Yan-yan Guo

An Increase in Synaptic NMDA Receptors in the Insular Cortex Contributes to Neuropathic Pain

Inhibiting NMDA receptor function in the insular cortex may prevent the development of neuropathic pain. Stopping the Pain Damage to the central or peripheral nervous system can trigger the development of neuropathic pain, which can manifest as painful sensations in response to stimuli that are not normally painful. Qiu et al. found that mice that had developed neuropathic pain after peripheral nerve injury showed changes in synaptic plasticity and increased abundance of synaptic NMDA receptors in the insular cortex, a region of the brain that is activated by acute and chronic pain. Using pharmacological inhibitors and transgenic mice, they mimicked these changes in vitro with insular cortical slices and thus identified the signaling pathway responsible. Mice injected with NMDA receptor inhibitors showed reduced behavioral signs of neuropathic pain after peripheral nerve injury. Thus, blocking NMDA receptor function in the insular cortex may prevent the development of neuropathic pain. 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 Tyr1472 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.

GluA1 Phosphorylation Contributes to Postsynaptic Amplification of Neuropathic Pain in the Insular Cortex

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.

Silibinin Prevents Autophagic Cell Death upon Oxidative Stress in Cortical Neurons and Cerebral Ischemia-Reperfusion Injury

Neuronal apoptosis and oxidative stress are involved in most of the neurodegenerative diseases, promoting neuron survival is critical for therapy. Silibinin (SLB), which is derived from the seeds of Silybinisus laborinum L., has been widely used as an antioxidant. Here we tested the neuroprotective effects of SLB and the involved molecular mechanisms. We demonstrated that SLB promoted neuron viability upon hydrogen peroxide (H2O2) challenge and reduced hypoxia/ischemia injury in the middle cerebral artery occlusion (MCAO) mouse model. SLB reversed the decreased level of procaspase-3 and balanced Bcl-2 and Bax expression upon H2O2 insult to inhibit cell apoptosis. Furthermore, SLB suppressed the activation of autophagy by decreasing microtubule-associated protein 1 light chain 3 (LC3-II) and Beclin-1 levels under oxidative stress accordingly. SLB phosphorylated protein kinase B (Akt-1) at Ser473 in a time- and dose-dependent manner. The inhibitor for phosphoinositide-3-kinase (PI3K) wortmannin abrogated SLB-induced phosphorylation of Akt-1 and mTOR, decreased the suppression of autophagy, and therefore abolished SLB-mediated neuroprotection. All the data suggested that SLB protected neurons by inhibiting both the mitochondrial and autophagic cell death pathways. This study opens new avenues for the use of SLB in treatment of central nervous system (CNS) diseases in which oxidative stress plays a major role in disease pathogenesis. Given that it occurs naturally with low toxicity and pleiotropic effects that benefit the nervous system, SLB acts potentially as a novel therapy for ischemic injury.

Anxiolytic-like effects of translocator protein (TSPO) ligand ZBD-2 in an animal model of chronic pain

The activation of Translocator protein (18xa0kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5xa0mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund’s adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-d-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.

Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro

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.

Activation of GPR30 attenuates chronic pain-related anxiety in ovariectomized mice.

Estrogen regulates neuroendocrine and inflammatory processes that play critical roles in neuroinflammation, anxiety, and chronic pain. Patients suffering from chronic pain often complain of anxiety. However, limited information is available regarding the neural circuitry of chronic pain-related anxiety and the related function of estrogen. Hindpaw injection of complete Freunds adjuvant (CFA) and chronic constriction injury (CCI) of the sciatic nerve induced notable pain sensitization and anxiety-like behavior in ovariectomized (OVX) mice. We found that the level of G-protein-coupled receptor 30 (GPR30), a membrane estrogen receptor, was significantly increased in the basolateral amygdala (BLA) of ovariectomized (OVX) mice suffering from chronic inflammatory and neuropathic pain. Subcutaneous injection or BLA local infusion of the GPR30 agonist G1 significantly reduced anxiety-like behavior in CFA-injected and CCI-OVX mice; however, this treatment did not alter the nociceptive threshold. GPR30 knock down by shRNA in the BLA of OVX mice inhibited the anxiolytic effects of GPR30 activation. G1 administration reversed the upregulation of GluR1 subunit in AMPA and NR2A-containing NMDA receptors and the downregulation of GABAA receptors in the BLA of CFA-injected and CCI-OVX mice. Electrophysiological recording revealed that GPR30 activation could prevent imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected OVX mice. In conclusion, GPR30 activation induced anxiolytic effects but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of GPR30 were partially due to maintaining the balance between excitatory and inhibitory transmissions in the BLA.

Neuroprotective effects of a novel translocator protein (18 kDa) ligand, ZBD-2, against focal cerebral ischemia and NMDA-induced neurotoxicity

Ligands of the translocator protein (18 kDa) (TSPO) have demonstrated rapid anxiolytic efficacy in stress responses and stress‐related disorders. This protein is involved in the synthesis of endogenous neurosteroids including pregnenolone, dehydroepiandrosterone, and progesterone. These neurosteroids promote γ‐aminobutyric acid‐mediated neurotransmission in the central neural system (CNS). A TSPO ligand, N‐benzyl‐N‐ethyl‐2‐(7,8‐dihydro‐7‐benzyl‐8‐oxo‐2‐phenyl‐9H‐purin‐9‐yl) acetamide (ZBD‐2) was recently synthesized. The purpose of the present study was to investigate the neuroprotective effects of ZBD‐2 and. In cultured cortical neurons, treatment with ZBD‐2 attenuated excitotoxicity induced by N‐methyl‐d‐aspartate (NMDA) exposure. It significantly decreased the number of apoptotic cells by downregulating GluN2B‐containing NMDA receptors (NMDARs), the ratio of Bax/Bcl‐2, and levels of pro‐caspase‐3. Systemic treatment of ZBD‐2 provided significant neuroprotection in mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that neuroprotection by ZBD‐2 is partially mediated by inhibiting GluN2B‐containing NMDA receptor‐mediated excitotoxicity.

Increased coupling of caveolin‐1 and estrogen receptor α contributes to the fragile X syndrome

Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the FMR1 gene. Interaction between estrogen receptor (ER) and lipid raft caveolae is critical for the estrogen signaling. Here, we tested the hypothesis that impaired ER–caveolae coupling contributes to the mental retardation of FXS.

Antidepressant-Like and Anxiolytic-Like Effects of ZBD-2, a Novel Ligand for the Translocator Protein (18 kDa)

Activation of translocator protein (18xa0kDa) (TSPO) plays an important role to mediate rapid anxiolytic efficacy in stress response and stress-related disorders by the production of neurosteroids. However, little is known about the ligand of TSPO on the anxiety-like and depressive behaviors and the underlying mechanisms in chronic unpredictable mild stress (UCMS) mice. In the present study, a novel ligand of TSPO, ZBD-2 [N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide] synthesized by our laboratory, was used to evaluate the anxiolytic and antidepressant efficacy and to elucidate the underlying mechanisms. ZBD-2 (3xa0mg/kg) significantly attenuated anxiety-like and depressive behaviors in the UCMS mice, which was blocked by TSPO antagonist PK11195 (3xa0mg/kg). Treatment of ZBD-2 reversed the decrease in biogenic amines (norepinephrine, dopamine, and serotonin) in the brain region of hippocampus in the UCMS mice. The decreases in TSPO, GluN2B-containing N-methyl-d-aspartate (NMDA) receptors, GluA1, p-GluA1-Ser831, p-GluA1-Ser845, PSD-95, and GABAA-a2 were integrated with the increases of CaMKII and iNOS levels in the hippocampus of the UCMS mice. ZBD-2 significantly reversed the changes of above proteins. However, ZBD-2 or PK11195 treatment did not affect the levels of GluN2A-containing NMDA receptors and the total levels of GAD67. Our study provides strong evidences that ZBD-2 has a therapeutic effect on chronic stress-related disorders such as depression and anxiety through regulating the biogenic amine levels and the synaptic proteins in the hippocampus.

Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model

Background Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. Results Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30u2009mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. Conclusions NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex.