Po-Wu Gean

A Role for the PI-3 Kinase Signaling Pathway in Fear Conditioning and Synaptic Plasticity in the Amygdala

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Valproic acid and other histone deacetylase inhibitors induce microglial apoptosis and attenuate lipopolysaccharide-induced dopaminergic neurotoxicity

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation using rodent primary neuron/glia or enriched glia cultures. Other histone deacetylase inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time- and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with SB or TSA caused a robust decrease in LPS-induced pro-inflammatory responses and protected DA neurons from damage in mesencephalic neuron-glia cultures. Taken together, our results shed light on a novel mechanism whereby HDACIs induce neuroprotection and underscore the potential utility of HDACIs in preventing inflammation-related neurodegenerative disorders such as Parkinsons disease.

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01-10 microM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC50 of 0.3 microM. In addition, the inhibitory effect induced by DA at a low concentration (0.1 microM) was antagonized by sulpiride (1-10 nM), a selective D2 dopaminergic receptor antagonist. However, D1 dopaminergic receptor antagonist SKF-83566 (1-5 microM) did not affect the blocking effect by DA 0.1 microM. Based on these findings, we conclude that DA at a low concentration (< or = 0.1 microM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D2, but not D1 dopaminergic receptors, located on the terminals of corticostriatal neurons.

The Role of the Amygdala in the Extinction of Conditioned Fear

The amygdala has long been known to play a central role in the acquisition and expression of fear. More recently, convergent evidence has implicated the amygdala in the extinction of fear as well. In rodents, some of this evidence comes from the infusion of drugs directly into the amygdala and, in particular, into the basolateral complex of the amygdala, during or after extinction learning. In vivo electrophysiology has identified cellular correlates of extinction learning and memory in the lateral nucleus of that structure. Human imaging experiments also indicate that amygdaloid activity correlates with extinction training. In addition, some studies have directly identified changes in molecular constituents of the basolateral amygdala. Together these experiments strongly indicate that the basolateral amygdala plays a crucial role in extinction learning. Interpreted in the light of these findings, several recent in vitro electrophysiology studies in amygdala-containing brain slices are suggestive of potential synaptic and circuit bases of extinction learning.

Extinction Training in Conjunction with a Partial Agonist of the Glycine Site on the NMDA Receptor Erases Memory Trace

Much evidence indicates that extinction training does not erase memory traces but instead forms inhibitory learning that prevents the expression of original memory. Fear conditioning induces long-term potentiation and drives synaptic insertion of AMPA receptors into the amygdala. Here we show that extinction training applied 1 h after training reversed the conditioning-induced increase in surface glutamate receptor subunit 1 (GluR1) in parallel with the inhibition of startle potentiation. However, if applied 24 h after training, extinction training reduced startle potentiation without influencing the GluR1 increase. We infused d-cycloserine (DCS), a partial agonist of the glycine site on the NMDA receptor, bilaterally into the amygdala 30 min before extinction training. This augmented the extinction training-elicited reduction in startle and reversed the conditioning-induced increase in GluR1. Delivery of five sets of tetanic stimulation (TS) to the external capsule produced a robust enhancement of synaptic responses in the lateral amygdala neurons that persisted for >2 h. Low-frequency stimulation applied 1 h after TS had no long-lasting effect on synaptic responses. The same treatments, however, induced depotentiation in the presence of DCS and reversed TS-induced increase in surface GluR1. Together, this study has two important findings: (1) whether a memory trace remains intact or is erased depends on the interval between conditioning and extinction training and (2) DCS facilitates the reversal of memory trace. DCS-induced augmentation of extinction and reversal of GluR1 surface expression are likely mediated by DCS-facilitated endocytosis of AMPA receptors.

Inhibition of N-type calcium currents by lamotrigine in rat amygdalar neurones

LAMOTRIGINE (LAG) is a new anticonvulsant drug for the treatment of partial and secondarily generalized seizures. The present study was aimed at elucidating the possible involvement of Ca2+ channels in the action of LAG using whole-cell patch clamp recordings in acutely dissociated amygdalar neurones. Whole-cell Ca2+ currents (ICa) were elicited by 200 ms step commands from −70 mV to −10 mV. Application of LAG reduced the ICa by an average of 40.3 ± 3.2%. The inhibition of ICa by LAG was markedly reduced or eliminated in the presence of the N-type Ca2+ channel blocker ω-cono-toxin-GVIA (1 μM). These results suggest that LAG may exert its anticonvulsant effect through inhibition of presynaptic N-type Ca2+ channels, thereby reducing glutamate release.

Serotonin depresses excitatory synaptic transmission and depolarization-evoked Ca2+ influx in rat basolateral amygdala via 5-HT1A receptors.

The actions of serotonin on rat basolateral amygdala neurons were studied with conventional intracellular recording techniques and fura‐2 fluorimetric recordings. Bath application of 5‐hydroxytryptamine (5‐HT or serotonin) reversibly suppressed the excitatory postsynaptic potential in a concentration‐dependent manner without affecting the resting membrane potential and neuronal input resistance. Extracellular Ba2+ or pertussis toxin pretreatment did not affect the depressing effect of 5‐HT suggesting that it is not mediated through activation of Gi/o protein‐coupled K+ conductance. The sensitivity of postsynaptic neurons to glutamate receptor agonist was unaltered by the 5‐HT pretreatment. In addition, the magnitude of paired‐pulse facilitation was increased in the presence of 5‐HT indicating a presynaptic mode of action. The effect of 5‐HT was mimicked by the selective 5‐HT1A agonist 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT) and was blocked by the selective 5‐HT1A antagonist 1‐(2‐methoxyphenyl)‐4[4‐(2‐phthalimido)butyl]piperazine oxadiazol‐3‐yl]methyl]phenyl]methanesulphonamide. In contrast, the selective 5‐HT2 receptor antagonist ketanserin failed to affect the action of 5‐HT. The effects of 5‐HT and 8‐OH‐DPAT on the high K+‐induced increase in [Ca2+]i were studied in acutely dissociated basolateral amygdala neurons. High K+‐induced increase in [Ca2+]i was blocked by Ca2+‐free solution and Cd2+ suggesting that Ca2+ entry responsible for the depolarizaton‐evoked increase in [Ca2+]i occurred through voltage‐dependent Ca2+ channels. Application of 5‐HT and 8‐OH‐DPAT reduced the K+‐induced Ca2+ influx in a concentration‐dependent manner. The effect of 5‐HT was completely abolished in slices pretreated with Rp‐cyclic adenosine 3′,5′‐monophosphothioate (Rp‐cAMP), a regulatory site antagonist of protein kinase A, suggesting that 5‐HT may act through a cAMP‐dependent mechanism. Taken together, these results suggest that functional 5‐HT1A receptors are present in the excitatory terminals and mediate the 5‐HT inhibition of synaptic transmission in the amygdala.

Regulation of amygdala-dependent learning by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol-3-kinase.

This study is designed to characterize the signal cascades by which brain-derived neurotrophic factor (BDNF) modulates long-term memory of fear conditioning. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis of tissue homogenates taken from fear-conditioned rats showed an increase in the amygdala of BDNF protein levels and its receptor TrkB phosphorylation. Bilateral administration of a TrkB ligand scavenger TrkB IgG and a Trk-specific tyrosine kinase inhibitor K252a to the amygdala impaired fear memory, as measured with fear-potentiated startle. Fear conditioning resulted in the association of Shc and TrkB, Shc and Ras, the increase in active Ras and phosphorylation of mitogen-activated protein kinase (MAPK). Treatment of amygdala slices with BDNF for 15 min increased the levels of active Ras, and MAPK and Akt phosphorylation. BDNF-induced MAPK phosphorylation was completely abolished by MEK inhibitors, and was partially inhibited by farnesyltransferase or phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors. On the other hand, BDNF-induced Akt phosphorylation was unaffected by farnesyltransferase or MEK inhibitors, but could be blocked by PI-3 kinase inhibitors. Together, these data suggest a requirement of BDNF for fear learning. The memory-enhancing effect of BDNF involves the activation of MAPK and PI-3 kinase. BDNF-induced MAPK phosphorylation in the amygdala is mediated via TrkB and the Shc-binding site. Shc binding to TrkB leads to activation of Ras, Raf, and MEK. In addition, BDNF could induce phosphorylation of MAPK via activation of PI-3 kinase.

The Role of Prefrontal Cortex CB1 Receptors in the Modulation of Fear Memory

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.

Transcriptional regulation of brain-derived neurotrophic factor in the amygdala during consolidation of fear memory.

We have demonstrated previously that brain-derived neurotrophic factor (BDNF) signaling in the amygdala is required for the consolidation of fear memory. This study is designed to characterize the signal cascades by which fear conditioning modulates transcriptional and translational expression of BDNF. Real-time reverse transcription-coupled polymerase chain reaction showed a significant increase in BDNF exon I- and III-containing mRNA in the amygdala of fear-conditioned rats, indicating that fear conditioning was capable of up-regulating BDNF mRNA. Bilateral administration of actinomycin D or anisomycin to the amygdala attenuated conditioning-induced increase in BDNF protein. Inhibitors for N-methyl-d-aspartate (NMDA) receptor, L-type voltage-dependent calcium channel (L-VDCC), adenylyl cyclase, cAMP-dependent protein kinase (PKA), and calcium/calmodulin-dependent kinase IV (CaMKIV) significantly reduced the increase. Moreover, DNA affinity precipitation and chromatin immunoprecipitation assays showed that phosphorylated cAMP response element-binding protein (p-CREB) binding activity in the proximal region of BDNF promoter I and III was significantly increased after fear conditioning. Intra-amygdala administration of cAMP response element decoy DNA before training impaired fear learning. Taken together, these results suggest that calcium influx through NMDA receptors and L-VDCCs during fear conditioning activates PKA and CaMKIV resulting in CREB phosphorylation. The phosphorylated CREB binds to BDNF promoter and up-regulates the expression of BDNF in the amygdala, which helps the consolidation of fear memory.