Angela Pignatelli

Neuronal differentiation potential of human adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells showed negative membrane potential (-60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after removal of differentiation medium. In view of these results and the easy availability of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.

Functional properties of dopaminergic neurones in the mouse olfactory bulb

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate‐limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH‐GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage‐dependent conductances: the two having largest amplitude were a fast transient Na+ current and a delayed rectifier K+ current. In addition, we observed three smaller inward currents, sustained by Na+ ions (persistent type) and by Ca2+ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na+ current and of a T‐type Ca2+ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin‐Huxley model of TH‐GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.

Calcium-independent synaptic transmission: artifact or fact?

The release of neurotransmitters at classical chemical synapses occurs via Ca2+ influx through voltage-dependent Ca2+ channels, which are opened following depolarization of presynaptic terminals. However, owing to a persistence or increase in the amount of transmitter released in preparations containing low concentrations of Ca2+, it has been proposed that transmitter release could also occur through a Ca(2+)-independent, carrier-mediated process. On the other hand, lowering extracellular [Ca2+] can actually promote Ca2+ influx through voltage-activated Ca2+ channels via a modification of the surface potential of plasma membranes. Therefore, the proposed Ca(2+)-independent transmitter release could be re-accommodated within the framework of the Ca2+ hypothesis of synaptic transmission by taking into account the surface-charge effects.

Cholinergic Modulation of Dopaminergic Neurons in the Mouse Olfactory Bulb

Considerable evidence exists for an extrinsic cholinergic influence in the maturation and function of the main olfactory bulb. In this study, we addressed the muscarinic modulation of dopaminergic neurons in this structure. We used different patch-clamp techniques to characterize the diverse roles of muscarinic agonists on identified dopaminergic neurons in a transgenic animal model expressing a reporter protein (green fluorescent protein) under the tyrosine hydroxylase promoter. Bath application of acetylcholine (1 mM) in slices and in enzymatically dissociated cells reduced the spontaneous firing of dopaminergic neurons recorded in cell-attached mode. In whole-cell configuration no effect of the agonist was observed, unless using the perforated patch technique, thus suggesting the involvement of a diffusible second messenger. The effect was mediated by metabotropic receptors as it was blocked by atropine and mimicked by the m2 agonist oxotremorine (10 muM). The reduction of periglomerular cell firing by muscarinic activation results from a membrane-potential hyperpolarization caused by activation of a potassium conductance. This modulation of dopaminergic interneurons may be important in the processing of sensory information and may be relevant to understand the mechanisms underlying the olfactory dysfunctions occurring in neurodegenerative diseases affecting the dopaminergic and/or cholinergic systems.

A potential reservoir of immature dopaminergic replacement neurons in the adult mammalian olfactory bulb

A significant fraction of the interneurons added in adulthood to the glomerular layer (GL) of the olfactory bulb (OB) are dopaminergic (DA). In the OB, DA neurons are restricted to the GL, but using transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter, we also detected the presence of TH-GFP+ cells in the mitral and external plexiform layers. We hypothesized that these could be adult-generated neurons committed to become DA but not yet entirely differentiated. Accordingly, TH-GFP+ cells outside the GL exhibit functional properties (appearance of pacemaker currents, synaptic connection with the olfactory nerve, intracellular chloride concentration, and other) marking a gradient of maturity toward the dopaminergic phenotype along the mitral–glomerular axis. Finally, we propose that the establishment of a synaptic contact with the olfactory nerve is the key event allowing these cells to complete their differentiation toward the DA phenotype and to reach their final destination.

Nonspecific Cation Current Associated with Native Polycystin-2 in HEK-293 Cells

Mutations in either PKD1 or PKD2 gene are associated with autosomal dominant polycystic kidney disease, the most common inherited kidney disorder. Polycystin-2 (PC2), the PKD2 gene product, and the related protein polycystin-L, function as Ca(2+)-permeable, nonselective cation channels in different expression systems. This work describes a nonspecific cation current (I(CC)) that is present in native HEK-293 cells and highly associated with a PC2-channel activity. The current is voltage dependent, activating for potentials that are positive to -50 mV and inactivating in a few milliseconds. It is sensitive to Cd(2+), Gd(3+), La(3+), SKF96365, and amiloride. After silencing of PC2 by RNA interfering, cells show a reduced current that is restored by transfection with normal but not truncated PC2. Consistently, I(CC) is abolished by perfusion with an anti-PC2 antibody. Furthermore, heterologous expression of the PC1 cytoplasmic tail significantly increases I(CC) peak amplitude compared with native cells. This is the first characterization of such a current in HEK-293 cells, a widely used expression system for ion channels. These cells, therefore, could be regarded as a suitable and readily accessible tool to study interactions between native PC2/PC1 complex and other membrane proteins, thus contributing to the understanding of autosomal dominant polycystic kidney disease pathogenesis.

CALCIUM-INDEPENDENT RELEASE OF NEUROTRANSMITTER IN THE RETINA : A COPERNICAN VIEWPOINT CHANGE

The release of synaptic transmitter in chemical synapses is brought about by Ca2+ influx through voltage-dependent Ca2+ channels opened by depolarisation of presynaptic terminals. However, in some preparations transmitter release persists or increases in low-Ca2+ media, and it has therefore been proposed that transmitter release could also occur through a Ca2+-independent, carrier mediated process. In particular it has been suggested that this may be the case for synaptic transmission between photoreceptors and second order neurones of the vertebrate retina. From our recent experiments on synaptic transmission from photoreceptors to horizontal cells of turtle and salamander retinas, it appears that lowering extracellular Ca2+ can actually promote Ca2+ influx through voltage-activated Ca2+ channels via a modification of surface potential of plasma membranes. On the basis of this apparently paradoxical effect of low Ca2+ media, it is possible to reaccommodate the so-called Ca2+-independent release within the framework of Ca2+-dependent synaptic transmission without invoking unconventional mechanisms.

Metabotropic glutamate receptors 1 and 5 differentially regulate bulbar dopaminergic cell function

Effects of activation of metabotropic glutamatergic receptors (mGluR) were investigated in mouse dopaminergic olfactory bulb neurons. After blockage of ionotropic receptors, focal application of glutamate or of group I/II mGluR agonist t-ACPD resulted in a depolarization, paralleled by an inward current in voltage-clamp conditions. The Group I agonist DHPG induced a depolarization, which could be largely blocked by mGluR1 antagonists. The DHPG action i) was prevented by buffering intracellular Ca(2+) with BAPTA and by a phospholipase C inhibitor; ii) was not affected by the block of Ca(2+) entry, and iii) was blocked by inhibitors of the Na(+)/Ca(2+) exchanger. These observations were interpreted as a mGluR1-mediated intracellular Ca(2+) release, followed by the activation of an electrogenic Na(+)/Ca(2+) exchanger. The mGluR5 agonist CHPG induced a hyperpolarization of membrane potential, resulting in a decrease of the spontaneous firing frequency. CHPG induced i) a decrease in membrane resistance; ii) an increase in the action potential repolarization rate, and iii) an increase in the amplitude of the afterhyperpolarization. This was interpreted as a mGluR5-mediated opening of a K(+) conductance. These data suggest that mGluR1 and mGluR5 play different and non-overlapping roles in the regulation of the excitability of bulbar dopaminergic neurons.

Looking over Toxin–K+ Channel Interactions. Clues from the Structural and Functional Characterization of α-KTx Toxin Tc32, a Kv1.3 Channel Blocker

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/β scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

The h-current in periglomerular dopaminergic neurons of the mouse olfactory bulb.

The properties of the hyperpolarization-activated cation current (Ih) were investigated in rat periglomerular dopaminergic neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. At 37 °C and minimizing the disturbance of the intracellular milieu with perforated patches, this current shows a midpoint of activation around −82.7 mV, with a significant level of opening already at rest, thereby giving a substantial contribution to the resting potential, and ultimately playing a relevant function in the control of the cell excitability. The blockage of Ih has a profound influence on the spontaneous firing of these neurons, which result as strongly depressed. However the effect is not due to a direct role of the current in the pacemaker process, but to the Ih influence on the resting membrane potential. Ih kinetics is sensitive to the intracellular levels of cAMP, whose increase promotes a shift of the activation curve towards more positive potentials. The direct application of DA and 5-HT neurotransmitters, physiologically released onto bulbar dopaminergic neurons and known to act on metabotropic receptors coupled to the cAMP pathway, do not modifythe Ih amplitude. On the contrary, noradrenaline almost halves the Ih amplitude. Our data indicate that the HCN channels do not participate directly to the pacemaker activity of periglomerular dopaminergic neurons, but influence their resting membrane potential by controlling the excitability profile of these cells, and possibly affecting the processing of sensory information taking place at the entry of the bulbar circuitry.