Loss of Visually Driven Synaptic Responses in Layer 4 Regular-Spiking Neurons of Rat Visual Cortex in Absence of Competing Inputs

Monocular deprivation (MD) during development shifts the ocular preference of primary visual cortex (V1) neurons by depressing closed-eye responses and potentiating open-eye responses. As these 2 p ..

Mild Inactivation of RE-1 Silencing Transcription Factor (REST) Reduces Susceptibility to Kainic Acid-Induced Seizures

RE-1 Silencing Transcription factor (REST) controls several steps in neural development by modulating the expression of a wide range of neural genes. Alterations in REST expression have been associated with the onset of epilepsy; however, whether such alterations are deleterious or represent a protective homeostatic response remains elusive. To study the impact of REST modulation on seizure propensity, we developed a tool for its negative modulation in vivo. The tool is composed of the paired-amphipathic helix 1 (PAH1) domain, a competitive inhibitor of REST activation by mSin3, fused to the light-oxygen-voltage sensing 2 (LOV2) domain of Avena sativa phototropin 1, a molecular switch to alternatively hide or expose the PAH1 inhibitor. We employed the C450A and I539E light-independent AsLOV2 variants to mimic the closed (inactive) and open (active) states of LOV2-PAH1, respectively. Recombinant AAV1/2 viral particles (rAAVs) allowed LOV2-PAH1 expression in HEK293T cells and primary neurons, and efficiently transduced hippocampal neurons in vivo. mRNA expression analysis revealed an increased expression of several neuronal genes in the hippocampi of mice expressing the open probe. AAV-transduced mice received a single dose of kainic acid (KA), a treatment known to induce a transient increase of REST levels in the hippocampus. Remarkably, mice expressing the active variant displayed a reduced number of KA-induced seizures, which were less severe compared to mice carrying the inactive probe. These data support the validity of our tool to modulate REST activity in vivo and the potential impact of REST modulation on epileptogenesis

Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression

Synapsin I controls synaptic maturation of long-range projections in the lateral amygdala in a targeted selective fashion

The amygdala, and more precisely its lateral nucleus, is thought to attribute emotional valence to external stimuli by generating long-term plasticity changes at long-range projections to principal cells. Aversive experience has also been shown to modify pre- and post-synaptic markers in the amygdala, suggesting their possible role in the structural organization of adult amygdala networks. Here, we focused on how the maturation of cortical and thalamic long-range projections occurs on principal neurons and interneurons in the lateral amygdala (LA). We performed dual electrophysiological recordings of identified cells in juvenile and adult GAD67-GFP mice after independent stimulation of cortical and thalamic afferent systems. The results demonstrate that synaptic strengthening occurs during development at synapses projecting to LA principal neurons, but not interneurons. As synaptic strengthening underlies fear conditioning which depends, in turn, on presence and increasing expression of synapsin I, we tested if synapsin I contributes to synaptic strengthening during development. Interestingly, the physiological synaptic strengthening of cortical and thalamic synapses projecting to LA principal neurons was virtually abolished in synapsin I knockout mice, but not differences were observed in the excitatory projections to interneurons. Immunohistochemistry analysis showed that the presence of synapsin I is restricted to excitatory contacts projecting to principal neurons in LA of adult mice. These results indicate that synapsin I is a key regulator of the maturation of synaptic connectivity in this brain region and that is expression is dependent on postsynaptic identity

Synapsins contribute to the dynamic spatial organization of synaptic vesicles in an activity-dependent manner.

The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity

Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy.

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation

Relaxation properties in classical diamagnetism

It is an old result of Bohr that, according to classical statistical mechanics, at equilibrium a system of electrons in a static magnetic field presents no magnetization. Thus a magnetization can occur only in an out of equilibrium state, such as that produced through the Foucault currents when a magnetic field is switched on. It was suggested by Bohr that, after the establishment of such a nonequilibrium state, the system of electrons would quickly relax back to equilibrium. In the present paper, we study numerically the relaxation to equilibrium in a modified Bohr model, which is mathematically equivalent to a billiard with obstacles, immersed in a magnetic field that is adiabatically switched on. We show that it is not guaranteed that equilibrium is attained within the typical time scales of microscopic dynamics. Depending on the values of the parameters, one has a relaxation either to equilibrium or to a diamagnetic (presumably metastable) state. The analogy with the relaxation properties in the Fermi Pasta Ulam problem is also pointed out

Fabrication of biocompatible free-standing nanopatterned films for primary neuronal cultures

Devising and constructing biocompatible devices for nervous system regeneration is an extremely challenging task. Besides tackling the issue of biocompatibility, biomaterials for neuroscience applications should mimic the complex environment of the extracellular matrix, which in vivo provides neurons with a series of cues and signals to guide cells towards their appropriate targets. In this work, a novel nanopatterned biocompatible poly-ε-caprolactone (PCL) film is realized to assist the attachment and growth of primary hippocampal neurons. Costly and time-consuming processes can be avoided using plasma-surface nanotexturing obtained by a mixed gas SF6/Ar at -5 °C. The intrinsic composition and line topography of nanopatterned PCL ensure healthy development of the neuronal network, as shown by confocal microscopy, by analysing the expression of a range of neuronal markers typical of mature cultures, as well as by scanning electron microscopy. In addition, we show that surface nanopatterning improves differentiation of neurons compared to flat PCL films, while no neural growth was observed on either flat or nanopatterned substrates in the absence of a poly-d-lysine coating. Thus, we successfully optimized a nanofabrication protocol to obtain nanostructured PCL layers endowed with several mechanical and structural characteristics that make them a promising, versatile tool for future tissue engineering studies aimed at neural tissue regeneration

REST/NRSF drives homeostatic plasticity of inhibitory synapses in a target-dependent fashion

The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron-specific genes. We showed that REST/NRSF downregulates glutamatergic transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation, triggers a homeostatic rearrangement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs in mouse cultured hippocampal neurons. Notably, this effect is limited to inhibitory-onto-excitatory neuron synapses, whose density increases at somatic level and decreases in dendritic regions, demonstrating a complex target- and area-selectivity. The upscaling of perisomatic inhibition was occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the Npas4 and Bdnf gene programs. On the opposite, the downscaling of dendritic inhibition was REST-dependent, but BDNF-independent. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at rescuing physiological levels of network activity in front of the ever-changing environment

Interfacing Graphene-Based Materials With Neural Cells

The scientific community has witnessed an exponential increase in the applications of graphene and graphene-based materials in a wide range of fields, from engineering to electronics to biotechnologies and biomedical applications. For what concerns neuroscience, the interest raised by these materials is two-fold. On one side, nanosheets made of graphene or graphene derivatives (graphene oxide, or its reduced form) can be used as carriers for drug delivery. Here, an important aspect is to evaluate their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. On the other side, graphene can be exploited as a substrate for tissue engineering. In this case, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation, which holds a great potential in regenerative medicine. In this review, we try to give a comprehensive view of the accomplishments and new challenges of the field, as well as which in our view are the most exciting directions to take in the immediate future. These include the need to engineer multifunctional nanoparticles (NPs) able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. We describe the state-of-the-art in the use of graphene materials to engineer three-dimensional scaffolds to drive neuronal growth and regeneration in vivo, and the possibility of using graphene as a component of hybrid composites/multi-layer organic electronics devices. Last but not least, we address the need of an accurate theoretical modeling of the interface between graphene and biological material, by modeling the interaction of graphene with proteins and cell membranes at the nanoscale, and describing the physical mechanism(s) of charge transfer by which the various graphene materials can influence the excitability and physiology of neural cells