Genetic and pharmacological investigation of α4-containing GABAA receptors in conditioned behaviours influenced by cocaine

α4-subunit containing GABAA receptors (α4-GABAARs) are often found co-assembled with δ-subunits in extrasynaptic locations on nucleus accumbens (NAc) medium spiny neurons (MSNs), were they mediate a tonic form of inhibition thought to control the excitability of the neuron. This thesis combines genetic and pharmacological techniques to explore the role of α4-GABAARs in locomotor and reward-conditioned behaviours. Activation of α4-GABAARs by systemic or intra-accumbal administration of THIP, a GABAAR agonist with a preference for δ-subunits, was able to reduce cocainepotentiated locomotor activity in wildtype but not GABAAR α4-subunit knockout mice. Similarly, the ability of repeated cocaine to induce behavioural sensitisation was unaffected in GABAAR α4-subunit knockout mice, but systemic THIP was able to reduce the sensitised increase in locomotor activity in wildtype but not knockout mice. α4-GABAARs are also able to modulate behavioural responses to reward-conditioned stimuli and their enhancement by cocaine. Deletion of GABAAR α4-subunits from dopamine D1-expressing neurons facilitated cocaine-CPP, and activation of α4- GABAARs on NAc D1-MSNs was able to attenuate cocaine-enhancement of cocaine CPP. Conversely, deletion of GABAAR α4-subunits from dopamine D2-expressing neurons increased CRf responding, and activation of α4-GABAARs on NAc D2-MSNs was able to attenuate cocaine-potentiation of CRf responding. These data also indicate that there is a dissociation in the NAc MSNs mediating cocaine-CPP and CRf responding. This may be explained by the different glutamatergic inputs needed to provide information about conditioned cues important for these behaviours. The data presented within this thesis indicate that α4-GABAAR-mediated inhibition of D1- and D2-expressing neurons plays an important physiological role in controlling behavioural responses to conditioned cues. Furthermore, NAc α4βδ GABAARs may provide a potential therapeutic target by which to limit the enhancement of locomotor and conditioned-behaviours by cocaine

Valence in the Nucleus Accumbens - identifying neural populations in appetitive and aversive responses

Dissertação de mestrado em Ciências da SaúdeO núcleo accumbens (NAc) é reconhecido como um componente essencial do circuito de recompensa, estando associado ao processamento de eventos recompensadores e aversivos e contribuindo para comportamentos motivados. O NAc é uma “interface límbico-motora”, e estudos mostram o seu envolvimento em codificar valência - o valor intrínseco de uma certa experiência e consequentes respostas emocionais e motivacionais. O NAc é constituído maioritariamente por neurónios espinhosos médios GABAérgicos (MSNs), divididos naqueles que expressam o recetor de dopamina D1 (D1-MSNs) e nos que expressam o recetor de dopamina D2 (D2-MSNs). Estas populações foram tradicionalmente segregadas anatomicamente (via direta vs indireta) e funcionalmente (recompensa e valência positiva vs aversão e valência negativa). No entanto, muitos estudos recentes desafiaram esta segregação simplista. Porém, é ainda incerto quais as populações neuronais que codificam valência no NAc, e esta é uma questão crucial na compreensão de distúrbios com défices emocionais. Nesta dissertação, avaliámos os níveis de ativação neuronal associados com estímulos de valência negativa (choque na pata) ou positiva (cocaína) no NAc, usando a amígdala basolateral (BLA) e central (CeA) como regiões controlo, usadas por codificarem valência. Também caracterizámos um vetor viral controlado por c-fos para estabelecer uma estratégia de marcação de ativação neuronal para trabalho futuro. Além disso, efetuámos ativação optogenética de neurónios responsivos a estímulos na BLA, usando o mesmo vetor, para induzir comportamentos de valência e validar a nossa metodologia. Os nossos dados mostram que o NAc core (NAcc) e a BLA contêm populações neuronais que respondem ao choque, medido por uma maior densidade de células c-fos+ em comparação os controlos. Porém, a cocaína não induziu alterações significativas de ativação neuronal. Quanto ao vetor (conduzindo expressão de channelrhodopsin-eYFP), determinámos que 16h pós-exposição a estímulo seria o período mais adequado para observar marcação viral. Ao usar este vetor e expor murganhos a estímulos positivos ou negativos, dados de densitometria de fluorescência mostraram apenas uma tendência para maior ativação neuronal na BLA após o choque na pata, sem efeitos no NAc, em comparação com animais controlo; sem efeitos devido à cocaína. Por último, ativação optogenética de neurónios responsivos a choque na pata da BLA induziu uma tendência de evasão num teste de preferência de lugar em tempo real (RTPP), contudo essa mesma ativação não induziu preferência de lugar condicionada (CPP). Embora os nossos dados mostrem ativação neuronal no NAcc e BLA em resposta a choque, a falta de diferenças na exposição a cocaína, junto com dados de densitometria e optogenética, indicam a necessidade de desenvolver novas ferramentas para marcar neurónios que codifiquem valência no NAc.The nucleus accumbens (NAc) is recognized as an essential component of the reward circuit, being associated with processing of both rewarding and aversive events, and contributing for motivated behaviours. The NAc is a “limbic-motor interface”, and evidence shows its involvement in valence encoding – the intrinsic value of a given experience and consequent emotional and motivational responses. The NAc is mainly constituted by GABAergic medium spiny neurons (MSNs), divided into those expressing dopamine receptor D1 (D1-MSNs) and those expressing dopamine receptor D2 (D2-MSNs). These populations have been traditionally segregated anatomically (direct vs indirect pathway) and functionally (reward and positive valence vs aversion and negative valence). However, many recent studies have challenged this simplistic segregation. Yet, it is still uncertain which neuronal populations encode valence in the NAc, and this is a crucial question in the understanding of disorders with emotional deficits. In this thesis work we evaluated the neuronal activation levels associated with negative- (footshock) or positive-valence stimuli (cocaine) in the NAc, using the basolateral (BLA) and central (CeA) amygdala as control regions, used because they encode valence. We also characterized a c-fos-driven viral vector in order to establish a neuronal activation labelling strategy for future work. Furthermore, we performed optogenetic activation of stimulus-responsive neurons in the BLA, using the same vector, to induce valence-specific behavioural responses and validate our methodology. Our data shows that the NAc core (NAcc) and the BLA contain neuronal populations that respond to shock, measured by a higher c-fos+ cell density in comparison to controls. However, cocaine induced no significant changes in neuronal activation. Regarding the vector (driving channelrhodopsin-eYFP expression), we found that 16h-post stimulus exposure would be the more adequate timeframe to observe neuronal labelling. When using this vector and exposing mice to positive or negative stimuli, densitometry fluorescence data showed only a tendency for higher neuronal activation in the BLA after footshock, with no effects in the NAc, in comparison with control animals; with no effects due to cocaine. Lastly, optogenetic activation of footshock-responsive neurons in the BLA induced a tendency for avoidance in a real time place preference test (RTPP), yet this same activation did not induce conditioned place preference (CPP). While our data show neuronal activation in the NAcc and BLA in response to shock, the lack of differences with cocaine exposure together with densitometry and optogenetics data, indicates the need to develop new tools to label valence encoding neurons in the NAc.O apoio financeiro foi prestado através de bolsas da Fundação Bial através dos projetos 30/2016 e 175/20; do Fundo Europeu de Desenvolvimento Regional (FEDER), no âmbito do projeto PTDC/MED-NEU/29071/2017 (REWSTRESS), através do COMPETE 2020; da Fundação “La Caixa” (ID100010434; LCF/PR/HR20/52400020); de fundos europeus da European Research Council (ERC) – ERC Consolidator “VALENCE”, No 101003187; da Plataforma de Microscopia Científica do ICVS, membro da PPBI - Portuguese Platform of Bioimaging (PPBI -POCI-01-0145-FEDER-022122); e por Fundos Nacionais, por meio da Fundação para a Ciência e Tecnologia (FCT) - projetos UIDB/50026/2020 e UIDP/50026/2020

Homeostasis Meets Motivation in the Battle to Control Food Intake.

Signals of energy homeostasis interact closely with neural circuits of motivation to control food intake. An emerging hypothesis is that the transition to maladaptive feeding behavior seen in eating disorders or obesity may arise from dysregulation of these interactions. Focusing on key brain regions involved in the control of food intake (ventral tegmental area, striatum, hypothalamus, and thalamus), we describe how activity of specific cell types embedded within these regions can influence distinct components of motivated feeding behavior. We review how signals of energy homeostasis interact with these regions to influence motivated behavioral output and present evidence that experience-dependent neural adaptations in key feeding circuits may represent cellular correlates of impaired food intake control. Future research into mechanisms that restore the balance of control between signals of homeostasis and motivated feeding behavior may inspire new treatment options for eating disorders and obesity

J Neurochem

Substance use disorder (SUD) is characterized, in part by behavior biased toward drug use and away from natural sources of reward (e.g., social interaction, food, sex). The neurobiological underpinnings of SUDs reveal distinct brain regions where neuronal activity is necessary for the manifestation of SUD-characteristic behaviors. Studies that specifically examine how these regions are involved in behaviors motivated by drug versus natural reward allow determinations of which regions are necessary for regulating seeking of both reward types, and appraisals of novel SUD therapies for off-target effects on behaviors motivated by natural reward. Here, we evaluate studies directly comparing regulatory roles for specific brain regions in drug versus natural reward. While it is clear that many regions drive behaviors motivated by all reward types, based on the literature reviewed we propose a set of interconnected regions that become necessary for behaviors motivated by drug, but not natural rewards. The circuitry is selectively necessary for drug seeking includes an Action/Reward subcircuit, comprising nucleus accumbens, ventral pallidum, and ventral tegmental area, a Prefrontal subcircuit comprising prelimbic, infralimbic, and insular cortices, a Stress subcircuit comprising the central nucleus of the amygdala and the bed nucleus of the stria terminalis, and a Diencephalon circuit including lateral hypothalamus. Evidence was mixed for nucleus accumbens shell, insular cortex, and ventral pallidum. Studies for all other brain nuclei reviewed supported a necessary role in regulating both drug and natural reward seeking. Finally, we discuss emerging strategies to further disambiguate the necessity of brain regions in drug- versus natural reward-associated behaviors.I01 BX004727/BX/BLRD VAUnited States/P20GM121310/University of Wyoming/DA012513/DA/NIDA NIH HHSUnited States/DP5 OD026407/OD/NIH HHSUnited States/DA003906/DA/NIDA NIH HHSUnited States/BX004727/U.S. Department of Veterans Affairs/DA046373/DA/NIDA NIH HHSUnited States/U01 DA045300/DA/NIDA NIH HHSUnited States/P20 GM121310/GM/NIGMS NIH HHSUnited States/R00 DA046522/DA/NIDA NIH HHSUnited States/R37 DA003906/DA/NIDA NIH HHSUnited States/P50 DA046373/DA/NIDA NIH HHSUnited States/R01 DA012513/DA/NIDA NIH HHSUnited States/K99 DA046522/DA/NIDA NIH HHSUnited States/R01 DA003906/DA/NIDA NIH HHSUnited States/DP5 OD026407/CD/ODCDC CDC HHSUnited States/DA046522/DA/NIDA NIH HHSUnited States/2022-06-01T00:00:00Z33420731PMC817815911425vault:3712

The Nuclear Transcription Factor CREB: Involvement in Addiction, Deletion Models and Looking Forward

Addiction involves complex physiological processes, and is characterised not only by broad phenotypic and behavioural traits, but also by ongoing molecular and cellular adaptations. In recent years, increasingly effective and novel techniques have been developed to unravel the molecular implications of addiction. Increasing evidence has supported a contribution of the nuclear transcription factor CREB in the development of addiction, both in contribution to phenotype and expression in brain regions critical to various aspects of drug-seeking behaviour and drug reward. Abstracting from this, models have exploited these data by removing the CREB gene from the developing or developed mouse, to crucially determine its impact upon addiction-related processes. More recent models, however, hold greater promise in unveiling the contribution of CREB to disorders such as addiction

The ventral basal ganglia, a selection mechanism at the crossroads of space, strategy, and reward

The basal ganglia are often conceptualised as three parallel domains that include all the constituent nuclei. The ‘ventral domain’ appears to be critical for learning flexible behaviours for exploration and foraging, as it is the recipient of converging inputs from amygdala, hippocampal formation and prefrontal cortex, putatively centres for stimulus evaluation, spatial navigation, and planning/contingency, respectively. However, compared to work on the dorsal domains, the rich potential for quantitative theories and models of the ventral domain remains largely untapped, and the purpose of this review is to provide the stimulus for this work. We systematically review the ventral domain’s structures and internal organisation, and propose a functional architecture as the basis for computational models. Using a full schematic of the structure of inputs to the ventral striatum (nucleus accumbens core and shell), we argue for the existence of many identifiable processing channels on the basis of unique combinations of afferent inputs. We then identify the potential information represented in these channels by reconciling a broad range of studies from the hippocampal, amygdala and prefrontal cortex literatures with known properties of the ventral striatum from lesion, pharmacological, and electrophysiological studies. Dopamine’s key role in learning is reviewed within the three current major computational frameworks; we also show that the shell-based basal ganglia sub-circuits are well placed to generate the phasic burst and dip responses of dopaminergic neurons. We detail dopamine’s modulation of ventral basal ganglia’s inputs by its actions on pre-synaptic terminals and post-synaptic membranes in the striatum, arguing that the complexity of these effects hint at computational roles for dopamine beyond current ideas. The ventral basal ganglia are revealed as a constellation of multiple functional systems for the learning and selection of flexible behaviours and of behavioural strategies, sharing the common operations of selection-by-disinhibition and of dopaminergic modulation

The Winding Road to Relapse: Forging a New Understanding of Cue-Induced Reinstatement Models and Their Associated Neural Mechanisms

In drug addiction, cues previously associated with drug use can produce craving and frequently trigger the resumption of drug taking in individuals vulnerable to relapse. Environmental stimuli associated with drugs or natural reinforcers can become reliably conditioned to increase behavior that was previously reinforced. In preclinical models of addiction, these cues enhance both drug self-administration and reinstatement of drug seeking. In this review, we will dissociate the roles of conditioned stimuli as reinforcers from their modulatory or discriminative functions in producing drug-seeking behavior. As well, we will examine possible differences in neurobiological encoding underlying these functional differences. Specifically, we will discuss how models of drug addiction and relapse should more systematically evaluate these different types of stimuli to better understand the neurobiology underlying craving and relapse. In this way, behavioral and pharmacotherapeutic interventions may be better tailored to promote drug use cessation outcomes and long-term abstinence

Involvement of the endogenous opioid and cannabinoid systems in addictive like behaviours

Grau académico estrangeiro - BiomedicinaABSTRACT: The increase incidence of obesity and eating disorders represents a major health problem in developed countries. The low rate of success of treatments to prevent or reverse obesity, and overeating that causes it, highlights the important behavioural alterations that are associated to this disease. These alterations seem to be mediated by modifications in the reward circuits that mimic changes occurring during addictive behaviour. Moreover, like drugs of abuse, obesity is associated with abnormal intake habits when maintaining diet that can endure vulnerability to relapse. In the present thesis, we have first investigated the involvement of the endogenous opioid system in the neurobiological mechanism underlying drug and food reinstatement, as a potential therapeutic target in these behavioural disorders. Moreover, we have investigated the relationships between overeating and behavioural addiction. Indeed, we have demonstrated that repeated operant training with palatable food promotes behavioural alterations, as well as epigenetic, proteomic and structural plasticity changes in the reward circuit reminiscent to those observed with drugs of abuse. Finally, we identified the cannabinoid receptor 1 and the delta opioid receptor as common neurobiological substrates underlying these alterations.RESUMEN: El aumento de la incidencia de la obesidad y de los trastornos de la alimentación representa un importante problema de salud en los países desarrollados. La baja tasa de éxito de los tratamientos disponibles para prevenir o revertir la obesidad y el fácil acceso a la comida obesogenica que lo causa, destacan la necesidad de encentrar dianas terapéuticas eficaces. Las importantes alteraciones conductuales que se asocian a esta enfermedad parecen estar mediadas por modificaciones en los circuitos de recompensa que imitan los cambios que ocurren durante el comportamiento adictivo. Por otra parte, al igual que las drogas de abuso, la obesidad se asocia con hábitos de ingesta anormales que pueden incrementar la vulnerabilidad a la recaída de búsqueda de comida. En la presente tesis, hemos investigado primero la implicación del sistema opioide endógeno en el mecanismo neurobiológico que subyace a la recaída del comportamiento de búsqueda de drogas y comida como una posible diana terapéutica en estos trastornos del comportamiento. En segundo lugar, hemos investigado las relaciones entre la sobre ingesta de comida palatable y la adicción conductual. De hecho, hemos demostrado que el entrenamiento operante repetido con comida palatable promueve alteraciones de la conducta, así como cambios epigenéticos, proteómicos y de plasticidad estructural en el circuito de la recompensa que recuerdan a los observados con las drogas de abuso. Es destacable señalar que hemos identificado el receptor cannabinoide 1 y el receptor delta opioide como sustratos neurobiológicos comunes que subyacen a estas alteraciones

Assessing the role of ghrelin and the enzyme ghrelin O-acyltransferase (GOAT) system in food reward, food motivation, and binge eating behavior.

Abstract The peripheral peptide hormone ghrelin is a powerful stimulator of food intake, which leads to body weight gain and adiposity in both rodents and humans. The hormone, thus, increases the vulnerability to obesity and binge eating behavior. Several studies have revealed that ghrelin's functions are due to its interaction with the growth hormone secretagogue receptor type 1a (GHSR1a) in the hypothalamic area; besides, ghrelin also promotes the reinforcing properties of hedonic food, acting at extra-hypothalamic sites and interacting with dopaminergic, cannabinoid, opioid, and orexin signaling. The hormone is primarily present in two forms in the plasma and the enzyme ghrelin O-acyltransferase (GOAT) allows the acylation reaction which causes the transformation of des-acyl-ghrelin (DAG) to the active form acyl-ghrelin (AG). DAG has been demonstrated to show antagonist properties; it is metabolically active, and counteracts the effects of AG on glucose metabolism and lipolysis, and reduces food consumption, body weight, and hedonic feeding response. Both peptides seem to influence the hypothalamic–pituitary–adrenal (HPA) axis and the corticosterone/cortisol level that drive the urge to eat under stressful conditions. These findings suggest that DAG and inhibition of GOAT may be targets for obesity and bingeing-related eating disorders and that AG/DAG ratio may be an important potential biomarker to assess the risk of developing maladaptive eating behaviors