Protective Role of NMDAR for Microwave-Induced Synaptic Plasticity Injuries in Primary Hippocampal Neurons

Background/Aims: The N-methyl-D-aspartic acid receptor (NMDAR) has been extensively studied for its important roles in synaptic plasticity and learning and memory. However, the effects of microwave radiation on the subunit composition and activity of NMDARs and the relationship between NMDARs and microwave-induced synaptic plasticity have not been thoroughly elucidated to date. Materials: In our study, primary hippocampal neurons were used to evaluate the effects of microwave radiation on synaptic plasticity. Structural changes were observed by diolistic (Dil) labeling and scanning electron microscopy (SEM) observation. Functional synaptic plasticity was reflected by the NMDAR currents, which were detected by whole cell patch clamp. We also detected the expression of NMDAR subunits by real-time PCR and Western blot analysis. To clarify the effects of microwave radiation on NMDAR-induced synaptic plasticity, suitable agonists or inhibitors were added to confirm the role of NMDARs on microwave-induced synaptic plasticity. Dil labeling, SEM observation, whole cell patch clamp, real-time PCR and Western blot analysis were used to evaluate changes in synaptic plasticity after treatment with agonists or inhibitors. Results: Our results found that microwave exposure impaired neurite development and decreased mRNA and protein levels and the current density of NMDARs. Due to the decreased expression of NMDAR subunits after microwave exposure, the selective agonist NMDA was added to identify the role of NMDARs on microwave-induced synaptic plasticity injuries. After adding the agonist, the expression of NMDAR subunits recovered to the normal levels. In addition, the microwave-induced structural and functional synaptic plasticity injuries recovered, including the number and length of neurites, the connections between neurons, and the NMDAR current. Conclusion: Microwave radiation caused neuronal synaptic plasticity injuries in primary hippocampal neurons, and NMDARs played protective roles on the damage process.

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Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

Journal of Neurophysiology ◽

10.1152/jn.1989.61.3.467 ◽

1989 ◽

Vol 61 (3) ◽

pp. 467-477 ◽

Cited By ~ 26

Author(s):

D. E. Meyers ◽

J. L. Barker

Keyword(s):

Patch Clamp ◽

Hippocampal Neurons ◽

Calcium Currents ◽

Tetraacetic Acid ◽

Whole Cell ◽

Inward Current ◽

Whole Cell Patch Clamp ◽

Voltage Dependent ◽

Cells Cultured ◽

In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

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Differential Roles of NMDA Receptor Subtypes NR2A and NR2B in Dendritic Branch Development and Requirement of RasGRF1

Journal of Neurophysiology ◽

10.1152/jn.00823.2009 ◽

2010 ◽

Vol 103 (4) ◽

pp. 1758-1770 ◽

Cited By ~ 45

Author(s):

Fernando J. Sepulveda ◽

Fernando J. Bustos ◽

Eveling Inostroza ◽

Felipe A. Zúñiga ◽

Rachael L. Neve ◽

...

Keyword(s):

Hippocampal Neurons ◽

Receptor Subtypes ◽

Dendritic Branch ◽

Spinal Cord Neurons ◽

Whole Cell Patch Clamp ◽

Electrophysiological Recordings ◽

Branch Formation ◽

The Individual

N-methyl-d-aspartate receptors (NMDARs) are known to regulate axonal refinement and dendritic branching. However, because NMDARs are abundantly present as tri-heteromers (e.g., NR1/NR2A/NR2B) during development, the precise role of the individual subunits NR2A and NR2B in these processes has not been elucidated. Ventral spinal cord neurons (VSCNs) provide a unique opportunity to address this problem, because the expression of both NR2A and NR2B (but not NR1) is downregulated in culture. Exogenous NR2A or NR2B were introduced into these naturally NR2-null neurons at 4 DIV, and electrophysiological recordings at 11 DIV confirmed that synaptic NR1NR2A receptors and NR1NR2B receptors were formed, respectively. Analysis of the dendritic architecture showed that introduction of NR2B, but not NR2A, dramatically increased the number of secondary and tertiary dendritic branches of VSCNs. Whole cell patch-clamp recordings further indicated that the newly formed branches in NR2B-expressing neurons were able to establish functional synapses because the frequency of miniature AMPA-receptor synaptic currents was increased. Using previously described mutants, we also found that disruption of the interaction between NR2B and RasGRF1 dramatically impaired dendritic branch formation in VSCNs. The differential role of the NR2A and NR2B subunits and the requirement for RasGRF1 in regulating branch formation was corroborated in hippocampal cultures. We conclude that the association between NR1NR2B-receptors and RasGRF1 is needed for dendritic branch formation in VSCNs and hippocampal neurons in vitro. The dominated NR2A expression and the limited interactions of this subunit with the signaling protein RasGRF1 may contribute to the restricted dendritic arbor development in the adult CNS.

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Absence of Thyroid Hormone Induced Delayed Dendritic Arborization in Mouse Primary Hippocampal Neurons Through Insufficient Expression of Brain-Derived Neurotrophic Factor

Frontiers in Endocrinology ◽

10.3389/fendo.2021.629100 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hiroyuki Yajima ◽

Izuki Amano ◽

Sumiyasu Ishii ◽

Tetsushi Sadakata ◽

Wataru Miyazaki ◽

...

Keyword(s):

Thyroid Hormone ◽

Neurotrophic Factor ◽

Hippocampal Neurons ◽

Brain Derived Neurotrophic Factor ◽

Mrna Levels ◽

Protein Levels ◽

Primary Hippocampal Neurons ◽

Developmental Impairment

Thyroid hormone (TH) plays important roles in the developing brain. TH deficiency in early life leads to severe developmental impairment in the hippocampus. However, the mechanisms of TH action in the developing hippocampus are still largely unknown. In this study, we generated 3,5,3’-tri-iodo-l-thyronine (T3)-free neuronal supplement, based on the composition of neuronal supplement 21 (NS21), to examine the effect of TH in the developing hippocampus using primary cultured neurons. Effects of TH on neurons were compared between cultures in this T3-free culture medium (-T3 group) and a medium in which T3 was added (+T3 group). Morphometric analysis and RT-qPCR were performed on 7, 10, and 14 days in vitro (DIV). On 10 DIV, a decreased dendrite arborization in -T3 group was observed. Such difference was not observed on 7 and 14 DIV. Brain-derived neurotrophic factor (Bdnf) mRNA levels also decreased significantly in -T3 group on 10 DIV. We then confirmed protein levels of phosphorylated neurotrophic tyrosine kinase type 2 (NTRK2, TRKB), which is a receptor for BDNF, on 10 DIV by immunocytochemistry and Western blot analysis. Phosphorylated NTRK2 levels significantly decreased in -T3 group compared to +T3 group on 10 DIV. Considering the role of BDNF on neurodevelopment, we examined its involvement by adding BDNF on 8 and 9 DIV. Addition of 10 ng/ml BDNF recovered the suppressed dendrite arborization induced by T3 deficiency on 10 DIV. We show that the lack of TH induces a developmental delay in primary hippocampal neurons, likely caused through a decreased Bdnf expression. Thus, BDNF may play a role in TH-regulated dendritogenesis.

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Vigabatrin Induces Tonic Inhibition Via GABA Transporter Reversal Without Increasing Vesicular GABA Release

Journal of Neurophysiology ◽

10.1152/jn.00856.2002 ◽

2003 ◽

Vol 89 (4) ◽

pp. 2021-2034 ◽

Cited By ~ 101

Author(s):

Yuanming Wu ◽

Wengang Wang ◽

George B. Richerson

Keyword(s):

Patch Clamp ◽

Gaba Receptors ◽

Hippocampal Neurons ◽

Gaba Release ◽

Tonic Inhibition ◽

Gabaergic Inhibition ◽

Gaba Transporter ◽

Whole Cell ◽

Ambient Gaba

Two forms of GABAergic inhibition coexist: fast synaptic neurotransmission and tonic activation of GABA receptors due to ambient GABA. The mechanisms regulating ambient GABA have not been well defined. Here we examined the role of the GABA transporter in the increase in ambient [GABA] induced by the anticonvulsant vigabatrin. Pretreatment of cultured rat hippocampal neurons with vigabatrin (100 μM) for 2–5 days led to a large increase in ambient [GABA] that was measured as the change in holding current induced by bicuculline during patch-clamp recordings. In contrast, there was a decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents mIPSCs with no change in their amplitude distribution, and a decrease in the magnitude of IPSCs evoked by presynaptic stimulation during paired recordings. The increase in ambient [GABA] was not prevented by blockade of vesicular GABA release with tetanus toxin or removal of extracellular calcium. During perforated patch recordings, the increase in ambient [GABA] was prevented by blocking the GABA transporter, indicating that the GABA transporter was continuously operating in reverse and releasing GABA. In contrast, blocking the GABA transporter increased ambient [GABA] during whole cell patch-clamp recordings unless GABA and Na+ were added to the recording electrode solution, indicating that whole cell recordings can lead to erroneous conclusions about the role of the GABA transporter in control of ambient GABA. We conclude that the equilibrium for the GABA transporter is a major determinant of ambient [GABA] and tonic GABAergic inhibition. We propose that fast GABAergic neurotransmission and tonic inhibition can be independently modified and play complementary roles in control of neuronal excitability.

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Oxidative Downmodulation of the Transient K-CurrentI A by Intracellular Arachidonic Acid in Rat Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.1999.82.1.508 ◽

1999 ◽

Vol 82 (1) ◽

pp. 508-511 ◽

Cited By ~ 15

Author(s):

Katrin Bittner ◽

Wolfgang Müller

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Arachidonic Acid ◽

Patch Clamp ◽

Hippocampal Neurons ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

Intracellular Glutathione

Membrane-permeable arachidonic acid (AA) is liberated in a Ca2+-dependent way inside cells. By using whole cell patch clamp we show that intracellular AA (1 pM) selectively reduces I A in rat hippocampal neurons, whereas extracellular application requires a 106-fold concentration. The nonmetabolized AA analogue ETYA mimics the effect of AA that is blocked by ascorbic acid or intracellular glutathione, suggesting an intracellular oxidative mechanism. We conclude that intracellular AA is extremely potent in reducing I A by an oxidative mechanism, particularly during oxidative stress.

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Neuronal megalin mediates synaptic plasticity—a novel mechanism underlying intellectual disabilities in megalin gene pathologies

Brain Communications ◽

10.1093/braincomms/fcaa135 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

João R Gomes ◽

Andrea Lobo ◽

Renata Nogueira ◽

Ana F Terceiro ◽

Susete Costelha ◽

...

Keyword(s):

Synaptic Plasticity ◽

Intellectual Disabilities ◽

Learning And Memory ◽

Neuronal Activity ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Genetic Disorder ◽

Cell Surface Receptor ◽

Neuronal Cultures

Abstract Donnai-Barrow syndrome, a genetic disorder associated to LRP2 (low-density lipoprotein receptor 2/megalin) mutations, is characterized by unexplained neurological symptoms and intellectual deficits. Megalin is a multifunctional endocytic clearance cell-surface receptor, mostly described in epithelial cells. This receptor is also expressed in the CNS, mainly in neurons, being involved in neurite outgrowth and neuroprotective mechanisms. Yet, the mechanisms involved in the regulation of megalin in the CNS are poorly understood. Using transthyretin knockout mice, a megalin ligand, we found that transthyretin positively regulates neuronal megalin levels in different CNS areas, particularly in the hippocampus. Transthyretin is even able to rescue megalin downregulation in transthyretin knockout hippocampal neuronal cultures, in a positive feedback mechanism via megalin. Importantly, transthyretin activates a regulated intracellular proteolysis mechanism of neuronal megalin, producing an intracellular domain, which is translocated to the nucleus, unveiling megalin C-terminal as a potential transcription factor, able to regulate gene expression. We unveil that neuronal megalin reduction affects physiological neuronal activity, leading to decreased neurite number, length and branching, and increasing neuronal susceptibility to a toxic insult. Finally, we unravel a new unexpected role of megalin in synaptic plasticity, by promoting the formation and maturation of dendritic spines, and contributing for the establishment of active synapses, both in in vitro and in vivo hippocampal neurons. Moreover, these structural and synaptic roles of megalin impact on learning and memory mechanisms, since megalin heterozygous mice show hippocampal-related memory and learning deficits in several behaviour tests. Altogether, we unveil a complete novel role of megalin in the physiological neuronal activity, mainly in synaptic plasticity with impact in learning and memory. Importantly, we contribute to disclose the molecular mechanisms underlying the cognitive and intellectual disabilities related to megalin gene pathologies.

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Distinct Role of Long 3′ UTR BDNF mRNA in Spine Morphology and Synaptic Plasticity in Hippocampal Neurons

Cell ◽

10.1016/j.cell.2008.05.045 ◽

2008 ◽

Vol 134 (1) ◽

pp. 175-187 ◽

Cited By ~ 420

Author(s):

Juan Ji An ◽

Kusumika Gharami ◽

Guey-Ying Liao ◽

Newton H. Woo ◽

Anthony G. Lau ◽

...

Keyword(s):

Synaptic Plasticity ◽

Hippocampal Neurons ◽

Bdnf Mrna ◽

Spine Morphology ◽

Distinct Role

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1,800 MHz Radiofrequency Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells

Frontiers in Public Health ◽

10.3389/fpubh.2021.771508 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yanqi Li ◽

Ping Deng ◽

Chunhai Chen ◽

Qinlong Ma ◽

Huifeng Pi ◽

...

Keyword(s):

Protein Expression ◽

Cell Viability ◽

Brain Development ◽

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neurite Length ◽

Primary Mouse ◽

Primary Hippocampal Neurons ◽

Neuro2a Cells

Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development.Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway.Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells.Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF-EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h.Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus.

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Expression and function of KV2-containing channels in human urinary bladder smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00447.2011 ◽

2012 ◽

Vol 302 (11) ◽

pp. C1599-C1608 ◽

Cited By ~ 15

Author(s):

Kiril L. Hristov ◽

Muyan Chen ◽

Serge A. Y. Afeli ◽

Qiuping Cheng ◽

Eric S. Rovner ◽

...

Keyword(s):

Smooth Muscle ◽

Patch Clamp ◽

Electrical Field Stimulation ◽

Bladder Smooth Muscle ◽

Rt Pcr ◽

Patch Clamp Technique ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca2+ imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5–30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

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