Biology subjects

Nardi, G.

Publications and source records attributed to Nardi, G..

6 recordsLinked to original sources

bioRxiv2026

High sensitivity imaging of intracellular Cl- reveals the spatiotemporal evolution of GABAergic chloride fluxes in vitro and in vivo

Fast GABAergic inhibition in the brain is mediated by chloride currents whose driving force depends on intracellular Chloride concentration ([Cl-]i). Resolving the spatiotemporal dynamics of [Cl-]i in neurons requires a sensor combining high Cl- affinity, pH independence, and photostability - properties no existing tool achieves simultaneously. Here we introduce iClima (improved Chloride Imaging), a ratiometric genetically encoded sensor with high Cl- affinity (Kd=3.5 mM at pH 7.2), pH-insensitive under physiological conditions, and markedly improved photostability compared to existing sensors. These properties enable dynamic Cl- imaging without simultaneous pH correction. We demonstrate that iClima resolves GABAA-driven Cl- transients in cultured neurons, detects discrete Cl- hotspots along dendrites, and captures sensory-evoked Cl- transients in vivo. Finally, exploiting the spectral compatibility of iClima with GCaMP6f, we measure in interleaved trials Cl- and Ca2+ transients in the same neurons in vivo, revealing a divergence between the somatic inhibitory drive and the neuronal excitation state as reported by Ca2+ dynamics.

neuroscience↗

bioRxiv2026

Sleep deprivation impairs information processing via dysregulation of chloride homeostasis in the prefrontal cortex

Sleep deprivation (SD) impairs information processing through alterations of prefrontal cortex (PFC) function, yet the molecular underpinnings of this process remain poorly understood. We previously showed that SD disrupts sensorimotor gating by elevating prefrontal levels of the neurosteroid allopregnanolone (AP), a positive allosteric modulator of GABA-A receptors. Here we identify a complementary, mechanistically independent process whereby SD alters GABA-A currents in the PFC of mice and rats. SD reduced membrane expression of the chloride exporter KCC2, leading to intracellular chloride accumulation and a depolarizing shift in GABA-A receptor reversal potential that weakened GABAergic inhibition. Pharmacological normalization of chloride homeostasis with bumetanide fully rescued SD-induced deficits in sensorimotor gating and information encoding. SD also upregulated BDNF, and intra-PFC antagonism of its receptor TrkB restored KCC2 expression and normalized information processing, identifying BDNF-TrkB signaling as an upstream driver of chloride dysregulation. Notably, blocking AP synthesis rescued behavioral deficits without correcting chloride imbalance, confirming mechanistic independence. Finally, combined administration of AP and a KCC2 blocker produced information-processing deficits akin to those induced by SD. These findings identify TrkB-dependent disruption of prefrontal chloride homeostasis as a druggable mechanism underlying sleep loss-induced cognitive dysfunction.

animal behavior and cognition↗

bioRxiv2024

Characterizing particle dynamics in live imaging through stochastic physical models and machine learning

Particle dynamics determine the orchestration of molecular signaling in cellular processes. A wide range of subdiffusive motions has been described at the cell interior and membrane, corresponding to different environmental constraints. However, the standard methods for motion analysis, embedded in a diffusion-based framework, lack robustness for capturing the complexity of stochastic dynamics. This work develops a classification method to detect the five main stochastic laws modeling particle dynamics accurately. The method builds on machine-learning techniques that use features properly designed to capture the intrinsic geometric properties of trajectories governed by the different processes. This guarantees the accurate classification of observed dynamics in an interpretable and explainable framework. The main asset of this approach is its capability to distinguish different subdiffusive behaviors making it a privileged tool for biological investigations. The robustness to localization error and motion composition is proven, ensuring its reliability on experimental data. Moreover, the classification of composed trajectories is investigated, showing that the method can uncover the paths mono-vs bi-dynamics nature. The method is used to study the dynamics of membrane receptors CCR5, involved in HIV infection. Comparing the basal state to an agonist-bound state which displays potent anti-HIV-1 activity, we show that the latter affects the natural dynamic state of receptors, thus clarifying the link between movement and receptor activation.

biophysics↗

bioRxiv2021

Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on {beta}-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

cell biology↗

bioRxiv2021

Diurnal variation in neuronal chloride levels and seizure susceptibility, in neocortex, reflecting changes in activity of chloride-cation-cotransporters

The main inhibitory synaptic currents, gated by gamma-aminobutyric acid (GABA), are mediated by Cl--conducting channels1-3, and are therefore sensitive to changes in the chloride electrochemical gradient. GABAergic activity dictates the neuronal firing range4,5 and timing6-9, which in turn influences the rhythms of the brain, synaptic plasticity, and flow of information in neuronal networks7,10-12. The intracellular chloride concentration [Cl-]i is, therefore, ideally placed to be a regulator of neuronal activity. Chloride levels have been thought to be stable in adult cortical networks, except when associated with pathological activation13-16. Here, we used 2-photon LSSmClopHensor imaging, in anaesthetized young adult mice13, to show that [Cl-] inside pyramidal cells shows a physiological diurnal rhythm, with an approximately 1.8-fold range, equating to an ~15mV positive shift in ECl at times when mice are typically awake (midnight), relative to when they are usually asleep (midday). This change of [Cl-]i alters the stability of cortical networks, as demonstrated by a greater than 3-fold longer latency to seizures induced by 4-aminopyridine at midday, compared to midnight. Importantly, both [Cl-]i and latency to seizure, in night-time experiments, were shifted in line with day-time measures, by inhibition of NKCC1. The redistribution of [Cl-]i reflects diurnal changes in surface expression and phosphorylation states of the cation-chloride-co-transporters, KCC2 and NKCC1, leading to a greatly reduced chloride-extrusion capacity at night (awake period). Our data demonstrate a means by which changes in the biochemical state of neurons are transduced into altered brain states.

neuroscience↗

bioRxiv2019

A Cre-amplifier to generate and detect genetic mosaics in vivo

Cre-Lox manipulation is the gold standard for cell-specific expression or knockout of selected genes. However, it is not unusual to deal with conditions of low Cre expression or transient activation, which can often go undetected by conventional Cre-reporters. We designed Beatrix, a general-purpose tool specifically devised to amplify weak Cre recombinase activity, and we used it to develop a powerful approach for the in vivo generation and detection of sparse mosaics of mutant and wild type cells.

neuroscience↗