Distinct engrams control fear and extinction memory.

Memories are stored in engram cells, which are necessary and sufficient for memory recall. Recalling a memory might undergo reconsolidation or extinction. It has been suggested that the original memory engram is reactivated during reconsolidation so that memory can be updated. Conversely, during extinction training, a new memory is formed that suppresses the original engram. Nonetheless, it is unknown whether extinction creates a new engram or modifies the original fear engram. In this study, we utilized the Daun02 procedure, which uses c-Fos-lacZ rats to induce apoptosis of strongly activated neurons and examine whether a new memory trace emerges as a result of a short or long reactivation, or if these processes rely on modifications within the original engram located in the basolateral amygdala (BLA) and infralimbic (IL) cortex. By eliminating neurons activated during consolidation and reactivation, we observed significant impacts on fear memory, highlighting the importance of the BLA engram in these processes. Although we were unable to show any impact when removing the neurons activated after the test of a previously extinguished memory in the BLA, disrupting the IL extinction engram reactivated the aversive memory that was suppressed by the extinction memory. Thus, we demonstrated that the IL cortex plays a crucial role in the network involved in extinction, and disrupting this specific node alone is sufficient to impair extinction behavior. Additionally, our findings indicate that extinction memories rely on the formation of a new memory, supporting the theory that extinction memories rely on the formation of a new memory, whereas the reconsolidation process reactivates the same original memory trace.

Involvement of cortical projections to basolateral amygdala in context-induced reinstatement of ethanol-seeking in rats.

Ethanol is the most consumed substance of abuse in the world, and its misuse may lead to the development of alcohol use disorder (AUD). High relapse rates remain a relevant problem in the treatment of AUD. Exposure to environmental cues previously associated with ethanol intake could trigger ethanol-seeking behavior. However, the neural mechanisms involved in this phenomenon are not entirely clear. In this context, cortical projections to the basolateral amygdala (BLA) play a role in appetitive and aversive learned behaviors. Therefore, we aimed to evaluate the activation of the cortical projections from the prelimbic (PL), orbitofrontal (OFC), and infralimbic (IL), to the BLA in the context-induced reinstatement of ethanol-seeking. Male Long-Evans rats were trained to self-administer 10% ethanol in Context A. Subsequently, lever pressing in the presence of the discrete cue was extinguished in Context B. After nine extinction sessions, rats underwent intracranial surgery for the unilateral injection of red fluorescent retrograde tracer into the BLA. The context-induced reinstatement of ethanol-seeking was assessed by re-exposing the rats to Context A or B under extinction conditions. Finally, we combined retrograde neuronal tracing with Fos to identify activated cortical inputs to BLA during the reinstatement of ethanol-seeking behavior. We found that PL, but not OFC or IL, retrogradely-labeled neurons from BLA presented increased Fos expression during the re-exposure to the ethanol-associated context, suggesting that PL projection to BLA is involved in the context-induced reinstatement of ethanol-seeking behavior.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Involvement of the ventral, but not dorsal, hippocampus in anxiety-like behaviors in mice exposed to the elevated plus maze: participation of CRF1 receptor and PKA pathway.

BACKGROUND: The hippocampus is a limbic structure involved in anxiety-like behaviors. We aimed to evaluate the role of the dorsal (DH) and ventral (VH) hippocampus in anxiety-like behaviors in the elevated plus maze (EPM). METHODS: We inhibited these brain regions using cobalt chloride (CoCl2: 1.0 nmol) microinjections. We also investigated the involvement of corticotropin-releasing factor (CRF) action and protein kinase A (PKA) pathway using intra-DH and intra-VH microinjections of the CRF1 receptor antagonist CP376395 (0, 3.0, or 6.0 nmol) and the PKA inhibitor H-89 (0, 2.5, or 5.0 nmol). RESULTS: The results indicated that intra-VH CoCl2 microinjection increased the percentage of time spent and entries in the open arms. The mice also exhibited fewer stretch attend postures in the protected area and increased percentage of open arm entries. Further, intra-VH injection of 3.0 nmol CP376395 increased time spent in the open arms. Intra-DH injection of 6.0 nmol CP376395 increased the frequency of unprotected head dipping, whereas intra-VH injection of 6 nmol CP376395 increased the frequency of protected head dipping. Intra-VH, but not intra-DH, microinjection of 2.5 nmol H-89 increased the percentages of open arm entries and time spent in the open arms. Microinjection of 2.5 and 5.0 nmol H-89 reduced the frequency of protected head dipping behavior. CONCLUSIONS: This study demonstrated that VH modulates anxiety-like behaviors in EPM. Moreover, CRF and the cAMP/PKA pathway seem to modulate these effects.

Discovery of Potent, Reversible, and Competitive Cruzain Inhibitors with Trypanocidal Activity: A Structure-Based Drug Design Approach.

A virtual screening conducted with nearly 4 000 000 compounds from lead-like and fragment-like subsets enabled the identification of a small-molecule inhibitor (1) of the Trypanosoma cruzi cruzain enzyme, a validated drug target for Chagas disease. Subsequent comprehensive structure-based drug design and structure-activity relationship studies led to the discovery of carbamoyl imidazoles as potent, reversible, and competitive cruzain inhibitors. The most potent carbamoyl imidazole inhibitor (45) exhibited high affinity with a Ki value of 20 nM, presenting both in vitro and in vivo activity against T. cruzi. Furthermore, the most promising compounds reduced parasite burden in vivo and showed no toxicity at a dose of 100 mg/kg. These carbamoyl imidazoles are structurally attractive, nonpeptidic, and easy to prepare and synthetically modify. Finally, these results further advance our understanding of the noncovalent mode of inhibition of this pharmaceutically relevant enzyme, building strong foundations for drug discovery efforts.

Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies.

BACKGROUND: Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise. METHODS: The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed. RESULTS: A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively). CONCLUSIONS: The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

Functional inactivation of the orbitofrontal cortex disrupts context-induced reinstatement of alcohol seeking in rats.

BACKGROUND: The high rate of relapse to drug use remains a central challenge to treating drug addiction. In human and rat models of addiction, environmental stimuli in contexts associated with previous drug use can provoke a relapse of drug seeking. Pre-clinical studies have used the ABA renewal procedure to study context-induced reinstatement of drug seeking. In the current study, we studied the role of the orbitofrontal cortex (OFC) in context-induced reinstatement to alcohol. METHODS: We trained male and female rats to self-administer alcohol in context A, extinguished drug-reinforced responding in a distinct context B, and assessed context-induced reinstatement in context A or B (control group). Next, we determined the effect of context-induced renewal of alcohol-seeking behavior on the expression of Fos (a neuronal activity marker) in the OFC. Finally, we determined the effect of reversible inactivation by GABAa and GABAb receptor agonists (i.e., muscimol and baclofen, respectively) in the OFC. RESULTS AND CONCLUSIONS: There were no differences between male and female rats in context-induced reinstatement of alcohol-seeking behavior. Re-exposure to Context A, but not Context B, reinstated alcohol-seeking behavior and increased expression of the neural activity marker Fos in the OFC. Reversible inactivation of the OFC with muscimol and baclofen attenuated context-induced reinstatement. Our data indicated that the OFC mediates context-induced reinstatement of alcohol-seeking behavior.

Amygdaloid involvement in the defensive behavior of mice exposed to the open elevated plus-maze.

Previous studies have shown that the exposure to an open elevated plus maze (oEPM, an EPM with all four open arms) elicits fear/anxiety-related responses in laboratory rodents. However, very little is known about the underlying neural substrates of these defensive behaviors. Accordingly, the present study investigated the effects of chemical inactivation of the amygdala [through local injection of cobalt chloride (CoCl2: a nonspecific synaptic blocker)] on the behavior of oEPM-exposed mice. In a second experiment, the pattern of activation of the basolateral (BLA) and central (CeA) nuclei of the amygdala was assessed through quantification of Fos protein expression in mice subjected to one of several behavioral manipulations. To avoid the confound of acute handling stress, 4 independent groups of mice were habituated daily for 10days to an enclosed EPM (eEPM) and, on day 11 prior to immunohistochemistry, were either taken directly from their home cage (control) or individually exposed for 10min to a new clean holding cage (novelty), an eEPM, or the oEPM. An additional group of mice (maze-naïve) was not subjected to either the habituation or exposure phase but were simply chosen at random from their home cages to undergo an identical immunohistochemistry procedure. Results showed that amygdala inactivation produced an anxiolytic-like profile comprising reductions in time spent in the proximal portions of the open arms and total stretched attend postures (SAP) as well as increases in time spent in the distal portions of the open arms and total head-dipping. Moreover, Fos-positive labeled cells were bilaterally increased in the amygdaloid complex, particularly in the BLA, of oEPM-exposed animals compared to all other groups. These results suggest that the amygdala (in particular, its BLA nucleus) plays a key role in the modulation of defensive behaviors in oEPM-exposed mice.

Stress induces behavioral sensitization, increases nicotine-seeking behavior and leads to a decrease of CREB in the nucleus accumbens.

Experimental evidence shows that exposure to stress engenders behavioral sensitization and increases drug-seeking and leads to intense drug taking. However the molecular mechanisms involved in these processes is not well known yet. The present experiments examined the effects of exposure to variable stress on nicotine-induced locomotor activation, cAMP-response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) activity and nicotine intravenous self-administration in rats. Male Wistar rats were exposed to variable stress that consisted of the exposure to different stressors twice a day in random order for 10 days. During this period the control group was left undisturbed except for cage cleaning. Ten days after the last stress episode, rats were challenged with either saline or nicotine (0.4 mg/kgs.c.) and the locomotor activity was recorded for 20 min. Immediately after behavioral recordings rats were sacrificed and their brains were removed to posterior western blotting analysis of CREB, phosphoCREB, ERK and phosphoERK in the nucleus accumbens. An independent set of control and stressed animals were subjected to an intravenous nicotine self-administration protocol. The break point during a progressive ratio schedule and nicotine intake patterns during a 24-hour binge was analyzed. Repeated variable stress caused a sensitized motor response to a single challenge of nicotine and decreased CREB in the nucleus accumbens. Furthermore, in the self-administration experiments previous stress exposure caused an increase in the break point and nicotine intake.

Ontogênese, estresse e dependência de substâncias psicoativas / Ortogenesis, stress and psychoactive substances addiction

O estudo da dependência de substâncias psicoativas apresentou grandes avanços conceituais nas últimas décadas. A evolução dos conceitos foi paralela às evidências científicas que têm revelado os aspectos comportamentais e os mecanismos neurais envolvidos nesse fenômeno. Contudo, um grande desafio que permanece na pesquisa sobre a dependência de substâncias psicoativas é a identificação de quais fatores são responsáveis pela transição do uso controlado para o uso compulsivo. Está demonstrado que muitas variáveis interagem para influenciar a probabilidade de que qualquer indivíduo inicie o uso abusivo de substâncias psicoativas ou se torne dependente. Nos últimos anos, o estresse tem sido destacado como um fator importante na iniciação, manutenção e recaída da utilização de substâncias psicoativas. Neste trabalho analisamos os conceitos e teorias da farmacodependência e as principais evidências comportamentais pré-clínicas que demonstram a relação entre estresse e a vulnerabilidade ao abuso e dependência de psicoestimulantes.

The investigation of the mechanisms of drug abuse and addiction showed great advances in the last decades. New concepts emerged from the scientific evidences on behavioral and neural aspects of this phenomenon. However, the biggest challenge for the future is the identification of which risk factors are implicated in the transition from controlled to compulsive drug use. Stress has been pointed as an important factor related to initiation, maintenance and relapse to drug use. In the present paper we discuss the concepts and theories of drug addiction, and the main behavioral pre-clinical evidences showing the relationship between stress and psychostimulant addiction.