Monica Pellegrini

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.

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.

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.

Two types of K+ channels in excised patches of somatic membrane of the leech AP neuron

The patch-clamp technique has been applied to the somatic membrane of the leech AP neurons. Ionic currents from single potassium channels were recorded in inside-out configuration. Two types of channels, sharing close values of conductance in symmetrical K+, were identified as distinct, according to their properties of rectification, Ca2+ sensitivity and voltage dependence. The channels designated as VCI exhibited an outward rectification and their gating was quite independent on changes of patch potential and of [Ca2+]i. The channels designated as VCD showed a linear I-V relationship and their activity was dependent on both the membrane potential and the intracellular [Ca2+].

The efferent vestibular neurons in the toad (Bufo bufo L.): their location and morphology. A horseradish peroxidase study

Retrogradely labeled neurons are observed in the central nervous system of the toad after peripheral application of peroxidase to the anterior and posterior stumps of the VIIIth nerve. These efferent vestibular neurons are localized in the brainstem only ipsilaterally to the treated nerve; they are restricted within a region close to the motor nucleus of the VIIth nerve, outside the vestibular nuclear complex, and are predominantly localized to the borders between the gray and white matter in an arrangement that seems to surround the motor nucleus of the VIIth nerve. No evidence was found for the existence of labeled Purkinje cells in the cerebellar cortex and therefore of a cerebellolabyrinthine pathway. The efferent vestibular neurons are medium-sized cells with two prominent dendrites, often oriented in a mediolateral direction. A comparative analysis between these neurons and the contiguous motoneurons of the VIIth nerve permitted differentiation of the two groups of neurons as to: distribution in a mediolateral direction, distribution in depth from the brainstem surface, longitudinal extension in the caudorostral direction and morphologic characteristics and dendritic arrangement of the neurons.

Use of conditional distributions in the analysis of ion channel recordings

A method to test the Markov nature of ion channel gating is proposed. It makes use of singly and doubly conditional distributions. The application of this method to recordings from single BK channels provides evidence that at least two states of the underlying kinetic scheme are left at a constant rate. Moreover, the probabilities, when leaving a state, of reaching another given state are shown to be constant for all the states of the system.

Stretch-activated cation channels of leech neurons exhibit two activity modes.

Single‐channel recordings were used to characterize two activity modes of stretch activated channels (SACs) in identified neurons of the leech. Clear‐cut differences in the activity pattern of SACs from freshly desheathed cell bodies and from cultured AP cells were observed. SACs of inside‐out patches, made by ‘gentle’ sealing and excised from cell bodies of freshly desheathed ganglia exhibited spike‐like (SL) activity, with a mean channel open time (MCOT) shorter than 10 ms. Fitting of dwell open‐time distributions revealed time constants shorter than 2 and 10 ms. This activity was characterized by a chord conductance of about 115 pS. SACs from cultured cells often displayed activity just after excision. MCOT exceeded 200 ms and the time constants of open‐time interval distributions were longer than 10 and 100 ms. Furthermore, this activity pattern was characterized by both sub‐ (about 80 and 40 pS) and super‐conductance (150 pS) levels, hence denoted as multiconductance (MC) mode. The percentage of open time spent at the main subconductance level (80 pS) was significantly higher in patches isolated from growth cones than in those from cell bodies of cultured neurons. The two activity modes (SL and MC) should belong to the same channel because both modes have a common main conductance value and exhibit outward rectification, stretch sensitivity and blockage by Gd3+ and gentamicin.

Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-µm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells.

Axotomy affects density but not properties of potassium leak channels, in the leech AP neurons

Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell body membrane. In a previous report we described single potassium channels contributing to the leak conductance in the soma membrane of AP cells. Here we compare both properties and density of single potassium leak channels in cell-free patches from normal and axotomized AP neurons. We show that properties such as single channel conductance, outward rectification, time constants of open and shut interval distributions and absence of inactivation do not significantly differ between normal and axotomized cells. On the other hand, we find that the number of channels per patch progressively increases with time after axotomy. We conclude that changes in density rather than alterations in properties of single channels can account for the increase in the resting potential, observed after axotomy.