CHOLINERGIC INTERNEURON MEDIATED ACTIVATION OF G-
PROTEIN COUPLED RECEPTORS IN THE DORSAL STRIATUM
by
APHRODITI A. MAMALIGAS
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Department of Neurosciences
CASE WESTERN RESERVE UNIVERSITY
August, 2018
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
Aphroditi A. Mamaligas
Candidate for the degree of Doctor of Philosophy.*
Thesis advisor:
Christopher Ford, PhD
Committee Chair:
David Friel, PhD
Committee Member:
Lynn Landmesser, PhD
Committee Member:
Evan Deneris, PhD
Date of Defense:
May 23, 2018
*We also certify that written approval has been obtained for any proprietary
material contained therein.
2 TABLE OF CONTENTS
List of figures...... 6
Acknowledgements...... 8
List of abbreviations...... 9
Abstract...... 12
Chapter 1...... 14
Introduction...... 15
Striatal microcircuitry...... 16
Striatal cholinergic interneuron activity and role in disease...... 19
ChI anatomy and distribution across the striatum...... 21
Striatal ACh release and signaling...... 22
Modulation of striatal inputs and interneurons...... 23
Cholinergic regulation of MSN muscarinic receptors...... 27
GPCR synapses and volume transmission in the central nervous system.29
Rationale...... 31
Figures and figure legends...... 33
3 Chapter 2...... 35
Nicotinic and opioid receptor regulation of striatal dopamine D2-
receptor mediated transmission...... 35
Abstract...... 36
Introduction...... 37
Experimental procedures...... 39
Results...... 42
Discussion...... 49
Figures and figure legends...... 55
Chapter 3...... 69
Spontaneous synaptic activation of muscarinic receptors by striatal
cholinergic interneuron firing...... 69
Abstract...... 70
Introduction...... 71
Experimental procedures...... 73
Results...... 78
Discussion...... 94
Figures and figure legends...... 100
4
Chapter 4...... 130
Cortical and thalamic inputs evoke cholinergic transmission in the
striatum...... 130
Abstract...... 131
Introduction...... 132
Experimental procedures...... 134
Results...... 138
Discussion...... 150
Figures and figure legends...... 153
Chapter 5...... 170
Discussion...... 171
Synaptic point-to-point transmission at M4 synapses...... 173
Unlaminated striatal structure and implications for ChI connectivity...... 174
Future directions...... 175
References...... 181
5 LIST OF FIGURES
• Chapter 1
o Figure 1.1……………………………………………………………….33
• Chapter 2
o Figure 2.1…………………………………………….…………………55
o Figure 2.2……………………………………………………………….58
o Figure 2.3……………………………………………………………….61
o Figure 2.4……………………………………………………………….63
o Figure 2.5……………………………………………………………….65
o Figure 2.6……………………………………………………………….67
• Chapter 3
o Figure 3.1…………………………………………………………….100
o Figure 3.2…………………………………………………………….103
o Figure 3.3…………………………………………………………….105
o Figure 3.4…………………………………………………………….108
o Figure 3.5…………………………………………………………….110
o Figure 3.6…………………………………………………………….113
o Figure 3.7…………………………………………………………….115
o Figure 3.8…………………………………………………………….117
o Figure 3.9…………………………………………………………….119
6 o Figure 3.10……………………………………………………………121
o Figure 3.11……………………………………………………………123
o Figure 3.12……………………………………………………………125
o Figure 3.13……………………………………………………………127
• Chapter 4
o Figure 4.1……………………………………………………………..153
o Figure 4.2……………………………………………………………..156
o Figure 4.3……………………………………………………………..159
o Figure 4.4……………………………………………………………..161
o Figure 4.5……………………………………………………………..164
o Figure 4.6……………………………………………………………..167
7 ACKNOWLEDGEMENTS
I would like to thank my mentor Chris Ford and the members of the Ford lab for their help, support, and contributions to my development as a scientist. I thank my thesis committee for their critiques, questions, and helpful suggestions at committee meetings. I thank Suhanti Banerjee, Kathy Lobur, and Michael
Grybko for their assistance with animal husbandry. I thank Yuan Cai and Michael
Grybko for assistance with recording experiments. I thank Sarah Zych for help with fluorescence imaging. I thank Ben Strowbridge for custom code used for the
2-photon microscope. I thank the CWRU Department of Neurosciences and the
Department of Physiology and Biophysics, as well as the University of Colorado
Denver Department of Pharmacology for their support and feedback during my graduate training. Finally, my thesis work was supported by NINDS R01
NS95809 and NIDA R01 DA35821 (to CPF).
8 LIST OF ABBREVIATIONS
AAV – adeno associated virus
ACh – acetylcholine
AChE – acetylcholinesterase aCSF – artificial cerebrospinal fluid
AP – action potential
ChAT – choline acetyltransferase
ChI – cholinergic interneuron
ChR2 – channelrhodopsin-2
CNS – central nervous system
CV – coefficient of variation
DAT – dopamine transporter dMSN – direct-pathway medium spiny neuron
EPSC – excitatory postsynaptic current
EPSP – excitatory postsynaptic potential
FSI – fast spiking interneuron
GIRK2 (Kir 3.2) – G-protein coupled inwardly rectifying potassium channel
GPCR – G-protein coupled receptor
GPe – globus pallidus (external)
GPi – globus pallidus (internal)
HCN – hyperpolarization-activated cyclic nucleotide-gated channel iMSN – indirect-pathway medium spiny neuron
9 IPSC – inhibitory postsynaptic current
IP3 – inositol trisphosphate
Kir 2 – inward rectifier potassium channel 2
KOR – kappa opioid receptor
LTD – long term depression
LTP – long term potentiation
LTSI – low threshold spiking interneuron
LRRK2 – leucine-rich repeat kinase 2 mAChR – muscarinic acetylcholine receptor
MOR – mu opioid receptor
MSN – medium spiny neuron
NAc – nucleus accumbens nAChR – nicotinic acetylcholine receptor
PD – Parkinson’s disease
Pf – parafascicular nucleus of the thalamus
PIP2 – phosphatidylinositol 4,5-bisphosphate
PKA – protein kinase A
PKC – protein kinase C
PLC – phospholipase C
PPR – paired pulse ratio
Pr – probability of release sEPSC – spontaneous excitatory postsynaptic current
10 sIPSC – spontaneous inhibitory postsynaptic current
SK – small conductance Ca2+-activated potassium channel
SNc – substantia nigra pars compacta
SNr – substantia nigra pars reticulata
TAN – tonically active neuron
TTX - tetrodotoxin uIPSC – unitary inhibitory postsynaptic current
VAChT – vesicular acetylcholine transporter
VTA – ventral tegmental area
2-AG – 2-arachinonoylglycerol
11 Cholinergic interneuron mediated activation of G-protein coupled
receptors in the dorsal striatum
Abstract
by
APHRODITI A. MAMALIGAS
The striatum serves as the main integration point for the basal ganglia circuit, which is critical for driving reward, movement, and associative behaviors.
While these basal ganglia inputs drive changes in striatal output, cholinergic interneurons are also strong modulators of striatal activity. These cells are thought to be important for behavioral flexibility and striatal-dependent learning.
Cholinergic interneurons modulate medium spiny neurons, the sole striatal output neuron, through multiple circuits. However, acetylcholine in this region activates receptors resulting in G-protein coupled receptor mediated modulation of medium spiny neurons. Due to the slow, multistep nature of G-protein coupled receptors and the lack of rapid acetylcholine measurement techniques, it has been challenging to investigate striatal acetylcholine transmission. The objective of this work was to examine nicotinic receptor-mediated dopamine release and D2 dopamine receptor activity as well as muscarinic M4 receptor activity on medium spiny neurons to determine the mechanisms of acetylcholine release in the striatum. This work used a combination of viral-mediated gene transfer,
12 electrophysiology, optogenetics, and immunohistochemical and 2-photon imaging to examine acetylcholine signaling in this region. First, I used viral overexpression of a G-protein inwardly rectifying potassium channel in medium spiny neurons to measure D2 receptor activity in the context of nicotinic- mediated dopamine release. I found that, although synchronous cholinergic interneuron firing is sufficient to drive dopamine release and subsequent D2 receptor activation, acetylcholine release does not modulate direct dopamine release from dopamine terminals. Next, I examined direct synaptic acetylcholine release at muscarinic synapses. I found, for the first time, that muscarinic receptors on medium spiny neurons could encode single action potentials and physiological firing patterns in cholinergic interneurons, that this occurred on a millisecond timescale, and that this receptor activation could change collateral transmission between striatal cells within milliseconds. Finally, I determined that excitatory inputs to the dorsal striatum from the cortex and thalamus differentially drove cholinergic interneuron firing, while evoking the same postsynaptic response at muscarinic synapses on medium spiny neurons. Overall, this work has shown that cholinergic interneuron firing can be encoded in receptors differentially in multiple different circuits and that postsynaptic receptor activity is rapid and stereotyped.
13
CHAPTER 1
INTRODUCTION
14 Introduction
Neuromodulation in the striatum is critical for information processing in the basal ganglia circuit. Inputs regulating excitability in this region arise from both motor and reward association areas such as the sensorimotor cortex, intralaminar thalamic nuclei, and other limbic regions, as well as neuromodulatory inputs from the midbrain (Gerfen, 1992). In addition to these regulatory inputs, cholinergic interneurons (ChIs) play an important role in modulation of striatal medium spiny neurons (MSNs), the principal output neurons of the striatum. ChIs, also called tonically active neurons or TANs, are the primary source of acetylcholine (ACh) release in the striatum (Woolf and
Butcher, 1981). Changes in ChI activity are correlated with aversive and rewarding salient stimuli in an animal’s environment (Atallah et al., 2014; Morris et al., 2004). Although these cells make up a small percentage of striatal neurons
(1-3%), they are critical for striatum-dependent associative behaviors (Bradfield et al., 2013; Stalnaker et al., 2016). Cholinergic transmission has been studied for decades, including in vivo and in vitro examination of ChI firing patterns, anatomy, role in striatal microcircuits, and post-synaptic receptor signaling.
Striatal ACh is known to activate G-protein coupled receptors (GPCRs) through multiple distinct circuits (Goldberg et al., 2012; Zhou et al., 2001).
Although previous work has studied ChI activity and the role of striatal
ACh receptors, little is known about the mechanisms of ACh release and GPCR activation at striatal synapses on MSNs. This work aims to examine the
15 dynamics synaptic ACh transmission at striatal cholinergic synapses and to determine how cholinergic receptors encode different ChI firing patterns. The objectives of this study are to examine 1) the role of ChI activity in activating D2 dopamine receptors following di-synaptic nicotinic receptor-mediated dopamine release, 2) the time-course of synaptic ACh transmission at striatal muscarinic synapses, 3) activation of muscarinic receptors in response to physiological ChI firing, and 4) excitatory drive of ACh release onto MSNs from convergent ChIs.
Striatal microcircuitry
As the integration point for the basal ganglia circuit, the striatum is a critical structure in information processing for movement, reward, and associative behaviors. In the dorsal aspect of the striatum, which is the focus of the present work, excitatory inputs from the sensorimotor cortex as well as from the parafascicular nucleus of the thalamus (Hintiryan et al., 2016; Hunnicutt et al.,
2016) as well as neuromodulatory dopamine inputs from the midbrain (Gerfen,
1992) converge to regulate excitability of striatal neurons (Figure 1.1). Unlike other brain structures that perform a larger amount of computational work, the striatum is largely unlaminated and disorganized. GABAergic MSNs comprise the vast majority of striatal neurons (~95%), half of which are direct-pathway MSNs
(dMSNs) and half of which are indirect-pathway MSNs (iMSNs) (Gerfen, 1992;
Kreitzer, 2009). These cells are distributed evenly throughout the striatum, as are all striatal cell types. Direct-pathway MSNs project to the substantia nigra pars
16 reticulata (SNr) and the internal aspect of the globus pallidus (GPi), leading to dis-inhibition of basal ganglia-recipient thalamic structures downstream and thus increased cortical activity (Goldberg et al., 2013; Mink, 1996; Sesack and Grace,
2010). Indirect pathway MSNs, on the other hand, inhibit cortical activity through inhibition of the external aspect of the globus pallidus, leading to GPi/SNr dis- inhibition, and thalamic activation (Goldberg et al., 2013; Mink, 1996; Sesack and
Grace, 2010). In addition to sending their projections to downstream basal ganglia structures, MSNs also exhibit a collateral inhibition network within the striatum whereby both dMSNs and iMSNs can directly inhibit one another (Koos et al., 2004; Taverna et al., 2008; Tecuapetla et al., 2007; Yamamoto et al.,
2013). Dorsal striatal activity through these distinct MSN circuits are important for creating action sequences, or action “chunking” (Jin et al., 2014). MSNs from these different pathways also display a different complement of receptors and peptide transmitters. Striatal dMSNs express D1 dopamine receptors, dynorphin, substance P, and M4 muscarinic receptors (Kreitzer, 2009). Indirect-pathway
MSNs express D2 dopamine receptors, A2A adenosine receptors, and enkephalin. Although it has been canonically thought that dMSNs and iMSNs correspond to the “GO” and “NO-GO” basal ganglia pathways, respectively
(DeLong, 1990; Gerfen, 1992; Kravitz et al., 2010; Tai et al., 2012), recent work has challenged the distinctive nature of these two cell types and pathways
(Tecuapetla et al., 2016). Instead, these two pathways seem to work in concert to
17 create behavioral ensembles, dependent on activity of both types of MSNs
(Klaus et al., 2017; Tecuapetla et al., 2016).
Despite the heterogeneity of these MSN populations in the striatum, they share a number of morphological as well as passive and active properties that have led to many studies treating them as a single population. As both dMSNs and iMSNs exhibit identical morphology, such as spiny dendrites, and share similar activity patterns, they were difficult to differentiate before the common use of Cre-driven fluorescent protein expression. MSNs have a low input resistance and resting membrane potential (~ -90 mV) in the absence of synaptic inputs
(Kreitzer, 2009), which is due to high expression of inwardly rectifying potassium
(Kir) channels (Nisenbaum et al., 1996). This hyperpolarized resting potential results in two different patterns of MSN activity in vivo. At rest, MSNs exist in the
“down” state, in which they remain hyperpolarized between -70 and -90 mV and are unable to fire action potentials (Wilson and Groves, 1981). Excitatory synaptic input from the cortex and thalamus allow for the transition of MSNs from their resting “down” state into a more excitable “up” state, allowing for action potential firing at threshold (Wilson and Groves, 1981; Wilson and Kawaguchi,
1996). In the striatum, neuromodulators can promote or inhibit transitions between down and up states.
In addition to MSNs, interneurons in the striatum modulate MSN activity.
GABAergic interneurons include low-threshold spiking interneurons (LTSIs) and fast-spiking interneurons (FSIs) (Koós and Tepper, 1999). Both types represent a
18 small percentage of striatal neurons (~ 1% for each type). These interneurons are inhibitory to MSN activity and can be subdivided into further classes of
GABAergic interneurons, including NPY-NGF expressing FSIs, which will be discussed in a later chapter (Koós and Tepper, 1999; Tepper et al., 2010).
Inhibition of GABAergic signaling in the striatum leads to dystonias and dyskinesias in multiple animal models (Gittis et al., 2011; Yamada et al., 1995;
Yoshida et al., 1991). Finally, striatal ChIs are strong modulators of MSN activity, and they will be reviewed in detail below.
Striatal cholinergic interneuron activity and role in disease
Cholinergic interneurons make up the remainder of striatal neurons (~1-
3%). Although there is recent evidence that other cholinergic nuclei in the brainstem project to the striatum (Dautan et al., 2014), it has long been widely accepted that ChIs are the major source of ACh in the striatum (Bolam, 1984;
Woolf and Butcher, 1981). ChIs are tonically active in the absence of synaptic input, both in the slice and in vivo, exhibiting pacemaker firing at approximately 1
- 10 Hz (Aosaki et al., 1995; Bennett and Wilson, 1999; Kreitzer, 2009). Both hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and small conductance (SK) potassium channels tightly regulate these firing patterns
(Bennett et al., 2000). In addition, they fire bursts of action potentials (~15 Hz) in response to both rewarding and aversive stimuli in an animal’s environment
(Aosaki et al., 1995; Atallah et al., 2014; Morris et al., 2004). Striatal inputs from
19 the parafascicular nucleus of the thalamus are thought to be critical for generating this bursting activity in vivo (Matsumoto et al., 2001). Although ChIs comprise only a small percentage of striatal neurons, increased ChI activity can evoke striatal-dependent associative behaviors (Bradfield et al., 2013; Stalnaker et al., 2016).
In addition, pathological changes can occur in ChIs during basal ganglia- dependent neurological diseases. Patients with Tourette’s syndrome display decreased numbers of ChIs, and ablating these cells causes symptoms mimicking the disorder (Kataoka et al., 2010; Xu et al., 2015). Further, in human patients with Huntington’s disease, there is a decrease in the activity of choline acetyltransferase (ChAT), the synthetic enzyme for ACh, as well as decreased concentration of striatal muscarinic receptors (Hiley and Bird, 1974; Suzuki et al.,
2001).
ChIs have also long been implicated in Parkinson’s disease (PD). During late stages of PD, ChIs can develop a-synuclein inclusions due to their high expression of leucine-rich repeat kinase 2 (LRRK2), mutations of which cause familial forms of PD (Mori et al., 2008). However, during the majority of PD disease progression, changes in ChI transmission create a hypercholinergic state, and this is recapitulated in rodent models of dopamine depletion (DeBoer et al., 1993; Lim et al., 2014). This increase in ACh concentration in the striatum could be caused by decreased expression of acetylcholinesterase (AChE), the strongly expressed degradative enzyme of ACh (Gonzales and Smith, 2015; Lim
20 et al., 2014). Although there is no overall change in ChI firing, ChI activity in response to salient stimuli is also hindered in primate PD models (Gonzales and
Smith, 2015). Finally, ChIs exhibit decreased connectivity with dMSNs but increased connectivity with iMSNs, causing a shift in basal ganglia signaling at the striatal level (Gittis et al., 2011; Salin et al., 2009).
This relationship between dopamine loss and changes in the striatal cholinergic system has led to the acetylcholine-dopamine balance hypothesis.
ChIs are capable of driving dopamine release through presynaptic nicotinic receptors (Cachope et al., 2012; Threlfell et al., 2012), while dopamine inhibits
ChI activity through D2 dopamine receptors expressed on ChIs (Chuhma et al.,
2014; Straub et al., 2014). This relationship leads to opposing firing patterns of dopamine neurons in the SNc and ChIs, in which ChIs burst before and after increases in dopamine neuron activity (Morris et al., 2004). The balance of both of these neuromodulators is thought to be important for MSN activity and transmission to downstream basal ganglia regions (Aosaki et al., 2010).
ChI anatomy and distribution across the striatum
Unlike other striatal neuron subtypes, ChIs are easily distinguishable due to their large soma size (15-20 µm), hyperpolarization induced sag, and tonic activity (Kreitzer, 2009; Lim et al., 2014). These neurons have highly branching axonal arbors, with some estimates of 500,000 presynaptic release sites along axons that can span up to 500 microns (Bolam, 1984; Contant et al., 1996;
21 Descarries et al., 2000). Due to these axonal structures, single ChIs are able to modulate large swaths of the striatum. In addition, their axons likely overlap, as
ChIs in the dorsal striatum are positioned, on average, between 40 and 70 microns away from one another (Matamales et al., 2016). Despite the far- reaching coverage of ChI terminals, only a small percentage are thought to make synaptic connections on MSNs (Bolam, 1984; Contant et al., 1996; Descarries et al., 2000). Nonetheless, tracing studies using rabies virus to trans-synaptically label connected neurons have found that approximately 3 ChIs synapse onto an area of 100 MSNs (Salin et al., 2009). Many convergent basal ganglia inputs to the striatum are subject to cholinergic regulation. ACh can activate both nicotinic
(nAChRs) and muscarinic receptors (mAChRs), which can both contribute to modulation of striatal output. These receptors are present on both striatal inputs as well as MSNs and other GABAergic interneurons. The striatum is largely unlaminated, so ChIs, as well as other striatal neuron types discussed above, are dispersed relatively evenly throughout this region. As a result, ACh can be released across the striatum to regulate output activity. Thus, ACh release is likely linked to striatal-dependent behaviors through multiple different circuits.
Striatal ACh release and signaling
ACh release plays a role in the majority of striatal circuits through various mechanisms. As a result, acetylcholinesterase expression is higher in this region than any other in the central nervous system (Hebb and Silver, 1961; Macintosh,
22 1941). Striatal ACh can activate ligand-gated nicotinic receptors present on dopamine inputs from the midbrain as well as on striatal GABAergic interneurons
(Cachope et al., 2012; English et al., 2011; Nelson et al., 2014a; Threlfell et al.,
2012), and these can evoke GABA and dopamine release onto MSNs (Figure
1.1). In addition to ligand-gated nicotinic receptors, metabotropic muscarinic receptors (mAChRs) are widespread throughout the striatum, expressed on excitatory inputs as well as ChIs and MSNs themselves (Goldberg et al., 2012;
Hersch et al., 1994) (Figure 1.1). Each muscarinic subtype is expressed in the striatum (Hersch et al., 1994). M1, M3, and M5 subtypes are Gq-coupled, and are thought to increase neuronal activity through mobilization of IP3 and release of intracellular Ca2+ stores, leading to depolarization (Caulfield, 1993; Goldberg et al., 2012). Inhibitory M2/M4 receptors are Gi/o-coupled, and their activation inhibits adenylyl cyclase, thus decreasing protein kinase A (PKA) and MAPK activity downstream (Caulfield, 1993; Goldberg et al., 2012). This combination of neuromodulatory receptors and modulation sites occurs through various ACh signaling pathways and thus evokes complex MSN regulation.
Modulation of striatal inputs and interneurons
ChI-mediated release of ACh at nicotinic sites on dopamine terminals and
GABAergic interneurons has been well-studied (Cachope et al., 2012; English et al., 2011; Nelson et al., 2014a; Sullivan et al., 2008; Threlfell et al., 2012; Zhang and Sulzer, 2004; Zhou et al., 2001). Synchronous ChI activation is required to
23 drive nAChR activity and subsequent di-synaptic transmitter release through these pathways (English et al., 2011; Nelson et al., 2014a; Threlfell et al., 2012). nAChR activity in response to synchronous ChI firing can drive both canonical dopamine release, measured extrasynaptically via fast-scan cyclic voltammetry, as well as GABA co-release from dopamine terminals across striatal subregions
(Cachope et al., 2012; Nelson et al., 2014a; Threlfell et al., 2012). nAChR activity on these dopamine inputs is strongly governed by receptor desensitization, as these receptors take tens of seconds to recover from desensitization (Cachope et al., 2012; Yu et al., 2009). Nicotinic receptors on dopamine terminals are thought to be desensitized at brain nicotine concentrations reached in habitual smokers
(250-300 nM) (Zhou et al., 2001). Because of this desensitization, previous studies have shown that nAChRs provide frequency dependent facilitation of dopamine release, such that they have a strong effect at low dopamine neuron firing rates and a weak effect during dopamine neuron bursting activity (Threlfell et al., 2012; Zhang and Sulzer, 2004).
In addition to dopamine inputs, nAChRs are also expressed on NPY-NGF expressing fast spiking striatal interneurons, and optogenetic activation of ChIs drives GABA release onto MSNs through this pathway (English et al., 2011;
Sullivan et al., 2008). Nicotinic-evoked activation of NPY-NGF interneurons also leads to lateral inhibition of ChIs across the striatum (English et al., 2011;
Sullivan et al., 2008). a4b2 nicotinic receptors are primarily responsible for ChI- evoked dopamine and GABA release through both dopamine terminals and
24 GABA interneurons, as DHbE, a4b2 subunit-specific nicotinic receptor antagonist, is sufficient to eliminate transmission through all pathways (Cachope et al., 2012; English et al., 2011; Nelson et al., 2014a; Threlfell et al., 2012). In addition, following chronic nicotine treatment, inhibition of a4b2 nicotinic receptors using DHbE antagonism or nicotine-mediated desensitization can also up-regulate synaptic glutamate release (Xiao et al., 2009). This increase in the frequency of glutamatergic spontaneous excitatory post-synaptic currents
(sEPSCs) results from decreased nicotinic-evoked dopamine release and subsequent activity of D2/D3 receptors on excitatory striatal inputs when nAChRs are desensitized (Xiao et al., 2009).
In addition to cholinergic regulation of other cell types in the striatum,
M2/M4 muscarinic receptors are expressed on ChIs as well (Hersch et al., 1994).
These Gi-coupled receptors inhibit ChI activity through cyclic AMP-independent inhibition of N and P-type Ca2+ channels (Ding et al., 2006; Yan and Surmeier,
1996) as well as through activation of G-protein coupled potassium channels
(Calabresi et al., 1998). Application of mAChR agonists results in low-frequency, irregular ChI firing, eventually leading to ChI quiescence (Calabresi et al., 1998;
Ding et al., 2006). Dopamine depletion eliminates this response through upregulation of RGS4 (Ding et al., 2006). As ChIs also express Gi/o-coupled D2 dopamine receptors, this suggests that there may be interactions between these
GPCR signaling pathways within ChIs.
25 Muscarinic receptors are also expressed on inputs to striatal cells, and can modulate their activity. M2/M4 muscarinic receptors are present on the axon terminals of excitatory inputs into the striatum, primarily those projecting from upstream cortical structures (Calabresi et al., 2000; Hersch et al., 1994; Malenka and Kocsis, 1988). Cholinergic activation of these presynaptic receptors result in a decrease in glutamatergic synaptic and field potentials as well as inhibition of long term potentiation of corticostriatal synapses onto MSNs (Calabresi et al.,
1998; Malenka and Kocsis, 1988; Pancani et al., 2014). Similarly, probability of release from glutamatergic afferents is decreased in the presence of muscarinic receptor agonists (Barral et al., 1999; Higley et al., 2009). Presynaptic muscarinic receptor activity also leads to a decrease in basal multi-vesicular release from striatal glutamatergic inputs, leading to an overall decrease in synaptic potency of excitatory afferents (Higley et al., 2009).
Finally, there is some evidence that muscarinic receptors can modulate dopamine release in the striatum (Foster et al., 2016; Shin et al., 2015). One set of experiments has determined that activation of M5 receptors, which are expressed on dopamine terminals, is capable of increasing optogenetically- evoked dopamine release as well as glutamate co-release from dopamine terminals (Hnasko et al., 2010; Shin et al., 2015; Stuber et al., 2010; Tecuapetla et al., 2010). The same study hypothesized that M2/M4 receptor activation inhibits dopamine release evoked through electrical stimulation due to autoreceptors present on ChIs (Shin et al., 2015). However, other work has
26 proposed that this occurs through alternative circuits. These studies suggest that synchronously evoked striatal ACh can drive M4 muscarinic receptor activity on
MSNs, which, through an unknown mechanism, evokes release of the endocannabinoid 2-arachinonoylglycerol (2-AG), leading to a decrease of dopamine release via activation of CB2 cannabinoid receptors presumably expressed on dopamine terminals (Foster et al., 2016).
Cholinergic regulation of MSN muscarinic receptors
Following ACh release in the striatum, muscarinic M1 and M4 receptors are also activated on MSNs themselves. Although M1 receptors are ubiquitously expressed on both MSN subtypes, M4 receptors are predominantly found on indirect pathway MSNs (Hersch et al., 1994; Kreitzer, 2009). ChIs typically form relatively few synapses onto spine necks, dendritic shafts, and somata of MSNs, and all populations of MSN mAChRs are localized in these regions. Muscarinic receptors are also located on spine heads at asymmetrical synapses (Hersch et al., 1994). Whereas inhibitory M4 receptors are Gi/o-coupled, as discussed previously, excitatory M1 receptors are Gq-coupled signal via IP3 and DAG signaling cascades. A small percentage of MSNs also express low levels of Gq- coupled M3 receptors (Hersch et al., 1994), which are also Gq-coupled. Overall, muscarinic agonism on MSNs evokes activation of multiple conductances, leading to enhanced MSN firing via increased membrane resistance (Galarraga et al., 1999). However, previous work looking at the effects of muscarinic receptors on excitability of MSNs has been confounded by the co-expression of
27 these two types of receptors on dMSN populations. As drugs that are selective between muscarinic receptor subtypes are not commercially available, M1 and
M4 activity have been historically challenging to parse apart. However, more recent work has aimed to dissect the functions of these receptors on MSNs.
In MSNs, M1 receptor activation results in upregulated phospholipase C
(PLC) activity, decreasing PIP2 and leading to decreased KCNQ and inward rectifier potassium channel (Kir2) open probability in MSNs (Goldberg et al.,
2012; Shen et al., 2005). This causes depolarization of the resting membrane potential as well as increased spiking during “up” states (Goldberg et al., 2012;
Shen et al., 2005). M1 activity also decreases L-type Ca2+ channel activity in
MSNs via protein kinase C (PKC), leading to decreased transmitter release
(Goldberg et al., 2012; Howe and Surmeier, 1995; Perez-Rosello et al., 2005).
Decreasing M1 receptor activation can also inhibit long term depression (LTD) at corticostriatal synapses, and this is alleviated through dopamine D2 receptor- mediated inhibition of ChI firing (Wang et al., 2006). M4 receptors, on the other hand, signal through Gai/o proteins, leading to a decrease in adenylyl cyclase, ultimately causing a decrease in MEK/ERK signaling (Goldberg et al., 2012). This can eventually lead to inhibition of long term potentiation (LTP) in dMSNs (Shen et al., 2015). M4 receptors are also thought to balance adenylyl cyclase activation that occurs through dopamine D1 receptors, which are Gs-coupled, allowing for alleviation of L-DOPA induced dyskinesias (Shen et al., 2015). In addition, the bg subunit of M4-associated G proteins can directly inhibit voltage
28 gated Ca2+ channel 2.2/2.3 activity. Knockout of M4 receptors in neurons expressing the D1 receptor results in hyperactivity and more rapid sensitization to psychostimulants in rodents (Jeon et al., 2010; Ztaou et al., 2016). These mice display decreased anxiety behaviors, and muscarinic antagonists are no longer capable of ameliorating L-DOPA induced dyskinesias in these animals (Ztaou et al., 2016). Overall, mAChR activity in striatal MSNs plays a complex role in MSN activity. Because of the opposing roles of M1 and M4 receptors and their different expression patterns, ACh release likely strikes a balance of activity in direct vs indirect pathway neurons.
GPCR synapses and volume transmission in the central nervous system
The tonic firing, broad branching axonal arbors, and lack of distinct synapses made by ChIs has led to the hypothesis that ACh in the striatum signals via volume transmission (Bennett and Wilson, 1999; Contant et al., 1996;
Descarries and Mechawar, 2000). Canonically, GPCR synapses in the central nervous system (CNS) are thought to signal in this way due to the intrinsically slow kinetics of downstream GPCR cascades. Volume transmission is characterized by transmitter release at axonal sites from which spillover of transmitter is required to activate postsynaptic receptors (Agnati et al., 2010;
Fuxe et al., 2012). Due to diffusion across the synaptic space, low concentrations of transmitter then stimulate receptors. Neurotransmitters at GPCR synapses,
29 including striatal ACh and dopamine, have been measured extracellularly in the low nanomolar range (Descarries et al., 2000). This differs from typical synaptic transmission at ligand-gated synapses, in which transmitter is released and diffuses 20-50 nm across the synaptic cleft, leading to direct activation of ligand- gated ion channels at a high concentration. It is thought that tonic firing of ChIs evokes consistent, low concentrations of ACh, whereas phasic ChI bursting results in slow changes in MSN G-protein activity (Descarries et al., 2000). More recent work has shown that not all GPCR synapses signal via volume transmission (Courtney and Ford, 2014; Ford et al., 2010; Marcott et al., 2014).
Somatodendritic dopamine release in the midbrain as well as dopamine release in the striatum signal in a more point-to-point manner (Courtney and Ford, 2014;
Ford et al., 2010; Marcott et al., 2014).
Despite the anatomical evidence for volume transmission at muscarinic synapses, there is no electrophysiological measure of muscarinic receptor activity at the ChI-MSN synapse. At other GPCR synapses in the CNS, including in the hippocampus, midbrain dopamine neurons, and neocortex, G-protein inwardly rectifying potassium channels (GIRK channels) often couple to Gi/o- coupled GPCRs (Bacci et al., 2004; Cruz et al., 2004; Dutar et al., 1999;
Newberry and Nicoll, 1985; Torrecilla et al., 2002). Due to the multistep nature of
GPCR signaling, GIRK-mediated synaptic currents activate more slowly than do ligand-gated synaptic currents, on the order of 50-200 ms to 10% onset
(Courtney and Ford, 2014; Marcott et al., 2014). Although it remains unclear how
30 ACh transmission occurs at muscarinic striatal synapses, we have overcome this limitation by overexpressing GIRK channels not endogenously expressed in
MSNs.
Rationale
Due to their relevance in behavior and disease, ChIs have been studied extensively over the past few decades. Their firing patterns, anatomy, and roles in reward and striatal circuit function have been analyzed. However, ACh signaling in the striatum remains largely unclear due to the lack of electrophysiological readout of transmitter release at cholinergic synapses. All studies analyzing striatal ACh have either relied on microdialysis or tritiated ACh, which both measure bulk ACh concentrations accumulated over tens of minutes or hours. My thesis work aims to address the mechanisms of ACh release in the striatum at GPCR synapses, including M4 muscarinic synapses on MSNs as well as disynaptic, nicotinic receptor-evoked D2 dopamine receptor activation.
Although millisecond measurement of these GPCRs in MSNs is not possible electrophysiologically, I will use viral overexpression of GIRK2 channels to test the hypotheses that 1) synchronous ChI firing is sufficient to drive nicotinic- mediated dopamine release at D2 receptor-containing synapses, 2) that M4 muscarinic receptors can encode firing patterns of individual ChIs via point-to- point synaptic transmission, and 3) that excitatory inputs can drive M4 activity through ChI activation. This will provide the first example of measurement of
31 synaptic actions of ACh at GPCR synapses in the CNS, providing a new concept of GPCR signaling and a different way to examine pathological changes in ACh transmission.
32 Figure 1.1. Cholinergic signaling and GPCR activity in striatal microcircuits.
Striatal circuit showing dMSNs and iMSNs as well as their connectivity with striatal interneurons (ChIs – blue, GABA interneurons – red) and striatal inputs
(glutamate – green; dopamine – purple). GPCRs relevant for the present work are highlighted. Muscarinic receptors are represented in blue, and dopamine receptors are represented in purple.
33
Figure 1.1
34 CHAPTER 2
Nicotinic and opioid receptor regulation of striatal dopamine D2-
receptor mediated transmission
Mamaligas A.A., Cai, Y., and Ford, C.P. (2016). Nicotinic and opioid receptor
regulation of striatal dopamine D2-receptor mediated transmission. Scientific
Reports 6, 37834.
35 Abstract
In addition to dopamine neuron firing, cholinergic interneurons (ChIs) regulate dopamine release in the striatum via presynaptic nicotinic receptors
(nAChRs) on dopamine axon terminals. Synchronous activity of ChIs is necessary to evoke dopamine release through this pathway. The frequency- dependence of disynaptic nicotinic modulation has led to the hypothesis that nAChRs act as a high-pass filter in the dopaminergic microcircuit. Here, we used optogenetics to selectively stimulate either ChIs or dopamine terminals directly in the striatum. To measure the functional consequence of dopamine release, D2- receptor synaptic activity was assessed via virally overexpressed potassium channels (GIRK2) in medium spiny neurons (MSNs). We found that nicotinic- mediated dopamine release was blunted at higher frequencies because nAChRs exhibit prolonged desensitization after a single pulse of synchronous ChI activity.
However, when dopamine neurons alone were stimulated, nAChRs had no effect at any frequency. We further assessed how opioid receptors modulate these two mechanisms of release. Bath application of the κ opioid receptor agonist U69593 decreased D2-receptor activation through both pathways, whereas the μ opioid receptor agonist DAMGO decreased D2-receptor activity only as a result of cholinergic-mediated dopamine release. Thus the release of dopamine can be independently modulated when driven by either dopamine neurons or cholinergic interneurons.
36 Introduction
Striatal dopamine is a critical component of basal ganglia dependent movement and motivational behaviors (Schultz, 2007). Dopamine neurons exhibit a stereotyped burst-firing pattern in response to rewarding stimuli and their cues (Grace and Bunney, 1984a, 1984b; Schultz, 2007) and provide synaptic input onto medium spiny neurons (MSNs), the main output neuron of the striatum (Kreitzer, 2009). In addition to dopamine neuron firing, striatal dopamine release can be driven by the activation of presynaptic nicotinic receptors
(nAChRs) on dopamine terminals (Cachope et al., 2012; Kress et al., 2014;
Quarta et al., 2007; Threlfell and Cragg, 2011; Zhou et al., 2001). The major source of acetylcholine (ACh) in the striatum originates from local cholinergic interneurons (ChIs) (Bolam, 1984). Recent work has shown that optogenetic stimulation of ChIs is sufficient to drive the release of dopamine (Cachope et al.,
2012; Threlfell et al., 2012), suggesting that synchronous activity of these striatal neurons evokes dopamine release independently of midbrain dopamine neuron impulse activity.
Studies using local electrical stimulation of the striatum have shown that inhibiting nicotinic receptors modulates the release of dopamine to a greater extent at low frequencies of stimulation (Rice and Cragg, 2004; Zhang and
Sulzer, 2004; Zhou et al., 2001). This work suggests that presynaptic nAChRs may function to enhance the contrast of dopamine release during different patterns of dopamine neuron firing (Rice and Cragg, 2004; Zhang and Sulzer,
37 2004). However, electrical stimulation simultaneously evokes dopamine release from both dopamine terminals and through ACh-mediated release. This pattern differs from the burst firing patterns of these two neurons, which occur out of phase. At rest, ChIs fire tonically at low frequencies (2-10 Hz) (Bennett and
Wilson, 1999; Kreitzer, 2009). However, in response to both rewarding and aversive salient stimuli, ChIs exhibit a pause in tonic firing followed by a burst pattern that opposes reward-evoked dopamine neuron burst firing (Atallah et al.,
2014; Morris et al., 2004). As a result, it still remains unclear how ChIs regulate dopamine release during when dopamine terminals are stimulated independently.
To test the role of nicotinic receptors in frequency-dependent dopamine release onto D2-receptors in MSNs, we used optogenetics to separate the contribution of cholinergic and dopaminergic dopamine release while recording
D2-receptor mediated inhibitory postsynaptic currents (D2-IPSCs).
Overexpressed G-coupled inwardly rectifying potassium (GIRK) channels provided an electrophysiological sensor of D2-receptor activation in striatal MSNs
(Marcott et al., 2014). Our results confirmed that nicotinic receptors potentiate dopamine release and subsequent D2-receptor activation in the striatum.
However, due to rapid desensitization, nAChRs only facilitated dopamine release on the first stimulus of a synchronous burst. In addition, tonic firing of ChIs did not affect the release of dopamine when dopamine terminals were directly stimulated. Lastly, we found that due to the distinct expression patterns of opioid
38 receptors in the striatum, mu opioid receptors (MORs) modulated only ChI- mediated dopamine transmission, while kappa opioid receptors (KORs) regulated transmission equally through both pathways.
Experimental Procedures
Stereotaxic injections: All animal procedures and protocols were approved by the
Institutional Animal Care and Use Committee at Case Western Reserve
University (CWRU IACUC, protocol # 2014-0012). All experiments were performed in accordance with the appropriate guidelines of the Institutional
Animal Care and Use Committee at Case Western Reserve University.
Wild-type C57BL6, ChAT-internal ribosome entry site-Cre heterozygote (ChAT-
Cre), or DAT-Cre transgenic heterozygote mice were injected at post-natal day
21. All animals were obtained from the Jackson Laboratory (Bar Harbor, ME).
Sterotaxic surgeries were performed using a model 1900 stereotax (Kopf).
Sterotaxic surgeries were performed under isoflurane anesthesia. Briefly, a small craniotomy was made using a 33-gauge drill bit above the desired coordinate. A small pulled glass pipette containing AAV was attached to a Nanoject II
(Drummond) and was then inserted to the appropriate depth. Injections were performed at a rate of 90 nl/minute. The coordinates used for striatal injections were (relative to bregma): AP +1.15 mm, ML -1.85 mm, DV -3.325 mm. A volume of 300 nL of AAV9.hSyn.tdTomato.T2A.mGIRK2-1-A22A.WPRE.bGH was injected into one hemisphere of all mice. To optogenetically activate ChIs, 300 nL
39 of AAV5.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH was combined with the
300 nL of AVV.GIRK2 and injected at the same time in ChAT-Cre mice. To optogenetically activate dopamine terminals, 500 nL of
AAV5.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH was separately injected into one side of the midbrain of DAT-Cre mice. Midbrain coordinates for the SNc/VTA
(relative to bregma) were: AP -2.3 mm, ML -0.55 mm, DV -4.7 mm. All AAVs were obtained from the University of Pennsylvania Viral Core. Animals were allowed to recover for ~2-4 weeks to allow for viral expression.
Slice preparation: Coronal slices (240 μM) containing the striatum were made in ice-cold cutting solution containing (in mM): 75 NaCl, 2.5 KCl, 6 MgCl2, 0.1
CaCl2, 1.2 NaH2PO4, 25 NaHCO3, 2.5 D-glucose, and 50 sucrose; bubbled with
95% O2 and 5% CO2. Slices were incubated at 35°C for at least 45 minutes in
ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2
NaH2PO4, 21.4 NaHCO3, and 11.1 D-glucose; and 10 μM MK-801 bubbled with
95% O2 and 5% CO2. Slices were transferred to a recording chamber and perfused with warm ACSF (34 ± 2°C) at 2 mL/minute. Perfusion solution contained picrotoxin (100 μM), DNQX (10 μM), CGP 55845 (300 nM), SCH
23390 hydrochloride (1 μM), and scopolamine (500 nM) to block GABAA, AMPA,
GABAB, dopamine D1 and muscarinic receptors respectively. MSNs were visualized using a BXWI51 scope (Olympus) with custom-built gradient contrast optics using a near-IR LED (Thor Labs).
40 Electrophysiology: Whole-cell recordings were performed using either Axopatch
200A or Axopatch 200B amplifiers (Molecular Devices). Membrane potentials were not corrected for liquid junction potentials. Patch pipettes (1.5-2 MΩ) were made from borosciliate glass capillary tubes (World Precision Instruments). Patch pipettes for MSNs contained (in mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl2,
10 HEPES(K), 10 BAPTA-tetrapotassium. Patch pipettes for ChIs contained (in mM): 135 D-gluconate(K), 10 HEPES(K), 0.1 CaCl2, 2 MgCl2, 0.1 EGTA. All internal patch solution contained: 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine (pH 7.35, 275 mOsm). Membrane potential was held at -60 mV during voltage clamp recordings. Cells were discarded if their series resistance exceeded 15 MΩ, as series resistance was not compensated. ChIs were identified by the presence of an h-current when stepped to -90 mV. Data was acquired with Axograph X (Axograph Scientific) at 5 kHz. Voltage clamp recordings were filtered to 2 kHz. Electrical stimulation (0.7 ms) was applied with a monopolar extracellular stimulating electrode filled with ACSF (World Precision
Instruments). Optogenetic stimulation was evoked with 2 ms widefield illumination (470 nm) using a custom made LED (470 nm Rebel LED Star Saber,
Luxeon Star). All drugs were applied via bath application unless otherwise specified. All recordings were made in the dorsal striatum.
Materials: DNQX, picrotoxin, MK-801, and naloxone were obtained from Ascent
Scientific. SCH 23390, CGP 55845, scopolamine hydrobromide, ambenonium, dihydro-b-erythroidine hydrobromide (DHβE), mecamylamine, sulpiride, and
41 DAMGO were obtained from Tocris Bioscience. K-methylsulfate was from Acros
Organic. BAPTA was from Invitrogen. All other chemicals were from Sigma-
Aldrich.
Statistics and analysis: Data are shown as mean ± SEM. Statistical significance was assessed using either Wilcoxon matched-pairs signed rank test, Mann-
Whitney test, or one-way ANOVA where appropriate (InStat 3.0, Graphpad).
Results
Activation of D2 receptors in MSNs
To examine D2-receptor activation in response to nicotinic receptor activity, we overexpressed G-protein coupled inwardly rectifying K+ (GIRK2) channels in striatal MSNs (Marcott et al., 2014). Injection of an adeno-associated virus (AAV) encoding GIRK2 and a soluble td-Tomato fluorophore resulted in expression of both proteins in MSNs (Mamaligas and Ford, 2016; Marcott et al.,
2014). After allowing three weeks for expression, coronal striatal slices were obtained from AAV-injected mice. Viral expression was observed in both direct and indirect pathway MSNs (Mamaligas and Ford, 2016; Marcott et al., 2014), but was restricted from ChIs likely as a result of poor efficiency of transfection of
ChIs using a synapsin promoter (Mamaligas and Ford, 2016). It was not examined whether other GABAergic interneurons expressed GIRK following AAV injection. All experiments in the present study were performed in the presence of antagonists for GABAA, GABAB AMPA, NMDA, dopamine D1, and muscarinic
42 receptors. Td-Tomato+ MSNs were voltage-clamped at a holding potential of -60 mV. As about half of striatal MSNs belong to the D2-receptor expressing indirect pathway (Gerfen, 1992), D2-receptor mediated GIRK2 currents were observed in roughly half of GIRK2+ MSNs (Marcott et al., 2014).
Electrical stimulation within the striatum evoked a D2-receptor mediated inhibitory post-synaptic current (D2-IPSC) in indirect pathway GIRK2+ MSNs that was attenuated by the α4β2 nicotinic receptor subtype antagonist DHβE (1 μM;
40.6 ± 5% inhibition, n = 5, p < 0.05, W = 28, Wilcoxon) (Figure 2.1A and 2.1B).
Saturating concentrations of the broad spectrum nicotinic antagonist mecamylamine similarly inhibited D2-IPSCs by roughly half (1 μM: 42.5 ± 2% inhibition, n = 5, W = -15; 10 μM: 46.8 ± 2 % inhibition, n = 6, W = -21; p < 0.05 for both concentrations, Wilcoxon) (Figure 2.1B). As α7 subunit-containing nicotinic receptors are not abundantly present in the dorsal striatum (Seguela et al., 1993), mecamylamine and DHβE produced similar inhibition of D2-IPSCs (p
> 0.05, One-way ANOVA). Bath application of ambenonium, an acetylcholinesterase inhibitor, also caused a decrease in the amplitude of D2-
IPSCs (100 nM; 32.2 ± 2% inhibition, n = 8, p < 0.01, W = -36, Wilcoxon).
Electrochemical studies have found that nicotinic receptor antagonism evokes a substantial decrease in dopamine release (Rice and Cragg, 2004; Zhang and
Sulzer, 2004; Zhou et al., 2001). Similarly, the observed ~ 40% decrease in electrically evoked D2-IPSCs shows that saturating concentrations of nicotinic receptor antagonists are insufficient to completely eliminate D2-receptor
43 activation. Thus, electrical stimulation both directly stimulates midbrain terminals and also drives the di-synaptic release of dopamine through the activation of presynaptic nAChRs on dopamine terminals at D2-receptor synapses.
To separate cholinergic-mediated dopamine release from direct dopamine terminal driven release, we employed an optogenetic approach. We injected a double-floxed virus encoding the light activated cation channel channelrhodopsin-2 (ChR2, AAV.DIO.ChR2.eYFP) into either the substantia nigra (SNc) of DAT-Cre mice or into the striatum of ChAT-IRES-Cre mice.
Photostimulation of either dopamine terminals (DAT-Cre:ChR2) or ChIs (ChAT-
Cre:ChR2) with a single flash of blue light (470 nm, 2 ms) was sufficient to evoke
D2-IPSCs in indirect pathway GIRK2+ MSNs (Figure 2.1C and 2.1D). In both cases, IPSCs were eliminated by the D2-receptor antagonist sulpiride (400 nM; n
= 4; p < 0.05, U = 4, Mann-Whitney) (Figure 2.1E) and tetrodotoxin (200 nM;
ChAT-Cre:ChR2: 16.1 ± 3 pA, n = 4; DAT-Cre:ChR2: 17.4 ± 3 pA, n = 5). DHβE
(1 μM) eliminated ChAT-Cre:ChR2 evoked D2-IPSCs (n = 10, p < 0.01 relative to control, W = -55, Wilcoxon) (Figure 2.1D and 2.1E) but did not alter DAT-
Cre:ChR2 evoked D2-IPSCs (n = 15, p > 0.05, W = -32, Wilcoxon; p < 0.0001 versus the inhibition of ChAT-Cre:ChR2 evoked IPSCs, U = 0, Mann-Whitney)
(Figure 2.1C and 2.1E). Thus, while synchronous activity of ChIs can drive the release of dopamine through nicotinic receptors (Cachope et al., 2012; Threlfell et al., 2012), the background tonic firing of ChIs did not modulate the release of dopamine when SNc terminals were directly activated.
44 We next examined whether a single action potential in a ChI was sufficient to evoke D2-receptor activation. To test this, we made paired recordings of ChIs
(current clamp) and GIRK2+-expressing MSNs (voltage clamp). ChIs were hyperpolarized to just below threshold during these recordings to prevent tonic firing. To increase the resolvable amplitude of paired IPSCs, dopamine transporters were inhibited with the dopamine transport blocker cocaine (10 μM).
While electrical stimulation evoked robust D2-IPSCs, single action potentials in
ChIs failed to elicit D2-IPSCs (Figure 2.1F and 2.1G). Single action potentials also did not evoke an IPSC when recorded in the presence of ambenonium (100 nM), which inhibits acetylcholinesterase thereby increasing the concentration of striatal ACh (4.7 ± 2 pA, n = 5). This confirms that multiple ChIs activated together are required to engage nicotinic receptor mediated dopamine release
(Threlfell et al., 2012).
Frequency dependence of cholinergic-induced dopamine release
Past work has demonstrated that nicotinic receptors provide stronger modulation of dopamine release during low frequency electrical stimulation
(Zhang and Sulzer, 2004). To examine this through the activation of D2- receptors, we compared the effect of a single electrical stimulus to bursts of stimuli (5 at 40 Hz). Bursts evoked larger amplitude D2-IPSCs than single stimuli
(burst 125.8 ± 5% of single stimulation amplitude, n = 14, p < 0.001, W = 91,
Wilcoxon) (Figure 2.2A and 2.2I) (Gonon and Buda, 1985). In addition, DHβE (1
45 μM) inhibited electrically evoked D2-IPSCs less when driven by bursts than single stimuli (single: 39.2 ± 4% inhibition, n = 4; burst: 13 ± 4% inhibition, n = 6; p < 0.01, Mann-Whitney) (Figure 2.2B and 2.2C). This indicates that ChI- mediated dopamine transmission contributed less during high frequency stimulation and is consistent with the high-pass filter model describing nicotinic modulation of dopamine release that has been seen before with electrical stimulation (Zhang and Sulzer, 2004).
In vivo, ChIs and dopamine neurons fire in opposing patterns during reward-learning tasks (Atallah et al., 2014; Morris et al., 2004). Phasic firing of dopamine neurons drives pauses in the tonic activity of ChIs through the activation of D2-receptors (Chuhma et al., 2014; Ding et al., 2010; Shen et al.,
2007; Straub et al., 2014). As ChIs and dopamine neurons do not fire bursts at the same time, we next examined the frequency dependence of dopamine release driven by optogenetic activation of dopamine terminals or ChIs individually. In DAT-Cre:ChR2 expressing mice, bursts of photostimulation of dopamine terminals evoked D2-IPSCs that were larger in amplitude relative to single flashes (DAT-Cre:ChR2: 149.2 ± 10% of single flash amplitude, n = 15, p <
0.0001, W = 120, Wilcoxon) (Figure 2.2D and 2.2I). DHβE (1 μM) did not alter the amplitude of DAT-Cre:ChR2-evoked D2-IPSCs (p > 0.05, n = 13-15, U = 72,
Mann-Whitney) (Figure 2.2E and 2.2F). Thus, tonic nicotinic receptor activity is insufficient to modulate dopamine release at D2-receptor synapses. In contrast, bursts of photostimulation of ChIs in ChAT-Cre:ChR2-expressing slices did not
46 potentiate D2-IPSCs (ChAT-Cre:ChR2: 96.8 ± 3% of single flash amplitude, n =
11, p > 0.05, W = -16, Wilcoxon) (Figure 2.2G and 2.2I). In all cases, DHβE (1
μM) eliminated ChI-driven D2-IPSCs (bursts: 4.0 ± 1% of baseline, p < 0.05, n =
5, W = -15) (Figure 2.2H). Thus, cholinergic-mediated dopamine release does not facilitate with bursts. As a result, nicotinic receptors only facilitate dopamine release during the first pulse of synchronous ChI burst activity.
To determine the kinetics of nAChR desensitization, we next examined the time course of recovery of D2-IPSCs. Paired pulse experiments revealed that
ChAT-Cre:ChR2 evoked IPSCs took significantly longer to recover than DAT-
Cre:ChR2 evoked IPSCs (Figure 2.3). As D2-IPSCs evoked by direct dopamine terminal stimulation recovered faster than D2-IPSCs evoked by ChI-stimulation,
(Figure 2.3B), the long recovery of ChAT-Cre:ChR2 evoked D2-IPSCs was likely not due to intrinsic properties of dopamine release. We found that ChAT-
Cre:ChR2 IPSCs evoked at a 1 second interpulse interval were almost completely abolished, while DAT-Cre:ChR2 IPSCs only decreased by half their amplitude (1 sec PPR: DAT 0.49 ± 0.02, ChAT 0.09 ± 0.01, n = 6 for both groups, p < 0.01, U = 0, Mann-Whitney). This difference in PPR suggests that nicotinic receptors on SNc terminals likely exhibit a slow recovery from desensitization in response to synchronous ACh release (Yu et al., 2009). Together, these data indicate that nicotinic receptors on dopamine terminals positively modulate D2- receptor transmission during synchronous ChI firing yet desensitize at high frequencies.
47
Opioid modulation of striatal D2-IPSCs in MSNs
Mu, kappa and delta opioid receptors are expressed in the striatum where they regulate transmission through multiple circuits (Banghart et al., 2015; Miura et al., 2008; Ponterio et al., 2013). Although mu and delta opioid agonists induce reward behavior (Devine and Wise, 1994), kappa opioid agonists are aversive in animal models (Shippenberg and Herz, 1986; Spanagel et al., 1994). Mu and delta receptors are both expressed postsynaptically on MSNs. Mu opioid receptors (MORs) are present on ChIs (Arenas et al., 1990; Jabourian et al.,
2005; Ponterio et al., 2013; Svingos et al., 2001) while kappa opioid receptors
(KORs) are on dopamine terminals (Schwarzer, 2009; Williams et al., 2001).
Both mu and kappa receptors have been shown to modulate striatal dopamine release (Britt and McGehee, 2008). To examine how these two opioid receptors regulate D2-IPSCs in MSNs, we applied agonists of either MORs or KORs while stimulating either dopamine terminals or ChIs. As MORs are located on CHIs, we found that bath application of the MOR antagonist DAMGO (1 μM) did not alter
DAT-Cre:ChR2 evoked D2-IPSCs (2.3 ± 5% inhibition, n = 5, p > 0.05, W = 7,
Wilcoxon) (Figure 2.4A) but strongly decreased the amplitude of D2-IPSCs evoked by ChI stimulation (42.4 ± 5% inhibition, n = 6, W = 21 p < 0.05,
Wilcoxon) (Figure 2.4B). During these experiments, we also observed an outward current of 157 pA in one of six MSNs resulting from MOR activation on the MSN itself. The inhibition of ChAT-Cre:ChR2 evoked D2-IPSCs by DAMGO
48 was reversed by the non-selective opioid receptor antagonist naloxone (1 μM, n
= 6; p < 0.05 vs control, W = 17, Wilcoxon). This indicates that MORs on ChIs selectively modulate dopamine transmission release through the nicotinic pathway (Britt and McGehee, 2008). In contrast, the KOR agonist U69593 inhibited D2-receptor activation from both pathways (42.2 ± 4% inhibition of DAT-
Cre:ChR2 evoked IPSCs, n = 6; 36.1 ± 6% inhibition of ChAT-Cre:ChR2 evoked
IPSCs, n = 8; W = 21, p < 0.05 for both groups) (Figure 2.5A and 2.5B). Thus,
KORs equally modulate dopamine release when either dopamine terminals or
ChIs are stimulated.
Discussion
As dopamine release in the striatum is critical for motivated behaviors and action selection, modulation of this release is an important regulator of striatal circuitry. Independent of dopamine neuron impulse activity, nicotinic receptors on striatal dopamine terminals also facilitate dopamine release (Cachope et al.,
2012; Threlfell et al., 2012). Using overexpressed GIRK2 channels in MSNs, we found that nicotinic receptors can modulate synaptic release of dopamine and subsequent D2-receptor activation. Direct comparison of D2-receptor activation in response to dopamine release driven through these two pathways revealed differential regulation of dopamine transmission.
Dopamine neurons typically fire at tonic low frequencies, and their firing switches to a bursting pattern in response to rewards and their cues (Grace and
49 Bunney, 1984a, 1984b; Schultz, 2007). Several studies have previously examined the role of nicotinic receptors as a high-pass filter of dopamine release at different firing frequencies using electrical stimulation (Rice and Cragg, 2004;
Threlfell et al., 2012; Zhang and Sulzer, 2004). This work has shown that nicotinic receptors strongly facilitate the release of dopamine at low stimulation frequencies (single pulse or 5 Hz) but have less of an effect (Zhang and Sulzer,
2004) or even depress dopamine release at higher frequencies (between 20 and
100 Hz) (Rice and Cragg, 2004). This nicotinic-mediated alteration of dopaminergic release probability was thought to result in contrast enhancement of dopamine release at different firing rates. However, dopamine neurons and
ChIs show opposing firing patterns in response to salient stimuli (Morris et al.,
2004) and striatal dopamine release induces a pause in ChI tonic activity
(Chuhma et al., 2014; Straub et al., 2014).Thus, it is unlikely that dopamine neurons and ChIs would be simultaneously and synchronously active in vivo. We found that, similar to the electrochemical measurements of Zhang and Sulzer
(2004), nicotinic receptors more strongly modulated dopamine release at low frequency electrical stimulation. However, by segregating these two pathways using optogenetics, we found that nicotinic receptors only act as high pass filters when both pathways are activated simultaneously. Selective stimulation of only dopamine terminals by activating ChR2 expressed in SNc axons of DAT-Cre mice revealed that D2-IPSCs exhibited no change in nicotinic receptor-mediated facilitation at any frequency examined. Thus, under conditions when dopamine
50 neurons and ChIs are not synchronously active, nicotinic receptors failed to modulate the release of dopamine that underlies the activation of D2-receptors.
High-affinity α4β2 nicotinic receptors on midbrain dopamine neurons rapidly desensitize in response to low concentrations of ACh or nicotine (Dani,
2015; Picciotto et al., 2008; Quick and Lester, 2002; Wooltorton et al., 2003). As such, it is thought that these receptors on dopamine terminals readily desensitize in response to the concentrations of ACh that are released when multiple ChIs are simultaneously active (Dani, 2015; Zhou et al., 2001, 2002). The reduced contribution of this di-synaptic pathway during high frequency cholinergic stimulation enables nicotinic receptors to only respond to the first synchronous stimulus of a burst (Cachope et al., 2012; Threlfell et al., 2012). Thus, trains of optogenetic ChI stimuli failed to evoke further dopamine facilitation through nicotinic receptors when compared to single stimulation.
As DAT-Cre:ChR2 evoked D2-IPSCs facilitated during high frequency optogenetic stimulation and recovered from paired-pulse depression at a faster rate than ChAT-Cre:ChR2 evoked IPSCs, our results reveal a ChI-mediated mechanism of depression rather than dopamine vesicle depletion or D2-receptor desensitization. Similar to the observed recovery of ChAT-Cre:ChR2 evoked
IPSCs, human α4β2 nAChRs expressed in heterologous systems can take ~20 seconds to recover their activity to normal levels in response to ACh and nicotine
(Yu et al., 2009). Further, ACh released onto MSNs at muscarinic synapses recovers within 10 seconds (Mamaligas and Ford, 2016), further suggesting that
51 cholinergic vesicle depletion is unlikely to be responsible for the observed paired pulse depression of ChAT-Cre:ChR2 evoked IPSCs.
ChIs are tonically active both in vivo and in the absence of synaptic inputs
(Bennett and Wilson, 1999), largely due to the presence of hyperpolarization- activated cyclic nucleotide gated (HCN) channels and small-conductance potassium (SK) channels (Bennett et al., 2000; Goldberg and Wilson, 2005). This has led to the idea that tonic ChI firing provides the striatum with a tone of ACh that activates postsynaptic receptors. We found that ACh tone in the slice provided little modulation of D2-receptor activation as DAT-Cre:ChR2 evoked
IPSCs showed little change in amplitude in response to nicotinic receptor antagonists. Importantly, tonic ChI activity does not release sufficient amounts of
ACh to desensitize nicotinic receptors, as optogenetic stimulation of ChIs could still evoke robust dopamine release. The lack of nicotinic modulation of DAT-
Cre:ChR2 evoked IPSCs may result from super-physiological release of dopamine in response to light activated ChR2 conductance in the axon terminal.
ChR2 is a light activated cation channel, stimulation of which causes an increased influx of Ca2+ in axon terminals compared with AP triggered release that has been shown to alter probability of release at other synapses (Zhang and
Oertner, 2007). As a result, one possibility may be that the effect of nicotinic receptors on dopamine terminals may have been occluded by ChR2-mediated release.
52 ACh tone may not affect dopamine release because acetylcholinesterase
(AChE), the degradative enzyme of ACh, is enriched in the striatum and highly efficient, effectively terminating ACh signaling before it can activate striatal nicotinic receptors (Zhou et al., 2001). Thus, in agreement with previous electrochemical studies (Threlfell et al., 2012), D2-IPSCs evoked from single action potentials in ChIs were unresolvable, whereas synchronous activity of
ChIs is required to evoke D2-IPSCs. As AChE is integral for phasic ACh transmission at muscarinic synapses (Mamaligas and Ford, 2016), it likely degrades ACh rapidly such that tonic ChI firing is insufficient to drive dopamine release through nicotinic receptors. Thus, AChE may have a major role in titrating nicotinic receptor activity in the striatum, as its activity decreases striatal
ACh while its inhibition desensitizes nicotinic receptors.
Within the striatum, mu, kappa and delta opioid receptors are widespread inhibitory modulators of striatal circuitry (Figure 2.6). Enkephalin is released from indirect pathway MSNs and activates delta receptors largely on indirect pathway
MSNs (Banghart et al., 2015; Kreitzer, 2009). In addition, striatal enkephalin can activate striatal MORs on MSNs (Banghart et al., 2015) and ChIs (Lendvai et al.,
1993). Dynorphin released primarily from direct pathway MSNs activates KORs to drive either reward or aversion in different striatal compartments (Al-Hasani et al., 2015). Electrochemical studies examining the effect of opioid receptors on striatal dopamine release have found that although KORs regulate dopamine release throughout the striatum, MOR regulation of dopamine release was
53 spatially segregated to specific areas (Britt and McGehee, 2008). We similarly saw that KORs decreased dopamine release from both dopamine terminals and through ChI-mediated release. The areas of MOR regulation that others have observed could potentially be delineated by the striatal patch and matrix, as
MORs are selectively expressed in the patch (Gerfen, 1992; Pert et al., 1976) as is acetylcholinesterase (Ragsdale and Graybiel, 1981). ChIs are thought to reside either in the matrix or on the interface of striatal patch and matrix compartments (Aosaki et al., 1995). However, they have far-reaching axons that cross the patch/matrix boundaries (Contant et al., 1996). It is possible that we reliably observe MOR-mediated inhibition of ChAT-Cre:ChR2 evoked D2-IPSCs because convergent ChIs activated during optogenetic stimulation extend their axons from MOR-expressing patches.
Together, this work suggests that the role of the burst-firing pattern of ChIs common following reward stimuli is to synchronize firing, allowing for transient, phasic dopamine release, rather than to alter gain of dopamine release in the striatum. This parallels in vivo microdialysis studies that have shown that nicotinic receptor antagonism specifically in the striatum causes a small decrease in dopamine concentration over time (Quarta et al., 2007). Based on our results, this change in striatal dopamine levels may be differentially gated through opioid receptors. These results further provide insight into the regulation of dopamine release through two different mechanisms.
54 Figure 2.1. Nicotinic receptor modulation of D2-receptor activation
A) Antagonism of α4β2 nicotinic receptors with DHβE (1 μM) attenuates electrically evoked D2-IPSCs. A single electrical stimulation (0.7 ms) was used to evoke the release of dopamine.
B) Quantification of electrically evoked D2-IPSC inhibition by the nAChR antagonists DHβE (1 μM) and mecamylamine (1 μM and 10 μM) (* = p < 0.05,
Wilcoxon matched-pairs signed rank and one-way ANOVA).
C) Optogenetic activation of dopamine terminals expressing ChR2 evokes D2-
IPSCs in GIRK2+ MSNs (DAT-Cre:ChR2, 2 ms flash, 470 nm light). D2-IPSCs evoked by photostimulation of dopamine terminals were unaffected by DHβE (1
μM, gray trace).
D) Synchronous optogenetic activation of striatal ChIs expressing ChR2 is sufficient to evoke D2-IPSCs in GIRK2+ MSNs (ChAT-Cre:ChR2, 2 ms flash, 470 nm light). D2-IPSCs evoked by photostimulation of ChIs were abolished by
DHβE (1 μM, gray trace).
E) Summary data quantifying the effect of DHβE (1 μM) and the D2-receptor antagonist sulpiride (400 nM) on D2-IPSCs optogenetically evoked by dopamine terminal stimulation (DAT-Cre:ChR2) or ChI stimulation (ChAT-Cre:ChR2) (** = p
< 0.01, Mann-Whitney).
F) In paired recordings between ChIs and GIRK2+ MSNs done in the presence of
10 μM cocaine, paired D2-IPSCs were not resolvable. Electrical stimulation of
55 the surrounding region evoked a robust D2-IPSC. ChIs were recorded in current clamp, while MSNs were recorded in voltage clamp.
G) Summary data illustrating the amplitudes of action potential evoked D2-IPSCs and electrically evoked D2-IPSCs in 10 μM cocaine (* = p < 0.05, Wilcoxon matched-pairs signed rank).
56
Figure 2.1
57 Figure 2.2. Frequency dependence of D2-IPSCs evoked by dopamine terminal or ChI stimulation
A) Representative traces of D2-IPSCs evoked by electrical stimuli. Illustrated are
IPSCs evoked by a single stimulus (black) and bursts (5 at 40 Hz, gray).
B) D2-IPSCs evoked by a single electrical stimulus are inhibited to a greater extent by DHβE (1 μM) than D2-IPSCs evoked with bursts of stimuli (5 at 40 Hz).
C) Quantification of DHβE inhibition of D2-IPSCs evoked by single or bursts of electrical stimuli (** = p < 0.01, Mann-Whitney).
D) Representative traces of D2-IPSCs evoked by photostimulation of dopamine terminals. Illustrated are IPSCs evoked by a single stimulus (black) and bursts (5 at 40 Hz, gray).
E) D2-IPSCs evoked by photostimulation of dopamine terminals (DAT-Cre:ChR2) show little inhibition by DHβE (1 μM).
F) Quantification of the lack of effect of DHβE (1 μM) on D2-IPSCs evoked by photostimulation of dopamine terminals. (Mann-Whitney).
G) Representative traces of D2-IPSCs evoked by photostimulation of ChIs.
Illustrated are IPSCs evoked by a single stimulus (black) and bursts (5 at 40 Hz, gray).
H) D2-IPSCs evoked by photostimulation of ChIs (ChAT-Cre:ChR2) are abolished in the presence of DHβE (1 μM).
I) Quantification of amplitude of D2-IPSCs evoked by bursts of stimulation normalized to the amplitude of single stimuli (* = p < 0.05, *** = p < 0.001,
58 Wilcoxon matched-pairs signed rank within group and Mann-Whitney between groups).
59
Figure 2.2
60 Figure 2.3. Paired pulse ratio shows slow recovery of D2-IPSCs evoked by photostimulation of ChIs
A) Representative traces illustrating optogenetic paired pulse stimulation of dopamine terminals (DAT-Cre:ChR2, top) and ChIs (ChAT-Cre:ChR2, bottom).
At a 5 second inter-pulse interval, D2-IPSCs evoked by ChI photostimulation have a lower paired pulse ratio relative to IPSCs evoked by photostimulation of dopamine terminals.
B) Quantification of paired pulse ratio (P2 / P1) for both DAT-Cre:ChR2 evoked
D2-IPSCs and ChAT-Cre:ChR2 evoked D2-IPSCs at inter-pulse intervals of 1, 3,
5, 10, 15, 20, 25, and 30 seconds (** = p < 0.01, *** = p < 0.001, Mann-Whitney).
61
Figure 2.3
62 Figure 2.4. Mu opioid receptors selectively regulate ChI-mediated dopamine release
A) The effect of the MOR agonist DAMGO (1 μM) on D2-IPSCs evoked by photostimulation of dopamine terminals (DAT-Cre:ChR2). Left: representative trace, middle: normalized quantification of D2-IPSC amplitude over the time, right: bar graph quantifying the amplitude of D2-IPSCs following bath application of DAMGO (1 μM), and naloxone (1 μM) (Wilcoxon matched-pairs signed rank).
B) The effect of the MOR agonist DAMGO (1 μM) on D2-IPSCs evoked by photostimulation of ChIs (ChAT-Cre:ChR2). Left: representative trace, middle: normalized quantification of D2-IPSC amplitude over the time, right: bar graph quantifying the amplitude of D2-IPSCs following bath application of DAMGO (1
μM), and naloxone (1 μM) (Wilcoxon matched-pairs signed rank).
63
Figure 2.4
64 Figure 2.5. Kappa opioid receptors inhibit D2-IPSCs evoked by both dopamine terminal and ChI stimulation
A) The effect of the KOR agonist U69593 (300 nM) on D2-IPSCs evoked by photostimulation of dopamine terminals (DAT-Cre:ChR2). Left: representative trace, middle: normalized quantification of D2-IPSC amplitude over the time, right: bar graph quantifying the amplitude of D2-IPSCs following bath application of U69593 (300 nM), and naloxone (1 μM) (* = p < 0.05, Wilcoxon matched-pairs signed rank).
B) The effect of the KOR agonist U69593 (300 nM) on D2-IPSCs evoked by photostimulation of ChIs (ChaT-Cre:ChR2). Left: representative trace, middle: normalized quantification of D2-IPSC amplitude over the time, right: bar graph quantifying the amplitude of D2-IPSCs following bath application of U69593 (300 nM), and naloxone (1 μM) (* = p < 0.05, Wilcoxon matched-pairs signed rank).
65
Figure 2.5
66 Figure 2.6. Microcircuit map of cholinergic and opioid modulation of dopamine release
Cholinergic terminals release ACh onto nicotinic receptors on dopamine terminals in the striatum. Synchronous ChI activity allows dopamine to be released through nicotinic-mediated facilitation. MORs present on ChI terminals modulate ChI-induced dopamine release, whereas KORs on dopaminergic terminals themselves regulate dopamine released through both pathways.
67
Figure 2.6
68 CHAPTER 3
Spontaneous synaptic activation of muscarinic receptors by
striatal cholinergic interneuron firing
Mamaligas A.A., and Ford C.P. (2016). Spontaneous synaptic activation of muscarinic receptors by striatal cholinergic interneuron firing. Neuron 91, 574-
586.
69 Abstract
Cholinergic interneurons (CHIs) play a major role in motor and learning functions of the striatum. As acetylcholine does not directly evoke postsynaptic events at most striatal synapses, it remains unclear how postsynaptic cholinergic receptors encode the firing patterns of CHIs in the striatum. To examine the dynamics of acetylcholine release, we used optogenetics and paired recordings from CHIs and medium spiny neurons (MSNs) virally overexpressing G-protein activated inwardly rectifying potassium (GIRK) channels. Due to the efficient coupling between endogenous muscarinic receptors and GIRK channels, we found that firing of individual CHIs resulted in monosynaptic spontaneous inhibitory post- synaptic currents (IPSCs) in MSNs. Paired CHI-MSN recordings revealed that the high probability of acetylcholine release at these synapses allowed muscarinic receptors to faithfully encode physiological activity patterns from individual CHIs without failure. These results indicate that muscarinic receptors in striatal output neurons reliably decode CHI firing.
70 Introduction
Cholinergic interneurons (CHIs) are the major source of ACh in the striatum (Bolam, 1984; Kawaguchi, 1993; Lim et al., 2014; Wilson et al., 1990).
While these cells represent a small population of striatal neurons (1 - 2%), their broad arborizations and tiled distribution provide dense ACh innervation throughout the striatum. CHI firing in vivo switches from tonic mode to a transient pause followed by a rebound increase that can be tightly locked to rewarding cues and related stimuli that occur during associative motor learning tasks
(Aosaki et al., 1994; Atallah et al., 2014; Morris et al., 2004). The timing of firing patterns in CHIs is associated with behaviors in response to expected rewards as well as aversive stimuli (Shimo and Hikosaka, 2001).
In the striatum, ACh signals through both ligand-gated ion channel nicotinic receptors and metabotropic G-protein coupled muscarinic receptors.
Through activation of these receptors, cholinergic transmission modulates the activity of multiple striatal circuits to regulate output activity from the striatum
(Goldberg et al., 2012; Koós and Tepper, 2002; Kreitzer, 2009; Lim et al., 2014).
Although many studies have examined the role of CHIs in striatal microcircuitry, the mechanisms by which ACh mediates synaptic transmission in the striatum still remain unclear. Striatal cholinergic transmission has often been studied by measuring the di-synaptic modulation of dopamine and GABA release. ACh mediated release of these transmitters involves activation of presynaptic nicotinic receptors on dopamine terminals or GABAergic interneurons and requires
71 synchronous activation of multiple CHIs (Cachope et al., 2012; Nelson et al.,
2014a; Threlfell et al., 2012). As a result of these indirect synaptic connections, it has been difficult to directly determine how the release of ACh as a result of CHI firing drives this modulation.
Muscarinic G-protein coupled receptors regulate the integration of synaptic inputs, plasticity, firing patterns, collateral connections and the transition to up-states of medium spiny neurons (MSNs), the output neuron of the striatum
(Ding et al., 2006; Goldberg et al., 2012; Higley et al., 2009; Perez-Rosello et al.,
2005; Yamamoto et al., 2013). In the striatum, muscarinic receptors do not directly couple to endogenous ion channels but instead indirectly alter excitability through 2nd messenger signaling cascades (Bernard et al., 1992; Calabresi et al.,
1998; Goldberg et al., 2012; Pakhotin and Bracci, 2007; Pisani et al., 2002; Shen et al., 2007; Sullivan et al., 2008). Due to the multistep processes underlying muscarinic signal transduction, it has been difficult to separate receptor activation from signaling, limiting the resolution of cholinergic signaling events onto MSNs.
The pacemaking of cholinergic interneurons (Bennett and Wilson, 1999) and broad axonal arborizations (Bolam, 1984; Perez-Rosello et al., 2005) have been thought to create a background tone of ACh that tonically drives muscarinic receptor activation. CHIs exhibit a range of firing patterns including regular and irregular tonic activity as well as bursts and pauses (Bennett and Wilson, 1999;
Bennett et al., 2000; Goldberg and Reynolds, 2011). However, it remains
72 unclear how the patterns of CHI firing and time course of ACh release activate cholinergic receptors on MSNs.
In the present study, G-protein activated potassium channels (GIRK2;
Kir3.2) were virally overexpressed in MSNs to provide a rapid electrophysiological readout of muscarinic receptor activation. We find that cholinergic interneurons make robust monosynaptic muscarinic connections with
MSNs. Using paired CHI-MSN recordings, we show that the firing of an individual
CHI is sufficient to drive a muscarinic receptor mediated synaptic event. Despite the intrinsically slow kinetics of GPCR signaling, the rapid dynamics of these synaptic events allows the firing of individual cholinergic interneurons to be discretely encoded within MSNs as spontaneous events as opposed to tonic muscarinic receptor activation. These properties allow for reliable cholinergic transmission in response to different patterns of CHI firing.
Experimental Procedures
Stereotaxic injections. All procedures were performed in compliance with guidelines of the Institutional Animal Care and Use Committee at Case Western
Reserve University. Both male and female wild-type C57BL6, ChAT-internal ribosome entry site-Cre heterozygote and D1-eYFP mice were injected at postnatal day 21. D1-eYFP mice were kindly provided by Dr. Veronica Alvarez at
NIAAA (NIH). All other animals were from The Jackson Laboratory, Bar Harbor,
ME. The injection coordinates were (relative to bregma): AP +1.15 mm, ML -1.85
73 mm, DV -3.35 mm. A volume of 300 nL of AAV9.hSyn.tdTomato.T2A.mGIRK2-1-
A22A.WPRE.bGH and/or 300 nL of AAV5.EF1a.DIO.hChR2(H134R)-
EYFP.WPRE.hGH or 300 nL of AAV1.EF1a.DIO.eNPHR3.0-EYFP.WPRE.hGH were injected into one hemisphere of the striatum. All AAVs were from the
University of Pennsylvania Viral Core unless otherwise specified. Animals were allowed to recover for ~1-4 weeks to allow for expression.
Immunohistochemistry. AAV9 injected mice were anesthetized and transcardially perfused with ice-cold PBS containing (in mM) 137 NaCl, 1.5
KH2PO4, 8 NaH2PO4, and 2.7 KCl (pH=7.4) followed by 4% paraformaldehyde in
PBS. Brains were rehydrated overnight in 30% sucrose. Slide-mounted sections were permeabilized with 0.1% Triton X-100 in PBS (PBS-T) and blocked in 5% normal donkey serum in PBS-T (2 hours, room temperature). Slides were washed and incubated with 1:200 goat anti-ChAT (Millipore) and/or rabbit anti-
Kir3.2 (Alomone Labs) antibodies, diluted in blocking buffer, for 72 hours (anti-
ChAT) at 4°C. Slides were then washed and incubated for 6 hours in 1:1000 donkey anti-goat 647 (ChAT staining) and washed. For GIRK staining, slides were then incubated for 2 hours in 1:500 donkey anti-rabbit 488 (Abcam).
Fluorescent confocal images were obtained using a Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss) with a 403 Plan-Neofluar, NA 1.3, oil- immersion lens. All images were processed using ImageJ, n = 5 animals examined.
74
Slice preparation. Striatal coronal slices (240 μm) were made in ice-cold sucrose cutting solution containing (in mM): 75 NaCl, 2.5 KCl, 6 MgCl2, 0.1 CaCl2, 1.2
NaH2PO4, 25 NaHCO3, 2.5 D-glucose, and 50 sucrose; bubbled with 95% O2 and
5% CO2.
Slices were incubated at 35°C for at least 45 minutes in ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and
11.1 D-glucose; and 10 μM MK-801 bubbled with 95% O2 and 5% CO2. Slices were transferred to the recording chamber and perfused with ACSF (34±2°C) at
2 mL/minute. Solutions contained DNQX (10 μM), picrotoxin (100 μM),
CGP55845 (300 nM), SCH 23390 hydrochloride (1 μM) and sulpiride (200 nM).
MSNs were visualized using a BXWI51 scope (Olympus) with IR gradient contrast optics and custom-made LEDs.
Electrophysiology. Recordings were performed using Axopatch 200A and
Axopatch 200B amplifiers (Molecular Devices). Membrane potentials were not corrected for liquid junction potentials. CHIs were identified by the presence of large h-current when stepped to -110 mV in voltage-clamp. Patch pipettes (1.5 -
2MΩ) for MSNs contained 115 mM K-methylsulphate, 20 mM NaCl, 1.5 mM
MgCl2, 10 mM HEPES(K), 10 mM BAPTA-tetrapotassium, 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine, pH 7.4, 275 mOsm. Pipettes for recordings of CHIs contained 135 mM D-gluconate(K), 10 mM HEPES(K), 0.1
75 mM CaCl2, 2 mM MgCl2, 0.1 mM EGTA, 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine, pH 7.4, 275 mOsm. All recordings were acquired with
Axograph X (Axograph Scientific) at 10 kHz. Voltage clamp and cell-attached recordings were filtered to 2 kHz. MSNs were held at a voltage of -60 mV. No series resistance compensation was used, and cells were discarded if their series resistance exceeded 15 MΩ. Light evoked ACh release was stimulated by wide-field illumination with 488 nm blue light (2 ms pulse, ~1.0 mW/mm2).
Halorhodopsin-mediated inhibition of CHI firing was induced by wide-field illumination with 570 nm green light (2 - 5 seconds, ~1.0 mW/mm2). All drugs were perfused via bath application unless otherwise noted. ACh was applied via iontophoresis (100 mM, 160 nA ejection, 25 - 50 ms; 1 pulse/min).
For paired dMSN-dMSN recordings, the presynaptic MSN internal solution contained: 135 mM D-gluconate(K), 10 mM HEPES(K), 0.1 mM CaCl2, 2 mM
MgCl2, 0.1 mM EGTA, 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine, pH 7.4, 275 mOsm. Presynaptic MSNs were held near -60 mV and APs were evoked by a 4ms injection of 800 pA. For recording the postsynaptic MSN, an internal solution containing 135 mM CsCl, 0.1 mM CaCl2,
2 mM MgCl2, 10 mM HEPES(K), and 0.1 mM EGTA was used. Postsynaptic
MSNs were held at -70 mV. GABAA IPSCs were recorded as inward currents
- (ECl = ~ 0 mV). Recordings were performed in the presence of SKF 38393 (10 nM).
76 In the subset of experiments in which muscarinic, GABAergic, and gluatamatergic transmission was assessed, postsynaptic MSNs were recorded with an internal solution containing: 57.5 mM K-methylsulphate, 67.5 KCl, 10 mM
NaCl, 50 μM CaCl2, 1.75 mM MgCl2, 10 mM HEPES(K), 5 mM BAPTA- tetrapotassium, 50 μM EGTA, 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine, pH 7.4, 275 mOsm (final Cl- concentration = 78.4 mM).
Materials. Picrotoxin, TTX, DNQX, and MK-801 were obtained from Ascent
Scientific. ACh, Vesamicol, Tropicamide, Ambenonium, CGP 55845, scopolamine hydrobromide, SKF 38393, and SCH 23390 were from Tocris
Bioscience. K-methylsulphate was from Acros Organic and BAPTA was from
Invitrogen. All other chemicals were from Sigma-Aldrich.
Statistics and Analysis. Data are shown as mean ± SEM. Statistical significance was determined using Pearson correlation, Wilcoxon match-pairs signed rank test, Mann-Whitney test, Kolmogorov-Smirnov test, or Tukey-Kramer test, where appropriate (InStat 3.0, Graphpad; Axograph Scientific). Statistical differences in
EC50 were determined by 95% confidence interval.
The predicted CV for sIPSCs was calculated using the following formula:
�(� × �� ) �� = �
where CVsIPSC and CVuIPSC represent the coefficients of variation for sIPSCs and uIPSCs, respectively, n represents the number of CHIs connected, and μuIPSC
77 and μtotalISPC represent the mean amplitudes of uIPSCs and the total population of uIPSCs and sIPSCs, respectively.
Results
Spontaneous muscarinic IPSCs in MSNs overexpressing GIRK2
To examine how muscarinic receptors encode cholinergic interneuron firing patterns in striatal output neurons, we virally overexpressed G-protein coupled inwardly rectifying K+ channels (GIRK2; Kir3.2) in MSNs (Marcott et al.,
2014). Injection of an adeno-associated virus (AAV) encoding tdTomato and
GIRK2 under a synapsin promoter into the dorsal striatum led to the co- expression of both GIRK2 and the soluble fluorophore tdTomato (Figure 3.1A)
(Marcott et al., 2014). Out of 409 tdTomato+ neurons, 366 co-expressed GIRK2
(Figure 3.1A and 3.1B). Expression was largely restricted from cholinergic neurons as limited GIRK2 immunoreactivity or tdTomato fluorescence could be detected in choline acetyltransferase (ChAT)-positive neurons (4 of 71 ChAT+ co- expressed GIRK2) (Figure 3.1A and 3.1B). Weak expression of GIRK2 in CHIs was likely due to the use of a synapsin promoter as we found that other AAVs using this promoter also failed to effectively transfect CHIs (Figure 3.2A). As seen previously (Marcott et al., 2014), no GIRK2 immunoreactivity could be detected in control uninjected striatal slices (Figure 3.2B).
To examine if exogenous GIRK2 channels could couple to muscarinic receptors (mAChRs) in MSNs, we performed whole-cell voltage clamp recordings
78 of tdTomato+ neurons in the dorsal striatum. MSNs were identified by their low input resistance, hyperpolarized resting membrane potential, and lack of hyperpolarization-activated current (Kreitzer, 2009; Marcott et al., 2014). After allowing 21 days for expression, application of the muscarinic agonist oxotremorine M (Oxo-M) (1 µm) evoked an outward current in tdTomato+ MSNs
(Figure 3.1C). Overexpressed GIRK2 channels couple efficiently to endogenous
Gai/o coupled GPCRs without altering the affinity of transmitter for the receptor
(Marcott et al., 2014). To confirm that overexpression of GIRK2 did not change the affinity of muscarinic receptors, we varied the extent of GIRK2 expression by recording from MSNs taken 3-5 days post AAV injection. In brain slices taken from mice > 21 days post-AAV injection, Oxo-M evoked larger outward currents in tdTomato+ neurons than in animals expressing GIRK for only 3-5 days (Figure
3.1D). Although the amplitude of Oxo-M induced outward currents were decreased under conditions of reduced expression, the EC50 of Oxo-M was unchanged (4 day expression: 431 nM, 95% confidence interval = 121 – 741 nM;
> 21 day expression 234 nM, 95 % confidence interval = 118 – 350 nM) (Figure
3.1D). Iontophoresis of exogenous ACh (100 µM, 100 ms) also evoked an outward current of 285 ± 84 pA in tdTomato+ MSNs (n = 8) (Figure 3.1E). The current was eliminated by the mAChR antagonist scopolamine (1 μM) (2 ± 1 pA, n = 5, p < 0.05, Wilcoxon) (Figure 3.1E). The current-voltage relationship in response to ACh showed inward rectification and a reversal potential near the predicted K+ equilibrium potential, consistent with activation of a GIRK
79 conductance (Figure 3.1F). Iontophoresis of ACh failed to evoke outward currents in control MSNs from the uninjected hemisphere (1 ± 1 pA, n = 27)
(Figure 3.1E). Together the results show that overexpressed GIRK2 channels couple to endogenous muscarinic receptors in MSNs without a change in the apparent affinity of receptors.
To isolate cholinergic transmission through muscarinic receptors, we
+ performed whole-cell recordings (Vh = - 60 mV) from GIRK2 MSNs in the presence of glutamate, GABA and dopamine receptor antagonists. In the absence of stimulation, recordings revealed spontaneous IPSCs (sIPSCs) that were abolished by the mAChR antagonists scopolamine (1 μM, n = 11, p <
0.001, Wilcoxon) or tropicamide (1 μM, n = 5, p < 0.05, Wilcoxon) (Figure 3.3A and 3.3F). The frequency of sIPSCs was similar to the range of frequencies reported for CHI pacemaker firing in slices (Bennett and Wilson, 1999; Wilson et al., 1990). While Gaq-coupled M1 mAChRs are expressed in all MSNs, Gai/o- coupled M4 mAChRs are expressed predominantly in direct pathway MSNs
(Bernard et al., 1992; Goldberg et al., 2012; Lim et al., 2014; Yan et al., 2001).
Spontaneous IPSCs occurred in roughly half of all MSNs (96 out of 202 GIRK2+
MSNs, Figure 3.3B). In a subset of experiments, we expressed GIRK2 in Drd1- eYFP mice that express YFP in direct pathway, D1-receptor-containing MSNs
(dMSN). We found that sIPSCs were present in all D1-eYFP neurons tested (10 out of 10 YFP+ neurons, Figure 3.3B). As GIRK channels preferentially couple to
80 Gai/o-coupled GPCRs (Lüscher and Slesinger, 2010), scopolamine-sensitive sIPSCs in dMSNs likely result from activation of muscarinic M4-receptors.
Spontaneous IPSCs occurred with a mean frequency of 1.4 ± 0.1 Hz
(2489 events, 32 cells) (Figures 3.3A and 3.3F) and displayed an average amplitude of 40 ± 4 pA (2865 events, 40 cells) that varied between 6 and 456 pA.
As the level of AAV-driven GIRK2 expression varies among MSNs (Marcott et al.,
2014), the amplitude of sIPSCs was not compared across neurons. However, the amplitude of events within each MSN was normally distributed (56 ± 4 pA, n = 51 events, single cell, p > 0.05, Chi-squared test) (Figure 3.3C) and did not correlate with 10 – 90% rise time (70 ± 1 ms, n = 51 events, single cell, r2 = 0.03, p = 0.21,
Pearson’s correlation) (Figure 3.3D). These results suggest that the rate of mAChR activation underlying sIPSCs is independent of the amount of ACh released.
To examine the mechanism underlying sIPSCs, we recorded events in tetrodotoxin (TTX, 200 nM). Application of TTX abolished sIPSCs (n = 5, p <
0.05, Wilcoxon), indicating that events were action potential dependent (Figure
3.3E and 3.3F). In the presence of TTX, it was not possible to resolve individual miniature IPSCs above the baseline noise. Limiting Ca2+ entry by using ACSF without added Ca2+ (n = 5, p < 0.05, Wilcoxon), disrupting the vesicular ACh transporter with vesamicol (2 µM, n = 5, p < 0.05, Wilcoxon), or blocking GIRK channels with Ba2+ (200 µM, n = 5, p < 0.05, Wilcoxon) eliminated sIPSCs
(Figure 3.3F). These results indicate that muscarinic sIPSCs resulted from the
81 activity dependent vesicular release of ACh. To confirm that ACh underlying sIPSCs originated from local CHIs, we selectively expressed the light activated cation channel, channelrhodopsin-2 (ChR2), in CHIs by co-injecting a Cre- dependent AAV (AAV.DIO.ChR2.eYFP) into the striatum of choline acetyltransferase-IRES-Cre transgenic mice (ChAT-Cre) (Figure 3.3G).
Photoactivation of ChR2-expressing CHIs (2 ms widefield light, 470 nm, ~1 mW)
(Figure 3.4A) resulted in scopolamine-sensitive muscarinic IPSCs in roughly half of all GIRK2+ MSNs examined (n = 8 of 19 neurons) (Figure 3.3H).
Optogenetically evoked IPSCs (oIPSCs) were larger in amplitude and longer in duration than sIPSCs (sIPSCs: 38 ± 6 pA, 197 ± 11 ms half width n = 8; oIPSCs:
517 ± 112 pA, 237 ± 14 ms half width, n = 8 neurons, p < 0.01 for amplitude and p < 0.05 for half width, Wilcoxon) (Figure 3.3I). Electrical stimulation (0.7 ms, 20
– 40 µA) also evoked large IPSCs that were longer than sIPSCs (302.1 ± 18.8 ms, n = 16, p < 0.05 vs sIPSC 20% width, Mann-Whitney). Thus, the synchronous activation of multiple CHI terminals led to a large increase in mAChR activation.
We confirmed that CHI firing was necessary to drive muscarinic sIPSCs using the light-driven Cl- pump, halorhodopsin, expressed in cholinergic interneurons. We co-injected a Cre-dependent AAV (AAV.DiO.eNpHR3.0-eYFP) into the dorsal striatum of ChAT-Cre mice along with AAV.GIRK2 (Figure 3.4B).
Wide-field illumination of the striatum with long pulses of green light (5 s, 530 nM) led to a silencing of CHI firing during current-clamp recordings (Figure 3.4C).
82 This resulted in a pause in sIPSCs in GIRK2+ MSNs (7.6 ± 2% of control, n = 5 neurons, p < 0.01, Wilcoxon) (Figure 3.3K). We found that while inhibiting CHI firing with halorhodopsin eliminated sIPSCs, there was no change in the holding current (99.4 ± 0.7% of control, n = 5 neurons, p > 0.05, Wilcoxon) (Figure 3.3K).
The lack of a standing outward current upon silencing CHI activity suggests that muscarinic receptors on MSNs encode tonic firing of CHIs as a series of phasic events rather than a sustained level of receptor activation.
Cholinergic interneuron firing evokes unitary IPSCs
Synchronous activation of cholinergic interneurons drives the release of dopamine and GABA in the striatum from SNc terminals via presynaptic nicotinic receptors (Cachope et al., 2012; Nelson et al., 2014a; Threlfell et al., 2012). As synchronous optogenetic activation of multiple CHIs evoked oIPSCs that were prolonged relative to sIPSCs (Figure 3.3I), we next performed paired recordings between cholinergic interneurons and GIRK2+ MSNs to determine if the firing of an individual CHI was sufficient to evoke a muscarinic receptor-mediated IPSC. MSNs were voltage clamped at -60 mV, and CHIs were recorded in current clamp configuration. CHIs were identified by their large size, lack of tdTomato fluorescence (Figure 3.1A), and presence of hyperpolarization activated inward current (Figure 3.4B). CHI-MSN pairs were separated by less than 200 µm.
Approximately 70% of recorded CHIs were synaptically connected to GIRK2+
MSNs that exhibited sIPSCs (62 of 89 paired attempts). Cholinergic interneurons
83 were hyperpolarized to just below threshold to prevent pacemaker firing. In synaptically coupled pairs, current injection (4 ms, 150 - 500 pA) triggered a single action potential (AP) in a CHI that was time locked to a unitary muscarinic IPSC
(uIPSCs) in the postsynaptic MSN (Figure 3.5A and 3.5B). Action potentials reliably evoked uIPSCs, as no failures were apparent in 560 events from 24 pairs
(Figure 3.5C). CHI-triggered uIPSCs activated following a lag of 39 ± 1 ms (time to
10%, n = 532 events, 24 pairs; 10 - 90% rise time: 76 ± 1 ms, n = 537 events, 24 pairs) (Figure 3.5B). The latency to activation is similar to that seen in isolated systems where saturating concentrations of agonist have been applied to evoke
GPCR mediated GIRK currents (Courtney and Ford, 2014; Ford et al., 2009;
Sodickson and Bean, 1996) and likely results from the intrinsic kinetics of the receptor/G-protein-GIRK signaling complex (Ford et al., 2009). Paired uIPSCs were abolished in the presence of scopolamine (1 µM) (control: 42.4 ± 19 pA, scopolamine: 3.4 ± 1 pA, n = 6 pairs, p < 0.05, Wilcoxon) (Figure 3.5D).
Subthreshold depolarizations of CHIs did not evoke IPSCs. Together the results indicate that individual CHIs make monosynaptic connections with MSNs and that following release, muscarinic receptors are exposed to a high concentration of
ACh.
In a subset of experiments, we made paired recordings in the absence of
GABA, and glutamate receptor antagonists. Unitary IPSCs were unaffected by blocking GABA, or glutamate receptors (uIPSC amplitude with antagonists: 111 ±
6% of control, n = 4 pairs, p > 0.05, Wilcoxon) (Figure 3.6A and 3.6B). In addition,
84 in the absence of antagonists, no paired GABA or glutamate synaptic events were observed from CHIs to MSNs (Figure 3.6A). This was in contrast to the fast inward synaptic current that was seen in MSNs following synchronous photo-activation of multiple CHIs via ChR2 (Figure 3.6C). This confirms that synchronous CHI activation is required to evoke the disynaptic release of GABA from dopamine terminals and GABAergic interneurons (English et al., 2011; Nelson et al., 2014a;
Tritsch et al., 2012) and the co-release of glutamate from cholinergic interneurons
(Higley et al., 2011; Nelson et al., 2014b) onto MSNs.
During paired recordings, we observed sIPSCs unpaired with firing of the recorded CHI (Figure 3.5A). Like sIPSCs, unitary IPSCs exhibited a range of amplitudes across MSNs. The average amplitude and duration of sIPSCs were similar to that of uIPSCs when compared across cells (p > 0.05 for all sIPSC vs uIPSC, Mann-Whitney) (Figure 3.5E). Within pairs of CHIs and MSNs uIPSCs exhibited little variability across events (coefficient of variation, CV = 0.27 ± 0.01, n
= 23 pairs) (Figure 3.5A and 3.5F). The stable amplitude of uIPSCs is consistent with the idea that ACh may be released from multiple CHI terminals after each AP as a result of their highly branched and varicose axonal arborizations (Bolam,
1984; Contant et al., 1996; Descarries and Mechawar, 2000). Within individual
MSNs, the amplitude of sIPSCs was much more variable when compared to paired uIPSCs (CV sIPSCs: 0.52 ± 0.01 CV: uIPSCs 0.27 ± 0.01, p = 0.0005, Wilcoxon)
(Figure 3.5F). As each CHI-MSN paired recording showed a low variability of uIPSCs, the increased CV of sIPSCs likely results from the fact that multiple CHIs
85 with a different number of intact synaptic connections in the slice synapse onto a given MSN. This idea is supported by the fact that in recordings made near the surface of the brain slice where axonal inputs from most CHIs would likely have been severed, MSNs exhibited low frequency consistent amplitude sIPSCs (CV =
0.29 ± 0.01, n = 6) suggestive of only a single CHI input (Figure 3.6D). In these cases sIPSCs were highly regular and the CV among events was similar to that of uIPSCs from paired recordings (p > 0.05, Mann-Whitney) (Figure 3.6D). Analysis of the CV from uIPSCs (0.27 ± 0.01) predicted that several CHIs may synapse onto each MSN. The predicted CV of spontaneous events when 3 CHIs were estimated to connect to each MSN (0.52) was in close agreement with that observed during recordings (0.52 ± 0.01). Together these results suggest that multiple CHIs are connected to a single MSN.
In paired recordings between CHIs and GIRK2+ MSNs we also found that the amplitude of uIPSCs was unaffected whether or not they were preceded by an unpaired sIPSC (isolated: 47 ± 7 pA, preceded: 52 ± 8 pA, n = 15 pairs of cells, p >
0.05, Wilcoxon, Figure 3.7A and 3.7B). This indicates that inputs from different
CHIs are likely independent. To further examine the connectivity between CHIs and MSNs, we next made loose, cell-attached recordings of CHIs while making whole cell voltage clamp recordings from GIRK2+ MSNs. Under these conditions, roughly 1/3rd of sIPSCs recorded could be attributed to the paired CHI (Figure
3.7C). In the cell illustrated in Figure 3.5C, 31% of IPSCs were paired and 69% were unpaired (Figure 3.7D). This suggests that possibly three CHIs were
86 synaptically coupled to that MSN. This estimation is likely an underestimate as some CHIs more superficial to the MSN recorded may have had axons severed during slicing. Across paired recordings, 38 ± 2% of all events were paired uIPSCs
(n = 18 pairs) (Figure 3.7E). Among cell-attached paired recordings, there was a wide distribution of the ratio between paired and total IPSCs. For individual pairs, between 23% and 55% of all sIPSCs recorded were contributed by a paired CHI (n
= 18 pairs, Figure 3.7F). This range indicates that between 1/5 and 1/2 of events were paired and suggests that in our slice recordings, a given MSN may receive input from between 2 and 5 CHIs (mean: 2.6 ± 0.2 CHIs per MSN) (Figure 3.7G).
This connectivity is similar to that predicted by analyzing the CV of events (Figure
3.5F, above). While CHIs with faster firing rates would contribute more to the apparent connectivity, these recordings suggest that in slices several CHIs independently converge onto each MSN.
Acetylcholinesterase limits the duration of mAChR activation
The striatum contains the highest levels of acetylcholinesterase (AChE) in the brain (Hebb and Silver, 1961; Macintosh, 1941). As the activation kinetics of sIPSCs (39 ± 1 ms lag till onset, n = 532 events) were greater than 2-fold faster than GIRK currents evoked when saturating concentrations (100 µM) of agonists were applied to other Gai/o-coupled GPCRs on membrane patches (Courtney and
Ford, 2014; Ford et al., 2009; Sodickson and Bean, 1996), the activation of muscarinic receptors underlying sIPSCs likely resulted from a local high
87 concentration of ACh. To determine the role of AChE in regulating the extent of muscarinic receptor activation, we applied a non-saturating concentration of the
AChE inhibitor ambenonium (10 nM). The amplitude (control: 48 ± 11, n = 274 events, n = 6 cells; ambenonium: 85 ± 14 pA, n= 278 events, n = 6 cells; p<0.0001,
Kolmogorov-Smirnov test) and half width (146 ± 8; ambenonium: 371 ± 23 ms n =
6 cells, p < 0.05, Wilcoxon) of sIPSCs increased in the presence of ambenonium
(Figure 3.8A – 3.8D). This suggests that due to efficient enzymatic degradation,
AChE limits the duration of muscarinic receptor activation.
Since silencing CHI firing with halorhodopsin did not lead to a change in holding current under control conditions (Figure 3.3J and 3.3K), we next used halorhodopsin to inhibit CHI firing while blocking AChE. In the presence of ambenonium (80 nM), photoactivation of halorhodopsin (5 s) led to a decrease in the baseline holding current (control: 2 ± 3 pA change, ambenonium 70 ± 13 pA change, n = 5 cells, p < 0.05, Wilcoxon, Figure 3.8F). A parallel depolarization of the membrane potential during recordings in current-clamp mode was also observed in the presence of ambenonium, but not in control conditions (Figure
3.9). This indicates that in brain slices, tonic receptor activation only occurs when
AChE is blocked. Thus despite the background tonic firing of CHIs, AChE functions to limit the extracellular tone of ACh to prevent the tonic activation of muscarinic receptors.
88 Cholinergic interneuron regulation of MSN output through muscarinic receptors
We found that muscarinic receptor activation led to the phasic activation of overexpressed GIRK channels, so we next wanted to test if muscarinic transmission could functionally regulate endogenous signaling cascades in dMSNs over similar timescales. The next experiments were performed without overexpressing GIRK. MSNs make recurrent synapses on neighboring MSNs
(Koos et al., 2004; Taverna et al., 2008; Tecuapetla et al., 2007). As muscarinic agonists inhibit GABA transmission between MSNs (Yamamoto et al., 2013), we next sought to examine whether the release of ACh from CHI could also transiently regulate GABA transmission through these collaterals. D1-receptor expressing dMSNs have a higher connectivity with other dMSNs than indirect pathway MSNs (iMSNs) (Taverna et al., 2008). We crossed D1-eYFP mice with
ChAT-Cre mice in order to both visualize dMSNs and express halorhodopsin in
CHIs using AAV.DiO.eNpHR3.0-eYFP. CHIs exhibit a rebound burst of APs following strong hyperpolarizations due to activation of an HCN conductance
(Straub et al., 2014; Wilson, 2005). In recordings from halorhodopsin expressing
CHIs, we found that following wide-field illumination (0.5 – 4 s; 530 nm; ~1 mW), termination of photostimulation led to a transient burst of 2-3 APs in CHIs that was time-locked to the end of the light pulse (latency from end of light pulse to first AP: 71 ± 8 ms; burst interspike interval: 64 ± 7 ms) (Figure 3.10A and
3.10B). We recorded dMSN-dMSN pairs in the presence of the nicotinic receptor
89 antagonist mecamylamine (5 µM) to isolate muscarinic transmission from CHIs.
In successfully coupled dMSN-dMSN pairs, we found that the amplitude of
GABAA IPSCs was decreased by triggering bursts of APs in CHIs 500ms prior to evoking GABAA IPSCs (16 ± 2 % decrease, n = 7 pairs, p < 0.05, Wilcoxon)
(Figure 3.10C and 3.10D). No inhibition was observed when repeating these experiments in the presence of the muscarinic antagonist scopolamine (1 µM)
(10 ± 9 % increase, n = 5 pairs, p > 0.05, Wilcoxon) (Figure 3.10E). Thus while overexpressed GIRK channels allow for rapid sensing of muscarinic receptor activation at cholinergic synapses, these results show that CHI firing also endogenously drives transient inhibition of GABA release from dMSN collateral terminals though these receptors on similar timescales.
Regulation of ACh release transmission at muscarinic synapses
As the probability of transmitter release (Pr) decreases at many synapses with repeated stimulations, we next examined whether CHI firing resulted in a short-term depression of muscarinic transmission. To assess the probability of
ACh release from CHIs onto GIRK2+ MSNs, we measured the paired pulse ratio
(PPR) of uIPSCs elicited by two action potentials. The amplitude of a second uIPSC was depressed relative to the first with short interpulse intervals (PPR 250 ms: 0.48 ± 0.04, n = 5 pairs, p < 0.01; PPR 500 ms: 0.6 ± 0.1, n = 5 pairs, p <
0.01, Mann-Whitney) and required several seconds for full recovery (PPR 7 s: 1
± 0.1, n = 5 pairs, p > 0.05, Mann-Whitney) (Figure 3.11A and 3.11D). Short-term
90 depression is often indicative of a change in the pre-synaptic release of transmitter that occurs at synapses with a high probability of release. However as both pre- and post-synaptic mechanisms can contribute to short-term depression, we also applied ACh using paired iontophoretic applications. The amplitude of the two resulting currents when ACh was applied with a 500 ms interpulse interval were the same (PPR: 1 ± 0.1, n = 3, p > 0.05, Wilcoxon), suggesting that the depression of uIPSCs was pre-synaptic in origin.
Cholinergic interneurons express muscarinic autoreceptors, which inhibit their activity. To determine if activation of presynaptic autoreceptors contributed to the reduction in ACh release, we replaced the GTP in the presynaptic intracellular solution with the GDP analog guanosine 5’-2-thiodiphosphate
(GDPbs) (0.6mM) to selectively block G-protein activation in CHIs without altering muscarinic signaling in postsynaptic MSNs. The GDP analog functions as a competitive antagonist of GTP binding to G-proteins. Dialysis of CHIs with
GDPbs (0.6 mM) for 6 minutes eliminated dopamine D2-receptor mediated
IPSCs (Figure 3.12) (Chuhma et al., 2014; Straub et al., 2014), confirming that
GDPbs blocked GPCR signaling in CHIs. We next repeated the PPR experiment after allowing for 8 – 10 minutes for GDPbs to fully dialyze CHIs. GDPbs dialysis of CHIs however did not alter the PPR of muscarinic uIPSCs at interspike intervals of 250 ms and 2 seconds when compared with control (intracellular
GTP) recordings (PPR 250 ms: 0.44 ± 0.04; PPR 2 seconds: 0.75 ± 0.03, n = 7 pairs, p > 0.05 relative to control, Mann-Whitney) (Figure 3.11B and 3.11C).
91 While the D2-receptors underlying D2-IPSCs likely reside at somatodendritic sites as opposed to muscarinic autoreceptors at CHI terminals, the lack of effect of GDPbs on muscarinic uIPSCs suggests that over the timescales examined, autoreceptors do not alter ACh release probability at muscarinic synapses.
To test whether any of the depression resulted from postsynaptic saturation of receptor signaling to G-proteins and GIRK channels, we repeated
PPR analysis in the presence of a low (~IC50) concentration of the muscarinic antagonist tropicamide (20 nM). Tropicamide reduced the amplitude of uIPSCs by approximately half (43 ± 14 %, n = 5, p < 0.05 Wilcoxon) (Figure 3.11D; inset).
Despite the reduction in amplitude, the PPR was unchanged (PPR 250 ms: control 0.48 ± 0.04, tropicamide 0.53 ± 0.05, n = 5 pairs each, p > 0.05, Mann-
Whitney; PPR 2 seconds: control 0.74 ± 0.01, n = 4, tropicamide 0.75 ± 0.03, n =
5 pairs, p > 0.05, Mann Whitney). Using GDPβS internal in conjunction with bath application of tropicamide (20 nM) produced no further reduction in uIPSC amplitude or the PPR (PPR 250 ms: 0.58 ± 0.04; PPR 2 seconds: 0.69 ± 0.05, n
= 4 pairs, p > 0.05 relative to control, tropicamide, and GDPβS groups, Mann-
Whitney). These results suggest that the observed depression does not result from activation of presynaptic autoreceptors or saturation of postsynaptic signaling cascades but instead may be due to intrinsic short-term depression of
ACh vesicle release.
CHIs are autonomous pacemakers that fire at low frequencies (Bennett and Wilson, 1999). To determine the implications of tonic CHI firing on
92 muscarinic transmission, we introduced trains of action potentials to CHIs at 2 Hz for 5 seconds (Figure 3.13A). Trains of action potentials evoked a marked depression of uIPSCs (Figure 3.13A). Despite the depression in amplitude, all action potentials in the train led to an IPSC such that no failures were observed
(Figure 3.13A, 2440 events, 6 pairs). At 2 Hz, the majority of the depression occurred after the first action potential of the train such that the amplitude of the
IPSC did not depress in subsequent events (p < 0.05, n = 6 pairs, Tukey-Kramer test) (Figure 3.13B). Coinciding with the decrease in amplitude, there was a decrease in variance of the amplitude of events across multiple trials (first pulse variance: 122 ± 44 pA2, tenth pulse: 33 ± 9 pA2, n = 6 pairs, p < 0.05, Wilcoxon)
(Figure 3.13C). The decrease in amplitude of events and the associated decrease in variance suggest that resting CHIs display a moderately high probability release profile, which lowers upon tonic activity. Recordings in which presynaptic GTP was replaced with GDPβS showed no differences in uIPSC amplitude relative to the first pulse in the train (n = 5 pairs, p > 0.05 for all events in the train, Mann-Whitney), indicating that autoreceptors do not underlie depression due to tonic firing. Directly testing the change in Pr with low extracellular Ca2+ (0.5 mM) was difficult because of the strong regulation of CHI pacemaker firing by Ca2+-activated potassium (SK) channels (Bennett and
Wilson, 1999). As a result, we found that in low extracellular Ca2+ (0.5 mM) there was dramatic increase in the frequency of sIPSCs that often summated, occluding resolvable uIPSCs.
93 Lastly we set out to examine how muscarinic transmission in MSNs is regulated during tonic cholinergic patterns of activity. CHIs can fire in both single spikes and burst patterns in response to salient stimuli (Goldberg and Reynolds,
2011; Morris et al., 2004; Schulz and Reynolds, 2013). While hyperpolarizing
CHIs to limit pacemaker firing, we found that a burst of action potentials (5 APs at
20 Hz) produced strong depression such that the burst-evoked uIPSCs were of similar amplitude to uIPSCs evoked with a single action potential (111 ± 6 % of single uIPSC, n = 9, p > 0.05, Wilcoxon, Figure 3.13D and 3.13E). In contrast, when IPSCs were depressed by evoking a train of 20 action potentials at 2 Hz, bursts of APs following a 1 sec pause led to an alleviation of depression (230 ±
14 % of last pulse, n = 6 pairs, p < 0.05, Wilcoxon) that was not observed with only a single action potential (120 ± 9 % of last pulse, p > 0.05, n = 6 pairs,
Wilcoxon) (Figure 3.13F – 3.13H). Rebound burst firing of CHIs in vivo is tightly locked to rewarding cues and associated stimuli that occur during reward-based learning tasks (Aosaki et al., 1994; Atallah et al., 2014; Morris et al., 2004).
Together these findings suggest that as a result of short-term depression, the extent of ACh release can be modulated in response to these patterns of firing.
Discussion
M4-receptors are the most highly expressed class of muscarinic receptors in the striatum (Bernard et al., 1992; Yan et al., 2001). These inhibitory receptors are localized primarily in dMSNs (Bernard et al., 1992; Yan et al., 2001), where
94 they decrease excitability (Howe and Surmeier, 1995) and oppose dopamine D1- receptors induction of glutamate long-term plasticity (Shen et al., 2015).
Increasing M4-receptor activity in vivo rescues L-DOPA induced impairments in synaptic plasticity and L-DOPA induced dyskinesia in animal models (Shen et al.,
2015). As animals lacking M4-receptors selectively in dMSNs exhibit increased locomotor activity and behavioral sensitization to psychostimulants (Jeon et al.,
2010), muscarinic GPCR signaling through these receptors is an important regulator of the striatonigral pathway. Despite the importance of these receptors in regulating striatal function, it has remained unclear how the release of ACh drives the activation of muscarinic receptors on MSNs. Using GIRK2 channels as a readout of muscarinic activation, our results show that muscarinic receptors on dMSNs receive independent, phasic synaptic inputs from CHIs. The firing of a single action potential in CHIs evoked unitary GIRK-mediated IPSCs in post- synaptic dMSNs. Our results also indicate that the physiological and anatomical characteristics of CHIs allow for consistently depressed ACh release during CHI tonic firing. Despite this depression, muscarinic M4-receptors reliably encode
ACh release evoked by physiological CHI firing patterns without failure. As a result of the depression of ACh release during physiological firing of CHIs, MSNs increase their dynamic range of receptor activation, potentially allowing for differential behavioral responses in response to influential stimuli. We found that in the absence of GIRK, muscarinic receptors also could rapidly regulate dMSN output locally in the striatum through an inhibition of axon collateral synapses.
95 Thus, the firing of CHIs may be endogenously encoded in striatal circuits through the transient inhibition of local circuits.
Although CHIs only comprise a small percentage of neurons in the striatum, their tiled distribution and extensive arborizations position them to modulate a large MSN population. Cholinergic terminals have been found to make sparse synaptic connections (~3% synaptic incidence) with MSN dendrites, occurring primarily at symmetrical synapses (Bolam, 1984; Contant et al., 1996;
Descarries and Mechawar, 2000). Despite this low connectivity, monosynaptic rabies-tracing studies that have mapped CHI inputs to MSNs have found CHIs are extensively connected with multiple dMSNs (Salin et al., 2009). Interestingly, the connections formed between CHIs and MSNs using rabies stands in contrast to similar studies that have examined SNc dopamine inputs onto MSNs (Wall et al., 2013). While SNc terminals provide abundant innervation to the striatum, monosynaptic rabies tracing approaches have found that only a small proportion of the total dopaminergic inputs are labeled (Wall et al., 2013). One possibility for the increased monosynaptic rabies labeling of cholinergic synapses may be that CHI terminals span shorter distances to MSN dendrites than DA synapses.
The symmetrical synapses formed between CHIs and MSNs where M4-receptors have been found (Hersch et al., 1994), may therefore be the cholinergic synapses labeled with trans-synaptic rabies mapping.
We found that CHI firing resulted in consistent amplitude muscarinic
IPSCs. The low failure rate, low coefficient of variation and high probability of
96 release of IPSCs suggest that each CHI action potential drives release of transmitter from many active release sites. The large number of active terminals likely allows for the overall stable amplitude of muscarinic events. This may be similar to other synapses at which multiple release sites and a high probability of release contribute to high fidelity post-synaptic receptor activation (Buhl et al.,
1994; Kraushaar and Jonas, 2000; Marty et al., 2011; Silver et al., 1998). During the course of these experiments we were unable to resolve miniature events resulting from spontaneous vesicle release. One possibility may be that the large number of active zones, in conjunction with the slow kinetics of relatively small events may have been below the level of detection. Future studies using mean- variance quantal analysis or noise deconvolution may elucidate the quantal characteristics of ACh release at this synapse.
ACh release exhibited robust paired pulse depression at these synapses.
This depression was presynaptic in nature, as decreasing postsynaptic receptor activity did not alter the paired pulse ratio. As a result of this depression, during pacemaker firing cholinergic transmission likely exists under a basally depressed state. A decrease in uIPSC variance coincided with the depression of ACh release, indicating that not all release sites are active during a given action potential (Clements and Silver, 2000; Scheuss and Neher, 2001). The decreased probability of release as a result of short-term plasticity could allow for a larger dynamic range of muscarinic receptor activation from a single CHI. During reward based learning tasks, pauses in CHI firing are followed by a rebound
97 burst (Aosaki et al., 1994; Atallah et al., 2014; Chuhma et al., 2014; Morris et al.,
2004; Straub et al., 2014). Thus, tonic depression of ACh release at basal CHI firing rates results in differential muscarinic receptor activation in response to burst firing, effectively allowing for bi-directional dynamics in this system and amplifying the range of receptor activation in response to salient cues.
The autonomous pacemaking of cholinergic interneurons (Bennett and Wilson,
1999) has been thought to create a background tone of ACh. The present results however show that the firing of individual CHIs evokes the local release of ACh in the striatum sufficient to evoke phasic receptor activation. We found CHI firing could transiently activate muscarinic receptor signaling either through GIRK channels or the endogenous inhibition of GABAergic collaterals. Thus, although cholinergic firing does not correlate with MSN firing at the cell body level (Adler et al., 2013), time-locked CHI activity can rapidly alter terminal transmitter release from dMSNs. This is similar to the rapid muscarinic receptor-mediated inhibition that has been observed at corticostriatal synapses onto MSNs (Pakhotin and
Bracci, 2007). Rather than directly modifying MSN excitability, muscarinic receptors may therefore evoke subtle changes in MSN activity. Similar to muscarinic receptors, dopamine receptors do not acutely regulate MSN excitability through direct interactions with ion channels (Gerfen and Surmeier,
2011; Kreitzer, 2009). Instead, recent studies have found that dopamine receptors can drive rapid effects through 2nd messenger signaling cascades to regulate integration and plasticity in MSNs on a short-latency millisecond
98 timescale (Swapna et al., 2016; Yagishita et al., 2014). Dopamine D1 receptors on dMSNs have been shown to promote spine growth in response to brief stimulation of dopamine release (Yagishita et al., 2014). Dopamine D1 receptors and muscarinic M4 receptors in dMSNs oppose one another’s downstream signaling activity (Shen et al., 2015). As dopamine receptors and muscarinic receptors can both signal transiently, the opposing firing patterns of dopamine neurons and CHIs likely shape the properties of transmitter release from dMSNs.
Because transient muscarinic receptor activation on dMSNs is involved in disconnecting the microcircuit through collateral inhibition in the striatum, CHIs could play an integral role in temporally segregated modulation of local striatal connectivity. Recent work studying the timing of striatal GPCR activation has begun to elucidate the rapid biochemical effects of receptor signaling. Our results show that firing of individual CHIs leads to temporally and spatially localized muscarinic receptor activation, providing insights into the initial stages activating these pathways. The faithful time locked response of muscarinic receptors on
MSNs could thus prove critical in controlling CHI-regulated behaviors.
99 Figure 3.1. Muscarinic receptors evoke an outward current in MSNs following viral mediated expression of GIRK2.
(A) Immunohistochemical image illustrating AAV induced expression of GIRK2 and tdTomato in striatal sections two weeks following injection of
AAV2/9.hSyn.tdTomato.T2A.GIRK2.WPRE into the dorsal striatum. Shown are tdTomato, GIRK2 and choline acetyl transferase (ChAT) immunoreactivity in striatal cells indicating that AAV drove the expression of GIRK2 in non- cholinergic striatal neurons. 20 µm scale bar.
(B) Quantification tdTomato+ and ChAT+ neurons expressing GIRK2 immunoreactivity.
(C) Example voltage-clamp recording from a tdTomato+ MSN. Bath application of the muscarinic agonist oxotremorine-M (1 µM) evoked an outward current.
(D) Concentration-response curves for the outward current produced from
GIRK2+ MSN by bath application of Oxo-M > 21-days (purple) or 4-days
(magenta) following AAV.GIRK2 injection into the striatum. There is an increase in the maximum effect produced by Oxo-M, but no change in the EC50. Error bars indicate ± SEM. n = 4 - 11 ** = p < 0.01, Mann-Whitney.
(E) Example trace from a tdTomato+ MSN in an AAV-injected hemisphere (top) and a control non-fluorescent MSN in a control uninjected slice (bottom).
Iontophoretic application of ACh (100 µM, 100 ms) evoked an outward current that was blocked by the muscarinic antagonist scopolamine (1 µM) (top). No current could be evoked in control MSNs from uninjected hemispheres (bottom).
100 (F) ACh-mediated current voltage relationship. 10mV voltage steps were given from -50 to -130 mV. Subtraction of the control traces from those recorded in
ACh reveals a current that shows inward rectification and reverses near the predicted K+ reversal potential.
101
Figure 3.1
102 Figure 3.2. Expression GIRK2 was largely restricted to non-cholinergic interneurons
(A) Immunohistochemical image two weeks following injection of
AAV5.hSyn.DIO.hM4Di.mCherry into the dorsal striatum of a ChAT-Cre mouse.
Shown are ChAT immunoreactivity but the lack of mCherry fluorescence illustrating the lack of expression of hM4Di.mCherry in CHIs. 20 µm scale bar.
(B) Immunohistochemical image illustrating from control, uninjected dorsal striatum. Shown are tdTomato, GIRK2 and choline acetyl transferase (ChAT) immunoreactivity. In control animals, no GIRK2 or tdTomato fluorescence was observed. 20 µm scale bar.
103
Figure 3.2
104 Figure 3.3. CHI firing evokes spontaneous muscarinic-IPSCs in GIRK2+
MSNs.
(A) Representative traces of sIPSCs in the absence of stimulation from a
GIRK2+ MSN (black) that were blocked by the muscarinic antagonist scopolamine (1 µM) (grey).
(B) Percentage of neurons displaying sIPSCs in GIRK2+ WT MSNs and D1- eGFP+ (dMSN) MSNs.
(C) Amplitude distribution of sIPSCs from the MSN shown in (A) (10 pA bin size).
(D) Rise time distribution of sIPSCs from the same neuron illustrating that rise time does not correlate with amplitude of event (n = 51 events, amplitude distribution: p > 0.05, Chi-squared test; amplitude vs rise: r2 = 0.03, p = 0.21,
Pearson correlation).
(E) Representative trace illustrating the sIPSCs were blocked by TTX (200 nM).
(F) Summary of pharmacological effects on sIPSCs frequency. sIPSCs were eliminated in the presence of scopolamine (1 µM), tropicamide (1 µM), Ba2+ (200
µM), vesamicol (2 µM), low Ca2+ (none added) and TTX (200 µM) (* = p < 0.05,
*** = p < 0.001, Wilcoxon matched-pairs signed rank).
(G) Representative immunohistochemical images showing selective expression of ChR2-eYFP in a ChAT+ neuron.
(H) Optogenetic activation of ChR2-expressing CHIs in the striatum evokes a muscarinic-IPSC in GIRK2+ MSNs. Representative trace illustrates that the light-
105 evoked IPSC (2 ms flash, 470 nm light) was blocked by the muscarinic antagonist scopolamine (1 µM).
(I) Summary data illustrating that optogenetic activation of multiple CHIs evokes muscarinic-IPSCs larger and longer in duration than muscarinic sIPSCs (n = 8 neurons, p < 0.01 for amplitude and p < 0.05 for duration, Wilcoxon matched- pairs signed rank).
(J) Optogenetic silencing of eNpHR-expressing CHIs in the striatum eliminates sIPSCs in GIRK2+ MSNs. Representative trace illustrates the effect of green light
(5 s, 530 nm). (K) Summary data illustrating that optogenetic silencing of CHIs eliminates sIPSCs yet does not alter the baseline holding current (frequency: p <
0.001, holding current: p > 0.05, Wilcoxon matched-pairs signed rank).
106
Figure 3.3
107 Figure 3.4. Expression of ChR2 and eNPHR in striatal cholinergic interneurons
(A) A 470 nm light pulse (2 ms) evokes an action potential in CHIs of ChAT-Cre mice expressing AAV.DIO.ChR2.
(B) Representative trace of a hyperpolarization induced inward current evoked by a 30 mV hyperpolarization in eNPHR.eYFP expressing CHIs recorded in ChAT-
Cre mouse. CHI were identified by the presence of a slow, hyperpolarization activated inward current.
(C) Pacemaker firing of CHIs is silenced by 530 nm light (5 seconds) in ChAT-
Cre animals expressing AAV.DIO.eNPHR.
108
Figure 3.4
109 Figure 3.5. Muscarinic receptors mediate unitary IPSCs at CHI-MSN synapses.
(A) In paired recordings between CHIs and GIRK2+ MSNs, an action potential in a current-clamped CHI elicits a unitary IPSC in a voltage-clamped MSN. MSNs were held at -60 mV. CHIs were hyperpolarized by negative DC current injection and depolarized to threshold with 4 ms 150 pA steps (bottom trace). Two uIPSCs (u) are shown that were time locked to the firing of the paired CHI and two sIPSCs (s) occurred that were unpaired with the firing of the recorded presynaptic CHI.
(B) Averaged MSN responses following single action potentials in the synaptically coupled CHI pair illustrated in (A).
(C) uIPSC amplitude histogram (mean = 113 ± 0.3, n = 40 uIPSCs, 10 pA bin size) and latency distribution histogram (time to 10% of peak, mean = 32.9 ±
0.09, n = 39 uIPSCs, 2 ms bin size) for the synaptically coupled CHI pair illustrated in (A). All action potentials resulted in a resolvable IPSC.
(D) Summary of average uIPSC amplitude that was blocked the muscarinic receptor antagonist scopolamine (1 µM) (n = 6, p < 0.05, Wilcoxon matched-pairs signed rank).
(E) No difference was found in the amplitude or duration between sIPSCs and uIPSCs in all CHI-GIRK2+ MSN paired recordings (Mann-Whitney).
110 (F) For each paired CHI-MSN recording, the coefficient of variation (CV) for uIPSCs was less than that of sIPSCs (n = 23 pairs, p < 0.001, Wilcoxon matched-pairs signed rank).
111
Figure 3.5
112 Figure 3.6. Firing of individual cholinergic interneurons do not evoke monosynaptic GABA or glutamate synaptic events.
(A) Paired uIPSCs recorded in the absence (black) or presence of GABA, or glutamate receptor antagonists (gray) are similar. In addition, no fast synaptic events were observed at the foot of the muscarinic IPSCs time locked with the firing of the CHI. Recordings were made in the presence of high Cl- solution (75 mM) solution so as to increase the outward driving force for Cl- to maximize resolving GABAa synaptic events. (B) Quantification of the amplitude of muscarinic uIPSCs in the absence and presence of picrotoxin (100 µM) and
DNQX (10 µM) to block GABAa or AMPA receptors.
(C) Representative trace from GIRK2+ MSN illustrating that optogenetic synchronous activation of multiple CHIs with ChR2 evokes large fast inward currents at the foot of muscarinic IPSCs. Recordings were made in the presence of high Cl- solution (78 mM) solution so as to increase the outward driving force for Cl-.
(D) Representative traces from 4 MSNs recorded near the surface of the brain slice illustrating consistent amplitude muscarinic sIPSCs.
113
Figure 3.6
114 Figure 3.7. Convergence and functional independence of CHI inputs onto
MSNs
(A) Two traces from a paired CHI-MSN recording separated by 5 s illustrating that the amplitude of uIPSCs were unaffected if immediately preceded by an unpaired sIPSC (grey box). CHIs were hyperpolarized by negative DC current injection and depolarized to threshold with 4 ms 150 pA steps to trigger uIPSCs
(bottom trace).
(B) Quantification of uIPSC amplitudes of isolated uIPSCs (not preceeded by an action potential for > 5s) and uIPSCs where a sIPSC occurred in < 1 s prior (n =
15 paired recordings, p > 0.05, Wilcoxon matched-pairs signed rank).
(C) Representative trace of cell-attached paired recordings showing paired and unpaired IPSCs. Dashed line indicate uIPSCs that are paired with the recorded
CHI.
(D) Quantified percentage of paired and unpaired IPSCs for the paired recording represented in (A).
(E) Group ratio of paired or unpaired IPSCs to the total number of IPSCs (n = 18 pairs, p < 0.0001, chi-square = 16.067, 1 DOF).
(F) Distribution histogram of paired IPSC ratios (0.1 bin size, n = 18 pairs).
(G) Number of CHIs connected to an MSN in each paired recording. Mean value represented by horizontal line (2.9 ± 0.2 CHIs, n = 18).
115
Figure 3.7
116 Figure 3.8. The extent of muscarinic receptor activation is regulated by the enzymatic degradation of ACh.
(A) Representative sIPSCs recorded under control conditions and in the presence of the AChE inhibitor ambenonium (10 nM).
(B) Averaged superimposed sIPSCs from the above cell under control conditions and in the presence of ambenonium (10 nM) illustrating that inhibiting AChE increases the amplitude and duration of sIPSCs.
(C) Summary of the effect of ambenonium (10 nM) on 10 – 90% rise time and duration (260 ± 24% control width) on sIPSCs (n = 6, p < 0.05, Wilcoxon matched-pairs signed rank).
(D) Amplitude distribution histograms (20 pA bin size) of sIPSCs in control and in the presence of ambenonium (10 nM) (p < 0.001, Kolmogorov-Smirnov test).
(E) Representative trace illustrating the effect of optogenetic silencing of eNpHR- expressing CHIs on muscarinic IPSCs in the presence of ambenonium (80 nM).
(F) Summary of the average baseline current amplitude measured before and after optogenetic silencing of eNpHR-expressing CHIs under control conditions and in the presence of ambenonium (80 nM) (p < 0.05, Mann-Whitney).
117
Figure 3.8
118 Figure 3.9. Silencing cholinergic firing leads to a depolarization of membrane potential in GIRK2+ MSNs only in when acetylcholinesterase is inhibited
(A) Current clamp recording from a GIRK2+ MSN from a ChAT-Cre mouse where AAV.DIO.eNPHR.eYFP has been injected. Wide field illumination of 530 nm light (5 seconds) leads to an inhibition of muscarinic inhibitory postsynaptic potentials (IPSPs) with no change in the baseline membrane potential (dashed line).
(B) In the presence of a subsaturating concentration of ambenonium (50 nM), to partially inhibit acetylcholinesterase activity, silencing CHI firing leads to an inhibition of muscarinic IPSCs as well as a depolarizing shift in the membrane potential.
(C) Quantification of the change in baseline membrane potential under control conditions and in the presence of ambenonium (50 nM) (p < 0.01, n = 3, students unpaired t-test).
119
Figure 3.9
120 Figure 3.10. Muscarinic inhibition of dMSN GABA collaterals
(A) Optogenetic silencing of eNpHR-expressing CHIs leads to a rebound burst of
APs upon termination of wide-field (530 nm) illumination.
(B) Quantification of the latency to 1st AP following termination of 530 nm light and quantification of the ISI of the first 2 APs in the initial rebound burst.
(C) Representative recording from a dMSN-dMSN paired recording in slices from a D1-eYFP x ChAT-Cre mouse expressing eNpHR in CHIs. An AP in the presynaptic dMSN triggered a monosynaptic GABAA IPSC in the postsynaptic dMSN. The amplitude of the GABAA IPSC was reduced by terminating wide-field
(530 nm) illumination 500 ms before triggering the AP in the presynaptic MSN.
Traces are the average of 6 sweeps for each condition. GABAA IPSCs were recorded in the presence of DNQX (10 µM) and SKF 38393 (10 nM).
(D) Quantification of the average amplitude of GABAA IPSCs under control conditions versus 500 ms following termination of 530 nm illumination (n = 7 pairs, p <0.05, Wilcoxon matched-pairs signed rank).
(E) Quantification of the average amplitude of GABAA IPSCs under control conditions versus 500 ms following termination of 530 nm illumination in the presence of scopolamine (1 µM) (n = 5 pairs, p >0.05, Wilcoxon matched-pairs signed rank).
121
Figure 3.10
122 Figure 3.11. Release properties of ACh at CHI-MSN muscarinic synapses
(A) Representative traces of uIPSCs evoked by paired pulse stimulation with 250 ms (above) and 500 ms (below) intervals. IPSCs evoked by the second action potential (AP) (grey) were calculated by subtracting the single pulse IPSC for intervals 100 and 250 ms apart.
(B) Representative traces illustrating that the PPR of uIPSCs was not altered when GTP was replaced with GDPbs (0.6 mM) in the intracellular solution.
(C) Summary data of PPRs of uIPSCs evoked at 250 or 2000 ms ISI with either
GTP or GDB s in the intracellular solution.
(D) PPR (P2 / P1) for uIPSCs plotted against ISIs of 100, 250, 500, 1000, 2000,
4000 and 7000 ms. PPR is illustrated for uIPSCs under control conditions
(black) and in the presence of an IC50 concentration of tropicamide (20 nM)
(grey). Inset: inhibition of evoked IPSCs by the IC50 concentration tropicamide
(20 nM) (p < 0.05, n = 5, Wilcoxon matched-pairs signed rank).
123
Figure 3.11
124 Figure 3.12. GDPbs abolishes dopamine D2-receptor mediated synaptic events in CHIs
(A) Voltage clamp recording from a CHI from a DAT-Cre mouse where
AAV.DIO.Chr2.eYFP was been injected into the substantia nigra. After allowing 3 weeks for ChR2 expression in midbrain dopamine neurons, optogenetic activation of dopamine terminals in the striatum evokes dopamine D2-receptor mediated IPSCs in CHI. D2-IPSCs could be evoked using a GDBbs-based internal immediately after breaking into whole-cell recording mode (black). In contrast, allowing 6 minutes for GDBbs to dialyze abolished D2-IPSCs.
Right: Quantification of the change in D2-IPSC amplitude between 1 and 6 minutes of dialyzing CHIs with GDBbs (p < 0.001, n = 3, students unpaired t- test).
125
Figure 3.12
126 Figure 3.13. Burst firing relieves depression at muscarinic synapses as a result of tonic firing of cholinergic interneurons.
(A) Representative trace of paired uIPSCs evoked by a 2 Hz train of 10 APs in a
CHI.
(B) Summary data of normalized uIPSCs amplitudes as a function of pulse number.
(C) Paired uIPSC amplitude variance as a function of pulse number for the 1st and 10th pulse in a train (2 Hz) (n = 6 p < 0.05, Wilcoxon matched-pairs signed rank).
(D) Bursts of APs (5 APs at 20 Hz) generate similar amplitude uIPSCs as a single APs when CHIs when tonic firing of CHIs is prevented.
(E) Quantification of uIPSC amplitudes when evoked by a single AP or bursts of
APs.
(F) Bursts of APs (5 APs at 20 Hz) generate larger amplitude uIPSCs than single
APs when transmission is depressed by tonic CHI firing (2 Hz). Tonic firing of
CHI was simulated by a inducing a train of 20 APs (2 Hz) in CHIs. A 1 s pause in firing was followed either by a single AP or a burst (5 at 20 Hz) of APs.
(G) Quantification of the amplitude of uIPSCs when evoked by a single AP or trains of APs following tonic firing (n = 5, p < 0.05, Wilcoxon matched-pairs signed rank).
127 (H) Quantification of the change in uIPSC amplitude evoked by single or bursts of
APs when compared to the amplitude of the last uIPSC in a train (n = 5, p < 0.05,
Wilcoxon matched-pairs signed rank).
128
Figure 3.13
129 CHAPTER 4
Cortical and thalamic inputs evoke cholinergic transmission in
the striatum
A version of this work will be submitted as:
Mamaligas, A.A., and Ford, C.P. (2018). Cortical and thalamic inputs evoke
cholinergic transmission in the striatum. In preparation.
130 Abstract
Striatal cholinergic interneurons (ChIs) modulate output activity of medium spiny neurons (MSNs), the major striatal projection neurons. Striatal inputs from the parafascicular nucleus of the thalamus drive ChIs to fire, whereas inputs from the motor cortex do not. It remains unclear how these changes in ChI firing are translated to muscarinic receptor activity on MSNs, as ChIs are convergent at
M4-expressing synapses. Using optogenetic stimulation of excitatory thalamic and cortical inputs onto ChIs, we found that thalamic inputs drive high probability
ChI firing due to presynaptic facilitation. Conversely, we found that a subset of
ChIs respond to stimulation of cortical inputs, despite no increase in ChI firing overall. Measuring M4 receptor activity in MSNs with overexpressed G-protein inwardly rectifying potassium channels (GIRK2), we found no difference in M4- receptor activity in response to these two stimuli. Despite the low number of ChIs that respond to cortical input, the convergence of these cells allows for consistent
ACh release.
131 Introduction
Acetylcholine (ACh) plays an important role in regulating activity in the striatum. Cholinergic interneurons (ChIs) are the major source of ACh within the striatum (Lim et al., 2014). Although ChIs comprise only a small percentage of striatal neurons (Kreitzer, 2009), increases in ChI activity can drive striatal dependent associative behaviors (Bradfield et al., 2013; Stalnaker et al., 2016), while pauses in ChI firing are sufficient to alleviate Parkinsonian symptoms
(Maurice et al., 2015). Release of ACh from ChIs modulates multiple cell types within the striatum, including the output cells of the striatum, medium spiny neurons (MSNs). Modulation of MSN activity occurs through presynaptic modulation via both nicotinic ACh receptors (nAChRs) and muscarinic receptors
(Cachope et al., 2012; English et al., 2011; Goldberg et al., 2012; Higley et al.,
2009; Mamaligas et al., 2016; Nelson et al., 2014a; Threlfell et al., 2012) as well as direct modulation of MSNs themselves via muscarinic receptors (Goldberg et al., 2012; Mamaligas and Ford, 2016). Of the five subtypes of muscarinic receptors, Gq/11-coupled M1-receptors are expressed on both direct pathway D1-
MSNs and indirect pathway D2-MSNs, while Gi/o-coupled M4-muscarinic receptors are primarily expressed on D1-MSNs (Hersch et al., 1994; Kreitzer,
2009). In D1-MSNs, ChIs make monosynaptic connections with MSNs at M4- muscarinic synapses, with multiple ChIs making independent synaptic connections onto a given MSN (Mamaligas and Ford, 2016). The activation of
M4-receptors on MSNs results in G-protein-mediated inhibition of Ca2+ channels
132 as well as decreased collateral activity between MSNs, leading to a decrease in excitability and inhibition of long term potentiation (Mamaligas and Ford, 2016;
Perez-Rosello, 2005; Yamamoto et al., 2013).
Although ChIs exhibit endogenous pacemaker activity, firing between 1-10
Hz (Bennett and Wilson, 1999; Graybiel et al., 1994), they display stereotyped
“burst-pause-burst” firing patterns in vivo in response to salient environmental stimuli (Aosaki et al., 1995; Graybiel et al., 1994; Morris et al., 2004). These transient bursts, lasting approximately 50 ms each (Aosaki et al., 1995; Apicella et al., 1997; Morris et al., 2004), are thought to be predominantly generated via excitatory inputs from the intralaminar thalamic nuclei (Ding et al., 2010;
Matsumoto et al., 2001; Threlfell et al., 2012). Previous work has determined that input from the parafascicular nucleus of the thalamus (Pf) is both correlated with and necessary for this stereotyped firing pattern in vivo (Matsumoto et al., 2001).
While the sensorimotor cortex comprises the other predominant excitatory input in the dorsal striatum (Gerfen, 1992), it sends stronger inputs to MSNs than ChIs
(Ding et al., 2008, 2010) Although motor cortex stimulation can increase ChI firing in vivo (Doig et al., 2014), cortical activity does not correlate strongly with
ChI firing patterns (Sharott et al., 2012). Similarly, work from brain slices has shown that while Pf inputs drive bursts of action potentials in ChIs, cortical inputs only weakly increase ChI firing above baseline (Ding et al., 2010).
While cortical and thalamic inputs are known to differentially drive ChI activity, it remains unclear how these changes in ChI firing drive ACh release at
133 MSN synapses. Using optogenetic activation of excitatory cortical and thalamic inputs to the striatum, we found that thalamic inputs indeed drive ChI firing more effectively than cortical inputs. However, there is a subset of ChIs that strongly respond to cortical inputs. As a result, both cortical and thalamic excitation consistently drove ACh release at muscarinic synapses onto direct pathway D1-
MSNs to a similar extent. We further determined that this ACh release is dependent on different populations of glutamate receptors on ChIs. Overall, our results suggest that convergence of ChIs at muscarinic synapses can create a disconnect between firing and postsynaptic receptor activity. Thus, cortical inputs drive ACh release more effectively than previously thought due to the selective innervation of cortical inputs to certain ChI populations.
Experimental Procedures
Stereotaxic Injections. All procedures were performed in accordance with the guidelines for the Institutional Animal Care and Use Committee at Case Western
Reserve University and University of Colorado Anschutz. Male and female
CaMKii-Cre heterozygote mice (generated from WT and B6.Cg-Tg(Camk2a- cre)T29-1Stl/J, Jackson Laboratory) were anesthetized with isoflurane and mounted in a stereotaxic frame (Kopf Instruments). They were then injected using a nanoject iii microinjector (Drummond Scientific) between postnatal day
21 and 28. 500 nL of AAV5.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH were injected into one hemisphere of either the parafascicular thalamus (coordinates
134 relative to bregma: AP -1.65, ML -0.85, DV -3.4) or the motor cortex (AP +1.1,
ML -1.45, DV -1.2). For experiments requiring expression of GIRK2, 400 nL of
AAV9.hSyn.tdTomato.T2A.mGIRK2-1-A22A.WPRE.bGH were also injected into the striatum (AP +1.15, ML -1.8, DV -3.3). Animals were allowed to recover for at least 3 weeks following surgery. All AAVs were from University of Pennsylvania
Viral Core.
Fluorescence Imaging. Following deep isoflurane anesthesia, AAV.ChR2- injected animals were transcardially perfused with phosphate buffered saline
(PBS) containing 137 mM NaCl, 1.5 mM KH2PO4, 8 mM NaH2PO4, and 2.7 mM
KCl (pH = 7.4), followed by 4% paraformaldehyde in PBS. Brains were rehydrated overnight in 30% sucrose solution and then frozen and embedded in
Neg-50 frozen section medium (Richard-Allan Scientific). Coronal slices (30 µm) were taken on a cryostat and directly mounted to the slides. Fluorescence images were then taken with a 10x objective using a Olympus BX61VS slide- scanning microscope.
Slice Preparation. Animals were transcardially perfused with ice-cold sucrose solution and then decapitated. Coronal slices containing the striatum (240 µm) were obtained in the same ice-cold sucrose cutting solution containing (in mM):
75 NaCl, 2.5 KCl, 6 MgCl2, 0.1 CaCl2, 1.2 NaH2PO4, 25 NaHCO3, 2.5 D-glucose,
50 sucrose, and 1 kyneurenic acid, bubbled with 95% O2 and 5% CO2. Slices were incubated at 34ºC for 1 hour in aCSF containing (in mM): 126 NaCl, 2.5
135 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and 11.1 D-glucose, bubbled with 95% O2 and 5% CO2. Slices were then transferred to the recording chamber and perfused with aCSF (34 ± 2ºC) at a rate of 2 mL/min. Solutions contained picrotoxin (100 µM). Striatal neurons were visualized using a BXWI51 microscope (Olympus) with IR gradient contrast optics and custom-made LEDs.
Electrophysiology. Recordings were performed using an Axopatch 200B amplifier
(Molecular Devices). ChIs were identified with a large h-current when stepped to
-90 mV in voltage clamp. Patch pipettes (1.5-2 MW) used for MSN recordings and current-clamp recordings in ChIs contained: 135 mM D-gluconic acid (K), 10 mM HEPES (K) 0.1 mM CaCl2, 2 mM MgCl2, 10 mM BAPTA, 10 mg/mL ATP, 1 mg/mL GTP, and 15 mg/mL sodium phospho-creatine, pH = 7.4, 275 mOsm.
Patch pipettes used for all ChI voltage clamp recordings contained: 135 mM
CsCl, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES (K), 0.1 mM EGTA, 10 mM
QX-314, 10 mg/mL ATP, 1 mg/mL GTP, and 15 mg/mL sodium phospho- creatine, pH = 7.4, 275 mOsm. All recordings were acquired with Axograph X
(Axograph Scientific) at 10 kHz. MSNs were held at a voltage of -60 mV. No series resistance compensation was used, and cells were discluded if their series resistance exceeded 15 MW. ChR2 excitation was evoked with 470 nm blue light
(2 ms per pulse, ~1.0 mW/mm2). All drugs were bath applied via perfusion unless otherwise noted. Glutamate was applied via iontophoresis (150 mM, 160 nA ejection, 200-400 ms; 1 pulse/2 minutes).
136 During voltage jump recordings, ChIs were held at -60 mV. ChIs were stepped to 0 mV and rested there for 3 seconds before the light stimulus. ChIs were then jumped back to -60 mV 5 ms after the light stimulus. These trials were interleaved with trials without the light stimulus, and the capacitive artifact was subtracted. During iontophoresis experiments, the delay between the stimulus and the jump from 0 mV to -60 mV was approximately 5 seconds.
2-Photon Imaging. Images were visualized with a BX5WI microscope (Olympus) and a home built 2-photon laser scanning microscopy system. This system used
XY galvanometer mirrors (6215, Cambridge Technology) and custom imaging software. A Mira 900 Ti:sapphire laser with a Verdi G10 pump laser (Coherent) was tuned to 800 nm. Epifluorescence signals were measured through a 60X water immersion objective (Olympus) using a T700LPXXR dichroic mirror,
ET680sp and ET620/60 filters (Chroma), and a H10721-20 photomultiplier tube
(Hamamatsu). A SR570 current preamplifier (Stanford Research Systems) converted output to voltage. The voltage was digitized using an NI PCI-6110 data acquisition board (National Instruments). Custom code (Ben Strowbridge) was used to generate bidirectional waveforms in Visual Studio and Python. Alexa 594
(20 µM) in the patch pipette allowed for visualization of ChIs. Internal solution for these experiments also contained: 135 mM CsCl, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES (K), 0.1 mM EGTA, 10 mM QX-314, 10 mg/mL ATP, 1 mg/mL GTP, and 15 mg/mL sodium phospho-creatine, pH = 7.4, 275 mOsm. For glutamate
137 iontophoresis, a thin-walled glass iontophoretic electrode was filled with glutamate (1 M) with sulforhodamine 101 (300 µM) and was dipped in a solution containing BSA-conjugated Alexa 594 (0.06%) for ~30 seconds. Glutamate was injected as an anion in 250 – 500 ms pulses. Glutamate leakage was prevented with a retention current of ~20 nA.
Materials. Picrotoxin was obtained from Abcam. QX314, NBQX, and AP5 were obtained from Tocris. BAPTA was from Invitrogen. All other chemicals were from
Sigma-Aldrich.
Statistics and Analysis. Data are shown as mean ± SEM. Statistical significance was determined using Wilcoxon matched-pair signed rank test, Mann-Whitney, 2-
Way ANOVA, Student’s paired t test, or Kruskal-Wallace tests where appropriate
(Prism, Graphpad).
Results
A subpopulation of ChIs increase firing in response to cortical stimulation
To examine the role of excitatory striatal inputs on ChI activity in the dorsal striatum, an adeno-associated virus (AAV) encoding a floxed channelrhodopsin and a eYFP fluorophore (AAV.DIO.ChR2.eYFP) was injected into either the M1 region of the motor cortex or the parafascicular nucleus of the thalamus (Pf) of
CaMKII-Cre heterozygous mice. Three weeks post-injection, neurons in both M1
138 and the Pf displayed robust YFP expression (Figure 4.1A and 4.1B). We performed whole cell voltage clamp recordings of ChIs in the striatum (Vh = -80 mV) and stimulated either cortical or thalamic inputs with a single pulse of blue light (2 ms, 470 nm). We found that optogenetically-evoked, AMPA receptor mediated excitatory post synaptic currents (EPSCs) were similar in amplitude when evoked from cortical and thalamic terminals (thalamus: -193 ± 70 pA, n =
10; cortex: -211 ± 50 pA, n = 7; p > 0.05, Mann-Whitney) (Figure 4.1C and 4.1D) and had similar AMPA/NMDA ratios (NMDA EPSCs measured at Vh = +40 mV; thalamus: 2.0 ± 0.3, n = 7; cortex: 1.6 ± 0.3, n = 8; p > 0.05, Mann-Whitney)
(Figure 4.1C and 4.1E). Thus, there were no differences in the magnitude of transmission at these two synapses for a single stimulus.
Previous work examining excitatory post-synaptic potentials (EPSPs) found that thalamic inputs facilitate, whereas cortical inputs depress (Ding et al.,
2010). As a result, bursts of thalamic excitation strongly drive ChI firing, whereas bursts of cortical excitation evoke only a modest increase in ChI activity (Ding et al., 2010). To confirm this, we performed whole cell current clamp recordings of
ChIs and recorded EPSPs in response to a burst of stimuli (5 stimuli, 50 Hz, 2 ms each). Although there was no overall difference in the amplitude of EPSCs on the first pulse (thalamus: 2.0 ± 0.5 mV, n = 14; cortex: 2.4 ± 0.9 mV, n = 15; p > 0.05,
Mann-Whitney) (Figure 4.1F and 4.1H), thalamic inputs indeed displayed robust facilitation, whereas cortical inputs depressed (thalamus 5th pulse/1st pulse: 2.1 ±
0.3, n = 10; cortex 5th pulse/1st pulse 1: 0.92 ± 0.07, n = 10; p < 0.001, Mann-
139 Whitney; across pulses p < 0.001, 2-Way ANOVA) (Figure 4.1F and 4.1G). Next, we examined the same question using cell-attached recordings of ChIs, allowing for observation of intrinsic firing patterns without disturbing the intracellular environment. Similar to previous findings, thalamic excitation (5 pulses, 50 Hz) increased the firing of all ChIs recorded during the 100 ms window following the stimulus compared to baseline (Figure 4.2A and 4.2C) (baseline probability: 0.24
± 0.06, stimulus probability: 0.77 ± 0.05, n = 13, p < 0.001, Wilcoxon matched pair signed rank) (Figure 4.2A and 4.2C). The baseline for each neuron, which also served as the threshold for “responding” ChIs, was determined by the mean action potential probability for each 100 ms window 2 seconds prior to the stimulus, plus 2 standard deviations. Conversely, cortical stimulation at the same frequency and intensity did not increase the overall firing of ChIs above baseline,
(baseline probability: 0.22 ± 0.02, stimulus probability: 0.28 ± 0.06, n = 29, p >
0.05, Wilcoxon matched pair signed rank) (Figure 4.2B and 4.2C). This confirms that thalamic activity more strongly drives ChI firing than does cortical input to the dorsal striatum.
We found, however, that while stimulation of cortical inputs did not alter the average firing of ChIs, a subpopulation of ChIs did show a robust increase in firing (9/29 ChIs recorded, average cortical responder AP probability 0.67 ± 0.1).
This was reflected in the greater overall coefficient of variation for action potential probability following cortical stimulation (thalamus: 0.25, n = 13; cortex: 1.16, n =
29) (Figure 4.3A). We also found that cortical responders displayed a shorter
140 latency to spike relative to ChIs receiving thalamic stimulation (cortical responders: 20.2 ± 6 ms, n = 10; thalamus: 44.74 ± 4 ms, n = 11; p < 0.01,
Mann-Whitney) (Figure 4.3B). Because cortical inputs consistently depress, we hypothesized that there is preferential connectivity to a subpopulation of ChIs, such that the amplitude of cortically-evoked EPSPs must be larger in responding
ChIs than in non-responders.
To examine differences between ChI responders and non-responders to cortical input, we recorded a ChI in cell-attached mode and activated cortical inputs to determine whether or not the recorded cell responded to the stimulus.
We found that in responding ChIs (neurons with stimulus AP probability above
0.75), EPSPs measured by re-patching the same neuron in current clamp were significantly larger in amplitude than in ChIs that did not respond (neurons with stimulus AP probability below 0.2) (responder: 2.6 ± 0.4 mV, n = 6; non- responder: 0.57 ± 0.2 mV, n = 6; p < 0.01, Mann-Whitney) (Figure 4.2D - 4.2F).
Further, responders displayed a greater area under the curve for cortically- evoked EPSPs relative to non-responders (responders: 0.17 ± 0.02 mV*s, non- responders: 0.03 ± 0.009 mV*s, n = 6 for each, p < 0.01, Kruskal-Wallace)
(Figure 4.2E and 4.2G). The area under the EPSP train accounts for summation of individual EPSPs due to the long membrane time constant of ChIs
(Kawaguchi, 1993; Wilson et al., 1990). The EPSP area was not significantly different between ChIs that responded to cortical inputs compared to ChIs in response to thalamic stimulation (thalamus: 0.29 ± 0.1 mV*s, n = 10, p > 0.05
141 relative to cortical responders, p < 0.05 relative to cortical non-responders,
Kruskal-Wallace) (Figure 4.2E and 4.2G). Overall, this suggests that, although cortical inputs do not increase ChI firing on average, there is a subset of neurons that responds similarly to both cortical and thalamic inputs.
Cortical and thalamic excitatory inputs target different locations on ChI dendrites
Anatomical examination of cortical and thalamic inputs onto ChIs has shown that, while thalamic inputs target soma and proximal dendrites, cortical inputs are localized on distal ChI dendrites (Lapper and Bolam, 1992). To test whether the distance of these inputs from the ChI cell body might result in differences in dendritic filtering, we performed a voltage jump experiment
(Pearce, 1993). Due to the extensive dendritic arbor of ChIs, filtering of synaptic currents may occur at more distal dendritic sites due to space clamp (Pearce,
1993). ChIs were held at 0 mV, the reversal potential for AMPA receptors, such that no current would flow through open AMPA receptors. A single flash stimulus was then delivered to evoke glutamate release from either thalamic or cortical inputs. Five milliseconds after the light pulse, the voltage was stepped to -60 mV
(Figure 4.4A). After subtracting the capacitive transient, the resulting current is due to the driving force generated following the jump through open AMPA receptors. The rate of activation is determined by the speed by which the current can be clamped and is independent of the kinetics of AMPA receptor activation.
142 Due to increased membrane time constant, distal inputs would be expected to have slower rates of activation. Using this protocol, we found that the tactivation was significantly slower for voltage-jump currents evoked from cortical inputs
(1.74 ± 0.4 ms, n = 9) than thalamic inputs (0.65 ± 0.2 ms, n = 8, p < 0.05, Mann-
Whitney) (Figure 4.4B and 4.4C). This suggests that cortical inputs are more distal than thalamic. As a control, to verify that the tactivation of voltage-jump currents are slower for inputs further from the soma, we used the same voltage protocol as above, this time using iontophoretic application of glutamate to distal and proximal dendrites. Exogenous glutamate was locally applied via an iontophoretic pipette imaged using 2-photon microscopy. The iontophoretic pipette was positioned near spines of the recorded ChI at different distances along the same dendritic branch (Figure 4.4D). Repeating the voltage jump experiment revealed that voltage-jump evoked currents could be clamped faster when glutamate was applied to proximal dendrites (Figure 4.4E). The t of activation for the more proximally evoked iontophoretic current (1.19 ± 0.3 ms) was faster than the distally evoked currents in the same cell (6.63 ± 2 ms, n = 5, p < 0.05, Student’s paired t test) (Figure 4.4F and 4.4G). Taken together, these data suggest that cortical and thalamic currents have differing dendritic integration due to the location of each input.
143
Cortical and thalamic inputs equally drive striatal cholinergic transmission
We next wanted to assess how these distinct ChI firing patterns drive the release of ACh and activation of muscarinic receptors at synapses onto MSNs.
To measure the activation of muscarinic receptors we virally overexpressed a G- protein coupled inwardly rectifying K+ channel (GIRK2) in MSNs. Endogenous
M4-muscarinic receptors couple to overexpressed GIRK2 channels, providing a rapid, direct readout of synaptic M4-receptor activation (Mamaligas and Ford,
2016). Using this approach, we previously showed that the pacemaker firing of
ChIs evokes ACh release that is encoded at these synapses as a series of individual spontaneous M4-muscarinic IPSCs in GIRK2-expressing direct pathway D1-MSNs (Mamaligas and Ford, 2016). The expression of GIRK2 does not affect MSN excitability or input resistance (Marcott et al., 2014) and expression is largely restricted from ChIs (Mamaligas and Ford, 2016).
An AAV encoding GIRK2 and a soluble tdTomato fluorophore driven by the synapsin promoter (AAV9.hSyn.tdTomato.mGIRK2) was injected into the striatum and AAV.DIO.ChR2.eYFP was injected into either motor cortex or the parafascicular thalamus. Whole cell voltage clamp recordings were made from
D1-MSNs in the dorsal striatum (Vh = -60 mV). As cortical and thalamic inputs directly synapse onto MSNs (Ding et al., 2010; Parker et al., 2016), photoactivation (5 stimuli at 50 Hz) of both terminals evoked AMPA EPSCs in
MSNs (Figure 4.5A – 4.5C). In GIRK+ D1-MSNs, these EPSCs were followed by
144 a slower muscarinic receptor mediated M4-IPSCs (Figure 4.5A – 4.5C). Due to the slower kinetics of these metabotropic M4-IPSCs relative to ligand-gated ion channel currents (Mamaligas and Ford, 2016), it was possible to separately resolve the direct input AMPA EPSCs from the disynaptic M4-IPSCs resulting from ACh release from ChIs. The amplitude of M4-IPSCs was similar when evoked by cortical and thalamic stimulation (thalamus: 185 ± 29 pA, n = 26; cortex: 186 ± 31 pA, n = 25; p > 0.05, Mann-Whitney) (Figure 4.5C). Bath application of NBQX (10 µM) and AP5 (50 µM), to block AMPA and NMDA receptors respectively, completely eliminated both glutamatergic EPSCs and disynaptic evoked muscarinic IPSCs in all recordings (thalamus: -0.33 ± 0.5% remaining, n = 6; cortex: 0.17 ± 1% remaining, n = 11). Consistent with the long latency of ChI firing when driven by thalamic inputs (Figure 4.3B), the time to peak of M4-IPSCs was also slower when evoked by thalamic stimulation
(thalamus: 249 ± 8 ms, n = 13; cortex: 202 ± 5 ms, n = 15; p < 0.001, Mann-
Whitney) (Figure 4.6C). However, we found that unlike the firing of ChIs, which was driven more strongly by thalamic inputs, both cortical and thalamic stimulation evoked equally robust M4-IPSCs in all MSNs examined (IPSC probability – thalamus: 0.96 ± 0.02, n = 14; cortex: 0.99 ± 0.006, n = 15; p > 0.05,
Mann-Whitney) (Figure 4.5D). As the release of ACh underlying M4-IPSCs is action-potential dependent (Mamaligas and Ford, 2016), this suggests that the subpopulation of ChIs that do respond to cortical stimulation drive cortically- evoked M4-IPSCs.
145 ChIs converge onto D1-MSNs at independent muscarinic synapses with approximately three ChIs synapsing onto one MSN (Mamaligas and Ford, 2016).
Across cell-attached recordings from ChIs, cells that do respond to excitatory stimuli from either region fire an action potential during approximately 70% of trials. MSNs are capable of integrating those convergent responses in an additive manner. When we performed minimal electrical stimulation of multiple ChIs converging onto a GIRK2+ MSN recorded in voltage clamp, these stimuli evoke
IPSCs that sum arithmetically (Figure 4.6A and 4.6B). This allows for the third of
ChIs that do respond to cortical input to regularly release ACh onto MSNs in response to increases in firing. Thus, convergence of tiled ChIs across the striatum, some of which respond to cortical input, provides reliable cholinergic signaling across striatal MSNs due to these stimuli. Overall, this suggests that convergence in ChI connectivity at muscarinic synapses on MSNs allows for reliable receptor activation even in situations in which ChI stimulation causes no average increase in firing.
Role of AMPA and NMDA receptors at cortical and thalamic ChI synapses
Following multiple excitatory stimuli, AMPA receptor-mediated depolarization of the post-synaptic cell is often sufficient to relieve NMDA receptor Mg2+ block, leading to increased activity during later pulses in the stimulus (Herron et al., 1986). Although we saw no differences in AMPA/NMDA ratios following single stimuli (Figure 4.1E), it is possible that multiple stimuli
146 would depolarize ChI dendrites sufficiently to unblock NMDA receptors. Because thalamic inputs facilitate, these inputs might be more likely to depolarize and unblock ChI NMDA receptors. To examine the role of different glutamate receptors on ChIs, we bath applied either NBQX (10 µM) to block AMPA receptors or AP5 (50 µM) to block NMDA receptors (Figure 4.5E – 4.5L).
Muscarinic M4-IPSCs evoked through thalamic stimulation showed a partial inhibition in the presence of NBQX (10 µM; 67.9 ± 8% remaining, n = 7) (Figure
4.5E and 4.5G), whereas AP5 (50 µM) completely eliminated thalamically-evoked
M4-IPSCs (0.42 ± 1% remaining, n = 5, p < 0.01, Mann-Whitney) (Figure 4.5F and 4.5G). Thus, even when AMPA receptors are blocked, NMDA receptors were still sufficient to evoke ChI activity. The peak time of thalamic-evoked
IPSCs also shifted in response to NBQX (control: 239 ± 8 ms, NBQX: 270 ± 10 ms, n = 7, p < 0.01, Wilcoxon matched pair signed rank) (Figure 4.6D). This suggests that, in the presence of NBQX, multiple thalamic stimuli are required to generate enough presynaptic facilitation to evoke NMDA receptor mediated depolarization and ChI firing. Action potential firing in ChIs followed a similar trend for both antagonists upon thalamic stimulation (NBQX: 41.6 ± 10% of baseline probability, n = 6; AP5: 14.6 ± 4% of baseline probability, n = 6; p <
0.05, Mann-Whitney) (Figure 4.6F and 4.6G). Although thalamically-evoked muscarinic M4-IPSCs were attenuated in the presence of NBQX, there was no change in IPSC probability under this condition (control: 1 ± 0, NBQX: 0.92 ±
0.08, n = 7, p > 0.05, Wilcoxon matched pair signed rank) (Figure 4.5H). This
147 likely results from ChI convergence because, when ChIs respond to the thalamic stimulus during approximately half of trials, M4-IPSCs should still be evoked consistently (Figure 4.6A and 4.6B).
Because blocking AMPA receptors inhibits thalamic-evoked muscarinic
M4-IPSCs while blocking NMDA receptors completely eliminates them, we hypothesized that AMPA receptors might serve to depolarize postsynaptic sites on ChIs, thus allowing for relief of Mg2+ block of NMDA receptors. To test this, we performed the same experiments testing thalamic-evoked M4-IPSCs, but in the presence of Mg2+-free solution. In this case, although NBQX strongly attenuated
EPSCs on MSNs, it did not affect M4-IPSCs (103 ± 3% of baseline amplitude,
IPSC probability = 1.0, n = 6) (Figure 4.6H and 4.6J). AP5, on the other hand, attenuated IPSC amplitude (14 ± 6% of baseline, n = 6) (Figure 4.6I and 4.6J).
This suggests that the role of AMPA receptors in this circuit may be to provide sufficient depolarization to unblock NMDA receptors, ultimately driving ChI firing and ACh release.
Next, we tested glutamate receptor contribution for cortical inputs onto
ChIs. Muscarinic M4-IPSCs evoked by optogenetic stimulation of the cortex were partially inhibited by both NBQX (10 µM; 30.2 ± 7% remaining, n = 5) (Figure 4.5I and 4.5K) and AP5 (50 µM; 54.2 ± 7% remaining, n = 8, p < 0.05 relative to
NBQX, Mann-Whitney) (Figure 4.5J and 4.5K). Inhibition of AMPA receptors had a stronger effect on the amplitude of M4-IPSCs than inhibition of NMDA receptors (Figure 4.5K) but neither antagonist decreased ChI firing probability
148 (NBQX: 0.90 ± 0.05, n = 5; AP5: 1 ± 0, n = 7; p > 0.05 for both, Wilcoxon matched pair signed rank) (Figure 4.5L). This suggests that both AMPA and
NMDA receptors contribute to cortically-evoked ACh release, but that AMPA receptors play a larger role. Because ChIs responding to cortical stimulation likely do so on the first pulse, it follows that both receptor types would play a role in generating sufficient conductance to drive ChI firing. However, AMPA receptors would have a stronger effect during the first pulse because of the Mg2+ block on NMDA receptors. When examining the effect of Mg2+-free solution on cortically-evoked M4-IPSCs, we found that NBQX only slightly inhibited M4-
IPSCs (89.0 ± 7% of baseline remaining, n = 6), whereas AP5 had a much stronger effect on M4-IPSCs (24.6 ± 14% of baseline remaining, n = 6, p < 0.01 relative to NBQX, Mann-Whitney) (Figure 4.6K - 4.6M), thus reversing the previous pattern of inhibition. In the absence of Mg2+, NMDA receptor activity is able to generate sufficient conductance to drive cortically-evoked ChI firing.
Together, these results suggest that both AMPA and NMDA receptors are important for ChI firing in response to glutamatergic inputs, and that AMPA receptors are responsible for depolarizing dendrites to allow for increased NMDA receptor activity during facilitating stimuli. AMPA receptor activity is thus more important for cortical inputs to ChIs than for thalamic inputs. Further, as summation of EPSCs from cortical inputs is not required to drive ChI firing,
AMPA and NMDA receptor interactions play a smaller role.
149 Discussion
Stimulation of excitatory inputs to the striatum is known to drive different firing patterns in ChIs, both in vivo and in ex vivo slices (Ding et al., 2010; Doig et al., 2014). However, it has been unclear how these firing patterns are integrated to evoke post-synaptic receptor activity. It has recently been determined that, unlike most GPCR synapses, single action potentials from ChIs release sufficient
ACh to evoke unitary inhibitory postsynaptic currents at M4-expressing synaptic sites in GIRK2-expressing MSNs, such that M4 receptors can read-out spontaneous ChI firing (Mamaligas and Ford, 2016). Further, M4 receptors are capable of encoding physiological firing patterns of convergent ChIs at independent synapses (Mamaligas and Ford, 2016). Using this system to examine the integration of ACh release at muscarinic synapses in response to excitatory stimulation, we found an apparent disconnect between ChI firing and
ACh release at synaptic sites on MSNs. Typically, a small average change in presynaptic neuron firing leads to small changes in transmitter release and subsequent postsynaptic receptor activity. However, in this microcircuit, a small number of ChIs responsive to excitatory input is sufficient to drive robust ACh release.
This disconnect likely results from the convergence of ChIs as well as the unlaminated architecture of the striatum. Because all neuron types are distributed relatively evenly throughout this region, the projections of an individual ChI is able to reach many MSNs. ChIs are known to have long, highly branched axons
150 (Bolam, 1984; Contant et al., 1996; Descarries and Mechawar, 2000), and this allows ChI firing to evoke consistent ACh release onto MSNs, at least within a distance of 200 µm (Mamaligas and Ford, 2016). As ChIs in the dorsal striatum are separated by a ~40 - 70 µm nearest neighbor distance on average
(Matamales et al., 2016), these neurons likely have overlapping axonal areas, resulting in synaptic convergence. These high-connectivity, no-failure synaptic connections thus release ACh such that muscarinic receptors on MSNs can encode firing of individual ChIs independently (Mamaligas and Ford, 2016).
Because of this, multiple ChIs increasing their firing during an excitatory stimulus results in muscarinic receptor activity that is consistent, even when each cell doesn’t respond to the stimulus during every trial, such as with cortical stimulation.
The heterogeneity in ChI firing responses to cortical inputs predominantly lies in preferential innervation of a certain population of ChIs that display larger
EPSPs. However, the amplitude of cortically-evoked EPSPs does not follow a binary distribution. Instead, there is a gradient of cortical EPSP amplitudes that seems to correspond to the distribution of ChI action potential probabilities. A similar gradient exists with thalamic inputs. Thalamically-evoked EPSPs in some
ChIs are sufficiently large that they might evoke firing with a single stimulus.
However, the majority of EPSPs are insufficient to drive firing upon the initial pulse. In this case, thalamic facilitation allows ChIs to eventually depolarize enough to fire. As cortical inputs depress, an evoked EPSP that begins below
151 threshold would never depolarize the ChI sufficiently to reach threshold. This range of EPSP amplitudes is transferred into a binary response that is still reflective of the scatter in the probability of responses.
This study provides evidence that cortical inputs drive much more robust
ChI activity and ACh release than previously thought. The architecture and geometry of the striatum allows for MSN integration of several ChIs, causing reliable ACh release in response to both major striatal excitatory inputs. As thalamic inputs from the parafascicular nucleus degenerate in even mild cases of
Parkinson’s disease (Smith et al., 2009, 2014), ACh release evoked through cortical activity could be overrepresented during an attempted movement. This could result in an increase in ACh release during movement and a decrease during associative behaviors, causing an imbalance of MSN activity. Indeed, although there is no overall effect on MSNs, lesion of the parafascicular thalamus itself leads to decreased ChI activity and impaired goal switching behaviors
(Bradfield et al., 2013). Animals with a parafascicular lesion struggled to suppress previous associations, continuing to perform previously learned actions despite devaluation of the reward (Bradfield et al., 2013). This suggests that, in a disease state, thalamic degeneration could evoke behavioral rigidity due to ACh imbalance.
152 Figure 4.1. Optogenetic activation of excitatory thalamic and cortical inputs evokes differential short-term plasticity in ChIs
(A,B) Injection schematic and representative fluorescent image of AAV-induced expression of floxed ChR2 and eYFP in the parafascicular nucleus of the thalamus (A) and motor cortex (B) in CaMKii-Cre mice. Scale bars represent 500
µm.
(C) Example traces of AMPA (Vh = -80 mV) and NMDA (Vh = +40 mV) receptor mediated currents, recorded in ChIs, evoked optogenetically (single pulse, 2 ms,
470 nm) via thalamic (purple) and cortical (orange) inputs.
(D) No difference in ChI AMPA EPSC amplitudes were found between thalamic and cortical inputs (thalamus: n = 10, cortex: n = 7, p > 0.05, Mann-Whitney).
Error bars indicate ± SEM.
(E) We found no difference in AMPA/NMDA ratios between thalamic and cortical inputs (thalamus: n = 7, cortex: n = 8, p > 0.05, Mann-Whitney). Error bars indicate ± SEM.
(F) Representative traces of current clamp recordings in ChIs illustrating facilitation of thalamic inputs (purple) and depression of cortical inputs (orange) in response to a burst stimulus (5 pulses, 50 Hz).
(G) Summary data displaying EPSP facilitation in thalamic inputs and EPSP depression in cortical inputs (n = 10 for each group, p < 0.001, 2-Way ANOVA).
Error bars indicate ± SEM.
153 (H) No difference in the amplitude of the first EPSP in the burst stimulus
(thalamus: n = 14, cortex: n = 15, p > 0.05, Mann-Whitney). Error bars indicate ±
SEM.
154
Figure 4.1
155 Figure 4.2. Differential responses of ChIs to thalamic and cortical stimuli
(A) Representative cell-attached recording of a ChI illustrating high average firing response rate due to thalamic burst stimulus (5 pulses, 50 Hz, 470 nm). Inset: zoom of stimulation period.
(B) Representative cell-attached recording of a ChI illustrating low average firing response due to cortical burst stimulus (5 pulses, 50 Hz, 470 nm). Inset: zoom of stimulation period.
(C) Summary distribution of action potential probability per trial within each ChI during 100 ms stimulus period. The dashed line represents the baseline action potential probability during the 2 seconds preceding the stimulus (mean probability for each 100 ms stimulus bin + 2 standard deviations). Thalamic inputs regularly evoke strong AP firing, whereas cortical inputs selectively activate a subset of ChIs during the same stimulus (thalamus: n = 13, cortex: n =
29, p < 0.001, Mann-Whitney). Error bars indicate ± SEM.
(D) Representative cell-attached recordings of ChIs illustrating responding
(orange) and non-responding (peach) neurons to the same cortical stimulus.
(E) Representative EPSPs from current clamp recordings corresponding to the
ChIs recorded in (D), as well as thalamically-evoked EPSPs (purple).
(F) Distribution of EPSP amplitudes from ChIs that respond and do not respond to cortical stimuli (n = 6 for both groups, p < 0.01, Mann-Whitney). Error bars indicate ± SEM.
156 (G) Distribution of EPSP area for thalamically-evoked EPSPs, cortical responder
EPSPs, and cortical non-responder EPSPs (thalamus: n = 10, cortical responders: n = 6, cortical non-responders: n = 6, Kruskal-Wallace). * indicates p
< 0.05; ** indicates p < 0.01. Error bars indicate ± SEM.
157
Figure 4.2
158 Figure 4.3. Characteristics of ChI AP firing in response to cortical and thalamic burst stimuli.
(A) Difference in coefficient of variation (CV) of action potential probability in ChIs in response to thalamic and cortical stimuli.
(B) Distribution of spike latencies for responding ChIs following thalamic or cortical stimulation (thalamus: n = 11, cortex: n = 10, p < 0.01, Mann-Whitney).
Error bars indicate ± SEM.
159
Figure 4.3
160
Figure 4.4. Differential dendritic integration of excitatory inputs at ChI synapses
(A) Example trace illustrating voltage jump protocol. The black trace represents an optogenetically-evoked EPSC continually recorded at Vh = -60 mV (single pulse, 2 ms, 470 nm). The red trace represents an EPSC in the same cell recorded at Vh = 0 mV and moved to Vh = -60 mV 5 ms following the same light stimulation. At 0 mV, although channels are open following glutamate release, there is no driving force. When the voltage is stepped to -60 mV, driving force is initiated and the current activates over a time that is related to the capacitance between the channels activated and the cell body.
(B) Averaged traces showing slower activation of cortically-evoked EPSCs (n =
9) vs thalamically-evoked EPSCs (n = 8) (single pulse, 2 ms, 470 nm).
(C) Quantification of slower t of activation (tact) of cortically-evoked (n = 9) vs thalamic-evoked (n = 8) EPSCs recorded in voltage jump protocol (p < 0.05,
Mann-Whitney). Error bars indicate ± SEM.
(D) Representative 2-photon microscopy image of a filled ChI (Alexa 594). An iontophoretic pipette with glutamate is positioned opposed to a distal section of the ChI dendrite. Scale bar 10 µm.
(E) Example trace illustrating iontophoresis during voltage jump protocol. The black trace represents an excitatory current, evoked via iontophoresis of glutamate, continually recorded at Vh = -60 mV. The red trace represents an
161 iontophoresis-evoked current in the same cell recorded at Vh = 0 mV and moved to Vh = -60 mV 250 ms.
(F) Representative traces showing slower activation of iontophoretic currents evoked at distal dendritic locations vs proximal locations.
(G) Quantification of slower tact of distally-evoked vs proximally-evoked iontophoretic currents recorded in voltage jump protocol (n = 5, p < 0.05,
Student’s paired t test). Error bars indicate ± SEM.
162
Figure 4.4
163 Figure 4.5. ChI response to excitatory stimuli evokes similar ACh release onto MSNs.
(A) Representative voltage clamp recording of an MSN over-expressing GIRK2 channels. Thalamic stimulation (5 pulses, 50 Hz, 470 nm) evokes AMPA EPSCs in MSNs followed by an M4 muscarinic receptor mediated IPSC resulting from
ChI firing.
(B) Representative trace from a GIRK2-expressing MSN, displaying that cortical stimulation evokes similar levels of ACh release to thalamic stimulation.
(C) Distribution of M4-IPSC amplitudes showing no difference in ACh release evoked between thalamic (n = 26) and cortical (n = 25) stimuli (p > 0.05, Mann-
Whitney). Error bars indicate ± SEM.
(D) Distribution of IPSC probabilities following the stimulus for each input. The dashed line represents the baseline IPSC probability during the 5 seconds preceding the stimulus (mean probability for each 250 ms stimulus bin + 2 standard deviations). The probability of thalamically-evoked (n = 14) and cortically-evoked (n = 15) IPSCs did not differ (n > 0.05, Mann-Whitney). Error bars indicate ± SEM.
(E) Representative trace showing inhibition of thalamically-evoked M4-IPSCs in the presence of NBQX (10 µM).
(F) Representative trace showing complete inhibition of thalamically-evoked M4-
IPSCs in the presence of AP5 (50 µM).
164 (G) Quantification of M4-IPSC inhibition due to glutamatergic antagonists indicates stronger role for NMDA receptors in thalamically-evoked ACh release
(NBQX: n = 7, AP5: n = 5). * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Error bars indicate ± SEM.
(H) Distribution of IPSC probability shows consistent thalamically-evoked ACh release during AMPA receptor antagonism but frequent failure when antagonizing NMDA receptors (NBQX: n = 7, p > 0.05; AP5: n = 5, p < 0.01;
Wilcoxon matched pair signed rank).
(I) Representative trace showing inhibition of cortically-evoked M4-IPSCs in the presence of NBQX (10 µM).
(J) Representative trace showing complete inhibition of cortically-evoked M4-
IPSCs in the presence of AP5 (50 µM).
(K) Quantification of M4-IPSC inhibition due to glutamatergic antagonists indicates stronger role for AMPA receptors in cortically-evoked ACh release
(NBQX: n = 5, AP5: n = 8). * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Error bars indicate ± SEM.
(L) Distribution of IPSC probability shows consistent cortically-evoked ACh release under all conditions (NBQX: n = 5, p > 0.05; AP5: n = 8, p > 0.05;
Wilcoxon matched pair signed rank).
(M) Schematic describing cortical and thalamic inputs and their glutamate receptor complement.
165
Figure 4.5
166 Figure 4.6. Characteristics of evoked M4-IPSCs.
(A) Representative traces illustrating the arithmetic summation (Stim 1 + 2) of
M4-IPSCs evoked via 2 separate minimal electrical stimuli, approximately 500
µm apart (Stim1 and Stim 2).
(B) Distribution of summed amplitudes (stim 1 amplitude + stim 2 amplitude) and amplitudes of simultaneous stimulation (stim 1 + 2 amplitude) (n = 3, p > 0.05,
Student’s paired t test).
(C) M4-IPSC peak time is slower in thalamically-evoked IPSCs (n = 13) than in cortically-evoked IPSCs (n = 15, p < 0.001, Mann-Whitney). Error bars indicate ±
SEM.
(D) Longer thalamically-evoked M4-IPSC peak time in the presence of NBQX (10
µM) (n = 7, p < 0.01, Wilcoxon matched pair signed rank). Error bars indicate ±
SEM.
(E) No difference in cortically-evoked M4-IPSC peak time in the presence of
NBQX (10 µM) (n = 5, p > 0.05, Wilcoxon matched pair signed rank). Error bars indicate ± SEM.
(F) Summary data showing AP probability in cell attached ChI recordings mimics pattern of inhibition seen in evoked M4-IPSCs (n = 6 for all groups, Wilcoxon matched pair signed rank). * indicates p < 0.05. Error bars indicate ± SEM.
(G) Representative trace showing no inhibition of thalamically-evoked M4-IPSCs in Mg2+-free artificial CSF (aCSF) during bath application of NBQX (10 µM).
167 (H) Representative trace showing complete inhibition of thalamically-evoked M4-
IPSCs in Mg2+-free aCSF during bath application of AP5 (50 µM).
(I) Quantification of M4-IPSC inhibition due to glutamatergic antagonists in Mg2+- free aCSF indicates stronger role for NMDA receptors in thalamically-evoked
ACh release (n = 6 for all groups). * indicates p < 0.05 and ** indicates p < 0.01.
Error bars indicate ± SEM.
(J) Normalized ChI AP probability in glutamatergic antagonists (drug AP probability/control AP probability) (n = 6 for all groups, Mann-Whitney). * indicated p < 0.05. Error bars indicate ± SEM.
(K) Representative trace showing slight inhibition of cortically-evoked M4-IPSCs in Mg2+-free aCSF during bath application of NBQX (10 µM).
(L) Representative trace showing strong inhibition of cortically-evoked M4-IPSCs in Mg2+-free aCSF during bath application of AP5 (50 µM).
(M) Quantification of M4-IPSC inhibition due to glutamatergic antagonists in
Mg2+-free aCSF indicates stronger role for NMDA receptors in cortically-evoked
ACh release under this condition (n = 6 for all groups). * indicates p < 0.05 and ** indicates p < 0.01. Error bars indicate ± SEM.
168
Figure 4.6
169
CHAPTER 5
DISCUSSION
170 Discussion
My thesis aimed to examine cholinergic transmission from ChIs in the striatum at GPCR synapses. Although previous studies have measured ChI firing patterns, anatomy, and relevant behaviors, none have been able to examine synaptic release of ACh at striatal GPCR synapses. Previous work has measured extracellular ACh over minutes to hours, but there has been no direct, monosynaptic readout of cholinergic signaling on a millisecond timescale. In my thesis work, I used a GIRK2 G-protein coupled potassium channel to measure synaptic ACh transmission at muscarinic and dopaminergic synapses on MSNs.
This tool allowed me to observe the mechanisms through which ACh signals in the striatum.
In Chapter 2, we examined nicotinic receptor-mediated dopamine release and subsequent D2-receptor activity on iMSNs in the dorsal striatum. We found that, although synchronous ChI activity was sufficient to evoke dopamine release, nicotinic receptors did not modulate optogenetically-evoked D2-IPSCs from dopamine terminals. Desensitization strongly governs nicotinic transmission on dopamine terminals, and this response recovers over a timecourse of approximately 30 seconds. Finally, we found that while KOR activation inhibits both dopamine terminal-evoked and ChI-evoked D2-IPSCs, MORs selectively inhibit ChI-evoked dopamine release. These results suggest that synchronous
ChI activity can drive D2 receptor activation, but that these two sub-circuits are both distinct in function and differentially modulated.
171 In Chapter 3, we found that single action potentials from ChIs are capable of robustly activating M4 muscarinic receptors on MSNs, measured as M4-
IPSCs. This provides a rare example of point-to-point synaptic transmission at
GPCR synapses in the CNS that occurs in less than 500 ms. Following ChI firing, a high concentration of ACh activates postsynaptic receptors, and this is governed by efficient AChE enzymatic activity. AChE also allows for phasic M4 receptor activation, degrading ACh before it can diffuse to create a broader tone.
The spontaneous tonic firing of ChIs is encoded in receptors at functionally independent synapses, and multiple convergent ChIs can activate M4 receptors.
A high probability of release at these synapses allows for stereotyped ACh release during tonic firing as well as increased M4 receptor activity during bursting patterns important in reward behavior. Finally, we determined that ChI stimulation inhibits collateral transmission between MSNs, and this occurs within the timecourse of M4-IPSCs. This work is the first examination of synaptic transmission at muscarinic synapses. Overall, it suggests that M4 receptors can distinctly encode convergent ChI firing during physiological activity patterns and that M4 receptor activity on these timescales is physiologically relevant.
In Chapter 4, we found that, although excitatory inputs to the dorsal striatum from the M1 motor cortex and the parafascicular thalamus evoke differential firing patterns in ChIs, convergence of ChIs onto MSNs allows for identical M4-IPSCs evoked from both inputs. While cortical inputs fail to evoke an overall increase in ChI firing, a sub-population of ChIs receives stronger inputs
172 from this region, leading to high probability ChI firing and subsequent M4 receptor activity. Further, we found that different populations of glutamate receptors at different sites along the ChI dendrite govern disynaptically-evoked
M4-IPSCs, such that NMDA receptors are critical for thalamic transmission whereas both AMPA and NMDA receptors are important for cortically-evoked
ACh release. These results suggest that, due to convergence of ChIs at muscarinic synapses, there can be a disconnect between ChI firing and cholinergic transmission. We have also shown that cortical inputs are more important in driving ChI activity than previously thought based on firing data alone.
Synaptic point-to-point transmission at M4 synapses
Previous work examining ACh transmission in the striatum has proposed that, because of low nanomolar concentrations of extrasynaptic ACh, ChI tonic firing patterns, broad axonal arbors, and ultrastructurally-measured release sites numbering in the hundreds of thousands, ChIs must release ACh via volume transmission (Bennett and Wilson, 1999; Contant et al., 1996; Descarries et al.,
2000). My work has shown that this mode of transmission is unlikely to underlie
ChI-mediated ACh release in the striatum. Although I have provided some evidence for ACh spillover occurring at muscarinic synapses during synchronous
ChI activity, this only occurs following electrical or optogenetic stimulation of
ChIs. Broad synchrony of ChI firing in this manner is unlikely during normal
173 striatal activity. Instead, my work suggests that each action potential during physiological ChI firing can be phasically encoded at postsynaptic muscarinic receptor sites on a millisecond timescale. Because of this tightly regulated, point- to-point transmission, both the tonic and bursting firing patterns of ChIs are capable of disinhibiting MSN collaterals during muscarinic receptor activation.
Unlaminated striatal structure and implications for ChI connectivity
The majority of brain regions important for integration of information, such as the hippocampus or cortex, are highly organized. These structures create signaling domains that allow for structured and systematic communication flow between neurons. However, the striatum is largely unlaminated, with all cell types exhibiting radial projections and disorganized connectivity between striatal neurons (Kawaguchi, 1993; Kawaguchi et al., 1990). Although there is emerging evidence for more specific architecture in the striatum resulting in synaptic organization (Straub et al., 2016), the majority of striatal synapses still occur relatively randomly. My work has found that ChIs follow this disorganized pattern.
Because of their radially distributed branching arbors (Contant et al., 1996), ChIs are capable of synapsing onto every cell within at least 200 µm. This provides a longer-range network of striatal communication, as ChIs are thought to project across striatal subregions.
174 Further, because ChIs are spaced between 40 and 70 µm away from one another, their terminal fields are highly overlapping. This results in a convergent network of ChIs that can activate postsynaptic receptors on the same MSN. The convergence of these cells allows MSNs to detect subtle changes in ChI firing that are difficult to assess looking at ChI activity itself. This could lead to ChI activity in different striatal subregions converging to modulate large swaths of
MSNs, creating different modalities of cell groups.
Future directions
While the work presented here has furthered the field of striatal cholinergic transmission, there are many opportunities for future work on mechanisms of ChI transmission.
Question 1: How do Parkinson’s disease models alter cholinergic transmission in the striatum?
As the ACh-dopamine balance hypothesis deems ChIs critical in balancing direct and indirect pathway activity in the striatum, and as the striatal cholinergic system develops pathological changes in PD, it is of particular interest to examine how cholinergic transmission is altered in Parkinsonian animal models.
Based on pathologies observed during previous studies, dopamine depletion evokes changes in ChI firing patterns as well as ACh concentration and AChE expression throughout the dorsal striatum (Gonzales and Smith, 2015). As M4-
175 mediated sIPSCs are dependent on ChI firing, the consequence of changes in firing patterns can be observed measuring amplitude and frequency of sIPSCs following dopamine depletion. Further, dopamine depletion alters M2/M4 muscarinic autoreceptor activity on ChIs, eliminating their inhibitory effect via upregulation of RGS4 (Ding et al., 2006). Although ChI paired pulse depression was not altered by presynaptic inhibition of G-protein signaling, it is possible that activation of presynaptic muscarinic autoreceptors might evoke differences in
ACh release at higher frequency ChI activity, and that dopamine depletion might modulate this process, changing the response of M4 receptors on MSNs to these firing patterns.
In addition to changes in ChI firing, increased extracellular ACh concentrations could result from increased amplitude or kinetics of ACh release in the striatum. Measurement of action potential-evoked unitary M4-IPSCs will help elucidate which of these is the case, if not both. Because AChE expression is diminished in PD (Gonzales and Smith, 2015; Lim et al., 2014), dopamine depletion will likely cause an increase in M4-IPSC timecourse. Using ambenonium, the AChE antagonist, can help determine the degree to which
AChE downregulation alters transmission at these synapses. Further, we can use this to determine whether the decrease in AChE following dopamine depletion evokes differential tonic activation of M4 receptors.
Finally, parafascicular inputs to the striatum show preferential but incomplete degeneration in PD patients and models (Henderson et al., 2000;
176 Smith et al., 2009, 2014). As ChIs are important in behavioral flexibility and the parafascicular nucleus is important in attention (Bradfield et al., 2013; Smith et al., 2011; Stalnaker et al., 2016), decreased signaling through this pathway could thus help explain some symptoms of PD patients, including behavioral and attentional rigidity (Henderson et al., 2000). Previous work has shown that excitatory thalamic inputs onto dMSNs exhibit selective degeneration in 6-OHDA models of dopamine depletion, while inputs to iMSNs remain unaffected (Parker et al., 2016). Although thalamic activity is critical for ChI burst pattern generation
(Matsumoto et al., 2001), it remains unclear how partial degeneration of these inputs would change ACh signaling. Using a combination of dopamine neuron lesions and GIRK2 readout of M4 activity, we could determine whether thalamically-evoked ChI activity and ACh release is altered in PD models. The decrease in dMSN excitability resulting from thalamic degeneration skews striatal output to the indirect pathway. Likely, if thalamic inputs are still sufficient to drive
ACh release following dopamine lesion, it will result in increased timecourse of receptor activation due to downregulated AChE. As ChI connectivity with iMSNs increases following dopamine depletion, while connectivity with dMSNs decreases (Salin et al., 2009), ACh release would cause a preferential increase in iMSN activity through M1 receptors. If this is the case, then cholinergic signaling would amplify the differences between direct and indirect pathway activity, causing more excitation in iMSNs.
177 Question 2: How does endogenous and exogenous MOR activity regulate ACh signaling at muscarinic synapses?
Striatal opioid receptors can drive reward behavior through multiple striatal pathways (Devine and Wise, 1994). Specifically, mu opioid receptors (MORs) are expressed on ChIs, where they inhibit ChI firing (Arenas et al., 1990; Jabourian et al., 2005; Ponterio et al., 2013; Svingos et al., 2001). Although these studies have examined ChI firing, none have been able to investigate the role of MORs on synaptic ACh release. To dissect the role of opioids in this circuit, three avenues of questioning can be used. First, we can examine the effects of MOR agonists and antagonists on fundamental properties ChI-MSN synaptic transmission. Second, we can examine the effect of chronic systemic administration of morphine at these synapses to determine if drugs of abuse play a role in MOR-mediated plasticity. Lastly, we could evoke release of endogenous enkephalin from iMSNs (Kreitzer, 2009). Here, it would be interesting to determine whether iMSNs release sufficient enkephalin to activate functionally activate MORs and to determine what the timecourse of that activation is. It is possible that MORs can signal rapidly, like muscarinic receptors do in this region.
For all of these questions, we could examine changes in amplitude or frequency of spontaneous and unitary M4-IPSCs or changes in ACh release during tonic and burst firing patterns. Also, based on crosstalk that may occur between
GPCRs on ChIs (Ding et al., 2006), we could examine the effect of different presynaptic GPCR signaling molecules.
178
Question 3: How do inputs to the ventral striatum drive cholinergic transmission?
While the dorsal striatum is important for movement, the ventral region of the striatum, or nucleus accumbens (NAc), is related to the limbic pathways in the basal ganglia circuits, important for reward and associative behaviors
(Grillner et al., 2005; Gruber et al., 2009). Inputs from the ventral hippocampus, prefrontal cortex, and amygdala all converge in the NAc (Britt et al., 2012;
Sesack and Grace, 2010) , but their role in driving activity in ChIs has not yet been elucidated. Further, it is unclear how different firing patterns are evoked or whether some of these inputs drive ChI activity that differs from postsynaptic M4 receptor activation.
In addition, drugs of abuse evoke input-specific synaptic changes that are specifically observed in dMSNs (Pascoli et al., 2011, 2014). Specifically, cocaine self-administration leads to increased EPSC amplitude, changes in input-specific striatal LTD, and altered AMPA/NMDA ratios in inputs from the ventral hippocampus and the prefrontal cortex, as well as increase Ca2+-permeable
AMPA subunit, GluA1, insertion at prefrontal corticostriatal synapses (Pascoli et al., 2011, 2014). It is possible that similar differences could occur at excitatory synapses onto ChIs, and that this could in turn alter ChI activity and ACh release.
Further, the balance of direct and indirect pathway activity skews toward the direct pathway following cocaine self-administration. Depending on the changes
179 that occur at glutamatergic ChI synapses, ACh could bring more balance to the striatal microcircuit or exacerbate the activity of the direct pathway.
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