Search

The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal β-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal β-oxidation in neural cells. publisher: Elsevier articletitle: Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency journaltitle: Neurobiology of Disease articlelink: http://dx.doi.org/10.1016/j.nbd.2016.06.012 content_type: article copyright: © 2016 Elsevier Inc. All rights reserved. ispartof: Neurobiology of Disease vol:94 pages:157-168 ispartof: location:United States status: published

We propose an upgraded version of our previously designed open-source lipid bilayer amplifier. This improved amplifier is now suitable both for the use in introductory courses in biophysics and neurosciences at the undergraduate level and for scientific research. Similar to its predecessor, the OpenPicoAmp-100k is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. It consists of the high-speed headstage, followed by voltage-gain amplifier with built-in 6-order Bessel filter. The amplifier has a bandwidth of 100 kHz in the presence of 100 pF input membrane capacitance and is capable of measuring ion channel current with amplitudes from sub-pA and up to ±4 nA. At the full bandwidth and with a 1 GΩ transimpedance gain, the amplifier shows 12 pArms noise with an open input and 112 pArms noise in the presence of 100 pF input capacitance, while at the 5 kHz bandwidth (typical in single-channel experiments) noise amounts to 0.45 pArms and 2.11 pArms, respectively. Using an optocoupler circuit producing TTL-controlled current impulses and using 50% threshold analysis we show that at full bandwidth the amplifier has deadtimes of 3.5 µs and 5 µs at signal-to-noise ratios(SNR) of 9 and 1.7, respectively. Near 100% of true current impulses longer than 5 µs and 6.6 µs are detected at these two respective SNRs, while false event detection rate remains acceptably low. The wide bandwidth of the amplifier was confirmed in bilayer experiments with alamethicin, for which open ion channel current events shorter that 10 µs could be resolved.

Amyloids β (Aβ) are a hallmark of Alzheimer's disease. They affect the communication between neurons. They can also bind to neuronal targets and thereby affect both intracellular signalling and neuronal electrical activity. During the onset of Alzheimer's disease, a positive feedback loop between Aβ40/42 and cytosolic calcium is thoughtto accelerate the progression of the disease. If intracellular calcium and Aβ reinforce themselves through this mechanism, one would expect that the neurons targets of Aβ may display an altered electrical activity caused by the increase in cytoplasmic calcium as it is known that there is a tight coupling between calcium dynamics and the electrical excitabilty. The aim of this work is to test this assumption when considering one of the privileged target of Aβ, the activity of extrasynaptic NMDA receptors. Our theoretical model is a simple description of neuronal electrical activity based on the Hodgkin-Huxley like formalism, including a term that corresponds to the activity of the NMDA receptor and a cytosolic calcium compartment. When the tonic activity of extrasynaptic NMDA receptors is increased, neurons are less excitable. This is a counterintuive result as NMDA receptors exert an excitatory effect. Further analysis show that this inhibitory effect is due to the activation of calcium-dependent potassium channels, which hyperpolarize the neurons. Activation of extrasynaptic NMDA receptors also provokes a marked increase in intracellular calcium concentration, thus reinforcing the feed-forward relation between Aβ production and calcium.

By means of pulsed Nuclear Magnetic Resonance and Magic Angle Spinning we have studied crystalline C60 × 0.2 C70 × 1.5(CHCl3). We present data of the spin lattice relaxation (T1) of 13C nuclei as a function of temperature between 140 and 310 K. Their analysis has been made in terms of the chemical shift anisotropy model. Our results show that the high temperature free rotation of C60 molecules is not suppressed and that there is no structural phase transition near 260 K as observed in pure crystals of C60.

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system, but their physiological functions remain poorly understood. Calretinin is expressed in cerebellar granule cells and calretinin deficient mice suffer from alterations in motor coordination. Using confocal microscopy, we demonstrate that calretinin deficient mice exhibit a significantly decreased density of granule cells at the level of the cerebellar cortex. Moreover, it has been shown that migration of granule cells is tighly associated with intracellular calcium fluctuations. Therefore, we hypothesize that the perturbation of the calcium dynamics in calretinin deficient mice may be the cause of the observed morphological alterations. To test this assumption, we are currently developping two strategies.First, using confocal microscopy and cerebellar microexplant cultures, we are studying calcium transients occuring during granule cell migration in wild type and calretinin knock-out mice. On the other hand, we are developping a dedicated computational model for [Ca2+]i transients that takes into account calcium fluxes through the plasma and ER membrane. This model will shed light on the possible mechanism responsible for the modulation by calretinin of calcium dynamics during granule cell migration.

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system, but their physiological functions remain poorly understood. Calretinin is highly expressed in cerebellar granule cells and calretinin deficient mice exhibit alterations in motor coordination. Using confocal microscopy, we demonstrate that the cerebellar cortex of calretinin deficient mice exhibit a significantly decreased granule cells density. Interestingly, it has been shown that the migration of granule cells is tightly associated with intracellular calcium oscillations . Therefore, we hypothesize that an alteration of these calcium oscillations in calretinin deficient mice could be involved in the observed morphological alterations. To test this assumption, we are currently developping two strategies. First, using confocal microscopy and cerebellar microexplant culture, we are studying calcium oscillations occuring during granule cell migration in the wild type control and calretinin knock-out mice. This allow us to characterize the impact of variations in calcium buffering capacity over neuronal development and on the generation of the calcium oscillations observed during the granule cell migration. On the other hand, we are developping a theoretical model to study the impact of calcium buffering modifications on the dynamics underlying the observed calcium oscillations . This dedicated computational model will shed light on the possible mechanism responsible for the modulation, by calretinin, of calcium oscillations during the granule cell migration.

Although people are generally interested in how the brain functions, neuroscience education is hampered by a lack of low cost and engaging teaching materials. To address this, we developed an open-source lipid bilayer amplifier which is appropriate for use in introductory courses in biophysics or neurosciences. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. This device can be used in simple experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionophores like gramicidin A. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.

We have investigated the detailed regulation of neuronal firing threshold by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch clamp recording in acute slice. Theoretical results show that, at similar free calcium concentration, increased calcium buffer concentration lowers the firing threshold of cerebellar granule cells. We show that this effect is a direct consequence of the major slowdown of calcium dynamics. Patch clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal alterations in the excitability threshold as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit a significative lower firing threshold. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing threshold and therefore that calcium-binding proteins may play a critical role in the information processing in the central nervous system.