Vincenzo Zimarino

Protein homeostasis in neurons and its pathological alterations.

In neuronal cells, proteins are synthesized on ribosomes from the genetic information encoded in DNA. In some instances translation takes place at the neuronal cell soma but in other it occurs at distal location, such as in a dendritic spine. Folding is usually initiated before the completion of protein synthesis and its outcome strictly depends on the local environment in which the nascent protein is submerged. Incompletely folded proteins and, more importantly, misfolded proteins are under the surveillance of several quality control systems that re-establish the correct conformation or initiate protein degradation. Regulation and maintenance of these systems is a vital issue for neuronal and glial cells, and impairments at different levels leads to neurodegenerative diseases.

Linear transformation of the encoding mechanism for light intensity underlies the paradoxical enhancement of cortical visual responses by sevoflurane

The mechanisms of action of anaesthetics on the living brain are still poorly understood. In this respect, the analysis of the differential effects of anaesthetics on spontaneous and sensory‐evoked cortical activity might provide important and novel cues. Here we show that the anaesthetic sevoflurane strongly silences the brain but potentiates in a dose‐ and frequency‐dependent manner the cortical visual response. Such enhancement arises from a linear scaling by sevoflurane of the power‐law relation between light intensity and the cortical response. The fingerprint of sevoflurane action suggests that circuit silencing can boost linearly synaptic responsiveness presumably by scaling the number of responding units and/or their correlation following a sensory stimulation.

A conditional transgenic reporter of presynaptic terminals reveals novel features of the mouse corticospinal tract

In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), synaptic alterations precede the demise of the neuronal cell, making synapses a useful vantage point from which to monitor the onset and progression of clinical signs and pathological changes. While murine models of ALS display many features in common with the clinical picture observed in patients, corticospinal tract (CST) involvement is usually less severe in mice than the picture observed in humans. In this paper we describe the characterization of a new conditional transgenic line obtained by targeted integration of a GFP-VAMP2 fusion gene into the Rosa26 locus, and devised to permit the detection of genetically defined presynaptic terminals in wild type mice and murine models of neural disorders. This reporter molecule is selectively enriched in presynaptic boutons, significantly reducing the background signal produced by fibers of passage. The specific features of this reporter line allow us to strongly support the view that murine CST terminals give rise to very few direct contacts with spinal motor neurons. Moreover, the evidence described here reveals the existence of previously uncharacterized, putative direct connections between CST presynaptic boutons and Renshaw neurons in the spinal cord. These results constitute a proof of concept for the potential application of this indicator line to morphological analyses of wild type and diseased synapses.

Effects of the Concomitant Activation of ON and OFF Retinal Ganglion Cells on the Visual Thalamus: Evidence for an Enhanced Recruitment of GABAergic Cells

A fundamental question in vision neuroscience is how parallel processing of Retinal Ganglion Cell (RGC) signals is integrated at the level of the visual thalamus. It is well-known that parallel ON-OFF pathways generate output signals from the retina that are conveyed to the dorsal lateral geniculate nucleus (dLGN). However, it is unclear how these signals distribute onto thalamic cells and how these two pathways interact. Here, by electrophysiological recordings and c-Fos expression analysis, we characterized the effects of pharmacological manipulations of the retinal circuit aimed at inducing either a selective activation of a single pathway, OFF RGCs [intravitreal L-(+)-2-Amino-4-phosphonobutyric, L-AP4] or an unregulated activity of all classes of RGCs (intravitreal 4-Aminopyridine, 4-AP). In in vivo experiments, the analysis of c-Fos expression in the dLGN showed that these two manipulations recruited active cells from the same area, the lateral edge of the dLGN. Despite this similarity, the unregulated co-activation of both ON and OFF pathways by 4-AP yielded a much stronger recruitment of GABAergic interneurons in the dLGN when compared to L-AP4 pure OFF activation. The increased activation of an inhibitory thalamic network by a high level of unregulated discharge of ON and OFF RGCs might suggest that cross-inhibitory pathways between opposing visual channels are presumably replicated at multiple levels in the visual pathway, thus increasing the filtering ability for non-informative or noisy visual signals.

Functional mapping of brain synapses by the enriching activity-marker SynaptoZip

Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals.Visualization of synaptic activity in the living brain is challenging. This study devises a simple and efficient scheme that reports synaptic vesicle recycling in vivo using SynaptoZip, a genetically encoded sensor of past synaptic activities.

18 Transcriptional Regulation of Heat Shock Genes

I. INTRODUCTION In all eukaryotes, transcriptional regulation of heat shock genes in response to elevated ambient temperatures is mediated by a common, positive, cis -acting sequence, the heat shock element (HSE), that is present in multiple copies upstream of the transcriptional start site. Originally defined as a 14-nucleotide sequence CT-GAA--TTC-AG (Pelham 1982), the HSE has recently been revised by further analysis to two or three modules consisting of alternating GAA or TTC blocks, arranged in alternating orientations and at 2-nucleotide intervals (Amin et al. 1988; Xiao and Lis 1988; see Lis et al., this volume). In this chapter, we focus on our current understanding of the transcriptional activator protein that stimulates the synthesis of heat shock mRNA through interaction with the HSE. Transcription factors that regulate some heat shock genes through cis elements that are unrelated to the HSE are not covered in this discussion. Initial evidence for a factor that interacts with the HSE came from studies of protein/DNA interactions in Drosophila cell nuclei, in which a factor, heat shock activator protein, was found to bind specifically to the HSE only upon heat shock stimulation (Wu 1984a,b); parallel studies using DNA-binding and in vitro transcription assays also identified a heat shock transcription factor that bound specifically to the HSE (Parker and Topol 1984). At the 1987 Cold Spring Harbor Meeting entitled “The Role of Heat Shock and Stress Response in Biology and Human Disease,” HSE-binding factors were given a general designation: heat shock factor (HSF). In the following discussion,...