Alessandro Formenti

Adult neural precursors isolated from post mortem brain yield mostly neurons: An erythropoietin-dependent process

This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4°C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.

Changes in extracellular Ca2+ can affect the pattern of discharge in rat thalamic neurons

1 The aim of this study was to investigate some of the cellular mechanisms involved in the effects caused by changes in extracellular Ca2+ concentration ([Ca2+]o). 2 Current‐ and voltage‐clamp experiments were carried out on acutely isolated thalamic neurons of rats. 3 Increasing [Ca2+]o alone induced a transition of the discharge from single spike to burst mode in isolated current‐clamped neurons. 4 Increasing [Ca2+]o caused the voltage‐dependent characteristics of the low voltage‐activated (LVA) transient Ca2+ currents to shift towards positive values on the voltage axis. Changing [Ca2+]o from 0.5 to 5 mM caused the inactivation curve to shift by 21 mV. 5 Extracellular Ca2+ blocked a steady cationic current. This current reversed at ‐35 mV, was scarcely affected by Mg2+ and was completely blocked by the non‐selective cation channel inhibitor gadolinium (10 μM). The effect of [Ca2+]o was mimicked by 500 μM spermine, a polyamine which acts as an agonist for the Ca2+‐sensing receptor, and was modulated by intracellular GTP‐γ‐S. 6 At the resting potential, both the voltage shift and the block of the inward current removed the inactivation of LVA calcium channels and, together with the increase in the Ca2+ driving force, favoured a rise in the low threshold Ca2+ spikes, causing the thalamic firing to change to the oscillatory mode. 7 Our data indicate that [Ca2+]o is involved in multiple mechanisms of control of the thalamic relay and pacemaker activity. These findings shed light on the correlation between hypercalcaemia, low frequency EEG activity and symptoms such as sleepiness and lethargy described in many clinical papers.

Responses of VPL thalamic neurones to peripheral stimulation in wakefulness and sleep

In unanaesthetized, undrugged, normally respiring cats extracellular recordings were obtained from ventroposterolateral thalamic units through different states of the sleep-waking cycle. An air-puff peripheral stimulation was used to activate the recorded neurones. State-dependent changes of the response of thalamic neurones were shown, comparing slow-wave sleep (SWS) to wakefulness (W). During SWS an increase was observed in the strength of the discharge suppression, which follows the excitatory peak in the typical response pattern. Also the cell excitability is further reduced in slow-wave sleep during the 150-200 ms period following an excitatory response, suggesting that an enhancement of the post-excitatory inhibition could be involved in the generation of the slow 5-6 Hz rhythms, observed during thalamic and cortical synchronization.

Effects of acetyl-l-carnitine on the survival of adult rat sensory neurons in primary cultures

Acetyl‐l‐carnitine produces a significant increase in the survival time‐course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 μm acetyl‐l‐carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost.

Inhibitory action of acetylcholine, baclofen and GTP-γ-S on calcium channels in adult rat sensory neurons

High- and low-voltage activated calcium channel currents (HVA and LVA) were inhibited by acetylcholine (10-100 microM) and baclofen (10 microM) in adult rat sensory neurons. This modulatory effect was present on dihydropyridine (nifedipine 1 microM) and/or omega-conotoxin (3.2 microM, 2-5 h incubation) insensitive components and was insensitive to holding potentials (Vh -50 to -90 mV). GTP-gamma-S (100 microM) prolonged calcium channel current activation in a time- and voltage-dependent manner. On the other hand, the current amplitude reduction induced by muscarinic and GABAB receptor activation, was not relieved by a 50-ms conditioning prepulse to +50 mV. This suggests the possibility of an alternative voltage-independent modulation mechanism.

Low-voltage activated calcium channels are differently affected by nimodipine

Voltage-dependent Ca2+ channels in adult rat sensory neurones were studied as far as their characterization and nimodipine effects are concerned, using patch-clamp whole-cell technique. Low-voltage activated (LVA) Ca2+ currents were identified according to activation and inactivation kinetics and sensitivity to amiloride. Nimodipine (10 nM) caused a decrease in LVA Ca2+ current amplitude (-40% +/- 2.2 s.e.m., n = 11 out of 30 with LVA Ca2+ currents). Conversely, in 6 neurones out of 30 nimodipine increased the Ca2+ current amplitude (+ 10% +/- 2). In some unaffected neurones (n = 5) nimodipine was also tested at higher concentrations (up to 5 microM) without any appreciable effect. In conclusion, nimodipine was partly able to block LVA calcium channels even at nanomolar concentrations. Supposing nimodipine acts directly on the channel, it can be assumed that there may be different types of LVA calcium channels in sensory neurones.

Medialis dorsalis thalamic unitary response to tooth pulp stimulation and its conditioning by brainstem and limbic activation

In nembutalized cats tooth pulp stimulation (TPS) was effective in exciting 18% of medialis dorsalis (MD) thalamic units. Facilitation of spontaneous MD unitary discharge followed high frequency stimulation of the lateral amygdala (25%), dorsal hippocampus (22%), mesencephalic reticular formation (20%) and septal nuclei (17%). Conditioning high-frequency stimulation of limbic and reticular structures, strongly facilitated the MD unitary responses to TPS. None of the thalamic neurons involved in nociception seems to project to the cerebral cortex. The conditioning effect on MD response of limbic and reticular stimulation suggests that these central structures may be involved in the modulation of the nociceptive input.

Calcium influx in rat thalamic relay neurons through voltage-dependent calcium channels is inhibited by enkephalin

High and low voltage-activated, transient (HVA and LVA,T) Ca2+ currents are crucial in determining the characteristic thalamic firing pattern, during the oscillatory mode. The modulatory effects induced by D-ala2-D-leu5-enkephalin (DADLE) on voltage-dependent Ca2+ channels have been investigated on acutely dissociated neurons from rat ventro-basal (VB) thalamus, by means of whole cell patch-clamp technique. DADLE (400 nM) reduced HVA Ca2+ channel currents in 37 out of 44 cells tested (-53 +/- 5.3% to 0 mV test potential, n = 24,). In 50% of the cases DADLE induced an effect which was persistent at all the potentials tested, i.e. a voltage-independent one. In the remaining neurons, the inhibition partially or totally disappeared on the currents evoked at the highest potentials. DADLE was also able to inhibit LVA Ca2+ channels (-40% in five out of 12 cells). In conclusion, thalamic relay neurons present opioid receptors negatively coupled to both HVA and LVA Ca2+ channels. The presence of two inhibitory effects of DADLE on the total HVA Ca2+ channels has been observed, and they are distinguishable on the basis of their sensitivity to voltage. It is suggested that Ca2+ current modulation may play a role in the production and tuning of the rhythmic burst discharge in these neurons.

Acrylate end-capped poly(ester-carbonate) and poly(ether-ester)s for polymer-on-multielectrode array devices: synthesis, photocuring and biocompatibility

Polymeric materials based on epsilon-caprolactone (CL), 1,5-dioxepan-2-one (DXO), and trimethylene carbonate (TMC) were prepared and evaluated as possible candidates for polymer-on-multielectrode (PoM) applications. CL was copolymerized with either DXO or TMC in the presence of the diol initiator 1,4-benzenedimethanol (BDM). The ring-opening polymerization experiments, carried out in bulk and using tin(II) catalysis, yielded the desired low molecular weight random copolymer diols, as evidenced by NMR, IR, MALDI-ToF MS, and DSC techniques. Upon reaction with acryloyl chloride, the corresponding diacrylate end-capped copolymers were obtained. The latter were characterized by NMR and IR spectroscopy, and their photocross-linking (in the presence of a UV initiator) was followed by ATR-FTIR spectroscopy. Transparent and soft thin films of the copoly(ether-ester) and copoly(ester-carbonate) diacrylates were prepared and cured under UV irradiation. The resulting polymeric films showed good biocompatibility properties as far as in vitro neural stem cells proliferation and differentiation to neurons and astrocytes are concerned. Noteworthy are the beneficial effects obtained upon preconditioning the copolymers by means of the cell-culture medium and the excellent properties shown particularly by the CL-TMC copolymer. Moreover, preliminary results show that microchannel formation by photocuring is possible with the synthesized polymers.

Spermine biphasically affects N-type calcium channel currents in adult dorsal root ganglion neurons of the rat.

Spermine (Spe) is a polyamine co-secreted with neurotransmitters. In this work its effects on N-type Ca2+ channel (CaV2.2) have been studied on adult sensory neurons of the rat by means of whole-cell patch-clamp. Spe exerted biphasic effects when added to the external solution: at 500 microM decreased N-type Ca2+ channel currents, reducing the maximum whole-cell conductance, shifting the activation curve to the right on the voltage axes and decreasing its slope; conversely, at lower concentration (500 nM) Spe induced completely opposite effects. In 62% of the neurons the inhibitory effects were accompanied by a slowing down of the activation kinetics relieved by a conditioning pre-pulse to +50 mV. The biphasic effects and their rapid onset and offset time course may be explained if multiple sites of action with a different affinity for Spe are present directly on the channel. The effects of Spe on HVA Ca2+ currents were strongly dependent on [Ca2+]ext, high [Ca2+] powerfully reducing Spe effects. This may be explained if we take into account that as Spe has four positive charges at physiological pH; it may compete with divalent cations for some negatively charged regulatory sites. In these experiments, Spe was effective at concentrations possibly reached in physiological conditions.