Developmental Regulation of Small-Conductance Ca²⁺-Activated K⁺ Channel Expression and Function in Rat Purkinje Neurons

Calcium transients play an important role in the early and later phases of differentiation and maturation of single neurons and neuronal networks. Small-conductance calcium-activated potassium channels of the SK type modulate membrane excitability and are important determinants of the firing properties of central neurons. Increases in the intracellular calcium concentration activate SK channels, leading to a hyperpolarization of the membrane potential, which in turn reduces the calcium inflow into the cell. This feedback mechanism is ideally suited to regulate the spatiotemporal occurrence of calcium transients. However, the role of SK channels in neuronal development has not been addressed so far. We have concentrated on the ontogenesis and function of SK channels in the developing rat cerebellum, focusing particularly on Purkinje neurons. Electrophysiological recordings combined with specific pharmacological tools have revealed for the first time the presence of an afterhyperpolarizing current (I_{AHP}) in immature Purkinje cells in rat cerebellar slices. The channel subunits underlying this current were identified as SK2 and localized by in situ hybridization and subunit-specific antibodies. Their expression level was shown to be high at birth and subsequently to decline during the first 3 weeks of postnatal life, both at the mRNA and protein levels. This developmental regulation was tightly correlated with the expression of I_{AHP} and the prominent role of SK2 channels in shaping the spontaneous firing pattern in young, but not in adult, Purkinje neurons. These results provide the first evidence of the developmental regulation and function of SK channels in central neurons

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells

Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVβ3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVβ5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for β3 and β5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation

Combining optogenetics with artificial microRNAs to characterize the effects of gene knockdown on presynaptic function within intact neuronal circuits

3siThe purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates shortterm synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.openopenThalhammer, Agnes; Jaudon, Fanny; Cingolani, Lorenzo A.*Thalhammer, Agnes; Jaudon, Fanny; Cingolani, Lorenzo A

PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage.

Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs

Multiple solutions to a magnetic nonlinear Choquard equation

We consider the stationary nonlinear magnetic Choquard equation [(-\mathrm{i}\nabla+A(x))^{2}u+V(x)u=(\frac{1}{|x|^{\alpha}}\ast |u|^{p}) |u|^{p-2}u,\quad x\in\mathbb{R}^{N}%] where $A\$is a real valued vector potential, $V$ is a real valued scalar potential$,$ $N\geq3$, $\alpha\in(0,N)$ and $2-(\alpha/N) <p<(2N-\alpha)/(N-2)$. \ We assume that both $A$ and $V$ are compatible with the action of some group $G$ of linear isometries of $\mathbb{R}^{N}$. We establish the existence of multiple complex valued solutions to this equation which satisfy the symmetry condition $$u(gx)=\tau(g)u(x)\text{\ \ \ for all}g\in G,\text{}x\in\mathbb{R}^{N},$$ where $\tau:G\rightarrow\mathbb{S}^{1}$ is a given group homomorphism into the unit complex numbers.Comment: To appear on ZAM

Nanotechnology approaches to self-organized bio-molecular devices

Abstract In this paper we briefly describe new strategies to exploit self-assembled solid-state biomolecular materials as active elements of electronic devices. Two basically different approaches are proposed: a top-down approach, where biomolecular semiconductors consisting of DNA basis are self-organized and interconnected by planar metallic nanopatterns, and a bottom-up approach, where single or ordered matalloproteins are immobilized in a nanocircuit realizing a hybrid covalently bound biologic–inorganic system. The transport characteristics of different devices such as diodes, photodetectors and metal–semiconductor–metal structures will be described

An observing system for the collection of fishery and oceanographic data

Fishery Observing System (FOS) was developed as a first and basic step towards fish stock abundance nowcasting/forecasting within the framework of the EU research program Mediterranean Forecasting System: Toward an Environmental Prediction (MFSTEP). The study of the relationship between abundance and environmental parameters also represents a crucial point towards forecasting. Eight fishing vessels were progressively equipped with FOS instrumentation to collect fishery and oceanographic data. The vessels belonged to different harbours of the Central and Northern Adriatic Sea. For this pilot application, anchovy (&lt;I&gt;Engraulis encrasicolus&lt;/I&gt;, L.) was chosen as the target species. Geo-referenced catch data, associated with in-situ temperature and depth, were the FOS products but other parameters were associated with catch data as well. MFSTEP numerical circulation models provide many of these data. In particular, salinity was extracted from re-analysis data of numerical circulation models. Satellite-derived sea surface temperature (SST) and chlorophyll were also used as independent variables. Catch and effort data were used to estimate an abundance index (CPUE &amp;ndash; Catch per Unit of Effort). Considering that catch records were gathered by different fishing vessels with different technical characteristics and operating on different fish densities, a standardized value of CPUE was calculated. A spatial and temporal average CPUE map was obtained together with a monthly mean time series in order to characterise the variability of anchovy abundance during the period of observation (October 2003&amp;ndash;August 2005). In order to study the relationship between abundance and oceanographic parameters, Generalized Additive Models (GAM) were used. Preliminary results revealed a complex scenario: the southern sector of the domain is characterised by a stronger relationship than the central and northern sector where the interactions between the environment and the anchovy distribution are hidden by a higher percentage of variability within the system which is still unexplained. &lt;br&gt;&lt;br&gt; GAM analysis showed that increasing the number of explanatory variables also increased the portion of variance explained by the model. Data exchange and interdisciplinary efforts will therefore be crucial for the success of this research activity

Regulation of dendritic spine length in corticopontine layer V pyramidal neurons by autism risk gene β3 integrin

The relationship between autism spectrum disorder (ASD) and dendritic spine abnormalities is well known, but it is unclear whether the deficits relate to specific neuron types and brain regions most relevant to ASD. Recent genetic studies have identified a convergence of ASD risk genes in deep layer pyramidal neurons of the prefrontal cortex. Here, we use retrograde recombinant adeno-associated viruses to label specifically two major layer V pyramidal neuron types of the medial prefrontal cortex: the commissural neurons, which put the two cerebral hemispheres in direct communication, and the corticopontine neurons, which transmit information outside the cortex. We compare the basal dendritic spines on commissural and corticopontine neurons in WT and KO mice for the ASD risk gene Itgb3, which encodes for the cell adhesion molecule beta 3 integrin selectively enriched in layer V pyramidal neurons. Regardless of the genotype, corticopontine neurons had a higher ratio of stubby to mushroom spines than commissural neurons. beta 3 integrin affected selectively spine length in corticopontine neurons. Ablation of beta 3 integrin resulted in corticopontine neurons lacking long (> 2 mu m) thin dendritic spines. These findings suggest that a deficiency in beta 3 integrin expression compromises specifically immature spines on corticopontine neurons, thereby reducing the cortical territory they can sample. Because corticopontine neurons receive extensive local and long-range excitatory inputs before relaying information outside the cortex, specific alterations in dendritic spines of corticopontine neurons may compromise the computational output of the full cortex, thereby contributing to ASD pathophysiology

Insulin resistance and HCV virologic response to peg-interferons (Peg-IFN) with ribavirin (RBV) in HIV/HCV co-infected patients

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Design of a beamline for soft and deep lithography on third generation synchrotron radiation source

ELETTRA is a third generation synchrotron radiation source. The energy spectrum allows the design of beamlines suitable for x-ray lithography from soft to hard x-ray wavelengths. An appropriate lithographic window for micro- and nanofabrication can be obtained by a combination of selected filters and mirrors. As the beamline is interfaced to a vertical x-ray stepper, a uniformity in the beam intensity better than 3% (3σ) in the horizontal direction has to be reached. The present beamline is designed by taking into account the main factors which can affect the beam quality, namely, thermal loading on mirrors and filters, slope errors, and surface roughness of the mirrors. The resulting lithographic resolution at soft x-ray wavelengths is better than 100 nm