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 Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing

2020 ◽  
Vol 27 (10) ◽  
pp. 2810-2827 ◽  
Author(s):  
Björn Friedhelm Vahsen ◽  
Vinicius Toledo Ribas ◽  
Jonas Sundermeyer ◽  
Alexander Boecker ◽  
Vivian Dambeck ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Axonal Degeneration ◽  
Dominant Negative ◽  
Focal Lesion ◽  
Pathological Feature ◽  
Nerve Crush ◽  
Cord Injury ◽  
Rat Cortical Neurons

Abstract Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

scholarly journals Dopamine modulates the integrity of the perisynaptic extracellular matrix at excitatory synapses

2019 ◽  
Author(s):  
Jessica Mitlöhner ◽  
Rahul Kaushik ◽  
Hartmut Niekisch ◽  
Armand Blondiaux ◽  
Christine E. Gee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Synaptic Activity ◽  
Excitatory Synapses ◽  
Intracellular Calcium Signaling ◽  
Rat Cortical Neurons ◽  
Synaptic Receptors

SummaryIn the brain, Hebbian-type and homeostatic forms of plasticity are affected by neuromodulators like dopamine (DA). Modifications of the perisynaptic extracellular matrix (ECM), controlling functions and mobility of synaptic receptors as well as diffusion of transmitters and neuromodulators in the extracellular space, are crucial for the manifestation of plasticity. Mechanistic links between synaptic activation and ECM modifications are largely unknown. Here, we report that neuromodulation via D1-type DA receptors can induce targeted ECM proteolysis specifically at excitatory synapses of rat cortical neurons via proteases ADAMTS-4 and -5. We show that receptor activation induces increased proteolysis of brevican (BC) and aggrecan, two major constituents of the adult ECM, in vivo and in vitro. ADAMTS immunoreactivity is detected near synapses, and shRNA-mediated knockdown reduced BC cleavage. We outline a molecular scenario how synaptic activity and neuromodulation are linked to ECM rearrangements via increased cAMP levels, NMDA receptor activation, and intracellular calcium signaling.

scholarly journals Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Masahiro Fuwa ◽  
Masaaki Kageyama ◽  
Koji Ohashi ◽  
Masaaki Sasaoka ◽  
Ryuichi Sato ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Nr2b Subunit ◽  
Neuroprotective Effects ◽  
Receptor Antagonism ◽  
Rat Brain Membranes ◽  
Rat Cortical Neurons ◽  
Orally Active ◽  
Related Compounds

AbstractIn addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

Mechanical perturbation of cultured cortical neurons reveals a stretch-induced delayed depolarization

1995 ◽  
Vol 74 (6) ◽  
pp. 2767-2773 ◽  
Author(s):  
S. J. Tavalin ◽  
E. F. Ellis ◽  
L. S. Satin
Keyword(s):  
Brain Injury ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Neuronal Firing ◽  
Neonatal Rat ◽  
Resting Membrane Potential ◽  
Cell Stretch ◽  
Cultured Cortical Neurons ◽  
Rat Cortical Neurons

1. An in vitro cellular model of injury was used to elucidate mechanisms contributing to traumatic brain injury (TBI). Neonatal rat cortical neurons cultured on a flexible silastic membrane were stretched rapidly and reversibly by a 50-ms pulse of pressurized air. 2. Sublethal cell stretch depolarized neuronal resting membrane potential by approximately 10 mV but only if cells were incubated for 1 h after injury. Stretch-induced delayed depolarization (or SIDD) returned to baseline values within 24 h. 3. SIDD was dependent on the degree of cell stretch and required neuronal firing, calcium entry, and N-methyl-D-aspartate receptor activation for its induction but not its maintainance. 4. Similarities between SIDD and TBI suggest that SIDD may play a role in brain injury.

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 Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

2022 ◽  
Author(s):  
Jinli Geng ◽  
Wenxiang Li ◽  
Yingjun Tang ◽  
Yunming Gao ◽  
Yitong Lu ◽  
...  
Keyword(s):  
Neural Development ◽  
Cortical Neurons ◽  
Intracellular Signaling ◽  
Sensory Response ◽  
Imaging Data ◽  
Membrane Excitability ◽  
Sensory Evoked

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca2+- dependent neuritogenesis). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations over an extended time frame. Chronic Ca2+ imaging data (≥1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca2+ activities would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca2+, including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo. For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca2+ activities are imaged and evaluated both in vitro and in vivo, providing new opportunities to monitor neural development or other chronic processes concurrently with Ca2+ dynamics.

scholarly journals Increased BDNF Protein Expression after Ischemic Or PKC Epsilon Preconditioning Promotes Electrophysiologic Changes That Lead to Neuroprotection

2014 ◽  
Vol 35 (1) ◽  
pp. 121-130 ◽  
Author(s):  
Jake T Neumann ◽  
John W Thompson ◽  
Ami P Raval ◽  
Charles H Cohan ◽  
Kevin B Koronowski ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cortical Neurons ◽  
Neuronal Excitability ◽  
Hippocampal Slices ◽  
Glucose Deprivation ◽  
Neuronal Firing ◽  
Trk Receptors ◽  
Bdnf Mrna

Ischemic preconditioning (IPC) via protein kinase C epsilon (PKCε) activation induces neuroprotection against lethal ischemia. Brain-derived neurotrophic factor (BDNF) is a pro-survival signaling molecule that modulates synaptic plasticity and neurogenesis. Interestingly, BDNF mRNA expression increases after IPC. In this study, we investigated whether IPC or pharmacological preconditioning (PKCε activation) promoted BDNF-induced neuroprotection, if neuroprotection by IPC or PKCε activation altered neuronal excitability, and whether these changes were BDNF-mediated. We used both in vitro (hippocampal organotypic cultures and cortical neuronal-glial cocultures) and in vivo (acute hippocampal slices 48 hours after preconditioning) models of IPC or PKCε activation. BDNF protein expression increased 24 to 48 hours after preconditioning, where inhibition of the BDNF Trk receptors abolished neuroprotection against oxygen and glucose deprivation (OGD) in vitro. In addition, there was a significant decrease in neuronal firing frequency and increase in threshold potential 48 hours after preconditioning in vivo, where this threshold modulation was dependent on BDNF activation of Trk receptors in excitatory cortical neurons. In addition, 48 hours after PKCε activation in vivo, the onset of anoxic depolarization during OGD was significantly delayed in hippocampal slices. Overall, these results suggest that after IPC or PKCε activation, there are BDNF-dependent electrophysiologic modifications that lead to neuroprotection.

scholarly journals Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 260 ◽  
Author(s):  
Jessica Mitlöhner ◽  
Rahul Kaushik ◽  
Hartmut Niekisch ◽  
Armand Blondiaux ◽  
Christine E. Gee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Synaptic Activity ◽  
Excitatory Synapses ◽  
Intracellular Calcium Signaling ◽  
Rat Cortical Neurons ◽  
Synaptic Receptors

In the brain, Hebbian-type and homeostatic forms of plasticity are affected by neuromodulators like dopamine (DA). Modifications of the perisynaptic extracellular matrix (ECM), which control the functions and mobility of synaptic receptors as well as the diffusion of transmitters and neuromodulators in the extracellular space, are crucial for the manifestation of plasticity. Mechanistic links between synaptic activation and ECM modifications are largely unknown. Here, we report that neuromodulation via D1-type DA receptors can induce targeted ECM proteolysis specifically at excitatory synapses of rat cortical neurons via proteases ADAMTS-4 and -5. We showed that receptor activation induces increased proteolysis of brevican (BC) and aggrecan, two major constituents of the adult ECM both in vivo and in vitro. ADAMTS immunoreactivity was detected near synapses, and shRNA-mediated knockdown reduced BC cleavage. We have outlined a molecular scenario of how synaptic activity and neuromodulation are linked to ECM rearrangements via increased cAMP levels, NMDA receptor activation, and intracellular calcium signaling.

scholarly journals Astrocytes Exhibit a Protective Role in Neuronal Firing Patterns under Chemically Induced Seizures in Neuron–Astrocyte Co-Cultures

2021 ◽  
Vol 22 (23) ◽  
pp. 12770
Author(s):  
Annika Ahtiainen ◽  
Barbara Genocchi ◽  
Jarno M. A. Tanskanen ◽  
Michael T. Barros ◽  
Jari A. K. Hyttinen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Microelectrode Arrays ◽  
Cell Types ◽  
Protective Role ◽  
Neuronal Firing ◽  
Neuronal Homeostasis ◽  
Chemically Induced ◽  
Rat Cortical Neurons

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer’s disease, and Parkinson’s disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

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