Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives.

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.

Primary Motor Cortex Transcranial Direct Current Stimulation Modulates Temporal Summation of the Nociceptive Withdrawal Reflex in Healthy Subjects.

OBJECTIVE: Transcranial direct current stimulation (tDCS) over the primary motor cortex (M1) has shown efficacy in a number of chronic pain conditions. Despite attempts to dissect the analgesic mechanisms, it is unknown whether M1 tDCS modulates the central integration of spinal nociception. To test this, we investigated the top-down modulation of spinal excitability using temporal summation (TS) of the nociceptive withdrawal reflex (NWR). METHODS: In this randomized, blinded, cross-over study, eight healthy subjects received electrically evoked TS of the NWR, which was delivered at 5 Hz at a threshold and a suprathreshold (1.1× threshold) to elicit TS resulting in different levels of pain. Subjects were asked to rate their pain after each stimulation. Changes in the TS of the NWR and pain ratings were investigated following 20 minutes of 2-mA anodal tDCS or sham stimulation applied over M1. RESULTS: Baseline recordings showed that TS of the NWR was induced with both threshold and suprathreshold stimulation. Suprathreshold stimulation was also associated with a higher pain intensity rating. After brain stimulation, there was no effect over the lower-intensity TS of the NWR or pain ratings in both the tDCS and sham conditions. However, tDCS reduced TS of the NWR and associated pain ratings following higher-intensity suprathreshold stimulation. CONCLUSIONS: These results indicate that M1 tDCS can indirectly modulate the central integration of suprathreshold nociceptive processing in the spinal cord. It is possible that the analgesic efficacy of tDCS is dependent on plasticity induced within pain pathways following repeated, high-intensity stimulation, which may explain the beneficial effects seen in chronic pain patients.