which by themselves had no effect on synaptic transmission. CPP,

DNQX disodium, chelerythrine chloride (Sigma-Aldrich, St. Louis, MO), E4CPG and

BDNF (PeproTech, Rocky Hill, NJ) were dissolved in 18 MΩ water. Bovine serum

37 albumin (BSA, Sigma-Aldrich, St. Louis, MO) was added to the BDNF solution at a concentration of 0.1g/L to reduce non-specific binding.

2.4. Data analysis

Off-line analysis was carried out using Clampfit 10 (Molecular Devices,

Sunnyvale, CA) and Prism 6 (GraphPad Software, La Jolla, CA). Statistical comparisons were made between average amplitudes of baseline responses and 9-

10 minutes after BDNF application (Chapter 3) or 27-35 minutes following theta burst stimulation (Chapter 4) using two-tailed Student’s paired t-test and/or one-way

ANOVA and Dunnett's Multiple Comparison Test unless otherwise stated. p < 0.05 was taken as a statistically significant effect. In individual examples, sweeps of evoked responses were averaged traces of five consecutive evoked IPSCs before and after 10 min of BDNF application (Chapter 3) or four consecutive evoked IPSCs during baseline and around 35 min post LTD induction (Chapter 4). Group data are reported as mean ± SEM.

Chapter 3

BDNF-endocannabinoid interactions at neocortical inhibitory synapses

require phospholipase C signaling

This chapter is a duplicate version of the publication: Zhao L and Levine ES, BDNF-endocannabinoid interactions at neocortical inhibitory synapses require phospholipase C signaling. J Neurophysiol. 2014 Mar;111(5):1008-15. doi: 10.1152/jn.00554.2013. LZ and ESL Designed Research, LZ Performed Research, LZ Analyzed Data, LZ and ESL Wrote the Paper.

Brain-derived neurotrophic factor (BDNF) is as an important modulator of excitatory and inhibitory synaptic transmission. During the past 20 years, much has been learned about the mechanism of action of BDNF at excitatory synapses

(reviewed in Carvalho et al. 2008; Gottmann et al. 2009). However, the current knowledge of the role and underlying mechanisms of BDNF in modulating inhibitory synaptic transmission is much less clear. The diverse effects of BDNF at inhibitory synapses depend on a variety of factors, including age of the animal, tissue preparation (slice vs. cell culture), brain region and cell type being studied, as well as BDNF treatment time course. For example, BDNF has been found to suppress inhibitory postsynaptic currents (IPSCs) in cerebellar granule cells (Cheng and Yeh

2003; 2005), and enhance postsynaptic GABA receptor responsiveness in Purkinje cells (Cheng and Yeh 2005). In cortical and hippocampal cell cultures, acute application of BDNF rapidly enhances miniature IPSC amplitude, followed by a prolonged suppression. This switch in the direction of effect was concurrent with

39 protein kinase C (PKC)-mediated phosphorylation (Jovanovic et al. 2004). In terms of the locus of BDNF effect, Frerking and colleagues found that the suppressive effect of BDNF on IPSCs is expressed presynaptically in the hippocampal CA1 region (Frerking et al. 1998), while several other studies indicated the involvement of postsynaptic tropomyosin-related kinase receptor B (trkB) receptors (Hewitt and

Bains 2006; Tanaka et al. 1997).

We have recently shown that at inhibitory synapses onto layer 2/3 cortical pyramidal neurons, acute application of BDNF rapidly suppresses GABAergic transmission via release of endocannabinoids from the postsynaptic pyramidal cell, which act in a retrograde manner to suppress presynaptic transmitter release

(Lemtiri-Chlieh and Levine 2010). This effect of BDNF is initiated by postsynaptic trkB signaling, as the effect is blocked when postsynaptic trkB receptors are selectively inhibited by intracellular loading of a tyrosine kinase inhibitor, or when endocannabinoid synthesis and release from the postsynaptic cell is prevented. The suppression of inhibitory transmission is expressed as a presynaptic decrease in

GABA release probability, because it is associated with changes in the paired-pulse ratio, the coefficient of variation, and the frequency of miniature IPSCs, and the

BDNF effect is also blocked by antagonists to the predominantly presynaptically- expressed CB1 receptor (Lemtiri-Chlieh and Levine 2010). However, the signaling pathway linking BDNF-trkB activation to endocannabinoid mobilization is not known.

The trkB receptor is the major receptor for BDNF signaling in the brain and mediates most of the effects of BDNF on synaptic transmission and plasticity. Upon binding to trkB receptors, BDNF stimulates at least three major downstream

40 intracellular signaling pathways via tyrosine phosphorylation, namely Ras/mitogen- activated protein kinase (MAPK) pathway, phosphatidylinositol 3-kinases (PI3K)/Akt pathway and the phospholipase Cγ (PLCγ) pathway. Activation of PLCγ leads to cleavage of phosphatidylinositol 4, 5-bisphosphate (PIP2) into second messengers inositoltriphosphate (IP3) and diacylglycerol (DAG). IP3 induces Ca2+ release from intracellular Ca2+ stores upon binding to its receptor and thus increases intracellular calcium concentration (reviewed in Huang and Reichardt 2003). DAG leads to activation of protein kinase C (PKC), which has been found to mediate some of the effects of BDNF at inhibitory synapses (Henneberger et al. 2002; Jovanovic et al.

2004). Activation of the Ras/MAPK and PI3K/Akt pathways are generally considered to be critical for neuronal survival and differentiation (reviewed in Huang and Reichardt 2003; Patapoutian and Reichardt 2001; Segal and Greenberg 1996), although activation of MAPK may also mediate some of the acute effects of BDNF on long-term potentiation and excitatory synaptic transmission (Jovanovic et al.

2000; Ying et al. 2002). The current study examines the potential intracellular signaling pathways that underlie BDNF-endocannabinoid interactions at inhibitory synapses onto layer 2/3 cortical pyramidal neurons.

3.1. The effect of BDNF on IPSC requires postsynaptic phospholipase-C

signaling

We first examined the effect of BDNF on inhibitory transmission in layer 2/3

PNs. As shown in the individual example in Fig. 3.1A, bath application of 20 ng/ml

(0.8 nM) BDNF rapidly reduced the peak amplitude of evoked IPSCs (eIPSCs). This effect persisted during BDNF application and typically showed little or no recovery up to 15 min post-washout of BDNF. Overall, peak eIPSC amplitude was significantly decreased to 75.6 ± 6.6 % of baseline after 10 min of BDNF exposure, as seen in the group time course in Fig. 3.1B (F(7,11)=5.698, p<0.05, n=8, one-way

ANOVA. Baseline, 896.8 ± 181.7 pA; BDNF, 712.4 ± 163.5 pA). Post hoc tests revealed a significant decrease after 4 min of BDNF treatment, which likely reflects penetration time of BDNF in the brain slice. In contrast, application of the vehicle solution had no significant effect on eIPSC amplitude (100.6 ± 3.0 %, n=3). We also confirmed that this effect of BDNF required activation of CB1 cannabinoid receptors.

As shown in Figs. 3.1C and D, the BDNF effect on evoked IPSC amplitude was completely blocked in the presence of the CB1 receptor antagonist AM251 (5 µM;

100.6 ± 6.5% of BL, n=5, AM251 baseline, 1564.0 ± 482.3 pA; BDNF + AM251,

1539.0 ± 437.9 pA).

shown in Figs. 3.2A and D, U-73122 prevented the effect of BDNF (97.3 ± 5.7% of baseline, n = 6, baseline, 814.1 ± 148.6 pA; BDNF, 825.8 ± 191.4 pA). In contrast, the inactive analog U-73343 (5 µM) did not block the BDNF effect (Figs. 3.2B and

D). After 10 min of exposure, BDNF reduced eIPSC amplitude to 77.0 ± 6.6% of baseline in the presence of U-73343 (Fig. 3.2D, p<0.05, n=9, paired t-test), similar to the effect of BDNF alone. The latency to onset of the BDNF effect in the presence of U-73343 was also similar to that with BDNF alone.

To further confirm the involvement of PLC signaling, we used another selective

PLC inhibitor, edelfosine (ET-18-OCH3 or ET-18, 5 µM). Similar to the result obtained with U-73122, the BDNF effect was completely abolished in the presence of ET-18 (Fig. 3.2C and D, 102.3 ± 4.9% of baseline). Exposure to each of the PLC inhibitors alone did not have any significant effect on baseline eIPSC amplitude (U-

73122, 118.6 ± 17% of BL, n=6; ET-18, 107.0 ± 17% of BL, n=4). The vehicles used to dissolve the PLC inhibitors also did not have any effect on eIPSC amplitudes

(0.08% DMSO for U-73122 and U-73343: 101.4 ± 1.2 % of BL, n = 3; 0.04% anhydrous ethanol for ET-18: 94.7 ± 3.0 % of BL, n = 4). To verify that the effect of

PLC inhibition is due to disruption of BDNF-trkB signaling in the postsynaptic cell, and not a disruption of presynaptic CB1R signaling, we tested the direct effect of a

CB1R agonist, WIN 55,212-2 (WIN, 5 μM), in the presence of U-73122. As seen in

Fig. 3.2D, exposure to WIN in the presence of the PLC inhibitor caused a significant suppression of eIPSC amplitude (74.8 ± 3.1 % of BL, n=5, p<0.05, paired t-test) which was similar to the effect of WIN alone (Lemtiri-Chlieh and Levine 2010).

3.2. The effect of BDNF is independent of PKC, MAPK and PI3K signaling

Activation of PLCγ leads to the generation of DAG and IP3. DAG is involved in several intracellular signaling pathways, including the DAG lipase (DGL)-dependent generation of the endocannabinoid 2-AG (Stella, Schweitzer et al. 1997). Previously, we have shown that the effect of BDNF is disrupted in the presence of the DGL inhibitor RHC 80267 (50 µM) (Lemtiri-Chlieh and Levine 2010), suggesting involvement of 2-AG. However, DAG also leads to the downstream activation of protein kinase C (PKC), which has been found to mediate some of the effects of

BDNF at inhibitory synapses (Henneberger, Juttner et al. 2002; Jovanovic, Thomas et al. 2004). Therefore, we also tested the possible involvement of PKC signaling.

We used two different selective PKC inhibitors, namely chelerythrine (Chel, 10 µM,

Weiner et al. 1997) and GF109203X (GF, 1 µM, Bellone and Luscher 2005). Neither drug blocked the BDNF effect (Chel, 63.9 ± 12.0% of BL, p<0.05, n=5; GF, 64.6 ±

9.5% of BL, p<0.05, n=5, paired t-test), as shown in individual examples and group data in Fig. 3.3. The latency and the magnitude of suppression by BDNF in the presence of either Chel or GF were not significantly different from the effect of

BDNF alone.

3.3. The effect of BDNF is independent of mGluR signaling

We also addressed whether the effect of BDNF could be mediated by glutamate, rather than a direct effect of BDNF-trkB signaling. BDNF can enhance presynaptic glutamate release (Numakawa, Matsumoto et al. 2001; Numakawa,

Yamagishi et al. 2002; Zhang, Fan et al. 2013), and glutamate can induce endocannabinoid release via activation of metabotropic glutamate receptors

(mGluRs) acting through PLCβ signaling (for reviews see Chevaleyre, Takahashi et al. 2006; Kano, Ohno-Shosaku et al. 2009). Thus we examined the BDNF effect in the presence of 500 µM E4CPG, a group I/group II mGluR antagonist (Perez et al.

2001). Blocking mGluR signaling did not prevent the effect of BDNF nor alter its time course (66.8 ± 2.5% of BL at the end of 10 min BDNF application, n=4, p<0.05, paired t-test, Fig. 3.5).

As a positive control to ensure that E4CPG effectively blocked mGluR activation, we also examined the effect of DHPG, a selective group I mGluR agonist, on inhibitory transmission. We found that DHPG (50 μM) caused a suppression of eIPSC amplitude, similar to the effect of BDNF (77.2 ± 7.5% of BL, p<0.05, n=8, paired t-test, Fig. 5C, black bar). This effect of DHPG was completely prevented by

E4CPG (103.6 ± 11.7% of BL, n=4, Fig. 5C, hatched bar). These results confirm the efficacy of the mGluR antagonist and indicate that the effect of BDNF on cortical inhibitory synaptic transmission is not dependent on glutamate-mediated mGluR activation. Because DHPG can also induce eCB release that is sensitive to DAGL inhibition (Galante and Diana 2004; Gregg et al. 2012), we examined the interaction between BDNF and DHPG using the same concentrations as above. DHPG

49 induced a stable suppression of eIPSC amplitude (73.5 ± 6.0% of BL, n=6), and subsequent addition of BDNF had no significant further effect on eIPSC amplitude

(94.3 ± 3.7% of DHPG baseline, n=6). Similarly, in the presence of BDNF, there was no effect of subsequent addition of DHPG (BDNF, 67.5 ± 5.3% of BL, DHPG,

100.9 ± 8.1% of BDNF, n=5). These results indicate that the effects of BDNF and

DHPG occlude each other, suggesting possible saturation of endocannabinoid signaling.

3.4. Summary and Discussion

Previously we have shown that the suppressive effect of BDNF on cortical layer 2/3 inhibitory synapses in somatosensory cortex is induced postsynaptically but expressed presynaptically. We have further shown that this effect requires eCB release from the postsynaptic cell and subsequent presynaptic CB1R activation

(Lemtiri-Chlieh and Levine 2010). In the present studies, we investigated the signaling pathways that underlie the CB1R-mediated synaptic effect of BDNF at inhibitory synapses onto layer 2/3 pyramidal neurons in somatosensory cortex from juvenile mice. Our results showed that activation of PLCγ is necessary for this effect of BDNF, because it was blocked by two different PLC inhibitors, U-73122 and ET-

18, while maintained in the presence of the inactive analog U-73343. In addition,

PLC inhibition did not alter the direct suppressive effect of a cannabinoid agonist.

Taken together, these results support a role for PLC signaling downstream of trkB activation. Furthermore, the CB1R-mediated synaptic effect of BDNF does not require PKC, MAPK or PI3K signaling because inhibition of these pathways did not prevent the effect of BDNF. Interestingly, this effect also does not require mGluR signaling, as the mGluR antagonist E4CPG did not block the BDNF effect on inhibitory transmission. Taken together with our previous study (Lemtiri-Chlieh and

Levine 2010), these results suggest that BDNF requires postsynaptic PLCγ signaling to induce endocannabinoid release, which leads to the CB1R-mediated synaptic effect of BDNF at inhibitory synapses.

Chapter 4

Role for Endogenous BDNF in Endocannabinoid-mediated Long-Term

Depression at Neocortical Inhibitory Synapses

This chapter is a duplicate version of the manuscript under revision: Zhao L and Levine ES, Role for Endogenous BDNF in Endocannabinoid-mediated Long-Term Depression at Neocortical Inhibitory Synapses. eNeuro, eN-NWR-0029-14. LZ and ESL Designed Research, LZ Performed Research, LZ Analyzed Data, LZ and ESL Wrote the Paper.

Endogenous cannabinoids (endocannabinoids, eCBs) are important regulators of synaptic function in the nervous system. In particular, endocannabinoids acutely modulate inhibitory and excitatory transmission throughout the forebrain, and mediate several forms of short-term plasticity at GABAergic and glutamatergic synapses, including deplolarization-induced suppression of inhibition (DSI) and excitation (DSE). During DSI or DSE, endocannabinoids are released from postsynaptic sites in response to depolarization-induced calcium influx and act retrogradely via presynaptic type-1 cannabinoid receptors (CB1Rs) to suppress transmitter release. Endocannabinoids also mediate specific types of long-term depression at excitatory and inhibitory synapses (for reviews see Cachope 2012;

Chevaleyre et al. 2006; Mackie 2008). These forms of plasticity are dependent on

CB1Rs, which are highly expressed throughout the forebrain, and are activated by both arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG), the two best-characterized endogenous ligands of CB1R. It has been shown that the on-demand synthesis and release of endocannabinoids in the hippocampus,

53 neocortex, cerebellum, and other areas can be stimulated by depolarization-induced calcium influx, as well as by activation of PLCβ triggered by Gq-protein coupled receptors, particularly metabotropic glutamate receptors (mGluRs) (see Castillo et al. 2012 for reviews; Hashimotodani et al. 2007a).

Recently it has been shown that brain-derived neurotrophic factor (BDNF) can also induce endocannabinoid release, part of a growing body of evidence for interactions between endocannabinoid signaling and BDNF. For example, there is evidence of eCB–BDNF interactions in visual cortex (Huang et al. 2008), hippocampus (Khaspekov et al. 2004; Roloff et al. 2010), and cerebellum (Maison et al. 2009). In support of this, there is strong co-localization of trkB receptors and

CB1R throughout the forebrain. Within the neocortex, trkB is predominantly localized to layers 2/3 and 5 (Cabelli et al. 1996; Fryer et al. 1996; Miller and Pitts

2000), and the highest levels of CB1R in the neocortex are also found in layer 2/3

(Egertova et al. 2003; Marsicano and Lutz 1999; Matsuda et al. 1993; Tsou et al.

1998). Recent studies in the neocortex have shown that endocannabinoid synthesis and release can be rapidly mobilized by BDNF-trkB signaling. In neocortical layer

2/3, acute application of BDNF rapidly suppresses GABAergic transmission via release of endocannabinoids from the postsynaptic pyramidal cell, which act in a retrograde manner to suppress presynaptic transmitter release (Lemtiri-Chlieh and

Levine 2010). This effect of BDNF is initiated by postsynaptic trkB signaling and requires downstream PLC signaling, and is independent of mGluR activation (Zhao and Levine 2014).

Endogenous BDNF has been shown to play a critical role in LTP at excitatory synapses, but its role at inhibitory synapses and its potential role in endocannabinoid-mediated synaptic plasticity are less clear. Because exogenous

BDNF can trigger endocannabinoid mobilization that suppresses GABA release at cortical synapses, we explored whether endogenous BDNF plays a role in long-term depression at cortical inhibitory synapses. In particular, we hypothesized that stimulation-induced release of BDNF, acting through trkB and downstream PLC signaling, can trigger endocannabinoid release to cause long-term depression at cortical inhibitory synapses.

4.1. Strong theta frequency burst stimulation induces eCB-dependent iLTD

Using theta-frequency burst stimulation (TBS), we examined iLTD at inhibitory synapses onto layer 2/3 cortical pyramidal neurons. Stimulation consisted of 7 trains of TBS (7X TBS) delivered with a 5s inter-train interval. Each TBS train contained

10 bursts (200 ms inter-burst interval), each burst consisted of 5 stimuli at 100 Hz.

This protocol induced a stable long-lasting suppression of inhibitory transmission

(Fig. 4.1A). As shown in Fig. 1B, eIPSC amplitude was reduced in all 7 cells tested at 35 min post-TBS (68.32 ± 4.2 % of baseline, p = 0.0005, n = 7, paired t-test; also see Fig. 4.2C). In 2 cells that lasted 60 min post-TBS, eIPSC amplitude was 65.11 ±

1.4 % of baseline, suggesting that suppression reached a stable plateau by 35 min post-induction.

4.2. iLTD in layer 2/3 of somatosensory cortex is independent of mGluR

signaling

Because many forms of eCB-mediated LTD require mGluR signaling

(Chevaleyre et al. 2006; Kano et al. 2009), we examined the effects of 7X TBS during bath application of the group I/group II mGluR antagonist E4CPG (500 µM).

Previously it has been shown that this concentration of E4CPG is sufficient to block the effect of bath application of an mGluR agonist on synaptic transmission in layer

2/3 (Zhao and Levine 2014). Interestingly, blocking mGluR signaling did not prevent

7X TBS from inducing iLTD at layer 2/3 inhibitory synapses (Fig. 4.2A). At 35 min post-induction, eIPSC amplitude was significantly reduced compared to baseline

(Fig. 4.2B & C, 53.88 ± 8.7% of baseline, p = 0.0001, n = 6, paired t-test). The amount of suppression was comparable to that caused by 7X TBS alone (Fig. 4.2C, p = 0.1447, unpaired t-test).

4.3. iLTD in layer 2/3 of somatosensory cortex requires endogenous BDNF

and phospholipase-C signaling

In addition to the mGluR/phospholipase-Cβ (PLCβ) signaling pathway that is known to induce eCB mobilization and release, recent studies have found that

BDNF/trkB signaling, via downstream PLCγ activation, can induce eCB release at inhibitory synapses in layer 2/3 (Lemtiri-Chlieh and Levine 2010; Zhao and Levine

2014). Furthermore, TBS can induce release of endogenous BDNF (Li et al. 2010;

Panja and Bramham 2014). We therefore examined whether endogenous BDNF plays a role in eCB-mediated iLTD in layer 2/3. The first set of experiments used

K252a (200 nM), a relatively specific inhibitor of autophosphorylation of trk tyrosine kinase receptors at this concentration (Berg et al. 1992; Hashimoto 1988; Knusel and Hefti 1992; Nye et al. 1992). As shown in Figs. 4.3A & D, iLTD produced by 7X

TBS was prevented by bath application of K252a (92.17 ± 11.8% of BL, p = 0.6175, n = 6, paired t-test; baseline, 0.73 ± 0.1 nA; 35 min-post, 0.68 ± 0.1 nA). K252a alone does not have any significant effect on eIPSC amplitude in layer 2/3 slices

(Lemtiri-Chlieh and Levine 2010). A role for BDNF-trkB signaling was confirmed with the selective trkB receptor antagonist, ANA-12 (10 µM) (Alder et al. 2013;

Cazorla et al. 2011; Santafe et al. 2014). As shown in Figs. 4.3B & D, bath application of ANA-12 completely blocked iLTD (101.7 ± 8.5% of BL, p = 0.5944, n

= 4, paired t-test; baseline, 1.22 ± 0.3 nA; 35 min-post, 1.30 ± 0.5 nA). ANA12 alone did not have any effect on inhibitory synaptic transmission (95.32 ± 2.5 % of baseline after 10 min application, n = 2).

It has been previously shown that BDNF induces eCB mobilization via PLC signaling, presumably PLCγ (Zhao and Levine 2014). We thus examined whether

PLC plays a role in iLTD in layer 2/3. As seen in Figs. 4.3C & D, in the presence of the broad spectrum PLC inhibitor U-73122 (2 μM), iLTD was completely abolished

(98.46 ± 7.1% of baseline, p=0.7368, n = 4, paired t-test; baseline, 1.08 ± 0.3 nA; 35 min-post, 1.06 ± 0.3 nA). U-73122 alone does not have any influence on eIPSC amplitude (data not shown). Taken together, these findings suggest that endogenous BDNF-trkB signaling and downstream activation of PLC, but not mGluR activation, is necessary for eCB-mediated iLTD at inhibitory synapses in layer 2/3 pyramidal neurons.

4.4. Exogenous BDNF facilitates iLTD induction

We next examined whether exogenous BDNF could facilitate iLTD induction by a subthreshold stimulus train. In contrast to the stable suppression caused by 7X

TBS, 3 trains of TBS (3X TBS) did not induce significant iLTD, as shown in the group data in Figs. 4.4A, B and F (102.7 ± 15.2% of baseline; p = 0.6832, n = 5, paired t-test). In fact, only 1 out of 5 cells showed any suppression with 3X TBS (Fig.

4.4B). We then examined the effects of BDNF alone on inhibitory synaptic transmission. It has previously been shown that BDNF at a concentration of 20 ng/mL (0.8 nM) significantly reduces inhibitory synaptic transmission (Lemtiri-Chlieh and Levine 2010; Zhao and Levine 2014). To determine a concentration of BDNF that does not by itself influence synaptic transmission, we examined the effect of 5

61 min bath-applied BDNF on eIPSC amplitude. Bath application of 0.2 nM (5 ng/mL)

BDNF for 5 min caused a suppression of eIPSC amplitude in 4 out of 5 cells, although this didn’t reach statistical significance (83.24 ± 10.5% of baseline, p =

0.2644, n = 5, paired t-test). At a concentration of 0.1 nM (2.5 ng/mL), BDNF also appeared to cause a slight suppression (89.38 ± 4.5% of baseline, n = 3). At 0.05 nM (1.25 ng/mL), BDNF had no acute effect on synaptic transmission (104.5 ± 8.8% of baseline; p = 0.7086, n = 6, paired t-test), as shown in Fig. 4C. In addition, 0.05 nM BDNF for 5 min had no effect on eIPSC amplitude measured 35 min post-BDNF application (Fig. 4.4F, 96.77 ± 9.1% of baseline, p = 0.6888, n = 4, paired t-test), indicating that brief application of 0.05 nM BDNF does not induce iLTD.

Interestingly, however, the combination of 3X TBS with 0.05 nM BDNF (applied for

5 min around the time of TBS) produced significant iLTD (Fig. 4.4D, E and F, 70.95

± 9.1% of baseline, p = 0.0305, n = 7, paired t-test), with 6 out of 7 cells showing suppression. The average amount of suppression produced by BDNF + 3x TBS was not significantly different than that produced by 7X TBS alone (Fig. 4.4F, p =

0.7978, unpaired t-test). These data suggest that a subthreshold concentration of

BDNF can synergize with weak TBS stimulation to induce iLTD at layer 2/3 inhibitory synapses.

4.5. Summary and discussion

In the present study, we investigated the interaction between BDNF and eCB signaling in long-term depression at inhibitory synapses (iLTD). We found that 7 trains of TBS induces stable eCB-mediated iLTD at cortical layer 2/3 pyramidal neurons, as inhibiting CB1R activation diminished this suppression. Surprisingly, this form of iLTD is independent of mGluR activation, in contrast to the most prevalent forms of eCB-mediated LTD (Bender et al. 2006; Chevaleyre and Castillo

2003; Chiu et al. 2010; Jiang et al. 2010; Lafourcade et al. 2007; Maejima et al.

2001). On the other hand, BDNF, which has also been shown to mobilize eCBs

(Lemtiri-Chlieh and Levine 2010; Zhao and Levine 2014) is required for this iLTD, as inhibiting trkB tyrosine kinase activity or blocking trkB receptor activation prevented TBS-induced iLTD. Consistent with previous findings that PLCγ is required for BDNF-induced eCB mobilization (Zhao and Levine 2014), the present results indicate that endogenous BDNF regulates eCB-mediated iLTD via PLC signaling, as the PLC inhibitor U-73122 diminished iLTD. We conclude that PLC inhibition disrupts BDNF-induced eCB release, rather than directly disrupting CB1R signaling, because the direct effect of a CB1R agonist on inhibitory transmission is not blocked by the PLC inhibitor (Zhao and Levine, 2014), Furthermore, we also found that a low concentration of exogenous BDNF that has no effects on its own, synergizes with 3 trains of TBS to induce CB1R-dependent iLTD. Similar to the signaling pathway that underlies endogenous BDNF-mediated eCB-iLTD, the combined effect of exogenous BDNF and weak TBS also requires PLC signaling.

Chapter 5

Discussion and Conclusions

5.1. Summary and interpretation of findings

The studies conducted in this dissertation examined BDNF-eCB interactions in the context of cortical inhibitory synaptic transmission. The first set of studies examined the intracellular signaling pathway underlying BDNF-induced eCB release.

Consistent with our previous results (Lemtiri-Chlieh and Levine 2010), we found that

BDNF acutely suppresses inhibitory synaptic transmission onto pyramidal neurons in layer 2/3 of somatosensory cortex. This effect requires activation of postsynaptic trkB receptors and presynaptic CB1Rs, suggesting that BDNF induces eCB release from the postsynaptic cell and subsequent activation of presynaptic CB1R. We showed that PLC, presumably PLCγ, is required for BDNF-induced eCB release, because the BDNF effect was blocked by the broad-spectrum PLC inhibitors U-

73122 and ET-18, but not the inactive analog U-73343.

We also examined other signaling pathways downstream of trkB receptor activation, namely mitogen-activated protein kinase (MAPK), phosphoinositide 3- kinase (PI3K), and protein kinase C (PKC). We found that pharmacological inhibition of these pathways did not block the effect of BDNF, indicating that BDNF- induced eCB release is independent of MAPK, PI3K, and PLC signaling.

Furthermore, we examined a role for mGluR signaling because BDNF can induce presynaptic glutamate release, and glutamate is known induce eCB release through

The second set of experiments examined whether endogenous BDNF plays a role in eCB-mediated synaptic plasticity, focusing on activity-dependent long-term depression at inhibitory synapses (iLTD). Using a theta-burst stimulation protocol, we identified a form of eCB-mediated iLTD in layer 2/3 that is independent of metabotropic glutamate receptor (mGluR) activation, in contrast to the previously established forms of eCB-mediated iLTD (Heifets et al. 2008; Jiang et al. 2010).

This novel form of iLTD requires endogenous BDNF signaling, because it is blocked by a trk tyrosine kinase inhibitor as well as by a selective trkB receptor antagonist.

This form of eCB-mediated iLTD also requires PLC signaling, consistent with the findings summarized above in which PLCγ is required for BDNF-induced eCB mobilization. In addition, eCB-mediated iLTD can be induced by combining a subthreshold concentration of exogenous BDNF with weak TBS stimulation that by itself is insufficient to induce iLTD. This synergized eCB-mediated iLTD also requires activation of CB1R and PLCγ signaling. Taken together, these findings suggest that theta-burst stimulation induces release of endogenous BDNF, which activates trkB-PLCγ signaling in the postsynaptic cell, thereby inducing eCB release and triggering presynaptic CB1R-dependent iLTD. These findings are summarized in Figure 5.2.

cortical synapses (Lemtiri-Chlieh and Levine 2010). Therefore different signaling mechanisms might be involved in mGluR-induced and BDNF-induced 2-AG release, even though they both require PLC signaling. On the other hand, it is possible that the other major endocannabinoid, anandamide (AEA) is also involved. It has been established that elevated intracellular calcium is needed for AEA synthesis (Cadas et al. 1996; Di Marzo et al. 1994). Moreover, an earlier study suggested an alternative pathway of AEA synthesis via PLC signaling (Liu et al. 2008). Further systematic studies will be needed to identify the specific endocannabinoid(s) released by BDNF.

In Figure 4.3, the 7 train TBS protocol induced a form of iLTD that requires

BDNF-trkB signaling. The source of endogenous BDNF in this form of iLTD is not clear. It is likely, however, that TBS induces endogenous BDNF release from presynaptic terminals. Previous studies have identified BDNF-containing vesicles in presynaptic terminals (Brigadski et al. 2005; Dean et al. 2012; Dieni et al. 2012;

Hartmann et al. 2001) and stimulation has been shown to induce the release of endogenous BDNF from glutamatergic terminals (Aloyz et al. 1999; Dean et al.

2012; Hartmann et al. 2001; Kohara et al. 2001). In the present studies, it is unclear whether release of BDNF from excitatory or inhibitory terminals (or both) are involved, as there is much evidence for BDNF release from excitatory terminals but little is known regarding BDNF release from inhibitory terminals (Reviewed in

Edelmann et al. 2013).

BDNF has also been localized to vesicles in postsynaptic dendrites (Aoki et al.

2000; Avwenagha et al. 2006) and has been shown to be released from dendrites

(Dean et al. 2012; Hartmann et al. 2001; Matsuda et al. 2009). Dendritic release of

BDNF can be triggered by 1) Ca2+ influx through ionotropic glutamate receptors or voltage-gated channels (VGCCs) (Hartmann et al. 2001); 2) Activation of Group I

2+ mGluR receptors, which subsequently triggers IP3-mediated Ca release from intracellular calcium store (Canossa et al. 2001); 3) Ca2+-dependent Ca2+ release from intracellular stores by ryanodine receptors (RyR) (Balkowiec and Katz 2002).

However, in the present studies, iLTD was induced in the presence of both ionotropic and metabotropic glutamate receptor antagonists, and postsynaptic cells were voltage-clamped at a hyperpolarized membrane potential, thus ruling out postsynaptic release via the known pathways. Astrocytes can also release BDNF, however this also requires mGluR signaling (Jean et al. 2008). Thus presynaptic release is the most likely source of the endogenous BDNF mediating the observed iLTD.

The location of the trkB receptors that are activated by endogenously-released

BDNF in the present studies is most likely on dendrites of postsynaptic pyramidal neurons in layer 2/3. There are high levels of trkB expression in postsynaptic dendrites in hippocampus and in cortex, particularly in layer 2/3 (Aoki et al. 2000;

Drake et al. 1999), although there is also evidence for trkB expression at presynaptic sites (Inagaki et al. 2008; Kohara et al. 2001; Xu et al. 2000).

Importantly, however, postsynaptic trkB receptor activation has been shown to be specifically required for exogenous BDNF-induced eCB release (Lemtiri-Chlieh and

Levine 2010).

In Chapter 4, eCB-mediated iLTD was induced using TBS protocols that are known to induce LTP at excitatory synapses, in which endogenous BDNF plays a critical role (Abidin et al. 2006; Aicardi et al. 2004; Lu et al. 2010). Interestingly, Lu and colleagues have shown that BDNF facilitates LTP by suppressing GABAergic inhibition and enhancing pyramidal neuron excitability (Lu et al. 2010). Thus, endogenous BDNF may enhance LTP and/or postsynaptic excitability at least in part by triggering eCB release which then induces long-term depression of inhibitory synapses, in addition to the direct effects of BDNF at excitatory synapses. In order to fully understand the net effect of a given stimulation protocol on cortical circuits, it will be important to consider simultaneous changes at both excitatory and inhibitory synapses.

5.2. Control experiments for pharmacological experiments

The results presented in this dissertation are heavily based on bath application of pharmacological reagents. With this approach, it is important to consider the appropriate control experiments for both negative and positive drug effects.

Generally, in experiments with negative drug effects, it is possible that 1) the drug did not have the desired pharmacological effect, 2) did not have access to the cell, 3) that the dose used was insufficient, or 4) that the drug was inactivated. Positive control experiments showing the effectiveness of the drug are needed to address these concerns, ideally using the same preparation and drug application method.

On the other hand, in experiments with positive drug effects, control experiments

72 are necessary to confirm compound selectivity and specificity, as well as distinguish on- and off-target effects. For a suppressive effect, it is also important to rule out the possibility of response run-down over time. Below I will talk about the positive and negative controls for the specific studies described in Chapters 3 & 4.

5.2.1. Controls for negative drug effects

Endocannabinoid synthesis and release has been reported to be triggered by mGluR activation in many previous studies (for reviews see Castillo et al. 2012;

Hashimotodani et al. 2007a). In our studies, however, the mGluR antagonist

E4CPG did not block the acute effect of BDNF in modulating inhibitory synaptic transmission (Figure 3.5), nor did it block eCB-mediated iLTD (Figure 4.2). The concentration we used has been shown to block mGluR-mediated signaling in similar preparations (Perez et al. 2001; Shpak et al. 2012) so it is unlikely that the concentration we used is insufficient to block mGluR. To verify that E4CPG was effectively blocking mGluR receptors in our slice preparation, we examined the effect of the selective mGluR agonist DHPG, which is known to induce a suppression of eIPSC amplitude. We were able to replicate the effect of DHPG in our slices, and we found that E4CPG completely blocked the effect of DHPG

(Figure 3.5), thus confirming the efficacy of E4CPG and validating the negative results of mGluR block on the BDNF effect and on iLTD.

We also found that the effect of BDNF is not prevented by antagonists to

MAPK, PI3K or PKC signaling (Figures 3.3, 3.4). The concentrations we used for these drugs (PD 98059, LY 294002, chelerythrine, and GF 109203X) were based

73 on studies in the literature using similar preparations where these drugs were shown to be effective in blocking MAPK, PK3K and PKC signaling respectively.

Specifically, Gottschalk and colleagues showed that BDNF is able to attenuate synaptic fatigue triggered by high frequency stimulation and 10 µM PD 98059 prevented this effect (Gottschalk et al. 1999). Shanley and colleagues showed that

10 µM LY 294002 blocked the effect of leptin in inhibiting Ca2+ transients in Mg2+- free medium (Shanley et al. 2002) . Weiner and colleagues showed that ethanol- induced increase of IPSC amplitude in CA1 pyramidal neurons is blocked by 10 µM chelerythrine (Weiner et al. 1997). Bellone and colleagues have shown that GF at 1

μm blocked DHPG-induced LTD in the VTA (Bellone and Luscher 2005). We did not replicate these experiments, but these positive control experiments could be used in our slices to strengthen the conclusion that these pathways are not involved in the effect of BDNF on inhibitory transmission. In our studies, we did, however, use two structurally different PKC inhibitors, chelerythrine and GF 109203X, and obtained the same negative result. Additional inhibitors, such as the MAPK inhibitor U-0126, together with its inactive analog U-0124, as well as the PI3K inhibitor wortmannin could be used to further validate the negative results described above.

An alternative approach to examine the role of the mGluR, MAPK, PI3K, and

PKC pathways is to use transgenic animals. For example, there are established conditional knockout mouse lines of MAPK (Hitz et al. 2007), PKC-δ (Zhang et al.

2007) and mGluR (Brody et al. 2003). We would predict that the effect of BDNF will remain the same in these conditional knockout animals, although genetic

74 manipulation has its own weaknesses, such as developmental deficits, which might influence the interpretation of the results.

5.2.2. Controls for positive drug effects

In our studies, we found that exogenous application of BDNF suppresses inhibitory transmission (Figure 3.1) and facilitates iLTD (Figure 4.4). To reduce possible nonspecific binding of BDNF, we included 0.1g/L BSA as a carrier protein in all BDNF solutions, as BDNF is known to be a sticky molecule (Anderson et al.

1995; Barde et al. 1982; Leibrock et al. 1989; Yan et al. 1994). We also showed that the effects of BDNF are not due to response rundown over time, as application of aCSF alone for the same amount of time did not have a significant effect on eIPSC amplitude (data not shown, but also see Lemtiri-Chlieh and Levine 2010). In addition, we found that BDNF had a dose-dependent effect on eIPSC amplitude, as shown in Figure 4.4, which further supports the specificity of this effect.

In our studies, we have found that the effects of BDNF on both synaptic transmission and eCB-iLTD are blocked by CB1R antagonists (Figures 3.2, 4.3, 4.5).

Prior to these studies, our lab has established the involvement of CB1R in the effect of BDNF with a series of negative control experiments (Lemtiri-Chlieh and Levine

2010). The effect of BDNF was blocked in the presence of AM251 or SR 141716A, two different CB1R antagonists. Furthermore, we also examined the potential role of

TRPV1 receptors because SR 141716A also targets TRPV1 receptors. However, the TRPV1 receptor antagonist capsazepine had no effect on BDNF-induced suppression of eIPSC amplitude, further confirming the involvement of CB1R.

Moreover, we have also found that BDNF and the CB1R agonist WIN occlude each other in suppressing eIPSC amplitude, indicating common downstream pathways between BDNF and WIN, which also supports the conclusion that the effect of

BDNF is mediated by CB1R, rather than non-selective effects of the CB1R antagonists.

The effect of BDNF (Figure 3.2) was also blocked by two different PLC inhibitors, U-73122 and ET-18. As a control, we used U-73343, the inactive analog of U-73122, which did not block the BDNF effect. These results indicate the specificity of PLC targeting. Moreover, the PLC inhibitors and the inactive analog by themselves did not have significant effects on eIPSC amplitude (data not shown), thus ruling out the possibilities that the effect of BDNF was masked, instead of blocked, by an enhancing effect on eIPSC amplitude of these drugs. To further address the concern of nonspecific targeting, we examined the direct effect of the

CB1R agonist (WIN) in the presence of U-73122. WIN induced a similar amount of suppression with or without U-73122 (Figure 3.2), indicating that PLC inhibitors are blocking postsynaptic eCB release instead of inhibiting presynaptic signaling events downstream of CB1R activation. Based on these studies in Figure 3.2, we thus concluded from Figures 4.3 & 4.5 that eCB-iLTD also requires PLC signaling.

In Figure 4.3, K252a and ANA-12 blocked iLTD. K252a is a non-selective protein kinase inhibitor, but it is a relatively specific inhibitor of autophosphorylation of trk tyrosine kinase receptors at the concentration used in the study (Berg et al.

1992; Hashimoto 1988; Knusel and Hefti 1992; Nye et al. 1992). ANA-12 is a selective trkB receptor antagonist that displays no effect on TrkA or TrkC (Cazorla

76 et al. 2011). In addition, similar results obtained with K252a and ANA-12 strengthened the specific targeting of trkB receptors. Another newly developed trkB- selective antagonist cyclotraxin-B could provide further support to this finding.

5.3. Limitations of the present studies

In Figure 3.2, we showed that the acute effect of BDNF on synaptic transmission is blocked by various PLC inhibitors. We concluded that the effect of

BDNF is mediated by PLCγ, as it is the only PLC isoform known to be activated by

BDNF signaling. However there are no pharmacological inhibitors that distinguish between the different isoforms of PLC. The best approach to address this question is perhaps to design PLCγ-specific short hairpin RNAs oligonucleotides (shRNAs) and transfect into an organotypic slice culture using biolistic transfection (gene gun).

Organotypic slice cultures retain to some extent the advantage of brain slices, in which the original cortical organization and connectivity are preserved, as well as the advantage of cell culture, in which genetic manipulations can be performed.

Biolistic transfection is a method that yields a relatively low rate of transfection

(Usachev et al. 2000), but this could be an advantage for electrophysiology experiments, allowing for recordings from transfected and non-transfected neighboring neurons. We predict that the effect of BDNF will be maintained in neurons transfected with PLCβ or scrambled shRNAs, but not those transfected with PLCγ shRNA.

Activation of postsynaptic trkB receptors and subsequent PLC signaling induces the synthesis and release of eCBs that act retrogradely on presynaptic

CB1Rs to trigger iLTD. The endogenous CB1R ligand that mediates iLTD in the present studies is not completely clear, but is likely to be 2-AG. Activation of PLCγ and PLCβ, downstream effectors of BDNF-trkB and mGluR, respectively, leads to cleavage of phosphatidylinositol 4, 5-bisphosphate (PIP2) into the second messengers inositoltrisphosphate (IP3) and DAG (Hashimotodani et al. 2007a;

Minichiello 2009), and DAG is converted to the endogenous cannabinoid 2-AG by the enzyme DAG-lipase (DGL). DGL has two isoforms, DGLα and DGLβ, both of which are able to convert DAG into 2-AG, although might be under unspecified different conditions (For review please see Alger and Kim 2011). It is not clear, however, which isoform ether . In fact, exogenous BDNF-induced eCB release is blocked by inhibiting DGL (Lemtiri-Chlieh and Levine 2010), and 2-AG has been shown to be the downstream effector of mGluR-dependent eCB release in many studies (for reviews see Katona and Freund 2012; Mackie 2008; Ohno-Shosaku and Kano 2014). However, a contributing role for anandamide cannot be ruled out, as several studies have shown a link between mGluR signaling and anandamide generation (Azad et al. 2004; Edwards et al. 2012; Huang and Woolley 2012;

Puente et al. 2011), presumably through PLC signaling (Liu et al. 2008). In addition, it has been established that elevated intracellular calcium is needed for AEA synthesis (Cadas et al. 1996; Di Marzo et al. 1994), and we have shown that the effect of BDNF requires elevation of intracellular Ca2+ in our previous study (Lemtiri-

Chlieh and Levine 2010).

Although creating shRNAs that target the specific synthesizing enzymes of 2-

AG and AEA could potentially compensate for the drawbacks of pharmacological approach, it is important to note that 1) in DGLα conventional knockout mice, basal

AEA level was decreased as well as basal 2-AG level (Tanimura et al. 2010); and 2) in NAPE-PLD knockout mice, NAPE is still able to convert to AEA through alternative pathways that are not clearly characterized (Leung et al. 2006; Liu et al.

2008). Thus shRNA may not be effective in addressing this question, and future studies combining better pharmacological tools with molecular genetic approaches will be needed to unequivocally identify the endocannabinoid(s) released through

BDNF-trkB signaling.

In Figure 4.3, we showed that eCB-iLTD is blocked by interfering with trkB signaling using K252a or ANA-12. We conclude that iLTD requires TBS-induced endogenous BDNF release, which binds to trkB receptors and leads to eCB release and eCB-iLTD. This conclusion is based on the established finding that TBS induces endogenous BDNF release (Aicardi et al. 2004; Balkowiec and Katz 2002;

Hartmann et al. 2001; Li et al. 2010). Although there is no evidence suggesting release of NT-4/5, which also binds to trkB receptors, after TBS stimulation, we are unable to rule out the possible involvement of NT-4/5 using the pharmacological approaches used in our studies. However, there are at least three ways to address this concern in future studies. First, this form of iLTD should be absent in the currently available BDNF Val66Met mouse line, which has deficits in activity- dependent BDNF release (Chen et al. 2006). Second, one could take advantage of the ELISA in situ method (Balkowiec and Katz 2002) to measure extracellular BDNF

79 after TBS stimulation. Notably, although the trkB-IgG fusion protein has been used in earlier studies as a “BDNF scavenger” (Amaral and Pozzo-Miller 2007; Cheng and Yeh 2003; Figurov et al. 1996), it does not provide conclusive answer in identifying the endogenous neurotrophin, as it in fact binds equally to neurotrophin-

4/5 (NT-4/5) and BDNF and with a slightly lower affinity to NT-3 (Chen et al. 1999).

Nevertheless, trkB-IgG could still be used as an alternative method to further strengthen the conclusion.

5.4. Functional impact of BDNF-eCB interactions on cortical synaptic

plasticity

In sensory cortices, information is processed by assemblies of neurons bundled into cortical columns (Hubel and Wiesel 1962; Mountcastle 1997).

According to the canonical model, sensory information transmission in the cortex starts from thalamic excitatory information entering layer 4, then relayed to layer 2/3, which then passed down to layer 5, the main output layer of cortical processing

(Gilbert 1993). This model, however, is oversimplified. As the knowledge of cortical circuit advances, we now know that pyramidal neurons do not just passively pass down information they received to the next layer, but actively integrate the information they receive from all synapses before projecting to the next cortical layer. In particular, layer 2/3 neurons ramify most extensively within layer (Bannister

2005) where they make frequent connections both intracolumnarly and transcolumnarly (Brecht et al. 2003; Dodt et al. 2003; Lubke and Feldmeyer 2007;

Lubke et al. 2003). In addition, Adesnik and Scanziani found that the activity in layer

2/3 generates lateral suppression of spiking in layer 2/3 pyramidal cells but feedforward facilitation in layer 5 pyramidal cells, suggested that layer 2/3 synaptic activity may have layer-specific modulation across different layers in the cortex

(Adesnik and Scanziani 2010). Thus the local microcircuit of layer 2/3 may play an important role in cortical information integration and processing.

The rich variety of local inhibitory interneurons is crucial for integrating and filtering incoming information, as well as shaping and fine tuning the spatiotemporal patterns of electrical signaling (Mendez and Bacci 2011). Understanding the local inhibitory microcircuits is important to understand the information process mechanism of cortical circuit. Compared to layer 5 PNs, however, much less is known about the synaptic integration of layer 2/3 (Larkum et al. 2007). Our studies of BDNF-eCB interaction and its role in modulating layer 2/3 inhibitory transmission provided a novel model in studying the signal integration of layer 2/3.

Endocannabinoid-dependent forms of plasticity are ideal models to study local microcircuit modulation of somatosensory cortex. CB1R immunoreactivity exhibits a laminar specific distribution, with highest levels in layer 2/3 and 5a, and physiological studies have shown that eCB signaling exert different functional roles in layer 2/3 and 5. In layer 2/3 of somatosensory cortex, depolarization-induced eCB release targets both inhibitory and excitatory synaptic transmission; however the net effect of a brief depolarization is an increase in pyramidal neuron excitability due to disinhibition. In contrast, in layer 5, depolarization-induced eCB release results in suppression of excitatory transmission (DSE) while the majority of inhibitory inputs

81 are cannabinoid insensitive. Thus, the net effect of a brief depolarization in layer 5 is a decrease in pyramidal neuron excitability (Fortin et al. 2004).

CB1Rs are predominantly expressed on presynaptic terminals of CCK basket cells (Bodor et al. 2005; Eggan et al. 2010; Marsicano and Lutz 1999), which form inhibitory synapses onto the perisomatic membrane of pyramidal neurons (Freund and Katona 2007; Savanthrapadian et al. 2014). Thus by selectively suppressing perisomatic inhibitory transmission and increasing pyramidal neuron excitability, eCB signaling may enhance the output from layer 2/3 to layer 5; on the other hand, cortical LTP could drive neural activity towards runaway excitation without proper scaling mechanism (Turrigiano and Nelson 2004). Suppressing spiking probability in layer 5 may provide a gating mechanism and keep the excitatory output under control. Thus eCB signaling in different cortical layers exhibits diverse effects, which could be related to the different roles each cortical layer plays in cortical information processing.

Cortical circuits are constantly shaped and refined by sensory experience during critical periods as well as in adulthood. Endocannabinoid-mediated long-term depression has been found to be important in shaping cortical circuits during the critical period of postnatal development (Huang et al. 2008; Jiang et al. 2010, see

Chapter 1.3.2 for detailed review about these two studies). In addition, BDNF has also been found to play important roles in experience-dependent refinement of cortical circuit. For example, Jiao and colleagues showed that sensory experience regulates cortical GABAergic circuits via activity-dependent expression of BDNF.

They found that knocking out activity-dependent BDNF expression (bdnf-KIV)

82 reduced GABA release from parvalbumin-expressing fast-spiking (FS) interneurons and decreased perisomatic inhibition. The disruption of perisomatic inhibition found in the bdnf-KIV mice can be mimicked by sensory deprivation in wild type animals, suggesting that postnatal experience controls cortical development through activity- driven BDNF expression (Jiao et al. 2011). The activity-dependent release of both eCB and BDNF makes them suitable models for studying the activity-dependent refinement of cortical microcircuits.

In summary, studying the layer- and input-specific effects of eCB signaling and its regulation by BDNF may provide insights in understanding the activity-dependent regulation of cortical information flow.

5.5. Conclusions and future directions for BDNF-eCB interactions

With 86 billion neurons, over 10 trillion synapses and a calculation speed of

10,000,000,000,000,000 calculations per second (Godwin and Cham 2012), as well as all the various information processing functions such as thinking, memorizing, problem solving and expressing emotions, it is without question that our brain is a very complicated but fascinating organ. As we can imagine, the mysteries of how our brain functions under normal and abnormal conditions are so complicated that obviously no single molecule can provide a detailed answer by itself. Interactions between different signaling systems should be the rule rather than a sporadic example.

The studies presented in this dissertation characterized the interactions between BDNF and eCB at layer 2/3 inhibitory synapses in the somatosensory cortex. First, we identified PLCγ as the signaling pathway that underlies the process of BDNF-induced eCB release. Further, we discovered a new form of endocannabinoid-mediated iLTD in which endogenous BDNF is required.

Many questions still remain to be answered following our discoveries. For example, an immediate question is identifying which eCB ligand is involved in the

BDNF-eCB interaction. Although we have some preliminary evidence suggesting the involvement of 2-AG (Lemtiri-Chlieh and Levine 2010), we are unable to rule out the possible involvement of AEA in our studies. In fact, identifying the eCB involved in any specific eCB-mediated process has been a challenge for studying endocannabinoid signaling ever since the establishment of eCB as synaptic modulators, and there is a longstanding inconsistency between findings of genetic and pharmacological manipulations (for review see Di Marzo 2011). In addition, because eCBs are fatty acids and synthesized on demand, current imaging and biochemical tools that are designed to track mobilization of proteins are difficult to apply to the study of eCBs. Development of better tools is needed to promote a more detailed understanding of the eCB system. In addition, in the iLTD described in Chapter 4, the source of endogenous BDNF release is unclear. Paired recording, high resolution imaging or time lapse microscopy techniques might be helpful to answer this question, as well as determining whether this iLTD is homosynaptic or heterosynaptic by nature.

The present studies focused on the role of BDNF in eCB-mediated long-term plasticity at inhibitory synapses. In addition to suppressing inhibitory transmission, endocannabinoids have been shown to suppress glutamate release in many brain areas, including cortical layer 5 (Fortin and Levine 2007), amygdala (Kodirov et al.

2010), cerebellum (Kreitzer and Regehr 2001) and hippocampus (Hoffman et al.

2010). Interestingly, BDNF potentiates excitatory synaptic transmission in these same brain areas by enhancing glutamate release (Madara and Levine 2008;

Schinder et al. 2000). It is not hard to imagine that in addition to their synergistic action at inhibitory synapses, BDNF and endocannabinoids may interact in modulating excitatory transmission, although the outcome may be more complicated, since BDNF and eCB modulate excitatory synaptic transmission in opposite ways (see Chapters 1.3 and 1.5, also see Alger 2002; Blum et al. 2002 for reviews). The outcome of their interaction at excitatory synapses might vary, depending on the brain region, which makes BDNF-eCB interaction a useful tool in studying the modulation of brain microcircuits. It will be also interesting to examine the role of BDNF in eCB-mediated synaptic plasticity at excitatory synapses (DSE and eCB-LTD). A recent study showed that exogenous BDNF enhances DSE in hippocampus via activation of the immediate-early gene homer1a (Roloff et al.

2010), however more studies are needed to have a full picture of their interactions at excitatory synapses.

In summary, although knowledge about BDNF and eCB systems alone has been greatly advanced through the past decade or two, BDNF-eCB interaction is still a relatively new concept. The interactions between BDNF and eCBs may have

85 significant roles in shaping synaptic plasticity and modulating the activity of brain microcircuits, as well as providing novel therapies for brain diseases. With the studies presented in this dissertation that used eCB-mediated synaptic plasticity as a model, I hope to shed some inspirations towards future studies of cortical circuit modulation.

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