Systemic administration of interleukin-1 beta activates select populations of central amygdala afferents

The central nucleus of the amygdala (CeA) is activated robustly by an immune challenge such as the systemic administration of the proinflammatory cytokine interleukin-1beta (IL-1beta). Because IL-1beta is not believed to cross the blood-brain barrier in any significant amount, it is likely that IL-1beta elicits CeA cell recruitment by means of activation of afferents to the CeA. However, although many studies have investigated the origins of afferent inputs to the CeA, we do not know which of these also respond to IL-1beta. Therefore, to identify candidate neurons responsible for the recruitment of CeA cells by an immune challenge, we iontophoretically deposited a retrograde tracer, cholera toxin b-subunit (CTb), into the CeA of rats 7 days before systemic delivery of IL-1beta (1 mug/kg, i.a.). By using combined immunohistochemistry, we then quantified the number of Fos-positive CTb cells in six major regions known to innervate the CeA. These included the medial prefrontal cortex, paraventricular thalamus (PVT), ventral tegmental area, parabrachial nucleus (PB), nucleus tractus solitarius, and ventrolateral medulla. Our results show that after deposit of CTb into the CeA, the majority of double-labeled cells were located in the PB and the PVT, suggesting that CeA cell activation by systemic IL-1beta is likely to arise predominantly from cell bodies located in these regions. These findings may have significant implications in determining the central pathways involved in generating acute central responses to a systemic immune challenge. J. Comp. Neurol. 452:288-296, 2002. (C) 2002 Wiley-Liss, Inc.

Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor

Psychological stressors trigger the activation of medullary noradrenergic cells, an effect that has been shown to depend upon yet-to-be-identified structures located higher in the brain. To test whether the amygdala is important in this regard, we examined the effects of amygdala lesions on noradrenergic cell responses to restraint, and also looked at whether any amygdala cells that respond to restraint project directly to the medulla. Ibotenic acid lesions of the medial amygdala completely abolished restraint-induced Fos expression in A1 and A2 noradrenergic cells. In contrast, lesions of the central amygdala actually facilitated noradrenergic cell responses to restraint. Tracer deposits in the dorsomedial (but not ventrolateral) medulla retrogradely labelled many cells in the central nucleus of the amygdala, but none of these cells expressed Fos in response to restraint. These data suggest for the first time that the medial amygdala is critical to the activation of medullary noradrenergic cells by a psychological stressor whereas the central nucleus exerts an opposing, inhibitory influence upon noradrenergic cell recruitment. The initiation of noradrenergic cell responses by the medial amygdala does not involve a direct projection to the medulla. Accordingly, a relay through some other structure, such as the hypothalamic paraventricular nucleus, warrants careful consideration.

Amygdala activation in response to 2D and 3D emotion-inducing stimuli

Studying changes in brain activation according to the valence of emotion-inducing stimuli is essential in the research on emotions. Due to the ecological potential of virtual reality, it is also important to examine whether brain activation in response to emotional stimuli can be modulated by the three-dimensional (3D) properties of the images. This study uses functional Magnetic Resonance Imaging to compare differences between 3D and standard (2D) visual stimuli in the activation of emotion-related brain areas. The stimuli were organized in three virtual-reality scenarios, each with a different emotional valence (pleasant, unpleasant and neutral). The scenarios were presented in a pseudo-randomized order in the two visualization modes to twelve healthy males. Data were analyzed through a GLM-based fixed effects procedure. Unpleasant and neutral stimuli activated the right amygdala more strongly when presented in 3D than in 2D. These results suggest that 3D stimuli, when used as “building blocks” for virtual environments, can induce increased emotional loading, as shown here through neuroimaging.

Elektrophysiologische Charakterisierung von Neuronen der lateralen Amygdala unter besonderer Berücksichtigung von Aktionspotentialmustern und Oszillationen

Magdeburg, Univ., Medizin. Fakultät, Diss., 2006

Genetische und epigenetische Einflüsse auf die Entwicklung der Amygdala und des emotionalen Verhaltens - Untersuchungen an drei Mäusestämmen

Embryo transfer, amygdala, neuropeptide Y, parvalbumin, maternal effect, genetic background, open field, elevated plus maze, acoustic startle response, intrauterine influences

Charakterisierung der Projektionen des ventralen CA1 und des Subiculums zum Nucleus basomedialis der Amygdala in der Maus und ihre Beziehungen zu GABAergen Interneuronen

Universität Magdeburg, Univ., Dissertation, 2015

Oxytocin enhances the inhibitory effects of diazepam in the rat central medial amygdala.

Oxytocin is a neuropeptide that can reduce neophobia and improve social affiliation. In vitro, oxytocin induces a massive release of GABA from neurons in the lateral division of the central amygdala which results in inhibition of a subpopulation of peripherally projecting neurons in the medial division of the central amygdala (CeM). Common anxiolytics, such as diazepam, act as allosteric modulators of GABA(A) receptors. Because oxytocin and diazepam act on GABAergic transmission, it is possible that oxytocin can potentiate the inhibitory effects of diazepam if both exert their pre, - respectively postsynaptic effects on the same inhibitory circuit in the central amygdala. We found that in CeM neurons in which diazepam increased the inhibitory postsynaptic current (IPSC) decay time, TGOT (a specific oxytocin receptor agonist) increased IPSC frequency. Combined application of diazepam and TGOT resulted in generation of IPSCs with increased frequency, decay times as well as amplitudes. While individual saturating concentrations of TGOT and diazepam each decreased spontaneous spiking frequency of CeM neurons to similar extent, co-application of the two was still able to cause a significantly larger decrease. These findings show that oxytocin and diazepam act on different components of the same GABAergic circuit in the central amygdala and that oxytocin can facilitate diazepam effects when used in combination. This raises the possibility that neuropeptides could be clinically used in combination with currently used anxiolytic treatments to improve their therapeutic efficacy.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

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.

Effects of amygdala-hippocampal stimulation on interictal epileptic discharges.

Deep brain stimulation (DBS) of different nuclei is being evaluated as a treatment for epilepsy. While encouraging results have been reported, the effects of changes in stimulation parameters have been poorly studied. Here the effects of changes of pulse waveform in high frequency DBS (130Hz) of the amygdala-hippocampal complex (AH) are presented. These effects were studied on interictal epileptic discharge rates (IEDRs). AH-DBS was implemented with biphasic versus pseudo monophasic charge balanced pulses, in two groups of patients: six with temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) and six with non lesional (NLES) temporal epilepsy. In patients with HS, IEDRs were significantly reduced with AH-DBS applied with biphasic pulses in comparison with monophasic pulse. IEDRs were significantly reduced in only two patients with NLES independently to stimulus waveform. Comparison to long-term seizure outcome suggests that IEDRs could be used as a neurophysiological marker of chronic AH-DBS and they suggest that the waveform of the electrical stimuli can play a major role in DBS. We concluded that biphasic stimuli are more efficient than pseudo monophasic pulses in AH-DBS in patients with HS. In patients with NLES epilepsy, other parameters relevant for efficacy of DBS remain to be determined.

Oxytocin selectively gates fear responses through distinct outputs from the central amygdala.

Central amygdala (CeA) projections to hypothalamic and brain stem nuclei regulate the behavioral and physiological expression of fear, but it is unknown whether these different aspects of the fear response can be separately regulated by the CeA. We combined fluorescent retrograde tracing of CeA projections to nuclei that modulate fear-related freezing or cardiovascular responses with in vitro electrophysiological recordings and with in vivo monitoring of related behavioral and physiological parameters. CeA projections emerged from separate neuronal populations with different electrophysiological characteristics and different response properties to oxytocin. In vivo, oxytocin decreased freezing responses in fear-conditioned rats without affecting the cardiovascular response. Thus, neuropeptidergic signaling can modulate the CeA outputs through separate neuronal circuits and thereby individually steer the various aspects of the fear response.

Stereological analysis of the rat and monkey amygdala

The amygdala is part of a neural network that contributes to the regulation of emotional behaviors. Rodents, especially rats, are used extensively as model organisms to decipher the functions of specific amygdala nuclei, in particular in relation to fear and emotional learning. Analysis of the role of the nonhuman primate amygdala in these functions has lagged work in the rodent but provides evidence for conservation of basic functions across species. Here we provide quantitative information regarding the morphological characteristics of the main amygdala nuclei in rats and monkeys, including neuron and glial cell numbers, neuronal soma size, and individual nuclei volumes. The volumes of the lateral, basal, and accessory basal nuclei were, respectively, 32, 39, and 39 times larger in monkeys than in rats. In contrast, the central and medial nuclei were only 8 and 4 times larger in monkeys than in rats. The numbers of neurons in the lateral, basal, and accessory basal nuclei were 14, 11, and 16 times greater in monkeys than in rats, whereas the numbers of neurons in the central and medial nuclei were only 2.3 and 1.5 times greater in monkeys than in rats. Neuron density was between 2.4 and 3.7 times lower in monkeys than in rats, whereas glial density was only between 1.1 and 1.7 times lower in monkeys than in rats. We compare our data in rats and monkeys with those previously published in humans and discuss the theoretical and functional implications that derive from our quantitative structural findings.

Voluntary Explicit versus Involuntary Conceptual Memory Are Associated with Dissociable fMRI Responses in Hippocampus, Amygdala, and Parietal Cortex for Emotional and Neutral Word Pairs.

Although functional neuroimaging studies have supported the distinction between explicit and implicit forms of memory, few have matched explicit and implicit tests closely, and most of these tested perceptual rather than conceptual implicit memory. We compared event-related fMRI responses during an intentional test, in which a group of participants used a cue word to recall its associate from a prior study phase, with those in an incidental test, in which a different group of participants used the same cue to produce the first associate that came to mind. Both semantic relative to phonemic processing at study, and emotional relative to neutral word pairs, increased target completions in the intentional test, but not in the incidental test, suggesting that behavioral performance in the incidental test was not contaminated by voluntary explicit retrieval. We isolated the neural correlates of successful retrieval by contrasting fMRI responses to studied versus unstudied cues for which the equivalent "target" associate was produced. By comparing the difference in this repetition-related contrast across the intentional and incidental tests, we could identify the correlates of voluntary explicit retrieval. This contrast revealed increased bilateral hippocampal responses in the intentional test, but decreased hippocampal responses in the incidental test. A similar pattern in the bilateral amygdale was further modulated by the emotionality of the word pairs, although surprisingly only in the incidental test. Parietal regions, however, showed increased repetition-related responses in both tests. These results suggest that the neural correlates of successful voluntary explicit memory differ in directionality, even if not in location, from the neural correlates of successful involuntary implicit (or explicit) memory, even when the incidental test taps conceptual processes.

Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala.

Accumulating evidence supports a role for brain-derived neurotrophic factor (BDNF) in depression. However, most of these studies have been performed in animal models that have a low face validity with regard to the human disease. Here, we examined the regulation of BDNF expression in the hippocampus and amygdala of rats subjected to the chronic mild stress (CMS) model of depression, a paradigm that induces anhedonia, a core symptom of depression. We found that exposure of rats to the CMS paradigm did not modulate BDNF mRNA expression in the hippocampus and amygdala. In addition, chronic administration of imipramine, which reversed CMS-induced anhedonia, did not alter BDNF mRNA expression in these limbic structures.

Neurophysiological effects of vasopressin and oxytocin in the central amygdala of the rat : a potential mechanism for the opposite modulation of anxiety and fear behavior

Abstract The amygdala is a group of nuclei in the temporal lobe of the brain that plays a crucial role in anxiety and fear behavior. Sensory information converges in the basolateral and lateral nuclei of the amygdala, which have been the first regions in the brain where the acquisition of new (fear) memories has been associated with long term changes in synaptic transmission. These nuclei, in turn, project to the central nucleus of the amygdala. The central amygdala, through its extensive projections to numerous nuclei in the midbrain and brainstem, plays a pivotal role in the orchestration of the rapid autonomic and endocrine fear responses. In the central amygdala a large number of neuropeptides and receptors is expressed, among which high levels of vasopressin and oxytocin receptors. Local injections of these peptides into the amygdala modulate several aspects of the autonomic fear reaction. Interestingly, their effects are opposing: vasopressin tends to enhance the fear reactions, whereas oxytocin has anxiolytic effects. In order to investigate the neurophysiological mechanisms that could underlie this opposing modulation of the fear behavior, we studied the effects of vasopressin and oxytocin on the neuronal activity in an acute brain slice preparation of the rat central amygdala. We first assessed the effects of vasopressin and oxytocin on the spontaneous activity of central amygdala neurons. Extracellular single unit recordings revealed two major populations of neurons: a majority of neurons was excited by vasopressin and inhibited by oxytocin, whereas other neurons were only excited by oxytocin receptor activation. The inhibitory effect of oxytocin could be reduced by the block of GABAergic transmission, whereas the excitatory effects of vasopressin and oxytocin were not affected. In a second step we identified the cellular mechanisms for the excitatory effects of both peptides as well as the morphological and biochemical mechanisms underlying the opposing effects, by using sharp electrode recordings together with intracellular labelings. We revealed that oxytocin-excited neurons are localized in the lateral part (CeL) whereas vasopressin excited cells are found in the medial part of the central amygdala (CeM). The tracing of the neuronal morphology showed that the axon collaterals of the oxytocin-excited neurons project from the CeL, far into the CeM. Combined immunohistochemical stainings indicated that these projections are GABAergic. In the third set of experiments we investigated the synaptic interactions between the two identified cell populations. Whole-cell patch-clamp recordings in the CeM revealed that the inhibitory effect of oxytocin was caused by the massive increase of inhibitory GABAergic currents, which was induced by the activation of CeL neurons. Finally, the effects of vasopressin and oxytocin on evoked activity were investigated. We found on the one hand, that the probability of evoking action potentials in the CeM by stimulating the basolateral amygdala afferents was enhanced under vasopressin, whereas it decreased under oxytocin. On the other hand, the impact of cortical afferents stimulation on the CeL neurons was enhanced by oxytocin application. Taken together, these findings have allowed us to develop a model, in which the opposing behavioral effects of vasopressin and oxytocin are caused by a selective activation of two distinct populations of neurons in the GABAergic network of the central amygdala. Our model could help to develop new anxiolytic treatments, which modulate simultaneously both receptor systems. By acting on a GABAergic network, such treatments can further be tuned by combinations with classical benzodiazepines. Résumé: L'amygdale est un groupe de noyaux cérébraux localisés dans le lobe temporal. Elle joue un rôle essentiel dans les comportements liés à la peur et l'anxiété. L'information issue des aires sensorielles converge vers les noyaux amygdaliens latéraux et basolatéraux, qui sont les projections vers différents noyaux du tronc cérébral et de l'hypothalamus, joue un rôle clef premières régions dans lesquelles il a été démontré que l'acquisition d'une nouvelle mémoire (de peur) était associée à des changements à long terme de la transmission synaptique. Ces noyaux envoient leurs projections sur l'amygdale centrale, qui à travers ses propres dans l'orchestration des réponses autonomes et endocrines de peur. Le contrôle de l'activité neuronale dans l'amygdale centrale module fortement la réaction de peur. Ainsi, un grand nombre de neuropeptides sont spécifiquement exprimés dans l'amygdale centrale et un bon nombre d'entre eux interfère dans la réaction de peur et d'anxiété. Chez les rats, une forte concentration de récepteurs à l'ocytocine et à la vasopressine est exprimée dans le noyau central, et l'injection de ces peptides dans l'amygdale influence différents aspects de la réaction viscérale associée à la peur. Il est intéressant de constater que ces peptides exercent des effets opposés. Ainsi, la vasopressine augmente la réaction de peur alors que l'ocytocine a un effet anxiolytique. Afin d'investiguer les mécanismes neurophysiologiques responsables de ces effets opposés, nous avons étudié l'effet de la vasopressine et de l'ocytocine sur l'activité neuronale de préparations de tranches de cerveau de rats contenant entre autres de l'amygdale centrale. Tout d'abord, notre intérêt s'est porté sur les effets de ces deux neuropeptides sur l'activité spontanée dans l'amygdale centrale. Des enregistrements extracellulaires ont révélé différentes populations de neurones ; une majorité était excitée par la vasopressine et inhibée par l'ocytocine ; d'autres étaient seulement excités par l'activation du récepteur à l'ocytocine. L'effet inhibiteur de l'ocytocine a pu être réduit par l'inhibition de la transmission GABAergique, alors que ses effets excitateurs n'étaient pas affectés. Dans un deuxième temps, nous avons identifié les mécanismes cellulaires responsables de l'effet excitateur de ces deux peptides et analysé les caractéristiques morphologiques et biochimiques des neurones affectés. Des enregistrements intracellulaires ont permis de localiser les neurones excités par l'ocytocine dans la partie latérale de l'amygdale centrale (CeL), et ceux excités par la vasopressine dans sa partie médiale (CeM). Le traçage morphologique des neurones a révélé que les collatérales axonales des cellules excitées par l'ocytocine projetaient du CeL loin dans le CeM. De plus, des colorations immuno-histochimiques ont révélé que ces projections étaient GABAergiques. Dans un troisième temps, nous avons étudié les interactions synaptiques entre ces deux populations de cellules. Les enregistrements en whole-cell patch-clamp dans le CeM ont démontré que les effets inhibiteurs de l'ocytocine résultaient de l'augmentation massive des courants GABAergique résultant de l'activation des neurones dans le CeL. Finalement, les effets de l'ocytocine et de la vasopressine sur l'activité évoquée ont été étudiés. Nous avons pu montrer que la probabilité d'évoquer un potentiel d'action dans le CeM, par stimulation de l'amygdale basolatérale, était augmentée sous l'effet de la vasopressine et diminuée sous l'action de l'ocytocine. Par contre, l'impact de la stimulation des afférences corticales sur les neurones du CeL était augmenté par l'application de l'ocytocine. L'ensemble de ces résultats nous a permis de développer un modèle dans lequel les effets comportementaux opposés de la vasopressine et de l'ocytocine sont causés par une activation sélective des deux différentes populations de neurones dans un réseau GABAergique. Un tel modèle pourrait mener au développement de nouveaux traitements anxiolytiques en modulant l'activité des deux récepteurs simultanément. En agissant sur un réseau GABAergique, les effets d'un tel traitement pourraient être rendus encore plus sélectifs en association avec des benzodiazépines classiques.