Mario Pellegrino

Piezoelectric Nanoparticle-Assisted Wireless Neuronal Stimulation.

Tetragonal barium titanate nanoparticles (BTNPs) have been exploited as nanotransducers owing to their piezoelectric properties, in order to provide indirect electrical stimulation to SH-SY5Y neuron-like cells. Following application of ultrasounds to cells treated with BTNPs, fluorescence imaging of ion dynamics revealed that the synergic stimulation is able to elicit a significant cellular response in terms of calcium and sodium fluxes; moreover, tests with appropriate blockers demonstrated that voltage-gated membrane channels are activated. The hypothesis of piezoelectric stimulation of neuron-like cells was supported by lack of cellular response in the presence of cubic nonpiezoelectric BTNPs, and further corroborated by a simple electroelastic model of a BTNP subjected to ultrasounds, according to which the generated voltage is compatible with the values required for the activation of voltage-sensitive channels.

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

Abstract Single Ca2+-activated K+ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca2+ content. After patch excision, the activity increased to levels determined by the Ca2+ concentration (0.5–10 µM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca2+ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca2+-activated K+ channels of human erythrocytes.

Two-photon polymerization of sub-micrometric patterned surfaces: investigation of cell-substrate interactions and improved differentiation of neuron-like cells

Direct Laser Writing (DLW) is an innovative tool that allows the photofabrication of high resolution 3D structures, which can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of submicrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 μm spaced submicrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 μm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the submicrometric patterned substrates. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as submicrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.

Calmodulin antagonists do not inhibit IKCa channels of human erythrocytes

Patch-clamp recordings were performed to study the effects of three calmodulin (CaM) antagonists on the gating of intermediate calcium-activated K(+) channels (IK(Ca)) of human erythrocytes. In the cell-attached configuration, both opening frequency and open probability of IK(Ca) channels were not significantly different in control cells and in those incubated with calmidazolium, trifluoperazine or W7. IK(Ca) channels in excised membrane patches, were normally activated by the calcium bathing the cytoplasmic side in the presence of CaM antagonists, at calcium concentrations ranging from 10(-7) to 10(-3) M. The activity of IK(Ca) channels, which had been previously up-modulated by an endogenous cAMP-dependent protein kinase, was not inhibited when perfused with CaM antagonists. The results presented in this study demonstrate that calmodulin antagonists do not inhibit the activity of native IK(Ca) channels of human erythrocytes. These data are in accordance with findings on the cloned IK(Ca) indicating that calmodulin is constitutively associated with these channels.

Response to axotomy of an identified leech neuron, in vivo and in culture

Membrane properties of identified leech neurons with non-spiking somata were studied after axotomy. Two distinct procedures were used: the section of ganglionic roots in vivo and the isolation of cell somata in culture. The results indicated that axotomized neurons progressively developed the excitability of somatic membrane, both in vivo and in culture.

Weak hydrostatic forces in far-scanning ion conductance microscopy used to guide neuronal growth cones

Scanning ion conductance microscopy (SICM) is currently used for high resolution topographic imaging of living cells. Recently, it has been also employed as a tool to deliver stimuli to the cells. In this work we have investigated the mechanical interaction occurring between the pipette tip and the sample during SICM operation. For the purpose, we have built a setup combining SICM with atomic force microscopy (AFM), where the AFM cantilever replaces the sample. Our data indicate that, operating in far-scanning mode with current decrease values below 2%, no force can be detected, provided that the level of the electrolyte filling the pipette is equal to that determined by the capillary tension. A filling level different from this value determines a hydrostatic pressure, a flux through the pipette tip and detectable forces, even in far-scanning mode. The absolute value of these forces depends on the pipette tip size. Therefore, a possible pitfall when using SICM for cell imaging is to imply zero-force working conditions. However the hydrostatic forces can be exploited in order to deliver weak mechanical stimuli and guide neuronal growth cones. Evidences of the effectiveness of this approach are herein given.

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.

Neural mechanisms underlying the segmental and generalized cord shortening reflexes in the leech

Summary1.The neural mechanisms responsible for contraction of the nerve cord sheath muscles were investigated in the leech,Hirudo medicinalis.2.Two kinds of cord contractions could be distinguished: one, of small amplitude and slow time course, is not preceded by discharge of the fast conducting system (FCS); the second has higher amplitude and faster time course and is always preceded by the discharge of the FCS (Fig. 2).3.Electric stimulation of the nerve cord before and after selective lesions of Faivres nerve or of the lateral connectives showed that “generalized” shortening of the nerve cord involving all the connectives requires firing of an intact FCS. Only “local” shortening, limited to the two connectives adjacent to the stimulating electrode, can be elicited after lesion of the FCS (Figs. 3, 4, 5).4.Impulse discharge of the primary mechanoceptive neurons T, P, and N causes local shortening without FCS firing. Firing of the T cell at a rate sufficient to activate the FCS transsynaptically, is always followed by generalized cord contraction (Fig. 6).5.Increases or decreases in the firing rate of the L motoneurone, either spontaneously occurring or experimentally imposed by altering its membrane potential, are consistently followed by contractions and relaxations of the nerve cord, respectively (Fig. 8).6.Single FCS action potentials are followed by all-or-none depolarizing PSPs in the L motoneurone, at a latency of less than 1 ms, and are able to fire the cell. After a single potential is fired by the FCS, the PSPs appearing in the L motoneurons located in different ganglia are separated by a latency corresponding to the conduction time of the FCS (Figs. 9, 10, 11). The PSPs are Mg++ resistant and are increased by hyperpolarization and decreased by depolarization of the post-synaptic membrane (Figs. 12, 13).7.It is concluded that the L motoneurone, which subserves body shortening, innervates also the muscle cells of the cord sheath and that the rôle of the FCS is to bring about the generalization of both body and cord shortening reflexes.

Use of scanning ion conductance microscopy to guide and redirect neuronal growth cones

Scanning ion conductance microscopy has been applied to neuronal growth cones of the leech either to image or to stimulate them. Growth cone advance was recorded in non-contact mode using a 2% ion current decrease criterion for pipette-membrane distance control. We demonstrate effective growth cone remodelling using a 5% criterion (near-scanning). Recurrent line near-scanning aligned growth cone processes along the scan line. The new membrane protrusions, marked by DiI, started a few minutes after scanning onset and progressively grew in thickness. Using scanning patterns suitable for connecting distinct growth cones, new links were consistently developed. Although the underlying mechanism is still a matter for investigation, a mechanical perturbation produced by the moving probe appeared to induce the process formation. Thanks to its deterministic and interactive features, this novel approach to guiding growth cones is a promising way to develop networks of identified neurons as well as link them with artificial structures.

Stretch-activated cation channels with large unitary conductance in leech central neurons

Stretch-activated cation channels were identified in the soma membrane of leech central neurons. These channels were almost silent under normal experimental conditions and were distinctly activated by application of negative pressure to the patch pipette. The channels exhibited a preferential selectivity for K+ and a slope conductance of about 200 pS, in symmetrical K+ solution. In cell-attached patches these cation channels were activated by cell swelling.