Live imaging of microtubule dynamics at excitatory presynaptic boutons in primary hippocampal neurons and acute hippocampal slices

Zn2+ is found in glutamatergic nerve terminals throughout the mammalian forebrain and has diverse extracellular and intracellular actions. The anatomical location and possible synaptic signaling role for this cation have led to the hypothesis that Zn2+ is released from presynaptic boutons, traverses the synaptic cleft, and enters postsynaptic neurons. However, these events have not been directly observed or characterized. Here we show, using microfluorescence imaging in rat hippocampal slices, that brief trains of electrical stimulation of mossy fibers caused immediate release of Zn2+ from synaptic terminals into the extracellular microenvironment. Release was induced across a broad range of stimulus intensities and frequencies, including those likely to induce long-term potentiation. The amount of Zn2+ release was dependent on stimulation frequency (1–200 Hz) and intensity. Release of Zn2+ required sodium-dependent action potentials and was dependent on extracellular Ca2+. Once released, Zn2+ crosses the synaptic cleft and enters postsynaptic neurons, producing increases in intracellular Zn2+ concentration. These results indicate that, like a neurotransmitter, Zn2+ is stored in synaptic vesicles and is released into the synaptic cleft. However, unlike conventional transmitters, it also enters postsynaptic neurons, where it may have manifold physiological functions as an intracellular second messenger.

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Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD

10.1101/2020.12.05.412981 ◽

2020 ◽

Author(s):

Jithin D. Nair ◽

Ellen Braksator ◽

Busra P Yucel ◽

Richard Seager ◽

Jack R. Mellor ◽

...

Keyword(s):

Ampa Receptor ◽

Ampa Receptors ◽

Hippocampal Neurons ◽

Hippocampal Slices ◽

Surface Expression ◽

Receptor Activation ◽

Kainate Receptors ◽

Receptor Surface Expression ◽

Acute Hippocampal Slices

AbstractHere we report that sustained activation of GluK2 subunit-containing kainate receptors leads to AMPA receptor endocytosis and a novel form of long-term depression (KAR-LTDAMPAR) in hippocampal neurons. The KAR-evoked loss of surface AMPA receptors requires KAR channel activity and is occluded by the blockade of PKC or PKA. Moreover, in acute hippocampal slices, kainate invoked LTD of AMPA EPSCs. These data, together with our previously reported KAR-LTPAMPAR, demonstrate that KARs bidirectionally regulate synaptic AMPARs and synaptic plasticity.

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Abundant GFP Expression and LTP in Hippocampal Acute Slices by In Vivo Injection of Sindbis Virus

Journal of Neurophysiology ◽

10.1152/jn.2001.86.2.1037 ◽

2001 ◽

Vol 86 (2) ◽

pp. 1037-1042 ◽

Cited By ~ 15

Author(s):

Massimo D'Apuzzo ◽

Georgia Mandolesi ◽

Gerald Reis ◽

Erin M. Schuman

Keyword(s):

Hippocampal Neurons ◽

Sindbis Virus ◽

Fluorescent Protein ◽

Hippocampal Slices ◽

Pyramidal Cells ◽

Long Term Potentiation ◽

Neuronal Structure ◽

Adult Rats ◽

Acute Hippocampal Slices

Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons.

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Rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices using adenoviral vectors

Molecular Brain Research ◽

10.1016/s0169-328x(96)00246-x ◽

1997 ◽

Vol 44 (1) ◽

pp. 171-177 ◽

Cited By ~ 12

Author(s):

Oliver Griesbeck ◽

Martin Korte ◽

Claude Gravel ◽

Tobias Bonhoeffer ◽

Hans Thoenen

Keyword(s):

Gene Transfer ◽

Hippocampal Neurons ◽

Hippocampal Slices ◽

Adenoviral Vectors ◽

Acute Hippocampal Slices ◽

Cultured Hippocampal Neurons

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ATRA-induced increase in intracellular Ca2+concentration in primary hippocampal neurons

Cell Biology International ◽

10.1042/cbi034s074b ◽

2010 ◽

Vol 34 (8) ◽

pp. S74-S74

Author(s):

Tingyu Li ◽

Xiaojuan Zhang ◽

Xuan Zhang ◽

Jian Hea ◽

Yang Bi Youxue Liu ◽

...

Keyword(s):

Hippocampal Neurons ◽

Primary Hippocampal Neurons

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Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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Schisandrin Restores the Amyloid β-Induced Impairments on Mitochondrial Function, Energy Metabolism, Biogenesis, and Dynamics in Rat Primary Hippocampal Neurons

Pharmacology ◽

10.1159/000507818 ◽

2021 ◽

pp. 1-11

Author(s):

Zhongyuan Piao ◽

Lin Song ◽

Lifen Yao ◽

Limei Zhang ◽

Yichan Lu

Keyword(s):

Energy Metabolism ◽

Cytochrome C ◽

Mitochondrial Biogenesis ◽

Mitochondrial Function ◽

Hippocampal Neurons ◽

Citrate Synthase ◽

Mitochondrial Permeability Transition ◽

Amyloid Β ◽

Mitochondrial Functions ◽

Primary Hippocampal Neurons

Introduction: Schisandrin which is derived from Schisandra chinensis has shown multiple pharmacological effects on various diseases including Alzheimer’s disease (AD). It is demonstrated that mitochondrial dysfunction plays an essential role in the pathogenesis of neurodegenerative disorders. Objective: Our study aims to investigate the effects of schisandrin on mitochondrial functions and metabolisms in primary hippocampal neurons. Methods: In our study, rat primary hippocampal neurons were isolated and treated with indicated dose of amyloid β1–42 (Aβ1–42) oligomer to establish a cell model of AD in vitro. Schisandrin (2 μg/mL) was further subjected to test its effects on mitochondrial function, energy metabolism, mitochondrial biogenesis, and dynamics in the Aβ1–42 oligomer-treated neurons. Results and Conclusions: Our findings indicated that schisandrin significantly alleviated the Aβ1–42 oligomer-induced loss of mitochondrial membrane potential and impaired cytochrome c oxidase activity. Additionally, the opening of mitochondrial permeability transition pore and release of cytochrome c were highly restricted with schisandrin treatment. Alterations in cell viability, ATP production, citrate synthase activity, and the expressions of glycolysis-related enzymes demonstrated the relief of defective energy metabolism in Aβ-treated neurons after the treatment of schisandrin. For mitochondrial biogenesis, elevated expression of peroxisome proliferator-activated receptor γ coactivator along with promoted mitochondrial mass was found in schisandrin-treated cells. The imbalance in the cycle of fusion and fission was also remarkably restored by schisandrin. In summary, this study provides novel mechanisms for the protective effect of schisandrin on mitochondria-related functions.

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Cdc42 Facilitates Axonogenesis by Enhancing Microtubule Stabilization in Primary Hippocampal Neurons

Cellular and Molecular Neurobiology ◽

10.1007/s10571-021-01051-0 ◽

2021 ◽

Author(s):

Ang Li ◽

Hui-Ming Zhu ◽

Yu Chen ◽

Fang Yan ◽

Zhong-Ying Liu ◽

...

Keyword(s):

Hippocampal Neurons ◽

Microtubule Stabilization ◽

Primary Hippocampal Neurons

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Neurotrophic Properties of Silexan, an Essential Oil from the Flowers of Lavender-Preclinical Evidence for Antidepressant-Like Properties

Pharmacopsychiatry ◽

10.1055/a-1293-8585 ◽

2020 ◽

Vol 54 (01) ◽

pp. 37-46

Author(s):

Kristina Friedland ◽

Giacomo Silani ◽

Anita Schuwald ◽

Carola Stockburger ◽

Egon Koch ◽

...

Keyword(s):

Essential Oil ◽

Hippocampal Neurons ◽

Day Treatment ◽

Neuronal Cell ◽

Antidepressant Activity ◽

In Vivo Models ◽

Antidepressant Effects ◽

Primary Hippocampal Neurons

Abstract Background Silexan, a special essential oil from flowering tops of lavandula angustifolia, is used to treat subsyndromal anxiety disorders. In a recent clinical trial, Silexan also showed antidepressant effects in patients suffering from mixed anxiety-depression (ICD-10 F41.2). Since preclinical data explaining antidepressant properties of Silexan are missing, we decided to investigate if Silexan also shows antidepressant-like effects in vitro as well as in vivo models. Methods We used the forced swimming test (FST) in rats as a simple behavioral test indicative of antidepressant activity in vivo. As environmental events and other risk factors contribute to depression through converging molecular and cellular mechanisms that disrupt neuronal function and morphology—resulting in dysfunction of the circuitry that is essential for mood regulation and cognitive function—we investigated the neurotrophic properties of Silexan in neuronal cell lines and primary hippocampal neurons. Results The antidepressant activity of Silexan (30 mg/kg BW) in the FST was comparable to the tricyclic antidepressant imipramine (20 mg/kg BW) after 9-day treatment. Silexan triggered neurite outgrowth and synaptogenesis in 2 different neuronal cell models and led to a significant increase in synaptogenesis in primary hippocampal neurons. Silexan led to a significant phosphorylation of protein kinase A and subsequent CREB phosphorylation. Conclusion Taken together, Silexan demonstrates antidepressant-like effects in cellular as well as animal models for antidepressant activity. Therefore, our data provides preclinical evidence for the clinical antidepressant effects of Silexan in patients with mixed depression and anxiety.

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Zinc Enhances the Inhibitory Effects of Strychnine-Sensitive Glycine Receptors in Mouse Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00500.2007 ◽

2007 ◽

Vol 98 (6) ◽

pp. 3666-3676 ◽

Cited By ~ 8

Author(s):

Hai Xia Zhang ◽

Liu Lin Thio

Keyword(s):

Action Potential ◽

Action Potentials ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Hippocampal Slices ◽

Glycine Receptors ◽

Inhibitory Effects ◽

Action Potential Firing ◽

Embryonic Mouse ◽

Potential Firing

Although extracellular Zn2+ is an endogenous biphasic modulator of strychnine-sensitive glycine receptors (GlyRs), the physiological significance of this modulation remains poorly understood. Zn2+ modulation of GlyR may be especially important in the hippocampus where presynaptic Zn2+ is abundant. Using cultured embryonic mouse hippocampal neurons, we examined whether 1 μM Zn2+, a potentiating concentration, enhances the inhibitory effects of GlyRs activated by sustained glycine applications. Sustained 20 μM glycine (EC25) applications alone did not decrease the number of action potentials evoked by depolarizing steps, but they did in 1 μM Zn2+. At least part of this effect resulted from Zn2+ enhancing the GlyR-induced decrease in input resistance. Sustained 20 μM glycine applications alone did not alter neuronal bursting, a form of hyperexcitability induced by omitting extracellular Mg2+. However, sustained 20 μM glycine applications depressed neuronal bursting in 1 μM Zn2+. Zn2+ did not enhance the inhibitory effects of sustained 60 μM glycine (EC70) applications in these paradigms. These results suggest that tonic GlyR activation could decrease neuronal excitability. To test this possibility, we examined the effect of the GlyR antagonist strychnine and the Zn2+ chelator tricine on action potential firing by CA1 pyramidal neurons in mouse hippocampal slices. Co-applying strychnine and tricine slightly but significantly increased the number of action potentials fired during a depolarizing current step and decreased the rheobase for action potential firing. Thus Zn2+ may modulate neuronal excitability normally and in pathological conditions such as seizures by potentiating GlyRs tonically activated by low agonist concentrations.

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