The present research reports the first comprehensive map of brain networks underlying latent inhibition learning, the first application of structural equation modeling to cytochrome oxidase data, and the first effects of methylene blue, a known metabolic enhancer, on latent inhibition. In latent inhibition, repeated exposure to a stimulus results in a latent form of learning that inhibits subsequent associations with that stimulus. As neuronal energy demand to form learned associations changes, so does the induction of the respiratory enzyme cytochrome oxidase. Therefore, cytochrome oxidase can be used as an endpoint metabolic marker of the effects of experience on regional brain metabolic capacity. Quantitative cytochrome oxidase histochemistry was used to map brain regions in mice trained on a tone-footshock fear conditioning paradigm with either tone preexposure (latent inhibition), conditioning only (acquisition), conditioning followed by tone alone (extinction), or no handling or conditioning (naïve). In normal latent inhibition, the ventral cochlear nucleus, medial geniculate, CA1 hippocampus, and perirhinal cortex showed modified metabolic capacity due to latent inhibition. Structural equation modeling was used to determine the causal influences in an anatomical network of these regions and others thought to mediate latent inhibition, including the accumbens and entorhinal cortex. An uncoupling of ascending influences between auditory regions was observed in latent inhibition. There was also a reduced influence on the accumbens from the perirhinal cortex in both latent inhibition and extinction. These results suggest a specific network with a neural mechanism of latent inhibition that involves sensory gating, as evidenced by modifications in metabolic capacity, effective connectivity between auditory regions, and reduced hippocampal influence on the accumbens. The effects of methylene blue on disrupted latent inhibition were also investigated. Reduced tone-alone presentations disrupted the latent inhibition effect and led to an increase in freezing behavior. Repeated low-dose administration of methylene blue decreased freezing levels and facilitated the disrupted latent inhibition effect. Methylene blue administration also resulted in changes in metabolic capacity in limbic and cortical regions. A unique functional neural network was found in methylene blue-restored latent inhibition that emphasized sensory gating of auditory information, attention processing, and cortical inhibition of behavior. / text

The ventral tegmental area (VTA) and its dopaminergic (DA) mesocorticolimbic projections are thought to be essential in the brain’s reward neurocircuitry. In humans and animal experimental subjects, mild electrical VTA stimulation increases dopamine levels and can induce euphoria. Paradoxically, aversive stimuli activate VTA neurons and forebrain DA activity, and excessive electrical stimulation of the VTA exaggerates fearfulness. Research suggests that experimental manipulation of either the amygdala or the VTA has similar effects on the acquisition and expression of Pavlovian conditioned fear. Recently it was demonstrated that electrical stimulation of the amygdala produced fear extinction deficits in rats. Fear extinction involves the progressive dissipation of conditioned fear responses by repeated non-reinforced exposure to a conditioned stimulus (CS). Maladaptive states of fear in fear-related anxiety disorders, such as post-traumatic stress disorders (PTSD) or specific phobias are thought to reflect fear extinction learning deficits. The primary purpose of the present study was to examine the effects of intra-VTA stimulation on fear extinction learning. Using fear-potentiated startle as a behavioural index of conditioned fear, it was found that 120 VTA stimulations paired or unpaired with non-reinforced CS presentations impaired the extinction of conditioned fear. This effect was not apparent in rats that received electrical stimulation of the substantia nigra (SN), suggesting that not all midbrain regions respond similarly. Electrical stimulation parameters did not have aversive affects because rats failed to show fear conditioning when electrical VTA stimulation was used as the unconditioned stimulus. Also, VTA stimulation did not alter conditioned fear expression in non-extinguished animals. Based on the results it is suggested that VTA activation disinhibited conditioned fear responding. Therefore, VTA neuronal excitation by aversive stimuli may play a role in fear-related anxiety disorders thought to reflect extinction learning deficits.

The mammalian genome encodes five collapsin response mediator protein (CRMP) isoforms. Cell culture studies have shown that the CRMPs mediate growth cone dynamics and neuron polarity through associations with a variety of signal transduction components and cytoskeletal elements. CRMP is also a member of a protein family including the presumably ancestral dihydropyrimidinase (DHP) protein that catalyzes the second step in pyrimidine degradation. In Drosophila, CRMP and DHP proteins are produced by alternatively spliced transcripts of the CRMP gene. The alternative protein forms have a 91% sequence identity, but unique expression patterns. CRMP is found exclusively in neuronal tissues and DHP is ubiquitously expressed in non-neuronal tissues. Comparative analysis of CRMP homologous sequences from insect taxa show CRMP alternative splicing is a common feature and probably represents the ancestral state of this gene family. To investigate the regulatory role of CRMP, loss-of-function mutations of CRMP that lack both proteins were isolated; homozygous animals display DHP-null phenotypes but exhibit no overt developmental or neurological defects. To determine possible interactions of Drosophila CRMP with signaling pathways in which mammalian CRMP has been shown to act, the UAS-GAL4 system was utilized. Phenotypes produced by misexpression of a variety of UAS signal transduction mediator responders were modified in a CRMP mutant background. The modification entails enhancement or suppression of a specific phenotype in a direction that corresponds to the hypothesized involvement of mammalian CRMP in signaling pathways that regulate growth cone dynamics. These data suggest that Drosophila CRMP has a role in cell signaling pathways similar to the role of the mammalian CRMPs. Furthermore, recent findings demonstrate that CRMP plays an important role in learning and memory of mice, leading to the assessment of new phenotypes in the Drosophila CRMP mutants. Tests utilizing the Pavlovian olfactory conditioning assay reveal that loss of CRMP function leads to significant learning, 3 hour memory, and long term memory deficits. Preliminary data also suggest that Drosophila CRMP may be required for normal circadian locomotor rhythms. Collectively, the data presented here demonstrate CRMP’s role in adult behavioral processes and regulating signaling events comparable to mammalian CRMP signaling.

The present thesis examined extinction of conditioned fear in the developing rat. In the adult rat, the hippocampus is thought to be important for the context-specificity of extinction. Because the hippocampus is a late-maturing structure, it was hypothesised that context-modulation of extinction may be different across development. The first series of experiments investigated reinstatement of extinguished fear in the developing rat (Chapter 2). The results showed that P24 rats exhibited context-specific reinstatement. On the other hand, P17 rats did not exhibit reinstatement of extinguished fear following a US reminder treatment. The failure to see reinstatement in P17 rats was not due to the reminder treatment being ineffective in these rats because the same treatment alleviated spontaneous forgetting in rat this age. The second series of experiments then examined the renewal effect and GABAergic involvement in extinction in P24 and P17 rats (Chapter 3). It was observed that P24 rats displayed renewal whereas P17 rats did not. Also, pre-test injection of FG7142 recovered extinguished fear in P24 rats but not in P17 rats, even across a range of doses. This failure to see any FG7142 effect on extinction in P17 rats was not due to the lack of responsiveness to this drug in these rats because FG7142 was found to be effective in alleviating spontaneous forgetting in rats this age. The third series of experiments then examined the effect of temporary inactivation of the amygdala on extinction and re-extinction in the developing rat (Chapter 4). It was observed that extinction retention is impaired in both P24 and P17 rats if the amygdala is inactivated during extinction training. Interestingly, when a CS that had been previously extinguished and then re-trained was re-extinguished, re-extinction was amygdala-independent if initial extinction occurred at 24 days of age but amygdala-dependent if initial extinction occurred at 17 days of age. That is, amygdala involvement in re-extinction was dissociated across development. Taken together, these experiments provide strong evidence for fundamental differences in mechanisms underlying fear extinction across development. The implications of the findings were discussed in light of the theoretical and neural models of extinction.

N-methyl-D-asparate (NMDA) receptors located within the basolateral complex of the amygdala are required for the consolidation and expression of Pavlovian conditioned fear. The events downstream of receptor activation that mediate these processes are not well defined. An intermediate step that may be of significance is the synthesis of the gas nitric oxide (NO). Nitric oxide is synthesised as a result of NMDA receptor activation and acts as an unconventional neurotransmitter freely diffusing across cell membranes interacting with its targets in a non-synaptic manner. The targets of NO include cellular components that play significant roles during the consolidation of conditioned fear and the neurotransmission associated with its expression. This implies that NO may be an important intermediary of NMDA receptor activation and both these processes. The current study sought to examine this possibility using fear potentiated startle to examine the expression of learned fear. Three experiments were conducted, fifty rats received intra-BSC microinfusions of the global nitric oxide synthase inhibitor L-NAME either prior to fear conditioning, fear testing, or examination of the shock sensitization of the acoustic startle affect. The results indicated that NO was indeed required for both the consolidation and expression of learned fear, whereas it was not required for shock enhanced startle responding. This study provides new information about the sub-cellular basis of conditioned fear, and highlights the pivotal role played by NO in processes associated with conditioned fear.

The ventral tegmental area (VTA) and its dopaminergic (DA) mesocorticolimbic projections are thought to be essential in the brain’s reward neurocircuitry. In humans and animal experimental subjects, mild electrical VTA stimulation increases dopamine levels and can induce euphoria. Paradoxically, aversive stimuli activate VTA neurons and forebrain DA activity, and excessive electrical stimulation of the VTA exaggerates fearfulness. Research suggests that experimental manipulation of either the amygdala or the VTA has similar effects on the acquisition and expression of Pavlovian conditioned fear. Recently it was demonstrated that electrical stimulation of the amygdala produced fear extinction deficits in rats. Fear extinction involves the progressive dissipation of conditioned fear responses by repeated non-reinforced exposure to a conditioned stimulus (CS). Maladaptive states of fear in fear-related anxiety disorders, such as post-traumatic stress disorders (PTSD) or specific phobias are thought to reflect fear extinction learning deficits. The primary purpose of the present study was to examine the effects of intra-VTA stimulation on fear extinction learning. Using fear-potentiated startle as a behavioural index of conditioned fear, it was found that 120 VTA stimulations paired or unpaired with non-reinforced CS presentations impaired the extinction of conditioned fear. This effect was not apparent in rats that received electrical stimulation of the substantia nigra (SN), suggesting that not all midbrain regions respond similarly. Electrical stimulation parameters did not have aversive affects because rats failed to show fear conditioning when electrical VTA stimulation was used as the unconditioned stimulus. Also, VTA stimulation did not alter conditioned fear expression in non-extinguished animals. Based on the results it is suggested that VTA activation disinhibited conditioned fear responding. Therefore, VTA neuronal excitation by aversive stimuli may play a role in fear-related anxiety disorders thought to reflect extinction learning deficits.

Due to abnormal functioning of the brain’s reward and prediction system patients with schizophrenia spectrum disorders are thought to assign salience to non-relevant objects and events and to form context-inappropriate associations. The brain’s ventral striatum is critical in the formation of associations, and aberrant associations are believed to create delusional content during psychosis. The study wanted to examine the neural response, particularly in the ventral striatum, combined with subjective reports as patients learn associations in an aversive Pavlovian conditioning paradigm. The stimuli were randomized and involved circles of different colors. The conditioned stimuli (CS+) was followed by an unconditioned stimuli (US), consisting of an unpleasant sound, in 50% of events. The unconditioned (CS-) stimuli was followed by a low, not unpleasant sound in 50% of events. The degree of striatal activation was thought to be associated with the severity of patient’s illness. Functional magnetic resonance imaging (fMRI) blood-oxygen-level dependent (BOLD) responses were examined in eleven unmedicated non-institutionalized patients with schizophrenia spectrum disorders and 15 matched healthy controls. No significant within group differences in neural or subjective response to the [CS+ > CS-] contrast were found. No significant associations between severity of illness and degree of striatal activation in response to CS+ or CS- were found. Significant differences in neural activation for the [CS+ > CS-] contrast were found in the ventral striatum, the right inferor frontal gyrus, and the right angular gyrus, with patients exhibiting stronger activation compared to controls. The results and implications are discussed along with suggestions for future research.

Les stimuli conditionnés (CS) guident les animaux vers des récompenses essentielles à leur survie, telle que la quête de nourriture et d'eau. Ils peuvent également promouvoir la poursuite excessive de récompense, comme dans l’addiction. La transmission glutamatergique dans l’amygdale basolatérale (BLA) régule les effets des CS. Cependant, le rôle des récepteurs glutamatergiques métabotropes du groupe II (mGlu2/3) de la BLA est inconnu. Les mGlu2/3 sont principalement localisés au niveau extrasynaptique sur les terminaisons neuronales. L’activation de ces récepteurs diminue la libération synaptique de glutamate. Ainsi, nous avons émis l’hypothèse que l'activation des mGlu2/3 dans la BLA diminue la capacité des CS à potentialiser la recherche de récompense via la réduction de la libération de glutamate. La méthode de transfert Pavlovien-à-instrumental (PIT) a été utilisée pour tester cette hypothèse. Nous avons d'abord confirmé un effet PIT chez des rats mâles. Les rats ont initialement appris à appuyer sur un levier pour obtenir de l’eau comme récompense. Ils ont ensuite appris qu'un stimulus auditif (CS+) prédit la livraison non-contingente d'eau, tandis qu’un stimulus auditif différent (CS-) ne signale aucune récompense. Le jour du test PIT, les rats ont pu appuyer sur le levier sous extinction (eau omise) et nous avons évalué l’influence des CS+ et CS- sur ce comportement. Les rats ont appuyé davantage sur le levier pendant le CS+ comparé au CS-, indiquant que le CS+ incite les rats à rechercher la récompense. Chez un nouveau groupe de rats mâles, les mêmes méthodes ont été appliquées avec un agoniste des mGlu2/3, le LY379268 (0, 3 ou 6 μg/hémisphère) injecté dans la BLA avant le test PIT. Le LY379268 a diminué la capacité du CS+ à inciter l’appui sur le levier. Dans une dernière étude, nous avons examiné l’influence d’une administration systémique de LY379268 (0, 0.3 ou 1 mg/kg, sous-cutanée) sur l’effet PIT chez un groupe de rats mâles et femelles. Les effets motivationnels du CS+ sur la recherche de récompense étaient similaires entre les rats mâles et femelles. De plus, l’injection systémique de LY379268 a diminué ces effets chez les deux sexes. Ces résultats indiquent que les mGlu2/3 régulent les propriétés motivationnelles des CS chez les deux sexes. / Conditioned stimuli (CS) guide animals towards rewards needed for survival, such as food and water. In parallel, they can also promote maladaptive reward seeking, as in addiction. Glutamate signaling within the basolateral amygdala (BLA) modulates the effects of cues. However, the role of metabotropic group II glutamate (mGlu2/3) receptors in the BLA is unknown. mGlu2/3 are localized predominantly extrasynaptically on presynaptic terminals. The activation of these receptors suppresses synaptic glutamate release. Thus, we hypothesized that activating BLA mGlu2/3 receptors would attenuate cue-triggered increases in incentive motivation for reward, via reduced glutamate release. The Pavlovian-to-Instrumental transfer (PIT) method was used to test this hypothesis. We first confirmed a PIT effect in a cohort of male rats. Rats initially learned to press a lever to obtain a water reward. Then, they learned that one auditory cue predicts noncontingent delivery of water (CS+), while a second different auditory cue does not (CS-). On PIT test day, the rats could lever press under extinction (water deliveries omitted), and we assessed changes in lever pressing in response to CS+ and CS-. The rats pressed more on the lever during CS+ versus CS-, indicating cue-triggered potentiation of incentive motivation. In a separate cohort of male rats, the methods were repeated with the mGlu2/3 agonist LY379268 (0, 3 or 6 μg/hemisphere) infused into the BLA prior to PIT testing. LY379268 abolished CS+ potentiated pressing on the water-associated lever. In a last study, we examined the influence of systemic administration of LY379268 (0, 0.3 or 1 mg/kg, subcutaneous) on PIT, in a cohort of female and male rats. We found that cue-triggered increases in incentive motivation was similar across sexes, and that systemic injection of LY379268 decreased this effect in both sexes. These results indicate that mGlu2/3 receptors mediate the motivational effects of cues in both sexes.

Pesquisas sobre interação entre contingências de treino Pavloviano (PV) e treino operante (OP) têm enfatizado a influência do treino Pavloviano sobre o responder operante, mas não uma interação entre ambos os tipos de condicionamento. Neste trabalho é proposto estudar a interação a partir da combinação dos estímulos de ambas as contingências de treino, de forma que eles atuem simultaneamente como variáveis independentes na produção de um fenômeno comportamental. Para isso, a manipulação da taxa de reforço correlacionada aos estímulos condicional (CS) e discriminativo (SD) e a combinação desses estímulos em procedimentos de discriminação com estímulo composto foram utilizados como estratégias experimentais. No Estudo 1, três experimentos foram realizados com o objetivo de verificar a relação linear entre a taxa de reforço (r) correlacionada aos estímulos e a taxa de resposta (R) em treinos de discriminação com estímulos compostos em ratos. Nos Experimentos 1 (treino PV) e 2 (treino OP), ratos passaram por treino com um estímulo composto AB100% e três estímulos elementais, B50%, C50% e D25%. No Experimento 1 uma solução aquosa de sacarose (SAC) era liberada, como estímulo incondicional (US), em 100% das tentativas de AB100%, em 50% das vezes em B50% e C50% e em 25% das tentativas com D25%, em esquema de tempo variável VT-10s. No Experimento 2 a mesma taxa de SAC correlacionada aos estímulos foi mantida, mas dependeu do responder em esquema variável VI-10 s. Após essa fase, em ambos os experimentos, tentativas de sonda com o estímulo A, em extinção, foi adicionada. As respostas avaliadas foram inserir a cabeça no bebedouro (RIC) no treino PV (Experimento 1) e pressão à barra (RPB) no OP (Experimento 2). No experimento 3, a r na presença do estímulo A100% foi 100% e de B50% e C50% foi 50%. O estímulo avaliado em sonda foi um composto BC. Os resultados dos Experimentos 1 e 2 mostraram que a R da Sonda A foi equivalente à R nos estímulos B50% e C50%, indicando uma relação linear entre r e R (i.e., rA = rAB-rB). No Experimento 3, a R na Sonda BC foi equivalente à R em A100%, indicando somação das taxas de reforço (i.e., rBC = rB + rC). Nos experimentos 4 e 5 (Estudo 2) os treinos PV e OP foram combinados. Ratos passaram por PV e OP na mesma sessão, como o objetivo de verificar a somação da taxa de reforço (i.e., US e Sr) a partir da combinação de CS e SD em condicionamento com estímulo composto. Nas tentativas de OP, RPB em VI-10 s foi seguido por SAC em 100% das vezes nas tentativas com o estímulo A100% e em 50% das vezes em C50%. Nas tentativas de treino PV, o estímulo B50% foi seguido por SAC em 50% das vezes, em VT-10s, com a barra ausente. Após essa fase, os estímulos B50% e um composto BC foram adicionados ao treino por dez sessões, mas apresentados em tentativas de sonda em extinção (Exp. 4); no Experimento 5, foram realizadas sete sessões apenas com a Sonda BC e mais quatro sessões com as Sondas B e BC. Os resultados do experimento 4 indicaram somação entre os estímulos B e C em relação à resposta RIC (R em Sonda BC equivalente à R em A100%), mas não para RPB. No experimento 5, houve ocorrência de somação entre os estímulos B e C em ambos os tipos de treino (R em BC foi equivalente à R em A100%, para ambas as respostas RIC e RPB). Os dados foram discutidos em termos da teoria da estimação da taxa (RET), somação da taxa de reforço envolvendo CS e SD combinados e valor preditivo dos estímulos durante o condicionamento / Researches on Pavlovian-operant training interaction have emphasized the influence of Pavlovian training on the operant responding, but not an interaction between both type of conditioning. In this work, it is proposed to study the interaction through the combination of the stimuli of both training contingencies, so that they act simultaneously as independent variables in the production of a behavioral phenomenon. For this, the manipulation of the reinforcement rate correlated to the conditional (CS) and discriminative (SD) stimuli, and the combination of these stimuli in the procedure of discrimination with compound stimulus were used as experimental strategies. In study1, three previous experiments were carried out with the objective of verifying the linear relationship between the reinforcement rate (r) correlated to stimuli and rate response (R) on compound stimulus discrimination in rats. In Experiments 1 (PV training) and 2 (OP training), rats were trained with a compound stimulus AB100% and three individual stimuli B50%, C50%, and D25%. In PV, the aqueous solution of sucrose (SUC) was delivered as the unconditional stimulus on 100% of trials with AB100%, 50% of times in B50%, and C50% and 25% of trials with D25%, on a variable time schedule VT-10 s. In the OP, the same SAC rate correlated to the stimuli was maintained but depended on the responding on a variable schedule VI-10 s. After this phase, in both experiments, probe trials with the stimulus A, in extinction, was added to each experimental session. The evaluated responses were a head entry (HE) in the PV training and lever pressing (LP) in OP training. In Experiment 3, rats received a similar training to Experiment 2, the stimulus A100% with r of 100% of trials, B50% and C50% with r of 50%. The probe stimulus was a compound BC. The results of Experiments 1 and 2 showed that R of Probe A was equivalent to R in B50% and C50% stimuli, indicating a linear relationship between r and R (i.e., rA = rAB-rB). In Experiment 3 (OP), R in Probe BC was equivalent to R in A100%, indicating summation of the reinforcement rates (i.e., rBC = rB + rC). The Experiments 4 and 5 (Study 2) were carried out with the combination of PV and OP training. Rats were trained in PV and OP procedures in the same session with the objective of verifying summation of reinforcement rates (i.e., US, and Sr) from the combination of CS and SD in conditioning with the compound stimuli. Rats were trained in PV and OP procedures in the same session, with the objective of verifying the summation of the reinforcement rate (i.e., US and Sr) from the combination of CS and SD in compound stimulus conditioning. In OP trials, LP on a VI-10s were followed by SUC on 100% of times in the A100% trials, and 50% of trials with C50%. In PV trials, the stimulus B50% was followed by SUC on 50% of trials on VT-10 s, without the lever. After this phase, B50% stimuli and a BC compound were added in OP contingency for ten sessions, in extinction (Exp 4); in Experiment 5, seven sessions were performed with only the Probe BC and four sessions with Probes BC and B. The results of Experiment 4 indicated evidence for summation in relation to HE (R in BC probe equivalent to R in A100%), but not for LP. In Experiment 5, there was the occurrence of summation in both types of training (R in BC was equivalent to R in A100%, for both HE and LP responses). The data were discussed in terms of Rate Estimation Theory (RET), the summation of the reinforcement rate involving combined CS and SD, and the predictive value of the stimuli during conditioning

In the field of drug addiction, relapse back to drug seeking and taking is the major unmet clinical need. The rate of relapse back to drug-taking is ~70-80% within a year of drug abstinence. Gaining a better understanding of the prolonged neuronal changes that have taken place during drug addiction may lead to the design of better anti-relapse therapies. It is now widely believed that one component of drug addiction is by aberrant learning and memory processes. To study this, we investigated synaptic changes caused by the development of drug-seeking behaviour in C57BL/6J mice. Mice were treated either with non-contingent morphine or trained to exhibit drug-seeking behaviour following morphine-induced conditioned place preference (CPP) training, hippocampal slices were taken from these animals and synaptic changes examined at the CA3-CA1 synapse using electrophysiological methods. Mice that underwent morphine CPP were demonstrated to exhibit a significant preference for the morphine paired compartment before ex vivo electrophysiological analysis. Using field recordings, both non-contingent morphine and morphine CPP treatments resulted in a reduced ability to undergo stimulus-induced LTP compared to their respective controls. Whole cell patch clamp was then utilised to further investigate these effects. Non-contingent morphine treatment resulted in both pre- and post-synaptic changes with an increased AMPA:NMDA receptor ratio, concurrent increases in cell size, and reductions in the release probability of both glutamate and GABA. Morphine CPP treatment resulted in a more variable increase in AMPA:NMDA receptor ratio (presumably by the same mechanism but in a more specific group of neurones) and GABA release probability was also decreased. There were no detected increases in cell size however, or any detected changes in glutamate release probability. These findings therefore reveal a set of synaptic adaptations in the hippocampus unique to morphine-induced behavioural change, and may provide targets for future intervention in drug addiction.