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Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl - ] int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na + -K + -2Cl - cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl - ] int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl - ] int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl - ] int . Other tested chloride channels or chloride transporters do not contribute to [Cl - ] int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K + -Cl - cotransporter change resting Bergmann glial [Cl - ] int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400., (© 2016 Wiley Periodicals, Inc.)

In rodents, the vomeronasal system controls social and sexual behavior. However, several mechanistic aspects of sensory signaling in the vomeronasal organ remain unclear. Here, we investigate the biophysical basis of a recently proposed vomeronasal signal transduction component-a Ca(2+)-activated Cl(-) current. As the physiological role of such a current is a direct function of the Cl(-) equilibrium potential, we determined the intracellular Cl(-) concentration in dendritic knobs of vomeronasal neurons. Quantitative fluorescence lifetime imaging of a Cl(-)-sensitive dye at the apical surface of the intact vomeronasal neuroepithelium revealed increased cytosolic Cl(-) levels in dendritic knobs, a substantially lower Cl(-) concentration in vomeronasal sustentacular cells, and an apparent Cl(-) gradient in vomeronasal neurons along their dendritic apicobasal axis. Together, our data provide a biophysical basis for sensory signal amplification in vomeronasal neuron microvilli by opening Ca(2+)-activated Cl(-) channels., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl- accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl- currents. The intracellular Cl- concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl- currents to signal generation differs between individual afferent neurons, and whether the specific Cl- levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl- homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl- concentrations and on the expression levels of Cl- transporters in rat DRG neurons., Results: We developed an in vitro assay for testing how inflammatory mediators influence Cl- concentration and the expression of Cl- transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 microM ATP, 0.9 microM bradykinin, and 1.4 microM PGE2 for 1-3 hours. Two-photon fluorescence lifetime imaging with the Cl--sensitive dye MQAE revealed an increase of the intracellular Cl- concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl- homeostasis toward a state of augmented Cl- accumulation, which is induced by a 1-3 hour treatment with inflammatory mediators., Conclusion: Our findings indicate that inflammatory mediators impact on Cl- homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl- levels and, hence, the driving force for depolarizing Cl- efflux. These findings corroborate current concepts for the role of Cl- regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl- concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl- currents in the presynaptic terminals. Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their afferent signals in the spinal cord are subject to modulation by Cl(-) currents. The underlying Cl(-) homeostasis of nociceptive neurons, in particular its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl(-) concentration, [Cl(-)]i, of somatosensory neurons in intact dorsal root ganglia of mice. Using two-photon fluorescence-lifetime imaging microscopy, we determined [Cl(-)]i in newborn and adult animals. We found that the somatosensory neurons undergo a transition of Cl(-) homeostasis during the first three postnatal weeks that leads to a decline of [Cl(-)]i in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation-chloride co-transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl(-) accumulation and extrusion is present. RT-PCR analysis showed that the transcription pattern of electroneutral Cl(-) co-transporters does not change during the maturation process. These findings demonstrate that dorsal root ganglion neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. This process parallels the developmental "chloride switch" in the central nervous system. However, while most CNS neurons achieve homogeneously low [Cl(-)]i levels - which is the basis of GABAergic and glycinergic inhibition - somatosensory neurons maintain a heterogeneous pattern of [Cl(-)]i values. This suggests that Cl(-) currents are excitatory in some somatosensory neurons, but inhibitory in others. Our results are consistent with the hypothesis that Cl(-) homeostasis in somatosensory neurons is regulated through posttranslational modification of cation-chloride co-transporters.

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca(2+)-permeable cation channels and Ca(2+)-gated Cl(-) channels, which conduct a depolarizing Cl(-) efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl(-), established by active accumulation of Cl(-) within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl(-)-sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl(-) concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl(-) concentration in the range of 40-50 mm, which is indicative of active Cl(-) accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl(-) uptake through the apical membrane counteracts Cl(-) depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl(-) accumulation, OSNs avoid the "chloride switch": they do not express KCC2, the main Cl(-) extrusion cotransporter operating in neurons of the adult CNS. Cl(-) accumulation provides OSNs with the driving force for the depolarizing Cl(-) current that is the basis of the low-noise receptor current in these neurons.