A novel integrated approach to estimate the mitochondrial content of neuronal cells and brain tissues.

BACKGROUND: Mitochondria and their dynamics fuel most cellular processes both in physiological and pathological conditions. In the central nervous system, mitochondria sustain synaptic transmission and plasticity via multiple mechanisms which include their redistribution and/or expansion to higher energy demanding sites, sustaining activity changes and promoting morphological circuit adaptations. NEW METHOD: To be able to evaluate changes in mitochondrial number and protein phenotype, we propose a novel methodological approach where the simultaneous analysis of both mitochondrial DNA and protein content is performed on each individual microsample, avoiding non-homogeneous loss of material. RESULTS: We validated this method on neuronal-like cells and tissue samples and obtained estimates for the mitochondrial/genomic DNA ratio as well as for the abundance of protein counterparts. When the mitochondrial content per cell was evaluated in different brain areas, our results matched the known regional variation in aerobic-anaerobic metabolism. When long-term potentiation (LTP) was induced on hippocampal neurons, we detected increases in the abundance of mitochondria that correlated with the degree of synaptic enhancement. CONCLUSIONS: Our approach can be effectively used to study the mitochondrial content andits changes in different brain cells and tissues.

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.

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

KEY POINTS: 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. ABSTRACT: General anaesthetics, which are expected to silence brain activity, often spare sensory responses. To evaluate differential effects of anaesthetics on spontaneous and sensory-evoked cortical activity, we characterized their modulation by sevoflurane and propofol. Power spectra and the bust-suppression ratio from EEG data were used to evaluate anaesthesia depth. ON and OFF cortical responses were elicited by light pulses of variable intensity, duration and frequency, during light and deep states of anaesthesia. Both anaesthetics reduced spontaneous cortical activity but sevoflurane greatly enhanced while propofol diminished the ON visual response. Interestingly, the large potentiation of the ON visual response by sevoflurane was found to represent a linear scaling of the encoding mechanism for light intensity. To the contrary, the OFF cortical visual response was depressed by both anaesthetics. The selective depression of the OFF component by sevoflurane could be converted into a robust potentiation by the pharmacological blockade of the ON pathway, suggesting that the temporal order of ON and OFF responses leads to a depression of the latter. This hypothesis agrees with the finding that the enhancement of the ON response was converted into a depression by increasing the frequency of light-pulse stimulation from 0.1 to 1 Hz. Overall, our results support the view that inactivity-dependent modulation of cortical circuits produces an increase in their responsiveness. Among the implications of our findings, the silencing of cortical circuits can boost linearly the cortical responsiveness but with negative impact on their frequency transfer and with a loss of the information content of the sensory signal.

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.

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.

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.

Constitutive nuclear import and stress-regulated nucleocytoplasmic shuttling of mammalian heat-shock factor 1.

Inducible expression of major cytosolic and nuclear chaperone proteins is mediated by the heat-shock transcription factor HSF1 that is activated by derepressive mechanisms triggered by transient heat stress and sustained proteotoxicity. Despite progress in defining essential aspects of HSF1 regulation, little is known about the cellular dynamics enabling this factor to mediate gene responses to cytosolic stress signals. We report that the inactive, stress-responsive form of HSF1 accumulates in the nucleus due to a relatively potent import signal, which can be recognized by importin-alpha/beta, and simultaneously undergoes continuous nucleocytoplasmic shuttling due to a comparatively weak, nonetheless efficient, export activity not involving the classical exportin-1 pathway. Strikingly, experimental stresses at physiological or elevated temperature reversibly inactivate the export competence of HSF1. Likewise, mutations mimicking stress-induced derepression impair export but not import. These findings are consistent with a dynamic process whereby exported molecules that are derepressed in an inductive cytosolic environment are recollected and pause in the nucleoplasm, replacing progressively the inactive pool. While steady-state nuclear distribution of the bulk of HSF1 ensures a rapid gene response to acute heat stress, our results suggest that the capture in the nucleus of molecules primed for activation in the cytosol may underlie responses to sustained proteotoxicity.

A small-scale survey identifies selective and quantitative nucleo-cytoplasmic shuttling of a subset of CREM transcription factors.

Elucidating dynamic aspects of intracellular localization of proteins is essential to decipher their functional interaction networks. Although transcription factors lacking a detectable cytoplasmic fraction have been generally considered compartmentalized in the nucleus, some were found to shuttle into the cytoplasm, suggesting functional interactions therein. To further investigate how common, specific and quantitative is this traffic, we have employed the heterokaryon assay for a small-scale survey of nuclear factors not previously tested for their nucleo-cytoplasmic motion. We show that a subset of cAMP response element (CRE) binding proteins of the CREM type shuttles within a biologically meaningful time frame, revealing a continuous flow into the cytoplasm that persists during signaling. Their dynamic behavior, not involving the classical Exportin-1 pathway, could be ascribed to C-terminal sequences, containing, in addition to the bZIP domain and the NLS, a nuclear export activity and an inhibitory activity at an adjacent site. Other proteins examined in this study either did not shuttle significantly or, like CREB and distinct CREM isoforms, shuttled with markedly delayed kinetics, denoting considerable selectivity of this traffic. These findings raise the possibility that events associated with bi-directional transport and periodic transit through the cytoplasm may modulate activities of select nuclear transcription factors.

Determination of the consensus binding sequence for the purified embryonic heat shock factor 2.

Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA-binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8-mer palindromic core 5'-TTCTAGAA-3'. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes.