Neurons in the insular cortex are activated by acute and chronic pain, and inhibition of neuronal activity in the insular cortex has analgesic effects. We found that in a mouse model in which peripheral nerve injury leads to the development of neuropathic pain, the insular cortex showed changes in synaptic plasticity, which were associated with a long-term increase in the amount of synaptic N-methyl-d-aspartate receptors (NMDARs), but not that of extrasynaptic NMDARs. Activation of cyclic adenosine monophosphate (cAMP)-dependent signaling enhanced the amount of synaptic NMDARs in acutely isolated insular cortical slices and increased the surface localization of NMDARs in cultured cortical neurons. We found that the increase in the amount of NMDARs required phosphorylation of the NMDAR subunit GluN2B at Tyr(1472) by a pathway involving adenylyl cyclase subtype 1 (AC1), protein kinase A (PKA), and Src family kinases. Finally, injecting NMDAR or GluN2B-specific antagonists into the insular cortex reduced behavioral responses to normally nonnoxious stimuli in the mouse model of neuropathic pain. Our results suggest that activity-dependent plasticity takes place in the insular cortex after nerve injury and that inhibiting the increase in NMDAR function may help to prevent or treat neuropathic pain.

17-␤-Estradiol (E2)is a steroid hormone involved in neuroprotection against excitotoxicity and other forms of brain injury. Through genomic and nongenomic mechanisms, E2 modulates neuronal excitability and signal transmission by regulating NMDA and non-NMDA receptors. However, the mechanisms and identity of the receptors involved remain unclear, even though studies have suggested that estrogen G-protein-coupled receptor 30 (GPR30) is linked to protection against ischemic injury. In the culture cortical neurons, treatment with E2 and the GPR30 agonist G1 for 45 min attenuated the excitotoxicity induced by NMDA exposure. The acute neuroprotection mediated by GPR30 is dependent on G-protein-coupled signals and ERK1/2 activation, but independent on transcription or translation. Knockdown of GPR30 using short hairpin RNAs (shRNAs) significantly reduced the E2-induced rapid neuroprotection. Patch-clamp recordings revealed that GPR30 activation depressed exogenous NMDA-elicited currents. Short-term GPR30 activation did not affect the expression of either NR2A-or NR2B-containing NMDARs; however, it depressed NR2B subunit phosphorylation at Ser-1303 by inhibiting the dephosphorylation of death-associated protein kinase 1 (DAPK1). DAPK1 knockdown using shRNAs significantly blocked NR2B subunit phosphorylation at Ser-1303 and abolished the GPR30-mediated depression of exogenous NMDA-elicited currents. Lateral ventricle injection of the GPR30 agonist G1 (0.2 g) provided significant neuroprotection in the ovariectomized female mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that fast neuroprotection by estradiol is partially mediated by GPR30 and the subsequent downregulation of NR2B-containing NMDARs. The modulation of DAPK1 activity by GPR30 may be an important mediator of estradiol-dependent neuroprotection.

The insular cortex (IC) is known to play important roles in higher brain functions such as memory and pain. Activity-dependent long-term depression (LTD) is a major form of synaptic plasticity related to memory and chronic pain. Previous studies of LTD have mainly focused on the hippocampus, and no study in the IC has been reported. In this study, using a 64-channel recording system, we show for the first time that repetitive low-frequency stimulation (LFS) can elicit frequency-dependent LTD of glutamate receptor-mediated excitatory synaptic transmission in both superficial and deep layers of the IC of adult mice. The induction of LTD in the IC required activation of the N-methyl-d-aspartate (NMDA) receptor, metabotropic glutamate receptor (mGluR)5, and L-type voltage-gated calcium channel. Protein phosphatase 1/2A and endocannabinoid signaling are also critical for the induction of LTD. In contrast, inhibiting protein kinase C, protein kinase A, protein kinase Mζ or calcium/calmodulin-dependent protein kinase II did not affect LFS-evoked LTD in the IC. Bath application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine produced another form of LTD in the IC, which was NMDA receptor-independent and could not be occluded by LFS-induced LTD. Our studies have characterised the basic mechanisms of LTD in the IC at the network level, and suggest that two different forms of LTD may co-exist in the same population of IC synapses.

Long-term potentiation of glutamatergic transmission has been observed after physiological learning or pathological injuries in different brain regions, including the spinal cord, hippocampus, amygdala, and cortices. The insular cortex is a key cortical region that plays important roles in aversive learning and neuropathic pain. However, little is known about whether excitatory transmission in the insular cortex undergoes plastic changes after peripheral nerve injury. Here, we found that peripheral nerve ligation triggered the enhancement of AMPA receptor (AMPAR)-mediated excitatory synaptic transmission in the insular cortex. The synaptic GluA1 subunit of AMPAR, but not the GluA2/3 subunit, was increased after nerve ligation. Genetic knock-in mice lacking phosphorylation of the Ser845 site, but not that of the Ser831 site, blocked the enhancement of the synaptic GluA1 subunit, indicating that GluA1 phosphorylation at the Ser845 site by protein kinase A (PKA) was critical for this upregulation after nerve injury. Furthermore, A-kinase anchoring protein 79/150 (AKAP79/ 150) and PKA were translocated to the synapses after nerve injury. Genetic deletion of adenylyl cyclase subtype 1 (AC1) prevented the translocation of AKAP79/150 and PKA, as well as the upregulation of synaptic GluA1-containing AMPARs. Pharmacological inhibition of calcium-permeable AMPAR function in the insular cortex reduced behavioral sensitization caused by nerve injury. Our results suggest that the expression of AMPARs is enhanced in the insular cortex after nerve injury by a pathway involving AC1, AKAP79/150, and PKA, and such enhancement may at least in part contribute to behavioral sensitization together with other cortical regions, such as the anterior cingulate and the prefrontal cortices.