RESUMO
Voltage-independent, Ca2+-activated K+ channels (KCa2.2, previously named SK2) are typically activated during a train of action potentials, and hence, are powerful regulators of cellular excitability by generating an afterhyperpolarizing potential (AHP) following prolonged excitation. In the acute in vitro epilepsy model induced in hippocampal brain slice preparations by exposure to the GABAA receptor blocker gabazine (GZ), the AHP was previously shown to be significantly decreased. Here, we asked the question whether KCa2.2 protein degradation occurs in this model and which pathways are involved. To this end, we applied either gabazine alone or gabazine together with inhibitors of proteasomal and lysosomal protein degradation pathways, Z-Leu-Leu-Leu-CHO (MG132) and chloroquine (CQ), respectively. Western blot analysis showed a significant decrease of total KCa2.2 protein content in GZ-treated slices which could be rescued by concomitant incubation with MG132 and CQ. Using HEK293 cells transfected with a green fluorescent protein-tagged KCa2.2 construct, we demonstrated that proteasomal rather than lysosomal degradation was involved in KCa2.2 reduction. We then recorded epileptiform afterdischarges at hippocampal Schaffer collateral-CA1 synapses and confirmed that the GZ-induced increase was significantly attenuated by both MG132 and CQ, with MG132 being significantly more effective than CQ. Epileptiform afterdischarges were almost prevented by co-application of protein degradation inhibitors. Furthermore, epileptiform afterdischarges could be re-established by using the KCa2.2 blocker UCL 1684 suggesting involvement of KCa2.2. We conclude that in GZ-induced acute epilepsy, KCa2.2 degradation by proteasomal rather than lysosomal pathways plays a major role in the generation of epileptiform afterdischarges.
Assuntos
Potenciais de Ação/fisiologia , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Piridazinas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Antagonistas GABAérgicos/farmacologia , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Masculino , Técnicas de Cultura de Órgãos , Proteólise/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Spatial learning and associating spatial information with individual experience are crucial for rodents and higher mammals. Hence, studying the cellular and molecular cascades involved in the key mechanism of information storage in the brain, synaptic plasticity, has led to enormous knowledge in this field. A major open question applies to the interdependence between synaptic plasticity and its behavioral correlates. In this context, it has become clear that behavioral aspects may impact subsequent synaptic plasticity, a phenomenon termed behavioral metaplasticity. Here, we trained control and pilocarpine-treated chronically epileptic rats of two different age groups (adolescent and adult) in a spatial memory task and subsequently tested long-term potentiation (LTP) in vitro at Schaffer collateral-CA1 synapses. As expected, memory acquisition in the behavioral task was significantly impaired both in pilocarpine-treated animals and in adult controls. Accordingly, these groups, without being tested in the behavioral training task, showed reduced CA1-LTP levels compared to untrained young controls. Spatial memory training significantly reduced subsequent CA1-LTP in vitro in the adolescent control group yet enhanced CA1-LTP in the adult pilocarpine-treated group. Such training in the adolescent pilocarpine-treated and adult control groups resulted in intermediate changes. Our study demonstrates age-dependent functional metaplasticity following a spatial memory training task and its reversal under pathological conditions.
Assuntos
Região CA1 Hipocampal/fisiopatologia , Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Comportamento Animal/fisiologia , Epilepsia/induzido quimicamente , Potenciação de Longa Duração/fisiologia , Pilocarpina , Ratos , Ratos Wistar , Memória Espacial/fisiologiaRESUMO
N-Methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) can be reversed by low-frequency stimulation (LFS) referred to as depotentiation (DP). We previously found GluN2B upregulated in CA1 neurons from post-status epilepticus (post-SE) tissue associated with an enhanced LTP. Here, we tested whether LFS-induced DP is also altered in pathological GluN2B upregulation. Although LTP was enhanced in post-SE tissue, LTP was significantly reversed in this tissue, but not in controls. We next tested the effect of the GluN2B subunit-specific blocker Ro 25-6981 (1 µM) on LFS-DP. As expected, LFS had no effect on synaptic strength in the presence of the GluN2B blocker in control tissue. In marked contrast, LFS-DP was also attained in post-SE tissue indicating that GluN2B was obviously not involved in depotentiation. To test for NMDA receptor-dependence, we applied the NMDA receptor antagonist D-AP5 (50 µM) prior to LFS and observed that DP was abolished in both control and post-SE tissue confirming NMDA receptor involvement. These results indicate that control Schaffer collateral-CA1 synapses cannot be depotentiated after fully established LTP, but LFS was able to reverse LTP significantly in post-SE tissue. However, while LFS-DP clearly required NMDA receptor activation, GluN2B-containing NMDA receptors were not involved in this form of depotentiation.
Assuntos
Hipocampo/fisiopatologia , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Receptores de N-Metil-D-Aspartato/fisiologia , Estado Epiléptico/fisiopatologia , Animais , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Fenóis/administração & dosagem , Pilocarpina , Piperidinas/administração & dosagem , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Estado Epiléptico/induzido quimicamente , Regulação para CimaRESUMO
Hyperpolarization-activated, cyclic nucleotide-gated nonselective (HCN) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity. We compared the influence of HCN channels on long-term depression (LTD) at the medial perforant path-granule cell synapse in early postnatal (P9-15) and adult (P30-60) rats. LTD was elicited in P9-15 slices using low-frequency stimulation (LFS, 900 pulses, 1 Hz; 80 ± 4% of baseline). Application of the specific HCN channel blocker ZD7288 (10 µM) before LFS significantly enhanced LTD (62 ± 4%; P < 0.01), showing HCN channels restrain LTD induction. However, when ZD7288 was applied after LFS, LTD was similar to control values and significantly different from the values obtained with ZD7288 application before LFS (81 ± 5%; P < 0.01), indicating that HCN channels do not modulate LTD expression. LTD in slices from adult rats were only marginally lower compared to those in P9-15 slices (85 ± 6%), but bath application of ZD7288 prior to LFS resulted in the same amount of LTD (85 ± 5%). HCN channels in adult tissue hence lose their modulatory effect. In conclusion, we found that HCN channels at the medial perforant path-granule cell synapse compromise LFS-associated induction, but not expression of LTD in early postnatal, but not in adult, rats.
Assuntos
Cardiotônicos/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/fisiologia , Via Perfurante/efeitos dos fármacos , Pirimidinas/farmacologia , Sinapses/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Técnicas In Vitro , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
PURPOSE: Epilepsy therapy is currently based on anti-seizure drugs that do not modify the course of the disease, i.e., they are not anti-epileptogenic in nature. Previously, we observed that in vivo casein kinase 2 (CK2) inhibition with 4,5,6,7-tetrabromotriazole (TBB) had anti-epileptogenic effects in the acute epilepsy slice model. METHODS: Here, we pretreated rats with TBB in vivo prior to the establishment of a pilocarpine-induced status epilepticus (SE) in order to analyze the long-term sequelae of such a preventive TBB administration. RESULTS: We found that TBB pretreatment delayed onset of seizures after pilocarpine and slowed down disease progression during epileptogenesis. This was accompanied with a reduced proportion of burst firing neurons in the CA1 area. Western blot analyses demonstrated that CA1 tissue from TBB-pretreated epileptic animals contained significantly less CK2 than TBB-pretreated controls. On the transcriptional level, TBB pretreatment led to differential gene expression changes of KCa2.2, but also of HCN1 and HCN3 channels. Thus, in the presence of the HCN channel blocker ZD7288, pretreatment with TBB rescued the afterhyperpolarizing potential (AHP) as well as spike frequency adaptation in epileptic animals, both of which are prominent functions of KCa2 channels. CONCLUSION: These data indicate that TBB pretreatment prior to SE slows down disease progression during epileptogenesis involving increased KCa2 function, probably due to a persistently decreased CK2 protein expression.
RESUMO
PURPOSE: We present a case of voltage-gated potassium channel (VGKC) complex antibody-positive limbic encephalitis (LE) harboring autoantibodies against Kv1.2. Since the patient responded well to immunotherapy, the autoantibodies were regarded as pathogenic. We aimed to characterize the pathophysiological role of this antibody in comparison to an antibody against the VGKC-associated protein contactin-associated protein-2 (CASPR2). METHODS: Stereotactic injection of patient sera (anti-Kv1.2-associated LE or anti-CASPR2 encephalopathy) and a control subject was performed into the hippocampus of the anesthetized rat in vivo, and hippocampal slices were prepared for electrophysiological purposes. Using extra- and intracellular techniques, synaptic transmission, long-term potentiation (LTP) and vulnerability to pro-epileptic conditions were analyzed. RESULTS: We observed that the slope of the field excitatory postsynaptic potential (fEPSP) was significantly increased at Schaffer collateral-CA1 synapses in anti-Kv1.2-treated and anti-CASPR2-treated rats, but not at medial perforant path-dentate gyrus synapses. The increase of the fEPSP slope in CA1 was accompanied by a decrease of the paired-pulse ratio in anti-Kv1.2, but not in anti-CASPR2 tissue, indicating presynaptic site of anti-Kv1.2. In addition, anti-Kv1.2 tissue showed enhanced LTP in CA1, but dentate gyrus LTP remained unaltered. Importantly, LTP in slices from anti-CASPR2-treated animals did not differ from control values. Intracellular recordings from CA1 neurons revealed that the resting membrane potential and a single action potential were not different between anti-Kv1.2 and control tissue. However, when the depolarization was prolonged, the number of action potentials elicited was reduced in anti-Kv1.2-treated tissue compared to both control and anti-CASPR2 tissue. In contrast, polyspike discharges induced by removal of Mg2+ occurred earlier and more frequently in both patient sera compared to control. CONCLUSION: Patient serum containing anti-Kv1.2 facilitates presynaptic transmitter release as well as postsynaptic depolarization at the Schaffer-collateral-CA1 synapse, but not in the dentate gyrus. As a consequence, both synaptic transmission and LTP in CA1 are facilitated and action potential firing is altered. In contrast, anti-CASPR2 leads to increased postsynaptic potentials, but without changing LTP or firing properties suggesting that anti-Kv1.2 and anti-CASPR2 differ in their cellular effects. Both patient sera alter susceptibility to epileptic conditions, but presumably by different mechanisms.
RESUMO
A common function of group III metabotropic glutamate receptors (mGluRs) located at the presynaptic site of a glutamatergic synapse is synaptic depression. Here, we studied synaptic depression mediated by group III mGluR activation at Schaffer collateral-CA1 (SC-CA1) synapses and associational-commissural-CA3 (AC-CA3) synapses by recording field excitatory postsynaptic potentials in the in vitro brain slice preparation. In order to gauge the impact of synaptic depression in chronically epileptic tissue, we compared rats after pilocarpine-induced status epilepticus (post-SE) with control animals. We observed that synaptic transmission at control AC-CA3 synapses was sensitive to the group III mGluR agonist L-AP4 (10µM), while there was no effect of this compound at SC-CA1 synapses in the same tissue. In contrast, synaptic depression at AC-CA3 synapses by L-AP4 was lost in chronically epileptic tissue, and we found a significant synaptic depression at SC-CA1 synapses in post-SE tissue by L-AP4 and by the mGluR8-selective agonist DCPG. The depression by L-AP4 and DCPG in CA1 was also demonstrated in immature control tissue suggesting developmental down-regulation of mGluR8 at this synapse as well as re-appearance of this isoform under pathological conditions. Quantitative real-time RT-PCR was used to identify mGluR isoforms and to assess their transcriptional changes in post-SE tissue. These analyses revealed down-regulation of mGluR4 and mGluR6 at AC-CA3 and up-regulation of mGluR8 at SC-CA1 synapses. We conclude that group III mGluR-mediated synaptic depression is differentially altered in chronically epileptic tissue by a bidirectional shift of the transcriptional level.
Assuntos
Epilepsia/metabolismo , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Animais , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Masculino , Neurotransmissores/farmacologia , Ratos Wistar , Sinapses/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Purpose: Autoantibodies against NMDA receptors (NMDAR) in the cerebrospinal fluid (CSF) from anti-NMDAR encephalitis patients have been suggested to be pathogenic since in previous studies using patient CSF, NMDAR-dependent processes such as long-term potentiation (LTP) were compromised. However, autoantibodies may represent a family of antibodies targeted against different epitopes, and CSF may contain further autoantibodies. Here, we tested the specificity of the autoantibody by comparing NMDAR-dependent and NMDAR-independent LTP within the same hippocampal subfield, CA3, using CSF samples from four anti-NMDAR encephalitis patients and three control patients. Methods: We performed a stereotactic injection of patient-derived cell-free CSF with proven presence or absence of NMDAR-antibodies into the rat hippocampus in vivo. Hippocampal brain slices were prepared 1-8 days after intrahippocampal injection, and NMDAR-dependent LTP at the associational-commissural (A/C) fiber-CA3 synapse was compared to NMDAR-independent LTP at the mossy fiber (MF)-CA3 synapse. Results: The LTP magnitude at A/C fiber-CA3 synapses in slices from control-CSF-treated animals (168 ± 8% n = 54) was significantly higher than LTP in slices from NMDAR-CSF-treated animals (139 ± 9%, n = 40; P = 0.015), although there was some variation between the individual CSF samples. We found residual LTP in NMDAR-CSF-treated tissue which could be abolished by the NMDAR inhibitor D-AP5. Moreover, the CA3 field excitatory postsynaptic potential (fEPSP) was followed by epileptiform afterpotentials in 5% of slices (4/78) from control-CSF-treated animals, but in 26% of slices (12/46) from NMDAR-CSF-treated animals (P = 0.002). Application of the LTP-inducing paradigm increased the proportion of slices with epileptiform afterpotentials, but D-AP5 significantly reduced the occurrence of epileptiform afterpotentials only in NMDAR-CSF-treated, but not in control tissue. At the MF synapse, no significant difference in LTP values of control-CSF and in NMDAR-CSF-treated tissue was observed indicating that NMDAR-independent MF-LTP is intact in NMDAR-CSF-treated tissue. Conclusion: These findings indicate that anti-NMDAR containing CSF impairs LTP at the A/C fiber-CA3 synapse, although there is substantial variation among CSF samples suggesting different epitopes among patient-derived antibodies. The differential inhibition of LTP at this synapse in contrast to the MF-CA3 synapse suggests the specificity and underlines the pathophysiological role of the NMDAR-antibody.
RESUMO
Autoimmune encephalitis is increasingly recognized in patients with otherwise unexplained encephalopathy with epilepsy. Among these, patients with anti-N-methyl D-aspartate receptor (NMDAR) encephalitis present epileptic seizures, memory deficits, and psychiatric symptoms. However, the functional consequences of such autoantibodies are poorly understood. In order to investigate the pathophysiology of this disease, we stereotactically injected either cerebrospinal fluid (CSF) from three anti-NMDAR encephalitis patients or commercially available anti-NMDAR1 into the dentate gyrus of adult female rats. Control animals were injected with either CSF obtained from three epilepsy patients (ganglioglioma, posttraumatic epilepsy, focal cortical dysplasia) lacking anti-NMDAR or saline. Intracellular recordings from dentate gyrus granule cells showed a significant reduction of the NMDAR-evoked excitatory postsynaptic potentials (NMDAR-EPSPs) in animals treated with anti-NMDAR. As a consequence of this, action potential firing in these cells by NMDAR-EPSPs was significantly impaired. Long-term potentiation in the dentate gyrus was also significantly reduced in rats injected with anti-NMDAR as compared to control animals. This was accompanied by a significantly impaired learning performance in the Morris water maze hidden platform task when the animals had been injected with anti-NMDAR antibody-containing CSF. Our findings suggest that anti-NMDAR lead to reduced NMDAR function in vivo which could contribute to the memory impairment found in patients with anti-NMDAR encephalitis.
Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato/complicações , Autoanticorpos/administração & dosagem , Giro Denteado/efeitos dos fármacos , Transtornos da Memória/etiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Adulto , Animais , Encefalite Antirreceptor de N-Metil-D-Aspartato/líquido cefalorraquidiano , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Humanos , Potenciação de Longa Duração , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Técnicas EstereotáxicasRESUMO
OBJECTIVE: To investigate the risk factors and clinical characteristics of deep venous thrombosis (DVT) and pulmonary thromboembolism (PTE). METHOD: The clinical manifestations and risk factors in 388 patients with DVT were retrospectively studied. RESULTS: The ratio of DVT cases in hospitalized patients had a trend of increasing in recent years. 80.4% of the patients with DVT had risk factors. The most common risk factors were age over 40 years old (88.9%), heart disease (43.0%), hypertension (35.1%), long term immobilization (21.1%), and infection (20.1%). The most common clinical presentations of DVT were swelling (66.2%) and pain (40.5%) of the affected extremity. Calf swelling by more than or equal to 1 cm compared with the asymptomatic leg was found in 87.2% patients. The site of DVT was more common in left extremities and proximal veins. PTE was found in 34.5% of the DVT cases and 74.6% of them were diagnosed prior to DVT. The ratio of PTE caused by DVT didn't show significant difference between left and right legs, neither between proximal and distal veins. CONCLUSIONS: The ratio of DVT cases in hospitalized patients was increasing. Most patients with DVT had risk factors. DVT was usually asymptomatic before PTE was complicated.