Letteratura scientifica selezionata sul tema "Fear-potentiated startle"

Ascending 5-HT projections from the median raphe nucleus (MRN), probably to the hippocampus, are implicated in the acquisition of contextual fear (background stimuli), as assessed by freezing behavior. Foreground cues like light, used as a conditioned stimulus (CS) in classical fear conditioning, also cause freezing through thalamic transmission to the amygdala. As the MRN projects to the hippocampus and amygdala, the role of this raphe nucleus in fear conditioning to explicit cues remains to be explained. Here we analyzed the behavior of rats with MRN electrolytic lesions in a contextual conditioning situation and in a fear-potentiated startle procedure. The animals received MRN electrolytic lesions either before or on the day after two consecutive training sessions in which they were submitted to 10 conditioning trials, each in an experimental chamber (same context) where they. received foot-shocks (0.6 mA, 1 sec) paired to a 4-sec light CS. Seven to ten days later, the animals were submitted to testing sessions for assessing conditioned fear when they were placed for five shocks, and the duration of contextual freezing was recorded. The animals were then submitted to a fear-potentiated startle in response to a 4-sec light-CS, followed by white noise (100 dB, 50 ms). Control rats (sham) tested in the same context showed more freezing than did rats with pre- or post-training MRN lesions. Startle was clearly potentiated in the presence of light CS in the sham-lesioned animals. Whereas pretraining lesions reduced both freezing and fear-potentiated startle, the post-training lesions reduced only freezing to context, without changing the fear-potentiated startle. In a second experiment, neurotoxic lesions of the MRN with local injections of N-methyl-D-aspartate or the activation of5-HT1Asomatodendritic auto-receptors of the MRN by microinjections of the5-HT1Areceptor agonist 8-hydroxy- 2-(di-n-propylamino)tetralin (8-OH-DPAT) before the training sessions also reduced the amount of freezing and the fear-potentiated startle. Freezing is a prominent response of contextual fear conditioning, but does not seem to be crucial for the enhancement of the startle reflex by explicit aversive cues. As fear-potentiated startle may be produced in posttraining lesioned rats that are unable to freeze to fear contextual stimuli, dissociable systems seem to be recruited in each condition. Thus, contextual fear and fear-potentiated startle are conveyed by distinct 5-HT-mediated circuits of the MRN.

The present thesis explored the neurobiological basis of three aspects of defense behaviors in humans. Positron emission tomography methodology was used, and changes in regional cerebral blood flow (rCBF) were measured as an index of neural activity. Firstly, brain function was studied in a group of patients suffering from combat-related posttraumatic stress disorder, using a symptom provocation paradigm with combat sounds in order to elicit fear. Exposure to auditory trauma reminders relative to neutral sounds was associated with increased rCBF in sensorimotor areas, the cerebellar vermis, the periaqueductal gray matter, and the right amygdala, whereas decreased activity was observed in the retrosplenial area of the posterior cingulate cortex. Secondly, the neural circuitry mediating the acoustic startle response and its habituation was studied in a group of healthy subjects. During acoustic startle stimulation as compared to a resting condition, increased rCBF was found in a medial posterior area of the pons corresponding to the nucleus reticularis pontis caudalis. As a result of startle repetition, altered activity was found in the cerebellum, pointing to its involvement in startle habituation. Thirdly, neural activity associated with startle modulation by phobic fear was studied in a group of subjects with specific animal phobias during exposure to pictures of their feared and non-feared objects, paired and unpaired with acoustic startle stimuli. As a result of startle potentiation, increased rCBF was found in the left amygdaloid-hippocampal region, and medially in the affective division of the anterior cingulate cortex. In conclusion, these results provide evidence for the involvement of limbic and paralimbic brain areas during fear provocation and fear-potentiated startle and for a similar neurocircuitry underlying startle in humans and animals.

Eleven experiments examined two late-developing learning and memory systems in rats: fear-potentiated startle (FPS) and the contextual regulation of latent inhibition and extinction. The first study was based on three previous developmental findings on FPS: (1) FPS to an odour CS emerges at postnatal day (PN) 23; Rats conditioned at PN16 to an odour CS express freezing but not FPS when tested at PN23, and (3) FPS to an odour CS trained at PN16 is activated if rats are also trained to a difference odour at PN23 (Yap, Stapinski, & Richardson, 2005). Yap et al. (2005) hypothesised that the activation effect only occurs if rats are given training to the second odour at an age when FPS has emerged. Study 1 assessed this hypothesis and trained the second odour CS at either PN23 or PN20. Contrary to expectations, the results of this study showed the activation effect for both groups of rats. Surprisingly, the results also revealed a significant FPS effect to the odour CS trained at PN20. Subsequent experiments examined this unexpected result, and found that learning to odour 1 at PN16 facilitated the age of onset for FPS at PN20. The results of Study 1 are discussed in relation to past findings on enrichment, cumulative learning, and neurobiological models of conditioned fear. The second section of this thesis (Studies 2 and 3) examined the context-specificity of two memory interference paradigms, latent inhibition and extinction, in developing rats. The studies found that both phenomena were context-specific at PN23-25 but not at PN16-18. Moreover, the results suggest that the context-specificity of both latent inhibition depended on the age of the rat during the second phase of training, but not their age during the first phase of training or their age at test. The implications of these findings for theoretical and neural models of learning, as well as the occurrence of latent inhibition and extinction during development are discussed.

The basolateral amygdala (BLA) contains NMDA, AMPA, and dopamine (DA) D1 and D2 receptors and neurobiochemical events within the amygdala mediate conditioned-fear-learning (CFL) and fear expression. Long-term potentiation (LTP) occurs in the amygdale during Pavlovian fear conditioning and is associated with fear-memory storage. CFL depends on NMDA, AMPA and dopaminergic receptor-mediated processes and enhanced amygdaloidal synaptic transmission facilitates fear-memory retrieval and makes the expression of conditioned fear possible. Since mesoamygdaloid DA receptors contribute to CFL and fear expression, Experiment 1A and 1D examined the impact of intra-BLA infusion of the DA D1 and D2 receptor antagonists SCH 23390 and raclopride L-tartrate on the acquisition of FPS in rats. Rats of the Wistar strain were bilaterally implanted with guide cannulae positioned 1.0 mm above the medial portion of the BLA. Approximately fourteen days later rats were assessed for baseline acoustic startle responding and assigned to drug-treatment groups. Forty-eight hours later rats were infused with either saline or the appropriate dopaminergic antagonist. The intra-BLA infusions occurred before five fear conditioning and testing blocks and were conducted to see if antagonism of DA receptors would prevent FPS acquisition. Retention testing for FPS took place forty-eight hours later. The results demonstrated that blockade of amygdaloid DA D1 and D2 receptors prevented the acquisition of FPS. The pretraining intra-BLA infusions of either raclopride or SCH 23390 disrupted the formation of long-term conditioned fear memories as rats treated with these DA antagonists failed to exhibit FPS on the retention test. Thus, the deficits in FPS displayed by SCH 23390 and raclopride-treated rats are likely due to the impact of these DA antagonists on associative learning and fear-memory consolidation processes. Experiment 2 demonstrated that fear-extinguished rats exposed to unsignalled footshocks displayed a reinstatement of FPS, but the exact neurobiochemicaI events involved in FPS reinstatement have not been elucidated. In contrast, fear-extinguished rats that received no unsignalled footshocks exhibited no FPS during final testing. Since unsignalled footshocks produced robust FPS reinstatement, Experiments 3A to 3D independently examined the effects of NMOA, AMPA, and DA D2 and D1 receptor antagonists on this phenomenon. Over a period of ten days, rats with cannulae targeting the BLA were base lined, fear-conditioned, pretested, fear-extinguished and then infused with either raclopride L-tartrate (8.0µg, 4.0 µg and 2.0µg), SCH 23390 (4.0 µg), (±)-2-amino-5-phosphonopentanoic acid {(APS); 2.5 µg and 1.25 µg}, 6-Cyno-7-nitroquinoxaline-2,3-dione disodium {(CNQX); 5.0 µg and 2.5 µg), or phosphate buffered saline (PBS) before exposure to five unsignalled footshocks. FPS reinstatement was assessed 24 hours later. Results from these experiments demonstrate that PBS-infused rats showed FPS reinstatement, whereas rats infused with AP5, CNQX, or the two higher doses of raclopride failed to exhibit FPS reinstatement. Intra-BLA SCH 23390 infusions did not appear to disrupt the reinstatement of FPS in Experiment 3B, however obstructed guide cannulae may have affected these results. In Experiment 3C, intra-BLA AP5 infusions made just before unsignalled footshock presentation, prevented rats from exhibiting FPS reinstatement during final testing. A similar effect on FPS reinstatement was produced by CNQX application to the BLA of rats in Experiment 3D. The overall findings of Experiment 3 suggest that DA D2 receptor antagonism and the glutamatergic receptor antagonists (AP5 and CNQX) impaired amygdaloid fear-memory reconsolidation and retrieval processes by preventing the re-excitation of neurons and pathways that had become established during fear-conditioning. It is speculated that these drugs may have interfered with excitatory synaptic transmission processes and neurobiological intracellular cascades within the amygdala and thus prevented FPS reinstatement from occurring. Two expression-control experiments (Experiments 4 and 5), revealed that the observed blockade of FPS reinstatement in Experiment 3 could not be attributed to the drugs simply blocking fear expression since infusion of raclopride, AP5, or CNQX into the BLA of non-extinguished rats 24 hours before final testing did not prevent rats from expressing FPS. Electrical stimulation (ES) of the human amygdala and temporal lobe region produces emotionally charged memory flash-backs and behaviours indicative of a central fear-state. ES of the rat amygdala is known to elevate acoustic startle amplitudes and enhance emotionality in rats and kindling of the rat amygdala exaggerates FPS and produces a variety of autonomic and behavioural fear responses. In rats, conditioned fear and LTP are induced by ES of amygdaloid afferents so it is possible that electrical excitation of BLA neurons can trigger FPS reinstatement. Experiments 7A and 7B were conducted to test the hypothesis that ES of BLA neurons can restore FPS responding in fear-extinguished rats. Thus, rats with bipolar electrodes implanted unilaterally in the BLA were baselined, fear conditioned, pretested and then assigned to one of five groups matched on FPS. Rats then received fear-extinction or no-extinction training, followed 48 hours later by either 100 unsignalled electrical stimulations of the BLA or no stimulation. Experimental groups included; [Extinction + Stimulation (N=12), Extinction + No stimulation (N=12), No Extinction + No stimulation (N= 12), No Extinction + Stimulation (N=11) and Extinction + Stimulation out of context (N=12)]. FPS reinstatement was assessed 24 hours later and BLA AD-current threshold were recorded four days after FPS reinstatement testing was completed. Experiment 7A demonstrated that all experimental groups exhibited FPS except the Extinction + No stimulation control group which displayed a robust extinction effect. Most importantly, the Extinction + Stimulation group exhibited statistically significant FPS reinstatement after ES of the amygdala. Experiment 7B demonstrated that ES of the amygdala in a context different to the startle apparatus also produced FPS reinstatement in fear-extinguished rats. However, the magnitude of this FPS reinstatement effect was much smaller than that obtained when rats received ES of the amygdala in the startle testing apparatus. The overall finding that ES of the BLA causes FPS reinstatement in fear-extinguished rats suggests that ES of the BLA resensitised fear-memory systems and restored FPS responding. This effect was likely achieved by exciting amygdaloid neurons and pathways containing the memory-trace of the CS-UCS association originally established during Pavlovian fear conditioning. Experiments 8 to 10 used the same protocol as Experiment 7A and investigated whether ES of other brain regions that share reciprocal connections with the amygdala would trigger FPS reinstatement in fear-extinguished rats. Rats in Experiments 8 to 10 had bipolar electrodes unilaterally implanted in the perirhinal/insular cortex (PRh/IC), dorsal periaqueductal gray (dPAG), ventral tegmental area (VTA) and ventral periaqueductal gray (vPAG). Experiment 8 revealed that ES of the PRh/IC produced a positive trend towards FPS reinstatement. Experiment 9 examined the impact of dPAG and VTA ES on FPS reinstatement. This experiment demonstrated that VTA but not dPAG ES produced a FPS reinstatement effect. This finding seems to support the research evidence that highlights the importance of mesoamygdaloid systems in mediating conditioned fear and stress responses. Experiment 10 assessed the impact of vPAG ES on FPS reinstatement and discovered that stimulation of this region did not trigger a restoration of FPS in fear-extinguished rats but it did seem to elevate overall acoustic startle responding during final testing, This finding would seem to indicate that ES of the vPAG likely increased contextual fear but not cue specific fear since acoustic startle amplitudes during both the noise-alone and the CS + noise conditions were elevated, The key finding that ES of the amygdala produces robust FPS reinstatement provides further proof that the amygdala and its afferent and efferent neural circuits are essential for fear-memory reconsolidation and conditioned fear expression and reinstatement.

O colículo inferior (CI) é uma estrutura envolvida primariamente com o processamento da informação acústica, porém participa também na integração dos aspectos sensoriais, autonômicos e comportamentais da reação de defesa frente a situações de ameaça. Além disso, essa estrutura apresenta alta concentração de receptores dopaminérgicos, sendo a dopamina um dos neuromoduladores mais ativos em mecanismos subjacentes a estados de medo e ansiedade. Desta forma, o objetivo do presente estudo foi avaliar o papel dos receptores dopaminérgicos (D1 e D2) do CI na expressão de respostas defensivas incondicionadas ou condicionadas. Para tanto, ratos Wistar machos (±270g, n=186) passaram por cirurgia estereotáxica para implante bilateral de cânulas-guia direcionadas ao CI. Esses animais receberam administração intra-CI de quimpirole (agonista D2), sulpirida (antagonista D2), SKF 38393 (agonista D1) ou SCH 23390 (antagonista D1), em diferentes doses, e foram submetidos aos testes do labirinto em cruz elevado (LCE) e ao campo aberto. Uma dose de sulpirida foi avaliada também no teste do sobressalto potencializado pelo medo (SPM). Quanto à avaliação das respostas defensivas no teste do LCE, foi observado que apenas a sulpirida diminuiu as entradas e o tempo despendido nos braços abertos, ou seja, causou um efeito pró-aversivo. As outras drogas não influenciaram essas respostas defensivas. Um comprometimento no desempenho motor foi observado pela administração intra-CI de quimpirole (diminuição nas entradas nos braços fechados) e de SCH 23390 (diminuição da locomoção no campo aberto). Quanto aos efeitos de sulpirida sobre as respostas defensivas no teste do SPM, nenhuma influência sobre a resposta de amplitude do sobressalto e sobre o congelamento foi constatada. Os dados obtidos apontam para o envolvimento da modulação dopaminérgica por meio de receptores da família D2 no CI na expressão de respostas incondicionadas de medo. Dopamina no CI parece, portanto, ser importante para regular a expressão dessas respostas. Por outro lado, não obtivemos evidências de que tal modulação no CI esteja envolvida na expressão de respostas condicionadas de medo. Portanto, a neurotransmissão dopaminérgica no CI sobre a expressão de respostas defensivas parece ocorrer por meio de receptores da família D2, com ação seletiva na modulação de respostas incondicionadas de medo.
The inferior colliculus (IC) is a structure primarily involved in acoustic information processing, but it also participates in the integration of the sensory, autonomic and behavioral aspects of the defensive reaction to threatening situations. Furthermore, this structure has a high concentration of dopamine receptors, and dopamine is one of the most active neuromodulators in the mechanisms underlying states of fear and anxiety. Thus, the aim of this study was to evaluate the role of IC dopamine receptors (D1 and D2) in the expression of unconditioned and conditioned defensive responses. For this purpose, male Wistar rats (±270 g, n=186) were implanted with bilateral guide cannuli directed to the IC. These animals received intraIC quinpirole (D2 agonist), sulpiride (D2 antagonist), SKF 38393 (D1 agonist) or SCH 23390 (D1 antagonist) at different doses, and were tested in the elevated plus maze (EPM) and the open field tests. A single dose of sulpiride was also evaluated in the fear potentiated startle test (FPS). In the EPM test, it was observed that only sulpiride decreased the numbers of entries and time spent in the open arms of the maze, suggesting an ansiogenic-like effect. The other drugs did not influence these defensive responses. Impairment in motor performance was observed with intraIC quimpirole (decrease in closed arm entries) and SCH 23390 (decrease in locomotion in the open field test). In the FPS test, no significant effects in the amplitude of the startle response and freezing behavior were observed. The data point to an involvement of IC dopaminergic D2-like receptors in the expression of unconditioned fear responses. Dopamine in the IC, therefore, seems to be important for regulating the expression of these responses. On the other hand, there was no evidence that this modulation in the IC is involved in the expression of conditioned fear responses. Therefore, the influence of the dopaminergic neurotransmission in the IC on the expression of defensive responses appears to occur via D2-like receptors, which selectively modulate unconditional fear responses.

O aumento do reflexo de sobressalto na presença de um estímulo que tenha sido previamente pareado a choques nas patas é tomado como índice de medo e nomeado sobressalto potencializado pelo medo (SPM). O congelamento, interrupção de todos os movimentos observáveis, exceto aqueles associados com a respiração, também tem sido utilizado como índice de medo em ratos. Um crescente número de evidências sugere o envolvimento de mecanismos dopaminérgicos em diferentes aspectos da memória afetiva, como sua formação, evocação e expressão. No entanto, resultados sobre como e por meio de quais receptores os mecanismos dopaminérgicos influenciam o medo têm sido inconsistentes. O presente estudo examina o envolvimento dos receptores dopaminérgicos na aquisição e na expressão do medo condicionado à luz. Para isso, foram analisados os efeitos do antagonista D1, SCH 23390, do agonista D1, SKF 38393, do antagonista D2, sulpirida, e do agonista D2, quimpirole, no SPM e no congelamento. A atividade motora dos animais também foi avaliada no teste do campo aberto. SCH 23390, SKF 38393, sulpirida e quimpirole, administrados antes do condicionamento, não produziram efeitos no SPM, mas SCH 23390 diminuiu o congelamento. As administrações de SCH 23390, SKF 38393 e sulpirida antes do teste também não produziram efeitos no SPM e no congelamento. Quimpirole, em doses que agem em receptores pré-sinápticos, causou uma redução significativa no SPM e no congelamento, quando administrado antes do teste. A ação das drogas não foi devida a efeitos não-específicos uma vez que elas não produziram efeitos no teste do campo aberto. Os resultados sugerem que mecanismos dopaminérgicos devem estar envolvidos tanto na aquisição, quanto na expressão do medo condicionado à luz. Receptores D1 pós-sinápticos parecem participar da aquisição do congelamento condicionado à luz-CS, mas não do SPM. Por outro lado, receptores D2 pré-sinápticos parecem estar envolvidos na expressão do medo condicionado à luz-CS.
The increase in the startle reflex in the presence of a stimulus that has been previously paired to footshock is taken as an index of fear and named fear potentiated startle (FPS). Freezing behavior, a cessation of all observable movements, except those associated with respiration, has also been used as an index of fear in rats. A growing body of evidence has suggested that dopaminergic mechanisms are implicated in different aspects of affective memory, namely its formation, expression or retrieval. However, the results of studies that have examined how, and through which receptors, dopaminergic mechanisms influence fear have been inconsistent. This work is aimed at examining the involvement of dopaminergic receptors in the acquisition and expression of conditioned fear to ligth-CS. We evaluated the effects of systemic administration of the D1 antagonist, SCH 23390, the D1 agonist, SKF 38393, the D2 antagonist, sulpiride, and the D2 agonist, quinpirole before and after conditioning on FPS and freezing. The motor activity of the animals was also evaluated in an open field test. SCH 23390, SKF 38393, sulpiride and quinpirole, injected before conditioning sessions, did not produce any effect on FPS, but SCH 23390 decreased freezing. Injections of SCH 23390, SKF 38393 and sulpiride before testing session did not produce any effect on FPS or freezing. Quinpirole, injected at doses acting at presynaptic level, caused significant reduction in FPS and freezing, when injected before testing. Drugs’ action was not due to nonspecific effects since they had no effect in the open field test. Our findings indicate that DA mechanisms are involved in the acquisition and expression of conditioned fear using light-CS. Dopaminergic mechanisms mediated by postsynaptic D1 receptors seem to be involved in the acquisition of conditioned freezing to light-CS, but not in FPS. On the other hand, dopaminergic mechanisms mediated by presynaptic D2 receptors seem to be involved in the expression of conditioned fear to light-CS.

OLIVEIRA, A.R. Envolvimento de receptores dopaminérgicos da área tegmental ventral e do complexo basolateral da amígdala na aquisição e na expressão do medo condicionado. 2010. 93 f. Tese (Doutorado) Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. O condicionamento Pavloviano é um dos paradigmas mais utilizados para estudar as bases biológicas das emoções, assim como da aprendizagem e memória. A dopamina (DA) é um dos principais neurotransmissores envolvidos na mediação de estados de medo e ansiedade. Um conjunto crescente de evidências dá suporte à hipótese de que a ativação da via mesocorticolímbica, proveniente de neurônios dopaminérgicos da área tegmental ventral (ATV), é particularmente sensível à estimulação aversiva. Entre as regiões inervadas por esta via, o complexo basolateral da amígdala (BLA) é um componente essencial dos circuitos neurais do medo condicionado. Assim, o presente estudo explorou o envolvimento de mecanismos DA da ATV e do BLA, através do uso de agonistas e antagonistas de receptores DA, na aquisição e expressão do medo condicionado à luz. Não houve efeito das drogas DA no sobressalto potencializado pelo medo (SPM), quando injetadas na ATV antes do condicionamento, indicando que os receptores DA da ATV não participam da aquisição do medo condicionado à luz. Ao contrário, quando injetado na ATV antes do teste, quimpirole (agonista D2) reduziu o SPM, enquanto as demais drogas não tiveram efeito. A administração de SCH 23390 (antagonista D1) no BLA não produziu efeitos no SPM, indicando que os receptores D1 do BLA não parecem envolvidos na expressão do SPM. Já a administração de sulpirida (antagonista D2) no BLA inibiu o SPM produzido pela luz. Além disso, a expressão do medo condicionado foi associada a um aumento do congelamento e dos níveis extracelulares de DA no BLA, ambos inibidos com a administração de quimpirole na ATV. A capacidade do quimpirole em diminuir o SPM e o congelamento condicionado parece ser resultado de sua ação em auto-receptores D2 da ATV. A ativação desses receptores diminui os níveis de dopamina em áreas que recebem terminações da via mesocorticolímbica. Os resultados com a sulpirida realçam a importância dos receptores D2 do BLA na expressão do medo condicionado Pavloviano.
OLIVEIRA, A.R. Involvement of dopaminergic receptors of ventral tegmental area and basolateral amygdala in the acquisition and expression of conditioned fear. 2010. 93 p. Thesis (Doctoral) Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. The Pavlovian fear conditioning is one of the most used paradigms to study the biological basis of emotion, as well as of learning and memory. Dopamine (DA) is one of the most important neurotransmitters involved in mechanisms underlying states of fear and anxiety. A growing body of evidence supports the hypothesis that excitation of the mesocorticolimbic pathway, originating from DA neurons in the ventral tegmental area (VTA), is particularly sensitive to fear-arousing stimuli. Among the forebrain regions innervated by this pathway, the basolateral amygdala (BLA) is an essential component of the neural circuitry of conditioned fear. The present study explored the involvement of VTA and BLA DA receptors, using DA agonists and antagonists, in the acquisition and expression of conditioned fear to a light conditioned stimulus (CS). None of the drugs used produced significant effects on fear-potentiated startle (FPS) when injected in VTA before conditioning, indicating that VTA DA receptors are not involved in the acquisition of conditioned fear to a light-CS. In contrast, when injected before the test session, intra-VTA quinpirole (D2 agonist) significantly reduced FPS, whereas the other drugs had no effect. Intra-BLA SCH 23390 (D1 antagonist) did not produce significant effects on FPS, indicating that BLA D1 receptors do not appear to be involved in the expression of FPS. On the other hand, intra-BLA sulpiride (D2 antagonist) inhibited FPS produced by light-CS previously paired with footshocks. Also, conditioned fear was associated with increased freezing and DA levels in the BLA, both inhibited by intra-VTA quinpirole. Quinpirole\'s ability to decrease FPS and conditioned freezing may be the result of an action on VTA D2 presynaptic autoreceptors. The activation of those receptors decreases dopamine levels in terminal fields of the mesocorticolimbic pathway. Sulpirides results stress the importance of BLA D2 receptors in the fear-activating effects of the Pavlovian conditioning.

A substância cinzenta periaquedutal dorsal (dPAG) e o hipotálamo medial (MH) são duas estruturas encefálicas que estão envolvidas na elaboração de estados aversivos e expressão de respostas defensivas. A estimulação elétrica da dPAG ou do MH produz uma série de respostas comportamentais que se assemelham às respostas defensivas induzidas pela presença de um predador. Esses mesmos comportamentos podem ser eliciados com a microinjeção local de agonistas glutamatérgicos nessas estruturas, indicando o envolvimento de aminoácidos excitatórios na expressão das respostas defensivas incondicionadas. Apesar disso, a participação destas estruturas no medo condicionado ainda é pouco conhecida. Assim, o objetivo deste estudo foi avaliar o envolvimento da mediação glutamatérgica da dPAG e de núcleos do MH núcleo anterior (AH) e núcleo pré-mamilar dorsal (PMd) na expressão do medo condicionado à luz. Para isso, foram avaliados os efeitos de agonistas e antagonistas glutamatérgicos (AMPA/Cainato e NMDA) administrados nessas estruturas no teste do sobressalto potencializado pelo medo (SPM) e na medida de congelamento condicionado. Ratos Wistar machos com cânulas-guias implantadas na dPAG, AH ou PMd foram submetidos ao condicionamento aversivo (pareamentos luz+choque). Vinte e quatro horas depois, esses animais receberam injeções intra-dPAG, AH ou PMd de NMDA ou ácido caínico (agonistas NMDA e AMPA/Cainato, respectivamente) ou AP7 ou NBQX (antagonistas NMDA e AMPA/Cainato, respectivamente) e foram submetidos ao teste do SPM. A resposta de congelamento condicionado foi avaliada na mesma sessão. Eventuais alterações motoras foram avaliadas no teste do campo aberto. A administração dos agonistas glutamatérgicos na dPAG promoveu efeitos pró-aversivos no SPM e congelamento condicionado. NBQX sozinho não produziu nenhum efeito significativo, ao passo que o AP7 diminuiu somente o congelamento condicionado. Entretanto, ambos os antagonistas bloquearam os efeitos dos respectivos agonistas. Já a administração dos agonistas e antagonistas glutamatérgicos no AH e PMd, em doses que não afetaram a atividade motora, não produziu efeitos significativos na resposta de congelamento condicionado e SPM. Os presentes resultados sugerem a participação de aminoácidos excitatórios da dPAG, mas não do MH, na expressão do medo condicionado à luz.
The dorsal periaqueductal gray matter (dPAG) and the medial hypothalamus (MH) are two brain structures that are involved in the elaboration of aversive states and expression of defensive responses. Electrical stimulation of the dPAG or MH produces a series of behavioral responses that resemble those defensive responses triggered in the presence of a predator. These same behaviors can be elicited with the local microinjection of glutamate agonists into these structures, indicating the involvement of excitatory amino acids in the expression of unconditioned fear responses. Nevertheless, the involvement of these structures in fear conditioning is still unknown. The objective of this study was to evaluate the involvement of glutamatergic mediation of the dPAG and MH nuclei anterior nucleus (AH) and dorsal pre-mammillary nucleus (PMd) in the expression of conditioned fear to the light. Thus, we evaluated the effects of glutamatergic agonists and antagonists (AMPA/Kainate and NMDA) administered into these structures in fear potentiated startle (FPS) and conditioned freezing responses to the light. Male Wistar rats with guide-cannulae implanted in the dPAG, AH or PMd were subjected to aversive conditioning (light+shock pairings). Twenty-four hours later, the animals were injected intra-dPAG, AH or PMd with NMDA or kainic acid (NMDA and AMPA/Kainate agonists, respectively) or AP7 or NBQX (NMDA and AMPA/Kainate antagonists, respectively) and were subjected to the FPS test. The conditioned freezing response was measured in the same session. Potential motor effects were evaluated with the open-field test. The administration of glutamate agonists into the dPAG promoted pro-aversive effects in the FPS and conditioned freezing. NBQX produced no significant effect per se, whereas AP7 only decreased conditioned freezing. Both antagonists blocked the effects of the respective agonist. On the other hand, the administration of glutamatergic agonists and antagonists into AH and PMd, in doses that did not affect motor activity, produced no significant effects on conditioned fear responses. The present results suggest the involvement of mechanisms mediated by excitatory amino acids of the dPAG, but not of the MH, in the expression of conditioned fear responses to light.

博士
國立成功大學
基礎醫學研究所
93
Learning and memory is the basis for the survival and development of creatures, however, aversive experience of fear leads to anxiety. With the changes of the society, anxious complications happen commonly in the modern life. Therefore, the problems of anxiety can be solved by an understanding of emotional fear in terms of its underlying cellular and molecular mechanisms. 　　NF-kB, originally identified as a regulator of immunoglobulin k light chain gene expression, is a DNA-binding factor that functions as a dimer. Recent studies indicate that NF-kB played an important role in the synaptic plasticity. Therefore, we used fear-potentiated startle paradigm to identify the role of NF-kB signaling pathway in memory formation. The results show that p50 and p65 subunits of NF-kB were selectively activated in the amygdala following fear conditioning through the signal-induced activation of PI-3 kinase, IKK and subsequent proteolytic degradation of IkB-a in the cytoplasm. This allows NF-kB to translocate into the nucleus where it binds to specific�nkB DNA consensus sequences in the enhancer region of�n�羠-responsive genes. Pharmacological blockade of NF-kB impairs fear memory in a dose-dependent manner. In in vitro slice preparation, bath application of kB decoy DNA attenuates tetanus-induced L-LTP in the amygdala. Therefore, a novel role of NF-kB in fear conditioning and synaptic plasticity has been demonstrated here. 　　CBP/p300 contains histone acetyltransferase that has been implicated in the regulation of gene expression. Recent studies show that the action of NF-kB is regulated by reversible acetylation. We found that the expression of acetyl-p65 subunit was selectively increased in the amygdala following fear conditioning through the increase associated with CBP (CREB binding protein). Pharmacological blockade of histone deacetylase further increase DNA binding activity of NF-kB and fear memory in a dose-dependent manner. In in vitro slice preparation, bath application of histone deacetylase inhibitor increases the degree of forskolin-induced L-LTP in the amygdala. Therefore, a novel role of NF-kB in fear conditioning and synaptic plasticity has been demonstrated. These results suggest that HDAC-mediated deacetylation functions as an intranuclear molecular switch culminating in the termination of NF-kB transcriptional response. 　　AMPA receptors mediate the majority of the fast excitatory synaptic transmission. One recently identified mechanism contributing to synaptic plasticity is the regulated trafficking of AMPA receptors in and out of synapses. Receptors with long cytoplasmic tails (GluR4/2 and GluR1/2) are driven into synapses in an activity-dependent manner. Our results show that synaptoneurosome membrane expression of GluR1 was selectively increased in the amygdala following fear conditioning through the signal-induced activation of PI-3 kinase, NMDA receptor and NF-kB. Pharmacological blockade of histone deacetylase further increases conditioning-induced membrane expression of GluR1 in NF-kB-dependent manner. Furthermore, fear training-induced increase in GluR1 was reversed when animal was exposed to the memory extinction protocol. The reversal of GluR1 increase was also blocked by D-APV and anisomycin treatment. The similar pattern of changes in GluR1 was observed in the amygdala slices after delivery of high-frequency stimulation (HFS) or HFS followed by low-frequency stimulation (LFS) that elicited long-term potentiation (LTP) and depotentiation respectively. These results suggest that long-term synaptic plasticity and memory formation are correlated with the changes in modification of GluR1 expression, and surface expression of GluR1 is a potential effector that contributes at least in part to the expression of fear memory.

碩士
國立成功大學
藥理學研究所
91
Accumulated evidence indicate that the amygdala is a crucial neuronal locus for the induction and expression of fear memory. Fear conditioning induces associative long-term potentiation in the amygdala and protein phosphorylation is a powerful regulator of long-term synaptic plasticity. Protein phosphorylation is also a direct and rapid modulator of exocytosis in neurons. Previous studies indicate that protein kinases play an important role in mediating the release of synaptic vesicles from the reserve pool and the synaptic-vesicle-associated phosphoprotein, synapsin I, is a key kinase substrate in this process. Hence, my study is to prove whether fear conditioning induces phosphorylation of synapsin I and whether this effect is subjected to regulate by memory extinction. In this study, I have shown that synapsin I is phosphorylated after fear training and phosphorylated synapsin I is subsequently dephosphorylated when animals receive extinction training. Previous studies in our laboratory demonstrated that L-CCG-induced long-term depotentiation ( LTD ) leads to a long-lasting decrease in transmitter release which was induced by presynaptically group II metabotropic glutamate receptor ( mGluRII )-mediated inhibition of Ca2+-sensitive adenylyl cyclase, resulting in a decrease in cAMP formation and PKA activation. Therefore, I used the mGluRII agonist, (2S, 2R,3R)-2-(2,3-Dicarboxycyclopropyl)glycine ( DCG-IV ), to examine its effect on the phosphorylation of synapsin I and used the antagonist, LY341495, to test whether it could reverse the effect of DCG-IV. The results revealed that DCG-IV decreased the phosphorylation of synapsin I that was reversed by LY341495 . Finally, I determined the different phosphorylation site of synapsin I in fear conditioning and found that the phosphorylation of PKA/CaMKI phosphorylation site, CaMKII phosphorylation site, and cdk5 phosphorylation site were increased following fear conditioning. By using the specific kinase inhibitor to block synapsin I phosphorylation, I found that these inhibitors reduced the phosphorylation of different synapsin I phosphorylation site and inhibited the fear-potentiated startle.