| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 90 === Learning and memory is the basis for the survival and development of creatures, however, aversive experience of fear is subject to anxiety. Therefore, The problems of anxiety can be solved by an understanding of emotional fear in terms of its underlying cellular and molecular mechanisms.
Accumulated evidence indicates that the amygdala is a crucial neuronal locus for the induction and expression of fear memory. And amygdaloid neurons are in excitatory states in fear conditioing rats. On the other hand, Ca2+ channels play an important role in excitatory neurons and the concentration of intracellular Ca2+ regulates the activation state of signalling factors.Therefore, all the modulatory factors which are involved in fear conditioning mechanisms will be investigated in this project. Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter in the mammalian peripheral and central nervous system. And buspirone used in clinic treatment is also a 5-HT1A receptor partial agonist. Previous studies showed that 5-HT could inhibit the fEPSP via the 5-HT1A receptors in the amygdaloid neurons, and the fear conditioning rats could be released after intraventricular infusion of 5-HT into the amygdala locus. Thus, the modulation of voltage-dependent calcium currents (Ica) by 5-HT hereby will be studied and rat acutely dissociated amygdala neurons will be investigated by using whole-cell patch-clamp recording techniques. The results show that 5-HT inhibits Ica in a concentration-dependent manner with a ED50 of ~ 1mM and a maximal inhibition of ~ 50%.And we found thatThe inhibition is mediated by 5-HT1A receptors. Pretreatment of neurons with the alkylating agent NEM or pertussis toxin markedly reduce the action of 5-HT. The modulation is partially reversed by strong depolarization and is not seen in cell-attached patches when the agonist is applied outside the recorded patch, suggesting a membrane-delimited G-protein-mediated signaling pathway. And the results show that P/Q-type and N-type VDCCs are mainly involved in the action of inhibition by 5-HT.
Stimulation of b-adrenergic receptor with isoproternol or activation of adenylyl cyclase with forskolin results in an enhancement of Ica.However, these results provide the first evidence showing a dominant effect of 5-HT-mediated inhibition over Iso-mediated enhancement of Ica.
Continuously, the intracellular signaling pathway will be studied further. Recently, it has been suggested that the changes of synaptic transmission in the LA are initiated by an influx of Ca++ into cells through NMDA receptors or L-type Ca++ channels, leading to the activation of protein kinases.However,PI3- kinase is one of the intracellular protein kinases that catalyzes the transfer of r-phosphate of ATP to the D-3 hydroxyl group of the inositol head group of phosphoinositides and also been suggested to participate in nerve growth factor (NGF) - and glial cell line-derived neurotropic factors (GDNFs) – mediated survival of sympathetic and spinal cord motoneurons. Although a recent study implied that PI3- kinase may play a role in the expression of LTP in hippocampal dentate gyrus, data conflicted as it was found that PI3- kinase -deficient mice displayed an enhanced hippocampal CAI LTP but with normal spatial memory. Therefore , fear conditioning rats will be used for identifying the role of PI3- kinase in fear memory formation.
Western blotting and extracellular recordings are applied with pharmacological techniques in the experiments. And the results show that PI-3 kinase is selectively activated in the amygdala following fear conditioning and pharmacological blockade of PI3-kinase impairs fear memory in a dose-dependent manner. In in vitro slice preparation, it shows that bath application of PI-3 kinase inhibitors attenuate tetanus-induced L-LTP in the amygdala. Therefore, a novel role of PI-3 kinase in fear conditioning and synaptic plasticity has been revealed here and the results also show that PI-3 kinase contributes to L-LTP and fear memory likely via the activation of MAPK and CREB. And additional immunohistochemical evidence also prove the role of PI-3 kinase in fear conditioning.
Fear extinction mechanism is important as well as fear conditioning mechanism .However, previous studies show that PI-3 kinase is an important role in the acquistion of fear, whether it also plays an essential role in fear extinction will be investigated further?As we know, animals that contact a cue (conditioned stimulus, CS), when repeatedly paired with a noxious stimulus (unconditioned stimulus, US), induce a fear response. However, after initial training, if the animals are exposed only to the cus (CS) without pairing with US, previously acquired response will gradually disappear. This reduction of conditioned response is known as fear extinction. However, the data show that the activation of PI-3 kinase is abated in fear-extinction rats, it shows that PI-3 kinase has been dephosphorylated. The result suggests that phosphatase seems involved in the event. Therefore, calcineurin, the common calcium-dependent neuronal phosphatase, will be investigated and the data show a significant increase in the expression of protein phosphatase calcineurin(including activities and protein expression). Another data show that bilateral infusion of calcineurin or protein synthesis inhibitors into the amygdala prevents the fear extinction. Thus, a signaling pathway played by calcineurin during extinction trials is important for the reversal or destabilization of original fear memory.
Taken together, all the results show that 5-HT can inhibit the Ca2+ currents by the regulation of N- and P/Q- type Ca2+ channels.And PI3-kinase plays an important role in the pathways of fear formation and fear extinction.On the other hand, calcineurin is also revealed as an important fear- supreesor factor. The findings of all the mechanisms are beneficial for pharmacological intervention of anxiety and posttraumatic stress disorders, on the other hand, they may provide some insight into the long-term memory formation in the brain.
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