Beyene, E. | Search bioRxiv

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

Physical restraint induces conditioned place aversion and region-specific c-Fos activation in mice

Linking environmental contexts with stressful experiences is critical for engaging adaptive responses necessary to avoid future threats. Yet, active context-dependent avoidance remains poorly understood. Here, we establish a restraint-induced conditioned place aversion (CPA) paradigm to examine how an acute physiological stressor acquires negative motivational value through contextual association. We found that mice repeatedly exposed to physical restraint in a contextually distinguishable chamber later avoid that location, demonstrating that restraint stress can drive learned aversion in the absence of continued exposure. To identify potential neuronal correlates underlying this learned association, we quantified c-Fos expression in several areas implicated in aversive motivation, emotional salience, and contextual encoding. We found that restraint within the context of the CPA paradigm was associated with increased c-Fos in the nucleus accumbens (NAc) and basolateral amygdala (BLA) while c-Fos expression increased in the ventral hippocampus in response to exposure to the contextual cues alone. These findings reveal region-specific activation in response to restraint stress and associated contextual cues. By connecting classical stress models and associative learning, this work provides a potential platform for further investigation of the neural mechanisms underlying stress-related negative affect and avoidance behaviors.

neuroscience↗