The majority of somatosensory DRG neurons express GABAA receptors (GABAAR) and depolarise in response to its activation based on the high intracellular chloride concentration maintained by the Na-K-Cl cotransporter type 1 (NKCC1). The translation of this response to peripheral nerve terminals in people is so far unclear. We show here that GABA (EC50 = 16.67μM) acting via GABAAR produces an influx of extracellular calcium in approximately 20% (336/1720) of isolated mouse DRG neurons. In contrast, upon injection into forearm skin of healthy volunteers GABA (1mM, 100μl) did not induce any overt sensations nor a specific flare response and did not sensitize C-nociceptors to slow depolarizing electrical sinusoidal stimuli. Block of the inward chloride transporter NKCC1 by furosemide (1mg/100μl) did not reduce electrically evoked pain ratings nor did repetitive GABA stimulation in combination with an inhibited NKCC1 driven chloride replenishment by furosemide. Finally, we generated a sustained period of C-fiber firing by iontophoretically delivering codeine or histamine to induce tonic itch. Neither the intensity nor the duration of histamine or codeine itch was affected by prior injection of furosemide. We conclude that although GABA can evoke calcium transients in a proportion of isolated mouse DRG neurons, it does not induce or modify pain or itch ratings in healthy human skin even when chloride gradients are altered by inhibition of the sodium coupled NKCC1 transporter.

Sepsis causes systemic inflammatory responses and acute lung injury (ALI). Despite modern treatments, sepsis-related ALI mortality remains high. Aqueous extract of Descuraniae Semen (AEDS) exerts anti-endoplasmic reticulum (ER) stress, antioxidant and anti-inflammatory effects. AEDS alleviates inflammation and oedema in ALI. Sodium-potassium-chloride co-transporter isoform 1 (NKCC1) is essential for regulating alveolar fluid and is important in ALI. The NKCC1 activity is regulated by upstream with-no-lysine kinase-4 (WNK4) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). This study aimed to investigate the effects of AEDS on lipopolysaccharide (LPS)-induced ALI model in A549 cells, considering the regulation of ER stress, WNK4-SPAK-NKCC1 cascades, inflammation and apoptosis. Cell viability was investigated by the CCK-8 assay. The expressions of the proteins were assessed by immunoblotting analysis assays. The levels of pro-inflammatory cytokines were determined by ELISA. The expression of cytoplasmic Ca2+ in A549 cells was determined using Fluo-4 AM. AEDS attenuates LPS-induced inflammation, which is associated with increased pro-inflammatory cytokine expression and activation of the WNK4-SPAK-NKCC1 pathway. AEDS inhibits the WNK4-SPAK-NKCC1 pathway by regulating of Bcl-2, IP3R and intracellular Ca2+. WNK4 expression levels are significantly higher in the WNK4-overexpressed transfected A549 cells and significantly decrease after AEDS treatment. AEDS attenuates LPS-induced inflammation by inhibiting the WNK4-SPAK-NKCC1 cascade. Therefore, AEDS is regarded as a potential therapeutic agent for ALI.

The master control of mammalian circadian rhythms is the suprachiasmatic nucleus (SCN), which is formed by the ventral and dorsal regions. In SCN neurons, GABA has an important function and even excitatory actions in adulthood. However, the physiological role of this neurotransmitter in the developing SCN is unknown. Here, we recorded GABAergic postsynaptic currents (in the perforated-patch configuration using gramicidin) to determine the chloride reversal potential (ECl) and also assessed the immunological expression of the Na-K-Cl cotransporter 1 (NKCC1) at early ages of the rat (postnatal days (P) 3 to 25), during the day and night, in the two SCN regions. We detected that ECl greatly varied with age and depending on the SCN region and time of day. Broadly speaking, ECl was more hyperpolarized with age, except for the oldest age studied (P20-25) in both day and night in the ventral SCN, where it was less negative. Likewise, ECl was more hyperpolarized in the dorsal SCN both during the day and at night; while ECl was more negative at night both in the ventral and the dorsal SCN. Moreover, the total NKCC1 fluorescent expression was higher during the day than at night. These results imply that NKCC1 regulates the circadian and developmental fluctuations in the [Cl-]i to fine-tune ECl, which is crucial for either excitatory or inhibitory GABAergic actions to occur in the SCN.

Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Ultrastructural studies of contusive spinal cord injury (SCI) in mammals have shown that the most prominent acute changes in white matter are periaxonal swelling and separation of myelin away from their axon, axonal swelling, and axonal spheroid formation. However, the underlying cellular and molecular mechanisms that cause periaxonal swelling and the functional consequences are poorly understood. We hypothesized that periaxonal swelling and loss of connectivity between the axo-myelinic interface impedes neurological recovery by disrupting conduction velocity, and glial to axonal trophic support resulting in axonal swelling and spheroid formation. Utilizing in vivo longitudinal imaging of Thy1YFP+ axons and myelin labeled with Nile red, we reveal that periaxonal swelling significantly increases acutely following a contusive SCI (T13, 30 kdyn, IH Impactor) versus baseline recordings (laminectomy only) and often precedes axonal spheroid formation. In addition, using longitudinal imaging to determine the fate of myelinated fibers acutely after SCI, we show that ∼73% of myelinated fibers present with periaxonal swelling at 1 h post SCI and ∼ 51% of those fibers transition to axonal spheroids by 4 h post SCI. Next, we assessed whether cation-chloride cotransporters present within the internode contributed to periaxonal swelling and whether their modulation would increase white matter sparing and improve neurological recovery following a moderate contusive SCI (T9, 50 kdyn). Mechanistically, activation of the cation-chloride cotransporter KCC2 did not improve neurological recovery and acute axonal survival, but did improve chronic tissue sparing. In distinction, the NKKC1 antagonist bumetanide improved neurological recovery, tissue sparing, and axonal survival, in part through preventing periaxonal swelling and disruption of the axo-myelinic interface. Collectively, these data reveal a novel neuroprotective target to prevent periaxonal swelling and improve neurological recovery after SCI.

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl- (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

The strength of inhibitory neurotransmission depends on intracellular neuronal chloride concentration, primarily regulated by the activity of cation-chloride cotransporters NKCC1 (Sodium-Potassium-Chloride Cotransporter 1) and KCC2 (Potassium-Chloride Cotransporter 2). Brain-derived neurotrophic factor (BDNF) influences the functioning of these co-transporters. BDNF is synthesized from precursor proteins (proBDNF), which undergo proteolytic cleavage to yield mature BDNF (mBDNF). While previous studies have indicated the involvement of BDNF signaling in the activity of KCC2, its specific mechanisms are unclear. We investigated the interplay between both forms of BDNF and chloride homeostasis in rat hippocampal neurons and in utero electroporated cortices of rat pups, spanning the behavioral, cellular, and molecular levels. We found that both pro- and mBDNF play a comparable role in immature neurons by inhibiting the capacity of neurons to extrude chloride. Additionally, proBDNF increases the endocytosis of KCC2 while maintaining a depolarizing shift of EGABA in maturing neurons. Behaviorally, proBDNF-electroporated rat pups in the somatosensory cortex exhibit sensory deficits, delayed huddling, and cliff avoidance. These findings emphasize the role of BDNF signaling in regulating chloride transport through the modulation of KCC2. In summary, this study provides valuable insights into the intricate interplay between BDNF, chloride homeostasis, and inhibitory synaptic transmission, shedding light on the underlying cellular mechanisms involved.

BACKGROUND: The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+-K+-2Cl--1 (NKCC1) and K+-2Cl--2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development.
METHODS: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA.
RESULTS: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway.
CONCLUSIONS: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

Bumetanide is used widely as a tool and off-label treatment to inhibit the Na-K-2Cl cotransporter NKCC1 in the brain and thereby to normalize intra-neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood-brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5-fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC50 (2.4 μM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain.