Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems, including forebrain dopamine-containing neurons. Recent evidence shows a strong correlation between neuropsychiatric disorders and BDNF hypofunction. The aim of the present study was to characterize the effect of low endogenous levels of BDNF on dopamine system function in the caudate-putamen using heterozygous BDNF (BDNF+/−) mice. Apparent extracellular dopamine levels in the caudate-putamen, determined by quantitative microdialysis, were significantly elevated in BDNF+/− mice compared to wildtype controls (12 vs. 5 nM, respectively). BDNF+/− mice also had a potentiated increase in dopamine levels following potassium (120 mM)-stimulation (10-fold) relative to wildtype controls (6-fold). Slice fast-scan cyclic voltammetry revealed that BDNF+/− mice had reductions in both electrically-evoked dopamine release and dopamine uptake rates in the caudate-putamen. Superfusion of BDNF led to partial recovery of the electrically-stimulated dopamine release response in BDNF+/− mice. Conversely, tissue accumulation of L-3,4-dihydroxyphenylalanine, extracellular levels of dopamine metabolites, and spontaneous locomotor activity were unaltered. Together, this study indicates that endogenous BDNF influences dopamine system homeostasis by regulating the release and uptake dynamics of presynaptic dopamine transmission.

Tight junctions form selectively permeable seals across the paracellular space. Both barrier function and selective permeability have been attributed to members of the claudin protein family, which can be categorized as pore-forming or barrier-forming. Here, we show that claudin-4, a prototypic barrier-forming claudin, reduces paracellular permeability by a previously unrecognized mechanism. Claudin-4 knockout or overexpression has minimal effects on tight junction permeability in the absence of pore-forming claudins. However, claudin-4 selectively inhibits flux across cation channels formed by claudins 2 or 15. Claudin-4-induced loss of claudin channel function is accompanied by reduced anchoring and subsequent endocytosis of pore-forming claudins. Analyses in nonepithelial cells show that claudin-4, which is incapable of independent polymerization, disrupts polymeric strands and higher order meshworks formed by claudins 2, 7, 15, and 19. This process of interclaudin interference, in which one claudin disrupts higher order structures and channels formed by a different claudin, represents a previously unrecognized mechanism of barrier regulation.

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Tight junctions form selectively‐permeable barriers that regulate paracellular flux. Individual members of the claudin protein family have been categorized as having either pore‐forming or sealing functions. For example, claudin‐2 forms paracellular cation‐selective pores. Conversely, overexpression of claudin‐4 can enhance the paracellular barrier, i.e., reduce permeability, in vitro; claudin‐4 has therefore been classified as a sealing claudin. Although specific residues that form paracellular pores, e.g., within claudin‐2, have been defined, the molecular mechanisms by which sealing claudins, e.g., claudin‐4, regulate the barrier have not. The goal of these studies was to determine the means by which the representative sealing protein claudin‐4 reduces paracellular permeability. METHODS Claudin‐4 was knocked out in Madin‐Darby Canine Kidney I (MDCK I) epithelial cells, which do not express claudin‐2 or other pore‐forming claudins, e.g., claudin‐15. These Cldn4‐KO MDCK I cells were stably transfected to inducibly express mCherry‐claudin‐4 with or without constitutive expression of EGFP‐claudin‐2. Transepithelial resistance (TER) and bi‐ionic potential measurements were used to assess barrier function. Protein anchoring and exchange were determined by Fluorescence Recovery After Photobleaching (FRAP). Widefield, confocal, and super‐resolution microscopy were used for live‐cell imaging of fluorescent claudin proteins in MDCK I and undifferentiated, fibroblast‐like cells (U2OS). RESULTS Surprisingly, claudin‐4 knockout had no effect on the paracellular barrier. Inducible mCherry‐claudin‐4 expression was also insufficient to modify TER. In contrast, EGFP‐claudin‐2 expression reduced TER 10‐fold (p<0.0001). Induction of mCherry‐claudin‐4 expression in EGFP‐claudin‐2‐expressing, but not claudin‐2‐deficient, MDCK I increased TER by 92±18% (p<0.001). FRAP analysis showed that mCherry‐claudin‐4 doubled the mobile fraction of tight junction‐associated EGFP‐claudin‐2 from 16±3% to 32±4% (p<0.001). In U2OS cells, mCherry‐claudin‐4 alone was incapable of forming tight junction‐like strands whereas EGFP‐claudin‐2 assembled into polymeric strands. Induction of mCherry‐claudin‐4 expression disrupted strands formed by EGFP‐claudin‐2, which was subsequently internalized and trafficked to LAMP2‐positive late endosomes. Although MitMAB (dynamin inhibitor) prevented EGFP‐claudin‐2 removal from the junction after mCherry‐claudin‐4 induction, monolayer TER was still increased by mCherry‐claudin‐4 expression. CONCLUSIONS These data refute the widely‐held view that claudin‐4 forms polymers that seal the paracellular pathway by demonstrating that i) neither knockout nor overexpression of claudin‐4 affect barrier function; and ii) claudin‐4 does not assemble into tight junction‐like strands (polymers). Instead, the results support an alternative model where claudin‐4 enhances barrier function by destabilizing and inhibiting flux via claudin‐2 pores. This challenges existing dogma regarding claudin function and reveals interclaudin antago...