Elevated prelimbic cortex-to-basolateral amygdala circuit activity mediates comorbid anxiety-like behaviors associated with chronic pain.

Chronic pain can cause both hyperalgesia and anxiety symptoms. However, how the two components are encoded in the brain remains unclear. The prelimbic cortex (PrL), a critical brain region for both nociceptive and emotional modulations, serves as an ideal medium for comparing how the two components are encoded. We report that PrL neurons projecting to the basolateral amygdala (PrLBLA) and those projecting to the ventrolateral periaqueductal gray (PrLl/vlPAG) were segregated and displayed elevated and reduced neuronal activity, respectively, during pain chronicity. Consistently, optogenetic suppression of the PrL-BLA circuit reversed anxiety-like behaviors, whereas activation of the PrL-l/vlPAG circuit attenuated hyperalgesia in mice with chronic pain. Moreover, mechanistic studies indicated that elevated TNF-α/TNFR1 signaling in the PrL caused increased insertion of GluA1 receptors into PrLBLA neurons and contributed to anxiety-like behaviors in mice with chronic pain. Together, these results provide insights into the circuit and molecular mechanisms in the PrL for controlling pain-related hyperalgesia and anxiety-like behaviors.

Astrocytic lactate dehydrogenase A regulates neuronal excitability and depressive-like behaviors through lactate homeostasis in mice.

Alterations in energy metabolism are associated with depression. However, the role of glycolysis in the pathogenesis of depression and the underlying molecular mechanisms remain unexplored. Through an unbiased proteomic screen coupled with biochemical verifications, we show that the levels of glycolysis and lactate dehydrogenase A (LDHA), a glycolytic enzyme that catalyzes L-lactate production, are reduced in the dorsomedial prefrontal cortex (dmPFC) of stress-susceptible mice in chronic social defeat stress (CSDS) model. Conditional knockout of LDHA from the brain promotes depressive-like behaviors in both male and female mice, accompanied with reduced L-lactate levels and decreased neuronal excitability in the dmPFC. Moreover, these phenotypes could be duplicated by knockdown of LDHA in the dmPFC or specifically in astrocytes. In contrast, overexpression of LDHA reverses these phenotypic changes in CSDS-susceptible mice. Mechanistic studies demonstrate that L-lactate promotes neuronal excitability through monocarboxylic acid transporter 2 (MCT2) and by inhibiting large-conductance Ca2+-activated potassium (BK) channel. Together, these results reveal a role of LDHA in maintaining neuronal excitability to prevent depressive-like behaviors.

Urolithin A Prevents Focal Cerebral Ischemic Injury via Attenuating Apoptosis and Neuroinflammation in Mice.

Neuroinflammation contributes to neuronal death in cerebral ischemia. Urolithin A (UA), a gut microbial metabolite of ellagic acid, has emerged as a potential anti-inflammatory agent. However, its roles and precise mechanisms in stroke remain unknown. Here we found that UA treatment ameliorated infarction, neurological deficit scores, and spatial memory deficits after cerebral ischemia. Furthermore, UA significantly reduced neuron loss and promoted neurogenesis after ischemic stroke. We also found that UA attenuated apoptosis by regulating apoptotic-related proteins. Meanwhile, UA treatment inhibited glial activation via affecting inflammatory signaling pathways, specifically by enhancing cerebral AMPK and IκBa activation while decreasing the activation of Akt, P65NFκB, ERK, JNK, and P38MAPK. Our findings reveal a key role of UA against ischemic stroke through modulating apoptosis and neuroinflammation in mice.

Social Company by a Receptive Mating Partner Facilitates Fear Extinction.

Fear extinction remains an unresolved challenge for behavioral exposure therapy in patients with post-traumatic stress disorder (PTSD). Previous reports have suggested that social support from either familiar or unfamiliar same-sex partners is beneficial to attenuating fear responses during fear extinction and renewal. Despite that, few studies have examined the effects of social support in advance on fear extinction and/or retrieval. It is also not clear whether social company by a receptive mating partner in advance facilitates fear extinction. In the present study, we address these questions by introducing a co-housing method, where fear-conditioned male mice are co-housed with or without a receptive mating partner prior to fear extinction. We found that while co-housing with an ovariectomized female mouse showed little effect on fear extinction or retrieval, social company by a receptive mating partner in advance dramatically facilitates fear extinction. In addition, the number of cFos-positive neurons in the basolateral amygdala (BLA) were also found to be reduced in male mice accompanied with receptive mating partner in response to fear extinction and retrieval, indicating diminished neuronal activation. Electrophysiological studies further showed that the excitability of excitatory neurons in BLA was decreased, which is probably due to the attenuated basal level of excitatory synaptic transmission. Together, our observations demonstrate an effect of social company by a receptive mating partner can facilitate fear extinction and afford a possible cellular mechanism.

The effect of ketogenic diet on behaviors and synaptic functions of naive mice.

INTRODUCTION: Beyond its application as an epilepsy therapy, the ketogenic diet (KD) has been considered a potential treatment for a variety of other neurological and metabolic disorders. However, whether KD promotes functional restoration by reducing the pathological processes underlying individual diseases or through some independent mechanisms is not clear. METHODS: In this study, we evaluated the effect of KD on a series of behaviors and synaptic functions of young adult naive mice. Wild-type C57BL/6J mice at age of 2-3 months were fed with control diet or KD for three months. Body weight and caloric intake were monitored throughout the experiments. We assessed behavioral performance with seizure induction, motor coordination and activity, anxiety level, spatial learning and memory, sociability, and depression. Synaptic transmission and long-term potentiation were also recorded. RESULTS: KD-fed mice performed equivalent to control-diet-fed mice in the behavioral tests and electrophysiological assays except exhibiting slower weight gain and increased seizure threshold. CONCLUSIONS: Our results contribute to the better understanding of effects of the KD on physiological behaviors and synaptic functions.

Neogenin in Amygdala for Neuronal Activity and Information Processing.

Fear learning and memory are vital for livings to survive, dysfunctions in which have been implicated in various neuropsychiatric disorders. Appropriate neuronal activation in amygdala is critical for fear memory. However, the underlying regulatory mechanisms are not well understood. Here we report that Neogenin, a DCC (deleted in colorectal cancer) family receptor, which plays important roles in axon navigation and adult neurogenesis, is enriched in excitatory neurons in BLA (Basolateral amygdala). Fear memory is impaired in male Neogenin mutant mice. The number of cFos+ neurons in response to tone-cued fear training was reduced in mutant mice, indicating aberrant neuronal activation in the absence of Neogenin. Electrophysiological studies show that Neogenin mutation reduced the cortical afferent input to BLA pyramidal neurons and compromised both induction and maintenance of Long-Term Potentiation evoked by stimulating cortical afferent, suggesting a role of Neogenin in synaptic plasticity. Concomitantly, there was a reduction in spine density and in frequency of miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents, suggesting a role of Neogenin in forming excitatory synapses. Finally, ablating Neogenin in the BLA in adult male mice impaired fear memory likely by reducing mEPSC frequency in BLA excitatory neurons. These results reveal an unrecognized function of Neogenin in amygdala for information processing by promoting and maintaining neurotransmission and synaptic plasticity and provide insight into molecular mechanisms of neuronal activation in amygdala.SIGNIFICANCE STATEMENT Appropriate neuronal activation in amygdala is critical for information processing. However, the underlying regulatory mechanisms are not well understood. Neogenin is known to regulate axon navigation and adult neurogenesis. Here we show that it is critical for neurotransmission and synaptic plasticity in the amygdala and thus fear memory by using a combination of genetic, electrophysiological, behavioral techniques. Our studies identify a novel function of Neogenin and provide insight into molecular mechanisms of neuronal activation in amygdala for fear processing.

TRPC6 contributes to LPS-induced inflammation through ERK1/2 and p38 pathways in bronchial epithelial cells.

receptor potential canonical (TRPC) channels are presently an emerging target for airway disorders. Recent evidence has indicated that TRPC6 as a member of the TRPC family plays an important role in airway inflammation, but its precise function in bronchial epithelial cells remains unclear. The aim of this study was to investigate the role of TRPC6 in Toll-like receptor 4 (TLR4)-mediated inflammation in human bronchial epithelial cells stimulated by endotoxin [lipopolysaccharide (LPS)]. Hyp9 is a simplified phloroglucinol derivative of hyperforin that highly selectively activates TRPC6 channels. The results show that the activation of TRPC6 by Hyp9 induced the production of interleukin (IL)-8 and IL-6. LPS was also able to induce the release of IL-8 and IL-6, which was significantly aggravated by Hyp9 and reduced by knockdown of TRPC6. Treatment with LPS not only chronically induced the expression of TRPC6 mRNA and protein in a TLR4-dependent manner but also acutely increased Ca2+ influx through TRPC6 channels. In addition, LPS-induced overexpression of TRPC6 and Ca2+ influx were associated with the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt. Importantly, TRPC6 was required for the activation of ERK1/2, p38, and NF-κB. In conclusion, these data reveal that LPS induced the overexpression of TRPC6 and TRPC6-dependent Ca2+ influx via the TLR4/PI3K/Akt pathway resulting in Ca2+ mobilization, which subsequently promoted the activation of ERK1/2, p38, and NF-κB and the inflammatory response in bronchial epithelial cells.

Store-operated Ca2+ channels blockers inhibit lipopolysaccharide induced astrocyte activation.

The destruction of calcium homeostasis is an important factor leading to neurological diseases. Store-operated Ca(2+) (SOC) channels are essential for Ca(2+) homeostasis in many cell types. However, whether SOC channels are involved in astrocyte activation induced by lipopolysaccharide (LPS) still remains unknown. In this study, we used LPS as an exogenous stimulation to investigate the role of SOC channels in astrocyte activation. Using calcium imaging technology, we first found that SOC channels blockers, 1-[h-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) and 2-aminoethyldiphenyl borate (2-APB), inhibited LPS induced [Ca(2+)]i increase, which prompted us to speculate that SOC channels may be involved in LPS induced astrocyte activation. Further experiments confirmed our speculation shown as SOC channels blockers inhibited LPS induced astrocyte activation characterized as cell proliferation by MTS and BrdU assay, raise in glial fibrillary acidic protein expression by immunofluorescence and Western Blot and secretion of interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) by ELISA. So, our studies showed that SOC channels are involved in LPS-induced astrocyte activation.

Neuroprotective Peptide humanin inhibits inflammatory response in astrocytes induced by lipopolysaccharide.

Humanin (HN) has been proved to be an extensive neuroprotective peptide against AD-related and unrelated insults, but little is know about the effect of HN in inflammation response. Current studies indicated the receptors of HN have a close relationship with immune system, which led us to hypothesize HN might have a role in inflammatory response. In this study, we used lipopolysaccharide (LPS) to induce astrocyte inflammation response. This model in vitro allowed us to study the effect of HN on the pure response of astrocyte without the exogenous influence between cells in vivo. Our results showed that 1.0 µg/ml LPS induced a significant activation of astrocyte, shown as the marked increase in the glial fibrillary acidic protein (GFAP) expression, the cell viability and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive living cells. Pretreatment with HN (5, 10, 20 µM) led to a significant inhibition in astrocyte overactivation in a concentration dependent manner. We also found pretreatment with HN decreased the level of proinflammatory cytokines, interleukin (IL)-6, IL-1ß and tumor necrosis factor α (TNFα) induced by LPS. Furthermore, we noticed HN couldn't completely reverse the above inflammatory injury. Our findings imply that HN partly antagonizes inflammation injury induced by LPS and the protective effect of HN on astrocyte is concentration-dependent.

Humanin protects cortical neurons from ischemia and reperfusion injury by the increased activity of superoxide dismutase.

The neuroprotective effects of superoxide dismutase (SOD) against hypoxia/reperfusion (I/R) injury and of humanin (HN) against toxicity by familial amyotrophic lateral sclerosis (ALS)-related mutant SOD led us to hypothesize that HN might have a role to increase the activity of SOD, which might be involved in the protective effects of HN on neuron against Alzheimer's disease-unrelated neurotoxicities. In the present study, we found that 4 h ischemia and 24 h reperfusion induced a significant increase in lactate dehydrogenase (LDH) release, malondialdehyde (MDA) formation and the number of karyopyknotic nuclei (4',6-diamidino-2-phenylindole dihydrochloride nuclear dyeing) and a decrease in the number of Calcein-AM-positive living cells and cell viability. Pretreatment of the cells with HN led to a significant decrease in LDH release, MDA formation and the number of karyopyknotic nuclei, and an increase in the number of Calcein-AM-positive living cells and cell viability in neurons treated with I/R. We also found a significant decrease in SOD activity in neurons treated with I/R only, while pre-treatment with HN before I/R induced a significant increase in the activity of SOD as compared with the I/R group. Our findings implied that HN protects cortical neurons from I/R injury by the increased SOD activity and that the protective effect of HN on neurons against I/R is concentration-dependent.

Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation.

BACKGROUND: Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells. RESULTS: In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death. CONCLUSION: These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.

Activation of group III metabotropic glutamate receptor reduces intracellular calcium in beta-amyloid peptide [31-35]-treated cortical neurons.

It is unknown whether amyloid beta-protein 31-35 (Abeta[31-35]) has effects similar to Abeta[1-40] and Abeta[25-35] on the intracellular calcium ([Ca(2+)]i) to induce a disruption of calcium homeostasis. In this study, we investigated the effects of Abeta[31-35] on [Ca(2+)]i in primary cultured cortical neurons using real time fluorescence imaging technique and the Ca(2+)-sensitive dye Furo-2/AM. It was found that Abeta[31-35] (25 microM) could induce a significant elevation in [Ca(2+)]i and a decrease in the average latency in the cortical neurons in a dose-dependent manner. To examine whether the activation of group III mGluRs could block the changes in [Ca(2+)]i and protect neurons from apoptosis induced by Abeta[31-35], we then investigated the effects of L: -serine-O-phosphate (L: -SOP) and (R,S)-4-phosphonophenylglycine ((R,S)-PPG), the selective agonists of group III metabotropic glutamate receptors (mGluRs), on [Ca(2+)]i and apoptosis in neurons treated by Abeta[31-35]. We demonstrated that L: -SOP or (R,S)-PPG (100 microM) treatment suppresses significantly the elevation of [Ca(2+)]i induced by Abeta[31-35] and also induces an almost complete recovery of both the fluorescence intensity and apoptotic cells (%) to the control level in the neurons. These results suggest that Abeta[31-35] may be the shortest sequence responsible for the neuronal toxicity of Abeta protein and that the neuroprotective role of the activation of group III mGluRs from the apoptosis induced by Abeta[31-35] might be partly due to its ability to inhibit the increased calcium influx, which results from Abeta[31-35].