scholarly journals KCC2-dependent Steady-state Intracellular Chloride Concentration and pH in Cortical Layer 2/3 Neurons of Anesthetized and Awake Mice

2017 ◽  
Author(s):  
Juan Carlos Boffi ◽  
Johannes Knabbe ◽  
Michaela Kaiser ◽  
Thomas Kuner
Keyword(s):  
Steady State ◽  
Cortical Neurons ◽  
Chloride Concentration ◽  
Cortical Layer ◽  
Model Systems ◽  
Adult Mice ◽  
Neuronal Inhibition ◽  
Wide Range ◽  
Intracellular Chloride Concentration

AbstractNeuronal intracellular Cl- concentration ([Cl-]i) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pHi) or cell volume. Up to date, neuronal [Cl-]i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl- indicator Superclomeleon (SCLM) to estimate the steady-state [Cl-]i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin as a ratiometric sensor to estimate the intracellular steady-state pH (pHi) of mouse cortical neurons in vivo. We estimated an average resting [Cl-]i of 6 ± 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pHi of 7.1 ± 0.1. Neither [Cl-]i nor pHi were affected by isoflurane anesthesia. We deleted the cation-Cl- co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl-]i to approximately 26 ± 8 mM, demonstrating that under in vivo conditions KCC2 produces low [Cl-]i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl-]i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.

scholarly journals High current optogenetic channels for stimulation and inhibition of primary rat cortical neurons

2017 ◽  
Author(s):  
Lei Jin ◽  
Eike Frank Joest ◽  
Wenfang Li ◽  
Shiqiang Gao ◽  
Andreas Offenhäusser ◽  
...  
Keyword(s):  
Neural Development ◽  
Cortical Neurons ◽  
Single Channel ◽  
Chloride Concentration ◽  
Neuronal Firing ◽  
High Current ◽  
Neuronal Inhibition ◽  
Rat Cortical Neurons

AbstractChR2-XXL and GtACR1 are currently the cation and anion ends of the optogenetic single channel current range. These were used in primary rat cortical neurons in vitro to manipulate neuronal firing patterns. ChR2-XXL provides high cation currents via elevated light sensitivity and a prolonged open state. Stimulating ChR2-XXL expressing putative presynaptic neurons induced neurotransmission. Moreover, stable depolarisation block could be generated in single neurons using ChR2-XXL, proving that ChR2-XXL is a promising candidate for in vivo applications of optogenetics, for example to treat peripheral neuropathic pain. We also addressed an anion channelrhodopsin (GtACR1) for the next generation of optogenetic neuronal inhibition in primary rat cortical neurons. GtACR1‘s light-gated chloride conduction was verified in primary neurons and the efficient photoinhibition of action potentials, including spontaneous activity, was shown. Our data also implies that the chloride concentration in neurons decreases during neural development. In both cases, we find surprising applications of these high current channels. For ChR2-XXL inhibition and stimulation are possible, while for GtACR1 the role of Cl−during neural development becomes a new optogenetic target.

scholarly journals Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

2020 ◽  
Author(s):  
Nicole M. Collette ◽  
Victoria H.I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

Possible Effects of Depolarizing GABAA Conductance on the Neuronal Input–Output Relationship: A Modeling Study

2005 ◽  
Vol 93 (6) ◽  
pp. 3504-3523 ◽  
Author(s):  
Kenji Morita ◽  
Kunichika Tsumoto ◽  
Kazuyuki Aihara
Keyword(s):  
Firing Rate ◽  
Cortical Neurons ◽  
Reversal Potential ◽  
Pyramidal Cells ◽  
Resting Potential ◽  
Input Output ◽  
Wide Range ◽  
The Relationship

Recent in vitro experiments revealed that the GABAA reversal potential is about 10 mV higher than the resting potential in mature mammalian neocortical pyramidal cells; thus GABAergic inputs could have facilitatory, rather than inhibitory, effects on action potential generation under certain conditions. However, how the relationship between excitatory input conductances and the output firing rate is modulated by such depolarizing GABAergic inputs under in vivo circumstances has not yet been understood. We examine herewith the input–output relationship in a simple conductance-based model of cortical neurons with the depolarized GABAA reversal potential, and show that a tonic depolarizing GABAergic conductance up to a certain amount does not change the relationship between a tonic glutamatergic driving conductance and the output firing rate, whereas a higher GABAergic conductance prevents spike generation. When the tonic glutamatergic and GABAergic conductances are replaced by in vivo–like highly fluctuating inputs, on the other hand, the effect of depolarizing GABAergic inputs on the input–output relationship critically depends on the degree of coincidence between glutamatergic input events and GABAergic ones. Although a wide range of depolarizing GABAergic inputs hardly changes the firing rate of a neuron driven by noncoincident glutamatergic inputs, a certain range of these inputs considerably decreases the firing rate if a large number of driving glutamatergic inputs are coincident with them. These results raise the possibility that the depolarized GABAA reversal potential is not a paradoxical mystery, but is instead a sophisticated device for discriminative firing rate modulation.

Chaotic Balanced State in a Model of Cortical Circuits

1998 ◽  
Vol 10 (6) ◽  
pp. 1321-1371 ◽  
Author(s):  
C. van Vreeswijk ◽  
H. Sompolinsky
Keyword(s):  
Cortical Neurons ◽  
Initial Conditions ◽  
Single Cells ◽  
Network Size ◽  
Short Time Scale ◽  
Strong Nonlinearity ◽  
Large Network ◽  
Wide Range ◽  
The Mean

The nature and origin of the temporal irregularity in the electrical activity of cortical neurons in vivo are not well understood. We consider the hypothesis that this irregularity is due to a balance of excitatory and inhibitory currents into the cortical cells. We study a network model with excitatory and inhibitory populations of simple binary units. The internal feedback is mediated by relatively large synaptic strengths, so that the magnitude of the total excitatory and inhibitory feedback is much larger than the neuronal threshold. The connectivity is random and sparse. The mean number of connections per unit is large, though small compared to the total number of cells in the network. The network also receives a large, temporally regular input from external sources. We present an analytical solution of the mean-field theory of this model, which is exact in the limit of large network size. This theory reveals a new cooperative stationary state of large networks, which we term a balanced state. In this state, a balance between the excitatory and inhibitory inputs emerges dynamically for a wide range of parameters, resulting in a net input whose temporal fluctuations are of the same order as its mean. The internal synaptic inputs act as a strong negative feedback, which linearizes the population responses to the external drive despite the strong nonlinearity of the individual cells. This feedback also greatly stabilizes the system's state and enables it to track a time-dependent input on time scales much shorter than the time constant of a single cell. The spatiotemporal statistics of the balanced state are calculated. It is shown that the autocorrelations decay on a short time scale, yielding an approximate Poissonian temporal statistics. The activity levels of single cells are broadly distributed, and their distribution exhibits a skewed shape with a long power-law tail. The chaotic nature of the balanced state is revealed by showing that the evolution of the microscopic state of the network is extremely sensitive to small deviations in its initial conditions. The balanced state generated by the sparse, strong connections is an asynchronous chaotic state. It is accompanied by weak spatial cross-correlations, the strength of which vanishes in the limit of large network size. This is in contrast to the synchronized chaotic states exhibited by more conventional network models with high connectivity of weak synapses.

Physiological Gain Leads to High ISI Variability in a Simple Model of a Cortical Regular Spiking Cell

1997 ◽  
Vol 9 (5) ◽  
pp. 971-983 ◽  
Author(s):  
Todd W. Troyer ◽  
Kenneth D. Miller
Keyword(s):  
Steady State ◽  
Cortical Neurons ◽  
Cortical Cell ◽  
Firing Frequency ◽  
Cell Physiology ◽  
State Behavior ◽  
Integrate And Fire ◽  
Wide Range ◽  
Visual Cortical

To understand the interspike interval (ISI) variability displayed by visual cortical neurons (Softky & Koch, 1993), it is critical to examine the dynamics of their neuronal integration, as well as the variability in their synaptic input current. Most previous models have focused on the latter factor. We match a simple integrate-and-fire model to the experimentally measured integrative properties of cortical regular spiking cells (McCormick, Connors, Lighthall, & Prince, 1985). After setting RC parameters, the postspike voltage reset is set to match experimental measurements of neuronal gain (obtained from in vitro plots of firing frequency versus injected current). Examination of the resulting model leads to an intuitive picture of neuronal integration that unifies the seemingly contradictory [Formula: see text] and random walk pictures that have previously been proposed. When ISIs are dominated by postspike recovery,[Formula: see text] arguments hold and spiking is regular; after the “memory” of the last spike becomes negligible, spike threshold crossing is caused by input variance around a steady state and spiking is Poisson. In integrate-and-fire neurons matched to cortical cell physiology, steady-state behavior is predominant, and ISIs are highly variable at all physiological firing rates and for a wide range of inhibitory and excitatory inputs.

scholarly journals Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 527-531 ◽  
Author(s):  
A K H MacGibbon ◽  
L F Blackwell ◽  
P D Buckley
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Liver Aldehyde Dehydrogenase

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 × 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.

Contribution of changes in the chloride driving force to the fading of I GABA in frog melanotrophs

2000 ◽  
Vol 278 (3) ◽  
pp. E430-E443 ◽  
Author(s):  
Frank le Foll ◽  
Olivier Soriani ◽  
Hubert Vaudry ◽  
Lionel Cazin
Keyword(s):  
Driving Force ◽  
Chloride Concentration ◽  
Reversal Potential ◽  
Resting Potential ◽  
Perforated Patch ◽  
Induced Currents ◽  
Patch Configuration ◽  
Intracellular Chloride Concentration ◽  
Current Reversal

Chloride redistribution during type A γ-aminobutyric acid (GABAA) currents ( I GABA) has been investigated in cultured frog pituitary melanotrophs with imposed intracellular chloride concentration ([Cl−]i) in the whole cell configuration or with unaltered [Cl−]i using the gramicidin-perforated patch approach. Prolonged GABA exposures elicited reproducible decaying currents. The decay of I GABAwas associated with both a transient fall of conductance ( g GABA) and shift of current reversal potential ( E GABA). The shift of E GABAappeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of I GABA was due to persistent shifts of E GABA as a result of g GABArecovering from one GABA application to another. In cells alternatively clamped at potentials closely flanking resting potential and submitted to a train of brief GABA pulses, a reversal of I GABA was observed after 150 s recording. It is demonstrated that, in intact frog melanotrophs, shifts of E GABA combine with genuine receptor desensitization to depress I GABA. These findings strongly suggest that shifts of E GABA may act as a negative feedback, reducing the bioelectrical and secretory responses induced by an intense release of GABA in vivo.

scholarly journals Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1295
Author(s):  
Nicole M. Collette ◽  
Victoria H. I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

In Vivo divisional Tracking System Reveals Cycling Heterogeneity in Long-Term Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 814-814
Author(s):  
Hitoshi Takizawa ◽  
Markus G Manz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Model Systems ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Tracking Model

Abstract Abstract 814 Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and give rise to all mature cells of hemato-lymphoid system for the lifetime of an individual. To ensure this, HSCs are kept at homeostatic levels in adult bone marrow. Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assay using 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., PLoS ONE 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic model systems (Wilson et. al., Cell 2008; Foudi et. al., Nat. Biotech. 2008). Based on the latter, it was suggested that one HSC pool turns over faster than another, dormant pool with very limited divisions during a lifetime. However, the fast cycling HSCs did not have long-term multilineage reconstitution capacity in lethally irradiated animals in contrast to dormant HSCs (Wilson et. al., Cell 2008; Foudi et.al., Nat. Biotech. 2008). From these experiments remained unclear, whether the faster cycling HSC loose long-term repopulation potential according to divisional history, or whether they represent progenitors with limited self-renewal potential, sharing a long-term HSC phenotype. Therefore, the dynamics of steady-state long-term HSC homeostasis and blood production remains to be determined. To address this directly, we set up an in vivo HSC divisional tracking assay. Here we show i.v. transfer of CFSE (carboxyfluorescein diacetate succinimidyl ester) -labeled HSCs into non-conditioned CD45.1/2 congenic F1 recipient mice that allows evaluation of steady-state HSC dynamics as CFSE distributes equally to daughter cells upon each cellular division. Sorted naïve CD4+CD62L+ T cells were used as non-dividing control cell population to determine the zero division CFSE staining level over time. Upon transfer of Lin-c-kit+Sca-1+ cells (LKS) into sublethally irradiated mice, all donor derived Lin-c-kit+ cells had divided >5 times after 3 weeks. However, transfer of LKS cells into non-irradiated mice revealed non-divided LKS cells in recipient bone marrow over 20 weeks. FACS analysis with HSC or progenitor specific marker expression showed that most of 0-2 time-divided and few of >5x divided LKS cells maintained a long-term HSC phenotype (CD150+, c-mpl+, CD34-). In order to test HSC potential, non- or >5x divided cells were sorted based on divisional history from primary recipients at different time points after transplantation, and competitively transplanted into lethally irradiated secondary recipients. At 3 weeks post primary transfer, single non-divided LKS cell was able to multi-lineage repopulate recipients, while 50 of >5x divided LKS cells showed no engraftment. Interestingly, both non- and >5x divided LKS cells at 7 or 12-14 weeks after primary transfer had long-term multilineage repopulating potential. Limiting dilution transplantation experiments demonstrated that HSC with long-term multilineage capacity (LT-HSC) were maintained at constant numbers that fit the numbers of free bone marrow niche space, with non-divided LT-HSC decreasing and >5x divided LT-HSC increasing with a constant division rate. We next tested the effects of hemato-immunological challenge on HSC cycling dynamics. Upon i.p. LPS injection into mice, previously transplanted with CFSE-labeled LKS, almost all LT-HSCs entered cell cycle within one week after challenge. These findings directly demonstrate that some LT-HSCs are quiescent for up to one fifth of the life-time of a mouse, while other LT-HSCs divide more actively, thus proving asynchronous LT-HSC division and contribution to hematopoiesis in steady-state. In addition, the results demonstrate that quiescent LT-HSCs are driven into division in response to naturally-occurring hematopoietic challenges, such as systemic bacterial infection. The CFSE-tracking model established here now allows to directly test the role of intrinsic versus environmental cues on cycling-dynamics of HSCs as well as leukemia initiating cells in steady-state and upon challenge on multiple genetic and different species background. Disclosures: No relevant conflicts of interest to declare.

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