De Luca Pasqualino, Mele Miranda, Tanqueiro Sara, Napoli Francesca, Butkevičiūtė Ugné, Souto Arthur C, Costa Rui O, Schwarz Alexander, Drexel Meinrad, Sebastião Ana M, Diógenes Maria J, Duarte Carlos B
CNC-UC - Center for Neuroscience and Cell Biology, and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal.
Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal.
J Biomed Sci. 2025 Sep 1;32(1):82. doi: 10.1186/s12929-025-01164-4.
Brain-derived neurotrophic factor (BDNF) is a key mediator of synaptic plasticity and memory formation in the hippocampus. However, the BDNF-induced alterations in the glutamate receptors coupled to the plasticity of glutamatergic synapses in the hippocampus have not been elucidated. In this work we investigated the putative role of GluN2B-containing NMDA receptors in the plasticity of glutamatergic synapses induced by BDNF.
The effects of BDNF on the surface expression of GluN2B-containing NMDA receptors was investigated in cultured hippocampal neurons and in hippocampal synaptoneurosomes by immunocytochemistry under non-permeabilizing conditions, using an antibody that binds to an extracellular epitope. Long term potentiation of hippocampal CA1 synapses was induced by using θ-burst stimulation. Epileptic seizures were induced using the Li-pilocarpine model of temporal lobe epilepsy. Pyk2 phosphorylation was assessed by western blot with a phosphospecific antibody.
Stimulation of hippocampal synaptoneurosomes with BDNF led to a significant time-dependent increase in the synaptic surface expression of GluN2B-containing NMDA receptors as determined by immunocytochemistry with colocalization with pre- (vesicular glutamate transporter) and post-synaptic markers (PSD-95). Similarly, BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors at the synapse in cultured hippocampal neurons, by a mechanism sensitive to the PKC inhibitor GӦ6983. The effects of PKC may be mediated by phosphorylation of Pyk2, as suggested by western blot experiments analyzing the phosphorylation of the kinase on Tyrosine 402. GluN2B-containing NMDA receptors mediated the effects of BDNF in the facilitation of the early phase of long-term potentiation (LTP) of hippocampal CA1 synapses induced by θ-burst stimulation, since the effect of the neurotrophin was abrogated in the presence of the GluN2B inhibitor Co 101244. In the absence of BDNF, the GluN2B inhibitor did not affect LTP. Surface accumulation of GluN2B-containing NMDA receptors was also observed in hippocampal synaptoneurosomes isolated from rats subjected to the pilocarpine model of temporal lobe epilepsy, after reaching Status Epilepticus, an effect that was inhibited by administration of the TrkB receptor inhibitor ANA-12.
Together, these results show that the synaptic accumulation of GluN2B-containing NMDA receptors mediate the effects of BDNF in the plasticity of glutamatergic synapses in the hippocampus.
脑源性神经营养因子（BDNF）是海马体中突触可塑性和记忆形成的关键介质。然而，BDNF诱导的与海马体谷氨酸能突触可塑性相关的谷氨酸受体变化尚未阐明。在这项研究中，我们调查了含GluN2B的N-甲基-D-天冬氨酸（NMDA）受体在BDNF诱导的谷氨酸能突触可塑性中的假定作用。
在非通透条件下，通过免疫细胞化学，使用与细胞外表位结合的抗体，研究BDNF对培养的海马神经元和海马突触体中含GluN2B的NMDA受体表面表达的影响。使用θ波爆发刺激诱导海马CA1突触的长时程增强。使用颞叶癫痫的锂-匹罗卡品模型诱导癫痫发作。通过用磷酸特异性抗体进行蛋白质印迹来评估酪氨酸蛋白激酶2（Pyk2）的磷酸化。
用BDNF刺激海马突触体导致含GluN2B的NMDA受体的突触表面表达随时间显著增加，这通过与突触前（囊泡谷氨酸转运体）和突触后标记物（突触后密度蛋白95，PSD-95）共定位的免疫细胞化学测定。同样，BDNF通过对蛋白激酶C（PKC）抑制剂GӦ6983敏感的机制，诱导培养的海马神经元突触中含GluN2B的NMDA受体的突触积累。蛋白质印迹实验分析激酶酪氨酸402位点的磷酸化表明，PKC的作用可能由Pyk2的磷酸化介导。含GluN2B的NMDA受体介导了BDNF在促进θ波爆发刺激诱导的海马CA1突触长时程增强（LTP）早期阶段的作用，因为在存在GluN2B抑制剂Co 101244的情况下，神经营养因子的作用被消除。在没有BDNF的情况下，GluN2B抑制剂不影响LTP。在达到癫痫持续状态后，从接受匹罗卡品颞叶癫痫模型的大鼠分离的海马突触体中也观察到含GluN2B的NMDA受体的表面积累，这种作用被TrkB受体抑制剂ANA-12给药所抑制。
总之，这些结果表明含GluN2B的NMDA受体的突触积累介导了BDNF在海马体谷氨酸能突触可塑性中的作用。
