Pharmacologically induced and stimulus evoked rhythmic neuronal oscillatory activity in the primary motor cortex in vitro

Parkinson's disease (PD) is associated with enhanced synchronization of neuronal network activity in the beta (15-30 Hz) frequency band across several nuclei of the basal ganglia (BG). Deep brain stimulation of the subthalamic nucleus (STN) appears to reduce this pathological oscillation, thereby alleviating PD symptoms. However, direct stimulation of primary motor cortex (M1) has recently been shown to be effective in reducing symptoms in PD, suggesting a role for cortex in patterning pathological rhythms. Here, we examine the properties of M1 network oscillations in coronal slices taken from rat brain. Oscillations in the high beta frequency range (layer 5, 27.8 +/- 1.1 Hz, n=6) were elicited by co-application of the glutamate receptor agonist kainic acid (400 nM) and muscarinic receptor agonist carbachol (50 mu M). Dual extracellular recordings, local application of tetrodotoxin and recordings in M1 micro-sections indicate that the activity originates within deep layers V/VI. Beta oscillations were unaffected by specific AMPA receptor blockade, abolished by the GABA type A receptor (GABAAR) antagonist picrotoxin and the gap-junction blocker carbenoxolone, and modulated by pentobarbital and zolpidem indicating dependence on networks of GABAergic interneurons and electrical coupling. High frequency stimulation (HFS) at 125 Hz in superficial layers, designed to mimic transdural/transcranial stimulation, generated gamma oscillations in layers 11 and V (incidence 95%, 69.2 +/- 7.3 Hz, n=17) with very fast oscillatory components (VFO; 100-250 Hz). Stimulation at 4 Hz, however, preferentially promoted theta activity (incidence 62.5%, 5.1 +/- 0.6 Hz, n=15) that effected strong amplitude modulation of ongoing beta activity. Stimulation at 20 Hz evoked mixed theta and gamma responses. These data suggest that within M1, evoked theta, gamma and fast oscillations may coexist with and in some cases modulate pharmacologically induced beta oscillations.

Synchronization processes in nonlinear systems and their relation to cortical oscillatory dynamics

This thesis was focused on theoretical models of synchronization to cortical dynamics as measured by magnetoencephalography (MEG). Dynamical systems theory was used in both identifying relevant variables for brain coordination and also in devising methods for their quantification. We presented a method for studying interactions of linear and chaotic neuronal sources using MEG beamforming techniques. We showed that such sources can be accurately reconstructed in terms of their location, temporal dynamics and possible interactions. Synchronization in low-dimensional nonlinear systems was studied to explore specific correlates of functional integration and segregation. In the case of interacting dissimilar systems, relevant coordination phenomena involved generalized and phase synchronization, which were often intermittent. Spatially-extended systems were then studied. For locally-coupled dissimilar systems, as in the case of cortical columns, clustering behaviour occurred. Synchronized clusters emerged at different frequencies and their boundaries were marked through oscillation death. The macroscopic mean field revealed sharp spectral peaks at the frequencies of the clusters and broader spectral drops at their boundaries. These results question existing models of Event Related Synchronization and Desynchronization. We re-examined the concept of the steady-state evoked response following an AM stimulus. We showed that very little variability in the AM following response could be accounted by system noise. We presented a methodology for detecting local and global nonlinear interactions from MEG data in order to account for residual variability. We found crosshemispheric nonlinear interactions of ongoing cortical rhythms concurrent with the stimulus and interactions of these rhythms with the following AM responses. Finally, we hypothesized that holistic spatial stimuli would be accompanied by the emergence of clusters in primary visual cortex resulting in frequency-specific MEG oscillations. Indeed, we found different frequency distributions in induced gamma oscillations for different spatial stimuli, which was suggestive of temporal coding of these spatial stimuli. Further, we addressed the bursting character of these oscillations, which was suggestive of intermittent nonlinear dynamics. However, we did not observe the characteristic-3/2 power-law scaling in the distribution of interburst intervals. Further, this distribution was only seldom significantly different to the one obtained in surrogate data, where nonlinear structure was destroyed. In conclusion, the work presented in this thesis suggests that advances in dynamical systems theory in conjunction with developments in magnetoencephalography may facilitate a mapping between levels of description int he brain. this may potentially represent a major advancement in neuroscience.

Induced visual illusions and gamma oscillations in human primary visual cortex

Using magnetoencephalography, we studied the spatiotemporal properties of cortical responses in terms of event-related synchronization and event-related desynchronization to a range of stripe patterns in subjects with no neurological disorders. These stripes are known for their tendency to induce a range of abnormal sensations, such as illusions, nausea, dizziness, headache and attacks of pattern-sensitive epilepsy. The optimal stimulus must have specific physical properties, and maximum abnormalities occur at specific spatial frequency and contrast. Despite individual differences in the severity of discomfort experienced, psychophysical studies have shown that most observers experience some degree of visual anomaly on viewing such patterns. In a separate experiment, subjects reported the incidence of illusions and discomfort to each pattern. We found maximal cortical power in the gamma range (30-60 Hz) confined to the region of the primary visual cortex in response to patterns of 2-4 cycles per degree, peaking at 3 cycles per degree. This coincides with the peak of mean illusions and discomfort, also maximal for patterns of 2-4 cycles per degree. We show that gamma band activity in V1 is a narrow band function of spatial frequency. We hypothesize that the intrinsic properties of gamma oscillations may underlie visual discomfort and play a role in the onset of seizures.

Oleate protects against palmitate-induced insulin resistance in L6 myotubes

Oleate has been shown to protect against palmitate-induced insulin resistance. The present study investigates mechanisms involved in the interaction between oleate and palmitate on insulin-stimulated glucose uptake by L6 skeletal muscle cells. L6 myotubes were cultured for 6 h with palmitate or oleate alone, and combinations of palmitate with oleate, with and without phosphatidylinositol 3-kinase (PI3-kinase) inhibition. Insulin-stimulated glucose uptake, measured by uptake of 2-deoxy-d-[3H]glucose, was almost completely prevented by 300 microm-palmitate. Cells incubated with oleate up to 750 micromol/l maintained a significant increase in insulin-stimulated glucose uptake. Co-incubation of 50-300 microm-oleate with 300 microm-palmitate partially prevented the decrease in insulin-stimulated glucose uptake associated with palmitate. Adding the PI3-kinase inhibitors wortmannin (10- 7 mol/l) or LY294002 (25 micromol/l) to 50 microm-oleate plus 300 microm-palmitate significantly reduced the beneficial effect of oleate against palmitate-induced insulin resistance, indicating that activation of PI3-kinase is involved in the protective effect of oleate. Thus, the prevention of palmitate-induced insulin resistance by oleate in L6 muscle cells is associated with the ability of oleate to maintain insulin signalling through PI3-kinase.

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Glucosamine-induced insulin resistance in L6 muscle cells

Background: Glucosamine increases flux through the hexosamine pathway, causing insulin resistance and disturbances similar to diabetic glucose toxicity. Aim: This study examines the effect of glucosamine on glucose uptake by cultured L6 muscle cells as a model of insulin resistance. Methods: Glucose uptake by L6 myotubes was measured using the non-metabolized glucose analogue 2-deoxy-D-glucose after incubation with glucosamine for 4 and 24 h, with and without insulin and several other agents (metformin, peroxovanadium and D-pinitol) that improve glucose uptake in diabetic states. Results: After 4 h, high concentrations of glucosamine (5 × 10-3 and 10-2 M) reduced basal and insulin-stimulated glucose uptake by up to 50%. After 24 h, the effect of insulin was completely abolished by 10-2 M glucosamine and reduced over 50% by 5 × 10-3 M glucosamine. Lower concentrations of glucosamine did not significantly alter glucose uptake. The effect of glucosamine could not be attributed to cytotoxicity assessed by the Trypan Blue test. Metformin, peroxovanadium and D-pinitol, each of which increased glucose uptake by L6 cells, did not prevent the decrease in glucose uptake with glucosamine. Conclusion: Glucosamine decreased insulin-stimulated glucose uptake by L6 muscle cells, providing a potential model of insulin resistance with similarities to glucose toxicity. Insulin resistance induced by glucosamine was not reversed by three agents (metformin, peroxovanadium and D-pinitol) known to enhance or partially mimic the effects of insulin. © 2004 Blackwell Publishing Ltd.

Palmitate induces insulin resistance in monocytes and increases expression of the integrin CD11b

Obesity and insulin resistance are important risk factors for atherosclerosis, and elevated level of plasma NEFA is a common feature in individuals with obesity and insulin resistance. Palmitate, one of the most abundant non-esterified SFA in plasma, has been reported to induce insulin resistance in adipose tissues and skeletal muscles and to cause an increased inflammatory response in monocytes. The present study investigated whether palmitate can induce insulin resistance in monocytes and its effect on monocyte adhesion molecular expression (CD11b). Insulin resistance was measured by in vitro uptake of insulin-stimulated 3H-labelled 2-deoxy-D-glucose into THP-1 cells, cell surface CD11b expression was measured by flow cytometry. The data showed that palmitate-induced insulin resistance in THP-1 monocytes was concentration and time dependent (Figure 1). The insulin-stimulated glucose uptake was significantly decreased in cells treated with 300 mM-palmitate compared with control cells (P<0.001) and was observed within 6 h, but was not a result of palmitate toxicity. There was no significant increase in caspase 3 activation (P>0.05). Treatment with 300 mM-palmitate for 24 h also caused a significant increase in surface CD11b expression in both U937 and THP-1 monocytic cell lines and human primary monocytes compared with the control (P<0.001). Both these effects were inhibited by co-incubation with Fumonisin B1, an inhibitor of de novo ceramide synthesis. In conclusion, these data show that palmitate, at physiological concentrations, can cause insulin resistance in monocytes and increase monocyte surface integrin CD11b expression, which is in part the result of the synthesis of ceramide.

The missing link:Analogous human and primate cortical gamma oscillations

Recent animal studies highlighting the relationship between functional imaging signals and the underlying neuronal activity have revealed the potential capabilities of non-invasive methods. However, the valuable exchange of information between animal and human studies remains restricted by the limited evidence of direct physiological links between species. In this study we used magnetoencephalography (MEG) to investigate the occurrence of 30-70 Hz (gamma) oscillations in human visual cortex, induced by the presentation of visual stimuli of varying contrast. These oscillations, well described in the animal literature, were observed in retinotopically concordant locations of visual cortex and show striking similarity to those found in primate visual cortex using surgically implanted electrodes. The amplitude of the gamma oscillations increases linearly with stimulus contrast in strong correlation with the gamma oscillations found in the local field potential (LFP) of the macaque. We demonstrate that non-invasive magnetic field measurements of gamma oscillations in human visual cortex concur with invasive measures of activation in primate visual cortex, suggesting both a direct representation of underlying neuronal activity and a concurrence between human and primate cortical activity. © 2005 Elsevier Inc. All rights reserved.

Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.

Effect of dopamine on synchronous neuronal oscillations in the globus pallidus-subthalamic nucleus network

Changes in the pattern of activity of neurones within the basal ganglia are relevant in the pathophysiology and symptoms of Parkinson’s disease. The globus pallidus (GP) – subthalamic nucleus (STN) network has been proposed to form a pacemaker driving regenerative synchronous bursting activity. In order to test whether this activity can be sustained in vitro a 20o parasagittal slice of mouse midbrain was developed which preserved functional connectivity between the STN and GP. Mouse STN and GP cells were characterised electrophysiologically by the presence or absence of a voltage sag in response to hyperpolarising current steps indicative of Ih and the presence of rebound depolarisations. The presence of evoked and spontaneous post-synaptic GABA and glutamatergic currents indicated functional connectivity between the STN and GP. In control slices, STN cells fired action potentials at a regular rate, activity which was unaffected by bath application of the GABAA receptor antagonist picrotoxin (50 μM) or the glutamate receptor antagonist CNQX (10 μM). Paired extracellular recordings of STN cells showed uncorrelated firing. Oscillatory burst activity was induced pharmacologically using the glutamate receptor agonist, NMDA (20 μM), in combination with the potassium channel blocker apamin (50 -100 nM). The burst activity was unaffected by bath application of picrotoxin or CNQX while paired STN recordings showed uncorrelated activity indicating that the activity is not produced by the neuronal network. Thus, no regenerative activity is evident in this mouse brain preparation, either in control slices or when bursting is pharmacologically induced, suggesting the requirement of other afferent inputs that are not present in the slice. Using single-unit extracellular recording, dopamine (30 μM) produced an excitation of STN cells. This excitation was independent of synaptic transmission and was mimicked by both the Dl-like receptor agonist SKF38393 (10 μM) and the D2-like receptor agonist quinpirole (10 μM). However, the excitation was partially reduced by the D1-like antagonist SCH23390 (2 μM) but not by the D2-like antagonists sulpiride (10 μM) and eticlopride (10 μM). Using whole-recordings, dopamine was shown to induce membrane depolarisation. This depolarisation was caused either by a D1-like receptor mediated increase in a conductance which reversed at -34 mV, consistent with a non-specific cation conductance, or a D2-like receptor mediated decrease in conductance which reversed around -100 mV, consistent with a potassium conductance. Bath application of dopamine altered the pattern of the burst-firing produced by NMDA an apamin towards a more regular pattern. This effect was associated with a decrease in amplitude and ll1crease in frequency of TTX-resistant plateau potentials which underlie the burst activity.

Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Zonal flow induced by longitudinal librations of a rotating cylindrical cavity

Longitudinal librations represent oscillations about the axis of a rotating axisymmetric fluid filled cavity. An analytical theory is developed for the case of a cylindrical cavity in the limit when the libration frequency is small in comparison with the rotation rate, but large in comparison with the inverse of the spin-up time. It is shown that through the nonlinear advection in the Ekman layers the librations cause the fluid to rotate more slowly. © 2010 Elsevier B.V. All rights reserved.

Aβ(1-42) induced metabolic effects in a stem cell derived neuron and astrocyte network.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.