Uptake of surface-functionalized thermo-responsive polymeric nanocarriers in corticospinal tract motor neurons.

Nanocarrier drug delivery systems are attractive options for targeted delivery of survival- and regeneration-enhancing therapeutics to neurons damaged by degenerative or traumatic central nervous system (CNS) lesions. Functional groups on nanocarrier surfaces allow derivatization with molecules to target specific cells but may affect cellular interactions and nanocarrier uptake. We synthesized differently sized -COOH and -NH2 surface functionalized polymeric nanocarriers (SFNCs) by emulsion copolymerization and assessed uptake by different cell types in mixed cortical cultures. Following 60-min incubation with SFNCs, mean intensity measurements of fluorescently labeled SFNCs indicated that corticospinal tract motor neurons (CSMNs) took up more COOH- or NH2- functionalized SFNCs with similar sizes (150 nm), compared to glia. However, larger diameter (750 nm) SFNCs were taken up at higher concentrations compared to smaller COOH-derivatized SFNCs (150 nm). These data suggest that larger SFNCs may provide an advantage for enhanced uptake by targeted neurons.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures.

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.

Simulating traumatic brain injury in vitro: developing high throughput models to test biomaterial based therapies.

Traumatic brain injuries are serious clinical incidents associated with some of the poorest outcomes in neurological practice. Coupled with the limited regenerative capacity of the brain, this has significant implications for patients, carers, and healthcare systems, and the requirement for life-long care in some cases. Clinical treatment currently focuses on limiting the initial neural damage with long-term care/support from multidisciplinary teams. Therapies targeting neuroprotection and neural regeneration are not currently available but are the focus of intensive research. Biomaterial-based interventions are gaining popularity for a range of applications including biomolecule and drug delivery, and to function as cellular scaffolds. Experimental investigations into the development of such novel therapeutics for traumatic brain injury will be critically underpinned by the availability of appropriate high throughput, facile, ethically viable, and pathomimetic biological model systems. This represents a significant challenge for researchers given the pathological complexity of traumatic brain injury. Specifically, there is a concerted post-injury response mounted by multiple neural cell types which includes microglial activation and astroglial scarring with the expression of a range of growth inhibitory molecules and cytokines in the lesion environment. Here, we review common models used for the study of traumatic brain injury (ranging from live animal models to in vitro systems), focusing on penetrating traumatic brain injury models. We discuss their relative advantages and drawbacks for the developmental testing of biomaterial-based therapies.

Noscapine Modulates Neuronal Response to Oxygen-glucose Deprivation/Reperfusion Injury Via Activation of Sigma-1 Receptor in Primary Cortical Cultures.

In the present study, we investigated the effects of noscapine (0.5-2 µM), an alkaloid from the opium poppy (Papaver somniferum), on primary murine cortical neurons exposed to 60 min oxygen-glucose deprivation (OGD) in the presence of 5 µM BD-1047, a selective sigma-1 receptor antagonist. The experiments were performed on cortical neurons after 11-16 days of culture. To initiate oxygen-glucose deprivation, the culture medium was transferred to glucose-free DMEM, and placed in a humidified incubation chamber containing a mixture of 95% N2 and 5% CO2 at 37 °C for 60 min. In order to explore the effect on neurons under oxygen-glucose deprivation in this condition, some cultures were pretreated with noscapine and BD1047 together, 24 h prior to OGD followed by 24 h recovery. Cell viability, nitric oxide (NO) production and intracellular calcium concentration ([Ca2+]i) levels were evaluated by MTT assay, the modified Griess method, and Fura-2, respectively. Pretreatment of the cultures with noscapine in the presence of BD1047 significantly increased cell viability and decreased NO generation in a dose-dependent manner compared to BD1047 alone. Pretreatment with 2 µM noscapine and BD-1047 was shown to decrease the rise in [Ca2+]i induced by sodium azide (NaN3) and glucose deprivation. We concluded that noscapine in the presence of BD1047 could protect primary cortical neurons after oxygen-glucose deprivation-induced cell injury but this effect was not complete. Our results indicate that neuroprotective effects of noscapine could be mediated partially through activation of sigma-1 receptor and by decreasing NO production and [Ca2+]i levels.

The antioxidant and neuroprotective effects of Zolpidem on acrylamide-induced neurotoxicity using Wistar rat primary neuronal cortical culture.

Zolpidem is an introduced medication for the therapy of sleeping disorders. Its pharmacological effects are consequently characterized by a quick onset and a half-life of 2.4 h. Previous studies revealed the antioxidant and neuroprotectant effects of zolpidem. In this research, we wanted to demonstrate the exact sub-cellular/molecular mechanism of this medication using the primary neuronal cortical culture. For this purpose, firstly, the cortical neurons were isolated from the postnatal Wistar rat pups. Thereafter, different neural toxicity endpoints caused by acrylamide including ROS formation, lipid peroxidation, mitochondrial membrane potential collapse, lysosomal membrane integrity, and apoptosis were determined. All of these parameters are upstream events of cellular apoptosis which justifies neurodegeneration involved in many diseases such as Alzheimer's and Parkinson's. Our results demonstrated that zolpidem at concentrations of 1 and 2 mM prevented all the acrylamide-induced above referenced neural toxic events leading to neuronal apoptosis. These results revealed that zolpidem has the antioxidant and neuroprotectant properties that make it a promising prophylactic agent for preventing neurodegenerative complications. Considering the important role of oxidative stress in the development or progression of diseases, if the medication used as a treatment of a disease has antioxidant properties at the same time, it will certainly have much greater healing effects.

Effect fingerprints of antipsychotic drugs on neural networks in vitro.

We compared the acute effect of typical (haloperidol) and atypical (aripiprazole, clozapine, olanzapine) antipsychotic drugs (APDs) on spontaneous electrophysiological activity of in vitro neuronal networks cultured on microelectrode arrays (MEAs). Network burst analysis revealed a "regularizing" effect of all APDs at therapeutic concentrations, i.e., an increase of network-wide temporal synchronization. At supratherapeutic concentrations, all APDs but olanzapine mediated a decrease of burst and spike rates, burst duration, number of spikes in bursts, and network synchrony. The rank order of potency of APDs was: haloperidol > aripiprazole > clozapine > olanzapine (no suppression). Disruption of network function was not due to enhanced cell death as assessed by trypan blue staining. APDs promoted distinct concentration-dependent alterations yielding acute effect fingerprints of the tested compounds. These effects were rather characteristic for individual compounds than distinctive for typical vs. atypical APDs. Thus, this dichotomy may be of value in distinguishing clinical features but has no apparent basis on the network or local circuitry level.

The role of neuron-glia interactions in the emergence of ultra-slow oscillations.

Ultra-slow cortical oscillatory activity of 1-100 mHz has been recorded in human by electroencephalography and in dissociated cultures of cortical rat neurons, but the underlying mechanisms remain to be elucidated. This study presents a computational model of ultra-slow oscillatory activity based on the interaction between neurons and astrocytes. We predict that the frequency of these oscillations closely depends on activation of astrocytes in the network, which is reflected by oscillations of their intracellular calcium concentrations with periods between tens of seconds and minutes. An increase of intracellular calcium in astrocytes triggers the release of adenosine triphosphate from these cells which may alter transmission at nearby synapses by increasing or decreasing neurotransmitter release. These results provide theoretical support for the emerging awareness of astrocytes as active players in the regulation of neural activity and identify neuron-astrocyte interactions as a potential primary mechanism for the emergence of ultra-slow cortical oscillations.

Corrigendum: A Simplified In vitro Experimental Model Encompasses the Essential Features of Sleep.

[This corrects the article on p. 315 in vol. 10, PMID: 27458335.].

A Simplified In vitro Experimental Model Encompasses the Essential Features of Sleep.

In this paper, we show that neuronal assemblies plated on Micro Electrode Arrays present synchronized, low frequency firing patterns similar to in vivo slow wave oscillations, which are a key parameter of sleep-like state. Although neuronal cultures lack the characteristic high-frequency waves of wakefulness, it is possible to modulate their spontaneous firing pattern through the administration of specific neurotransmitters such as acetylcholine. We thus stimulated the cortical cultures with an agonist of acetylcholine receptor, Carbachol, which caused a desynchronization of the spontaneous firing of the cultures. We recorded and monitored the cultures for a period of over 31 h. We analyzed the electrophysiological signals by exploiting novel methodological approaches, taking into account the different temporal scales of the recorded signals, and considering both spikes and local field potentials. Supporting the electrophysiological analysis results, gene expressions of targeted genes showed the activation of specific markers involved in sleep-wake rhythms. Our results demonstrate that the Carbachol treatment induces desynchronization of neuronal activity, altering sleep-like properties in an in vitro model.

Neuroprotective effect of noscapine on cerebral oxygen-glucose deprivation injury.

BACKGROUND: The present study aims to investigate the effect of noscapine (0.5-2.5 µM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. METHODS: Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively. RESULTS: The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 µM noscapine. Increasing noscapine concentration up to 2.5 µM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 µM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 µM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 µM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD. CONCLUSIONS: We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.

Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice.

Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.

Synaptic dynamics contribute to long-term single neuron response fluctuations.

Firing rate variability at the single neuron level is characterized by long-memory processes and complex statistics over a wide range of time scales (from milliseconds up to several hours). Here, we focus on the contribution of non-stationary efficacy of the ensemble of synapses-activated in response to a given stimulus-on single neuron response variability. We present and validate a method tailored for controlled and specific long-term activation of a single cortical neuron in vitro via synaptic or antidromic stimulation, enabling a clear separation between two determinants of neuronal response variability: membrane excitability dynamics vs. synaptic dynamics. Applying this method we show that, within the range of physiological activation frequencies, the synaptic ensemble of a given neuron is a key contributor to the neuronal response variability, long-memory processes and complex statistics observed over extended time scales. Synaptic transmission dynamics impact on response variability in stimulation rates that are substantially lower compared to stimulation rates that drive excitability resources to fluctuate. Implications to network embedded neurons are discussed.

Extracting functionally feedforward networks from a population of spiking neurons.

Neuronal avalanches are a ubiquitous form of activity characterized by spontaneous bursts whose size distribution follows a power-law. Recent theoretical models have replicated power-law avalanches by assuming the presence of functionally feedforward connections (FFCs) in the underlying dynamics of the system. Accordingly, avalanches are generated by a feedforward chain of activation that persists despite being embedded in a larger, massively recurrent circuit. However, it is unclear to what extent networks of living neurons that exhibit power-law avalanches rely on FFCs. Here, we employed a computational approach to reconstruct the functional connectivity of cultured cortical neurons plated on multielectrode arrays (MEAs) and investigated whether pharmacologically induced alterations in avalanche dynamics are accompanied by changes in FFCs. This approach begins by extracting a functional network of directed links between pairs of neurons, and then evaluates the strength of FFCs using Schur decomposition. In a first step, we examined the ability of this approach to extract FFCs from simulated spiking neurons. The strength of FFCs obtained in strictly feedforward networks diminished monotonically as links were gradually rewired at random. Next, we estimated the FFCs of spontaneously active cortical neuron cultures in the presence of either a control medium, a GABA(A) receptor antagonist (PTX), or an AMPA receptor antagonist combined with an NMDA receptor antagonist (APV/DNQX). The distribution of avalanche sizes in these cultures was modulated by this pharmacology, with a shallower power-law under PTX (due to the prominence of larger avalanches) and a steeper power-law under APV/DNQX (due to avalanches recruiting fewer neurons) relative to control cultures. The strength of FFCs increased in networks after application of PTX, consistent with an amplification of feedforward activity during avalanches. Conversely, FFCs decreased after application of APV/DNQX, consistent with fading feedforward activation. The observed alterations in FFCs provide experimental support for recent theoretical work linking power-law avalanches to the feedforward organization of functional connections in local neuronal circuits.

Extracellular HMGB1 Released after Zinc-treatment Induces Neuronal Death in Primary Cortical Cultures / 대한해부학회지

As a nonhistone DNA-binding protein, high mobility group box 1 (HMGB1) is released in large amounts into the extracellular space immediately after ischemic insult and plays a role in the release of proinflammatory cytokines. Here, we the examined cytokine-like or signaling molecule-like function of extracellular HMGB1 in primary cortical cultures. We found that a large amount of HMGB1 was released following zinc-induced neuronal cell death in primary cortical cultures and that this extracellular HMGB1 might aggravate neuronal damage. The conditioned media collected from zinc-treated primary cortical cultures decreased neuronal cell survival to 69.6+/-1.4% of control values when added to fresh primary cortical cultures. In contrast, treatment with HMGB1-depleted conditioned media produced by cultures treated with an HMGB1 siRNA-expression vector suppressed the induction of neuronal death. A mutant HMGB1 siRNA-expression vector did not suppress the induction of neuronal death, demonstrating a role of HMGB1 in neuronal death. Moreover, HMGB1-depletion in media conditioned by cotreatment with anti-HMGB1 antibody or with anti-RAGE antibody, a potential receptor for HMGB1, recovered neuronal cell survival to 81.0+/-4.0% and 79.0+/-4.0%, respectively, when added to fresh primary cortical cultures. These results indicate that extracellular HMGB1 released after zinc treatment induces neuronal death, which might aggravate zinc toxicity.