Lynette C. Daws - US grants

The serotonin (5-HT) transporter (SERT) is responsible for terminating serotonergic neurotransmission by high-affinity uptake of 5-HT from extracellular fluid (ECF) and is therefore critical in determining concentrations of 5-HT in ECF. Drugs that act at the SERT, such as selective serotonin reuptake inhibitors, ameliorate several neuropsychiatric disorders, including depression and certain anxiety disorders. The SERT is also a primary site of action of drugs of abuse such as "Ecstasy" (MDMA). However, there is remarkable variability in the response of individuals to these drugs and the reason for this is not clear. For example, some individuals respond well to antidepressant treatment while others do not. Also, certain people will overdose on MDMA at a dose that will only mildly affect others. Allelic variations in the SERT have now been identified that influence SERT activity and may be associated with a number of psychiatric disorders and/or to an individual's response to drug treatment. The main goal of this proposal is to study the functional and adaptive consequences of genetically- induced reductions in the expression of the SERT and 5-HT1B receptor on the clearance of 5-HT from ECF in vivo. Using in vivo high-speed chronoamperometry we have found that the 5-HT1B autoreceptor can regulate clearance of 5-HT from ECF and also, that alterations in the density of the SERT can change the clearance rate of 5-HT. This proposal will take advantage of two lines of mice with null mutations of the SERT or the 5-HT1B receptor gene (the SERT knockout (KO) and 5-HT1B receptor KO mice), to gain new insight into both the regulation of the SERT and the effect of altered gene expression on drug sensitivity in these animals in vivo. It is predicted that in heterozygotes of both KO strains (which express 50 percent fewer SERTs and 5-HT1B receptors respectively), clearance rates of 5-HT will not differ from wild-type mice under basal conditions, but defects will be revealed in response to pharmacologic challenge. Our general hypothesis is that changes in the kinetics of 5-HT clearance as an adaptive consequence of reduced SERT or 5-HT1B receptor density will alter the sensitivity of these mice to psychotropic drugs. Changes in sensitivity will be indexed by the ability of these drugs to influence the kinetics (KT and Vmax) of 5-HT clearance in the three genotypes of each KO strain. High-speed chronoamperometry will be used to measure 5-HT clearance in vivo. Quantitative autoradiography will be used to assess the effect of genetic mutation of the SERT on 5-HT1B receptor density and vice- versa. Alterations in the uptake of 5-HT by these mice and in the response of these mice to psychotropic drugs will provide information important to our better understanding of the pathobiology of neuropsychiatric disorders and addiction as well as to the selection of drug treatment in individuals with allelic variations of the SERT.

DESCRIPTION (provided by applicant): Drug abuse is a prevalent psychiatric disorder with immense negative public health consequences. As a result, understanding its neural underpinnings is a focus of intense research. The dopamine (DA) transporter (DAT) is a primary site of action of drugs of abuse such as amphetamine and is critical in regulating DA neurotransmission by high affinity transport of DA into the terminal. Understanding how the DAT is regulated is therefore of fundamental importance to studies of amphetamine abuse. In this regard, there is converging evidence that insulin can produce profound regulatory control of DAT activity. It is known for example that both food restriction as well as experimentally-induced diabetes have major effects on behavioral responses to amphetamine. The significance of these observations is underlined by the high co-morbidity of eating disorders and drug abuse. Recently it has been reported that rates of DA uptake are decreased in hypoinsulinemic rats and that insulin applied intracerebroventricularly to rats or to cells stably transfected with the DAT increases DA uptake. Importantly, it seems that insulin can interfere with the action of amphetamine at the DAT and can prevent amphetamine-induced internalization of the DAT. Of particular importance to the present proposal is that diabetic rats with a history of amphetamine self-administration show increased rather than decreased DA uptake. Insulin and its signaling pathways may therefore represent a novel target for the development of new treatments for drug abuse. To date no studies have assessed the role of insulin in controlling DAT activity in vivo. Because of the potential impact of insulin in promoting the abuse potential of drugs the proposed study will use an innovative approach, high-speed chronoamperometry, to measure insulin-dependent changes in DA efflux and clearance in vivo. Because a major signaling mechanism that underlies insulin's cellular actions is stimulation of phosphatidylinositol (PI)-3 kinase these studies will also test the hypothesis that DA effiux and clearance will be increased and decreased respectively, upon activation and inhibition of PI-3 kinase. Importantly, these studies will determine the relationship between the action of amphetamine and insulin status as they relate to DAT activity. Our general hypothesis is that the kinetics of DA efflux and clearance as well the locomotor stimulatory effects of amphetamine will be tightly correlated with insulin status. Restoration of insulin to normal levels will restore normal responses. The results obtained here will not only improve our fundamental understanding of DAT regulation but importantly the neural circuitry controlling reward and motivation, as well as provide a framework for a larger RO1 application. In turn, these studies may help to illuminate the neural mechanisms underlying the high co-morbidity of eating disorders and drug abuse.

DESCRIPTION (provided by applicant): The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by high affinity uptake of released serotonin. It is a key site of action for many psychotherapeutic and addictive drugs. The level of 5-HTT expression varies in humans according to whether an individual carries the short (s) variant of the 5-HTT promoter region polymorphism (5-HTTLPR). Carriers of the s allele express ~50% fewer 5-HTTs than those homozygous for the long (/) allele. Carriers of the s allele appear to be more prone to a variety of psychiatric disorders, including depression and drug abuse, and are often resistant to treatment with selective serotonin reuptake inhibitors (SSRIs) compared to individuals homozygous for the / allele. To study the neurochemical underpinnings of the relationship between 5-HTT genotype and drug effect, we have made use of mice with a null mutation of the 5-HTT. Heterozygote 5-HTT mutants resemble carriers of the s allele in that they express 50% fewer 5-HTTs and clear serotonin from extracellular fluid more slowly than wild-type mice. The null mutants lack 5-HTTs. One of our most intriguing findings is that 5-HTT mutant mice are more sensitive to the serotonin clearance inhibiting effect of "Ecstasy" (MDMA) and alcohol where greatest inhibition of serotonin clearance occurs in null mutants. Thus, these drugs inhibit serotonin clearance but via a 5-HTT independent mechanism. Importantly, this mechanism has presumably undergone compensatory upregulation in 5-HTT mutants. The studies proposed here seek to identify these alternative mechanisms for serotonin transport. It is known that the dopamine and norepinephrine transporters take up serotonin, but preliminary data indicate that at least in the CA3 region of hippocampus these transporters do not play a significant role in serotonin clearance in 5-HTT mutant mice. By contrast, blockers of the organic cation (OCT) and proline (PROT) transporters profoundly inhibit serotonin clearance in 5-HTT mutant mice. Here we will determine the relationship between genetically defined deficiencies in 5-HTT expression and (1) OCT and PROT expression, (2) OCT and PROT function and (3) behavior to assess changes in effectiveness of SSRIs and blockers of the OCT and PROT in preclinical measures for antidepressant efficacy and treatment of alcoholism. We will also investigate OCT and PROT as sites of action for antidepressant and abused drugs and how their efficacy varies with 5-HTT genotype. In addition to increasing our fundamental understanding of serotonergic neurotransmission, these studies may lead to novel treatments for affective and addictive disorders that are better tailored to the individual. The clinical implications for upregulation of alternative mechanisms for serotonin uptake when 5-HTT expression is compromised are far reaching. That is, drugs targeted at OCTs or PROT may represent first line or adjunctive therapies for individuals who do not respond well to treatment with SSRIs.

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how changes in insulin signaling regulate the actions of amphetamine (AMPH). Dopamine (DA) transporters (DATs), which largely control DA clearance, are targets for psychostimulants such as AMPH and cocaine. By acting on the DAT, AMPH attenuates DA clearance efficiency and induces reverse transport of DA. As a consequence, AMPH increases synaptic DA levels and enhances dopaminergic transmission, with profound effects on behavior. Exciting new experiments suggest that insulin, through phosphatidylinositol 3 kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. Consistent with these data, in experimentally-induced diabetic rats, where insulin signaling is impaired, DA clearance measured by in vivo chronoamperometry is reduced, as is the ability of AMPH to cause DA efflux. This project will combine biochemistry, biophysics, imaging and in vivo chronoamperometry to elucidate the relationships between changes in PI3K signaling in brain and changes in AMPH-induced increases in extracellular DA. Therefore, the key issues to resolve include how PI3K signaling regulates AMPH-induced DA efflux and to determine, in vivo, whether perturbations in PI3K signaling caused by changes in food intake and disease states such as diabetes regulate the ability of AMPH to increase extracellular DA levels. Interestingly, repeated systemic AMPH, which parallels social intake in humans, overrode the ability of hypoinsulinemia to reduce the effect of acute AMPH. This phenomenon appears to be mediated by D2 receptors. Therefore, we will also evaluate how signaling pathways activated by repeated AMPH exposures (e.g. D2 receptor through ERK1/2 activation) restore the acute actions of AMPH. The proposed studies address the following Specific Aims: 1) To define how PI3K signaling regulates AMPH-induced DA efflux. 2) To demonstrate, in vivo, that hypoinsulinemia or insulin resistance induced by changes in diet, reduce DA clearance and AMPH-induced DA efflux and, ex vivo that these modifications are regulated by DAT trafficking. 3) To determine whether in hypoinsulinemic and insulin resistant rats, repeated administration of AMPH restores AMPH-induced DA efflux by stimulating D2 receptors and consequently ERK1/2. These studies will illuminate pathways that may contribute to the development of psychostimulant abuse. PUBLIC HEALTH RELEVANCE: Stimulant abuse and potentially other dopamine-related pathologies such as schizophrenia and motor disorders (e.g. Parkinson's disease), are a tremendous public health burden. The dopamine transporter, which regulates extracellular brain dopamine, is the major molecular target of several psychoactive drugs, including amphetamine and cocaine. This proposal will analyze how perturbations of insulin signaling induced by diet and diseases states, such as diabetes, regulate dopamine clearance and the ability of amphetamine to increase extracellular brain dopamine, which can lead to addiction. Defining how insulin signaling affects dopamine neurotransmission may help to explain the mechanistic basis of how food intake regulates dopamine neurotransmission and drug abuse.

DESCRIPTION (provided by applicant): Depression and related disorders are a major public health problem, compounded by the fact that at least half of patients are not effectively treated by currently available medications. Among the most commonly prescribed is the class of selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act to inhibit 5-HT transporter (SERT) mediated 5-HT uptake. The increase in extracellular 5-HT that follows is thought to be critical for initiation of the cascade of downstream events needed for therapeutic effects. Although SERT is the major player regulating high-affinity 5-HT uptake, there is emerging evidence for an important role of organic cation transporter-3 (OCT3) and possibly the plasma membrane monoamine transporter (PMAT) in taking up 5-HT in brain. This raises the possibility that lack of therapeutic response following SERT blockade could be due to significant 5-HT uptake by OCT3 (and/or PMAT). Our studies using decynium-22 (D-22), a blocker of both OCT3 and PMAT, lend support to this idea. For example, D-22 augments the effect of an SSRI, fluvoxamine, to inhibit 5-HT uptake and to produce antidepressant-like effects in wildtype mice. Moreover, D-22 produces these effects also when given alone in mice that lack, or have reduced SERT expression. Thus, the antidepressant-like effect of D-22 appears to be most pronounced when SERT is either pharmacologically or genetically inactivated. We also found that OCT3 expression (but not PMAT) is increased in mice with a constitutive reduction of SERT, suggesting a compensatory role for OCT3. One important aspect of the proposed studies will be to examine the possibility that this also occurs after chronic treatment with SSRIs, which is known to reduce SERT expression. In addition to 5-HT, OCT3 (and PMAT) can transport norepinephrine (NE) and dopamine (DA), neurotransmitters also linked to the therapeutic action of current antidepressants. Taken together, the goals of the proposed studies are to (1) validate OCT3 (and/or PMAT) as the site where D-22 produces its antidepressant-like effect; (2) determine the relative importance of inhibition of 5-HT, NE and DA uptake in producing the antidepressant-like effect of D-22, and (3) examine the therapeutic potential of D-22 by studying its effect on biogenic amine uptake and antidepressant-like activity after its chronic administration. The results of these studies will help to establish OCT3 (and/or PMAT) as a novel target for the discovery of drugs with improved therapeutic potential, as well as provide a mechanism that can, at least in part, account for poor therapeutic response to current antidepressant drugs.

DESCRIPTION (provided by applicant): The Serotonin Club, founded in 1987, is an international association for biomedical scientists who are interested in research on any aspect of serotonin. An important objective of the Club is to facilitate the involvement of young investigators, comprising students, post-doctoral fellows and junior researchers (less than 3 years past their post-doctoral fellowship) in an international high-quality scientific meeting with serotonin as its central theme. Serotonin has been implicated in substance abuse, pain, cardiovascular and gastrointestinal function, neural development as well as a host of psychiatric diseases, including addiction. A diverse audience is attracted by including not only the neuroscience of serotonin, and the neurotransmitter systems on which it impinges, but also the role of serotonin in peripheral function. Thus, our goal is to bring together researchers with a common interest in serotonin such that the depth of its functions, and importantly, new discoveries, can be communicated. A new initiative of the Serotonin Club Meetings is to provide a focus and build a strong emphasis on substance abuse, as it relates to serotonin's interactions with other neurotransmitter systems, and importantly, to understand serotonin's role in the disease of addiction and how this system can be targeted for the treatment of substance abuse. A key goal of the 2012 meeting will be to foster this new initiative by inclusion of symposia directly related to substance abuse. Importantly, a major goal of the Serotonin Club is to provide a dynamic forum to help develop the careers of young investigators in the field. As such, we plan to include ten recipients of NIDA Travel Awards as featured speakers, one in each of ten programmed symposia. In addition these young investigators will i) be part of a mentoring program, ii) be able to network with senior researchers, including leaders in the field, at many occasion (e.g. poster sessions, breaks, lunches, and dinner banquet); and iii) have their work published in the conference proceedings. Selection will be based on the applicant's credentials and on the relevance of their abstract to drug abuse and related disorders. The first meeting of the Serotonin Club for which funds are sought will be held July 10-12, 2012 in Montpellier, France. The primary aim of this R13 application is to provide financial support to these young investigators to enable them to attend and participate in the 2012 Serotonin Club Meeting as well as to support future young investigators to attend subsequent meetings in 2014 and 2016.

DESCRIPTION (provided by applicant): Although eating disorders are complex and their etiology unclear, dysregulation of dopamine neurotransmission is a consistent finding. A vital regulator of dopamine neurotransmission is the dopamine transporter (DAT). DAT terminates dopamine neurotransmission by uptake of dopamine from extracellular fluid and because of this DAT is a primary determinant of both the strength and duration of dopamine signaling. In spite of its critical role in maintaining dopamine homeostasis, there have been no investigations of DAT function, per se, in eating disorders. Using neurochemical, cellular and behavioral approaches, we have shown that DAT activity is highly sensitive to diet and food intake, strongly supporting the notion that DAT activity is dysregulated in individuals with eating disorders. However, studies so far have been in adult, male rodents. Anorexia, bulimia and binge eating are most common in teenage girls. What is lacking in our fundamental understanding of DAT is how its activity varies as a function of age and sex and, importantly, how age and sex interact to contribute to disordered eating. In turn, how does aberrant DAT activity contribute to disordered eating? Here we will begin to fill these critical knowledge gaps. We will make use of the activity-based anorexia (ABA) model in rats to determine how age, sex and eating disorder influence DAT function and how manipulations of DAT activity modify eating behavior. The ABA model recapitulates key characteristics of anorexia in humans, including reduced food intake in the presence of hunger, weight loss, hyperactivity, and increased insulin sensitivity. Importantly, in this model rats must choose between eating and another rewarding condition, exercise. Thus, food intake is controlled by the rat and not artificially by the experimenter, offering a powerful translational model system. We will explore phosphotidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK), regulators of DAT activity that we have identified as putative targets to restore normal DAT function, as novel targets for therapeutic intervention to prevent, or lessen the severity of ABA. Given the well-established role of dopamine in reward and motivation, these studies will not only provide mechanistic insight into dysregulation of dopamine neurotransmission in eating disorders, but also other illnesses, including addiction and depression, which are often co-morbid with eating disorders.

? DESCRIPTION (provided by applicant): Depression is a major public health problem for which the majority of patients are not effectively treated. This problem is exacerbated further in children and adolescents for whom only two antidepressant drugs are currently approved. Both belong to the selective serotonin (5-HT) reuptake inhibitor (SSRI) class of antidepressant, and act by blocking high-affinity uptake of 5-HT from extracellular fluid via the serotonin transporter (SERT). The therapeutic utility of SSRIs is thought to be triggered by downstream events that occur in response to their ability to increase extracellular levels of 5-HT. However, our studies using adult mice show that the ability of SSRIs to inhibit 5-HT uptake is greatly limited by the presence of non-SERT, decynium-22 (D22) sensitive transporters for 5-HT. Thus, by preventing extracellular 5-HT rising to therapeutically useful levels, non-SERT transporters provide a mechanistic basis for limited therapeutic efficacy of SSRIs. D22 inhibits activity of organic catio transporters (OCTs) and the plasma membrane monoamine transporter (PMAT). OCTs and PMAT are expressed in adult brain, but their impact on serotonergic neurotransmission may be even greater in juvenile and adolescent brain, particularly if their expression and activity is disproportionately greater than SERT. However, little is known about expression or function of SERT in juvenile and adolescent brain, and nothing is known about the expression and function of OCTs and PMAT at these young ages. Not surprisingly, nothing is known about the relation among SERT, OCTs and PMAT in juvenile, adolescent, and adult brain and antidepressant response. The goals of the proposed studies are to fill these critical gaps in knowledge by (1) providing a systematic profile for SERT expression and function in juvenile, adolescent, and adult mice and importantly, determining how expression and function of OCTs and PMAT varies with that of SERT, and (2) determining how the antidepressant-like response to blockers of these transporters differs among juvenile, adolescent, and adult mice. Our preliminary data support the hypothesis that OCTs and/or PMAT play a more prominent role in 5-HT uptake during childhood and adolescence than in adulthood, and may be useful targets for antidepressant drugs, especially in this young population. Studies proposed here will afford new insight into mechanisms regulating 5- HT uptake in brain during childhood and adolescence, compared with adulthood. Given the strong link between dysfunction in 5-HT signaling and many psychiatric disorders, depression being prominent among them, elucidating mechanisms controlling 5-HT uptake in children and adolescents compared with adults will further our understanding of the etiological bases for these disorders and importantly, will guide the development of improved treatments.

ABSTRACT Co-abuse of cocaine and alcohol is one of the most common, and dangerous drug pairings, as evidenced by their concurrent use being a major cause for emergency hospitalization. Thus, this drug combination is not only a serious health threat to the individual user, but a major public health burden. Currently, there are no effective treatments for addiction to cocaine and ethanol, underscoring the vital need to understand the mechanistic basis of this highly addictive drug pairing in order to discover new targets for therapeutic intervention. It is well-known that cocaine and ethanol each increase extracellular levels of dopamine (DA), serotonin (5-HT), and norepinephrine (NE), biogenic amine neurotransmitters that are strongly linked to the rewarding properties of drugs. Cocaine does this by inhibiting the high-affinity, low-capacity transporters for these neurotransmitters, DAT, SERT, and NET, respectively. However, the mechanisms by which ethanol does so are unclear. It is known that ethanol inhibits uptake of DA, 5-HT, and NE, however our published data, together with literature evidence, show this inhibition to be DAT-, SERT-, and NET-independent. Organic cation transporter 3 (OCT3) is a low-affinity, high-capacity transporter for DA, 5-HT, and NE, and is emerging as an important player in regulation of biogenic amine homeostasis. Interestingly, recent reports show that corticosterone, a blocker of OCT3, enhances cocaine-induced DA signaling and potentiates reinstatement of cocaine seeking via an OCT3-dependent mechanism. Moreover, we found that OCT3 expression is increased in mice lacking SERT (-/-), and that ethanol, and corticosterone, both inhibit 5-HT clearance in SERT-/- mice to a much greater extent than in their wild-type counterpart. Together, these findings raise the possibility that ethanol may interact with OCT3 to inhibit uptake of biogenic amines, thereby increasing the addictive properties of cocaine, and propagating the concurrent use of these drugs. To this end, the studies proposed in this exploratory R21 will test the overarching hypothesis that one mechanism by which ethanol increases extracellular DA, 5-HT and NE is by inhibition of their uptake via OCT3, and that this inhibition enhances the increase in biogenic amines produced by cocaine, which blocks their uptake via DAT, SERT and NET. Importantly, we will determine the OCT3-dependency of ethanol?s ability to enhance the rewarding properties of cocaine. We will use pharmacological and genetic approaches, combined with in vivo neurochemistry, and behavioral assays relevant for reward. Regardless of the outcome of these exploratory studies, results will fill fundamental knowledge gaps about the mechanism(s) through which ethanol inhibits uptake of biogenic amines and enhances rewarding effects of cocaine. Results from these studies will improve our understanding of mechanisms that make the abuse potential of concurrent alcohol and cocaine use so high. Importantly, these studies will form an essential platform on which to base larger scale studies probing novel molecular targets, putatively OCT3, for medications to treat abuse of alcohol and cocaine.

Medications to help treat addiction exist for many major drugs of abuse, but not for psychostimulants, such as amphetamine, and its congeners. They are also lacking for increasingly used synthetic drugs designed to mimic the actions of known psychostimulants. Both known and new psychoactive substances continue to pose a major and increasing public health threat. To develop effective treatments, the mechanisms by which these stimulants produce their abuse-related effects need to be fully understood. Many stimulants interact with the dopamine (DA) transporter (DAT), which is thought to mediate their abuse-related effects. However, strategies targeting DAT have yielded little to no benefit in the treatment of psychostimulant addiction, raising the possibility that these stimulants have significant actions elsewhere to modulate dopaminergic neurotransmission. Consistent with this, a rapidly growing literature supports a prominent role for organic cation transporter 3 (OCT3) in regulating dopaminergic neurotransmission. Our preliminary data support this idea, showing that an OCT3 inhibitor, decynium-22 (D22), inhibits amphetamine-evoked hyperlocomotion and DA release in vivo, effects that were lost in constitutive OCT3 knockout (KO, -/-) mice. Furthermore, amphetamine-induced substrate efflux could be inhibited by D22 in a manner independent of cocaine-sensitive transporters. These data raise the exciting possibility that OCT3 is a critical player in the actions of amphetamine, which may help to explain why DAT-based therapeutics have not been successful in treating amphetamine abuse. Our intention is to submit an R01 to build on these exciting findings, but before doing so, additional preliminary data are needed. First, we need to determine if potential compensation in constitutive OCT3-/- mice accounts for the lack of difference in their locomotor and DA releasing responses to amphetamine compared with wild-type (OCT3+/+) mice. To do this, OCT3 floxed mice have recently been generated at the University of Texas Health Science Center at San Antonio (UTHSCSA). We will cross these mice with a commercially available Cre line to generate a tamoxifen inducible global OCT3 KO. In this way, we can temporally control OCT3 KO, and in future studies, use different Cre lines to create brain region specific inducible KOs. We will use these mice to test the hypothesis that amphetamine-induced DA release, locomotion, and stereotypy will be attenuated in inducible OCT3 KO mice compared with control mice. Moreover, if OCT3 is to be a useful target in the treatment of amphetamine abuse, we need to demonstrate that OCT3 is important in mediating the rewarding and reinforcing effects of amphetamine. To this end, we will use conditioned place preference (CPP), and self-administration in mice to test the hypotheses that the rewarding and reinforcing effects of amphetamine are less in inducible OCT3 KO mice than control mice, and that D22 will attenuate development of CPP to amphetamine and amphetamine self-administration in control mice, but not in the inducible OCT3 KO. These proposed studies will provide data essential for an R01 submission, and will begin to fill crucial knowledge gaps about the role of OCT3 in abuse-related effects of amphetamine.

Dysfunctional serotonergic neuromodulation in mood-regulating circuits underlies many psychiatric diseases, thus understanding regulation of serotonin (5-HT) transmission is of fundamental importance. The 5-HT transporter (SERT) clears 5-HT from extracellular fluid with high-affinity, and is considered a primary controller of the strength and duration of 5-HT signaling. Our studies have revealed that organic cation transporter 3 (OCT3), a low-affinity, but high-capacity transporter of monoamines, plays a critical role in 5-HT clearance as well. Though circuits modulating arousal and emotion are highly complex, processing within the basolateral amygdala (BLA) is considered essential, especially for fear conditioning. The BLA receives dense input from 5-HT neurons in dorsal raphe nucleus (DRN), and BLA principal neurons have numerous fear-regulatory outputs, including dense projections to medial entorhinal cortex (mEC), which serves as a gateway for fear memory information flow into and out of hippocampus. Like SERT, OCT3 is highly expressed in BLA, ideally positioning these transporters to powerfully control extracellular 5-HT and its local neuro-modulatory efficacy. Proposed studies test the hypothesis that 5-HT clearance by OCT3 and SERT in BLA facilitates acquisition and consolidation of fear memory by buffering the rise of 5-HT that normally restrains BLA-mEC neuronal activation by excitatory fear memory-promoting limbic inputs. We posit that fear conditioning stimuli, which lead to fear memory, co-activate limbic and DRN 5-HT inputs to BLA along with activating the hypothalamic-pituitary-adrenal stress axis. OCT3 is potently inhibited by corticosterone, indicating that diminished OCT3 clearance allows 5-HT to rise high enough during fear conditioning to activate 5-HT receptors and effectively buffer limbic excitation of BLA-mEC neurons, decreasing their output and reducing fear memory. We will use state-of-the-art conditional gene deletion strategies to separately and collectively deplete SERT and OCT3 from DRN neurons, combined with optogenetic activation and inhibition of DRN 5-HT neurons projecting directly to BLA. AAV shRNA will be used to knockdown SERT and/or OCT3 on all cell types in BLA. These approaches will be used to determine the relative contributions of SERT and OCT3 to 1) 5-HT clearance in BLA in vivo using high-speed chronoamperometry; 2) 5-HT modulation of BLA-mEC neuronal activity using in vivo single neuron and whole-cell patch clamp recording in brain slices; 3) fear conditioning behavior. Because of their important roles in fear conditioning and 5-HT signaling in BLA, we will interrogate the functional contributions of 5-HT2A and 5-HT1A receptors in this circuit. Serotonergic neurotransmission potently modulates behavior, and its dysregulation is strongly implicated in psychiatric diseases. Proposed, discovery driven, studies will provide unprecedented mechanistic insights into the role 5-HT, and its regulation by SERT and OCT3, play specifically within the DRN-BLA-mEC fear conditioning hub.