Biology subjects

Ratto, G. M. M.

Publications and source records attributed to Ratto, G. M. M..

3 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↗

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↗