The tendency of users to relapse severely hinders adequate treatment of addiction. Physical and psychological stressors often contribute to difficulties in maintaining behavior change, and may play a significant role in relapse. We have previously shown that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Hence, chronic stress substantially increased BNST PACAP levels, intra-BNST PACAP infusions produced the behavioral and endocrine consequences of stressor exposure, and BNST PACAP antagonism blocked many of the consequences of chronic stress. In the present set of studies, we investigated the role of BNST PACAP in stress-induced reinstatement of cocaine seeking. Rats self-administered cocaine (3mg/ml; 0.5mg/ig/infusion, i.v.) for 1hr daily over 10 days, which was followed by extinction training in which lever pressing no longer resulted in cocaine delivery. In the first experiment we showed that intra-BNST PACAP infusion (1 μg; 0.5 μl per side) reinstated previously extinguished cocaine seeking behavior. In the second experiment intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38 (1 μg; 0.5 μl per side) blocked stress-induced reinstatement. Hence, stressor exposure (5 sec 2mA footshock) caused significant reinstatement of cocaine seeking behavior, which was blocked by intra-BNST PACAP6-38 infusion. Overall, these data suggest that BNST PACAP systems mediate stress-induced reinstatement to drug seeking. Understanding the neuropharmacology of BNST PACAP in stress-induced reinstatement and the role of PACAP systems may lead to viable targets for relapse prevention.

Rationale: Evidence indicates that drug-paired stimuli can evoke drug-craving leading to drug-seeking and repeated relapse periods can influence drug-seeking behaviors. Objectives: The present study examined (1) the effect of an interaction between repeated deprivation cycles and excitatory conditioning stimuli (CS+) on ethanol (EtOH)-seeking; (2) the effects of EtOH-paired cue-exposure in a non-drug-paired environment on subsequent conditioning in a drug-paired environment; and (3) the temporal effects of conditioned cues on subsequent EtOH-seeking. Methods: Adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS− associated with the absence of EtOH (extinction training), and a neutral stimulus (CS0) presented in a neutral non-drug-paired environment. The rats underwent four deprivation cycles or were non-deprived, following extinction they were maintained in a home cage for an EtOH-free period, and then exposed to no cue, CS+, CS−, or CS0 to assess the effect of the conditioned cues on EtOH-seeking behavior. Results: Repeated deprivations enhanced and prolonged the duration of CS+ effects on EtOH-seeking. Presentation of the CS− in a non-drug-paired environment blocked the ability of a CS+ to enhance EtOH-seeking in a drug-paired environment. Presentation of the CS+ or CS− in a non-drug-paired environment 2 or 4 h earlier significantly altered EtOH-seeking. Conclusion: Results indicated an interaction between repeated deprivation cycles and CS+ resulted in a potentiation of CS+ evoked EtOH-seeking. In addition, a CS− may have therapeutic potential by providing prophylactic protection against relapse behavior in the presence of cues in the drug-using environment.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2017-10-09T19:23:49Z No. of bitstreams: 1 DissPP.pdf: 2336243 bytes, checksum: 6713ce2a544f0c9590312b61ae159504 (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-31T13:11:51Z (GMT) No. of bitstreams: 1 DissPP.pdf: 2336243 bytes, checksum: 6713ce2a544f0c9590312b61ae159504 (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-31T13:11:59Z (GMT) No. of bitstreams: 1 DissPP.pdf: 2336243 bytes, checksum: 6713ce2a544f0c9590312b61ae159504 (MD5) / Made available in DSpace on 2017-10-31T13:16:14Z (GMT). No. of bitstreams: 1 DissPP.pdf: 2336243 bytes, checksum: 6713ce2a544f0c9590312b61ae159504 (MD5) Previous issue date: 2017-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Context-induced reinstatement of drug seeking is an animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in prelimbic cortex has been implicated in context-induced reinstatement drug seeking. Here, we report a new and feasible training procedure for context-induced reinstatement of alcohol seeking to explore the role of prelimbic cortex in context-induced relapse to alcohol seeking. Long-Evans rats were first given home-cage access to 10% ethanol. Using a saccharin fading technique, rats were first trained to self-administer 10% ethanol in one context. Next, lever pressing in the presence of the discrete cue was extinguished in a different context. Subsequently, context-induced reinstatement of drug seeking was assessed by re-exposing rats to the drug-associated or extinction context under extinction conditions. Re-exposure to the alcohol-associated context reinstated alcohol seeking and increased expression of the neural activity marker Fos in the prelimbic prefrontal cortex. The percentage of neural activation in this brain region was 3.4 % in extinction context and 7.7 % in drug associated context. Reversible inactivation of neural activity in prelimbic prefrontal cortex using GABA agonists muscimol and baclofen attenuated context-induced reinstatement. Based on previous findings of the literature that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in prelimbic cortex and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the GABAα5 gene and decrease in the AMPA receptor subunit genes GluR1 and GluR2 in only Fos-positive neurons. Our results demonstrate an important role of prelimbic cortex in context-induced reinstatement of alcohol seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons. / Uma das principais dificuldades relacionada ao tratamento da dependência de etanol é o alto índice de reincidência ao uso dessa substância. Neste sentido, a exposição ao contexto associado ao uso de etanol é a principal causa de recaída. Evidencias mostram que a recaída envolve comportamentos de aprendizado associativo. Essas associações são armazenadas por pequenos grupamentos neurais conhecidos como “neuronal ensembles”. A recaída induzida pelo contexto pode ser estudada através de um modelo animal conhecido como “ABA renewal”. Neste procedimento, ratos são treinados a autoadministrarem etanol em um determinado contexto. Após o aprendizado, os roedores são submetidos ao procedimento de extinção desse comportamento, em um ambiente com diferenças táteis, visuais e sonoras do contexto do treino. A reinstalação é avaliada através da exposição do animal ao contexto inicial. O objetivo do presente estudo foi: a) comparar o desempenho de ratos Wistar e Long-Evans no protocolo de reinstalação da autoadministração de etanol induzida pelo ambiente; b) investigar a participação do córtex pré-frontal medial nesse comportamento; c) investigar a presença de alterações na expressão gênica em “neuronal ensembles” do córtex pré-límbico, relacionadas a reinstalação da autoadministração de etanol. Observamos quea exposição ao contexto previamente associado a autoadministração de etanol foi capaz de reinstalar esse comportamento em ratos Long-Evans e Wistar. Sendo que a linhagem de ratos Long-Evans apresentou melhor desempenho no teste de reinstalação da autoadministração de etanol induzida pelo contexto quando comparado aos ratos Wistar. Demonstramos que a reinstalação da autoadministração de etanol induzida pelo contexto promoveu aumento da ativação neuronal do córtex pré-límbico. Notamos ainda, que uma pequena porcentagem de neurônios foi ativada no córtex pré-límbico durante a reinstalação da autoadministração de etanol induzida pelo contexto (~7,7%). Evidenciamos que a inibição farmacológica do córtex pré-límbico atenuou a reinstalação da autoadministração de etanol induzida pelo contexto. E por fim, encontramos que a reinstalação da autoadministração de etanol foi associada a redução na expressão de GluR1 e GluR2 (subunidades do receptor glutamatérgico AMPA) e ao aumento na expressão de GABAα5 (subunidade do receptor GABAérgico GABA A) em neurônios seletivamente ativados no córtex pré-límbico durante o teste comportamental. Concluímos que a reinstalação da autoadministração de etanol induzida pelo ambiente é mediada por alterações na expressão gênica em “neuronal ensembles” ativados pela associação entre o efeito reforçador do etanol e o contexto em que essa substância de abuso foi administrada.