O crack, subproduto da cocaína, introduzida na década de 80 no Brasil, é um potente estimulador do Sistema Nervoso Central (SNC), desde a sua introdução no país, verificou-se que os efeitos no convívio social e familiar do usuário são quase imediatos, levando a sociedade e o estado a terem uma preocupação maior com o seu uso e controle. Devido à magnitude dos efeitos da abstinência das substâncias psicoativas (no caso o crack), a recaída apareceu como um fator comum no cotidiano dos usuários, para analisar esse fato se propôs na década de 70 um modelo de tratamento denominado Prevenção de Recaída (PR), baseado no modelo compensatório (Terapia cognitivo-comportamental) em que não há julgamento ou atribuição de culpa ao indivíduo pelo uso da droga, mas o leva à reflexão sobre seus atos e a se responsabilizar pelas mudanças de comportamento. Assim o objetivo desse estudo foi identificar os determinantes intrapessoais e interpessoais do processo de recaída em usuários de crack, além de identificar o perfil sociodemográfico, o inicio do uso e padrão de uso de crack e outras drogas (lícitas e ilícitas). Trata-se de um estudo qualitativo de teor descritivo. A pesquisa ocorreu em um CAPS ad II em uma cidade do interior de Minas Gerais e a amostra foi de conveniência. Foram realizadas entrevistas semi- estruturas em duas etapas, com intervalo de três meses cada uma com os mesmos participantes, foi aplicada a análise de conteúdo no tratamento dos dados qualitativos. Os dados sociodemográficos concordaram com a literatura nacional; exceto no que tange a média de idade. O início do consumo de drogas ocorreu com o uso de drogas lícitas na adolescência e a relação dos usuários de crack com as demais SPA (lícitas e ilícitas) mostrou-se ambivalente, não sendo reconhecidas como \"drogas\", particularmente o álcool, percebido também como gatilho ou porta de entrada para o uso do crack, já as recaídas apareceram atreladas a fatores intrapessoais e interpessoais, sendo que eles se retroalimentam, criando um círculo vicioso. O estudo mostrou que, no atendimento ao usuário de crack deve-se trabalhar com todas as facetas do uso ou do motivo da recaída, e identificar juntamente com o usuário, os gatilhos de recaída e a rede de apoio de que dispõe, fortalecendo-o na busca de mecanismos de autocontrole do uso / The crack, a byproduct of cocaine, introduced in the 80s in Brazil, is a potent stimulator of the central nervous system (CNS), since its introduction in the country, it was found that the effects on social and family life of the user are almost immediate , leading the society and the state to have a major concern with its use and control. Due to the magnitude of the effects of abstinence l of psychoactive substances (in this case crack), relapse appeared as a common factor in the daily lives of users, to analyze this fact was proposed in the 70\'s a model of treatment called Relapse Prevention (RP) based on the compensatory model (cognitive behavioral therapy) in which there is no judgment or blaming the individual for drug use, but leads to reflection on their actions and take responsibility for behavior change. Therefore the aim of this study was to identify intrapersonal and interpersonal determinants of relapse process in crack users, and identify the sociodemographic profile, the start of use and crack cocaine use pattern and other drugs (licit and illicit). This is a qualitative study of descriptive content. The research took place in a CAPS II ad in a city in Minas Gerais and the sample was a convenience. Semi-structures interviews were conducted in two stages, with an interval of three months each with the same participants were given the content analysis in the treatment of qualitative data. Socio-demographic data agreed with the national literature; except with respect to mean age. The start of drug use occurred with the use of illicit drugs in adolescence and crack users compared with other SPA (legal and illegal) proved to be ambivalent, not being recognized as \"drugs\", particularly alcohol use has also noticed as a trigger or gateway to crack use, since relapses appeared linked to intrapersonal and interpersonal factors, and they feed back, creating a vicious circle. The study showed that, in the care of crack users must work with all aspects of the use or reason of relapse, and identify with the user, the relapse triggers and the support network available to it, strengthening it in the pursuit of self- control mechanisms usage

In the field of forensic toxicology, several challenges exist with quantification analysis of cocaine and metabolites in post mortem samples. Cocaine can prove difficult to detect and quantify in blood, urine, and soft tissues following extensive decomposition. Alternative matrices, such as hair, nails, and bone could prove useful in detecting chronic drug use in post-mortem toxicology cases. Detection and quantification of drugs in complex matrices is difficult to accomplish due to time-consuming extraction processes, and inability to detect an analyte at trace levels. Further, analysis of drugs in hard tissues, such as hair and bone, has only been attempted in recent years. Even fewer studies have investigated detection of drugs following decomposition of remains, specifically outdoor decomposition. The objective of this study was to develop a robust extraction and clean up methodology, in which a homogenization step precedes, to efficiently extract drugs from complex matrices, reach a target limit of detection (LOD) and to maintain instrument performance using multidimensional chromatography. Multi-dimension chromatography platform such as two dimensional liquid chromatography tandem mass spectrometry ( 2D-LC/MS/MS,) offers options not compatible with single dimension v units. With large volume injection capabilities of aqueous and organic extracts, the analytical process be reduced from multiple hours to minutes. All rat specimens used for this study fell under an Institutional Animal Care and Use Committee (IACUC) protocol. The rodents underwent a 10-12 weeks chronic intravenous self-administration of cocaine. This was followed by a six-week period of abstinence, followed again by a three-week period of cocaine self-administration before being euthanized. Average daily dosages for each rat fell within a range of 13-19 mg/kg. A total of 14 cocaine positive rats were placed outside and above ground in the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A) for a period of 12 months. All recoverable skeletal samples were collected for testing. Drug free control rat bones were also acquired by placing drug-free rats outdoors, above ground, until full decomposition occurred. In this study, a method analyzing cocaine and its major metabolites benzoylecgonine and ecgonine methyl ester was developed. After homogenization of whole bones, the extraction process was performed using a mixed mode reversed-phase/ion exchange sorbent. The use of a 2D LC/MS/MS technology eliminates the need for a lengthy evaporation step in the extraction method. The chosen 2D LC/MS/MS used in this application was identified using a 6x6 automated method development protocol. The manual extraction of the bone samples was completed in less than an hour. The analysis was performed using 100μL of the final organic solvent (MeOH) extracts. vi The limit of quantitation (LOQ) for cocaine and benzoylecgonine was measured at 0.05ng/g (0.05ng/mL or 50pg/g) of sample material and the LOQ for ecgonine methyl ester was measured at 0.1ng/g (0.1 ng/mL or 100pg/g). The extraction method for cocaine proved to give a linear dynamic range of 2.5 orders of magnitude (0.05 ng/g to 10ng/g with an R2 = 0.998. The micro extraction protocol combined with a multi-dimension chromatography used in this study decreased sample preparation time without sacrificing the quality seen with current single dimension chromatography techniques. The procedure developed in this study can be utilized on bone and completed in less than an hour before injection into the 2D-LC/MS/MS system.

The neural mechanisms mediating one-trial and multi-trial behavioral sensitization during early ontogeny are poorly understood. The purpose of this thesis was to assess the importance of D2-like receptors for the induction of cocaine- and methamphetamine-induced one-trial and multi-trial behavioral sensitization during the middle and late preweanling period. In a series of four experiments, rats were injected with saline or the selective dopamine D2-like receptor antagonist raclopride 15 min prior to treatment with the indirect dopamine agonists cocaine or methamphetamine. Acute control groups received two injections of saline. The pretreatment regimens occurred on either PND 16 or PND 20 (one-trial behavioral sensitization) or PND 13-16 or PND 17-20 (multi-trial behavioral sensitization). On PND 17 or PND 21, rats were challenged with either cocaine or methamphetamine and sensitized responding was assessed. With only a single exception, both one -trial and multi-trial cocaine- and methamphetamine-induced sensitization was evident on PND 17 and PND 21. Importantly, the D2-like receptor antagonist raclopride did not prevent the induction of cocaine- or methamphetamine-induced one-trial behavioral sensitization. In regards to multi-trial behavioral sensitization, raclopride failed to inhibit cocaine -induced sensitized responding on PND 17 and PND 21. Interestingly, higher doses of raclopride (0.5 and 1 mg/kg) were able to prevent the induction of multi-trial methamphetamine-induced sensitization on PND 17. Therefore, D2-like receptor antagonism differentially affected methamphetamine -induced behavioral sensitization depending on whether a one-trial or multi-trial paradigm was employed. When considered together, these results suggest that the neural mechanisms underlying the methamphetamine -induced behavioral sensitization of preweanling rats differs depending on the type of experimental paradigm (one- vs multi-trial) being used. Other potential explanations (i.e., nonspecific antagonist effects, impact of contextual conditioning, etc.) for this interesting effect are presented in the Discussion.

Major depressive disorder (MDD) is commonly diagnosed prior to adulthood, and when left untreated, may result in negative consequences that extend into adulthood. It is estimated that children and adolescents who suffer from MDD are more likely to develop conduct and anxiety disorders, and that up to 25% eventually develop substance abuse disorder. Consequently, this has resulted in a disproportionate increase in the prevalence of antidepressants prescribed to populations below 20 years of age, many of whom remain treatment-resistant. Recently, the non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist, ketamine, has been shown to alleviate symptoms of MDD in individuals that suffer from treatment-resistant depression. However, little is known about the potential long-term consequences of ketamine exposure during early development. This is important to examine at the preclinical level, given ketamine’s abuse potential, and the diffuseness of glutamatergic innervation within the reward circuit. We first demonstrate that juvenile ketamine treatment enhances preference for natural reward (i.e., 1% sucrose) three weeks post ketamine administration. In order to determine whether these long lasting increases in reward sensitivity extend to drugs of abuse, we examined whether adolescent ketamine treatment leads to an increased preference for cocaine later in life. Specifically, male c57BL/6 mice were exposed to ketamine (0 or 20 mg/kg) either during adolescence (postnatal days [PD] 35-49) or adulthood (PD70-84), and later assessed for sensitivity to cocaine (0.0, 2.5, 5.0, 7.5, or 10.0 mg/kg) place conditioning (CPP). We show that, when compared to saline pretreated controls, adult mice treated with ketamine during adolescence display an enhanced preference for environments previously paired with moderate doses of cocaine (5.0, 7.5, and 10.0 mg/kg). In contrast, mice that are pretreated with ketamine during adulthood do not show an enhanced preference for cocaine later in life. This study is the first to show that ketamine exposure during adolescence, but not adulthood, leads to a heightened preference for cocaine in adulthood. Future research is needed in order to determine to what extent ketamine exerts age-dependent functional modification of the reward circuit, and the mechanisms by which these responses are mediated.

The prefrontal cortex (PFC) is considered a critical node in the neural circuitry underlying drug-seeking behaviors. However, the mechanisms by which this region influences drug seeking and whether or not the lateral PFC mediates cocaine or heroin seeking are questions that have yet to be answered. To expand on the role of the PFC in drug seeking, rats were trained on either heroin or cocaine self-administration for a minimum of 12 days before undergoing extinction training and subsequent reinstatement tests (cued and drug-prime). All pharmacological manipulations were delivered immediately prior to reinstatement testing and were targeted at either the ventral region of the medial PFC, the infralimbic cortex (IL), the anterior portion of the medial PFC, the medial orbitofrontal cortex (mOFC), the anterior region of the insular cortex, the dorsal agranular insular cortex (AId), or the posterior region of the insular cortex, the posterior insular cortex (PIc). In chapter 1, D1 and D2 antagonists were administered into the IL and mOFC prior to cued and cocaine-prime reinstatement. Although previous studies found that the IL inhibits cocaine seeking, blocking D1 receptor activity in this region reduced cued reinstatement and had no effect on cocaine-prime reinstatement, indicating that the IL can promote cocaine seeking under certain circumstances. In contrast, blocking D1 receptors in the mOFC reduced all forms of reinstatement that were examined. Blocking D2 receptors in either region had no effect on cocaine seeking. Our data are the first to demonstrate a role for the mOFC in cocaine seeking and suggest that although the IL and mOFC lie immediately adjacent to one another, they play distinct roles in mediating cocaine seeking. In chapter 2, we pharmacologically inactivated the AId and PIc via a GABA agonist administered immediately prior to both cocaine and food seeking. Reversible inactivation of the AId reduced cued reinstatement but had no effect on cocaine-prime reinstatement. In contrast, inactivating the PIc had no effect on any form of cocaine seeking. Additionally, blocking the AId during cued and food-prime reinstatement had no effect on food seeking, indicating the role of the AId in reinstatement is specific to cocaine seeking and not general motivated behavior. Additionally, blocking CRF1 receptors in the AId blocked cued reinstatement, suggesting a possible mechanism whereby the AId is influencing cocaine seeking. These data are the first to establish a role for the AId in cocaine seeking and demonstrate that although the PIc influences alcohol and nicotine seeking, it does not mediate cocaine seeking. Chapter 3 further examined the role of the AId in cocaine seeking and expanded the influence of the insular cortex in drug seeking to heroin. AId D1 receptor blockade reduced both cued and cocaine-prime reinstatement following extinction training, whereas D2 receptor blockade had no effect on cocaine seeking. These results establish a role for the AId in cocaine-prime reinstatement, as pharmacological inactivation showed no role for the AId in cocaine-induced drug seeking. Additionally, blocking the AId during heroin seeking potentiated cued reinstatement whereas blocking the PIc during heroin seeking reduced cued reinstatement. These results demonstrate a role for the insular cortex in heroin seeking that has never been shown before and further explain how the AId may be influencing cocaine seeking.

Prenatal cocaine exposure places infants at risk for developmental delays, particularly language delays (Chapman, 2000; Lester, LaGasse, & Seifer, 1998), which increase the likelihood that they will require special education services as children and show poor developmental outcomes (Delgado, Vagi, & Scott, 2006). This study used second-order latent growth curve modeling to identify the trajectory of language development of infants from 12 to 36 months of age who had completed a three-year early intervention program. This study also investigated the predictive ability of six factors related to the child, family, and intervention. Child factors included sex, birthweight, and cognitive ability at 12 months, a family factor included mother's education level, and intervention factors included intervention type and age at enrollment. Results revealed that language growth across the two-year period was quadratic, with growth declining initially, then accelerating after 24 months, compared to the nationally normed sample upon which standardized language scores were based. Being a girl, being enrolled in the program soon after birth, and having higher cognitive ability at 12 months predicted higher initial language ability. Intervention group predicted growth in language, such that both intervention groups predicted less or no initial decline compared to the primary care non-intervention group, and the center-based intervention group showed faster acceleration after 24 months compared to the home-based intervention group. Implications of these results on the research literature and practical applications were discussed.

The positive link between children's joint attention skills and subsequent language outcomes is well established. There is also abundant evidence that responsive caregiver behaviors lead to optimal language outcomes. Though directive behaviors are generally considered detrimental to children's growth, specific types of directive behaviors which extend or build upon a child's behavior are thought to promote children's learning. No study has examined how caregiver behaviors interact with children's joint attention skills to affect subsequent language. Therefore, the objective of the present study was to examine how three different caregiver behaviors (Responsiveness, Supportiveness, and Intrusiveness) measured at 18 months moderated the relation between joint attention (also measured at 18 months) and language in two separate samples (a 24-month and a 36-month outcome sample). Intrusiveness was a significant moderator of the relation between RJA and 24-month language. RJA was a significant predictor of 24-month receptive and expressive language only in children whose caregivers had a low to moderate level of intrusiveness. Understanding the child and caregiver factors that promote or hinder children's language outcomes in children at risk for delay may help inform and target interventions that will help improve children's school readiness outcomes.

<p>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.</p> / Dissertation

Cocaine (CA) exposure during pregnancy causes long-lasting negative effects on fetal brain development and eventually results in motor dysfunction or changes in learning and memory performance. £^-amino-butyric acid (GABA) is the primary inhibitory neurotransmitter in the adult brain and undergo a switch from excitatory to inhibitory during early postnatal period. The excitatory/inhibitory switch is resulted in the relative temporal expression of K+-Cl- cotransporter 2 (KCC2). GABA is the neurotransmitter in the rat was born from excitement to inhibition and until the growth of thirty days have completely inhibitory. Here we test the effect of CA prenatal exposure on the expression of KCC2 in prefrontal cortex (recognition), hippocampus (memory), VTA (reward) and nucleus accumbens (reward). Protein expression profile of control or prenatal CA treated groups were evaluated by western blot in 2 days interval from postnatal day (PND) 8 to 30. The expression of KCC2 was time-dependently enhanced from PND 8 and reaches its maximal expression around PND 18 in prenatal CA exposure groups. The time-dependent profile of KCC2 expression in prefrontal cortex and NAc was significantly delayed in prenatal CA exposure group. We then correlate the KCC2 expression and the cocaine sensitivity by locomotor activity assay. We found group A shows a higher sensitivity to cocaine than group B in control rats. Surprisingly, group A of prenatal cocaine reduce the sensitive to cocaine to a similar extend like group B in control rats, suggesting prenatal exposure of cocaine might enhance the KCC2 expression. Furthermore, age range of A group (PND 22~27) and B group (PND 29~34) to repeated cocaine exposure resulted in up-regulation of KCC2 expression in B group earlier than A group. We found that the KCC2 expressions of repeated cocaine exposure in B group were higher than A group. In other words, in the B group, the inhibitory effect of GABA was significant and the locomotor activity was relatively slow. Therefore, the A group was more easy be cocaine addiction than B group. We next explore the signaling mechanism underlying cocaine exposure-induced KCC2 expression inhibition. Brain slices were incubated with cocaine with or without dopamine receptor antagonists and KCC2 expression was evaluated by western blot. Either SCH23390 (dopamine D1-receptor inhibitor) or eticlopride (dopamine D2-receptor inhibitor) significantly hamper the inhibition of KCC2 expression by cocaine in normal slices. However, only D2 antagonist eticlopride but not SCH23390 is effective reverse cocaine-induced KCC2 expression inhibition. Overall, results from our current studies provide a further insight into the molecular mechanism of cocaine-induced synaptic modification.

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