Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

Evaluating feeding as unconditioned stimulus for conditioning of an endocrine effect in Nile tilapia

This study tested the adequacy of feeding as an unconditioned stimulus (US) to condition an endocrine response (plasma cortisol increase) in the cichlid fish Nile tilapia (Oreochromis niloticus). In a first study, conditioning was confirmed in grouped fish in the only experiment using single-held Nile tilapia. In this test a conditioned stimulus (CS - aeration off) was associated with a stressor (air emersion for 2 min - US). We then assessed whether several events of paired CS-US resulted in a conditioned endocrine response (CR), in this case an increase in plasma cortisol after presentation of the CS only. Before testing feeding as US, the postprandial or social holding condition for feeding effects on cortisol levels was tested. Nile tilapia showed increased cortisol after feeding associated to social context (grouped fish), but not to food only (single-held fish). In a third study, feeding was tested as US in an experiment similar to the first study but an increase in feeding-induced cortisol could not be conditioned. The absence of CR suggests that the stressor affects acquisition of this response, which may be a consequence of stimulus intensity or biological relevance. This study expands the recently reported Pavlovian conditioning paradigm for endocrine response in fish. (C) 2007 Elsevier B.V. All rights reserved.

Effects of stimulus modality and task condition on blink startle modification and on electrodermal responses

Participants in Experiments 1 and 2 performed a discrimination and counting task to assess the effect of lead stimulus modality on attentional modification of the acoustic startle reflex. Modality of the discrimination stimuli was changed across subjects. Electrodermal responses were larger during task-relevant stimuli than during task-irrelevant stimuli in all conditions. Larger blink magnitude facilitation was found during auditory and visual task-relevant stimuli, but not for tactile stimuli. Experiment 3 used acoustic, visual, and tactile conditioned stimuli (CSs) in differential conditioning with an aversive unconditioned stimulus (US). Startle magnitude facilitation and electrodermal responses were larger during a CS that preceded the US than during a CS that was presented alone regardless of lead stimulus modality. Although not unequivocal, the present data pose problems for attentional accounts of blink modification that emphasize the importance of lead stimulus modality.

Extracellular serotonin level in the basolateral nucleus of the amygdala and dorsal periaqueductal gray under unconditioned and conditioned fear states: An in vivo microdialysis study

Serotonin (5-HT) plays a key role in the neural circuitry mediating unconditioned and conditioned fear responses related to panic and generalized anxiety disorders. The basolateral nucleus of the amygdala (BLA) and the dorsal periaqueductal gray (dPAG) appear to be mainly involved in these conditions. The aim of this study was to measure the extracellular level of 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in the BLA and dPAG during unconditioned and conditioned fear states using in vivo microdialysis procedure. Thus, for the unconditioned fear test, animals were chemically stimulated in the dPAG with semicarbazide, an inhibitor of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase. For the conditioned fear test, animals were subjected to a contextual conditioned fear paradigm using electrical footshock as the unconditioned stimulus. The results show that the 5-HT and 5-HIAA level in the BLA and dPAG did not change during unconditioned fear, whereas 5-HT concentration, but not 5-HIAA concentration, increased in these brain areas during conditioned fear. The present study showed that the 5-HT system was activated during conditioned fear, whereas it remained unchanged during unconditioned fear, supporting the hypothesis that 5-HT has distinct roles in conditioned and unconditioned fear (dual role of 5-HT in anxiety disorders). (C) 2009 Elsevier B.V. All rights reserved.

Neural mechanisms of stimulus generalization in auditory fear conditioning

Dissertation presented to obtain the Ph.D degree in Neuroscience Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Unconditioned responses and functional fear networks in human classical conditioning.

Human imaging studies examining fear conditioning have mainly focused on the neural responses to conditioned cues. In contrast, the neural basis of the unconditioned response and the mechanisms by which fear modulates inter-regional functional coupling have received limited attention. We examined the neural responses to an unconditioned stimulus using a partial-reinforcement fear conditioning paradigm and functional MRI. The analysis focused on: (1) the effects of an unconditioned stimulus (an electric shock) that was either expected and actually delivered, or expected but not delivered, and (2) on how related brain activity changed across conditioning trials, and (3) how shock expectation influenced inter-regional coupling within the fear network. We found that: (1) the delivery of the shock engaged the red nucleus, amygdale, dorsal striatum, insula, somatosensory and cingulate cortices, (2) when the shock was expected but not delivered, only the red nucleus, the anterior insular and dorsal anterior cingulate cortices showed activity increases that were sustained across trials, and (3) psycho-physiological interaction analysis demonstrated that fear led to increased red nucleus coupling to insula but decreased hippocampus coupling to the red nucleus, thalamus and cerebellum. The hippocampus and the anterior insula may serve as hubs facilitating the switch between engagement of a defensive immediate fear network and a resting network.

L-Allylglycine dissociates the neural substrates of fear in the periaqueductal gray of rats

The dorsal (dPAG) and ventral (vPAG) regions of the periaqueductal gray are well known to contain the neural substrates of fear and anxiety. Chemical or electrical stimulation of the dPAG induces freezing, followed by a robust behavioral reaction that has been considered an animal model of panic attack. In contrast, the vPAG is part of a neural system, in which immobility is the usual response to its stimulation. The defense reaction induced by the stimulation of either region is accompanied by anti nociception. Although GABAergic mechanisms are known to exert tonic inhibitory control on the neural substrates of fear in the dPAG, the role of these mechanisms in the vPAG is still unclear. The present study examined defensive behaviors and antinociception induced by microinjections of an inhibitor of gamma-aminobutyric acid synthesis, L-allylglycine (L-AG; 1, 3, and 5 mu g/0.2 mu l), into either the dPAG or vPAG of rats subjected to the open field and tail-flick tests. Passive or tense immobility was the predominant behavior after L-AG (1 or 3 mu g) microinjection into the vPAG and dPAG, respectively, which was replaced with intense hyperactivity, including jumps or rearings, after injections of a higher dose (5 mu g/0.2 mu l) into the dPAG or vPAG. Moreover, whereas intra-dPAG injection of 3 mu g L-AG produced intense antinociception, only weak antinociception was induced by intra-vPAG injections of 5 mu g L-AG. These findings suggest that GABA mechanisms are involved in the mediation of antinociception and behavioral inhibition to aversive stimulation of the vPAG and exert powerful control over the neural substrates of fear in the dPAG to prevent a full-blown defense reaction possibly associated with panic disorder. (C) 2009 Elsevier Inc. All rights reserved.

Selective involvement of GABAergic mechanisms of the dorsal periaqueductal gray and inferior colliculus on the memory of the contextual fear as assessed by the fear potentiated startle test

The inferior colliculus (IC) together with the dorsal periaqueductal gray (dPAG), the amygdala and the medial hypothalamus make part of the brain aversion system, which has mainly been related to the organization of unconditioned fear. However, the involvement of the IC and dPAG in the conditioned fear is still unclear. It is certain that GABA has a regulatory role on the aversive states generated and elaborated in these midbrain structures. In this study, we evaluated the effects of injections of the GABA-A receptor agonist muscimol (1.0 and 2.0 nmol/0.2 mu L) into the IC or dPAG on the freezing and fear-potentiated startle (FPS) responses of rats submitted to a context fear conditioning. Intra-IC injections of muscimol did not cause any significant effect on the FPS or conditioned freezing but enhanced the startle reflex in non-conditioned animals. In contrast, intra-dPAG injections of muscimol caused significant reduction in FPS and conditioned freezing without changing the startle reflex in non-conditioned animals. Thus, intra-dPAG injections of muscimol produced the expected inhibitory effects on the anxiety-related responses, the FPS and the freezing whereas these injections into the IC produced quite opposite effects suggesting that descending inhibitory pathways from the IC, probably mediated by GABA-A mechanisms, exert a regulatory role on the lower brainstem circuits responsible for the startle reflex. (C) 2008 Elsevier Inc. All rights reserved.

Conditioned inhibition of autonomic Pavlovian conditioning in humans

The present study aimed to demonstrate conditioned inhibition of Pavlovian conditioning of autonomic responses in humans. Subjects (N = 21) were presented initially with four geometric shapes (A, B, C and D). An electric shock served as the unconditioned stimulus (US) during acquisition. Conditional stimuli lasted for 8 s and US onset coincided with CS offset. Subjects were trained with A-US, C-US, and AC-US pairings and AB alone and B alone presentations. The subsequent summation test consisted of C-US pairings and CB alone and CD alone presentations. Conditioning was evident in self-reported US expectancy and first and second interval electrodermal responses. Evidence for conditioned inhibition during the summation test was found in US expectancy and second interval electrodermal responses. (C) 1997 Elsevier Science B.V.

Latent inhibition and schizophrenia: Pavlovian conditioning of autonomic responses

Latent inhibition (LI) is an important model for understanding cognitive deficits in schizophrenia, Disruption of LI is thought to result from an inability to ignore irrelevant stimuli. The study investigated LI in schizophrenic patients by using Pavlovian conditioning of electrodermal responses in a complete within-subject design. Thirty-two schizophrenic patients, ( 16 acute. unmedicated and 16 medicated patients) and 16 healthy control subjects (matched with respect to age and gender) participated in the study. The experiment consisted of two stages: preexposure and conditioning. During preexposure two visual stimuli were presented, one of which served as the to-be-conditioned stimulus (CSp +) and the other one was the not-to-be-conditioned stimulus (CSp -) during the following conditioning ( = acquisition). During acquisition. two novel visual stimuli (CSn + and CSn -) were introduced. A reaction time task was used as the unconditioned stimulus (US). LI was defined as the difference in response differentiation observed between proexposed and non-preexposed sets of CS + and CS -. During preexposure. the schizophrenic patients did not differ in electrodermal responding from the control subjects, neither concerning the extent of orienting nor the course of habituation. The exposure to novel stimuli at the beginning of the acquisition elicited reduced orienting responses in unmedicated patients compared to medicated patients and control subjects, LI was observed in medicated schizophrenic patients and healthy controls. but not in acute unmedicated patients. Furthermore LI was found to be correlated with the duration of illness: it was attenuated in patients who had suffered their first psychotic episode. (C) 2002 Elsevier Science B.V. All rights reserved.

Dynamics of the recruitment of lateral amygdala neurons following successive fear learning to different conditional stimuli

In fear conditioning, an animal learns to associate an unconditioned stimulus (US), such as a shock, and a conditioned stimulus (CS), such as a tone, so that the presentation of the CS alone can trigger conditioned responses. Recent research on the lateral amygdala has shown that following cued fear conditioning, only a subset of higher-excitable neurons are recruited in the memory trace. Their selective deletion after fear conditioning results in a selective erasure of the fearful memory. I hypothesize that the recruitment of highly excitable neurons depends on responsiveness to stimuli, intrinsic excitability and local connectivity. In addition, I hypothesize that neurons recruited for an initial memory also participate in subsequent memories, and that changes in neuronal excitability affect secondary fear learning. To address these hypotheses, I will show that A) a rat can learn to associate two successive short-term fearful memories; B) neuronal populations in the LA are competitively recruited in the memory traces depending on individual neuronal advantages, as well as advantages granted by the local network. By performing two successive cued fear conditioning experiments, I found that rats were able to learn and extinguish the two successive short-term memories, when tested 1 hour after learning for each memory. These rats were equipped with a system of stable extracellular recordings that I developed, which allowed to monitor neuronal activity during fear learning. 233 individual putative pyramidal neurons could modulate their firing rate in response to the conditioned tone (conditioned neurons) and/or non- conditioned tones (generalizing neurons). Out of these recorded putative pyramidal neurons 86 (37%) neurons were conditioned to one or both tones. More precisely, one population of neurons encoded for a shared memory while another group of neurons likely encoded the memories' new features. Notably, in spite of a successful behavioral extinction, the firing rate of those conditioned neurons in response to the conditioned tone remained unchanged throughout memory testing. Furthermore, by analyzing the pre-conditioning characteristics of the conditioned neurons, I determined that it was possible to predict neuronal recruitment based on three factors: 1) initial sensitivity to auditory inputs, with tone-sensitive neurons being more easily recruited than tone- insensitive neurons; 2) baseline excitability levels, with more highly excitable neurons being more likely to become conditioned; and 3) the number of afferent connections received from local neurons, with neurons destined to become conditioned receiving more connections than non-conditioned neurons. - En conditionnement de la peur, un animal apprend à associer un stimulus inconditionnel (SI), tel un choc électrique, et un stimulus conditionné (SC), comme un son, de sorte que la présentation du SC seul suffit pour déclencher des réflexes conditionnés. Des recherches récentes sur l'amygdale latérale (AL) ont montré que, suite au conditionnement à la peur, seul un sous-ensemble de neurones plus excitables sont recrutés pour constituer la trace mnésique. Pour apprendre à associer deux sons au même SI, je fais l'hypothèse que les neurones entrent en compétition afin d'être sélectionnés lors du recrutement pour coder la trace mnésique. Ce recrutement dépendrait d'un part à une activation facilité des neurones ainsi qu'une activation facilité de réseaux de neurones locaux. En outre, je fais l'hypothèse que l'activation de ces réseaux de l'AL, en soi, est suffisante pour induire une mémoire effrayante. Pour répondre à ces hypothèses, je vais montrer que A) selon un processus de mémoire à court terme, un rat peut apprendre à associer deux mémoires effrayantes apprises successivement; B) des populations neuronales dans l'AL sont compétitivement recrutées dans les traces mnésiques en fonction des avantages neuronaux individuels, ainsi que les avantages consentis par le réseau local. En effectuant deux expériences successives de conditionnement à la peur, des rats étaient capables d'apprendre, ainsi que de subir un processus d'extinction, pour les deux souvenirs effrayants. La mesure de l'efficacité du conditionnement à la peur a été effectuée 1 heure après l'apprentissage pour chaque souvenir. Ces rats ont été équipés d'un système d'enregistrements extracellulaires stables que j'ai développé, ce qui a permis de suivre l'activité neuronale pendant l'apprentissage de la peur. 233 neurones pyramidaux individuels pouvaient moduler leur taux d'activité en réponse au son conditionné (neurones conditionnés) et/ou au son non conditionné (neurones généralisant). Sur les 233 neurones pyramidaux putatifs enregistrés 86 (37%) d'entre eux ont été conditionnés à un ou deux tons. Plus précisément, une population de neurones code conjointement pour un souvenir partagé, alors qu'un groupe de neurones différent code pour de nouvelles caractéristiques de nouveaux souvenirs. En particulier, en dépit d'une extinction du comportement réussie, le taux de décharge de ces neurones conditionné en réponse à la tonalité conditionnée est resté inchangée tout au long de la mesure d'apprentissage. En outre, en analysant les caractéristiques de pré-conditionnement des neurones conditionnés, j'ai déterminé qu'il était possible de prévoir le recrutement neuronal basé sur trois facteurs : 1) la sensibilité initiale aux entrées auditives, avec les neurones sensibles aux sons étant plus facilement recrutés que les neurones ne répondant pas aux stimuli auditifs; 2) les niveaux d'excitabilité des neurones, avec les neurones plus facilement excitables étant plus susceptibles d'être conditionnés au son ; et 3) le nombre de connexions reçues, puisque les neurones conditionné reçoivent plus de connexions que les neurones non-conditionnés. Enfin, nous avons constaté qu'il était possible de remplacer de façon satisfaisante le SI lors d'un conditionnement à la peur par des injections bilatérales de bicuculline, un antagoniste des récepteurs de l'acide y-Aminobutirique.

Sex differences in the neurobiology of fear conditioning and extinction: a preliminary fMRI study of shared sex differences with stress-arousal circuitry.

BACKGROUND: The amygdala, hippocampus, medial prefrontal cortex (mPFC) and brain-stem subregions are implicated in fear conditioning and extinction, and are brain regions known to be sexually dimorphic. We used functional magnetic resonance imaging (fMRI) to investigate sex differences in brain activity in these regions during fear conditioning and extinction. METHODS: Subjects were 12 healthy men comparable to 12 healthy women who underwent a 2-day experiment in a 3 T MR scanner. Fear conditioning and extinction learning occurred on day 1 and extinction recall occurred on day 2. The conditioned stimuli were visual cues and the unconditioned stimulus was a mild electric shock. Skin conductance responses (SCR) were recorded throughout the experiment as an index of the conditioned response. fMRI data (blood-oxygen-level-dependent [BOLD] signal changes) were analyzed using SPM8. RESULTS: Findings showed no significant sex differences in SCR during any experimental phases. However, during fear conditioning, there were significantly greater BOLD-signal changes in the right amygdala, right rostral anterior cingulate (rACC) and dorsal anterior cingulate cortex (dACC) in women compared with men. In contrast, men showed significantly greater signal changes in bilateral rACC during extinction recall. CONCLUSIONS: These results indicate sex differences in brain activation within the fear circuitry of healthy subjects despite similar peripheral autonomic responses. Furthermore, we found that regions where sex differences were previously reported in response to stress, also exhibited sex differences during fear conditioning and extinction.

Fruit flies learn to avoid odours associated with virulent infection.

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.

Attempts to control pain prioritize attention to signals of pain: an experimental study

Clinical evidence suggests that a persistent search for solutions for chronic pain may bring along costs at the cognitive, affective, and behavioral level. Specifically, attempts to control pain may fuel hypervigilance and prioritize attention towards pain-related information. This hypothesis was investigated in an experiment with 41 healthy volunteers. Prioritization of attention towards a signal for pain was measured using an adaptation of a visual search paradigm in which participants had to search for a target presented in a varying number of colored circles. One of these colors (Conditioned Stimulus) became a signal for pain (Unconditioned Stimulus: electrocutaneous stimulus at tolerance level) using a classical conditioning procedure. Intermixed with the visual search task, participants also performed another task. In the pain-control group, participants were informed that correct and fast responses on trials of this second task would result in an avoidance of the Unconditioned Stimulus. In the comparison group, performance on the second task was not instrumental in controlling pain. Results showed that in the pain-control group, attention was more prioritized towards the Conditioned Stimulus than in the comparison group. The theoretical and clinical implications of these results are discussed.

Álcool e comportamento: efeitos na aprendizagem e memória

Excessive alcohol consumption is responsible for many harmful effects on individuals and society. Despite years of research, the mechanisms by which alcohol affects neurological functions and the exact causes of cognitive impairment related to long-term use are unknown. In this sense, this master study proposed to observe how different doses of alcohol affect the addiction response and the learning ability of two fish species: Betta splendens and Danio rerio, the latter a commonly model due to organizational and functional characteristics shared with mammals. For this, different concentrations of ethanol (0%, 0.1%, 0.25%, 1% and 1.5%) were used in acute, chronic and withdrawal treatments. We tested the fish in three experimental protocols: 1) alcohol addiction potential using conditioned place preference, 2) associative conditioning using light as unconditioned stimulus and food as conditioned stimulus and 3) spatial learning using a maze without cues. For the alcohol addiction potential, preference between two different places in a shuttle box was tested before and after alcohol exposure (chronic and acute). In this test, the animals intoxicated by 0.1% did not change behavior, while animals receiving 1% and 1.5% alcohol changed the initial preference to the side where they received alcohol For the associative conditioning, the results show that the groups undergoing low dose (0.1%), both in chronic and withdrawal treatment, learned the task faster than control; groups under 0.25 and 1% alcohol withdrawal learned the task after control; groups chronically intoxicated with these doses did not learn the task. For the spatial learning test, fish submitted to acute and chronic treatments decreased the time to exit the maze; there were significant differences in the animal s performance in a dose-dependent pattern. This difference was not observed for the withdrawal treatment. Given these results, we conclude that the effects of alcohol on learning are dependent on the dosage. Furthermore, low doses of alcohol seem to maximize animal performance on learning tasks and do not alter their seeking behavior, while higher doses induced addition and hinder learning