Gian Michele Ratto

Molecular basis of plasticity in the visual cortex.

Sensory experience is known to shape the maturation of cortical circuits during development. A paradigmatic example is the effect of monocular deprivation on ocular dominance of visual cortical neurons. Although visual cortical plasticity has been widely studied since its initial discovery by Hubel and Wiesel >40 years ago, the description of the underlying molecular mechanisms has lagged behind. Several new findings are now beginning to close this gap. Recent data deepen our knowledge of the factors involved in the intercellular communication and intracellular signaling that mediate experience-dependent plasticity in the developing visual cortex. In addition, new findings suggest a role for the extracellular matrix in inhibition of ocular-dominance plasticity in the adult visual cortex.

Long‐term Survival of Retina Optic Nerve Section in Adult Ganglion Cells Following bcl‐2 Transgenic Mice

The bcl‐2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl‐2 in adult transgenic mice can protect the axotomited ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice˜65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, <10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl‐2 protein in central neurons is a very effective strategy to ensure long‐term survival in axotomized cells.

An Excitatory Loop with Astrocytes Contributes to Drive Neurons to Seizure Threshold

Studies in rodent brain slices suggest that seizures in focal epilepsies are sustained and propagated by the reciprocal interaction between neurons and astroglial cells

In vivo distribution and toxicity of PAMAM dendrimers in the central nervous system depend on their surface chemistry.

Dendrimers have been described as one of the most tunable and therefore potentially applicable nanoparticles both for diagnostics and therapy. Recently, in order to realize drug delivery agents, most of the effort has been dedicated to the development of dendrimers that could internalize into the cells and target specific intracellular compartments in vitro and in vivo. Here, we describe cell internalization properties and diffusion of G4 and G4-C12 modified PAMAM dendrimers in primary neuronal cultures and in the CNS of live animals. Confocal imaging on primary neurons reveals that dendrimers are able to cross the cell membrane and reach intracellular localization following endocytosis. Moreover, functionalization of PAMAMs has a dramatic effect on their ability to diffuse in the CNS tissue in vivo and penetrate living neurons as shown by intraparenchymal or intraventricular injections. 100 nM G4-C12 PAMAM dendrimer already induces dramatic apoptotic cell death of neurons in vitro. On the contrary, G4 PAMAM does not induce apoptotic cell death of neural cells in the sub-micromolar range of concentration and induces low microglia activation in brain tissue after a week. Our detailed description of dendrimer distribution patterns in the CNS will facilitate the design of tailored nanomaterials in light of future clinical applications.

Dynamic regulation of ERK2 nuclear translocation and mobility in living cells

The extracellular signal-regulated protein kinase ERK1/2 is a crucial effector linking extracellular stimuli to cellular responses: upon phosphorylation ERK [also known as mitogen-activated protein kinase P42/P44 (MAPK)] concentrates in the nucleus where it activates specific programs of gene expression. Notwithstanding the importance of this process, little is known about the modalities, time course and regulation of ERK exchange between nucleus and cytoplasm in living cells. We visualized the dynamic of nuclear translocation by expressing low levels (<150 nM) of fluorescently tagged ERK2 in living fibroblasts. Time-lapse imaging demonstrated that nuclear concentration can change bidirectionally with a time constant of a few minutes. The increase of nuclear concentration requires continuous MEK (also known as MAPK kinase) activity upstream of ERK and is rapidly reduced by the operation of phosphatases. We measured quantitatively the speed of ERK2 shuttling between nucleus and cytoplasm and determined that shuttling accelerated after ERK activation, becoming fast enough not to be rate-limiting for translocation. Finally, we demonstrated that ERK2 did not diffuse freely in the nucleus and that diffusion was further impeded after phosphorylation, suggesting the formation of complexes of low mobility. These results show that nucleocytoplasmic trafficking of ERK2 and its mobility are dynamically regulated in living cells.

Acute physiological response of mammalian central neurons to axotomy: ionic regulation and electrical activity

The transection of the axon of central neurons has dramatic consequences on the damaged cells and nerves. Injury activates molecular programs leading to a complex repertoire of responses that, depending on the cellular context, include activation of sprouting, axonal degeneration, and cell death. Although the cellular mechanisms started at the time of lesion are likely to shape the changes affecting injured cells, the acute physiological reaction to trauma of mammalian central neurons is not completely understood yet. To characterize the physiology of the acute response to axonal transection, we have developed a model of in vitro axotomy of neurons cultured from the rodent cortex. Imaging showed that axotomy caused an increase of calcium in the soma and axon. Propagation of the response to the soma required the activation of voltage‐dependent sodium channels, since it was blocked by tetrodotoxin. The electrophysiological response to axotomy was recorded in patched neurons kept in the current clamp configuration: injury was followed by vigorous spiking activity that caused a sodium load and the activation of transient calcium currents that were opened by each action potential. The decrease of the electrochemical gradient of sodium caused inversion of the Na‐Ca exchanger that provided an additional mean of entry for calcium. Finally, we determined that inhibition of the physiological response to axotomy hindered the regeneration of a new neurite. These data provide elements of the framework required to link the axotomy itself to the downstream molecular machinery that contributes to the determination of the long‐term fate of injured neurons and axons.

High-performance and site-directed in utero electroporation by a triple-electrode probe

In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here, we present a new in utero-electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum.

The short-time structural plasticity of dendritic spines is altered in a model of Rett syndrome

The maturation of excitatory transmission comes about through a developmental period in which dendritic spines are highly motile and their number, form and size are rapidly changing. Surprisingly, although these processes are crucial for the formation of cortical circuitry, little is known about possible alterations of these processes in brain disease. By means of acute in vivo 2-photon imaging we show that the dynamic properties of dendritic spines of layer V cortical neurons are deeply affected in a mouse model of Rett syndrome (RTT) at a time around P25 when the neuronal phenotype of the disease is still mild. Then, we show that 24h after a subcutaneous injection of IGF-1 spine dynamics is restored. Our study demonstrates that spine dynamics in RTT mice is severely impaired early during development and suggest that treatments for RTT should be started very early in order to reestablish a normal period of spine plasticity.

Activity‐regulated cell death contributes to the formation of ON and OFF α ganglion cell mosaics

At maturity, ON and OFF α ganglion cells in the cat retina are arrayed in regular mosaics, with adjacent cells commonly forming ON‐OFF pairs. In the present study, we investigated the role of activity‐mediated ganglion cell death in the formation of such cellular patterns. Because direct measures of ganglion cell mosaics are problematic in the developing retina, we examined the distributions of ON and OFF α cells in the postnatal cat retina by assessing the degree to which cells in closest proximity were of opposite sign (i.e., ON‐OFF pairs). Computer simulations demonstrated that superimposition of two regular distributions results in a high incidence (∼90%) of opposite sign pairs. This is also the case for ON and OFF α cells in the mature retina, reflecting the high degree of regularity exhibited by this cell class. In contrast, during the first postnatal month, α cells displayed a much lower incidence of opposite sign pairs (∼60%), comparable to the superimposition of two simulated random distributions.

The N-terminal domain of ERK1 accounts for the functional differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output.