REGULATION OF PRESYNAPTIC FUNCTION BY SODIUM

PERMEABLE ION CHANNELS AT THE CALYX OF HELD

AN ABSTRACT

SUBMITTED ON THE NINETEENTHDAY OF APRIL2021

TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

OF THE SCHOOL OF SCIENCEAND ENGINEERING

OF TULANE UNIVERSITY

FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

BY

DAINAN LI Approved: --�--4+· _-_z..__?__ - __ Hai Huang, Ph.D., Advisor

Jeff�eyG. Tasker, Ph.D.

Laura A. Schrader, Ph.D. �OJ-1 Andrea Zsombok, Ph.D. ABSTRACT

Synaptic strength, which is described as the amplitude of postsynaptic response upon a presynaptic spike, is essential for reliable synaptic transmission. Previous work has revealed a presynaptic cytosolic Na+-dependent regulation on vesicular glutamate content and miniature excitatory postsynaptic current (mEPSC) amplitude via activating vacuolar Na+/H+ exchangers

(NHEs) expressed on the synaptic vesicles, suggesting a presynaptic determinant of quantal size for synaptic strength (Goh et al., 2011; Huang and Trussell, 2014). Manipulation of the presynaptic

Na+ at the calyx of Held synapse with up and down regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity, which induced a small change of cytosolic resting Na+ level, bidirectionally changed the quantal size (Huang and Trussell, 2014). However, it remains unknown how spike activities control intracellular Na+ at the terminals and how the fluctuation of presynaptic Na+ during activities modulates quantal content and contributes to synaptic strength. I studied these questions using the calyx of Held, a giant glutamatergic synapse in the auditory brainstem that allows direct pre- and postsynaptic recordings and manipulation of presynaptic cytosolic environment. With two-photon Na+ imaging, I found that presynaptic Na+ substantially accumulated during spike firing in a frequency and duration-dependent manner. This spike-induced elevation of presynaptic Na+ gradually increased EPSC amplitude by solely affecting vesicular glutamate filling, which was further confirmed as increased amplitude of asynchronous released vesicles, but without affecting the size of readily releasable pool or neurotransmitter release probability. This Na+-dependent modulation of EPSC amplitude resulted in a change of the reliability of transferring presynaptic spike to postsynaptic firing. Finally, blockade of NHEs reduced both EPSC amplitude and reliability of synaptic signaling, suggesting that NHEs are required for presynaptic Na+ regulation of synaptic transmission. Recent studies demonstrated that a TTX- and Cs+-resistant, non-inactivation cation channel

NALCN (Na+ leak channel, non-selective), characterized as a major Na+ leak channel, is widely expressed in the . I asked whether NALCN channel is also expressed in the and if so how it controls intracellular Na+ and synaptic transmission.

Immunostaining with antibodies against NALCN revealed the expression of this channel at the calyceal terminals. In line with a role of NALCN in controlling the cell excitability, calyces with conditional knockout (cKO) of NALCN exhibited a more hyperpolarized resting membrane potential compared with the wildtype (WT) calyces. Blockade of NALCN with a non-specific

NALCN blocker gadolinium (Gd3+) induced a reduction of basal Na+ level and mEPSC amplitude

(quantal size) in the WT but not in cKO group, suggesting the involvement of presynaptic NALCN channels in regulating the vesicular glutamate content. More importantly, two-photon Ca2+ imaging showed that NALCN channels were permeable to Ca2+, and Gd3+ decreased the basal Ca2+ level in WT but not cKO calyces. The Ca2+ permeability was further confirmed by reduced sensitivity of mEPSC frequency in response to increased extracellular Ca2+ concentration in cKO and reduced initial release probability in response to application of Gd3+ to block NALCN channels in WT group. Finally, Gd3+ induced a stronger reduction of EPSC amplitude in WT group compared to cKO group. Overall, these data indicate that NALCN channels regulate glutamate transmission through modulation of both quantal size and initial release probability. REGULATIONOF PRESYNAPTIC FUNCTION BY SODIUM

PERMEABLE ION CHANNELS AT THE CALYX OF HELD SYNAPSE

A DISSERTATION

SUBMITTED ON THE NINETEENTHDAY OF APRIL2021

TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

OF THE SCHOOL OF SCIENCE AND ENGINEERING

OF TULANE UNIVERSITY

FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

DAINAN LI Approved: __.....,_{k --'---·�----- Hai Huang, Ph.D., Advisor

� ( l'ya,, JeffreyG. Tasker, Ph.D. c::Zt..� dLaura A. Schrader, Ph.D. �a/2 Andrea Zsombok, Ph.D. © Copyrights by Dainan Li, 2021

All Rights Reserved ACKNOWLEDGMENT

It has been seven years since the Department of Cell and Molecular Biology at

Tulane University provided the stage for me to pursue my Ph.D. degree. I deeply appreciate this chance and this whole Ph.D. training has become one of the most valuable experience in my life. This is a long journey and the only reason I can survive is that I received tremendous supports from my mentors, colleagues, family, and friends.

First and foremost, I would like to express my special thanks of gratitude to my

Ph.D. advisor, Dr. Hai Huang. As an excellent and rigorous electrophysiologist, he taught me in many aspects of doing research including basic knowledge, experimental skills, scientific thinking, and troubleshooting since the first day I joined the lab. I have greatly benefited from his suggestions, such as how to understand and learn more information from publications, how to design appropriate experiments to address scientific questions, and how to fully understand and analyze experimental results without missing new discoveries.

Without Dr. Huang’s constant guidance and support, I will never finish my dissertation and reach my goal.

I also would like to gratefully thank my committee members, Dr. Jeffrey Tasker,

Dr. Laura Schrader, and Dr. Andrea Zsombok for their invaluable feedbacks and suggestions, which kept my research work in pace. I not only took their classes to build my basic background of neuroscience, but more importantly received advice from them to help me better understand my work and be confident to explore more scientific questions. I especially want to thank Dr. Tasker for letting me use the equipment and materials in his lab and being a source of motivation.

ii Next, I would like to thank all the current and previous members in the Huang lab for creating a lovely environment that I always feel relax and joyful during my stay in the lab. I want to express my thanks to Dr. Yihui Zhang for all the experimental discussion, technical assistance, and encouragement. I would like to thank Yun Zhu, who helped me to finish the capacitance measurement experiment and shared a lot of useful experimental tricks to me. Many thanks go to Youad Darwish for staying with me in the lab in a lot of days, to Dr. Inga Kristaponyte for helping with my dissertation and sharing tasty chocolate cake flavor beer, and to Tianhao Wu for taking care of our animals.

Then, I would like to thank all the faculty and Ph.D. students in the CMB for their encouragement and useful advice I received after my annual student presentation. I also would like to thank the nice people in our CMB office, Marnie Elsky, John Drwiega, and

Jonathan Flack for their sweet help in our daily work.

Last, but certainly not least, I would like to thank my family and friends for their constant support. Special thanks to my husband Xin Fu, who always tried to make me happy during my tough time.

iii Table of Contents

ACKNOWLEDGMENT ...... ii

LIST OF FIGURES ...... vii

CHAPTER 1: INTRODUCTION ...... 1

1.1 Synaptic Transmission Between Neurons ...... 1

1.2 Strength of Synaptic Transmission ...... 3

1.2.1 Readily releasable vesicles (n) ...... 5

1.2.2 Release probability (pr) ...... 6

1.2.3 Quantal size (q) ...... 8

1.2.4 The role of Ca2+ for synaptic strength ...... 10

1.3 Research Model: The Calyx of Held Synapse ...... 11

1.4 Formation of Quanta at Glutamatergic Synapse ...... 14

1.5 Presynaptic Determinants of Quantal Size in Glutamatergic Synapse ...... 16

1.5.1 Expression level of VGLUTs ...... 16

1.5.2 Cl- gradient ...... 17

1.5.3 Cation/H+ exchanger ...... 18

1.6 Overview of Thesis ...... 21

CHAPTER 2 MATERIALS AND METHODS ...... 23

2.1 Mouse lines ...... 23

2.2 Slice Preparation ...... 23

2.3 Whole-Cell Recordings ...... 24

iv 2.4 Perforated Patch-Clamp Recordings ...... 26

2.5 Two-Photon Na+ and Ca2+ Imaging ...... 26

2.6 Membrane Capacitance Measurement ...... 28

2.7 Immunohistochemistry ...... 29

2.8 Drugs ...... 29

2.9 Analysis ...... 30

CHAPTER 3: SPIKE ACTIVITY REGULATES VESICLE FILLING AT A

GLUTAMATERGIC SYNAPSE...... 31

3.1 Abstract ...... 31

3.2 Introduction ...... 32

3.3 Results ...... 33

3.3.1 Spikes control presynaptic cytosolic Na+ concentration ...... 33

3.3.2 Presynaptic Na+ regulates glutamatergic transmission ...... 38

3.3.3 Na+-dependent regulation of synaptic transmission is not due to change in AP waveform or Ca2+

influx ...... 42

3.3.4 Presynaptic Na+ does not affect the readily releasable vesicle pool or release probability ...... 46

3.3.5 Presynaptic Na+ controls vesicular glutamate content ...... 48

3.3.6 Presynaptic Na+ is required for reliable signal transmission ...... 50

3.3.7 NHE activity promotes synaptic transmission and signaling reliability ...... 54

3.4 Discussion ...... 56

v CHAPTER 4: PRESYNAPTIC NALCN CHANNELS MODULATE SYNAPTIC

STRENGTH THROUGH CONTROLLING VESICLE FILLING AND BASAL

CALCIUM ...... 62

4.1 Abstract ...... 62

4.2 Introduction ...... 63

4.2.1 Na+ leak conductance in neurons ...... 63

4.2.2 Structure and characteristics of Na+ leak channel NALCN ...... 64

4.2.3 The NALCN-FAM155A-UNC80-UNC79 Complex...... 66

4.2.4 Pharmacology of NALCN channel ...... 68

4.2.5 Function of NALCN channel ...... 69

4.2.6 Introduction for this chapter ...... 72

4.3 Results ...... 74

4.3.1 Identification of the expression of NALCN at the calyx of Held synapse ...... 74

4.3.2 NALCN channels contribute to Na+ leak currents and RMP in the calyceal terminals ...... 78

4.3.3 NALCN channels regulate presynaptic basal Na+ level and quantal size ...... 81

4.3.4 NALCN channels are Ca2+ permeable and control presynaptic basal Ca2+ level ...... 85

4.3.5 NALCN channels control initial release probability and glutamate release ...... 87

4.4 Discussion ...... 91

CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS ...... 97

5.1 The Role of Na+ in Synaptic Transmission ...... 97

5.2 Specific Function of NALCN Channels on the Axon Terminal ...... 99

BIBLIOGRAPHY...... 101

vi LIST OF FIGURES

Figure 1.1 Model for Na+/H+ exchanger-dependent facilitation of vesicular glutamate loading ...... 20

Figure 3.1 Presynaptic spikes control cytosolic Na+ concentration ...... 35

Figure 3.2 Presynaptic spikes robustly increase cytosolic Na+ at heminode and terminal in both immature and mature calyces ...... 37

Figure 3.3 Presynaptic Na+ regulates the EPSC amplitude ...... 40

Figure 3.4 Presynaptic Na+ regulates EPSC amplitude in response to presynaptic ...... 44

Figure 3.5 Presynaptic Na+ does not affect spike-evoked Ca2+ rise and decay ...... 45

Figure 3.6 Presynaptic Na+ does not affect the readily releasable pool size or release probability ...... 47

Figure 3.7 Presynaptic Na+ regulates aEPSC amplitude ...... 49

Figure 3.8 Presynaptic Na+ level contributes to reliable synaptic transmission ...... 52

Figure 3.9 NHE activity is required for reliable synaptic signaling ...... 55

Figure 4.1 Immunostaining of NALCN channels at the calyx of Held ...... 77

Figure 4.2 NALCN channels mediate a small inward current and induce a hyperpolarization of RMP in the calyx of Held ...... 80

Figure 4.3 NALCN channels regulate presynaptic basal Na+ level and quantal size 83

Figure 4.4 NALCN channels are Ca2+ permeable and control presynaptic basal Ca2+ level ...... 86

Figure 4.5 NALCN channels control initial release probability and glutamate release

...... 89

vii CHAPTER 1: INTRODUCTION

1.1 Synaptic Transmission Between Neurons

Since the first demonstration of discontinuity between neurons by Cajal and the discovery of specialized junctions called , where the inter-neuronal communications primarily happen, a thorough knowledge of the synaptic transmission becomes fundamentally crucial to understand the normal functions of the brain. Two different types of synapses for synaptic transmission have been revealed: electrical synapse which is made of gap junctions to connect pre- and postsynaptic neurons, and chemical synapse which releases neurotransmitters for signal transmission. In this thesis, I will focus exclusively on chemical synapses, the most common type in the mammalian brain.

At chemical synapses, there is no cytoplasmic continuity between pre- and postsynaptic elements, instead, they are separated by the synaptic cleft. The presynaptic side of the synapse is usually an axon terminal, while the postsynaptic density containing enriched neurotransmitter receptors can be on the , or even another axon of neurons. Once action potentials (APs) arrive at the presynaptic terminal, voltage-gated Ca2+

2+ (CaV) channels are activated by membrane . Ca influx through CaV channels triggers neurotransmitter release. The released neurotransmitters present in the synaptic cleft bind to their receptors on the postsynaptic site to initiate downstream responses.

Since the discovery of chemical synaptic transmission, researchers have identified different types of neurotransmitters, most of which can be classified into three main

1 categories: small molecule amino acids, amines, and large molecule peptides. The most common neurotransmitters in the central nervous system (CNS) are amino acids, with glutamate as the most abundant excitatory neurotransmitter, and gamma-aminobutyric acid

(GABA), and glycine as inhibitory neurotransmitters, while different neurons in the central and periphery nervous systems release different neurotransmitters for specific functions.

Neurotransmitters can be recognized by their specific receptors which are embedded in the postsynaptic density. Although more than 100 different neurotransmitter receptors have been discovered, they fall into two groups: transmitter-gated ion channels and metabotropic receptor (or G-protein coupled receptors). Each neurotransmitter may have more than one corresponding receptors for different functions.

The neurotransmitter release from presynaptic terminals is mediated by the fusion of neurotransmitter-filled synaptic vesicles (SVs) with plasma membrane. This mechanism was first discovered and reported in frog’s neuromuscular junction (NMJ) in 1950s (Fatt and Katz, 1952). Spontaneously occurring postsynaptic potentials with very small and constant average amplitude were recorded in the presence of low extracellular Ca2+ concentration, and were described as “miniature” endplate potentials (mEPP) by Fatt and

Katz (1952). These were further referred as quanta by del Castillo and Katz as the least unit for endplate potential (EPP) in response to nerve stimulus (Del Castillo and Katz, 1954).

Quantal nature of neurotransmitter release has been rigorously tested by different methods such as , electron microscope, membrane capacitance measurement, and optical analysis of labeled synaptic vesicles, in both vertebrate and invertebrate nervous system. The thought that each quantum equals one SV has been widely appreciated as the

2 fundamental theory for understanding chemical signal transmission (Rossi et al., 1994;

Hammond, 2015).

The quanta hypothesis further states that there are plenty of SVs stored in the presynaptic terminal and each of them has a probability to release. Still at frog NMJ, the release probability of SV was very low at rest, while in response to a presynaptic nerval impulse and in the presence of sufficient extracellular Ca2+ concentration, a large number of SVs released almost simultaneously to generate a corresponding evoked EPP. Lowering the extracellular Ca2+ to different levels could gradually reduce the EPP amplitude but its amplitude was constantly a composition of multiple quanta (Del Castillo and Katz, 1954).

Similar “step-wise” manner was also observed in evoked postsynaptic potentials (PSP) in

CNS neurons (Kuno, 1964). Hence, evoked PSPs consist of discrete units of quantal size in a binomial mode (simplify the probability as success or failure outcome with repeated times), and the fluctuations in PSP amplitude could be predicted with Poisson distribution when the total number of vesicles is very large and release probability is low. These studies not only demonstrate that Ca2+ is one of the prerequisites for the release of vesicular neurotransmitter from the presynaptic terminal, but more importantly provide a statistical analysis method to study the probabilistic nature of vesicle release (Kuno, 1964; Katz,

1971).

1.2 Strength of Synaptic Transmission

Synapses are not static, so the strength of synaptic transmission (or synaptic strength), defined as the size of postsynaptic response that is generated by the presynaptic activity, is a fundamental element for signal propagation in neuronal networks (Atwood and Karunanithi, 2002; Branco and Staras, 2009). Considering the quantitative estimation

3 of synaptic strength, the recorded postsynaptic current (PSC) corresponding to single presynaptic AP, is the most useful parameter (Schneggenburger et al., 2002). However, the

PSC is the final result of synaptic transmission. Previous steps include depolarization of presynaptic terminal by arrived APs, elevation of intracellular Ca2+ level, and fusion of

SVs. During repetitive firing, recycling of SVs is also involved. Hence, successfully obtaining the PSC depends on multiple variables from the whole process. The quantal hypothesis, combined with the binomial model of analysis of quantal neurotransmitter release, provides a conceptual and experimental basis for understanding the synaptic responses. Synaptic strength, described as PSC, is fundamentally determined by three parameters: the number of readily releasable synaptic vesicles (‘n’), the probability of vesicular neurotransmitter release by AP (release probability, ‘pr’), and the size of the postsynaptic current elicited by neurotransmitter release from each single vesicles (quantal size, ‘q’) (Del Castillo and Katz, 1954; Quastel, 1997). Their relationship can be described with a simple equation:

PSC = n × pr × q

All three quantal parameters should be taken into account when studying the synaptic strength. On the one hand, during synaptic activity, one or more of these three parameters may change, which thereby cause a change of synaptic strength. On the other hand, collection and releasing of SVs from the presynaptic terminal, as a defining feature of chemical synapses, needs to be efficiently maintained based on these three main parameters for reliable synaptic transmission.

4 1.2.1 Readily releasable vesicles (n)

The replenishment and mobility of SVs are important for sustained synaptic activity.

At any type of synapse, not all the SVs are released following an AP. Instead, SVs reside in different “vesicle pools” and participate in an exocytosis-endocytosis cycle which allows them to be used repeatedly and efficiently during ongoing synaptic transmission (Rizzoli and Betz, 2005; Xue et al., 2013). Only a small fraction of vesicle population is docked on the intracellular side of active zone membrane and ready to be released with Ca2+ influx.

These immediately available vesicles upon stimulation are generally stored in a vesicle pool called the readily releasable pool (RRP). Another two “non-readily releasable” pools are called recycling pool and reserve pool. They contain most of the SVs, but may only maintain release during intense stimulation (Rizzoli and Betz, 2005).

Vesicle pools have been carefully investigated in different systems, such as

Drosophila Melanogaster and frog NMJ, calyx of Held synapse, and hippocampal boutons, by using different techniques including electrophysiological recordings, and electron and fluorescence microscopy. The RRP can be depleted rapidly with a train of high frequency stimulation, tens of milliseconds strong depolarization pulse, or an elevation of extracellular K+ concentration, which provides a possible way to estimate the size of RRP.

RRP size is variable and primarily determined by presynaptic factors. In most cases, the depletion and replenishment of RRP during prolonged firing are regulated by Ca2+ or Ca2+- binding proteins (Borst and Soria van Hoeve, 2012). For example, Ca2+ promotes refilling of the rapidly released vesicles in the RRP during prolonged activities, presumably via -dependent mechanism (Sakaba and Neher, 2001; Thanawala and Regehr,

2013). During short-term potentiation at the calyx of Held, an increase of RRP size in the

5 course of vesicle replenishment is important to scale up the synaptic responses and it is modulated by Ca2+-dependent PKC/munc13-1 pathway (Lou et al., 2008; Chen et al., 2013;

Chu et al., 2014). Other Ca2+-independent mechanisms have also been reported, such as the regulation of cAMP via the Epac pathway (Kaneko and Takahashi, 2004).

1.2.2 Release probability (pr)

For each AP, not all the vesicles in the RRP are necessarily immediately released.

Each vesicle has a certain likelihood to release, which is defined as the release probability

(pr). Release probability not only fundamentally determines the reliability and efficacy of a synapse for relaying presynaptic AP signal to postsynaptic neuron, but also dynamically changes in a use-dependent manner for shaping the way that the synapse optimally adapts its strength.

In most synapses that receive multiple inputs, it is difficult to directly measure release probability. Investigators have developed multiple ways for relative comparison, estimation or even calculation of release probability, depending on the type of synapses.

Quantal analysis and paired-pulse ratio (PPR) are two of the most acceptable ways for monitoring the change of release probability, based on electrophysiological methods

(Branco and Staras, 2009). Quantal analysis, as a statistical procedure mentioned above, was first developed in frog NMJ and further refined and applied to the synapses in the CNS

(Del Castillo and Katz, 1954; Redman, 1990). Quantal analysis also can be combined with optical methods to overcome many difficulties that existed in the classic way due to the heterogeneity of release probability (Oertner et al., 2002; MacDougall and Fine, 2019).

Moreover, the degree of miniature vesicular release is generally believed to correlate with evoked response since intracellular Ca2+ concentration influencing the frequency of

6 miniature activity has been discovered in different types of neurons. The frequency of miniature events therefore turns out as an indicator for evoked release probability (Prange and Murphy, 1999). PPR is defined as the ratio of the amplitude of the second postsynaptic current to that of the first one in responding to two closely spaced presynaptic APs. PPR reflects two types of synapses: facilitating synapses that have greater amplitude at the second postsynaptic response (PPR > 1) and appear to have smaller initial release probability; depressing synapses that exhibit decreased postsynaptic response in amplitude following the first response (PPR < 1), which results from higher initial release probability

(Xu-Friedman and Regehr, 2004). It is known that release probability is sensitive to presynaptic Ca2+ level and can be manipulated by changing extracellular Ca2+ concentration (Dodge and Rahamimoff, 1967). Cumulative evidence has demonstrated an inverse relation of PPR to the initial release probability by operating a synapse from facilitation-type to depression-type with increased extracellular Ca2+ concentration

(Dobrunz and Stevens, 1997; Zucker and Regehr, 2002). For some highly specialized synapses, such as calyx of Held, release probability can be accurately calculated after determining the RRP size (Schneggenburger et al., 1999), which allows investigators quantitatively studying the synaptic strength.

In most studies of release probability and synaptic function, the measured release probability only represents a basal averaged value. However, release probability is dynamic all the time, highly dependent on the continuous synaptic activity under physiological conditions. Ca2+ is always considered as the most important determinant for release probability. Changes in presynaptic Ca2+ concentration or the Ca2+ sensitivity of vesicle fusion by affecting their molecular coupling may result in a change of release probability

7 (Branco and Staras, 2009). For example, Ca2+-dependent PKC-mediated post-tetanic potentiation at the calyx of Held enhanced vesicle release prominently by increasing release probability before hearing onset (Chu et al., 2014). Phorbol esters-induced increased transmitter release through PKC/munc13 pathway had no effect on presynaptic

Ca2+ current but largely increased the Ca2+ sensitivity of vesicle fusion (Hori et al., 1999;

Lou et al., 2005).

The intrinsic heterogeneity of release probability also exists in a single axon and is more evident with ultrastructural analysis. In the hippocampal glutamatergic terminals, release probability, the number of CaV channels and docked vesicles showed a tight positive correlation with the active zone area, which therefore can be used to predict the size of synaptic response (Holderith et al., 2012). In the calyx of Held, the vesicles in the

RRP can be further classified into two distinct subset populations based on their release probability: some vesicles are readily releasable in response to a presynaptic AP as a fast component, whereas others are reluctant to be released and defined as a slow component

(Wolfel et al., 2007). Thus, the number of CaV channels and the distance between CaV channels and docked vesicles diversify release probability in the terminal for expanding the synaptic plasticity.

1.2.3 Quantal size (q)

Typically, the average amplitude of miniature events recorded from the postsynaptic neuron is considered as the quantal size. It has become clear that quantal size could change bidirectionally as a functional formation of synaptic strength. These changes were demonstrated to be determined by the number and sensitivity of receptors on the postsynaptic membrane (Bredt and Nicoll, 2003). For example, early investigation of

8 homeostatic plasticity in cultured visual cortical neurons described activity-induced change of mEPSC amplitude. Chronic blockade of neuronal activities with TTX or AMPAR inhibitor increased the mEPSC amplitude, whereas blockade of GABAA-mediated inhibition with its blocker bicuculline for inducing more neuronal activates eventually reduced the mEPSC amplitude. These activity-dependent changes in mEPSC amplitude were mainly due to postsynaptic alternations of receptor number (Turrigiano et al., 1998).

A large body of evidence supported the view that besides postsynaptic effects on quantal size, the total amount of neurotransmitter stored in one SV can also vary (Takamori,

2016). In many cases, a single SV is unable to saturate all postsynaptic receptors, indicating a strong possibility of presynaptic regulation of quantal size. In the calyx of Held synapse, increasing the cytosolic glutamate concentration with direct patch onto presynaptic calyceal terminal is followed by an increased quantal amplitude, providing direct evidence for nonsaturation of postsynaptic AMPARs (Ishikawa et al., 2002). Fluorescence optical methods used in hippocampal synapses also described multi-quantal release, implying that individual quantal release is far from saturation (Conti and Lisman, 2003). Early studies have demonstrated that experimental modification, such as genetic manipulation, of presynaptic terminals could change quantal size (Atwood and Karunanithi, 2002). Some evidence also suggested that altered neuronal activities could up- or downregulate the expression level of vesicular transporters or activities of neurotransmitter cycle-related enzymes, and hence lead to bidirectional change of quantal size (Turrigiano and Nelson,

2004; Wilson et al., 2005). More importantly, under physiological conditions, quantal size also could be manipulated. For example, a natural behavior (high crawling activities) in

Drosophila induced a larger quantal size and enhanced synaptic transmission through a

9 PKA- and actin-dependent pathway during vesicle replenishment (Steinert et al., 2006). In the calyx of Held synapse, generation of post-tetanic potentiation by a high-frequency AP train required an increase of quantal size via PKC-dependent mechanism or

Ca2+/-mediated compound vesicle fusion (He et al., 2009; Xue and Wu,

2010).

In summary, the presynaptic mechanisms underlying quantal size can be classified into two types: either affecting the concentration of neurotransmitters in a single vesicle, or in some reported cases, mediating compound fusion between vesicles to generate giant vesicles with no change of neurotransmitter concentration. Both mechanisms have been carefully studied in certain types of synapses (Wu et al., 2007; He et al., 2009; Goh et al.,

2011).

1.2.4 The role of Ca2+ for synaptic strength

Ca2+ plays an essential role in synapses that undergo Ca2+-dependent exocytosis.

As a secondary messenger and direct trigger, Ca2+ cooperates vesicle release and synaptic strength through the action of Ca2+ and Ca2+-binding proteins that are responsible for vesicular fusion, and regulation of release probability and RRP size (Thanawala and

Regehr, 2013). Since Ca2+ concentration in the presynaptic terminal is critical for overall synaptic strength, there is a large number of pre- and postsynaptic factors that can modify the intraterminal Ca2+ concentration via different mechanisms including the number, type

2+ 2+ and function of CaV channels, the presynaptic AP waveform-dependent Ca entry, Ca buffer capabilities, and postsynaptic retrograde messengers (Borst and Sakmann, 1999;

Chen and Regehr, 1999; Meinrenken et al., 2003; Catterall and Few, 2008; Neher and

Sakaba, 2008).

10 Ca2+-dependent vesicle fusion normally requires high Ca2+ cooperativity, but subtle change of basal Ca2+ level is also important although received less attention. Previous work on the calyceal terminal showed that activation of presynaptic ionotropic glycine receptors by glycine spillover from postsynaptic MNTB neurons could trigger a small depolarization of presynaptic RMP. This gentle depolarization enhanced the intraterminal Ca2+ concentration and thereby increased the initial release probability (Turecek and Trussell,

2001). The phenomenon of depolarized RMP associated with increased release probability was also observed with blockade of presynaptic KCNQ5 channel, which mediates a K+ current and can be activated even below RMP at the calyx (Huang and Trussell, 2011).

One possible source that serves to the gradual rise of the presynaptic Ca2+ level above the

2+ resting level is an extremely small Ca current generated by P/Q type CaV channels at a subthreshold voltage (Awatramani et al., 2005). Whether other sources are also involved for the regulation of basal Ca2+ level and release probability are not entirely clear.

1.3 Research Model: The Calyx of Held Synapse

To simplify the analysis of the above described three fundamental factors that regulate synaptic strength, the calyx of Held, a giant glutamatergic synapse in the auditory brainstem is an ideal model, because of its unique electrophysiological accessibility, as well as its simple ‘single calyceal input’ synaptic connection (Quastel, 1997; Baydyuk et al., 2016).

The calyx of Held synapse is located in the medial nucleus of the

(MNTB) of auditory brainstem. The globular bushy cell in the ventral projects its single axon to the contralateral MNTB principal neuron to form this giant axosomatic synapse (Borst and Soria van Hoeve, 2012). As an extreme example in the

11 mammalian CNS, the calyx of Held synapse shares basic biophysical properties of fast synaptic transmission with other small CNS synapses (Taschenberger et al., 2002). Hence, taking advantage of its special structure and properties, neuroscientists were able to unveil the mechanisms that contribute to synaptic transmission and short-term plasticity in the past 30 years (Sakaba et al., 2002).

Firstly, the extraordinary size of axosomatic connection allows direct whole-cell patch-clamp recordings on both presynaptic terminal and postsynaptic principal neurons

(Forsythe, 1994). This electrophysiological accessibility provides a possibility to study in detail about each step of the whole process of signal transduction, from measurement of presynaptic APs and calcium currents to postsynaptic corresponding events. More importantly, whole-cell presynaptic recordings allow manipulation of the intraterminal content by dialyzing different patch solutions. The EPSCs recorded in the postsynaptic soma can be relatively easily clamped and have minimal jitter based on somatic innervation, which are appropriate for basic biophysical analysis of neurotransmitter release. Moreover, release of glutamate from calyceal terminal activates both AMPAR and NMDAR on the postsynaptic MNTB neurons. However, the size of NMDAR conductance is strongly downregulated after hearing onset, while AMPARs are more concentrated and mediate larger EPSC during maturation (Borst and Soria van Hoeve, 2012). As a fast-gating glutamate receptor, AMPAR has faster kinetics compared to NMDAR, thus it contributes to the well-timed signal transmission and is more suitable to be used for measurement of transmitter release (Trussell, 1999; Sakaba et al., 2002). Lastly, as early as postnatal day 4

(P4), most of MNTB principal neurons consistently receive only a single large calyceal input (Hoffpauir et al., 2006). These large calyceal terminals harbor hundreds of active

12 zones, and each individual active zone can release multiple SVs simultaneously. Hence, this synaptic structure is specialized for ensuring that each presynaptic AP reliably and precisely drives one postsynaptic AP by generating a large EPSC (Meyer et al., 2001;

Taschenberger et al., 2002).

Based on the simple connection, multiple methods have been used to quantitively estimate the quantal parameters including the total number of readily releasable vesicles

(the size of RRP) and the release probability at the calyx of Held synapse. As mentioned early, both parameters are predominantly regulated on presynaptic side and important for basal transmission and synaptic strength. For RRP, strong stimulation, such as a train of high frequency stimuli (100 Hz, 0.5 s) or 20-30 ms depolarization pulse (from -80 mV to

+10 mV), is able to deplete all the vesicles in the RRP, which can be carried out by

AMPAR-mediated EPSCs or capacitance jump (plasma membrane capacitance change due to vesicle fusion), respectively (Sun and Wu, 2001; Sakaba et al., 2002). Release probability can be further estimated after determining the RRP, as the ratio of single AP- induced response to RRP size (Schneggenburger et al., 1999). Each method has been carefully studied by different research groups and widely appreciated as an accurate estimation of RRP and release probability.

Calyceal terminals undergo rapid ultrastructural changes throughout the postnatal development until they reach the adult-like maturation. The whole developmental transformation is necessary for the refinement of calyx of Held from an immature synapse, which has been extensively utilized as a model system for studying synaptic transmission and short-term plasticity, to a mature synapse that maintains fast and faithful relay of acoustic signal information even at near-1kHz frequencies. The postnatal changes include

13 but are not limited to the changes of morphology, more concentrated and tightly coupled

P/Q-type CaV channels with docked vesicles, narrower AP waveform, increased vesicles number and size, as well as more dominant AMPARs (Borst and Soria van Hoeve, 2012).

Nevertheless, the ultrastructural and functional changes that occur during the postnatal weeks optimize mature synapse to follow prolonged high-frequency firing with high- fidelity, but the basic release machineries for Ca2+-dependent exocytosis remain the same in both immature and mature synapses (Taschenberger et al., 2002).

1.4 Formation of Quanta at Glutamatergic Synapse

During sustained , synaptic vesicles filled with neurotransmitters are released by calcium-dependent exocytosis, and then retrieved by endocytosis and refilled for next round of release (Sudhof, 2004). In the exocytosis-endocytosis cycle, neurotransmitters are simultaneously synthesized in the cytoplasm of axon terminal and then concentrated into the SVs by the activity of specific vesicular neurotransmitter transporters. In the case of glutamate, the glutamine-glutamate cycle has been well established (Edwards, 2007). After release from axon terminal, free glutamate in the synaptic cleft is primarily taken up by nearby through the Na+-dependent excitatory amino acid transporters (EAAT1 or EAAT2) and then converted into glutamine by the activity of enzyme glutamine synthetase. Transfer of glutamine back to axon terminals is sequentially processed by the activities of Na+-dependent system N and A transporters (SN and SA) on the and neuron, respectively. Then, the intraterminal glutamine is converted into glutamate by the enzyme glutaminase and further packaged into synaptic vesicles by vesicular glutamate transporters (VGLUTs).

14 VGLUT, which belongs to the solute carrier 17 (SLC17) gene family, was first successfully cloned as a brain-specific Na+-dependent inorganic phosphate transporter

(BNPI) and further identified as a glutamatergic neuron-specific vesicle-bound transporter for glutamate loading (Bellocchio et al., 2000; Takamori et al., 2000). All known vesicular transporters including VGLUTs transport the cytoplasmic neurotransmitters into SVs with an exchange of intravesicular H+, which is fueled by a H+ electrochemical gradient (H+) across the vesicle membrane. H+ is generated by the vacuolar-type H+-ATPase (V-

ATPase) on the vesicle membrane which pumps H+ into the SV by harnessing the energy released from ATP hydrolysis (Stevens and Forgac, 1997). There are two components of

H+: the chemical gradient (or pH gradient, pH) and inside positive membrane potential

(electrical gradient, ). The transport of different neurotransmitters relies on either one gradient component or both of them (Edwards, 2007). The evaluation of glutamate uptake in the purified rat brain SVs demonstrated that the activity of VGLUTs is driven primarily by the  (Maycox et al., 1988b).

There are three isoforms of VGLUTs in the mammalian brain, and the most conspicuous difference among those three VGLUTs is the regional distribution profile. The two widespread isoforms VGLUT1 and VLGUT2, exhibit highly distinct and complementary expression patterns in the adult brain. VGLUT1 is highly concentrated in the cortex, hippocampus, and cerebellum, whereas VGLUT2 predominates in thalamus, hypothalamus, and brainstem (Fremeau et al., 2001). The expression level of these two major transporters also shows developmental change. For example, in the calyx of Held synapse, VGLUT1 displays a greatly upregulated expression with age, while VGLUT2 remains at the same expression level (Billups, 2005). In the developing mouse cerebellum,

15 there is an age-dependent subtype switch from VGLUT2 to VGLUT1 at parallel fiber-

Purkinje cell synapses (Miyazaki et al., 2003). The minor isoform VGLUT3 was normally detected in neurons that are better known to release other neurotransmitters, such as striatal cholinergic interneurons and even glia (Edwards, 2007). Although these three isoforms are linked with different modes of glutamatergic transmission, they share similar transport characteristics for glutamate uptake.

1.5 Presynaptic Determinants of Quantal Size in Glutamatergic Synapse

As mentioned above, presynaptic regulation on quantal size is also a main contributor to the synaptic strength. Detailed studies in the calyx of Held synapse have investigated different sources of quantal variation and found that vesicular glutamate concentration, rather than vesicle volume, is the major presynaptic source for quantal variation (Wu et al., 2007). Hence, during the formation of quanta on the axon terminal, there are different presynaptic determinants to modulate the final concentration of vesicular glutamate, including but not limited to, the expression level of VGLUTs, the activity of V-

ATPase, the surrounding ions such as Cl-, and other transporters on the SV membrane

(Takamori, 2016).

1.5.1 Expression level of VGLUTs

Since quantal glutamate release depends on glutamate loading into SVs by

VGLUTs, the expression level of VGLUTs is critical to determine vesicular glutamate content. It has been reported in many cases that inactivation/deletion of VGLUTs results in a reduction of quantal size (Fremeau et al., 2004; Wojcik et al., 2004; Wilson et al., 2005;

Moechars et al., 2006). During development, the calyx of Held synapse expresses both

VGLUT1 and VGLUT2 isoforms and undergoes a strong increased expression of

16 VGLUT1, while maintains a constant expression of VGLUT2 (Billups, 2005). This overall increased VGLUTs level is associated with an increased mEPSC amplitude, which reflects an increased vesicular content since postsynaptic AMPARs are not saturated (Yamashita et al., 2003; Yamashita et al., 2009). Strikingly, deletion of VGLUT1 in the calyceal terminals did not cause a significant change of quantal size, probably due to a compensatory effect of VGLUT2 on the same SVs, but VGLUT1-deficient calyces showed slower SV refilling which eventually affected the reliability of synaptic transmission

(Nakakubo et al., 2020).

1.5.2 Cl- gradient

The establishment of either pH or , which are the two components of the H+ electrochemical gradient generated by V-ATPase, requires sustained activity of V-ATPase by inhibiting the formation of another component. For pH, Cl- is always considered as the major anion to dissipate  and keep the activity of V-ATPase.

The first report of VGLUT not only revealed its function as a glutamate/H+ exchanger for glutamate uptake, but also showed a channel-like chloride conductance, which is one of the major permeation pathways of Cl- through SVs (Bellocchio et al., 2000;

Schenck et al., 2009). Cumulative studies in the isolated SVs demonstrated a biphasic dependence on Cl- of VGLUTs. In the absence of extravesicular Cl-, although providing a favorable condition for positive vesicular membrane potential , which is the primarily reliance of the activity of VGLUTs, the efficacy of glutamate loading into SVs was low.

Increasing the Cl- level to ~4 mM greatly enhanced the glutamate uptake, while further increasing the Cl- level would induce a shift from  to pH and then inhibited VGLUT

17 activities (Juge et al., 2006; Martineau et al., 2017). Therefore, Cl- may function as an allosteric activator for VGLUTs and regulate glutamate content in SVs (Juge et al., 2010).

1.5.3 Cation/H+ exchanger

During vesicular glutamate transport, the accumulation of anion glutamate (glu-) in the SVs will acidify the intravesicular environment and dissipate the , which further suppresses the activity of both V-ATPase and VGLUTs. To eliminate the increased pH that is induced by glutamate entry, SVs express coupled cation/H+ exchanger, which can replace the intravesicular H+ with cation. By taking advantage of pH to drive cation uptake, cation/H+ exchanger neutralizes the extra pH and simultaneously generates more

 with cation, providing a favorable environment for glutamate uptake. This cation/H+ exchange mechanism was first demonstrated in glutamatergic SVs by Edwards lab (Goh et al., 2011). They estimated the uptake of 3H-glutamate in isolated SVs in a bath solution containing high concentration of Na+, K+, or NHE impermeable cation choline, and found that both Na+ and K+ stimulated vesicular glutamate uptake. With whole-cell presynaptic recordings at the calyx of Held, substituting the intracellular [K+] or [Na+] by large cation

NMDG+ gradually reduced the mEPSC amplitude, indicating presynaptic cation concentration affected quantal size. Reduced mEPSC amplitude was also observed when

Na+(K+)/H+ exchangers (NHEs) blocker EIPA was applied intracellularly even with high level of Na+ or K+, suggesting an involvement of NHEs (Figure 1.1) (Goh et al., 2011;

Huang and Trussell, 2014).

So far, 9 isoforms have been identified in the mammalian NHE superfamily: NHE1-

5 are expressed on the plasma membrane and only recognized Na+; NHE6-9 are mainly distributed on the intracellular endosomes and the cation selectivity extends to both Na+

18 and K+ (Donowitz et al., 2013). Plasma membrane NHEs transport Na+ into the cytoplasm based on its driving force across the membrane in exchange of H+ efflux, which is important for regulating cytosolic pH, osmotic homeostasis, and cell volume (Orlowski and Grinstein, 2004; Brett et al., 2005). In contrast, the activity of endosomal NHEs is coupled to the pH gradient generated by V-ATPase on the endosomes. Luminal H+ flowing out drives cytoplasmic cation Na+ and K+ to transport into endosome. There are two endosomal subtypes, NHE6 and NHE9, are highly expressed in the brain area. Mutations of NHE6 or NHE9 have been reported that are associated with multiple neurological diseases including autism, attention deficit hyperactivity disorder, Christianson syndrome, and Angelman syndrome (Gilfillan et al., 2008; Ouyang et al., 2013; Kondapalli et al., 2014;

Ullman et al., 2018). Notably, immunostaining analysis in brain slices and western blot on purified SVs indicated the localization of NHE6 on the SVs (Ouyang et al., 2013;

Preobraschenski et al., 2014), and more recent work identified that NHE6, but not NHE9, is responsible for the regulation of glutamate uptake in SVs (Lee et al., 2021b).

19 Figure 1.1

Figure 1.1 Model for Na+/H+ exchanger-dependent facilitation of vesicular glutamate loading

(A) vesicular glutamate transporter (green) primarily utilizes , which is one of the two components of H+ electrochemical gradient generated by V-ATPase (blue), to transport glutamate (glu-) into vesicle but simultaneously increases the other component pH. (B) the vesicular Na+/H+ exchanger (red) promotes the glutamate uptake by replacing the intravesicular H+ with cation Na+ or K+ to convert pH to .

(modified from Goh et al., 2011)

20 1.6 Overview of Thesis

Previous studies have demonstrated that the presynaptic HCN channel, which is permeable to both Na+ and K+ but carries Na+ as the major ion when activated during hyperpolarization, is the main source for maintaining basal Na+ level and regulating vesicle filling through the NHE-dependent pathway (Goh et al., 2011; Huang and Trussell, 2014).

Inhibition of HCN channels at the calyx of Held synapse reduced the basal Na+ level and mEPSC amplitude, whereas activation of HCN channels induced an elevation of cytosolic

Na+ level followed with an increased mEPSC amplitude (Huang and Trussell, 2014). In

+ this thesis, I studied the contribution of other Na -permeable channels, NaV channel and sodium leak channel NALCN, to vesicular glutamate filling and overall synaptic strength via controlling of presynaptic Na+ level. In Chapter 1, I briefly reviewed the fundamental presynaptic factors that determine synaptic strength: the total number of readily releasable vesicles, the release probability, and total amount of neurotransmitter release from a single vesicle. The third factor, which is termed quantal size, is the main focus of this thesis. I further reviewed several presynaptic determinants of quantal size, especially the mechanism of vesicular NHE-dependent facilitation of glutamate uptake, which is the fundamental premise of presynaptic Na+ regulation on quantal size. In Chapter 2, I summarized all the materials and methods I used in my experiments. In Chapter 3, I presented experimental evidence that presynaptic spike induced a large increase of cytosolic Na+ level. This strong elevation of Na+ was important for maintaining reliable synaptic transmission by solely affecting vesicle filling of glutamate, which turned out as an increased quantal size, but had no effect on release probability, readily releasable pool size or presynaptic Ca2+ entry and decay. This chapter was modified from two articles

21 published in The Journal of Neuroscience (Li et al., 2020; Zhu et al., 2020). In Chapter 4,

I studied another Na+-permeable channel called sodium leak channel NALCN. More detailed information about NALCN channel is covered in the introduction. The experimental results revealed a functional expression of NALCN channels on the calyceal terminal, which mediated a small inward current which efficiently regulated presynaptic

RMP, basal cytosolic Na+ level, and quantal size. Strikingly, NALCN channels also exhibited Ca2+ permeability in brain slices. It influenced initial release probability through modulation of basal Ca2+ level. Hence, presynaptic NALCN channels are able to regulate synaptic strength via both modulation of glutamate content and release probability. In

Chapter 5, I summarized the conclusions and their possible application to other systems.

I also discussed future directions regarding the function of presynaptic Na+ and NALCN channels.

22 CHAPTER 2 MATERIALS AND METHODS

2.1 Mouse lines

All animal handling and procedures were approved by the Institutional Animal Care and Use Committee of Tulane University and followed U.S. Public Health Service guidelines. All experiments were performed from mice of either sex aged postnatal day 8-

18. In Chapter 3, C57BL/6J mice were used for all experiments. In Chapter 4, mouse strains include the floxed Nalcn transgenic mice (shared by Dr. Dejian Ren at University of Pennsylvania), Krox20cre (Jackson Laboratory, www.jax.org/strain/025744), and Atoh-

1cre (Jackson Laboratory, www.jax.org/strain/011104).

2.2 Slice Preparation

Coronal brainstem slices containing the MNTB were prepared, being similar to previously described (Zhang and Huang, 2017). Briefly, mice brainstems were dissected and 210 μm (for P8-12 mice) or 190 μm (for P14-18 mice) sections were sliced using a vibratome (VT1200S, Leica) in ice-cold, low-Ca2+, low-Na+ saline containing the following (in mM): 230 sucrose, 10-25 glucose, 2.5 KCl, 3 MgCl2, 0.1 CaCl2, 1.25

NaH2PO4, 25 NaHCO3, 0.4 ascorbic acid, 3 myo-inositol, and 2 Na-pyruvate, bubbled with

95% O2/5% CO2. Slices were immediately incubated at 32°C for 15–20 min and subsequently stored at room temperature in normal artificial cerebrospinal fluid (aCSF) containing the following (in mM): 125 NaCl, 10-25 glucose, 2.5 KCl, 1.8 MgCl2, 1.2

CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 0.4 ascorbic acid, 3 myo-inositol, and 2 Na-pyruvate, pH 7.4 bubbled with 95% O2/5% CO2 before use.

23 2.3 Whole-Cell Recordings

Slices were transferred to a recording chamber and perfused with normal aCSF (2-

3 ml/min) warmed to ~32 °C by an in-line heater (Warner Instruments). Neurons were visualized using an Olympus BX51 microscope with a 40× or 60× water-immersion objective and custom infrared Dodt gradient contrast optics. Whole-cell patch-clamp recordings were performed with a Multiclamp 700B amplifier (Molecular Devices) except for the capacitance measurements. Pipettes pulled from thick-walled borosilicate glass capillaries (WPI) had open tip resistances of 3–5 MΩ and 2–3 MΩ for the pre- and postsynaptic recordings, respectively. Series resistance (4-15 MΩ) was compensated by up to 70% (bandwidth 3 kHz) except mEPSCs recordings. Liquid junction potentials were measured for all solutions, and reported voltages are appropriately adjusted.

In Chapter 3, to record presynaptic action potentials (AP) under different Na+ concentration, pipette solution containing the following (in mM): 60 K-methanesulfonate,

20 KCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Tris3-GTP, 14 Tris2-phosphocreatine, 5 glutamate, as well as 40 NMDG-methanesulfonate (for Na+-free solution), 10 Na- methanesulfonate + 30 NMDG-methanesulfonate (for 10 mM Na+ solution), or 40 Na- methanesulfonate (for 40 mM Na+ solution). Solutions were adjusted to pH 7.3 with KOH

(290 mOsm) and the final K+ for all solutions was around 92 mM. For presynaptic voltage- clamp recordings with different Na+ concentration, pipette solution containing the following (in mM): 70 Cs-methanesulfonate, 20 CsCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP,

0.3 Tris3-GTP, 10 Tris2-phosphocreatine, 5 glutamate, as well as 40 NMDG- methanesulfonate (for Na+-free solution), 10 Na-methanesulfonate + 30 NMDG- methanesulfonate (for 10 mM Na+ solution), or 40 Na-methanesulfonate (for 40 mM Na+

24 solution). Solutions were adjusted to pH 7.3 with CsOH (310-315 mOsm). To isolate presynaptic Ca2+ currents in response to voltage steps, 10 mM TEA-Cl, 2 mM 4-AP and 1

µM tetrodotoxin were added to aCSF, substituting for NaCl with equal osmolarity. For

EPSC recordings, postsynaptic pipette solution containing the following (in mM): 130 Cs- methanesulfonate, 10 CsCl, 10 HEPES, 5 EGTA, 4 Mg-ATP, 0.3 Tris3-GTP, 5 Na2- phosphocreatine, 4 QX-314-Cl (290 mOsm, pH 7.3 with CsOH). 5 μM (R)-CPP, 50 μM picrotoxin, and 1 μM strychnine were added in the aCSF to block NMDA, GABA, and glycine receptors, respectively. 2 mM kynurenic acid and 100 μM cyclothiazide were also applied in the aCSF to block AMPA receptor saturation and desensitization, respectively

(Barnes-Davies and Forsythe, 1995; Huang and Trussell, 2011). For postsynaptic spiking recordings, pipette solution containing the following (in mM): 135 K-gluconate, 10 KCl,

10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Tris3-GTP, 7 Na2-phosphocreatine (290 mOsm, pH

7.3 with KOH). To record the asynchronous release, extracellular Ca2+ was replaced by

Sr2+ (7.5 mM). Presynaptic APs were evoked by afferent fiber stimulation with a bipolar stimulating electrode placed close to the midline of slices.

In Chapter 4, for presynaptic voltage-clamp recordings, pipettes contained the following (in mM): 110 Cs-methanesulfonate, 20 CsCl, 1 MgCl2, 10 HEPES, 4 Mg-ATP,

0.3 Tris3-GTP, 3 Na2-phosphocreatine, and 10 Tris2-phosphocreatine (290 mOsm, pH 7.3 with CsOH). To isolate presynaptic Na+ leak currents, 10 mM TEA-Cl, 2 mM 4-AP were added to aCSF, substituting for NaCl with equal osmolarity to block Kv channels. In

NMDG replacement experiment, 115 mM NaCl was replaced by NMDG-Cl in the aCSF with equal osmolarity, and pH value was carefully adjusted by HCl. For EPSC recordings, postsynaptic pipette solution contained the following (in mM): 130 Cs-methanesulfonate,

25 10 CsCl, 10 HEPES, 5 EGTA, 4 Mg-ATP, 0.3 Tris3-GTP, 5 Na2-phosphocreatine, 4 QX-

314-Cl (290 mOsm, pH 7.3 with CsOH). Recording aCSF contained 10 μM (±)-CPP, 50

μM picrotoxin, 1 μM strychnine, and 2 mM CsCl to block NMDA, GABAA, glycine receptors, and HCN-induced effects, respectively. To record mEPSCs, 0.5 μM tetrodotoxin

(TTX) was added together with previous blockers in the aCSF. To record evoked EPSCs, a bipolar electrode was positioned above the fibers where between the midline and MNTB for afferent fiber stimulation (4 nA, 0.1 ms), and 2 mM kynurenic acid was applied to block

AMPA receptor saturation. 100 μM cyclothiazide was only applied to block side effects when Gd3+-induced effects were monitored (Lei and MacDonald, 2001).

2.4 Perforated Patch-Clamp Recordings

Perforated whole-cell patch-clamp recordings were used during prolonged high- frequency postsynaptic recordings. The pipette solution was similar to that for conventional whole-cell postsynaptic spiking recordings, while gramicidin (final concentration of 60

µg/ml) was added immediately before use. The tip of the recording pipette was first filled with gramicidin-free solution and then back-fill with gramicidin-containing solution. The degree of perforation was monitored after formation of gigaohm seal and recordings were started after overall access resistance dropped to below 30 MΩ.

2.5 Two-Photon Na+ and Ca2+ Imaging

A Galvo multiphoton microscopy system (Scientifica) with a Ti:sapphire pulsed laser (Chameleon Ultra II; Coherent) was used for two-photon Na+ and Ca2+ imaging

(Bender et al., 2010). Whole-cell patch-clamp recordings were performed with a

Multiclamp 700B amplifier and pClamp software (Molecular Devices). The laser was tuned to 800 nm for Na+ imaging and 810 nm for Ca2+ imaging, and epifluorescence signals

26 were captured through 60×, 1.0 NA objectives and a 1.4 NA oil immersion condenser

(Olympus). Fluorescence was split into red and green channels using dichroic mirrors and band-pass filters. Data were collected in frame-scan or line-scan modes using SciScan

(Scientifica). Drug application or presynaptic spikes-induced corresponding Na+ and Ca2+ signals were recorded under two-photon imaging.

For Na+ imaging, pipette solution containing the following (in mM): 110 K- methanesulfonate, 20 KCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Tris3-GTP, 5 Na2- phosphocreatine (290 mOsm; pH 7.3 with KOH), while 1 mM SBFI and 15 μM Alexa 594 were added. Solution was adjusted to pH 7.3 with KOH (290 mOsm). To avoid the perfusion of cytoplasmic Na+ during resting and spike-evoked Na+ signals measurement, pipettes were subsequently detached after whole-cell dialysis with SBFI and Alexa 594 dyes when the fluorescence intensities are stable and Na+ signals were monitored after waiting at least 10 min. Standard calibration methods were used to measure absolute Na+ concentrations (Rose, 2012; Huang and Trussell, 2014). After loading fluorescent dyes through whole-cell recordings, pipettes were detached for resting [Na+] and calibration.

The solutions for in situ calibration of SBFI fluorescence contained (in mM): 20 KCl, 25 glucose, 10 HEPES, 130 (K-gluconate + Na-gluconate) and adjusted to pH 7.4 with KOH.

3 μM gramicidin D, 10 μM monensin, and 50 μM ouabain were added into the calibration solutions before experiments. Calibration data are normalized and fitted by the equation:

+ + (ΔG/R) = (G/R)max × [Na ]i/([Na ]i + Kapp), where G/R is the ratio of green fluorescence relative to red fluorescence; (ΔG/R) is the change in fluorescence ratio measured at a given

+ + [Na ]i divided by that at 0 mM [Na ]i; (G/R)max is the maximal change in fluorescence ratio and Kapp is the apparent Kd of SBFI.

27 For Ca2+ imaging under different Na+ concentrations with spike activities, pipette solution containing the following (in mM): 70 K-methanesulfonate, 20 KCl, 10 HEPES, 4

Mg-ATP, 0.3 Tris3-GTP, 10 Tris2-phosphocreatine, 5 Glutamate, as well as 40 NMDG- methanesulfonate (for Na+-free solution), 10 Na-methanesulfonate + 30 NMDG- methanesulfonate (for 10 mM Na+ solution), or 40 Na-methanesulfonate (for 40 mM Na+ solution). All solutions were adjusted to 290 mOsm, pH 7.3 with KOH. 250 μM Fluo-5F and 20 μM Alexa 594 were added to the pipette solution before the experiments. Data are expressed as Δ(G/R)/(G/R)max × 100%, where (G/R)max was the maximal fluorescence in saturating Ca2+ (Spratt et al., 2019). For Ca2+ imaging with bath application of Gd3+, pipette solution containing the following (in mM): 130 K-gluconate, 20 KCl, 4 MgATP, 0.3 Tris-

GTP, 7 Na2-phosphocreatine, 10 HEPES, and adjusted to 290 mOsm, pH 7.3 with KOH.

100 μM Oregon Green Bapta-1 (OGB-1) and 20 μM Alexa 594 were added to the pipette solution before use. Data are expressed as Δ(G/R)/(G/R)0 × 100%, where (G/R)0 is the baseline fluorescence, and Δ(G/R) is the transient fluorescence response.

2.6 Membrane Capacitance Measurement

Whole-cell patch-clamp recordings were made with an EPC-10 USB double amplifier controlled by Patchmaster software (HEKA) to record presynaptic calcium current and membrane capacitance (Cm) response. The calyx of Held terminals were voltage-clamped at holding potential of −80 mV with a sinusoidal wave (60 mV peak-to- peak amplitude at 1 kHz) superimposed (Sun and Wu, 2001). Tips of patch pipettes were coated with dental wax to reduce capacitance. Series resistance (6-20 MΩ) was electronically compensated by up to 75% (10 μs lag). The calyceal terminals were depolarized from –80 mV to +10 mV for 1 ms and 30 ms to mimic the AP-induced release

28 and to deplete the releasable pool, respectively. Recordings were performed at 32oC. Data were obtained within 20 min after break-in and sampled at 100 kHz. (Performed by Yun

Zhu)

2.7 Immunohistochemistry

P14 wild-type and KO mice were anesthetized with isoflurane and perfused intracardially with 5 ml ice-cold phosphate buffer solution (PBS, pH = 7.4) followed by ice cold 10 ml 4% paraformaldehyde (PFA) in PBS. Brains were then isolated, postfixed in the 4% PFA in PBS overnight, and cryopreserved with 30% sucrose in PBS for 48 h at

4°C. The 45-μm-thick brainstem slices containing MNTB were collected with a cryostat

(Leica) and incubated in blocking solution (1% BSA, and 0.1% Triton X-100 in PBS) for

45 mins at room temperature. The slices were then incubated for 24 hr at 4°C with primary antibodies against NALCN (polyclonal rabbit, 1:75/100, Alomone labs) and vGlut1

(polyclonal guinea pig, 1:1000, Synaptic systems) in blocking solution. Slices were then rinsed with PBS (3 x 5min) and incubated with secondary antibodies (Alexa 488 goat anti- guinea pig IgG, Alexa 647 donkey anti-rabbit IgG, 1:1000, Invitrogen) for 2 hr at room temperature. Finally, Slices were rinsed in PBS (3 x 5min), mounted on gel-coated slides, and coverslipped with DAPI fluoroshield mounting medium (Sigma) to be ready to use.

Images were captured using confocal microscope (Nikon A1) and analyzed in Nikon

Analysis software and ImageJ (NIH).

2.8 Drugs

Drugs were obtained from Tocris (H-89), Alomone Labs (tetrodotoxin, CPP, cyclothiazide), Invitrogen (SBFI, Fluo-5F, Oregon Green Bapta-1, and Alexa Fluor 594), and all others from Sigma-Aldrich.

29 2.9 Analysis

Analysis Data were analyzed using Clampfit (Molecular Devices), Patchmaster

(HEKA), Igor (WaveMetrics), Prism 6 (GraphPad) and ImageJ (NIH). Data are expressed as mean ± SEM. Statistical significance was established using paired or unpaired t tests, and ANOVA as noted, with p < 0.05 indicating a significant difference.

30 CHAPTER 3: SPIKE ACTIVITY REGULATES VESICLE FILLING

AT A GLUTAMATERGIC SYNAPSE

3.1 Abstract

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na+ substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking.

The activity-induced elevation of cytosolic Na+ activated vesicular Na+/H+ exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronously released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na+ increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na+/H+ exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na+ accumulated in the terminals, activated vesicular Na+/H+ exchanger, and regulated glutamate loading as a function of the level of vesicle release.

31 3.2 Introduction

High-frequency firing neurons are widely distributed throughout the central nervous system, including the brainstem, cerebellum, thalamus, hippocampus, and neocortex (McCormick et al., 1985; Chen and Regehr, 1999; Rudy and McBain, 2001;

Taschenberger et al., 2002; Hu and Jonas, 2014). Accordingly, synaptic vesicles need to be recycled and refilled rapidly to support the high-frequency synaptic signaling (Rizzoli and Betz, 2005; Edwards, 2007; Farsi et al., 2017). Accumulating evidence has uncovered the mechanisms of vesicle fusion and recycling (Heuser and Reese, 1973; Klingauf et al.,

1998; Wang and Kaczmarek, 1998; Sudhof, 2004); however, the control of the contents of synaptic vesicles has received considerably less attention (Balmer and Trussell, 2016).

Recent studies showed that synaptic vesicles expressing Na+/H+ monovalent cation exchanger (NHE) activity can convert the pH gradient into an electrical potential required by the vesicular glutamate transporter (Goh et al., 2011; Preobraschenski et al., 2014). Na+ flux through HCN channels enhances presynaptic Na+ concentration and thus promotes synaptic vesicle filling with glutamate (Huang and Trussell, 2014). However, how spike activity controls the presynaptic Na+ dynamics and how accumulated Na+ modulates synaptic transmission are unknown. Using the mouse calyx of Held, a giant glutamatergic synapse in the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem that permits direct pre- and postsynaptic recordings and manipulation of the presynaptic cytosolic environment, we showed that glutamate loading is facilitated by intracellular Na+ over the physiological concentration range. During high-frequency signaling, when large amounts of glutamate are released, Na+ accumulates in terminals, activates NHE, facilitates glutamate uptake into synaptic vesicles, thus accelerating vesicle refilling and sustaining

32 reliable synaptic transmission. Therefore, presynaptic cytosolic Na+ works as a signaling ion to coordinate glutamate loading as a function of the level of vesicle release.

3.3 Results

3.3.1 Spikes control presynaptic cytosolic Na+ concentration

Changes in cytosolic Na+ during presynaptic firing were assayed using Na+ imaging with two-photon laser scanning microscopy. Immature calyceal terminals from P9-11 mice were loaded via whole-cell recordings with the Na+ indicator SBFI (1 mM) and the volume marker Alexa 594 (15 μM). Standard calibration methods (Rose, 2012; Huang and Trussell,

2014) were used to measure the absolute [Na+] (Figure 3.1A). The presynaptic [Na+] at the resting state was 15.8 ± 2.1 mM (n = 4), which is similar to previous estimations in neuron and non-neuron cells (Huang and Trussell, 2014; Meyer et al., 2019). APs were evoked by afferent fiber stimulation and propagated to the presynaptic terminals. Upon 100 Hz stimulation for 1 s, presynaptic cytosolic [Na+] increased by 5.7 ± 1.1 mM, which decayed to the control level with a time constant of 12.2 ± 1.0 s (n = 5; Figure 3.1B). After ensuring reliable AP evocation by afferent fiber stimulation, the recording pipettes were subsequently detached from the calyces, and the Na+ signals upon stimulations at different frequencies were measured. Spiking at 10 Hz for 60 s reversibly increased the [Na+] by

12.2 ± 1.7 mM (n = 7; Figure 3.1C). Na+ transients were gradually augmented as increasing spike frequency: 20 Hz for 30 s increased the [Na+] by 16.3 ± 2.0 mM (n = 8; Figure 3.1D) and 100 Hz for 20 s increased the [Na+] by 55.6 ± 5.9 mM (n = 8; Figure 3.1E). Therefore, spike activities efficiently increase the presynaptic cytosolic Na+ concentration in an activity-dependent manner (Figure 3.1F).

33 + Na flows into the cytosol through NaV channels during the firing of AP, while the location of NaV channels in the calyx of Held is still under debate (Leao et al., 2005; Huang and Trussell, 2008; Kim et al., 2010; Sierksma and Borst, 2017). To test the whether the global [Na+] increase at the calyceal terminal is strongly based on the diffusion from axon or is comparable at both axon and terminal, calyces with a complete heminode were used to monitor the Na+ changes at different locations under line-scans and frame-scans (Figure

3.2A). Upon 10 s stimulation at 100 Hz, Na+ accumulated into the whole calyx terminal

(Figure 3.2B-C). The Na+ rise and decay kinetics at axon heminode and calyceal terminal overlapped, while Na+ increase at the calyceal terminal (37.4 ± 4.8 mM) was slightly smaller than that of the axon heminode (42.7 ± 4.0 mM) (P = 0.009, n = 6). These experiments were done in prehearing (P8-12) calyces at 32 oC. Previous studies showed that temperature influences AP waveform and thus the Na+ influx (Kushmerick et al., 2006) and Na+ extrusion through Na+/K+-ATPase (Kim et al., 2007). We then repeated the Na+ imaging experiment in calyces of hearing mice (P13-16) at 35-37oC (Figure 3.2E-G).

Similar results were obtained. The Na+ increased by 32.2 ± 5.6 mM at the calyceal terminal and 39.2 ± 6.8 mM at the heminode (P = 0.04, n = 5, Figure 3.2G). Therefore, spike activity substantially increases the cytosolic Na+ concentration in both axon heminode and presynaptic terminal of both prehearing and hearing mice at physiological temperatures.

34 Figure 3.1

Figure 3.1 Presynaptic spikes control cytosolic Na+ concentration

(A) In situ calibration of SBFI fluorescence under two-photon microscopy. Upper: Single optical section of a calyx of Held terminal filled with SBFI (green) and Alexa 594 (red) through recording pipette. Pipette was subsequently detached for resting [Na+]

+ + measurement and [Na ] calibration. Lower: Change in fluorescence with [Na ]i (n = 4).

The fitted curve yielded a Kapp of 25.7 mM and (G/R)max of –0.86 for SBFI (see Materials

+ and Methods). The resting [Na ]i was shown as the insert. (B) The same calyx of Held in

(A) filled with SBFI and Alexa 594 under whole-cell recording. Spikes were evoked at 100

35 Hz for 1 s by afferent fiber stimulation. An increase in [Na+] was visualized under line- scan and frame-scan modes. (C-E) After detaching recording pipettes from calyces, Na+ signals in response to increasing spike frequencies were measured. Spikes at 10 Hz (C), 20

Hz (D), and 100 Hz (E) induced different changes in intracellular Na+ concentration. (F)

Summary results of presynaptic Na+ increases under different stimulations. Error bars, ±

S.E.M.

36 Figure 3.2

Figure 3.2 Presynaptic spikes robustly increase cytosolic Na+ at heminode and terminal in both immature and mature calyces

(A) Maximum intensity montage of a P11 calyx of Held recorded at 32oC with SBFI and

Alexa 594 using two-photon microscopy. (B) Spikes were evoked at 100 Hz for 10 s by afferent fiber stimulation. (C) Corresponding Na+ increase was detected at both the preterminal axon heminode (red) and calyceal terminal (blue). (D) Summary plot of cytosolic Na+ increase at the axon and terminal in P8-12 calyces at 32 oC. (E-F) Similar to

A and C, while a P14 calyx was recorded at 37oC. (G) Summary plot of cytosolic Na+ increase at the axon and terminal in P13-16 calyces at 35-37oC. Statistical significance was assessed using a two-tailed, paired Student t-test. *P < 0.05, **P < 0.01. Error bars, ±

S.E.M.

37 3.3.2 Presynaptic Na+ regulates glutamatergic transmission

A previous study showed that presynaptic cytosolic Na+ promotes vesicular glutamate uptake and increases mEPSC amplitude (Huang and Trussell, 2014). Here we performed simultaneous pre- and postsynaptic whole-cell recordings to examine how presynaptic Na+ influences AP-driven synaptic transmission. We dialyzed the cytosolic contents of presynaptic calyces and simultaneously measured AMPA receptor-mediated

EPSCs in MNTB principal neurons in the whole-cell mode. The calyces were recorded with pipette solutions containing 0 mM, 10 mM, or 40 mM Na+. 5 mM glutamate was added in the presynaptic solutions to maintain cytosolic glutamate (Ishikawa et al., 2002;

Huang and Trussell, 2014). Pairs of presynaptic APs (10 ms intervals) were evoked every

15-20 s by afferent fiber stimulation and the resulting EPSCs were continuously recorded

(Figure 3.3A-B). Immediately after presynaptic break-in to whole-cell mode, no difference was detected in EPSC amplitude among different Na+ concentrations (P = 0.49, one-way

ANOVA). With the 10 mM Na+ solution presynaptically, the EPSC amplitude remained unchanged over 20 min of recording (104 ± 2%; P = 0.16, n = 6). When the calyceal terminals were manipulated with Na+-free pipette solution, the EPSC amplitude gradually declined by 23 ± 4% over the 20 min period (P = 0.003, n = 5), whereas increasing the presynaptic [Na+] to 40 mM induced a 39 ± 8% increase in the EPSC amplitude (P = 0.002, n = 8) (Figure 3.3B-C). When normalized to the values observed immediately after break- in, the EPSC amplitude was clearly smaller in the Na+-free solution than in 10 mM Na+ (P

= 0.0001) and higher in 40 mM Na+ compared to 10 mM Na+ (P = 0.003, unpaired t-test;

Figure 3.3C). The presynaptic AP amplitudes remained stable over the recording period in all conditions (Figure 3.3A), presumably because Na+ dialysis is much faster than its

38 function on vesicular glutamate transport (Goh et al., 2011; Xue et al., 2013). Since the change in Na+ is restricted to the nerve terminal and does not affect the postsynaptic neuron, these data indicate that the changes in EPSC amplitude reflected an alteration in presynaptic glutamate release. No apparent change of paired-pulse ratio (Figure 3.3D) was observed in each group, suggesting that the change in EPSC amplitude is unlikely caused by the change from release probability.

39 Figure 3.3

Figure 3.3 Presynaptic Na+ regulates the EPSC amplitude

(A) Pairs of presynaptic APs induced by afferent fiber stimulation when 0 mM (left), 10 mM (middle), or 40 mM Na+ (right) was present in the presynaptic pipette solution. Traces recorded within 2 min of break-in (black) and after 15 min (red) of recording are

40 superimposed. (B) Postsynaptic currents in response to the presynaptic APs from (A). (C)

Left: time course of the normalized amplitudes of the first EPSC during paired-pulse recordings. Each point represents the average of 1 min recordings. Right: Relative amplitudes of the first EPSC after 15 min dialysis of the calyces with different Na+ concentrations. Amplitudes were normalized to the amplitudes measured within 2 min of break-in. (D) Presynaptic Na+ does not affect the paired-pulse ratio. Bar graphs of paired- pulse ratios measured within 2 min of break-in and after 15 min of recording were compared with presynaptic [Na+] of 0 mM (P = 0.12), 10 mM (P = 0.20), and 40 mM (P =

0.49). Statistical significance was assessed using a two-tailed, paired Student t-test. **P <

0.01, ***P < 0.001. Error bars, ± S.E.M.

41 3.3.3 Na+-dependent regulation of synaptic transmission is not due to change in AP waveform or Ca2+ influx

Intracellular Na+ concentration influences the Na+ driving force, and further affects

AP waveform, Ca2+ influx and fusion of synaptic vesicles. We then used 1 ms depolarizing pulses from –80 mV to +10 mV to mimic the presynaptic AP and trigger glutamate release

(Figure 3.4). No significant differences were detected in Ca2+ currents either among groups

(P = 0.89, one-way ANOVA) or within the recording period in all conditions (P > 0.46, two-way ANOVA; Figure 3.4A). While the EPSC amplitude remained relatively stable over 20 min of recording when the presynaptic solution contained 10 mM Na+ (95 ± 2%;

P = 0.06, n = 5), the EPSC amplitude gradually declined by 20 ± 3% (P = 0.002, n = 5) with the Na+-free solution and increased by 32 ± 4% with 40 mM Na+ (P = 0.0002, n = 5;

Figure 3.4B-C). No change in the paired-pulse ratio (Figure 3.4D) was observed in any group. This result was similar to that of the AP-triggered release, confirming that the presynaptic Na+-induced change in EPSC amplitude is not due to changes in AP waveform.

To directly test whether cytosolic Na+ affects Ca2+ influx and decay during spiking activity, we made two-photon Ca2+ imaging with Fluo-5F loaded into the presynaptic terminal (Figure 3.5). Calyces were recorded with pipette solutions containing 0, 10 or 40 mM Na+. A burst of 10 APs evoked a rapid Ca2+ rise of similar concentrations at all levels

+ of Na tested, as indicating by the fluorescence increases of 29.3 ± 0.9% of (G/R)max in

Na+-free( n = 8), 30.3 ± 1.5% in 10 mM Na+ (n = 7), and 31.3 ± 1.6% in 40 mM Na+ (n =

6) pipette solutions (Figure 3.5C). The Ca2+ signals decayed to the background level within seconds and were not different among groups. The Ca2+ decay time constants were 0.56 ±

0.06 s for 0 mM Na+, 0.60 ± 0.07 s for 10 mM Na+, and 0.61 ± 0.05 s for 40 mM Na+

42 solutions (P = 0.80, ANOVA test; Figure 3.5D). Thus, presynaptic Na+ does not directly affect Ca2+ influx or decay.

Figure 3.4

43 Figure 3.4 Presynaptic Na+ regulates EPSC amplitude in response to presynaptic depolarization

(A-B) Presynaptic calcium currents (A) and corresponding postsynaptic responses (B) induced by pairs of 1 ms presynaptic depolarizations from –80 mV to +10 mV when 0 mM

(left), 10 mM (middle), or 40 mM (right) Na+ was present in the presynaptic pipette solutions. Recordings made within 2 min of break-in (black) and after 15 min (red) are superimposed. (C) Left: time course of the changes in EPSC amplitude. Each point represents the average of 1 min recordings. Right: Relative amplitudes of the first EPSC at

15-20 min of recordings with different presynaptic Na+ concentrations. Amplitudes were normalized to the amplitudes measured within 2 min of break-in. (D) Presynaptic Na+ does not affect the paired-pulse ratio in response to presynaptic depolarization. Bar graphs of paired-pulse ratios measured within 2 min of break-in and after 15 min of recording were compared with presynaptic [Na+] of 0 mM (P = 0.95), 10 mM (P = 0.69), and 40 mM (P =

0.25). Statistical significance was assessed using a two-tailed, paired Student t-test. **P <

0.01, ***P < 0.001. Error bars, ± S.E.M.

44 Figure 3.5

Figure 3.5 Presynaptic Na+ does not affect spike-evoked Ca2+ rise and decay

(A) Single optic section of the calyx with attached patch pipette. (B) Presynaptic Ca2+ transients induced by 10 spikes at 100 Hz when dialyzed with pipette solution containing

0mM, 10 mM, or 40 mM Na+. (C-D) Summary plots of relative Ca2+ rise and decay time with different intracellular Na+ concentrations. Error bars, ±SEM.

45 3.3.4 Presynaptic Na+ does not affect the readily releasable vesicle pool or release probability

We next studied the mechanisms of presynaptic Na+-dependent regulation of glutamatergic synaptic transmission. Measurement of membrane capacitance (Cm) allows direct detection of exocytosis of synaptic vesicles at the calyx of Held with high temporal resolution (Sun and Wu, 2001). Typically, a 1-ms depolarization from –80 mV to +10 mV

2+ can elicit a Ca influx and capacitance change (∆Cm) is equivalent to a single AP, while a

30-ms step depolarization is sufficient to deplete the whole readily releasable pool at calyceal terminals (Wu and Borst, 1999; Fedchyshyn and Wang, 2005). We found the ∆Cm evoked by 1-ms or 30-ms step depolarization showed no difference across the different

[Na+] groups (P = 0.63 and 0.81, respectively, ANOVA test; Figure 3.6). Release probability, defined as the ratio of single AP-evoked ∆Cm by the readily releasable pool, was not different among these groups (P = 0.39). These data suggest that the presynaptic

[Na+] does not affect the presynaptic readily releasable pool size or the release probability.

(The capacitance measurement experiments were performed by Yun Zhu)

46 Figure 3.6

Figure 3.6 Presynaptic Na+ does not affect the readily releasable pool size or release probability

2+ (A-B) Sampled presynaptic Ca currents (ICa) (A) and Cm responses (B) induced by a 1- ms (black) or 30-ms (red) depolarizations from –80 mV to +10 mV with presynaptic pipette solutions containing 0 mM (left), 10 mM (middle), or 40 mM (right) Na+. The corresponding membrane conductance (Gm) and series conductance (Gilfillan et al.) are shown to confirm the recording stability. (C) Group data show that the presynaptic [Na+] does not affect the Cm responses or release probability. Error bars, ± SEM.

47 3.3.5 Presynaptic Na+ controls vesicular glutamate content

Previous study has demonstrated that presynaptic Na+ regulated vesicular glutamate content through NHE-dependent mechanism at resting status (Huang and Trussell, 2014).

However, recent studies showed that synaptic vesicles undergoing spontaneous and evoked fusion might derive from different pools (Fredj and Burrone, 2009; Chanaday and Kavalali,

2018). We recorded the amplitude of asynchronous EPSCs (aEPSCs) in response to single

AP, since evoked synchronous release and asynchronous release share the same set of vesicles (Kaeser and Regehr, 2014). Single AP-triggered aEPSCs were induced by replacing extracellular Ca2+ with Sr2+ and recorded from the postsynaptic MNTB neurons

(Figure 3.7). Immediately after presynaptic break-in to whole-cell mode (within 2 min), the aEPSCs showed no significant difference in amplitude in any of the groups (P = 0.20, one-way ANOVA test). Dialyzed with 10 mM Na+ pipette solution, the aEPSC amplitude remained stable for over 10 min (98 ± 2%; P = 0.50, n = 5). The aEPSC amplitude was significantly reduced by 15 ± 2% (P = 0.003; n = 5) with 0 mM Na+ and increased by 17 ±

2% (P = 0.0005; n = 6) with 40 mM Na+ presynaptically. We also tested how 8-Br-cAMP, a cell membrane-permeant cAMP analog that activates HCN channels and increases presynaptic Na+ levels (Huang and Trussell, 2014), affects the aEPSC amplitude. To avoid the possible PKA phosphorylation-induced change of AMPA receptor function (Esteban et al., 2003), 10 µM H-89 was added into the postsynaptic pipette solution to inhibit kinase activation. Bath application of 8-Br-cAMP increased the aEPSC amplitude by 19 ± 2% (P

= 0.0006; n = 6). The difference in aEPSC amplitude suggested that alteration of the presynaptic Na+ level affected vesicular glutamate loading for both spontaneous and evoked release.

48 Figure 3.7

Figure 3.7 Presynaptic Na+ regulates aEPSC amplitude

(A) Example trace of prolonged period of aEPSCs following an initial evoked EPSC in response to a single stimulation. The insert shows an expanded trace of the asynchronous release (blue). (B) Left, example traces of asynchronous events within 2 min (black) or >

10 min (red) after presynaptic break-in with a 0 mM, 10 mM, or 40 mM Na+ patch pipette solution. The bottom trace shows the recordings before and 10 min after application of 8-

Br-cAMP; 10 µM H-89 was present in the postsynaptic pipette solution to inhibit 8-Br- cAMP induced kinase activation. (C) Bar graphs of the change in aEPSC amplitude in the

0 mM (P = 0.003, n = 5), 10 mM (P = 0.50, n = 5), 40 mM Na+ (P = 0.0005, n = 6), or 8-

Br-cAMP (P = 0.0006, n = 6) groups. Statistical significance was assessed using a two- tailed paired Student t test. **P < 0.01; ***P < 0.001. Error bars, ± S.E.M.

49 3.3.6 Presynaptic Na+ is required for reliable signal transmission

High-frequency signals of each globular bushy cell reliably transmit to a target

MNTB principal neuron through the calyx of Held synapse, with few or no failures (Mc

Laughlin et al., 2008; Lorteije et al., 2009). We next asked whether the cytosolic Na+- dependent modulation of EPSCs affects the reliability of signal transmission from the presynaptic calyx of Held to the postsynaptic MNTB neuron. Paired pre- and postsynaptic action potentials were recorded in response to calyceal fiber stimulation. Fiber stimuli at

200 Hz reliably evoked APs in the presynaptic terminals during the whole-cell recordings in 0, 10, and 40 mM Na+ conditions (Figure 3.8A). Immediately after presynaptic break-in to whole-cell mode, most of the triggered presynaptic spikes correlated with postsynaptic spikes in the MNTB neurons, with only a few failures in the late phase of the stimulation

(Figure 3.8B). When the calyceal terminals were dialyzed with a 10 mM Na+ solution, no significant difference in the reliability of postsynaptic AP was observed over recording durations of >10 min (P = 0.46, n = 8, Figure 3.8B-E). The probability of failure increased gradually with time when the presynaptic solution was Na+-free. After 10 min of presynaptic dialysis, the reliability was greatly reduced, and presynaptic release in the late part of the stimulus train was unable to drive postsynaptic APs (P = 0.01, n = 6, Figure

3.8B-E). In contrast, when the calyceal terminal was dialyzed with a 40 mM Na+ solution, presynaptic spikes transmitted to the postsynaptic neuron showed less failures and higher firing probability after repeated stimuli trains, indicating that higher presynaptic Na+ level enhanced reliable synaptic transmission at the calyx of Held synapse (P = 0.004, n = 8,

Figure 3.8B-E). It was notable that, for those events where the MNTB principal neuron failed to fire an AP, a subthreshold EPSP was observed, indicating that the presynaptic

50 spikes caused reliable release, but the synaptic current was not big enough to trigger a postsynaptic spike. Thus, the presynaptic Na+ level facilitates vesicular glutamate uptake and eventually boosts reliable synaptic transmission.

51 Figure 3.8

Figure 3.8 Presynaptic Na+ level contributes to reliable synaptic transmission

(A) Example traces of 50 presynaptic APs immediately (black) and 10 min (red) after break-in with 0 mM, 10 mM, and 40 mM Na+ in the presynaptic pipette solution. APs were

52 evoked by 200 Hz afferent fiber stimulation. (B) Postsynaptic spiking in MNTB principal neurons in response to the presynaptic firing in (A). (C) Raster plots of spikes evoked by

200 Hz, 250 ms stimulus trains repeated with 60 s intervals. Presynaptic spikes are shown at the top in red. (D) Summary plots of average postsynaptic firing probability in response to 50 presynaptic action potentials at 200 Hz. (E) Overall change in the postsynaptic firing probability between the first 2 min and after 10 min, with 0 mM, 10 mM, or 40 mM presynaptic Na+. Statistical significance was assessed using a two-tailed paired Student t test. *P < 0.05, ** P < 0.01. Error bars, ± SEM.

53 3.3.7 NHE activity promotes synaptic transmission and signaling reliability

Synaptic vesicles express Na+/H+ monovalent cation exchanger (NHE) that converts the pH gradient into an electrical potential required by the vesicular glutamate transporter and promotes synaptic vesicle filling with glutamate (Goh et al., 2011; Huang and Trussell, 2014; Preobraschenski et al., 2014). Since presynaptic spikes substantially increase the presynaptic Na+ level, we asked if NHE activity is required in maintaining the highly reliable synaptic transmission. Under perforated patch-clamp recordings, we were able to record EPSCs and postsynaptic APs over a long duration (>30 min). Prolonged presynaptic fiber stimulation evoked reliable and stable EPSCs. Incubating EIPA (100 µM), an NHE specific blocker, reduced the EPSC amplitude to 54.8 ± 6.8% (P = 0.003, n = 5,

Figure 3.9A, C). Meanwhile, the postsynaptic firing started to fail after 6 min incubation of EIPA; the overall firing probability reduced to 21.2 ± 8.6% (P = 0.003, n = 4, Figure

3.9B, D). A subthreshold EPSP was always observed when the MNTB neuron failed to fire an AP, indicating the presynaptic spikes invaded into the terminals while the glutamate contents were not enough to trigger a postsynaptic spike. Decrease of both EPSC amplitude and firing probability confirms Na+ and NHE activity is required in maintaining reliable synaptic signaling at high frequency.

54 Figure 3.9

Figure 3.9 NHE activity is required for reliable synaptic signaling

(A) Example traces of EPSC recordings before and 10 min after incubation of 100 µM

EIPA upon 20 Hz presynaptic stimulation. (B) Summary graph of EPSC amplitude with

EIPA application. (C) Example traces of postsynaptic APs recordings before and 10 min after applying EIPA. (D) Summary graph shows that EIPA reduced reliability of postsynaptic firing. Statistical significance was assessed using a two-tailed paired Student t test. ** P < 0.01. Error bars, ± SEM.

55 3.4 Discussion

In this study, we found a substantial accumulation of Na+ in the presynaptic terminal during high-frequency signaling. The presynaptic Na+ facilitated glutamate uptake into synaptic vesicles without changing the readily releasable pool size or release probability. Our results reveal a mechanism by which AP-driven Na+ influx controls the strength of synaptic transmission by modulating vesicular content. During high-frequency synaptic signaling, when large amounts of glutamate are released, Na+ accelerates vesicle replenishment and sustains synaptic transmission, representing a novel cellular mechanism that supports reliable synaptic transmission at high-frequency in the central nervous system.

Glutamate is the principal excitatory neurotransmitter in the brain and is involved in most aspects of brain function (Mayer and Armstrong, 2004). The concentration of glutamate into synaptic vesicles involves plasma membrane excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs). EAATs are high- affinity, Na+-coupled transporters that recycle glutamate and glutamine from the extracellular space to the cytoplasm; the function of these transporters has been extensively studied (Amara and Fontana, 2002). VGLUTs transport glutamate from the cytoplasm into synaptic vesicles for subsequent release by exocytosis, but we still know very little about the basic mechanisms that regulate vesicular glutamate transport, in part because classical biochemical approaches have limitations in revealing the complex ionic basis for the loading of neurotransmitter into vesicles while electrophysiological methods are hard to apply to the study of synaptic organelles (Balmer and Trussell, 2016). Glutamate receptors are not saturated by synaptically released glutamate (Ishikawa et al., 2002; Conti and

Lisman, 2003; Sargent et al., 2005); therefore, changes in the amount of glutamate released

56 per synaptic vesicle have the potential to control synaptic strength. Fusion of a single vesicle induces a quantal response, and the size of the quantum varies at most synapses.

The variation of mEPSC amplitude (quantal size) is determined by vesicular glutamate concentration rather than vesicle volume (Wu et al., 2007), indicating a crucial role of vesicular glutamate uptake in determining synaptic strength. Vesicular transporters drive neurotransmitter accumulation using the energy of the proton electrochemical gradient

+ (ΔμH+) produced by the vacuolar H -ATPase (V-ATPase) (Edwards, 2007). The ΔμH+ reflects both electrical potential (Δψ) and chemical concentration gradient (ΔpH). Previous studies demonstrated that glutamate uptake into synaptic vesicles by the VGLUTs is dependent mostly on Δψ rather than ΔpH (Maycox et al., 1988a; Tabb et al., 1992).

However, glutamate entry acidifies synaptic vesicles and reduces the capacity of V-ATPase to create the Δψ required for VGLUT activity, thereby stalling the uploading of glutamate.

Recent work showed that synaptic vesicles express a Na+(K+)/H+ monovalent cation exchanger (NHE) activity that converts ΔpH into Δψ and promotes synaptic vesicle filling with glutamate. Manipulating presynaptic [K+] changed the mEPSC amplitude even when the glutamate supply was constant, indicating that the synaptic vesicle NHE regulates glutamate release and synaptic transmission (Goh et al., 2011). Another study showed that

K+ facilitates glutamate transport by directly acting on VGLUT binding sites and confirmed that the NHE is responsible for Na+ effects on vesicular glutamate uptake

(Preobraschenski et al., 2014). Indeed, presynaptic Na+ is more potent than K+ in facilitating glutamate uptake and a small change in presynaptic [Na+] affects the mEPSC amplitude even in the presence of normal [K+]. Na+ influx through plasma membrane HCN channels affects presynaptic Na+ concentration, regulates glutamate uptake, and thus

57 controls mEPSC amplitude (Huang and Trussell, 2014). Here we found that Na+ facilitated synaptic transmission by facilitating glutamate uptake into synaptic vesicles without changing the readily releasable pool size or release probability. Recent studies suggested the isoform NHE6, which has been found in both GABAergic and glutamatergic synaptic vesicles with spectrometry experiments (Gronborg et al., 2010) and in purified SVs with western blotting (Preobraschenski et al., 2014), is the candidate for Na+-dependent presynaptic modulation of quantal size . At rest, both knockout of NHE6 and knockdown of its synaptic localization protein secretory carrier membrane protein 5 (SCAMP5) resulted in a hyperacidification of SVs and reduction of quantal size (Lee et al., 2021b).

During prolonged presynaptic firing, such as chemical long-term potentiation in cultured hippocampal neurons, NHE6 recruitment by SCAMP5 was strongly enhanced in both existing and newly formed boutons (Lee et al., 2021a). Hence, presynaptic Na+ facilitated vesicular glutamate refilling via NHE6-dependent pathway play a critical role in modulating synaptic efficacy and maintaining reliable synaptic transmission.

NaV channels are expressed in hippocampal mossy fiber boutons (Engel and Jonas,

2005), while the location of NaV channels in the calyx of Held is more complicated (Leao et al., 2005; Ford et al., 2015; Berret et al., 2016; Sierksma and Borst, 2017; Xu et al.,

2017). The morphology of the calyceal terminals and the expression of NaV channels undergo dramatic development change around hearing onset. NaV channel is expressed on the calyceal terminals of prehearing rodents (Sierksma and Borst, 2017; Xu et al., 2017) and the Na+ currents recorded in calyces with incised axon show comparable amplitude

(Huang and Trussell, 2008). In more mature animals, NaV channel is more concentrated at the preterminal heminode region (Ford et al., 2015; Berret et al., 2016; Xu et al., 2017)

58 although lower expression was detected in the calyces of hearing rats (Sierksma and Borst,

2017). Consistently, about 30% of the outside-out patches of P8-10 calyx have Na current

(Leao et al., 2005) and it decreases to about 10% in P13-17 calyx of rat (Berret et al., 2016).

We showed here that spike activity efficiently and substantially increased the cytosolic Na+ concentration in both axon heminode and presynaptic terminal, the overall [Na+] reached to 50 mM after 10 s of 100 Hz spiking (Figure 3.1). Our result is comparable to a previous study (Berret et al., 2016). The Na+ increase was slightly lower in hearing calyces at 35-

37oC than in prehearing calyces at 32oC, presumably due to a developmental change of

+ + NaV channel expression and temperature-dependence of AP waveform and Na /K -

ATPase activity (Taschenberger and von Gersdorff, 2000; Kushmerick et al., 2006; Kim et al., 2007; Xu et al., 2017). Moreover, the Na+ rise and decay kinetics at axon heminode and calyceal terminal overlapped, while Na+ increase at the calyceal terminal was slightly

(12%–18%) smaller than that of the axon heminode, presumably due to NaV channel distribution. Although HCN is prominent in controlling the resting Na+ concentration, spikes are much more potent in regulating presynaptic Na+ accumulation than HCN channels during activity. Blocking HCN channels, which took over 10 min to reach an equilibrated concentration, reduced the resting Na+ concentration by about 5 mM (Huang and Trussell, 2014). The calyx Na+ increase upon 10 s of 100 Hz firing (Figure 3.1) is 5 times larger than the overall contribution of HCN channel at resting membrane potentials.

Since HCN channels are deactivated by depolarizations during spiking, their contribution to the overall presynaptic cytosolic Na+ would be even smaller (Huang and Trussell, 2014).

The calyx recorded in brain slices does not fire spontaneously, however, it fires in vivo at frequencies of 71 ± 11 Hz in the absence of sound and up to 352 ± 34 Hz with 80 dB tones

59 (Lorteije et al., 2009). Therefore, the presynaptic [Na+] would be substantially higher in vivo than that of the slice preparations.

Globular bushy cells fire APs reliably and precisely synchronize to sound. High- frequency signals of globular bushy cells are reliably transmitted to the target MNTB neurons through the calyx of Held synapse (von Gersdorff and Borst, 2002). Several cellular mechanisms have been established that are important to support neurotransmission at such high rates, including presynaptic ion channels that enable reliable presynaptic spike waveform and calcium influx; a large readily releasable pool, many release sites, and low release probability that enhance the release reliability; and fast kinetics of postsynaptic

AMPA-type glutamate receptors that allow fast and faithful transmission to the postsynaptic MNTB neurons (Taschenberger and von Gersdorff, 2000; Taschenberger et al., 2002; Borst and Soria van Hoeve, 2012; Xue et al., 2013). We found that the cytosolic

Na+-dependent facilitation of vesicular glutamate uptake contributes to the reliability of synaptic transmission at the calyceal synapse. The reliability of AP propagation from calyx to MNTB dropped to about 50% after a few spikes at 200Hz (Figure 3.8D), which is much lower than that in in vivo conditions (Mc Laughlin et al., 2008; Lorteije et al., 2009). This could be explained by the lower presynaptic [Na+] in slice preparation that calyces do not fire spikes spontaneously. In the intact brain, however, the calyx fires at high frequencies, which would elevate the presynaptic [Na+] to over 50 mM (Figure 3.1, 3.2). The elevated

[Na+] facilitates vesicular glutamate transport and the bigger quantal size ensures reliable synaptic transmission. Indeed, increasing the presynaptic [Na+] to 40 mM in the slice preparation rescued the reliability of high-frequency transmission (Figure 3.8C-D, right panel).

60 The strength of synaptic transmission is determined by the readily releasable vesicle pool, release probability, and quantal size. Although the readily releasable vesicle pool and release probability reflect presynaptic properties, it is generally accepted that the changes in quantal size indicate postsynaptic alterations in neurotransmitter receptor interactions.

Our results show that during high-frequency spiking activity, intracellular Na+ is elevated in the terminals and altered the quantal size, which provides a novel presynaptic mechanism to control synaptic strength through changes in the concentration of transmitter in synaptic vesicles without changing the readily releasable vesicle pool or release probability. Since this presynaptic change of synaptic transmission does not affect paired- pulse ratio, our results suggest caution in interpreting studies which use paired-pulse ratio to determine pre- or postsynaptic changes. This activity-dependent modulation of vesicular content provides a novel mechanism of synaptic plasticity, and these findings have potentially universal implications for the regulation of synaptic efficacy in the central nervous system, especially for neurons that fire high-frequency spikes.

61 CHAPTER 4: PRESYNAPTIC NALCN CHANNELS MODULATE

SYNAPTIC STRENGTH THROUGH CONTROLLING VESICLE

FILLING AND BASAL CALCIUM

4.1 Abstract

Na+ leak channel non-selective (NALCN) is a constitutively active cation channel that widely expressed in the central nervous system (CNS) to control the resting membrane potential (RMP) and neuronal excitability. However, the distribution and possible functions on axon terminals remain unknown. Here, we show that NALCN channels were expressed on the glutamatergic calyx of Held terminal and modulated synaptic transmission. NALCN channels mediated a small tonic inward current which efficiently regulated the RMP and the basal Na+ and Ca2+ levels. Blockade of NALCN channels with trivalent ion gadolinium (Gd3+) decreased the basal Na+ level by 4.1 mM and reduced the amplitude of miniature excitatory postsynaptic currents (mEPSCs) by 9% in wild-type (WT) group but not in conditional NALCN knockout (cKO) group. Furthermore, compared to no effects on cKO group, WT group exhibited a 16% decrease of the basal Ca2+ level, increased paired-pulse ratio (PPR) with Gd3+ application, and higher sensitivity of mEPSP frequency in response to increased extracellular Ca2+ concentration, suggesting that

NALCN is permeable to Ca2+ and important for setting the initial release probability.

Finally, blocking NALCN channels induced a 17% more reduction of evoked EPSC

(eEPSC) amplitude in WT group, reflecting a NALCN-mediated change of glutamatergic transmission. Together, these data demonstrate that NALCN channels are expressed on the

62 calyceal terminals and influence synaptic strength by regulating presynaptic RMP, controlling quantal size through a Na+-dependent pathway, and modulating initial release probability through regulation of basal Ca2+ level.

4.2 Introduction

4.2.1 Na+ leak conductance in neurons

At rest, neurons maintain a steady and negative membrane potential, known as resting membrane potential (RMP). Theoretically, RMP can be quantitatively calculated by the Goldman-Hodgkin-Katz voltage equation based on K+, Na+, and Cl- concentration and permeability inside and outside of plasma membrane under physiological conditions

(Hodgkin and Katz, 1949). Since the neuronal membrane at rest has a great K+ leak conductance (Hodgkin and Huxley, 1946), K+ efflux from the cytoplasm induces a tonic

+ outward current and drives the RMP very close to the equilibrium potential of K (EK, ~ -

90 mV). However, the measured RMP in most mammalian neurons is in a range of about

-50 to -80 mV, which is close but never reaches to EK. This difference suggested an existence of a steady countervailing conductance that balances K+ leak conductance and

+ + + keeps the RMP above EK, and it is believed to be Na conductance. In contrast to K , Na is over 10 times more concentrated outside of neurons, leading to the ENa far depolarizing from the RMP (~ +60 mV). Hence, although the Na+ leak conductance is very small compared to K+ leak conductance (only 4% in squid giant axon) (Hodgkin and Katz, 1949), it plays an important role in modifying the RMP and neuronal excitability.

Na+ leak conductance is maintained by different Na+-permeable ion channels on

+ the plasma membrane. Firstly, TTX-sensitive persistent Na current (INaP) and resurgent

+ + Na current (INaR) generated by voltage-gated Na (NaV) channels, which are activated at a

63 subthreshold potential (Taddese and Bean, 2002; Bean, 2005). In some particular neuronal regions, such as the nerve terminal, INaP exhibits a more negative activation voltage to regulate the resting properties of the synapse (Huang and Trussell, 2008). Moreover, some neurons also express hyperpolarization-activated cation channels (HCNs, Ih current), which conduct both Na+ and K+ around RMP and this conductance is further enhanced with hyperpolarization (Robinson and Siegelbaum, 2003). Although NaV and HCN channels have ubiquitous distribution in the brain (Moosmang et al., 1999; Yu and Catterall, 2003), both of them are highly sensitive to voltage and contributing to basal Na+ conductance cannot be accounted as the major function of either of them. In addition to NaV and HCN channels, recent studies revealed a TTX-resistant, non-inactivating cation channel NALCN that also contributes to resting Na+ permeability. NALCN channels exhibit less dependence on voltage and are characterized as major background Na+ conductance (Lu et al., 2007;

Chua et al., 2020).

4.2.2 Structure and characteristics of Na+ leak channel NALCN

NALCN channel was first cloned and described as a four repeat protein related to

NaV and CaV channels around 20 years ago (Lee et al., 1999). It represents a distinct branch of the ion channel superfamily that consists of four functionally homologous repeated domains (DI – IV), with each formed by 6 transmembrane segments (S1 – S6). Other members in this superfamily are NaV channel family and CaV channel family. The detailed structure of NALCN channel remains unclear until three recent publications provided substantial pharmacological and structural information to understand the NALCN channels

(Kang et al., 2020; Kschonsak et al., 2020; Xie et al., 2020). The overall structure of

NALCN channels has similar domain arrangement compared to typical pore-forming

64 subunit of eukaryotic NaV and CaV channels: a central ion-conducting pore domain formed by the S5 and S6 helices from all four repeats, while S1 – S4 voltage-sensing domains

(VSDs) are packed around outside of the pore.

Nevertheless, several unique structural features of NALCN channel have been highlighted to distinguish it from other voltage-gated channels in the superfamily. Firstly,

NALCN has an atypical conformation of VSD, which senses the changes of membrane potential and control the movement of S6-activation gate. In the canonical voltage-gated ion channels, functional VSDs need consecutive positive gating charges (GCs) on the S4 helix and charge-transfer center (CTC) at the bottom of each VSD (Tao et al., 2010; Wu et al., 2016). Although the structures of DI-VSD and DII-VSD are conserved, suggesting functional VSDs may exist in NALCN, a defective CTC in DIII-VSD and reduced GCs in

DIV-VSD indicate at least a weaker voltage sensitivity of NALCN. Secondly, the key residues at the selectivity filter (SF) of NALCN, EEKE-motif (E280 (DI), E554 (DII),

K1115 (DIII), and E1389(DIV)), is different from the DEKA-motif in NaV channel and

EEEE-motif in CaV1 & 2 channel or EEDD-motif in CaV3 channel. On the one hand, the arrangement of charged residues in SF motif of NALCN is more similar to that of NaV channel (both contain a positive charged lysine residue), suggesting a stronger Na+ selectivity of NALCN than Ca2+ (Kang et al., 2020). On the other hand, the overall structure of two-pore helices-linked SF is closer to that of CaV channels, providing a probability that the SF of NALCN still preserves its permeability to Ca2+ (Xie et al., 2020).

Early electrophysiological studies showed that heterologously expressed NALCN alone in HEK293 cells displayed a constitutive ‘leak’ current with a linear current-voltage

(I-V) relationship, indicating a voltage-insensitive property of NALCN channels. By

65 scanning the permeability to different cations under bi-ionic conditions, NALCN was determined as a nonselective cation channel with the following selectivity sequence: Na+ >

K+ ~Cs+ > Ca2+ (Lu et al., 2007). However, more recent studies revealed some novel properties of NALCN channels that question this conclusion (Chua et al., 2020; Kschonsak et al., 2020). Firstly, considerable leak currents could only be recorded with co-expression of NALCN-FAM155A-UNC79-UNC80 tetramer in cultured cells (Egan et al., 2018;

Bouasse et al., 2019; Chua et al., 2020). Secondly, although the NALCN channel tetramer was constitutively active, it exhibited a maximal current at -40 mV in response to voltage steps, indicating that its constitutive activation is modulated by voltage. Thirdly, consistent with early studies, the NALCN channel complex was permeable to cations but not anions.

However, it was only selective for small monovalent cations with a permeability sequence of Na+ > K+ > Cs+. Divalent cation Ca2+ was barely permeable and extracellular Ca2+ at physiological concentrations even showed an inhibitory effect. So far, both early and recent electrophysiological results are supported at least partially by structural basis of NALCN channels, hence, more experiments need to be performed to understand the characteristics of NALCN channel, especially under in vitro and in vivo physiological conditions.

Together, based on the electrophysiological and structural studies, NALCN is characterized as a non-inactivating cation channel. Even though its voltage sensitivity and

Ca2+ permeability is still under debate, it is clear that the major ion going through the channel at RMP is Na+, which generates a “true” sodium leak current as its name indicates.

4.2.3 The NALCN-FAM155A-UNC80-UNC79 Complex

Typical NaV and CaV channels consist of multiple subunits. In addition to the essential pore-forming subunit, associated auxiliary subunits are also important for

66 channel’s basic function, albeit they do not directly participate in ion conductance. As mentioned above, recent findings suggested that functional NALCN channel required co- expression with FAM155A, UNC80, and UNC79. All these three proteins are highly conserved cross species, and mutation of any of them results in a dysfunction of NALCN channel (Jospin et al., 2007; Ren, 2011; Xie et al., 2013; Xie et al., 2020).

Although stable hetero-tetrameric complex of NALCN-FAM155-UNC80-UNC79 was failed to obtain, a stable core complex of NALCN-FAM155A was purified for structural analysis. The transmembrane protein FAM155A (family with sequence similarity 155, member A) binds tightly on the extracellular side of NALCN DI, DIII, and

DIV with its cysteine-rich domain (CRD). The binding residues of NALCN are distinct from NaV and CaV channels and these interacting residues of NALCN and FAM155A are highly conserved and electrically complementary, suggesting a strong binding specificity and interaction stability of NALCN-FAM155A. NLF-1, the homolog of FAM155A in C. elegans, was previously demonstrated as a novel endoplasmic reticulum (ER) resident protein that promoted axon delivery of NCA (homolog of NALCN), as well as UNC79 and

UNC80. Loss of NLF-1 in C. elegans or knockdown of nlf-1 gene in mouse cortical neuron cultures both resulted in a reduced Na+ leak current and a hyperpolarized RMP, which were same as the electrophysiological phenotypes observed in NCA/NALCN deficits (Xie et al.,

2013). Human FAM155A was detected on the plasma membrane alone and may contribute to proper folding and translocation of NALCN (Chua et al., 2020).

Another two large and well conserved proteins UNC80 and UNC79 showed sequence similarity neither with other auxiliary subunits in the 4  6 TMs superfamily nor predicted transmembrane segments, but have been considered as subunits of functional

67 NALCN channelosome with genetic, biochemical, electrophysiological evidence. Firstly, homologs of Nalcn gene were identified in C. elegans and Drosophila, where their mutants yielded a similar phenotype to unc79 and unc80 mutants (Jospin et al., 2007). Targeted mutations of either unc80 or unc79 gene in mice also exhibited similar phenotypes including severe apnea and neonatal lethality, with those observed in Nalcn null (Lu et al.,

2010; Ren, 2011). Secondly, removing NALCN from the brain lysates that contain all brain proteins also depleted UNC80 and UNC79 protein levels, suggesting an interaction among these three proteins. Finally, Nalcn and unc80 genes exhibit coincidental expression patterns in different brain areas, and recordings of robust NALCN-mediated current needs at least co-expression of UNC80, indicating that they may function together (Jospin et al.,

2007; Wie et al., 2020).

In addition, several functional domains of UNC80 have been determined. Those include the UNC79-interacting domain in the C-terminal side, NALCN-interacting domain in the N-terminal side and C-terminal soma-retention domain for NALCN complex trafficking to and (Wie et al., 2020). There is no direct evidence for

UNC79-NALCN association, but UNC80 was reported as a bridge for the connection of

UNC79 and NALCN (Lu et al., 2009; Lu et al., 2010). Nevertheless, knockout either of these two proteins could drastically reduce another protein level, suggesting the requirement of each other for stability and function of NALCN channel.

4.2.4 Pharmacology of NALCN channel

Since NALCN channel belongs to the 4  6 TMs superfamily, compounds including several NaV and CaV channel antagonists and modulators, divalent and trivalent metal cations and general neurotoxins, have been tested for evaluation of NALCN pharmacology

68 in NALCN-transfected cells. NALCN-mediated current is not sensitive to NaV channel blockers such as TTX or other neurotoxins. Some general CaV channel blockers, such as

Cd2+, Co2+, and verapamil, can partially block the NALCN-mediated current but only with high concentrations (Lu et al., 2007). The divalent cations Ca2+, Mg2+ and Zn2+ show relatively strong inhibition of NALCN channels but cannot be further used for pharmacological studies due to their complicated roles in the nervous system (Chua et al.,

2020; Kschonsak et al., 2020). The trivalent ion Gd3+, which is also considered as a high- affinity antagonist of the stretch-activated ion channel (SACs) (Yang and Sachs, 1989), effectively blocks NALCN-mediated current with an IC50 of 1.4 M (Lu et al., 2007;

Kschonsak et al., 2020). A recent study found that L-703606, originally developed as a potent antagonist for the human neurokinin-1 (NK1) receptor (Cascieri et al., 1992), inhibited NALCN-mediated current with low concentration in both transfected cells and midbrain dopaminergic neurons (Hahn et al., 2020). Gd3+ has been used in cultured neurons and brain slices as a non-specific blocker of NALCN channels, but other potential effects, such as alternations of postsynaptic AMPAR, inhibition of calcium channels, and inhibition of intracellular Ca2+ signaling, cannot be pharmacologically separated (Lei and

MacDonald, 2001; Baykara et al., 2019).

4.2.5 Function of NALCN channel

Functional NALCN channel homologs have been found in various animal species including C. elegans (homolog NCA-1/2), snails, D. melanogaster (homolog 1U), mouse, and humans. NALCN channels are also highly conserved in mammals, human and rat share

99% sequence similarity (Ren, 2011). This conserved sequence indicates that NALCN channels possibly maintain essential functional properties. Despite the inconsistent

69 conclusions regarding voltage sensitivity and Ca2+ permeability from different research groups, it is clear that the major contribution of NALCN is to basal Na+ conductance and neuronal excitability (Lu et al., 2007). Furthermore, NALCN channels are modulated by hormones, and extracellular Ca2+ level (Lu et al., 2009; Lu et al., 2010).

Several diseases related to dysfunctional channel have been carefully studied and extensively discussed (Al-Sayed et al., 2013; Aoyagi et al., 2015).

The first functional study of NALCN channel is from Ren lab (Lu et al., 2007).

They identified NALCN as a non-inactivating cation channel that is responsible for a TTX- resistant Na+ leak current in the cultured mouse hippocampal neurons. Blockade or knockout of NALCN induced a ~10 mV hyperpolarization, indicating its contribution to the regulation of the RMP and neuronal excitability. This functional property has been

+ further demonstrated in the CO2/H -sensitive neurons in the mouse retrotrapezoid nucleus,

GABAergic neurons from substantia nigra pars reticulate (SNr), midbrain dopaminergic neurons, and spinal projection neurons in the spinal cord for neurons’ spontaneous firing- related functions (Lutas et al., 2016; Shi et al., 2016; Yeh et al., 2017; Ford et al., 2018).

The activity of NALCN can be modulated by neurotransmitters, neuropeptides, and extracellular Ca2+ with different mechanisms. In the midbrain dopaminergic neurons, both

NALCN-mediated Na+ leak current and spontaneous firing rate were strongly reduced by activation of D2 dopamine receptors and GABAB receptors, which are inhibitory G protein- coupled receptors (Philippart and Khaliq, 2018). NALCN channels also can be activated by peptide neurotransmitters substance P (SP) and neurotensin with an involvement of G protein-coupled receptors but through Src family kinases-dependent pathway rather than direct activation of G protein (Lu et al., 2009). This phenomenon has been observed in the

70 hippocampus, ventral tegmental area (VTA), and respiratory network including preBötzinger complex (preBötC) and retrotrapezoid nucleus (Oertner et al.) (Yeh et al.,

2+ 2+ 2017). In addition, extracellular Ca ([Ca ]e) was determined as a blocker for the NALCN channels even at physiological level. The mechanism probably can be explained by potential binding of Ca2+ to SF of NALCN (Chua et al., 2020), or the dependence of calcium-sensing G protein-coupled receptor (CaSR). In the cultured mouse hippocampal

2+ neurons, lowering the [Ca ]e from physiological condition (1.2 mM) to 0.5 mM boosted the NALCN-mediated current. This signal pathway required the presence of CaSR to detect

2+ and transduce changes of [Ca ]e, and intracellular proteins UNC80 and UNC79 to activate

NALCN channels (Lu et al., 2010).

So far, NALCN mutations (including mutant in NALCN, FAM155A, UNC80, and

UNC79) in various animal species, including C. elegans, Drosophila, and mice, have clearly elucidated an essential role of NALCN complex. The most common phenotype induced by NALCN mutations is the defects in rhythmic behaviors. Mutations in any one of Nalcn, unc80, and unc79 genes exhibited a “fainter” locomotion phenotype resulting from disrupted rhythmic movements in C. elegans and Drosophila. Loss of one or more of

UNC80, UNC79, and FAM155A homologs caused a reduced localization and stabilization of NALCN in axons and eventually failed to sustain the rhythmic locomotion (Humphrey et al., 2007; Jospin et al., 2007; Xie et al., 2013). In mammals, rhythmic activity is highly related to breathing. NALCN KO mouse pups have severe apnea, identified as disrupted respiratory rhythm, and die within 24 h after birth. It is perhaps due to the reduced activities resulting from the deficiency of NALCN function in the respiration network (Lu et al.,

2007; Shi et al., 2016; Yeh et al., 2017). Circadian rhythm is another type of rhythmic

71 behavior. The mutation of NALCN led to the abnormal coupling between light and locomotion and was first reported in Drosophila and then also discovered in mice with reduced rapid eye movement sleep (Nash et al., 2002; Funato et al., 2016).

In humans, NALCN dysfunction has been reported as a cause of multiple diseases including severe hypotonia, speech impairment, cognitive delay, intellectual disability, and developmental delay in kids (Al-Sayed et al., 2013; Aoyagi et al., 2015). Hence, understanding the functional properties of highly disease-associated channel NALCN, is important for discovering treatments for NALCN channelopathies.

4.2.6 Introduction for this chapter

At the nerve terminals, the distribution of ion channels may differ compared to those on the soma. More importantly, presynaptic ion channels may have additional functions, such as controlling neurotransmitter release and synaptic strength (Huang and

Trussell, 2008, 2011; Hu and Bean, 2018). Previous studies have reported that elevation of presynaptic cytosolic Na+ level through either presynaptic HCN channels or repeated spike activity facilitated synaptic vesicle filling at a glutamatergic synapse by activating vesicular

Na+/H+ exchangers (NHEs) for the activity of vesicular glutamate transporter (VGLUT)

(Goh et al., 2011; Huang and Trussell, 2014; Li et al., 2020). These studies revealed a functional role of presynaptic Na+ for vesicular glutamate uptake and reliable synaptic transmission. Another cation that has received an extensive attention at the axon terminals

2+ 2+ is Ca . Besides an elevation of intracellular Ca through CaV channels being the prerequisite for the synchronous exocytosis of synaptic vesicles (Schneggenburger and

Neher, 2000), subtle alterations in basal Ca2+ level are also crucial for controlling the initial

72 probability of neurotransmitter release (Awatramani et al., 2005). However, ion channels for setting the basal Ca2+ level have not been carefully elucidated.

The sodium leak channel NALCN, has been identified as a non-inactivation cation channel that is predominantly expressed throughout the brain (Lee et al., 1999; Lu et al.,

2007). NALCN-mediated sodium leak current regulates RMP and neuronal excitability (Lu et al., 2007), which has been demonstrated to be involved in multiple essential behaviors including respiration, circadian rhythm, and locomotion in various animal species (Jospin et al., 2007; Xie et al., 2013; Cochet-Bissuel et al., 2014; Funato et al., 2016; Lutas et al.,

2016; Shi et al., 2016; Yeh et al., 2017). Whether NALCN channels are expressed at the axon terminals to regulate presynaptic basal Na+ level and thereby modulate vesicular content remains to be determined. In comparison to its well-established Na+ permeability, both electrophysiological studies and protein structure analysis in NALCN-transfected cells showed conflicting evidence for its Ca2+ permeability (Lu et al., 2007; Chua et al.,

2020; Kang et al., 2020; Kschonsak et al., 2020; Xie et al., 2020). Nevertheless, whether

NALCN is selective to Ca2+ in brain tissue has not been examined yet.

Here, we reported a functional expression of NALCN channels on the mouse calyx of Held synapse. Consistent with their major contribution, NALCN channels maintain the basal Na+ current and RMP in the calyceal terminal. Blockade of NALCN with Gd3+ reduced basal cytosolic Na+ concentration as well as mEPSC amplitude, while no distinguishable change was observed in conditional NALCN KO calyces (cKO). Further, using two-photon calcium imaging, we showed that blocking NALCN reduced basal Ca2+ level, indicating Ca2+ permeability of NALCN in acute brain slices. Small amounts change of basal Ca2+ level on the presynaptic terminal by NALCN was sufficient to lower release

73 probability, which was estimated by paired-pulse ratio (PPR). Thus, owing to its permeability to Na+ and Ca2+, presynaptic NALCN channels control glutamatergic transmission by modulating both vesicular neurotransmitter uptake and probability of neurotransmitter release.

4.3 Results

4.3.1 Identification of the expression of NALCN at the calyx of Held synapse

To determine the expression of NALCN channels at the calyceal terminal, we generated conditional NALCN knockout mice specifically in the auditory brainstem due to neonatal lethality caused by whole brain deletion (Lu et al., 2007). A floxed Nalcn mouse line (Nalcnflox) was crossed with specific Cre-driver mouse lines, Krox20Cre or Atoh-1Cre to conditionally remove Nalcn at the calyx of Held synapse. Both of these Cre-driver mouse lines mainly express Cre-recombinase in the auditory brainstem where the calyx of Held synapse is located, with few exceptions in other areas of the CNS (Voiculescu et al., 2000;

Yang et al., 2010). We first validated the specificity of calyceal labelling of these two Cre mouse lines by crossing with a Cre reporter Ai14-tdTomatoloxP/loxP mouse line. The tdTomato signal in the calyx was overlapped with the presynaptic marker VGLUT1, suggesting that both Cre lines are specific for targeting the calyceal terminals (Figure 4.1E,

F).

Immunohistochemical method was next used to determine the expression of

NALCN in the calyces of wildtype mice (Nalcnflox, as WT) and conditional NALCN knockout mice (Nalcnflox::Krox20Cre or Nalcnflox::Atoh-1Cre, as cKO). The presence of

NALCN was detected on presynaptic structures with anti-NALCN antibody, which colocalized with presynaptic marker VGLUT1 (Figure 4.1A, C; arrowheads). This

74 colocalization was not observed in the cKO calyces (Figure 4.1B, D). These data indicate that NALCN channels are expressed on the calyceal terminals and are successfully deleted in the cKO group.

75 Figure 4.1

76 Figure 4.1 Immunostaining of NALCN channels at the calyx of Held

(A-D) Left: Immunofluorescence signals of VGLUT1 (green) were used as a presynaptic marker. Middle: immunofluorescence signals of NALCN (red) were observed in both pre- and postsynaptic area in WT calyces (A, C), but only in postsynaptic area in cKO calyces

(B: Nalcnflox::Krox20Cre; D: Nalcnflox::Atoh-1Cre). Right: color merge of VGLUT1 and

NALCN signals showed colocalization (arrowheads) in WT (A, C), but not in cKO calyces

(B, D). (E-F) Krox20 and Atoh-1 driven Cre activities were determined at the calyx of Held synapse by crossing with reporter line. Presynaptic marker VGLUT1 signals (green, left) were colocalized with tdTomato fluorescence signals (red, middle), suggesting that both the Krox20Cre and Atoh-1Cre mouse lines can be used to generate conditional NALCN KO mice specifically in the auditory brainstem (merge, right).

77 4.3.2 NALCN channels contribute to Na+ leak currents and RMP in the calyceal terminals

Given that NALCN channels lack specific blockers (Lu et al., 2007; Kschonsak et al., 2020), the NALCN-mediated current (INALCN) can be measured as a change in holding current with Na+ substitution (lowering the extracellular [Na+] from 153.25 mM to 28.25 mM) under voltage clamp recordings. The previous studies determined that presynaptic

HCN and persistent Na+ are two main types of Na+-permeable channels which are active around RMP at the calyx of Held to regulate the resting properties (Huang and Trussell,

2008, 2014). To determine the contribution of NALCN to the overall basal Na+ leak current, we first isolated basal Na+ leak current in the WT calyces using a Cs+-based pipette solution and extracellularly incubation of 2 mM 4-AP and 10 mM TEA-Cl to block K+ conductance.

Then, 0.5 μM TTX and 2 mM CsCl were subsequently applied to block persistent Na+ current (INaP) and HCN current (Ih), respectively. To further isolated INALCN, extracellular

Na+ (115 mM) was replaced with equimolar concentration of a larger ion NMDG+.

Blockade of INaP and Ih induced a tonic outward holding current of 14.1 pA ± 1.6 pA, and switching to NMDG+-containing bath solution further increased the outward current by

14.6 pA ± 1.1 pA (n = 9, Figure 4.2A, B). In the cKO calyces, Na+ substitution with

NMDG+ had significantly smaller effect (7.4 pA ± 1.6 pA, P = 0.0023, n = 11, Figure 4.2A,

B), while the holding current that is sensitive to TTX and Cs+ was not significantly changed compared to that in the WT calyces (17.2 pA ± 1.4 pA, P = 0.15, Figure 4.2B). The residual

NMDG+-sensitive current in cKO calyces may be due to a mild change of junction potential

(2-3 mV) during Na+ substitution, functional changes of Na+-dependent transporters on plasma membrane resulting from a change of driving force of Na+, alternative cation

78 channels that are active near RMP, or incomplete Cre-driven knockout of NALCN. Taken together, NALCN underlies a basal TTX- and Cs+- resistant Na+ leak current on the calyceal terminal.

Additionally, the non-specific NALCN blocker Gd3+ was also used to assess the

NALCN-mediated current at the calyx of Held. The effects of Gd3+ on holding current include multiple components due to some known or unknown targets. Thus, after pharmacologically blocking NaV, KV, and HCN channels, a voltage step protocol at a negative voltage range (from -120 mV to -60 mV in steps of 10 mV within 1 s) was performed before and after bath application of 100 µM Gd3+ at a holding potential of -80 mV. The averaged current values were relatively linear throughout the tested voltage range.

In WT calyces, 4 of 5 cells displayed a smaller current response after Gd3+ application (P

= 0.023, n = 5, Figure 4.2C), while no effect of Gd3+ was observed in cKO calyces (P =

0.26, n = 7, Figure 4.2D). Notably, WT calyces also showed an increased holding current after Gd3+ application, indicating a blockade of a tonic inward current (Figure 4.2C). These findings are consistent with the NMDG+ replacement results.

Basal Na+ leak current counteracts basal K+ current to maintain the RMP. It is therefore expected that NALCN channels may influence presynaptic RMP. Indeed, the

RMP measured under current clamp mode from WT calyces on average was -74.2 ± 0.5 mV (n = 26), whereas knocking out of NALCN in the calyces significantly hyperpolarized the RMP to -77.5 ± 0.7 mV (P = 0.0005, n = 11, unpaired t-test; Figure 4.2E). Thus, the

NALCN-mediated inward Na+ current is small but important for maintaining presynaptic

RMP.

79 Figure 4.2

Figure 4.2 NALCN channels mediate a small inward current and induce a hyperpolarization of RMP in the calyx of Held

(A) Example traces showing the effect of Na+ substitution with NMDG+ on holding current in WT (top) and cKO (bottom) calyces. INaP and Ih were blocked by TTX and CsCl respectively before NMDG+ replacement. (B) Summary graph of holding current changes in WT and cKO calyces. cKO calyces showed a reduced NMDG+-sensitive current. (C-D)

Example traces of current responses to the voltage steps before and after Gd3+ application in WT (C) and cKO (D) calyces. Voltage step protocol: from -120 mV to -60 mV in steps of 10 mV within 1 s at a holding potential of -80 mV. (E) cKO calyces had a more hyperpolarized RMP compared to WT calyces. **P < 0.01, *** P < 0.001. Error bars, ±

SEM.

80 4.3.3 NALCN channels regulate presynaptic basal Na+ level and quantal size

Previous studies reported that presynaptic Na+ regulates glutamate loading into synaptic vesicles through a vesicular NHE-dependent pathway (Goh et al., 2011). Since cytosolic Na+ level can be dynamically regulated by Na+-permeable ion channels (Huang and Trussell, 2014; Li et al., 2020), it is expected that NALCN channels may also modulate presynaptic basal Na+ level and quantal size. To test the effect of NALCN channels on cytosolic Na+ concentration, whole-cell recordings from calyces were made and cytosolic

Na+ transients were assayed using two-photon laser-scanning microscopy. The calyceal terminals were loaded with the volume marker Alexa594 (15 µM) and Na+ indicator SBFI

(1 mM) via patch pipettes. After both dyes diffused into the terminal, the pipette electrode was carefully withdrawn to allow the cell membrane to reseal. In the presence of TTX and

+ Cs to block INaP and Ih, respectively, frame-scan mode was made at the calyces every 10-

20 s to monitor the changes in fluorescence signal during bath application of 20 µM Gd3+ to block NALCN. We found that Gd3+ wash-in reduced the resting [Na+] by 4.1  0.6 mM

(n = 8, Figure 4.3A, B) in the WT calyces, but had no effect in the cKO group (0.0 ± 0.7 mM, n = 6, Figure 4.3A, B), indicating that Gd3+ negatively changed presynaptic basal Na+ level via blockade of NALCN (P = 0.0011, unpaired t-test).

To test whether a reduced basal Na+ level by blocking NALCN channels at the terminal decreases the quantal size, which is represented as mEPSC amplitude, we monitored the change of mEPSC amplitude during bath application of non-specific

NALCN blocker Gd3+ in both WT and cKO groups. As we expected, within 25 min of whole-cell recordings, adding of Gd3+ to the bath solution gradually reduced the mEPSC amplitude to 91.4 ± 1.1% and shifted the cumulative frequency distribution of mEPSC to

81 the left in WT cells (P= 0.0007, n = 6, paired t-test, Figure 4.3C-E), while both the amplitude and cumulative frequency distribution remained stable in cKO cells during the entire recording (100.3 ± 1.7%, P = 0.86, n = 9, paired t-test, Figure 4.3C-E). The 9% change of mEPSC amplitude with blockade of NALCN channels is comparable with the effect of presynaptic HCN channels (Huang and Trussell, 2014), since both channels maintain the basal Na+ level. No significant difference of mEPSC amplitude was observed between WT and cKO groups (WT: 50.0 ± 2.0 pA, n = 16; cKO: 47.0 ± 2.3 pA, n = 21; P

= 0.35, unpaired t-test, Figure 4.3F), probably because the potential difference between groups is masked by the variation of mEPSC amplitude.

82 Figure 4.3

Figure 4.3 NALCN channels regulate presynaptic basal Na+ level and quantal size

(A) Left: Single optic section of P10 WT calyx recorded with two-photon microscopy.

Right: example traces of cytosolic [Na+] transients in response to bath application of Gd3+.

(B) Summary graph of cytosolic [Na+] change in WT and cKO groups. (C) Sample traces of mEPSC recordings in control condition (left) and in the presence of Gd3+ (right) in both

WT and cKO cells. (D) Cumulative frequency distributions from the same cells in (C).

Gd3+ shifted the curve to the left only in the WT cell. (E) Time course of normalized mEPSC amplitude from WT (black) and cKO (red) groups in response to Gd3+ application

(time point of Gd3+ wash-in is labeled with a dotted line). Each point represents a 1 min of

83 recording. Right panel shows relative mEPSC amplitude at 20-25 min of recording normalized to the one at 0-5 min. (F) Summary graph of mEPSC amplitude in WT and cKO groups. Statistical significance was assessed using a two-tailed paired or unpaired

Student t-test. **P < 0.01. Error bars ± SEM.

84 4.3.4 NALCN channels are Ca2+ permeable and control presynaptic basal Ca2+ level

Unlike Na+ permeability, the Ca2+ selectivity of NALCN channels remains controversial in NALCN-transfected cultured cells (Lu et al., 2007; Kschonsak et al., 2020), and has not been tested in brain slices. To explore whether NALCN channels are permeable to Ca2+ and affect basal Ca2+ level, two-photon Ca2+ imaging with calcium indicator Oregon green BAPTA-1 (OGB-1) was performed. After OGB-1 (100 μM) and volume marker dye

Alexa 594 (20 μM) were loaded into calyces with patch pipette, the changes of Ca2+ transients were monitored before and after 20 μM Gd3+ application to the bath solution.

Application of Gd3+ remarkedly reduced basal Ca2+ level by 15.6 ± 3.1% in WT calyces (n

= 7). In parallel experiments with cKO calyces, the ability of Gd3+ to reduce basal Ca2+ was completely abolished (+0.04 ± 4.5 %, n = 7), which suggests that the Gd3+-induced decrease of basal Ca2+ level is mediated by NALCN channels (P = 0.015, unpaired t-test,

Figure 4.4A, B). To further confirm the Ca2+ permeability of NALCN channels, we studied the effect of increased Ca2+ influx on mEPSC frequency by increasing extracellular Ca2+

2+ concentration ([Ca ]e). In the WT group, mEPSC frequency was strongly enhanced by

2+ 2+ 46.1 ± 6.1% in 2.4 mM [Ca ]e as compared with 1.2 mM [Ca ]e (n = 5), while cKO group only exhibited an increase of 12.2 ± 1.8% (n = 7), indicating that NALCN channel is a major source for Ca2+ influx at resting status (P = 0.0001, unpaired t-test, Figure 4.4C, D).

Overall, these data provide evidence that NALCN channels on the calyceal terminal are permeable to Ca2+ and control basal Ca2+ level.

85 Figure 4.4

Figure 4.4 NALCN channels are Ca2+ permeable and control presynaptic basal Ca2+ level

(A) Left: Single optic section of the calyx; Right: presynaptic Ca2+ transients affected by bath application of Gd3+ in WT and cKO calyces. (B) Summary graph of Ca2+ signal in response to Gd3+ application in WT and cKO groups. (C) Sample traces of mEPSC

2+ recordings in 1.2 mM (top) and 2.4 mM (bottom) [Ca ]e in both WT and cKO cells. (D)

2+ mEPSC frequency in 2.4 mM [Ca ]e being normalized to the one recorded with 1.2 mM

2+ [Ca ]e in both WT and cKO groups. Statistical significance was assessed using a two- tailed unpaired Student t-test. * P < 0.05, *** P < 0.001. Error bars, ± SEM.

86 4.3.5 NALCN channels control initial release probability and glutamate release

For NALCN channels, the permeability to Ca2+ received little attention in the soma.

However, Ca2+ flux through NALCN channels on the presynaptic terminal may play a crucial role in neurotransmitter release. In line with results showing that the release probability is sensitive to presynaptic resting Ca2+ concentration, depolarizing membrane potential from -80 to -60 mV increased the intraterminal Ca2+ level by 50-100 nM at the calyx of Held, and this subtle rise in the basal Ca2+ level was sufficient to increase the probability of glutamate release (Awatramani et al., 2005). To examine the contribution of

NALCN-mediated basal Ca2+ influx, we assessed the paired-pulse ratio (PPR), which is sensitive to a change of release probability with an inverse relation (Zucker and Regehr,

2002). With incubation of Gd3+, all recorded WT cells showed increased PPR, indicating a reduced initial release probability (P = 0.0009, n = 7, paired t-test, Figure 4.5B, C), whereas increased PPR was only observed in 2 of 7 cKO cells with unchanged average value (P = 0.55, n = 7, paired t-test, Figure 4.5B, C). In the comparison between groups, cKO group exhibited a slightly higher averaged PPR value and wider distribution in plotted histogram, but no significant change was obtained (WT: 0.68  0.04, n = 29; cKO: 0.72 

0.04, n = 48; P = 0.48, unpaired t-test, Figure 4.5E). Overall, these results suggest that

NALCN channels control the basal Ca2+ influx and initial release probability at the calyx of Held synapse.

Since blockade of NALCN channels affected both quantal size and release probability, we would expect a decreased glutamatergic transmission, which could be inferred from reduced evoked EPSC (eEPSC) amplitude. Bath application of Gd3+, however, dramatically decreased the eEPSC amplitude in both groups. This unexpected

87 phenomenon could be explained by Gd3+-induced inhibition of AMPARs (Lei and

MacDonald, 2001). More importantly, Gd3+ induced a 16.5% larger decrease of eEPSC amplitude in WT cells compared to cKO cells (WT: 57.1 ± 6.3%, n = 7; cKO: 73.6 ± 7.3%, n = 7, Figure 4.5D), which is larger than the difference observed in mEPSC amplitude between groups (9%, Figure 4.3E). This stronger reduction of eEPSC amplitude in WT group suggests that the NALCN channels alter presynaptic glutamate release via modulation of both quantal size and release probability. Notably, although no significant difference in the averaged amplitudes was observed between WT and cKO groups, the eEPSC amplitude in cKO cells exhibited a left-shifted distribution with more smaller values, also indicating a higher possibility to induce failures during synaptic transmission

(WT: 5.0 ± 0.5 nA, n = 29; cKO: 4.7 ± 0.5 nA, n = 48; P = 0.67, unpaired t-test, Figure

4.5F).

88 Figure 4.5

Figure 4.5 NALCN channels control initial release probability and glutamate release

(A) Example traces of a pair of eEPSCs from control and cKO calyces before (black) and after (red) bath application of Gd3+. (B) Time course of normalized PPR from WT (black) and cKO (red) groups in response to Gd3+ application (time point of Gd3+ wash-in is labeled with a dotted line). Each point represents 1 minute of recording. Right panel shows relative

PPR at 20-25 min recordings normalized to the recording at 0-5 min. (C) The changes of

PPR before and after Gd3+ application in WT and cKO groups. (D) Time course of normalized eEPSC amplitude from both WT (black) and cKO (red) groups in response to

Gd3+ application. Each point represents 1 minute of recording. Right panel shows relative

89 eEPSC amplitude at 20-25 min recordings normalized to the one at 0-5 min. (E-F) Left: summary graphs of PPR (E) and eEPSC amplitude (F) in WT and cKO groups. No significant difference was observed between groups in either analyzed parameters. Right: histograms of relative distribution in WT and cKO groups that fitted with Gaussian function. Statistical significance was assessed using a two-tailed paired or unpaired Student t test. ** P < 0.01, *** P < 0.001. Error bars, ± SEM.

90 4.4 Discussion

In this study, we reported a functional expression of NALCN channels on a glutamatergic synapse in the CNS. NALCN channels, which were identified by immunohistochemical, electrophysiological, and pharmacological methods, combined with conditional knockout animals, showed specific functions on presynaptic terminal for the modulation of vesicular content and initial probability of glutamate release due to its permeability to Na+ and Ca2+.

Northern blot analysis of NALCN showed that its mRNA was expressed predominantly in the rat and human brain (Lee et al., 1999). Consistent with the mRNA expression, functional NALCN channels have been found in certain types of neurons in various brain regions including the hippocampus, retrotrapezoid nucleus, substantia nigra, ventral tegmental area, and spinal cord (Lu et al., 2007; Lutas et al., 2016; Shi et al., 2016;

Yeh et al., 2017; Ford et al., 2018). In transfected cultured hippocampal neurons, NALCN was detected not only in soma, but also in dendrites and axons, suggesting a wide-spread distribution of NALCN in a single neuron (Wie et al., 2020). As a widely expressed channel, it was not surprised that NALCN channels were also identified in the calyx of Held synapse with immunostaining (Figure 4.1). Unfortunately, electrophysiological study of NALCN channels in the brain slices is currently extremely challenging due to the absence of specific blocker and the small size of NALCN-mediated current which almost reaches the detection limit in whole-cell patch clamp recordings (Ren, 2011). To overcome these technical challenges, we isolated a small TTX and Cs+-resistant inward current (14.6 pA in WT and reduced to 7.4 pA in cKO calyces) by replacing the extracellular Na+ with equimolar concentration of a larger cation NMDG+ (Figure 4.2A, B). In previous studies, presynaptic

91 HCN and persistent Na+ channels have been functionally characterized on the calyceal terminals (Huang and Trussell, 2008, 2014). Blockade of either of them could induce a ~3 mV hyperpolarization of RMP, indicating that both channels contribute to basal Na+ conductance and RMP. However, both HCN and persistent Na+ are highly sensitive to voltage with a relatively high density. Despite the small current generated by NALCN channels at rest, it resulted in a 3.3 mV more hyperpolarized RMP in the cKO calyxes

(Figure 4.2G), which probably due to its extremely small conductance. Hence, together with presynaptic HCN and persistent Na+ currents, NALCN, as one of the components of basal Na+ current, also regulate presynaptic RMP.

To determine the presynaptic functions of NALCN channels, we used its non- specific blocker Gd3+ combined with cKO animals to isolate the NALCN-mediated changes on the calyx of Held synapse. Trivalent ion Gd3+ has been used for blocking

NALCN channels in cultured neurons and in vitro studies, but other known effects, such as alternations of postsynaptic AMPAR, inhibition of calcium channels, transient receptor potential (TRP) channels and intracellular Ca2+ signaling, and other unknown effects could also be involved (Bourne and Trifaro, 1982; Yang and Sachs, 1989; Lei and MacDonald,

2001; Baykara et al., 2019). Although we found a trend of differences between WT and cKO groups, these differences did not reach a statistical significance (Figure 4.3F, 4.5E,

F). The NALCN deletion mediated changes might be masked by the variation between individual neurons. To rule out this variation and to eliminate non-specific effects from

Gd3+, we analyzed the Gd3+-sensitive changes in both WT and cKO groups. The differences between these two groups in the presence of Gd3+ were then considered to be mediated by

NALCN channels.

92 Previous studies have revealed a mechanism that presynaptic cytosolic Na+ is able to regulate the amount of glutamate in single vesicle via a vesicular NHE-dependent pathway (Goh et al., 2011). Spikes are potent in facilitating presynaptic Na+ accumulation during neuronal activity, while HCN channel was considered to be prominent in controlling the resting Na+ level (Huang and Trussell, 2014; Li et al., 2020). Here, we further tested the contribution of NALCN channels to quantal size based on its Na+ permeability. With two-photon Na+ imaging, our results showed that bath application of Gd3+ to block NALCN in WT calyces reduced the resting Na+ level by 4.1  0.6 mM in the presence of TTX and

Cs+ (Figure 4.3A, B). Consistent with a decreased cytosolic Na+ level, a ~9% reduction of mEPSC amplitude was obtained in WT compared to cKO cells when Gd3+ was applied in the bath (Figure 4.3D-E). These data indicated an important role of NALCN channels in modulating glutamate uptake through a Na+-dependent pathway.

An early electrophysiological study in NALCN-transfected HEK293 cells reported that NALCN is a nonselective cation channel, which is permeable not only to monovalent cation Na+ and K+, but also to divalent cation Ca2+ with less selectivity (Lu et al., 2007).

However, more recent studies showed evidence to challenge its Ca2+ permeability with co- expression of NALCN-FAM155A-UNC80-UNC79 complex in HEK293 cells. Instead, extracellular Ca2+ could function as a blocker at physiological concentrations (Chua et al.,

2020; Kschonsak et al., 2020). So far, both results can be at least partially supported by the structural analysis of NALCN. The key residue at the selectivity filter (SF) of NALCN are

EEKE-motif, which is different from the DEKA-motif in NaV channel and EEEE-motif in

CaV1 & 2 channels or EEDD-motif in CaV3 channel, although all of these three types of channels come from the same 4  6 TMs superfamily (Lu et al., 2007; Ren, 2011). On one

93 hand, the arrangement of charged residues of SF motif in NALCN is more similar to that

+ in NaV channel (both contain a positive charged lysine residue), suggesting a stronger Na selectivity (Kang et al., 2020). On the other hand, the overall structure of two-pore helices- linked SF is more identical to that of CaV channels, suggesting a probability that SF of

NALCN still preserves its permeability to Ca2+ (Xie et al., 2020). Despite the controversy, direct in vitro or in vivo evidence is missing, and it could not rule out the possibility that

Ca2+ permeability of NALCN channels might be regulated intracellularly. Taking into account the extremely small size of overall NALCN-mediated current, we tested its Ca2+ permeability with two-photon calcium imaging. The reduced basal Ca2+ level after bath application of Gd3+ was only observed in WT but not in cKO calyces, indicating a strong probability of NALCN’s permeability to Ca2+ (Figure 4.4A, B). Furthermore, cKO group showed a much smaller response of mEPSC frequency when extracellular Ca2+ concentration was increased, further demonstrating the regulation of Ca2+ influx through

NALCN channels (Figure 4.4C, D).

An obvious hypothesis, although has not been fully tested, is that the long-lasting steady-state intracellular Ca2+ level is strictly balanced by the inwardly Ca2+ “leak” and anti-ionic gradient transport (Rizzuto and Pozzan, 2006). The cytosolic free Ca2+ is maintained tightly at the range of 50-100 nM, while the extracellular Ca2+ is about 1.2 mM, which is ten thousand times higher. This steep gradient of Ca2+ across the cell membrane is believed to be manipulated by the actions of Ca2+- ATPase, Na+/Ca2+ exchanger (NCX) and Na+/Ca2+-K+ exchanger (NCKX) (Carafoli and Brini, 2000; Dong et al., 2001; Kim et al., 2005). However, the sources for Ca2+ “leak” into the cytoplasm are still unclear.

Intracellular Ca2+ storage organelles such as endoplasmic reticulum (ER) and mitochondria

94 may contribute to the Ca2+ flux into cytoplasm (Baker et al., 2013). For example, G-protein coupled receptors (GPCRs) can trigger Ca2+ efflux from ER by indirectly activating the inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), which are two ligand-gated Ca2+ channels. These pathways may function as a regulator for oscillations of cytosolic free Ca2+ (Dhyani et al., 2020). However, GPCR signal transductions are triggered by extracellular stimuli, unlikely providing a constitutively “true” Ca2+ leak. In addition, inhibition of the Ca2+-ATPase led to a depletion of Ca2+ in the intracellular stores and a transient increase of cytosolic free Ca2+, but did not affect the long-lasting steep gradient of Ca2+ across the plasma membrane (Rizzuto and Pozzan, 2006), indicating that intracellular Ca2+ stores are not necessary for basal Ca2+ level. Hence, it is more likely that the Ca2+ leaks from the cell membrane are the main source for setting up the basal calcium level. Indeed, a variety of CaV channels have been well characterized in different neurons.

Although high voltage activated CaV channels require strong depolarization to be open and

2+ trigger Ca influx, low voltage activated CaV channel, T-type CaV, has a “window current” around RMP for Ca2+ entry (Perez-Reyes, 2003), which can be considered as an important

2+ source for basal Ca . Besides CaV channels, other nonselective cation channels may also be involved. Novel Ca2+-permeable cation channels include glutamate-gated ion channels

(NMDA receptor and subtype AMPA receptor), transient receptor potential (TRP) channels, and acid-sensing ion channels (ASICs) (Clapham et al., 2001; Xiong et al., 2004;

Fortin et al., 2010). However, all these channels need to be activated by ligands or specific factors, which largely restrict their regulation on basal Ca2+ permeability. In the present study, we observed a Gd3+-sensitive reduction of basal Ca2+ level with two-photon calcium imaging and decreased release probability, which was estimated by increased PPR in

95 whole-cell recordings (Figure 4.4, Figure 4.5A-C). These Gd3+-sensitive changes were only observed in WT group but not obvious in cKO group, indicating that those changes were mediated by NALCN channels. Therefore, NALCN, as a widely expressed channel in the brain, is one of the best candidates for constitutively generating subtle but essential

Ca2+ influx for basal Ca2+ level. In particular, Ca2+ level on the axon terminal is highly related with neurotransmitter release and overall synaptic strength, which provides additional potential function of NALCN channels on the synapse.

In comparison with WT group, the eEPSC amplitude, which represents overall glutamatergic transmission at the synapse, showed slightly though not significantly smaller averaged value in cKO group. However, the relative distribution of eEPSC amplitude exhibited more evident differences between groups when a histogram was plotted (Figure

4.5F). The cells with smaller eEPSC amplitude shared higher percentage in cKO group:

25% (12 of 48 cells) recorded cKO cells had an eEPSC amplitude smaller than 2 nA, while only 2 of 29 cells in the WT group showed such small amplitude. We would expect a strong influence of NALCN channels on the initial glutamate release. Also, bath application of

Gd3+ induced a stronger reduction of eEPSC amplitude in WT group (Figure 4.4D), which presumably resulted from a combined effect of reduced quantal size and initial release probability. Moreover, more recent studies reported that NALCN channel exhibited a voltage-dependence manner with a maximal inward current at -40 mV in response to voltage steps, although its voltage sensitivity is not as strong as NaV and CaV channels

(Chua et al., 2020; Kschonsak et al., 2020). Thus, NALCN channel may be involved more during sustained synaptic activities.

96 CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS

5.1 The Role of Na+ in Synaptic Transmission

Since over one hundred years ago, Sydney Ringer first discovered abnormal long- lasting time of isolated organs with NaCl and invented the Ringer’s solution for in vitro experiments on tissues and organs, Na+, K+, Ca2+, and Cl- have been appreciated as major and essential ions cross the cell membrane. Later, a major role in controlling cell excitability of each cation (Na+, K+, and Ca2+) in the Ringer’s solution has been discovered.

Due to sustained activity of Na+/K+ pump on the plasma membrane, Na+ and K+ levels are opposite maintained as high cytosolic [K+] and high extracellular [Na+] for stabilizing resting membrane potential and generating a favorable condition for firing action potentials.

Previous works found an alternative functional role of Na+ and K+ during synaptic transmission at glutamatergic synapses. They reported that Na+(K+)/H+ exchanger (NHE) on the synapse vesicles membrane could efficiently convert ΔpH into Δψ by replacing the intravesicular H+ with Na+ or K+. Δψ could be further used for the activity of VGLUT thereby facilitated vesicular glutamate uptake (Goh et al., 2011; Preobraschenski et al.,

2014). With simultaneous pre- and postsynaptic whole-cell recordings at the calyx of Held synapse, manipulating presynaptic Na+ or K+ level both resulted in a change of quantal size, further supporting the importance of Na+ and K+ for glutamate loading via NHE-dependent pathway in acute brain slices (Goh et al., 2011; Huang and Trussell, 2014). In this thesis, my colleagues and I found that cytosolic Na+ could reach relatively high level at the axon terminal during presynaptic firing (Figure 3.1, 3.2). This elevation of Na+ increased

97 postsynaptic response by solely affecting vesicle refilling through NHE to maintain reliable synaptic transmission.

Although both Na+ and K+ can affect the quantal size through NHE-dependent pathway, presynaptic Na+ may be more powerful than K+ in facilitating glutamate uptake especially during sustained neuronal activity. Firstly, the resting intracellular K+ level is much higher than Na+ and relatively stable, while the intracellular Na+ level may dramatically change during activities of different Na+-permeable ion channels.

Manipulating the presynaptic Na+ at relatively high level during whole-cell recordings could gradually increase the quantal size even when intracellular K+ was maintained at physiological concentration in the calyx of Held, suggesting that quantal content is sensitive to the change of intracellular Na+ level (Huang and Trussell, 2014). Then, besides replacing the intravesicular H+ through NHE, K+ can directly bind to the cation binding site of VGLUT for K+/H+ exchange, indicating that K+ associated with VGLUT is enough for glutamate uptake, while Na+ associated with NHEs is an alternative pathway to maintain the ionic and charge balance during glutamate transport (Preobraschenski et al.,

2014). However, this alternative pathway may become more important during sustained activities. Prolonged presynaptic firing may lead to a local ‘high Na+, low K+’ cytosolic environment, as well as a loss of releasable vesicles. Thus, high presynaptic Na+ is critical for facilitating vesicular refilling, offsetting possible loss of synaptic strength during repetitive firing and maintaining reliable synaptic transmission.

This mechanism that presynaptic Na+ accumulation during high-frequency synaptic firing accelerates vesicular glutamate refilling by promoting NHE activity, not only can be applied in the specialized large glutamatergic synapses, but may also play an important

98 role at small central synapses, where vesicle supply is the rate-limiting step during ongoing synaptic activities. For example, in the hippocampal synapses, considering the limited vesicle number in the readily releasable pool (RRP) and relatively slow vesicle replenishment through vesicle mobilization from reserve and recycling vesicle pools, rapid recovery of RRP with retrieved vesicles may become the key determinant of synaptic reliability (Kim et al., 2020). In this case, the impact of presynaptic Na+-dependent facilitation on vesicular glutamate refilling is even stronger during sustained synaptic transmission.

5.2 Specific Function of NALCN Channels on the Axon Terminal

NALCN channels share the similar properties on regulating basal Na+ level and

RMP with HCN channels, hence it is not surprising that NALCN channels expressed on the axon terminals can also modulate quantal size by promoting the activity of vesicular

NHE through a Na+-dependent way at resting status. More importantly, more recent studies about NALCN channels provided detailed electrophysiological and structural evidence for its voltage sensitivity (Chua et al., 2020; Kschonsak et al., 2020). Despite the voltage sensor domains (VSDs) in the NALCN contain less voltage-sensing residues compared to NaV and CaV channels, FAM155A approaches the DII-VSD to possibly support functional

VSDs in the NALCN (Kschonsak et al., 2020). Consistently, a maximal inward current was detected at -40 mV when co-expressed NALCN-FAM155A-UNC80-UNC79 complex in the transfected cells (Chua et al., 2020). The voltage-dependent manner of NALCN channels indicates that NALCN may be more constitutively active at depolarized membrane potential. In addition, repetitive firing may induce a local reduction of extracellular Ca2+ concentration and thereby activate NALCN channels. If so, during

99 prolonged synaptic firing, NALCN channels may work together with NaV channels as a

+ compensation of NaV channel inactivation to maintain a relatively high cytosolic [Na ] environment, which is important for vesicular glutamate refilling.

Direct evidence is still missing for the function of NALCN channels during neuronal activity. Taking advantage of the conditional NALCN knockout animal at the calyx of Held synapse, reliability of synaptic transmission can be tested during prolonged high-frequency stimulation in both WT and cKO groups. We expected to observe earlier or more failures of corresponding postsynaptic firing when NALCN channels were deleted at the axon terminals.

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