Confocal imaging of cytosolic Ca2+ and fuzzy clustering reveal the circuit topology details underlying synchronization in hippocampal neurons

2018 ◽  
Author(s):  
Sarpras Swain ◽  
Priyanka Devi Pantula ◽  
Kishalay Mitra ◽  
Lopamudra Giri
Keyword(s):  
Fuzzy Clustering ◽  
Hippocampal Neurons ◽  
Confocal Imaging ◽  
Circuit Topology

Confocal Imaging and k-Means Clustering of GABAB and mGluR Mediated Modulation of Ca2+ Spiking in Hippocampal Neurons

2018 ◽  
Vol 9 (12) ◽  
pp. 3094-3107 ◽  
Author(s):  
Sarpras Swain ◽  
Rishikesh Kumar Gupta ◽  
Kasun Ratnayake ◽  
Pantula Devi Priyanka ◽  
Ranjana Singh ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Confocal Imaging

scholarly journals Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

2001 ◽  
Vol 154 (2) ◽  
pp. 355-368 ◽  
Author(s):  
Kristina D. Micheva ◽  
Ronald W. Holz ◽  
Stephen J. Smith
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Resting Cells ◽  
Ph Domain ◽  
Cellular Processes ◽  
Vesicle Recycling ◽  
Green Fluorescent

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

scholarly journals Neuronal activity controls transsynaptic geometry

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Oleg O. Glebov ◽  
Susan Cox ◽  
Lawrence Humphreys ◽  
Juan Burrone
Keyword(s):  
Electron Microscopy ◽  
Neuronal Activity ◽  
Hippocampal Neurons ◽  
Super Resolution ◽  
Synaptic Cleft ◽  
Synaptic Function ◽  
Confocal Imaging ◽  
Relative Positioning ◽  
Spatial Coupling ◽  
Presynaptic Vesicle

Abstract The neuronal synapse is comprised of several distinct zones, including presynaptic vesicle zone (SVZ), active zone (AZ) and postsynaptic density (PSD). While correct relative positioning of these zones is believed to be essential for synaptic function, the mechanisms controlling their mutual localization remain unexplored. Here, we employ high-throughput quantitative confocal imaging, super-resolution and electron microscopy to visualize organization of synaptic subdomains in hippocampal neurons. Silencing of neuronal activity leads to reversible reorganization of the synaptic geometry, resulting in a increased overlap between immunostained AZ and PSD markers; in contrast, the SVZ-AZ spatial coupling is decreased. Bayesian blinking and bleaching (3B) reconstruction reveals that the distance between the AZ-PSD distance is decreased by 30 nm, while electron microscopy shows that the width of the synaptic cleft is decreased by 1.1 nm. Our findings show that multiple aspects of synaptic geometry are dynamically controlled by neuronal activity and suggest mutual repositioning of synaptic components as a potential novel mechanism contributing to the homeostatic forms of synaptic plasticity.

GABAC ρ1 Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons

2001 ◽  
Vol 86 (5) ◽  
pp. 2605-2615 ◽  
Author(s):  
Qing Cheng ◽  
Paul M. Burkat ◽  
John C. Kulli ◽  
Jay Yang
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Short Pulse ◽  
Gaba Release ◽  
Ruthenium Red ◽  
Confocal Imaging ◽  
Imaging Data ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent

The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABAC receptor ρ1 subunit or a mutant GABAA receptor β2 subunit lacking picrotoxin sensitivity [β2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABAC receptor-mediated and picrotoxin-resistant GABAA receptor-mediated GABA-evoked currents in ρ1- and β2(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg2+, and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABAC receptors in ρ1-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ρ1 receptors or failure of the ρ1 subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABACreceptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ρ1 neurons proved that a brief GABA transient is sufficient to activate ρ1 receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABACreceptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in β2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed ρ1immunofluorescence restricted to the soma, whereas punctate β2(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ρ1 subunit, the GABACreceptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of β2(mut) subunits into subsynaptic GABAA receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.

scholarly journals Inhibition of AKT Signaling Alters βIV Spectrin Distribution at the AIS and Increases Neuronal Excitability

2021 ◽  
Vol 14 ◽  
Author(s):  
Jessica Di Re ◽  
Wei-Chun J. Hsu ◽  
Cihan B. Kayasandik ◽  
Nickolas Fularczyk ◽  
T. F. James ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Hippocampal Neurons ◽  
Protein Composition ◽  
Neuronal Firing ◽  
Confocal Imaging ◽  
Control Treatment ◽  
Support Vector ◽  
Protein Distribution ◽  
Primary Hippocampal Neurons ◽  
Pathway Inhibition

The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing βIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on βIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in βIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

scholarly journals TPEN attenuates amyloid-β25–35-induced neuronal damage with changes in the electrophysiological properties of voltage-gated sodium and potassium channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Wen-bo Chen ◽  
Yu-xiang Wang ◽  
Hong-gang Wang ◽  
Di An ◽  
Dan Sun ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Neuronal Damage ◽  
Amyloid Β ◽  
Zinc Ion ◽  
Confocal Imaging ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Voltage Gated ◽  
Age Related ◽  
Sodium And Potassium

AbstractTo understand the role of intracellular zinc ion (Zn2+) dysregulation in mediating age-related neurodegenerative changes, particularly neurotoxicity resulting from the generation of excessive neurotoxic amyloid-β (Aβ) peptides, this study aimed to investigate whether N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+-specific chelator, could attenuate Aβ25–35-induced neurotoxicity and the underlying electrophysiological mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of hippocampal neurons and performed single-cell confocal imaging to detect the concentration of Zn2+ in these neurons. Furthermore, we used the whole-cell patch-clamp technique to detect the evoked repetitive action potential (APs), the voltage-gated sodium and potassium (K+) channels of primary hippocampal neurons. The analysis showed that TPEN attenuated Aβ25–35-induced neuronal death, reversed the Aβ25–35-induced increase in intracellular Zn2+ concentration and the frequency of APs, inhibited the increase in the maximum current density of voltage-activated sodium channel currents induced by Aβ25–35, relieved the Aβ25–35-induced decrease in the peak amplitude of transient outward K+ currents (IA) and outward-delayed rectifier K+ currents (IDR) at different membrane potentials, and suppressed the steady-state activation and inactivation curves of IA shifted toward the hyperpolarization direction caused by Aβ25–35. These results suggest that Aβ25–35-induced neuronal damage correlated with Zn2+ dysregulation mediated the electrophysiological changes in the voltage-gated sodium and K+ channels. Moreover, Zn2+-specific chelator-TPEN attenuated Aβ25–35-induced neuronal damage by recovering the intracellular Zn2+ concentration.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

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

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

Immunohistochemical localization of lysosomal cysteine protease cathepsins B and L in monkey hippocampal neurons after transient ischemia

1999 ◽  
Vol 19 (3) ◽  
pp. 302-310
Author(s):  
Yukihiko Kohda ◽  
Katsuhiro Tsuchiya ◽  
Junkoh Yamashita ◽  
Masaki Yoshida ◽  
Takashi Ueno ◽  
...  
Keyword(s):  
Cysteine Protease ◽  
Hippocampal Neurons ◽  
Immunohistochemical Localization ◽  
Transient Ischemia ◽  
Lysosomal Cysteine Protease
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