In der vorliegenden Dissertation wurden die Folgen einer SPRED2-Defizienz in einem Knockout Mausmodell untersucht. Dabei wurde insbesondere die mögliche Verbindung zur Zwangsstörung, einer psychiatrischen Erkrankung beleuchtet. Das SPRED2-Protein kommt im menschlichen Körper in zahlreichen Geweben vor, besonders im Hirn wurde eine ubiquitäre Expression nachgewiesen und ein Zusammenhang mit der Neurogenese und neuronaler Differenzierung vermutet. Seine regulatorische Funktion besteht in einer inhibitorischen Wirkung auf den BDNF/TrkB-ERK-Signalweg, welcher u.a. für die Transkription neuronaler Gene verantwortlich ist. Die verwendeten SPRED2-defizienten Mäuse wurden durch Insertion eines Gene-Trap Vektors in das Spred2-Gen generiert. Die Insertion verhindert letztendlich die korrekte Translation des Proteins. Von der durch weitere Verpaarung entstehenden SPRED2-Knockout Mauslinie wurden ausschließlich männliche Tiere verwendet. Im Rahmen einer SPRED2-KO-Studie von der AG Schuh des Physiologischen Instituts der Universität Würzburg, die u.a. die Entgleisung der HHNA mit resultierendem erhöhten Stresshormonspiegel und eine Dysregulation des Mineralhaushaltshormons Aldosteron zeigte, wurden bei den Versuchstieren zwanghafte Verhaltensmuster beobachtet. Daraufhin wurden elektrophysiologische Messungen durchgeführt, die auf eine Anomalie in der synaptischen Übertragung zwischen Thalamus und Amygdala hindeuteten. Erhöhte Effizienz und Erregbarkeit der amygdaloiden Neuronen führten zu der morphologischen Untersuchung, die im Rahmen dieser Arbeit durchgeführt wurden. Da die Afferenzen des Thalamus vorwiegend in den lateralen Kern der Amygdala projizieren, wurde zunächst dieser betrachtet. Ziel der Untersuchung war es, Erkenntnisse darüber zu erlangen, ob der Knockout des SPRED2-Proteins in Mäusen zu einer veränderten Morphologie der Neuronen der lateralen Amygdala führt. Falls dies der Fall sein sollte, könnte damit zumindest ansatzweise das zwanghafte Verhalten der SPRED2-defizienten Mäusen erklärt werden. Die Hirne der Versuchstiere wurden nach der Golgi-Cox-Imprägnierung nach Glaser und Van der Loos und der Einbettung in Celloidin in 150 μm dicke Scheiben geschnitten und anschließend mithilfe eines Hellfeld-Mikroskops und des Neurolucida-Systems analysiert. Quantitativ erfasst und analysiert wurden pyramidale Klasse 1-Neuronen der lateralen Amygdala inklusive absoluter Anzahl und Dichte der Spines an ihren Dendriten. Die Untersuchung zeigte bei SPRED2-KO-Mäusen eine signifikante Erhöhung der mittleren Länge des apikalen Dendriten in Branch order 3 und eine tendenzielle Erhöhung der Gesamtzahl der Spines an den Dendriten in Branch order 1-3 gegenüber den Wildtyp-Mäusen. Daraus lässt sich folgern, dass ein Knockout des SPRED2-Proteins sich auf die Morphologie der Neuronen der lateralen Amygdala auswirkt. Die erhöhte mittlere Länge des apikalen Dendriten in Branch order 3 und die tendenziell erhöhte Spine-Anzahl korrelieren mit der gesteigerten synaptischen Übertragung und Erregbarkeit an amygdaloiden pyramidalen Neuronen. Auf molekularer Ebene kann die Hyperaktivität der lateralen Amygdala als Folge der fehlenden Inhibition des BDNF/TrkB-ERK-Signalwegs und der dadurch veränderten Expression zahlreicher synaptischer Proteine diskutiert werden. Die veränderte Morphologie der Neuronen in der lateralen Amygdala kann eine Ursache für das zwanghafte Verhalten der Mäuse sein, jedoch ist anzunehmen, dass Zwangsstörungen nicht bloß eine monokausale Ursache haben. Diese Arbeit identifiziert SPRED2 als neuen Regulator der Morphologie und Aktivität von Synapsen und die Amygdala als wichtige Hirnregion bei der Entstehung von Zwangsstörungen. SPRED2 ist somit ein vielversprechender Angriffspunkt für andere und spezifischere Untersuchungen der Hirnfunktion und eine potenzielle genetische Ursache für weitere neurologische Erkrankungen. / In this present dissertation, the consequences of SPRED2-deficiency in a knockout mouse model have been investigated. In particular, the possible connection to the obsessive-compulsive disorder was examined. The SPRED2 protein is found in many tissues in the human body. Especially in the brain, ubiquitous expression was found and a connection to neurogenesis and neuronal differentiation was suspected. Its regulatory function is an inhibitory effect to the BDNF/TrkB-ERK signaling pathway, which amongst others is responsible for the transcription of neuronal genes. The SPRED2-deficient mice used were generated by insertion of a gene trap vector into the Spred2 gene. The insertion ultimately prevents the correct translation of the protein. From the SPRED2 knockout mouse line only male animals were used. As part of a SPRED2-KO study by AG Schuh of the Physiological Institute of the University of Würzburg, which showed, inter alia, the derailment of HHNA resulting in increased stress hormone levels and a dysregulation of the mineral household hormone aldosterone, obsessive behaviors were observed in the experimental animals. Subsequently, electrophysiological measurements were performed indicating an abnormality in synaptic transmission between thalamus and amygdala. Increased efficiency and excitability of the amygdaloid neurons led to the morphological investigation, which were accomplished in the context of this work. Since the afferents of the thalamus predominantly project into the lateral nucleus of the amygdala, it was first considered. The aim of the study was to find out if the knockout of the SPRED2 protein in mice leads to an altered morphology of neurons of the lateral amygdala. If so, it could at least somewhat explain the compulsive behavior of SPRED2-deficient mice. The brains of the test animals were cut into 150 μm slices and, after Golgi-Cox impregnation according to Glaser and Van der Loos, embedded in celloidin and then analyzed using a bright field microscope and the Neurolucida system. Quantitatively, pyramidal class 1 neurons of the lateral amygdala were recorded and analyzed, including the absolute number and density of the spines at their dendrites. The study showed a significant increase in the mean length of the apical dendrites in branch order 3 in SPRED2-KO mice and a tendency to increase the total number of spines on the dendrites in branch order 1-3 compared to the wild-type mice. It can be concluded that a knockout of the SPRED2 protein affects the morphology of the neurons of the lateral amygdala. The increased mean length of the apical dendrites in branch order 3 and the tendency to increased spine counts correlate with the increased synaptic transmission and excitability of amygdaloid pyramidal neurons. At the molecular level, the hyperactivity of the lateral amygdala may be discussed as a consequence of the lack of inhibition of the BDNF/TrkB-ERK pathway and the resulting altered expression of numerous synaptic proteins. The altered morphology of the neurons in the lateral amygdala may be a cause of the compulsive behavior of the mice, but it can be assumed that obsessive-compulsive disorder does not merely have a monocausal cause. This work identifies SPRED2 as a new regulator of morphology and activity of synapses and the amygdala as an important brain region in the development of obsessive-compulsive disorder. SPRED2 is thus a promising target for other and more specific studies of brain function and a potential genetic cause for other neurological disorders.

Thesis advisor: Gorica Petrovich / Novel foods and novel environments both impact consumption, but their interaction is poorly understood, especially how this interaction varies across habituation and by sex. Prior studies found that placement in a novel context suppressed consumption of a novel food across habituation in a two-choice paradigm with familiar food, and there were neural correlates in the amygdala of consumption under novelty during the first exposure. The current study extended these findings using a paradigm with only a novel food. We placed adult male and female rats in a novel or familiar environment and measured their consumption of a novel, palatable food across four habituation sessions and a final test session. We collected brain tissue after the test session to measure Fos induction with immunohistochemistry during the final exposure to novelty. Fos induction was measured in the central nucleus of the amygdala and the nuclei of the basolateral complex. We found that placement in a novel context suppressed consumption of a novel food at every time point. During the test, Fos induction was elevated in groups tested in the novel context in the medial part of the central nucleus and all nuclei of the basolateral complex except the anterior part of the basolateral nucleus despite the test being the fifth exposure to the novel stimuli. Parts of the central nucleus and nuclei of the basolateral complex showed sex-specific elevations in Fos induction in females regardless of the testing context. Correlations of Fos induction across regions showed that novel context tested groups had similarly elevated Fos induction throughout the central nucleus and basolateral complex, unlike their familiar context tested counterparts. Females had more correlations of Fos induction than males regardless of testing context. These results demonstrated that habituation to eating a novel food is prolonged in a novel environment compared to a familiar environment. Notably, Fos induction remained high in the novel context groups after multiple exposures to novelty. These behavioral and neural findings demonstrate that unfamiliar environments remain salient throughout the process of habituation. / Thesis (MA) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology and Neuroscience.

SUMOylation is a type of post-translational protein modification similar to ubiquitination and it involves the covalent attachment of a small ubiquitin-like modifier (SUMO) protein to the lysine residue of a target substrate. While there is strong evidence for the role of protein ubiquitination in the formation of fear-based memories, few studies have been conducted examining the role that SUMOylation plays in this same process. The amygdala is the main site of storage for emotional memories and there is strong evidence that protein ubiquitination is critical for fear memory formation in this region. However, it has not previously been studied whether protein SUMOylation in the amygdala is also involved in fear memory formation. Additionally, although there is evidence to support sex differences in ubiquitin signaling during fear memory formation in the amygdala, whether males and females differ in their need for protein SUMOylation during fear memory formation has not been investigated. We have found significant sex differences in protein SUMOylation in the amygdala both at baseline (rest) and during fear memory formation. Western blot analysis revealed higher resting levels of SUMOylated proteins in females when compared to males, though both sexes showed global increases following fear conditioning. A SUMOylation-specific proteomic analysis discovered that only females had increased protein targeting with SUMO following fear conditioning, with four proteins being identified that gained SUMOylation modifications, the main target being a heat shock protein. One heat shock protein in males was identified as having lower SUMOylation levels following fear conditioning. This suggests sex differences in the interaction and targeting of proteins by SUMOylation following fear conditioning. We also inhibited the function of the only E2 conjugase for SUMOylation, Ube2i, via siRNA in the amygdala and found impaired fear memory in males but enhanced fear memory in females, though the latter only occurred under high siRNA concentrations. Interestingly, western blot analysis revealed that knockdown of Ube2i caused an increase in protein SUMOylation levels in females but a decrease in males, indicating that compensation is likely occurring in females. This suggests that in females, protein SUMOylation may be critical for basal cellular functioning, which precludes us from directly determining its role in fear memory formation. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation. / Master of Science / SUMOylation is a modification of protein which plays a key role in various biological processes and is similar to the protein modification process called ubiquitination, which has been implicated in the formation of fear-based memories for traumatic events. Despite this and the established role of SUMOylation in genomic stability, cell proliferation, and migration, less is known about its role in the process of memory formation. Importantly, ubiquitination and SUMOylation of proteins often work in tandem to regulate cell signaling and recent evidence suggests that SUMOylation may also be involved in fear memory formation. However, the role of protein SUMOylation in regulating fear memory formation in the amygdala, the primary site of storage for emotional memories, has never been directly examined. Additionally, there is also a significant gap in the literature regarding whether sex differences exist for the requirement of protein SUMOylation in fear memory formation. We have found that there are significant differences between the sexes regarding protein SUMOylation during fear memory formation in the amygdala. Western blot analysis showed that females have higher resting (baseline) levels of SUMOylated proteins in the amygdala compared to males, though both sexes showed global increases in protein SUMOylation following fear conditioning. In addition, a proteomic analysis revealed that four proteins in females gained a SUMOylation modification following fear conditioning. In contrast, one protein was identified in males which lost a SUMOylation modification, together suggesting unique targeting of proteins by SUMOylation across sexes during fear memory formation. Further, when the function of an essential enzyme for protein SUMOylation was inhibited in vivo, fear memory in males was impaired but enhanced in females. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation.

No description available.

BACKGROUND: Mild cognitive impairment (MCI) is a clinical condition that is characterized by mild changes in cognition. The amnestic form of MCI (aMCI) primarily affects memory and is thought to represent a stage between healthy aging and Alzheimer’s disease (AD). The medial temporal lobe (MTL) and the limbic system are two areas of the brain that have been implicated in the amnestic form of MCI. While MCI represents a risk factor for AD, it does not always lead to dementias. Being a carrier of the APOE Ɛ4 allele has also shown to increase chances of progression from MCI to AD. OBJECTIVE: To determine whether the subnuclei of the amygdala, along with other specific regions within the MTL, can differentiate between cognitively normal individuals and age-matched subjects with aMCI. METHODS: T1-weighted magnetic resonance imaging (MRI) data from two sources, the Boston University Alzheimer’s Disease Center (BU-ADC) and the Alzheimer’s Disease Neuroimaging Initiative (ADNI), was compiled for cross-sectional analysis. 95 scans in total from 45 cognitively normal participants and 50 diagnosed with aMCI were analyzed and the volumes of interest were automatically generated by the developmental version of FreeSurfer v6.0. To evaluate how well the volumes could predict either group membership (i.e. control group or MCI group) or APOE Ɛ4 status (i.e. carrier or noncarrier), the variables were assessed by nominal logistic regression models. RESULTS: Six of the nine nuclei of the amygdala had significantly reduced volumes in the aMCI group compared to controls. The whole amygdala and the perirhinal cortex also demonstrated reduced volumes in the aMCI group compared to the control group. The whole amygdala was a good predictor of group membership (R2 = 0.1386, whole model test chi square = 18.21558, p = 0.0004), but none of the subnuclei were good predictors individually. A model containing the 9 nuclei, the entorhinal cortex, and the perirhinal cortex provided a good fit for predicting APOE Ɛ4 status fit (R2 = 0.3000, whole model test chi square = 36.29563, p = 0.0002) and the best predictor was the corticoamygdaloid transition area of the amygdala. CONCLUSIONS: The results of our study confirm previous findings of reduced whole amygdala volume and add to the limited literature of reduced perirhinal cortex and amygdaloid nuclei volumes in MCI compared to healthy controls. To the best of our knowledge, this was the first time the automatic segmentation atlas was used to analyze the volumes of nine subnuclei of the amygdala in a population of aMCI. Our model testing the volume of the whole amygdala accurately predicted aMCI subjects with 58% accuracy and controls with 70% accuracy; the accuracy rose to 69% when the entorhinal cortex and the perirhinal cortex were added to the model to predict aMCI subjects from controls. Additionally, the model for predicting APOE Ɛ4 status identified noncarriers of the allele at 85% accuracy. Future studies should consider increasing the sample size to better assess small ROIs and assess for differences in the separate hemispheres.

Local inhibitory microcircuit of amygdala is an active component in processing emotional information. Despite prominent evidence of its importance, our understanding of GABAergic cell types, their connectivity and role in amygdala network is limited. The aim of this thesis is to understand connectivity and physiology of two specific components of GABAergic microcircuit of amygdala: so-called intercalated cells and neuropeptide Y (NPY) expressing interneurons. Intercalated cells (ITCs) of the amygdala are clusters of GABAergic neurons that surround the basolateral complex of amygdala (BLA). There is growing evidence suggesting that ITCs are required for the expression of fear extinction. The main intercalated nucleus (Im) is the largest of the ITC clusters and could be also important for emotional processing. Using patch-clamp whole-cell recordings paired with subsequent anatomical analysis I described basic physiology and anatomy of neurons within the Im. I found that these neurons share common characteristics to earlier described neurons within the medial ITC cluster, yet they can be divided into three distinct groups. Next, I provided anatomical and functional evidence that Im neurons project to central and basal nucleus of amygdala and that they are reciprocally connected with medial and lateral ITCs clusters. I found that Im neurons receive excitatory inputs from BLA as well as cortex; next I verified that heterogeneous inputs do not interact with each other. I have shown that the Im neurons express both AMPA and NMDA receptors, suggesting that they may undergo NMDA-dependent plasticity. I have reported that dopamine hyperpolarizes Im neurons via dopamine receptor 1, therefore providing a cellular substrate for disinhibition of the amygdala at the systemic level. Thus, the Im is likely to be an additional site of integration of the distributed network underlying acquisition, expression and extinction of conditioned fear. In another project, I report novel interneuron type of the BLA and call it neurogliaform cell (NGFC) of amygdala. I used a mouse line expressing green fluorescent protein (GFP) under NPY promoter and patch clamp technique combined with pharmacology and electron microscope analysis. I performed paired recordings between presynaptic NPY-GFP positive (+) cells and postsynaptic principal neurons (PNs). Presynaptic NPY-GFP+ neurons display small soma and short dendrites embedded in a cloud of highly arborized axon. I showed that NPY-GFP+ cells are source of GABAA receptor-mediated slow inhibitory postsynaptic currents (IPSCs, decay time constant > 30 ms) evoked in PNs and in themselves (autapses). These slow IPSCs are known in literature as GABAA,slow. My results indicate that the slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. Physiologically-relevant in vivo firing re-played in NPY+-NGFCs in vitro evoked a transient depression of the IPSCs. Presynaptic GABAB receptors controlled the strength of this short-term plasticity. Interestingly, synaptic contacts made by NGFCs showed close appositions, without identifiable classical synaptic structures, between presynaptic boutons of the recorded cells and postsynaptic profiles. Thus, volume transmission of GABA is likely to be generated by this interneuron of the amygdala. NPY+-NGFC is a novel interneuron type of the BLA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Cocaine- and amphetamine-regulated transcript (CART), as its name implies, was initially identified as an upregulated transcript in response to psychostimulant administration. Consequently, it has been posited to play a role in psychostimulant abuse and dependence. Spurred on by the finding that a polymorphism in the CART gene was associated with alcoholism, we initiated studies designed to elucidate the role of CART peptide in alcohol dependence. We first investigated the functional significance of CART peptide in alcohol dependence in vivo using a CART KO mouse. We found that CART KO mice had a significant decrease in ethanol consumption that could not be attributed to differences in total intake, taste perception, metabolism, or sensitivity to ethanol. In vitro we found that CART peptide facilitated NMDA receptor-mediated currents in central amygdala neurons. Given the emerging role of CART peptide in anxiety and stress, we decided to examine basal and stress-induced anxiety behaviors in CART KO mice. Under basal and acute stress conditions, CART KO mice did not differ in anxiety-like behaviors from WT mice; however, in response to a stressor, CART KO mice exhibited a potentiated corticosterone response. Using chronic intermittent ethanol exposure (CIE), we tested CART KO and WT mice for common signs of ethanol dependence including an escalation of volitional consumption and the presence of withdrawal-induced anxiety. We further investigated glutamatergic neuroadaptations within the central amygdala of CART KO and WT mice following CIE exposure and early withdrawal. CIE increased ethanol consumption and anxiety-like behaviors in mice of both genotypes but to a lower extent in CART KO mice. Electrophysiologically, CIE enhanced spontaneous excitatory postsynaptic currents in both genotypes and decreased the probability of presynaptic release in WT mice only. We believe that these electrophysiological neuroadaptations contribute to the development of ethanol dependence and may mediate withdrawal-induced anxiety behaviors. Overall, these studies indicate a role for CART peptide in alcohol dependence and specifically in modulating ethanol withdrawal-induced anxiety. / text

Depression is a complex psychiatric illness that affects a large proportion of the population. Many researchers make use of preclinical animal models to study the behavioural and neurobiological characteristics of this disease. However, although a bias towards maladaptive thinking patterns and emotional responses is a cardinal symptom of depression, these symptoms have been rarely considered in preclinical models. One way to investigate maladaptive thinking is through the use of fear conditioning paradigms. Fear conditioning evaluates emotional memory by assessing a rodent’s ability to associate neutral cues with an aversive experience. It requires the activation of brain structures critically involved in emotion-related learning and memory processes, most notably the hippocampus and amygdala, to successfully learn the task. The primary goal of this dissertation was to gain a better understanding of the consequences of repeated corticosterone injections—a validated preclinical model of depression-- on emotionally driven behaviour, the involvement of the hippocampus and amygdala in mediating these behaviours, and whether the antidepressant, fluoxetine, can prevent the effects of corticosterone on these behaviours. To begin, in Chapter 2 I confirmed that the depressogenic effects of corticosterone in the forced swim test, which is a traditional behavioural assay for depression in rodents, are not due to procedural differences or non-specific motor effects. I then investigated the impact of repeated corticosterone injections on the learning and memory of delay and contextual fear conditioning. I examined whether altering the order in which rats recall context versus tone cued fear associations determines the magnitude of corticosterone’s effect on conditioned fear. I found that corticosterone dose-dependently increased freezing to contextual cues whereas freezing to tone cues was increased regardless of dose. Furthermore, the order of the presentation of context versus tone cues during recall determined whether corticosterone produced significant enhancements in freezing. In Chapter 4, I investigated whether neuronal activity in the hippocampus and amygdala after recall of contextual or tone cued fear was associated with the effects of corticosterone found in Chapter 3. Recall of contextual cues was associated with neuronal activity in specific sub regions of the amygdala without any observed changes in the hippocampus. In Chapter 5, I investigated whether repeated corticosterone injections would also enhance the learning and memory of trace fear conditioning, a task that is heavily reliant on the hippocampus. I found that corticosterone increased freezing during recall of trace cues and enhanced the acquisition of trace cues. The results from this chapter, taken together with the results from chapters 3 and 4, suggest that repeated corticosterone exposure readily enhances learning and memory processes that evoke emotional arousal. In Chapter 6, I asked whether repeated treatment with the antidepressant, fluoxetine, could prevent increased fear learning produced by repeated corticosterone injections. I found that fluoxetine decreased freezing behaviour in corticosterone rats during recall of tone cues. Overall, the results of this dissertation further our understanding of the effects of corticosterone on learning and memory tasks that evoke emotional arousal, support the use of fear conditioning as a measure of depression-like behaviour, and demonstrate that repeated corticosterone injections reliably produce a depressive phenotype in rats.

No description available.

Mevinphos (Mev) is an orgnophosphate insectide used for suicidal purposes in Taiwan; seizure and cardiovascular depression are commom syptoms observed in organophosphate-poisoned patients. The amygdala (AMG) is part of the limbic system and the basolateral nucleus of AMG (BLA) is one of the most seizure-prone brain structures. The central neucleus of AMG (CeA) is thought to play a central role in behavioral, physiological response and cardiovascular regulation. However, detailed mechanisms in Mev-induced seizure and cardiovascular depression by an action on AMG are lacking. Based on electroencephalographic (EEG) activity to indicate neuronal electrical activity and arterial blood pressure (AP) and heart rate (HR) to indicate cardiovascular responses, the present study investigated whether Mev acts on AMG to elicit seizure or cardiovascular depression. Microinjection of Mev into BLA of adult male Sprague-Dawley (SD) rats maintained under propofol anesthesia increased EEG activity in AMG, cortex and CA3 of hippocampus leading to seizure initiation; however AP, HR, respiration rate (RR) and the power density of low-frequency (LF) component of AP was not significantly changed. Microinjection of Mev into BLA also time-dependently increased protein level and mRNA of cytokines interleukin (IL)-12, IL-13, tumor suppressor factor alpha (TNF£\) and interferon gamma (IFN£^) and cyclooxygenase (COX) activity in AMG. Microinjection of Mev into CA3 induced less seizure activity in cortex and CA3 than that induced by microinjection of Mev into BLA. In addition, microinjection Mev into CA3 did not induce seizure in AMG. These results suggest that Mev acted on BLA to initiate limbic seizures. Intraperitoneal injection of muscarinic receptor antagonist atropine (ATR), which can pass the blood-brain barrier (BBB), activator of GABAergic neurotransmission midazolam (MDZ) or antiinflaamatory agent pentoxifylline (PTX) and Lisofylline (LSF), but not muscarinic receptor antagonist atropine methyl nitrate (AMN), which can not pass BBB, inhibited Mev-induced seizure and increase of cytokines in AMG by an action on BLA. Microinjection of ATR, COX-1 inhibitor naproxen (NPX) or COX-2 inhibitor NS-398, antiserum against receptor of IL-12, IL-13, TNF£\ or IFN£^, but not nicotinic receptor antagonist mecamylamine (MEL), into BLA inhibited Mev-induced seizure and increase of cytokines and COX activity in AMG by an action on BLA. However, caspase 3 activity and DNA fragmentation at AMG were not changed by microinjection of Mev into BLA. Microinjection of Mev into CeA induced a decrease in AP and RR leading to cardiovascular depression and an increase of power desity of LF, accompanied with insignificant HR and EEG activity change. Microinjection of Mev into CeA induced the time-dependently increase of caspase 3 activity and DNA fragmentation leading to apoptosis in AMG. Microinjection of ATR or caspase 3-dependent apoptosome inhibitor NS-3694, but not MEL, into CeA inhibited cardiovascular depression and the increase of caspase 3 activity and DNA fragmentation induced by Mev action on CeA. However, the levels of cytokines were not changed by Mev treatment. Intravenous injection of Mev did not induce changes of partial pressure of oxygen, blood flow and the level of superoxide anion in AMG. In addition, microinjection of Mev into BLA or CeA did not affect the level of superoxide anion in AMG. These results suggest that AMG mediates the initiation of seizure and cardiovascular depression induced by Mev. Furthermore, inflammation in BLA and apoptosis in CeA individually play an important role in Mev-induced seizure and cardiovascular depression.