The K+-Cl− cotransporter KCC2 is responsible for maintaining low Cl− concentration in neurons of the central nervous system (CNS), essential for postsynaptic inhibition through GABAA and glycine receptors. While no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1–9. Recent reports indicate that enhancing KCC2 activity may be the favoured therapeutic strategy to restore inhibition and normal function in pathological condition involving impaired Cl− transport10–12. We designed an assay for high-throughput screening which led to the identification of KCC2 activators that reduce [Cl−]i. Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl−]i. CLP257 restored impaired Cl− transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalised stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of Pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

All organisms possess innate behavioural and physiological programmes that ensure survival. In order to have maximum adaptive benefit, these programmes must be sufficiently flexible to account for changes in the environment. Here we show that hypothalamic CRH neurons orchestrate an environmentally flexible repertoire of behaviours that emerge after acute stress in mice. Optical silencing of CRH neurons disrupts the organization of individual behaviours after acute stress. These behavioural patterns shift according to the environment after stress, but this environmental sensitivity is blunted by activation of PVN CRH neurons. These findings provide evidence that PVN CRH cells are part of a previously unexplored circuit that matches precise behavioural patterns to environmental context following stress. Overactivity in this network in the absence of stress may contribute to environmental ambivalence, resulting in context-inappropriate behavioural strategies.

BackgroundThe measurement of mechanosensitivity is a key method for the study of pain in animal models. This is often accomplished with the use of von Frey filaments in an up-down testing paradigm. The up-down method described by Chaplan et al. (J Neurosci Methods 53:55–63, 1994) for mechanosensitivity testing in rodents remains one of the most widely used methods for measuring pain in animals. However, this method results in animals receiving a varying number of stimuli, which may lead to animals in different groups receiving different testing experiences that influences their later responses. To standardize the measurement of mechanosensitivity we developed a simplified up-down method (SUDO) for estimating paw withdrawal threshold (PWT) with von Frey filaments that uses a constant number of five stimuli per test. We further refined the PWT calculation to allow the estimation of PWT directly from the behavioral response to the fifth stimulus, omitting the need for look-up tables.ResultsThe PWT estimates derived using SUDO strongly correlated (r > 0.96) with the PWT estimates determined with the conventional up-down method of Chaplan et al., and this correlation remained very strong across different levels of tester experience, different experimental conditions, and in tests from both mice and rats. The two testing methods also produced similar PWT estimates in prospective behavioral tests of mice at baseline and after induction of hyperalgesia by intraplantar capsaicin or complete Freund’s adjuvant.ConclusionSUDO thus offers an accurate, fast and user-friendly replacement for the widely used up-down method of Chaplan et al.

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Na v 1.