Browsing by Author "Kuhn, Cynthia Moreton"

Adolescence is a behaviorally well-defined developmental period during which experimentation with illicit drugs such as marijuana is common. While the lasting effects of adolescent marijuana use have been studied in humans and in animal models, relatively little is known about the acute response to marijuana in adolescents. It is known that adolescent rats are more impaired by the psychoactive ingredient in marijuana, delta-9 tetrahydrocannabinol (THC), than adults in a water maze spatial learning task. However, what causes this greater sensitivity to THC-induced learning impairment is not understood. We characterized adolescent (postnatal day 30-35) and adult (postnatal day 70-75) rat cannabinoid CB1 receptor number, distribution, and functional coupling in the hippocampus, the brain which may be the site at which THC impairs spatial learning impairment. Next, we elucidated the time course of hippocampal CB1 receptor desensitization in adolescents and adults in response to daily treatment with 10 mg/kg THC. Finally, we characterized the development of tolerance to the learning impairment caused by THC in adolescent and adult rats by pre-treating them for five days with 10 mg/kg THC, and measuring learning performance in the Morris water maze. Our results indicate that agonist stimulation of the CB1 receptor in adolescent hippocampus produces less functional coupling to G proteins than adults. Also, adolescent hippocampal CB1 receptors desensitize less rapidly in response to 10 mg/kg THC treatment than those in adults. Finally, adolescent rats do not become tolerant to the learning impairment effects of 10 mg/kg THC after five days of pre-treatment, while adults do. We conclude that adolescents may be more impaired by THC than adults as a result of more slowly desensitizing hippocampal CB1 receptors, which may be due to

lesser functional CB1-G protein coupling in adolescents.

Adolescence is a critical period for drug addiction in humans. Most lifelong drug addiction is initiated during adolescence and the progression from initial drug use to the expression of addictive behaviors occurs more rapidly during adolescence than in adulthood. The purpose of this work was to examine if the adolescent brain uniquely responds to the addictive stimulant cocaine. This was accomplished by comparing the following measures in adolescent and adult male rats: locomotor responses to cocaine across a range of doses in two acute cocaine binge models, plasma cocaine and brain concentrations, locomotor responses to apomorphine, the relative magnitude of locomotor sensitization induced by a single high dose of cocaine (40 mg/kg), and cocaine-induced c-fos and zif268 expression. We determined that young adolescent (PN 28) rats had greater stereotypy responses to all doses of a repeated dose cocaine binge (15 mg/kg), the highest dose of an escalating dose binge (25 mg/kg), and low dose apomorphine. In addition to showing exaggerated acute locomotor responses to cocaine, young adolescents demonstrated a form of intrabinge sensitization that was absent in adults. Exaggerated adolescent locomotor responses could not be attributed to cocaine metabolism as we did not observe greater cocaine plasma or brain concentrations in adolescents compared to adults. A single high dose of cocaine (40 mg/kg) induced more ambulatory and stereotypy sensitization in young adolescents than adults. Further, the magnitude of the acute locomotor response to cocaine predicted the magnitude of locomotor sensitization in individual adolescents. We also showed that cocaine dose-dependently caused age-specific increases in the expression of the plasticity-associated immediate early genes c-fos and zif268: low dose (10 mg/kg) cocaine caused greater increases in striatal c-fos expression in adolescents whereas high dose (40 mg/kg) cocaine caused greater increases in striatal c-fos and zif268 expression in adults. Both doses of cocaine stimulated bigger increases in cortical zif268 expression in adults compared to adolescents. Finally, we demonstrated that the coordinated expression of striatal c-fos and zif268 develops during adolescence: there was no correlation between striatal c-fos and zif268 expression in individual adolescents but a strong correlation was seen in adults. The results of these experiments demonstrate that adolescents have unique molecular responses to acute cocaine and may help explain how adolescents show unique adaptive changes following continued cocaine use.

One of the outstanding goals of neuroscience is to understand how neural circuits are assembled to produce context appropriate behavior. In an ever changing environment, it is critical for animals to be able to flexibly respond to different stimuli to optimize their behavioral responses accordingly. Oviposition, or the process of choosing where to lay eggs, is an important behavior for egg-laying animals, yet the neural mechanisms of this behavior are still not completely understood. Here, we use the genetically tractable organism, Drosophila melanogaster, to investigate how the brain decides which substrates are best for egg deposition. We show that flies prefer to lay eggs away from UV light and that induction egg-laying correlates with increased movement away from UV. Both egg-laying and movement aversion of UV are mediated through R7 photoreceptors, but only movement aversion is mediated through Dm8 amacrine neurons. We then identify octopaminergic neurons as being potential modulators of egg-laying output. Collectively, this work reveals new insights into the neural mechanisms that govern Drosophila egg-laying behavior.

Sex differences in drug addiction are mediated in part by effects of the ovarian hormone estradiol (E2) within the ascending dopamine (DA) system from the midbrain to the striatum. Estradiol enhances the effects of psychostimulants, but the exact underlying mechanisms are unknown. Mice could serve as an ideal genetically-tractable model for mechanistic studies into sex and hormone effects within the DA system but have been under-utilized. This study sought to: 1) characterize psychostimulant-induced behavior in mice as an indirect but quantifiable measure of DA neurotransmission, and 2) elucidate the mechanism underlying E2's enhancement of psychostimulant effects in females using surgical, pharmacological, and genetic manipulations. The spontaneous behavior of mice during habituation to a novel environment and after the psychostimulants d-amphetamine (AMPH; 1, 2.5, and/or 5 mg/kg) and cocaine (COC; 5, 15, and/or 30 mg/kg) were assessed in open field chambers using both automated photobeam interruptions and behavioral observations. Behaviors were assessed in the following groups of mice: intact males and females; ovariectomized mice replaced with either E2 for 2 days or 30 minutes or with estrogen receptor-selective agonists; and female mice lacking either ERα (αERKO) or ERβ (βERKO) versus wildtype (WT) littermates. Brain psychostimulant concentrations and tissue content of DA and its metabolites were determined at the time of maximum behavioral stimulation. Psychostimulants induced behavioral activation in mice including both increased locomotion as detected with an automated system and a sequence of behaviors progressing from stereotyped sniffing at low doses to patterned locomotion and rearing at high doses. Intact female mice exhibited more patterned locomotion and a shift towards higher behavior scores after psychostimulants despite having lower AMPH and equivalent COC brain levels as males. Actively ovariectomized mice exhibited fewer ambulations and lower behavior scores during habituation and after psychostimulants than Sham females. Two days but not 30 minutes of E2 replacement restored COC-induced behavioral responses to Sham levels. ERα-selective PPT replacement in ovariectomized mice and genetic ablation of ERα in αERKO mice altered COC-stimulated behavior. Immunohistochemistry revealed that midbrain DA neurons in mice express ERβ but not ERα, and that non-DA cells in the midbrain and the striatum express ERα. These results indicate that E2 enhances COC-stimulated locomotion in mice through an indirect effect of ERα. ERα may alter behavior through presynaptic effects on DA neuron activity and/or through postsynaptic effects on transcription and signal transduction pathways within striatal neurons.

The appropriate evaluation of reward and punishment is critical to mammalian survival, and its disruption contributes to numerous psychiatric illnesses. Few animal models capture this tension and rely on learned fear rather than assessment of affective valence. In contrast, conditioned taste aversion (CTA) is a behavioral paradigm in which a palatable substance is paired with an aversive visceral experience, generally an injection of a nausea-inducing agent such as lithium chloride (LiCl), to produce aversion to the substance at subsequent exposure. CTA utilizes a critical decision-making mechanism for assessing the relative danger or safety of a stimulus. This process has broad implications in human psychology and mental health disorders, including anorexia nervosa, anxiety, and post-traumatic stress disorder. Although these disorders all show female predominance, understanding the neural circuits activated in CTA is fundamentally unstudied in females.The purpose of the present study was to deepen our understanding of both the behavioral outputs and neural mechanisms underlying reinforcement versus aversion in male and female rats. We used ultrasonic vocalizations (USVs) as a means of assessing affective valence. We found striking sex differences in this behavior. Males produced 55 kHz USVs, associated with a positive emotional valence, when anticipating a reward, and inhibited these calls when the reward was devalued with LiCl. Females produced 55 kHz based on their estrous cycle, but were more likely to make 22 kHz vocalizations, associated with a negative emotional valence, when anticipating the devalued stimulus. We measures c-fos response to each of the acute and conditioned stimuli during CTA. C-fos responses were similar in males and females after boost or LiCl. Females engaged the gustatory cortex more than males during the reinforcing task and males engaged the amygdala more than females in both the reinforcing and devalued tasks. We used a new technology, RNAScope, to identify a population of dopamine receptor-expressing neuronal subtypes that are activated in the process of balancing reward and aversion during CTA. We found that the central nucleus of the amygdala (CeA) responds to hedonic and aversive stimuli in a cell-specific manner. The basolateral amygdala (BLA), in contrast, did not show receptor-type cell specificity. The BLA showed a strong response in the females regardless of treatment group. In contrast, males engaged the BLA when treated with LiCl and during expression of the CTA tasks. The BLA processes affective valence and current studies suggest it is necessary for supporting motivated behavior. The present study suggests that males and females differentially process the affective valence of a stimulus to produce the same goal-directed behavior. Further studies on the mechanisms underlying these differences will broaden our understanding of how the amygdala differentially processes reward and aversion in males and females, which could result in new therapeutic targets for the treatment of a variety of neurobiological and psychiatric disorders. We also used a separate but complementary behavioral conditioning paradigm, conditioned nausea, as a model of chemotherapy-induced anticipatory nausea. Twenty-five to fifty % of chemotherapy patients will develop anticipatory nausea and vomiting (ANV), in which symptoms occur in anticipation of treatment. ANV is triggered by environmental cues and shows little response to traditional antiemetic therapy, suggesting unique neural pathways mediate this response. We used a rat model of ANV that pairs a novel context (test cage) with a high dose of the emetic drug lithium chloride (LiCl) to produced conditioned nausea behaviors in the LiCl-paired environment even in the absence of the toxin. Male and female rats were treated on days 0, 2, 4, and 6. On day 8, they were placed in the context and we measured gaping, an analogue of human vomiting. To identify brain regions associated with acute LiCl and ANV, we measured c-fos activation by immunohistochemical staining on days 0 and 8. We found both male and female rats exhibited gaping at equal rates but showed remarkable sex differences in their c-fos response. Acute LiCl activated brain regions responsive to aversive visceral stimuli, including the central nucleus of the amygdala (CeA), nucleus of the solitary tract (NTS), and area postrema (AP) that were not active during ANV. ANV activated c-fos expression in the frontal cortex and insula of males but not females. These data suggest that therapies such as ondansetron which target the AP are not effective in ANV because it is not a site activated during the ANV response. Further studies aimed at characterizing the cell types activated in the conditioned nausea response will help identify novel therapeutic targets for the treatment of this condition, improving both quality of life and outcomes for patients undergoing chemotherapy.

Fibroblast growth factor homologous factors (FHFs) are non-canonical members of the fibroblast growth factor family (FGF11-14) that were initially discovered to bind and regulate neuronal and cardiac voltage-gated Na+ channels. Loss-of-function mutations that disrupt interaction between FHFs and Na+ channels cause spinocerebellar ataxias and cardiac arrhythmias such as Brugada syndrome. Although recent studies in brain of FHF knockout mice suggested novel functions for FHFs beyond ion channel modulation, it is unclear whether FHFs in the heart serve additional roles beyond regulating cardiac excitability. In this study, we performed a proteomic screen to identify novel interacting proteins for FGF13 in mouse heart. Mass spectrometry analysis revealed an interaction between FGF13 and a complex of cavin proteins that regulate caveolae, membrane invaginations that organize protective signaling pathways and provide a reservoir to buffer membrane stress. FGF13 controls the relative distribution of cavin 1 between the plasma membrane and cytosol and thereby acts as a negative regulator of caveolae. In inducible, cardiac-specific Fgf13 knockout mice, cavin 1 redistributed to the plasma membrane and stabilized the caveolar structural protein caveolin 3, leading to an increased density of caveolae. In a transverse aortic constriction model of pressure overload, this increased caveolar abundance enhanced cardioprotective signaling through the caveolar-organized PI3 kinase pathway, preserving cardiac function and reducing fibrosis. Additionally, the increased caveolar reserve provided mechanoprotection, as indicated by reduced membrane rupture in response to hypo-osmotic stress. Thus, our results establish FGF13 as a novel regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling, and suggest that inhibition of FHFs in the adult heart may have cardioprotective benefits in the setting of maladaptive hypertrophy.

Infections are most commonly identified by microscopy, culturing the organism, or testing the patients blood for antigens or antibodies. These methods are unreliable in bacteria that persist in a non-dividing, metabolically inactive dormant state, leading to treatment delays and an increased risk of developing chronic morbidities. Borrelia burgdorferi (B. burgdorferi), the causative spirochete in Lyme Borreliosis, is an example of a stealth pathogen difficult to culture from blood, capable of evading the host immune system, and under adverse growth conditions in host tissue, can survive in a dormant state. Despite early diagnosis and treatment, 20-35% of patients with Lyme Borreliosis experience chronic symptoms, the etiology of which remains unknown due to the lack of accurate diagnostics to demonstrate the presence of a persistent infection. In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we characterize HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G) within B. burgdorferi, the bacteria responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen, potentially solving both the problem of diagnosis and treatment.

Dopamine regulates movement, cognition and the rewarding effects of addictive drugs. Sex differences mediated by gonadal hormones affect each of these processes. An extensive literature suggests that estrogen augments dopaminergic function. Our laboratory found that female rats exhibit increased locomotor stimulation in response to cocaine and greater cocaine-induced dopamine overflow compared to males, sex differences that emerge in early adulthood. Currently, the underlying mechanisms for these differences are poorly understood. I hypothesized that female rats would have more dopamine neurons in midbrain regions and that ovarian hormones would exert trophic effects on dopamine neurons. Immunohistochemical and stereological techniques were used to quantitate the number of cells in the SNpc and VTA of male and female rats and mice to assess: (1) if sex differences in dopamine neuron number exist and when they emerge, (2) how gonadal hormones influence dopaminergic cell number and dopamine-mediated behaviors (3) the role of specific hormone receptors in the effects on cell number (4) the possibility that dopamine neuron number is directly linked to cocaine-stimulated behavior and electrically-stimulated dopamine release and that these responses to cocaine are mediated through gonadal hormone modulation of midbrain dopamine neuron number. I discovered sex differences in midbrain dopamine neuron number; adult female rodents have more neurons in the SNpc and VTA. We also found that gonadectomy in adulthood reduced midbrain dopamine neuron number in females and increased neuron number in males, establishing the trophic effects of estrogen in the intact midbrain and possible suppressive effects of androgens. Treatment with agonists for estrogen receptor subtypes alpha and beta and androgen receptor reversed the effects of gonadectomy on cell number in females and males, respectively. In an effort to bridge cocaine-stimulated behavior and cell number in sham ovariectomized and ovariectomized females, we discovered cocaine-stimulated behavior, dopamine release and SNpc cell density were positively correlated in intact female rats, an effect that is lost with ovariectomy. This dissertation demonstrates that estrogen is critical for the maintenance of dopaminergic cell populations that enhance behavioral responses to psychostimulants in females, thereby contributing to the observed sex differences.

Adolescence is the period of transition between childhood and adulthood, and is characterized across mammalian species by changes in behavior that include increases in risk taking, novelty/sensation seeking, and social behavior. Immaturity of the central serotonergic system during adolescence could contribute to risk taking behavior by resulting in lower avoidance of aversive stimuli in adolescents than adults. The purpose of this dissertation was to investigate whether immature serotonergic function could contribute to adolescent risk taking. We studied pre- and postsynaptic serotonergic function and circuit-level mechanisms relevant to risk taking behavior using behavioral and neurochemical approaches.

Serotonergic modulation of behavior was assessed in adult (67-74 day old) and adolescent (28-34 day old) male rats in the novelty induced hypophagia (NIH), elevated plus maze, (EPM), and light/dark (LD) tests for anxiety-like behavior. Serotonin depletion with the synthesis inhibitor p-chlorophenylalanine (PCPA) produced anxiolytic effects only in adult rats in the NIH test and in both age groups in the EPM. These data showed that some serotonin-mediated behavioral inhibition is present during adolescence. However, adolescent rats were less sensitive than adults to the anxiogenic effects of the serotonin releasing drugs fenfluramine and methylenedioxymethamphetamine (MDMA) and the serotonin uptake inhibitor fluoxetine in the LD test, suggesting that serotonin is not as effective at inhibiting behavior in adolescents as it is in adults.

Microdialysis conducted in medial prefrontal cortex (mPFC) showed that adolescent rats exhibited lower increases in extracellular serotonin after treatment with the releasing drug fenfluramine, but not the uptake inhibitor fluoxetine. Further investigation of presynaptic serotonin function in adults and adolescents revealed that adolescent rats have lower tissue serotonin content than adults in several forebrain regions, but similar rates of serotonin synthesis, density of serotonin transporter (SERT)-immunoreactive innervation, and SERT radioligand binding. These data suggest that adolescents may have a lower increase in extracellular serotonin than adults after a releasing drug, but not an uptake inhibitor, due to lower tissue serotonin stores. Lower serotonin stores may limit the ability of a releasing drug to increase extracellular serotonin, but are unlikely to affect response to an uptake inhibitor. These findings also indicate that extracellular serotonin does not completely account for lower serotonin-mediated behavioral inhibition in adolescents.

Since presynaptic serotonin function did not explain age differences in the anxiogenic effects of indirect serotonin agonists, we investigated postsynaptic serotonin signaling by testing the behavioral effects of serotonin receptor agonists in the LD test. Adolescent rats were less sensitive than adults to the anxiogenic effects of the 5-HT_1A agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH DPAT) in the LD test, but not to the 5-HT₂ agonist meta-chlorophenylpiperazine (mCPP). No age differences were observed in 3H-8-OH DPAT binding in prefrontal cortex, amygdala, or hippocampus between adolescents and adults, and infusion of 8-OH DPAT into mPFC (prelimbic cortex), ventral hippocampus, or basolateral amygdala was unable to replicate the systemic effects of 8-OH DPAT. These data suggest that lower adolescent sensitivity to the anxiogenic effects of 8-OH DPAT is not due to age differences in receptor expression, and show that 5-HT_1A stimulation in mPFC, ventral hippocampus, and basolateral amygdala alone is not sufficient to mimic the effects of systemic 8-OH DPAT.

We tested the circuit-level effects of fluoxetine and 8-OH DPAT, since stimulating 5-HT_1A receptors in single brain regions failed to reproduce age differences in systemic 8-OH DPAT administration. Both drugs activated regions of the amygdala more in adults than adolescents, and 8-OH DPAT also produced greater prefrontal cortical activation in adults. Fluoxetine produced greater expression of the immediate early gene c-Fos in regions of the extended amygdala in adult rats, and 8-OH DPAT produced greater activation of the lateral orbital cortex and central amygdala in adult rats. Lower activation of cortical and amygdala brain regions could underlie the lower behavioral effects of these drugs in adolescents, as these brain regions are important in mediating behavioral inhibition and anxiety-like behavior. These data are also consistent with human studies showing immature cortical and amygdala function during adolescence.

This dissertation shows that adolescents are less sensitive than adults to serotonin mediated behavioral inhibition, and that this may be due to immature activation of neural circuits modulated by the 5-HT_1A receptor between the prefrontal cortex and amygdala. This immature serotonin mediated behavioral inhibition could contribute to adolescent risk taking, drug abuse, and increased risk for suicidality during SSRI therapy for depression and mood disorders.

Herein I describe studies I have undertaken that were aimed at understanding the mechanisms of achieving β-arrestin-biased signaling at the dopamine D2 receptor (D2R), methods for studying downstream mediators of β-arrestin-biased signaling, and the development of a mouse model of schizophrenia that could test the efficacy of β-arrestin-biased D2R ligands. Each of these studies will improve our understanding of how to better tailor drugs to treat schizophrenia. I have employed a wide variety of in vitro and in vivo methods ranging from bioluminescent resonance energy transfer (BRET) to adeno-associated viral delivery of neuronal actuators. Ultimately, I was able to demonstrate that the D2R can achieve β-arrestin biased signaling through its ability to directly recruit the G protein-coupled receptor kinase 2 (GRK2). Next, I developed a BRET-based approach to study interactions of GPCR-β-arrestin complexes and applied this to the D2R. Finally, I have laid the ground work for a mouse model of schizophrenia capable of generating dopamine circuit imbalances hypothesized to occur in schizophrenia as a means to test β-arrestin-biased D2R ligands.