Ryan T. LaLumiere, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of the proposed experiments is to improve the understanding of the neurobiology underlying emotionally influenced memory consolidation. The role of the dopaminergic system in the basolateral amygdala (BLA) and nucleus accumbens (NAc) during such consolidation is not fully understood. The specific aims of the proposed experiments are to determine whether dopamine (DA) receptor activation in the BLA is necessary after training for inhibitory avoidance (IA), whether the amount of DA released after IA training correlates with retention, and whether DA receptor activation in the NAc is necessary for memory modulation by the BLA. The proposed experiments will use animals with cranial cannula implantations to investigate this question. The animals will be trained and tested on an inhibitory avoidance task. Post-training manipulations or measurements of DA levels will be made. From the results, inferences will be drawn about the role of the dopaminergic system in these two structures during memory consolidation. These findings may provide insight into disorders of memory, such as posttraumatic stress disorder, as well as into the connections between emotionally influenced memory consolidation and addiction processes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The specific aims of the proposed experiments are: 1) To determine how NAC1 over expression in the nucleus accumbens (NAc) of rats affects the acquisition, maintenance, and reinstatement of cocaine self- administration. 2) To determine how self-administration of cocaine affects NAc protein levels and localization of the 20S proteasome, the E3 ligase Cul3, and poly-ubiquitinated proteins as well as the activity of the proteasome. 3) To determine how the loss of the NAC1 gene in knockout mice affects cocaine self- administration, the protein levels and localization of the above proteins, and proteasome activity. Experiments will use self-administration as the model of addiction and rats will receive AAV-NAC1 infusions to investigate over expression. The NAc from rats and mice will be dissected and sub fractionated to retrieve the cytosolic, nuclear, and postsynaptic density fractions. Some fractions will then undergo proteasome activity assays. Other fractions will undergo immunoblotting to determine fraction levels of the 20S subunit, Cul3, and poly-ubiquitinated proteins. Together, these findings will provide important new information regarding the neuroplastic changes induced by cocaine addiction.

DESCRIPTION (provided by applicant): Cocaine addiction is a serious problem afflicting the United States, and treatment remains imperfect. Although the study of cocaine self-administration and reinstatement in rats has provided much knowledge of cocaine addiction, relatively few studies have explored the extinction of self-administration behavior in rats. Nonetheless, the extinction of cocaine self- administration provides a major potential pharmacotherapeutic target in the effort to reduce relapse to cocaine use. Therefore, the long-term objective of the proposed work is to investigate the neurobiology of extinction of cocaine self-administration and determine whether infralimbic cortical activity can be modulated to influence memory consolidation of extinction training. Previous findings, together with preliminary data, suggest that a circuit involving the infralimbic cortex regulates the memory consolidation of extinction of cocaine self-administration. The proposed experiments will use male Sprague-Dawley rats that will undergo cocaine self- administration followed by extinction. Rats will receive microinjections immediately AFTER extinction sessions on days 1-5 of extinction training. Specific Aim #1 will examine how inactivation of the infralimbic cortex immediately after extinction training sessions influences the retention of the extinction training. Specific Aim #2 will investigate whether blockade of 2- adrenergic receptors in the infralimbic cortex immediately after extinction training sessions impairs memory consolidation of that training. Specific Aim #3 will examine whether activation of 2-adrenergic receptors in the infralimbic cortex immediately after extinction training sessions enhances memory consolidation of that training. Together, these experiments will provide important new information on the circuitry underlying the learning and memory of extinction of cocaine self-administration and how 2-adrenergic receptor blockade and activation in the infralimbic cortex modulate memory consolidation of extinction training. Cocaine addiction remains a significant problem, in large part due to the high rate of relapse following treatment. The proposed research will investigate the neurobiology of the extinction of cocaine self-administration in rats. This research will have a significant impact on public health as the extinction process is a potential pharmacotherapeutic target in the treatment of drug addiction.

DESCRIPTION (provided by applicant): Understanding the neurobiology of memory consolidation is crucial to the development of treatments for memory-related disorders, such as post-traumatic stress disorder and phobias. Considerable evidence indicates that the basolateral amygdala (BLA) interacts with a variety of forebrain structures to influence the consolidation for different types of learning, but previous work has been unable to examine the roles of specific pathways from the BLA in the memory consolidation for distinct types of learning. The recent development of optogenetics now enables optical control over structures, as well as their projections to efferent regions, with excellent temporal and spatial precision. Th long-term objective of the proposed research is to use optogenetics to investigate the neural circuitry, and especially the projections connecting structures, during memory consolidation. The present R21 will develop the use of optogenetics in the study of memory consolidation as a foundation for future research investigating larger neural circuits. In the present experiments, the basolateral amygdala (BLA) will be virally transduced to express the depolarizing cation channel channelrhodopsin-2 (ChETA) or the hyperpolarizing outward proton pump archaerhodopsin-3 (ArchT). Because the opsins are expressed along the entire cell membrane, including the axons, illumination can also be provided to structures, such as the nucleus accumbens (NA), that receive BLA input to control that specific pathway. In the present experiments, rats will be trained in a modified contextual fear conditioning (CFC) task that separates the context learning from the footshock learning. Thus, the current proposal will use optogenetic control over the BLA and its projections to the NA to determine how these neural circuits influence consolidation for different kinds of learning. Aim 1 will investigate how optogenetic control of the BLA influences the consolidation of the footshock vs. context learning in the modified CFC task. In Aim 1, we will use a variety of different illumination parameters to stimulate (via ChETA expression) or inhibit (via ArchT expression) BLA activity immediately after training. Aim 1's findings will provide critical knowledge about how optogenetic control of BLA glutamatergic neuron activity influences memory consolidation. Aim 2 will examine how optical control over the BLA's projections to the NA (shell vs. core) influences the consolidation of context vs. footshock learning. Aim 2 will take advantage of the ability of optogenetics to control specific projection pathways by illuminating opsin-expressing BLA fibers in the NAshell or core. These findings will answer previously intractable questions about the pathways from the BLA to the NAshell and core during the consolidation of different aspects (context vs. footshock) of CFC learning. Together, these experiments will provide a critical foundation in the application of optogenetics to the understanding of the neural circuits involved in memory consolidation and answer major questions regarding the circuits from the BLA to the NA during consolidation for different types of learning.

DESCRIPTION (provided by applicant): The study of cocaine self-administration and the reinstatement of cocaine seeking in rats has provided much knowledge regarding the mechanisms underlying cocaine addiction and relapse. Yet few studies have explored the neurobiology of the extinction of cocaine-seeking behavior in rats, despite the fact that extinctio provides a major potential therapeutic target in the effort to reduce relapse to cocaine use. Thus, the long-term objective of the proposed work is to investigate the neural circuitry in rats mediating the suppression of cocaine seeking that develops from extinction training after cocaine self-administration. Our previous work suggests that a circuit involving the infralimbic cortex and nucleus accumbens shell is involved in the suppression of cocaine-seeking behavior, findings that parallel those from human studies regarding the human homologue of the infralimbic cortex. Based on our work and others' findings, we have developed a neural circuit that enables activity in the infralimbic cortex to inhibit activity in the ventral tegmental area va the rostromedial tegmental nucleus and the lateral habenula and, thereby, suppress cocaine seeking. The proposed experiments will examine specific parts of the neural circuit to determine their role in the suppression of cocaine seeking. In the experiments, male Sprague-Dawley rats will undergo cocaine self-administration followed by extinction training and, in some experiments, cue-induced reinstatement testing. Aim 1 will examine whether the lateral habenula suppresses cocaine-seeking behavior after extinction training and whether it interacts with the infralimbic cortex to do so. Rats will receive bilateral microinjections of either GABA agonists to inactivate the structure or the AMPA receptor potentiator PEPA to activate the structure. The microinjections will be given into the structures either before an extinction sessio or a cue-induced reinstatement session in order to assess how the neural structures interact to suppress cocaine-seeking. Aim 2 will use a similar approach to Aim 1 in order to examine whether the rostromedial tegmental nucleus suppresses cocaine-seeking and how it interacts with the infralimbic cortex to do so. Aim 2 will also determine whether the lateral habenula and the rostromedial tegmental area interact with each other to regulate cocaine seeking. Aim 3 will use a complementary approach to examining the neural circuit underlying the suppression of cocaine-seeking. Specifically, Aim 3 will use optogenetic inhibition of axonal projections from the infralimbic cortex to determine which projection pathways from the structure are involved in the suppression of cocaine seeking. This approach is of particular importance because it can reveal the specific pathways, and not just the structures themselves, that are crucial to a particular behavior. Together, the proposed experiments will provide converging lines of evidence regarding this novel circuit and its role in suppressing cocaine seeking. Moreover, the results will furnish critical new knowledge on the neural mechanisms underlying the suppression of cocaine-seeking behavior that will enable the development of new treatments for cocaine addiction.

DESCRIPTION (provided by applicant): Drugs of abuse, such as cocaine, produce long-lasting synaptic adaptations that increase the compulsive nature of addiction, undermine self-control, and increase the likelihood of relapse. Identifying and understanding the molecules that regulate these synaptic changes may suggest novel therapies. Recently, we found that acid-sensing ion channels (ASICs) and brain pH play critical roles in the synaptic plasticity thought to underlie addiction. Our findings suggest that ASIC1a is activated during synaptic transmission in medium-spiny neurons (MSNs) of the nucleus accumbens (NAc), a site firmly implicated in addiction-related behavior. Genetically deleting ASIC1a in mice led to a number of synaptic changes paralleling those previously observed following cocaine withdrawal. Consistent with these synaptic effects, disrupting ASIC1a in mice throughout the body or specifically in the NAc increased conditioned place preference (CPP) to cocaine and to morphine, indicating important behavioral consequences that generalize to multiple drugs of abuse. Confirming the NAc as a key site of ASIC1a action in cocaine-dependent behavior, restoring ASIC1a expression to the NAc of ASIC1a-/- mice reversed the synaptic abnormalities and normalized cocaine CPP. We also tested synaptic and behavioral effects of ASIC1a in rats and found results similar to those in mice. In rats, overexpressing ASIC1a in the NAc doubled the ASIC-mediated synaptic current, and significantly reduced cocaine self- administration. Together, these observations indicate that ASIC1a inhibits addiction-related behavior. Furthermore, these results suggest the hypothesis that ASIC1a and brain pH might be targeted to reduce the synaptic changes underlying addiction and relapse. To test this hypothesis, we propose to explore genetic and pharmacological approaches to increase ASIC1a function at synapses and to determine their ability to affect cocaine-related synaptic physiology and behavior in mice and rats. The planned studies capitalize on novel insight into the roles of ASICs and pH in synaptic transmission, and take advantage of state-of-the-art electrophysiological approaches and an innovative collaboration between principal investigators with extensive experience in ASICs, brain pH, and drug-related behavior. Our planned behavioral analyses include models of craving/relapse using long-access cocaine self-administration in rats, widely considered one of the best models of addiction because animals control their own drug intake, thus facilitating assessment of various stages of drug-seeking behavior. Because ASIC1a structure and function in rodents are nearly identical to those in humans, these studies will be highly relevant to the human brain. Moreover, the knowledge gained through these experiments will inform innovative strategies to interrupt addictive behaviors by targeting ASICs and/or brain pH.

? DESCRIPTION (provided by applicant): Prior studies examining multiple memory systems in the brain have had great difficulty elucidating the specific neural connections that influence the consolidation and synaptic plasticity for different kinds of learning. The present proposal will utilize optogenetic manipulations of specific pathways connecting different brain regions in rats immediately after the rats have undergone different kinds of learning. Specifically, this proposal will build upon the evidence that the basolateral amygdala (BLA) influences consolidation for many different kinds of learning, including spatial/contextual, emotional, and cued-response learning, whereas other regions are involved in memory for more discrete forms of learning. Additional evidence indicates that the BLA influences consolidation through regulation of synaptic plasticity in these other brain regions, including modulation of activity-regulated cytoskeletal-associated protein (Arc), which has been shown to mediate some of the BLA's effects on memory consolidation. Based on these prior findings, the present studies will investigate how distinct BLA projections to different parts of the hippocampus and caudate modulate memory consolidation and Arc expression in downstream brain regions following different kinds of learning. Follow-up experiments will determine whether the increased Arc expression is necessary for the memory modulation. To do so, the current proposal will utilize optogenetic control of activity in specific BLA projections immediately after different learning tasks to directly examine in each Aim: 1) how the candidate pathways influence consolidation for the specific type of learning and 2) how the pathways influence Arc expression in downstream regions and whether the Arc expression is necessary for the memory modulation. Aim 1 will determine how BLA projections to different parts of the hippocampal formation influence consolidation and Arc expression for the context vs. footshock learning in a modified contextual fear conditioning task. Aim 2 will determine how BLA projections to part of the hippocampal formation and the caudate influence consolidation and Arc expression for spatial vs. cued-response learning. In both aims, optogenetic stimulation/inhibition will be given to each candidate pathway immediately after the relevant training to determine its role in influencing consolidation. Moreover, each aim will examine whether such optogenetic manipulations alter Arc expression in different downstream structures and whether reducing Arc levels in those regions prevents the memory-modulating effects of the optogenetic manipulations, providing a critical complementary set of experiments that will improve our model for understanding how the BLA influences memory and plasticity-related proteins such as Arc in other regions. The findings from these studies will provide, for the first time, knowledge for how the specific pathways from the BLA to other brain regions influence memory consolidation and synaptic plasticity. Moreover, these experiments will provide a first step to creating an understanding of the functional connections within the brain underlying consolidation processes.

Relapse to heroin use remains a significant challenge in the treatment of heroin addiction, yet our understanding of those neural systems that enable individuals to inhibit heroin seeking and relapse remains poor. The long-term goal of our laboratory is to identify the neural circuits and the changes in those circuits that underlie the inhibition of heroin-seeking behavior, using rat models of heroin seeking. Previous work with cocaine seeking has suggested that the infralimbic cortex is involved in the extinction and inhibition of cocaine seeking. Yet, the role of this prefrontal region in opioid seeking has been unclear, as studies have produced findings indicating that the infralimbic promotes and inhibits opioid seeking. As increasing evidence suggests that different projections from prefrontal regions can mediate distinct functions, we have hypothesized that the ability of the infralimbic to either promote or inhibit heroin seeking is related to the specific projections from this region to the nucleus accumbens shell (NAshell) vs. the amygdala. In addition, we have recently developed preliminary data suggesting that the anterior portion of the insular cortex (rostral agranular insular cortex, RAIC) inhibits heroin seeking. However, other studies with other drugs of abuse indicate that the RAIC also appears to promote drug-seeking behavior. Thus, it seems likely that, as with the IL, the key to understanding the role of the RAIC in heroin seeking depends on targeting different projections. Indeed, like the infralimbic, the RAIC projects to the amygdala, a region that prior studies have found to be involved in promoting reinstatement to heroin seeking. However, the RAIC also provides an input to the infralimbic itself, thus providing a mechanism for how the RAIC may influence and interact with the infralimbic during the extinction and inhibition of heroin seeking. The proposed studies, therefore, will examine these circuits during heroin seeking and will include approaches that have not, to our knowledge, been used in studies of heroin seeking. In particular, our studies will examine the neurophysiological correlates of heroin seeking in the RAIC and IL via ensemble recordings in both regions simultaneously. The findings from this work will likely reveal neuronal subpopulations in both regions related to the extinction and/or inhibition of heroin seeking. Moreover, our proposed work will use optogenetic and chemogenetic manipulations of the specific projections from the infralimbic to the NAshell and amygdala and from the RAIC to the infralimbic cortex and amygdala. Specifically, these manipulations will allow us to interrogate the role of each pathway in the encoding of the extinction learning and the reinstatement of heroin seeking. As a result, this proposal will provide converging lines of evidence regarding this circuitry and the inhibition of heroin seeking. The findings from the studies will furnish critical new basic knowledge of the neural systems underlying the suppression of heroin seeking that will potentially lead to the development of more effective treatments that strengthen such systems in heroin- addicted individuals.

Relapse to cocaine use remains a significant challenge in the treatment of cocaine addiction, yet our understanding of those neural systems that enable individuals to inhibit cocaine seeking and relapse remains poor. The long-term goal of our laboratory is to identify the neural circuits and the changes in those circuits that underlie the inhibition of cocaine-seeking behavior, using rat models of cocaine seeking. The current proposal builds upon the findings we have obtained in recent years as well as new approaches we have developed in our laboratory. In our studies, rats undergo cocaine self-administration, extinction training and reinstatement testing of their cocaine seeking. We have found that the infralimbic cortex is a central component of the systems involved in the extinction and inhibition of cocaine-seeking behavior. Activity in the infralimbic cortex is necessary for the normal encoding of the extinction learning as well as extinction expression. For example, infralimbic activation inhibits cocaine seeking following extinction training. However, the larger circuit in which the infralimbic cortex performs these functions remains unclear. In particular, as evidence suggests functional heterogeneity within the infralimbic cortex in terms of its role in cocaine seeking, understanding the larger circuitry may provide insight into these issues. The proposed work will focus on the specific pathways projecting into and out of the infralimbic that account for its role in extinction and inhibition of cocaine seeking. The work will include approaches that have not, to our knowledge, been used in studies of cocaine seeking and, therefore, will enable notable progress in our knowledge of these systems. In particular, our studies will use multi-site recordings of neural activity during cocaine seeking to understand how a network of brain regions coordinates behavior. Moreover, the proposed studies will use optogenetic approaches to examine how different pathways mediate different aspects of the extinction and inhibition of cocaine seeking. The findings from this work will reveal those specific pathways and network activity related to the IL with regard to the extinction/inhibition of cocaine seeking. Furthermore, the proposed work will both 1) examine changes in IL-based circuits as a consequence of cocaine self-administration and extinction and 2) manipulate these circuits to determine how they functionally control the inhibition of cocaine seeking. The findings from the studies will furnish critical new basic knowledge of the neural systems underlying the suppression of cocaine seeking that will potentially lead to the development of more effective treatments that strengthen such systems in cocaine-addicted individuals.