DELINEATING MIDBRAIN CIRCUITS UNDERLYING MOTIVATED BEHAVIORS Alice Stamatakis A ...

175 Pages·2014·4.65 MB·English

DELINEATING MIDBRAIN CIRCUITS UNDERLYING MOTIVATED BEHAVIORS

Alice Stamatakis

A dissertation submitted to the faculty at the University of North Carolina at Chapel
Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
the Graduate School (Neurobiology Curriculum).

Chapel Hill
2014

Approved by:

Garret D. Stuber
Thomas L. Kash
Regina M. Carelli
Spencer L. Smith
Fulton T. Crews
© 2014
Alice Stamatakis
ALL RIGHTS RESERVED

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ABSTRACT
Alice Stamatakis: Delineating midbrain circuits underlying motivated behaviors
(Under the direction of Garret Stuber)

Lateral habenula (LHb) neurons convey aversive and negative reward
conditions through potent indirect inhibition of ventral tegmental area (VTA)
dopaminergic neurons. Although the LHb and VTA reciprocally project to each other,
the electrophysiological properties and the behavioral consequences associated with
selective manipulations of this circuit are unknown. We found that exposure to
aversive stimuli in mice increased LHb excitatory drive onto RMTg neurons.
Furthermore, optogenetic activation of this pathway promoted active, passive and
conditioned behavioral avoidance. Thus, activity of LHb efferents to the midbrain is
aversive, but can also serve to negatively reinforce behavioral responding. Aspects
of this behavioral phenotype were recapitulated by optogenetically activating lateral
hypothalamic (LH) glutamatergic inputs to the LHb, suggesting that the LH may be an
important upstream contributor to aversive signaling of LHb neurons. Optogenetic
activation of VTA dopaminergic inputs to the LHb resulted in no detectable dopamine
release in LHb brain slices. Instead, stimulation produced GABA-mediated inhibitory
synaptic transmission, which suppressed the firing of postsynaptic LHb neurons in
brain slices and increased the spontaneous firing rate of VTA dopaminergic neurons
in vivo. Furthermore, in vivo activation of this pathway produced reward-related
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phenotypes that were dependent on intra-LHb GABA receptor signaling. These
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results suggest that noncanonical inhibitory signaling by these hybrid dopaminergic-
GABAergic neurons acts to suppress LHb output under rewarding conditions.
Collectively, these data demonstrate that the LHb and midbrain interact in a
reciprocal manner and implicate the VTA’s projection to the LHb as a key node in the
classical midbrain reward circuit.

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ACKNOWLEDGEMENTS

First and foremost I would like to thank my mentor, Garret Stuber, for his
guidance, patience, and brilliant scientific insight. I would like to thank all of the
members of the Stuber lab, past and present, especially Dennis Sparta, Grace Blair,
Shanna Resendez, and Josh Jennings. Josh, your inherent curiosity, resilient work
ethic, and passion for science are out of this world and truly inspiring. I would also
like to thank my dissertation committee, Regina Carelli, Fulton Crews, Thomas Kash,
and Spencer Smith, for their time, mentorship, and excellent feedback. Finally, this
process could not be possible without the support from all of my family and friends.

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TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………..…………... viii
LIST OF ABBREVIATIONS………………….…………………………………………… x
CHAPTER 1: GENERAL INTRODUCTION ………………….…………………..……. 1
History and function of dopamine………………….……………..………..……. 1
Composition and heterogeneity of the VTA…………………………….………. 3
VTA circuitry………...…………………………………………………………...… 6
Role of the lateral habenula in motivated behaviors …………...….....……… 21
Relationship between neural circuits underlying motivated behaviors and
neuropsychiatric diseases……………………………………………………...…22

Overview of optogenetics……………………….…………………………...……23
Dissertation…………………………………………………………………………33
CHAPTER 2: ACTIVATION OF THE LATERAL HABENULA INPUTS TO THE
VENTRAL MIDBRAIN PROMOTES BEHAVIORAL AVOIDANCE………..…………36

Introduction...…………………………………………………….……………..… 36
Methods………………………………………………..……………..…………….37
Results……………………………………………………….………...……...……43
Discussion………………………….………………………….……..…………… 47
Figures………...……………………………………………….…………..……… 49
CHAPTER 3: A UNIQUE POPULATION OF VENTRAL TEGMENTAL AREA
NEURONS INHIBITS THE LATERAL HABENULA TO PROMOTE REWARD…….59

Introduction ……………………………………………..….………………….…59
Methods………………………………………………………………………….... 61
Results……………………………………………………….……….....………… 75
Discussion………………………….………………………….……..……….……87
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Figures………...……………………………………………….………………….. 91
CHAPTER 4: A MAJOR GLUTAMATERGIC LATERAL HABENULA AFFERENT
FROM THE LATERAL HYPOTHALAMUS NEGATIVELY
CONTROLS FEEDING AND REWRAD………………………………..……………..110

Introduction ………………………………………………….………………… 110
Methods…………..……………………………………….……...…………...….112
Results……………………………………………………….………...………… 119
Discussion………………………….………………………….………………… 124
Figures………...……………………………………………….………………… 127
CHAPTER 5: GENERAL DISCUSSION…..………………………...……….………. 135
REFERENCES…..……………………………………………...…….………...……….143

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LIST OF FIGURES

Figure 2.1: The LHb sends a functional glutamatergic projection to the
RMTg……......................................................................................................49

Figure 2.2: Acute unpredictable foot shock exposure enhances LHb-to-RMTg
glutamate release….......................................................................................51

Figure 2.3: Activation of LHb inputs to the RMTg produces active and
conditioned behavioral avoidance ….............................................................53

Figure 2.4: Activation of LHb inputs to the RMTg promotes negative
reinforcement …............................................................................................ 55

Figure 2.5: Activation of LHb inputs to the RMTg disrupts positive
reinforcement………………………………………………………………...…….57

Figure 3.1: THVTA neurons project to the LHb…....................................................... 91

Figure 3.2: THVTA-LHb neurons are a distinct population of neurons..........................94

Figure 3.3: THVTA-LHb neurons are located in the medial-anterior portion of
the VTA……...................................................................................................95

Figure 3.4: THVTA-LHb neurons exhibit distinct electrophysiological
characteristics ……........................................................................................97

Figure 3.5: THVTA-LHb neurons express lower amounts of mRNA for
dopaminergic markers than THVTA-NAc neurons……...................................... 99

Figure 3.6: THVTA-LHb neurons do not release detectable levels of dopamine in
the LHb……................................................................................................. 100

Figure 3.7: THVTA-LHb neurons release GABA in the LHb.........................................101

Figure 3.8: GABA-negative asymmetric THVTA-LHb::ChR2 synapse.........................103

Figure 3.9: In vivo optical stimulation of THVTA-LHb terminals suppresses
RMTg activity…….........................................................................................104

Figure 3.10: In vivo optical stimulation of THVTA-LHb terminals enhances
spontaneous firing of THVTA neurons…….....................................................106

Figure 3.11: Activation of THVTA-LHb terminals produces reward-related
behavioral phenotypes.................................................................................108

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Figure 4.1: Genetic ablation of LH glutamatergic neurons increases
caloric intake and weight gain……...............................................................127

Figure 4.2: The anterior LH sends a glutamatergic projection to the LHb…….......129

Figure 4.3: Characterization of neurotransmitter release from
VGlut2LH-LHb::ChR2 terminals ……...............................................................131

Figure 4.4: Optogenetic modulation of VGlut2LH-LHb::ChR2 circuit
bidirectionally modulates feeding and reward-related behaviors…………..133

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LIST OF ABBREVIATIONS

3V Third ventricle
AAV Adeno-associated virus
aCSF Artificial cerebral spinal fluid
Arch Archaerhodopsin
BAC Bacterial artificial chromosome
BLA Basolateral amygdala
BNST Bed nucleus of the stria terminalis
CamKIIα Calcium/calmodulin-dependent protein kinase II α
CART Cocaine- and amphetamine-regulated transcript
CHAT Choline acetyltransferase
ChR2 Channelrhodopsin-2
DA Dopamine
DAT1 Dopamine transporter
DG Dentate gyrus
DMH Dorsal medial hypothalamus
DRD2 Dopamine receptor D2
DRN Dorsal raphe nucleus
EN Entopenduncular nucleus
eYFP Enhanced yellow fluorescent protein
FSCV Fast scan cyclic voltammetry
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