Although appropriate exercise is beneficial for enhancing brain functions, high-intensity exercise (HIE)-induced cognitive dysfunction is causing more and more concerns nowadays. In the present study, we observed the effects of high-intensity treadmill running on the spatial learning of the adult Sprague Dawley male rats in Y-maze (n = 16 per group), and investigated its possible electrophysiological and molecular mechanisms by examining in vivo hippocampal long-term potentiation (LTP), central inflammatory responses, and JNK/p38/ERK signal pathway. The Y-maze active avoidance test showed that high-intensity treadmill running impaired spatial learning ability of rats, with increased error times and prolonged training time in recognizing safety condition. Associated with the cognitive dysfunction, the induction and maintenance of hippocampal LTP were also impaired by the HIE. Furthermore, accompanied by elevated levels of inflammatory factors IL-1β, TNF-α, and iNOS, overactivation of microglia and astrocytes was also found in the CA1 region of hippocampus in the excessive exercise group, indicating an inflammatory response induced by HIE. In addition, Western blot assay showed that the phosphorylation of JNK/p38/ERK proteins was enhanced in the exercise group. These results suggest that exercise stress-induced neuronal inflammatory responses in the hippocampus are associated with HIE-induced cognitive deficits, which may be involved in the upregulation of the JNK/p38/ERK pathway. © 2016 Wiley Periodicals, Inc.
Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.