Basolateral amygdala (BLA) neurons are excited by conditioned cues after appetitive or aversive Pavlovian conditioning, and these neuronal excitations are considered to be a substrate for cue- outcome associative learning. However, an equal or greater number of BLA neurons can be inhibited by conditioned cues, and the role and mechanism of this inhibitory signal are not understood. Here we show that 77% of BLA neurons demonstrate long-lasting inhibition during a reward-predictive cue that is positively correlated with both learning and extinction of reward seeking. Optogenetic activation of BLA projection neurons during cue presentation impaired conditioned behavior, consistent with cue-evoked inhibition in BLA causally contributing to this behavior. Pharmacological inactivation of the orbitofrontal cortex (OFC) and optogenetic inactivation of OFC axon terminals in BLA also reduced conditioned behavior, with pharmacological inactivation of OFC additionally diminishing BLA cue-evoked inhibitions. Together, these data reveal a new role for BLA neuronal inhibitions in conditioned behavior, and suggest that this signal is mediated in part by OFC axon projections to BLA, which may activate local interneurons.

While the ventral tegmental area (VTA) and its dopamine (DA) projections have been implicated strongly in reward processing, and more tentatively in fear conditioning, the response time course of all of the many types of VTA neurons to rewards, aversive events, and cues predictive of such outcomes is still unclear. To this end, extracellular recordings of VTA neural activity were conducted during appetitive and aversive Pavlovian conditioning in awake, behaving rats. Two populations of VTA neurons were excited by a visual reward-predictive cue: a Congruent population excited by reward, as well as an Incongruent population inhibited by reward. The Congruent population displayed cue response characteristics often ascribed to presumed DA neurons, such as learning-related enhancement, extinction-related decrement, correlation with motivation, and a negative reward prediction error signal. The Incongruent population did not display these cue response characteristics, and may encode the sensory salience of the cue. In the Pavlovian fear conditioning procedure, phasic responses to visual and auditory cues predicting footshock delivery were found. The proportion of cue inhibitions, and the strength of cue excitations, encoded behaviorally expressed cue-elicited fear. Conditioned phasic responses to appetitive and aversive cues were very similar, and most cells excited by the fear-conditioned cue were excited during reward consumption. Taken together, our findings suggest that VTA neurons whose responses change with learning are not generally selective for the hedonic valence of events, but may instead reflect the motivational salience of both rewarding and aversive events.

Much of the behavior of humans and other animals is directed towards seeking out edible, social, cognitive or drug rewards. Dopamine, a neurotransmitter released by a handful of cells in the brain, is known to be essential for reward-seeking behavior. Yet despite decades of focused investigation, the nature of its influence remains a matter of considerable debate. The precise roles that dopamine neurons play are likely to depend on their afferent and efferent connectivity, the timing and length of neural activation, and the features of the behavior under investigation. Accordingly, it is becoming increasingly appreciated that delineating the specific contributions of dopamine neurons to cellular, circuit, and systems-level phenomena will require more sophisticated control over their patterns of activity than conventional electrophysiological and pharmacological techniques can provide.

Recently developed optogenetic tools hold great promise for disentangling these complex issues. In Chapter 2, I describe the development and characterization of a novel transgenic rat line that permits selective opsin expression in ventral tegmental area dopamine neurons, as well as other catecholamine neurons in the rat brain. I then utilize this tool to determine if temporally-precise patterns of dopamine neuron activity are causally related to appetitive behaviors. In Chapter 3, I present data from a series of experiments designed to clarify the role of ventral tegmental area dopamine neurons, or their major efferent projection to the nucleus accumbens, in positive reinforcement. Then in Chapter 4, I describe experiments designed to determine if ventral tegmental area dopamine neurons are capable of supporting associative cue-reward learning. Collectively, my results demonstrate that ventral tegmental dopamine neurons are sufficient to reinforce instrumental responding, and that their ability to influence reward-seeking behavior is at least partly due to the fact that they can drive associative learning directly. As such, they represent an important advance in our understanding of the neural basis of learned appetitive behaviors.

Flexible behavior in response to a changing environment is required for survival, and is the physical manifestation of communication among the prefrontal cortex and different subcortical systems. The basolateral amygdala (BLA), a crucial component of the reward circuitry mediating behavioral choices, is thought to encode the value of discrete cues in the environment. In particular, the BLA is thought to be required in updating the current cue-reward association. Previous studies have shown that the BLA is involved in flexible behavior, demonstrated using reversal learning tasks, where the subject has to inhibit responding to a previously rewarded cue and start responding to a previously unrewarded cue. However, the precise role of the BLA's contribution to inhibiting responding to a previously rewarded cue or learning to respond to a previously unrewarded cue remains unclear. Furthermore, it is not known if the BLA contributes to higher order rule learning that would occur over several reversals. Therefore, we examined the role of the BLA in a 4-choice odor discrimination reversal task across several reversal phases. We found that excitoxic lesions of the BLA impaired reversal learning compared to sham-lesioned mice. BLA-lesioned mice returned more frequently to the previously rewarded odor, indicating that the BLA primarily plays a role in inhibiting response to a previously rewarded cue instead of learning a new cue-reward association. Furthermore, since the BLA acts in concert with the prefrontal cortex to produce flexible behavior, understanding the dynamics of structural connectivity from the BLA to higher-order cortical regions involved in reversal learning is the first step to elucidate how amygdalocortical networks function. To develop our understanding of the development of these critical reward circuit connections, we used two-photon in vivo imaging to compare structural dynamics of axonal boutons of cortical-projecting BLA neurons in adult mice. We found that amygdala axons terminating in the dorsomedial prefrontal cortex are cortical-like in terms of their structural plasticity, and they are highly similar to orbitofrontal axons terminating in the same region. The work in this thesis elucidates a more precise role of the BLA in guiding flexible behavior, while also for the first time defining the baseline structural plasticity of axons from a limbic region projecting to a fronto-cortical region.

Alcoholism is a devastating disease afflicting roughly 17 million Americans. While this figure is staggering, it is all the more striking that 87% of the population can drink without developing dependence, given the addictive potential of alcohol. This suggests the existence of mechanisms which curb excessive alcohol intake and prevent the majority of alcohol drinkers from developing dependence. Previously we identified a homeostatic pathway in the dorsal striatum in which the brain-derived neurotrophic factor (BDNF) is upregulated by ethanol and subsequently acts to reduce ethanol intake (McGough et al., 2004). The central aim of this dissertation is the elucidation of the molecular mechanism underlying striatal BDNF homeostasis, as well as the determination of the functional significance of BDNF homeostasis with respect to ethanol consumption and the progression to addiction.

Here I demonstrate a complete signaling pathway in which ethanol treatment of striatal neurons increases BDNF protein, resulting in the phosphorylation of its receptor TrkB and subsequent activation of the MAPK pathway. Importantly, I show that ethanol increases production of the downstream effector, preprodynorphin, via a BDNF- and MAPK-dependent mechanism.

In addition, I show that in vivo conditional deletion of BDNF increases ethanol intake. Conversely, increasing BDNF expression in brain regions including the striatum decreases ethanol intake via a dynorphin-dependent mechanism. I also demonstrate that activation of dynorphin's receptor, the kappa opioid receptor, blocks acquisition of ethanol conditioned place preference, indicating that the BDNF/dynorphin pathway may inhibit the formation of ethanol-related memories.

Finally, I reveal a role for the BDNF homeostatic pathway in the progression to addiction. Specifically, I show that BDNF homeostasis breaks down throughout the corticostriatal circuit after extensive experience with ethanol. Critically, BDNF homeostasis does not recover following two weeks of abstinence from ethanol, indicating that inhibition of corticostriatal BDNF may be a hallmark of the addictive process which underlies the propensity to relapse.

Taken together, these data establish an integral role of BDNF in the dorsal striatum, via its downstream effector dynorphin, in regulating ethanol intake and preventing the progression to addiction.

Midbrain dopamine (DA) neurons are phasically activated in response to unexpected reward presentation and are depressed by reward omission; these phasic responses are proposed to contribute to learning. In addition, because DA release in the nucleus accumbens precedes initiation of instrumental actions, and DA receptor blockade in the nucleus accumbens decreases effortful behavioral responding, DA is thought to be necessary to initiate and maintain motivated responding. Hence, midbrain DA signaling may be critically involved in multiple, temporally-specific, behavioral processes. Notably, previous findings were based on long-term perturbations such as dopamine depletion and receptor antagonism, which do not assess the contribution of DA to behavior on short time scales. Therefore, in this study, we used in vivo optogenetic techniques to inhibit midbrain DA neurons of mice in real time to investigate how the precise timing of DA neural activity contributes to the decision to exert effort and to maintain cue-reward associations. We found that inhibiting DA neurons while mice were engaged in a bout of operant nose-poke responses decreased the probability of continued nose-poke behavior. Conversely, we found that inhibiting DA neurons while mice were engaged in off-task behavior decreased the probability of initiating a nose-poke response. Finally, we found that inhibiting DA neurons during reward consumption extinguished learned cue-reward associations, but did not alter effortful responding. We conclude that DA is required at multiple specific phases in behavior to differentially support reward prediction, and both initiation and maintenance of motivation to respond for reward.

Several lines of evidence suggest that the amygdala is important for emotional arousal. However, whether overlapping or segregated populations of cells in the amygdala contribute to appetitive and aversive emotional arousal remains unclear. Here we test two predictions of the hypothesis that overlapping populations of cells in the amygdala contribute to appetitive and aversive emotional arousal: neuronal activity in the amygdala during appetitive and aversive arousal should tend to be similar, and the activity of cells with similar changes in activity during appetitive and aversive emotional arousal should be correlated with measures of arousal.

Consistent with the hypothesis that overlapping populations of cells contribute to appetitive and aversive emotional arousal, we show that a large proportion of amygdala cells (26%) has qualitatively similar changes in activity during conditioned appetitive and aversive emotional arousal, despite the different sensory modalities of the eliciting stimuli. Furthermore, when the entire population of recorded cells is considered together, neuronal activity is more similar than expected by chance during conditioned appetitive and aversive emotional arousal. The basolateral region (BLA) and central nucleus (CeN) of the amygdala had similar proportions of "Same" cells, suggesting that overlapping populations of cells contribute to appetitive and aversive emotional arousal in both regions. Interestingly, however, the CeN had a greater proportion of cells with opposite changes in activity during the conditioned appetitive and aversive stimuli than the BLA, suggesting that neuronal activity in the CeN has a greater degree of affective specificity.

We also found that a large proportion of cells in the amygdala, including over half of the "Same" cells, had activity that was correlated with blood pressure--a measure of emotional arousal that is sensitive to lesions of the amygdala in both appetitive and aversive settings.

Together, our results suggest that the amygdala contributes to appetitive and aversive emotional arousal through the recruitment of overlapping populations of cells that respond similarly to appetitive and aversive stimuli.

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