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Objectives: To explore the role of microRNA-21-5p (miR-21-5p) in hypoxia/reoxygenation- (H/R-) induced HT22 cell damage., Methods: The hypoxia/reoxygenation (H/R) model was established in mouse neuronal cells HT22. Cell Counting Kit-8 (CCK-8) and qRT-PCR were used to determine the effects of H/R treatment on cell viability and miR-21-5p expression. HT22 cells were transfected with miR-21-5p mimic or negative control (NC) followed by the induction of H/R; cell viability, apoptosis, and SOD, MDA, and LDH activities were detected. Besides, the apoptosis-related proteins including BAX, BCL2, cleaved caspase-3, and caspase-3 as well as proteins of EGFR/PI3K/AKT signaling pathways were measured by Western blot. To verify the target relation between cytoplasmic polyadenylation element binding protein 3 (CPEB3) and miR-21-5p, luciferase reporter gene experiment was performed. After cotransfection with miR-21-5p mimic and CPEB3 plasmids, the reversal effects of CPEB3 on miR-21-5p in H/R damage were studied., Results: H/R treatment could significantly reduce the cell viability ( P < 0.05) and miR-21-5p levels ( P < 0.05) in HT22 cells. After overexpressing miR-21-5p, cell viability was increased ( P < 0.05) under H/R treatment, and the apoptosis rate and the levels of apoptosis-related proteins were suppressed (all P < 0.05). Furthermore, SOD activity was increased ( P < 0.05), while MDA and LDH activity was decreased (both P < 0.05). Besides, miR-21-5p could restore the activation of the EGFR/PI3K/AKT signaling pathway inhibited by H/R treatment (all P < 0.05). The luciferase reporter gene experiment verified that CPEB3 is the target of miR-21-5p ( P < 0.05). When coexpressing miR-21-5p mimic and CPEB3 in the cells, the protective effects of miR-21-5p under H/R were reversed (all P < 0.05), and the activation of the EGFR/PI3K/AKT pathway was also inhibited (all P < 0.05)., Conclusion: This study showed that miR-21-5p may regulate the EGFR/PI3K/AKT signaling pathway by targeting CPEB3 to reduce H/R-induced cell damage and apoptosis., Competing Interests: The authors declare that they have no conflict of interests., (Copyright © 2021 Bin Wang et al.)

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction, deterioration of neuronal transmission, and consequently neuronal death. Although there is no treatment for AD, exposure to enriched environment (EE) in mice, as well as physical and mental activity in human subjects have been shown to have a protective effect by slowing the disease's progression and reducing AD-like cognitive impairment. However, the molecular mechanism of this mitigating effect is still not understood. One of the mechanisms that has recently been shown to be involved in neuronal degeneration is microRNAs (miRNAs) regulation, which act as a post-transcriptional regulators of gene expression. miR-128 has been shown to be significantly altered in individuals with AD and in mice following exposure to EE. Here, we focused on elucidating the possible role of miR-128 in AD pathology and found that miR-128 regulates the expression of two proteins essential for synaptic transmission, SNAP-25, and synaptotagmin1 (Syt1). Clinically relevant, in 5xFAD mouse model for AD, this miRNA's expression was found as downregulated, resembling the alteration found in the hippocampi of individuals with AD. Interestingly, exposing WT mice to EE also resulted in downregulation of miR-128 expression levels, although EE and AD conditions demonstrate opposing effects on neuronal functioning and synaptic plasticity. We also found that miR-128 expression downregulation in primary hippocampal cultures from 5xFAD mice results in increased neuronal network activity and neuronal excitability. Altogether, our findings place miR-128 as a synaptic player that may contribute to synaptic functioning and plasticity through regulation of synaptic protein expression and function., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Although mulberry fruit possesses some biological activities, it is not known how it protects neuronal cells in neurodegenerative diseases. Here, we examined whether mulberry fruit extract (MFE) protected neuronal cells against oxidative stress-induced neurodegeneration. Methods: In this experiments, glutamate challenged hippocampal neuronal HT-22 cell lines as an in vitro model and scopolamine-induced memoty-impairment mice model were used. Results: MFE improved cell viability and glutathione level as well as reducing reactive oxygen species level in glutamate-treated HT-22 cells. Additionally, MFE suppressed apoptotic bodies and mitochondrial depolarization through regulating expression of apoptosis-related proteins. Furthermore, MFE elevated expression of p-TrkB, p-Akt, p-CREB, BDNF, and antioxidant enzymes as well as nuclear translocation of Nrf2. In contrast, the inclusion of K252a, a TrkB inhibitor, or MK-2206, an Akt selective inhibitor, neutralized the neuroprotective actions of MFE. Separately, MFE attenuated scopolamine-induced amnesia via regulating the activities of enzymes related with cholinergic function and the antioxidant system in mice. Additionally, MFE protected neuronal cells in the hippocampal CA1 and CA3 regions in brain of mice. Conclusions: MFE protects neuronal cells against oxidative stress-induced apoptosis through upregulating the expression of BDNF and antioxidant enzymes by stabilizing the activation of the TrkB/Akt pathway. Such an effect of MFE, which includes rich polyphenols, may provide information for its application as a food supplement for the prevention and treatment of neurodegenerative diseases.

Apoptosis of hippocampal neurons is one of the mechanisms of hippocampal atrophy in posttraumatic stress disorder (PTSD), and it is also an important cause of memory impairment in PTSD patients. Endoplasmic reticulum stress (ERS) mediated by activated transcription factor 6α (ATF6α)/site 1 protease (S1P)/S2P is involved in cell apoptosis, but it is not clear whether it is involved in hippocampal neuron apoptosis caused by PTSD. A PTSD rat model was constructed by the single prolonged stress (SPS) method. The study was divided into three parts. Experiment 1 included the control group, SPS 1 d group, SPS 7 d group, and SPS 14 d group. Experiment 2 included the control group, SPS 7 d group, SPS 7 d + AEBSF group, and control + AEBSF group. (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) is an ATF6α pathway inhibitor). Experiment 3 included the control group, SPS 4 d group, SPS 4 d + AEBSF group, and control + AEBSF group. The protein and mRNA expression levels of ATF6α, glucose-regulated protein (GRP78), S1P, S2P, C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of PTSD rats were detected by immunohistochemistry, Western blotting and qRT-PCR. Apoptosis of hippocampal neurons was detected by TUNEL staining. In experiment 1, the protein and mRNA expression of ATF6α and GRP78 increased gradually in the SPS 1 d group and the SPS 7 d group but decreased in the SPS 14 d group (P < 0.01). In experiment 2, compared with that in the control group, the protein and mRNA expression of ATF6α, GRP78, S1P, S2P, CHOP, and caspase-12 and the apoptosis rate were significantly increased in the SPS 7 d group (P < 0.01). However, the protein and mRNA expression of ATF6α, GRP78, S1P, S2P, CHOP, and caspase-12 and the apoptosis rate were significantly decreased after AEBSF pretreatment (P < 0.01). In experiment 3, compared with that in the control group, the protein and mRNA expression of ATF6α, GRP78, S1P, S2P, CHOP, and caspase-12 and the apoptosis rate were increased in the SPS 14 d group (P < 0.05). However, the protein and mRNA expression of ATF6α, GRP78, S1P, S2P, CHOP, and caspase-12 and the apoptosis rate were decreased after AEBSF pretreatment (P < 0.05). SPS induced apoptosis of hippocampal neurons by activating ERS mediated by ATF6α, suggesting that ERS-induced apoptosis is involved in the occurrence of PTSD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Postoperative cognitive impairment and nervous system damage caused by anesthetics seriously affect patient's postoperative recovery. Recent study has revealed that CTRP6 could alleviate apoptosis, inflammation and oxidative stress of nerve cells, thereby relieving nervous system damage induced by cerebral ischemia reperfusion. However, whether CTRP6 could relieve sevoflurane induced central nervous system injury is unclear. We stimulated C57BL/6 mice with sevoflurane and injected CTRP6 overexpression adenovirus vector. Next, H&E staining and TUNEL assays were performed to examine the effect of CTRP6 on sevoflurane induced injury of central nervous system. Finally, we isolated primary nerve cells of hippocampus. Flow cytometry and commercial kits were used for the detection of apoptosis and ROS levels of these cells. Western blotting was used for the detection of the expression level of p-Akt in central nervous tissues and primary cells. Results showed that sevoflurane induced injury and apoptosis of central nervous tissues. Overexpression of CTRP6 relieved apoptosis and injury of these tissues. CTRP6 inhibited the expression of cleaved caspase-3 and cleaved PARP in these tissues. Sevoflurane promoted apoptosis of primary cells and enhanced the expression of ROS and MDA in these cells. Overexpression of CTRP6 alleviated apoptosis and suppressed production of ROS and MDA in these cells. In addition, CTRP6 also enhanced the expression of p-Akt in primary cells. Taken together, our results suggested that CTRP6 relieved sevoflurane induced injury of central nervous tissues by promoting the expression of p-Akt. Therefore, the targeted drug of CTRP6 should be explored for the remission of these symptoms.

CPT1C, which is expressed in hippocampus, influences ceramide level, endogenous cannabinoid and oxidation process, as well as plays an important role in various brain functions such as learning. This study aimed to investigate the role of CPT1C in Alzheimer's disease (AD) and its underlying mechanism. We established a model of Alzheimer's disease in vitro by exposing primary hippocampal neurons to beta-Amyloid peptide fragment 25-35 (Aβ 25-35 ). The cell viability, lactate dehydrogenase (LDH) level, expressions of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected using Cell Counting Kit-8 (CCK-8), LDH assay, ROS kits, malondialdehyde (MDA) kits and SOD kits, respectively. Moreover, the expression of oxidative stress-related proteins as well as the expressions of amyloid precursor protein (App), p-Tau andβ-site APP-cleaving enzyme1 (Bace-1) were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot. Tunel and western blot were adopted to detect apoptosis as well as its related proteins. After the treatment of peroxisome proliferators-activated receptor alpha (PPARα), CPT1C expression was detected with the application of RT-qPCR and western blot. CPT1C expression was reduced in Aβ 25-35 -induced HT22 cells. Overexpression of CPT1C relieved cell viability and toxic injury as well as attenuated oxidative stress, apoptosis and expression levels of AD marker proteins. Moreover, higher doses of PPARα agonist activate the expression of CPT1C in Aβ 25-35 -induced HT22 cells. In conclusion, CPT1C alleviates Aβ 25-35 -induced oxidative stress, apoptosis and deposition of AD marker proteins in hippocampal neurons, suggesting that CPT1C has favorable effects on alleviating AD and participates in PPARα activation.

Bupivacaine (BUP) is a long-acting amide local anesthetic that may induce strong neurotoxicity and neurological complications. In this study, we elucidate the influence of microRNA-494-3p (miR-494-3p) in BUP-induced neurotoxicity in primary mouse hippocampal neuronal cells. In this study, primary hippocampal neurons were isolated from neonatal C57BL/6 mice. The isolated neurons were treated with various doses of BUP. MTT assay was conducted to analyze neuronal viability. Gene expression measurement was done by RT-qPCR. The impact of miR-494-3p in BUP-mediated neural injury was examined using TUNEL, flow cytometry, western blotting, and ROS activity detection. The regulatory relationship between miR-494-3p and cyclin-dependent kinases 4 and 6 (CDK6) was identified using a luciferase reporter assay. BUP treatment led to neurotoxicity and miR-494-3p upregulation in primary cultured hippocampal neurons. Functionally, miR-494-3p depletion alleviated neuronal apoptosis and oxidative damage induced by BUP. We verified that miR-494-3p targeted and negatively modulated CDK6. MiR-494-3p depletion also activated PI3K/AKT signaling by elevating CDK6 expression in BUP-treated neurons. Furthermore, CDK6 knockdown or PI3K/AKT inactivation attenuated the neuroprotective role of miR-494-3p depletion. Silencing miR-494-3p exerts neuroprotective function in hippocampal neuronal cells against BUP-induced injury by the CDK6-PI3K/AKT pathway., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Could learning that uses cognitive control to judiciously use relevant information while ignoring distractions generally improve brain function, beyond forming explicit memories? According to a neuroplasticity hypothesis for how some cognitive behavioural therapies are effective, cognitive control training (CCT) changes neural circuit information processing 1-3 . Here we investigated whether CCT persistently alters hippocampal neural circuit function. We show that mice learned and remembered a conditioned place avoidance during CCT that required ignoring irrelevant locations of shock. CCT facilitated learning new tasks in novel environments for several weeks, relative to unconditioned controls and control mice that avoided the same place during reduced distraction. CCT rapidly changes entorhinal cortex-to-dentate gyrus synaptic circuit function, resulting in an excitatory-inhibitory subcircuit change that persists for months. CCT increases inhibition that attenuates the dentate response to medial entorhinal cortical input, and through disinhibition, potentiates the response to strong inputs, pointing to overall signal-to-noise enhancement. These neurobiological findings support the neuroplasticity hypothesis that, as well as storing item-event associations, CCT persistently optimizes neural circuit information processing., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol 4,5 -bisphosphate (PIP 2 ) depletion in non-excitable cells identical to agonist-induced activation of the G q11 coupled receptors. PIP 2 is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca 2+ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP 2 depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1-5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM 1 ). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP 2 depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP 2 reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: This study aimed to investigate whether electroacupuncture (EA) promotes the survival and synaptic plasticity of hippocampal neurons by activating brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase (TrkB)/extracellular signal-regulated kinase (Erk) signaling, thereby improving spatial memory deficits in rats under SD., Methods: In vivo, Morris water maze (MWM) was used to detect the effect of EA on learning and memory, at the same time Western blotting (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to explore the plasticity of hippocampal neurons and synapses, and the expression of BDNF/TrkB/Erk signaling. In vitro, cultured hippocampal neurons were treated with exogenous BDNF and the TrkB inhibitor K252a to confirm the relationship between BDNF/TrkB/Erk signaling and synaptic plasticity., Results: Our results showed that EA mitigated the loss of hippocampal neurons and synapses, stimulated hippocampal neurogenesis, and improved learning and memory of rats under SD accompanied by upregulation of BDNF and increased phosphorylation of TrkB and Erk. In cultured hippocampal neurons, exogenous BDNF enhanced the expression of synaptic proteins, the frequency of the postsynaptic currents, and the phosphorylation of TrkB and Erk; these effects were reversed by treatment with K252a., Conclusions: Electroacupuncture alleviates SD-induced spatial memory impairment by promoting hippocampal neurogenesis and synaptic plasticity via activation of BDNF/TrkB/Erk signaling, which provided evidence for EA as a therapeutic strategy for countering the adverse effects of SD on cognition., (© 2021 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

G protein-gated inwardly rectifying K + (GIRK) channels are critical mediators of excitability in the heart and brain. Enhanced GIRK-channel activity has been implicated in the pathogenesis of supraventricular arrhythmias, including atrial fibrillation. The lack of selective pharmacological tools has impeded efforts to investigate the therapeutic potential of cardiac GIRK-channel interventions in arrhythmias. Here, we characterize a recently identified GIRK-channel inhibitor, VU0468554. Using whole-cell electrophysiological approaches and primary cultures of sinoatrial nodal cells and hippocampal neurons, we show that VU0468554 more effectively inhibits the cardiac GIRK channel than the neuronal GIRK channel. Concentration-response experiments suggest that VU0468554 inhibits G βγ -activated GIRK channels in noncompetitive and potentially uncompetitive fashion. In contrast, VU0468554 competitively inhibits GIRK-channel activation by ML297, a GIRK-channel activator containing the same chemical scaffold as VU0468554. In the isolated heart model, VU0468554 partially reversed carbachol-induced bradycardia in hearts from wild-type mice but not Girk4 -/- mice. Collectively, these data suggest that VU0468554 represents a promising new pharmacological tool for targeting cardiac GIRK channels with therapeutic implications for relevant cardiac arrhythmias. SIGNIFICANCE STATEMENT: Although cardiac GIRK-channel inhibition shows promise for the treatment of supraventricular arrhythmias, the absence of subtype-selective channel inhibitors has hindered exploration into this therapeutic strategy. This study utilizes whole-cell patch-clamp electrophysiology to characterize the new GIRK-channel inhibitor VU0468554 in human embryonic kidney 293T cells and primary cultures. We report that VU0468554 exhibits a favorable pharmacodynamic profile for cardiac over neuronal GIRK channels and partially reverses GIRK-mediated bradycardia in the isolated mouse heart model., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The purpose of our study is to determine the protective effects of mitophagy enhancers against phosphorylated tau (P-tau)-induced mitochondrial and synaptic toxicities in Alzheimer's disease (AD). Mitochondrial abnormalities, including defective mitochondrial dynamics, biogenesis, axonal transport and impaired clearance of dead mitochondria are linked to P-tau in AD. Mitophagy enhancers are potential therapeutic candidates to clear dead mitochondria and improve synaptic and cognitive functions in AD. We recently optimized the doses of mitophagy enhancers urolithin A, actinonin, tomatidine, nicotinamide riboside in immortalized mouse primary hippocampal (HT22) neurons. In the current study, we treated mutant Tau expressed in HT22 (mTau-HT22) cells with mitophagy enhancers and assessed mRNA and protein levels of mitochondrial/synaptic genes, cell survival and mitochondrial respiration. We also assessed mitochondrial morphology in mTau-HT22 cells treated and untreated with mitophagy enhancers. Mutant Tau-HT22 cells showed increased fission, decreased fusion, synaptic & mitophagy genes, reduced cell survival and defective mitochondrial respiration. However, these events were reversed in mitophagy enhancers treated mTau-HT22 cells. Cell survival was increased, mRNA and protein levels of mitochondrial fusion, synaptic and mitophagy genes were increased, and mitochondrial fragmentation is reduced in mitophagy enhancers treated mTau-HT22 cells. Further, urolithin A showed strongest protective effects among all enhancers tested in AD. Our combination treatments of urolithin A + EGCG, addition to urolithin A and EGCG individual treatment revealed that combination treatments approach is even stronger than urolithin A treatment. Based on these findings, we cautiously propose that mitophagy enhancers are promising therapeutic drugs to treat mitophagy in patients with AD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

β -Asarone is the main constituent of Acorus tatarinowii Schott and exhibits important effects in diseases such as neurodegenerative and neurovascular diseases. Icariin (ICA) is a major active ingredient of Epimedium that has attracted increasing attention because of its unique pharmacological effects in degenerative disease. In this paper, we primarily explored the effects of the combination of β -asarone and ICA in clearing noxious proteins and reversing cognitive deficits. The accumulation of damaged mitochondria and mitophagy are hallmarks of aging and age-related neurodegeneration, including Alzheimer's disease (AD). Here, we provide evidence that autophagy/mitophagy is impaired in the hippocampus of APP/PS1 mice and in A β 1-42-induced PC12 cell models. Enhanced mitophagic activity has been reported to promote A β and tau clearance in in vitro and in vivo models. Meanwhile, there is growing evidence that treatment of AD should be preceded by intervention before the formation of pathological products. The efficacy of the combination therapy was better than that of the individual therapies applied separately. Then, we found that the combination therapy also inhibited cell and mitochondrial damage by inducing autophagy/mitophagy. These findings suggest that impaired removal of defective mitochondria is a pivotal event in AD pathogenesis, and that combination treatment with mitophagy inducers represents a potential strategy for therapeutic intervention., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Nanbu Wang et al.)

The ability to generate new hippocampal neurons throughout adulthood and successfully integrate them into existing neural networks is critical to cognitive function, while disordered regulation of this process results in neurodegenerative or psychiatric disease. Consequently, identifying the molecular mechanisms promoting homeostatic hippocampal neurogenesis in adults is essential to understanding the etiologies of these disorders and developing therapeutic interventions. For example, recent evidence identifies a strong association between metabolic function and adult hippocampal neurogenesis. Hippocampal neural stem cell (NSC) fate dynamically fluctuates with changes in substrate availability and energy status (AMP/ATP and NAD + /NADH ratios). Furthermore, many metabolic hormones, such as insulin, insulin-like growth factors, and leptin exhibit dual functions also modulating hippocampal neurogenesis and neuron survivability. These diverse metabolic inputs to NSC's from various tissues seemingly suggest the existence of a system in which energy status can finely modulate hippocampal neurogenesis. Supporting this hypothesis, interventions promoting energy balance, such as caloric restriction, intermittent fasting, and exercise, have shown encouraging potential enhancing hippocampal neurogenesis and cognitive function. Overall, there is a clear relationship between whole body energy status, adult hippocampal neurogenesis, and neuron survival; however, the molecular mechanisms underlying this phenomenon are multifaceted. Thus, the aim of this review is to analyze the literature investigating energy status-mediated regulation of adult neurogenesis in the hippocampus, highlight the neurocircuitry and intracellular signaling involved, and propose impactful future directions in the field., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Solifenacin (Vesicare ® , SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH 3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K + current (I K(M) ) with effective EC 50 value of 0.34 μM. The activation time constant of I K(M) was concurrently shortened in the SOL presence, hence yielding the K D value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of I K(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of I K(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated I K(M) ; however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in I K(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K + (K M ) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH 3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the I K(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with K M channels and hence in stimulating I K(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH 3 or mHippoE-14 cells.

Ethnopharmacological Relevance: Shuxuening injection (SXNI) is a Chinese medicine of Ginkgo biloba L. leaves extract (GBE), which is widely used clinically for cardiovascular diseases such as stroke and myocardial infarction, but the pharmacological mechanism of its therapeutic effect is not fully understood., Aim of the Study: Preclinical studies suggested that inhibition of neuronal apoptosis effectively improves brain damage after ischemic stroke. The purpose of this study was to investigate the inhibitory effect of SXNI on neuronal apoptosis in post-stroke mice and its underlying mechanism., Materials and Methods: A mouse cerebral ischemia-reperfusion injury (CIRI) model was constructed by middle cerebral artery occlusion (MCAO) and treated with 3 mL/kg SXNI. TUNEL and immunohistochemistry experiments were performed on brain slices on the 7th day after stroke. The protein was extracted from the hippocampus region of the brain for western-blot assay. To simulate the in vivo ischemia-reperfusion process, the hippocampal neuron cell line HT-22 was subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro, and 200 μg/mL SXNI was administered. The HT-22 cells were then studied by RT-PCR and immunocytochemistry., Results: In vivo, SXNI treatment significantly reduced hippocampal neuronal apoptosis. Immunohistochemistry showed that SXNI inhibited the activation of Caspase-3 protein in the hippocampus after ischemic stroke. Western blot analysis further confirmed that SXNI regulated the expression of the antagonizing protein pair Bax and Bcl-2 to exert anti-apoptotic effect in addition to reducing the expression of Cleaved-Caspase-3 in the hippocampus. In vitro, 200 μg/mL SXNI treatment significantly improved HT-22 apoptosis caused by OGD/R. Further RT-PCR and immunocytochemistry study showed that 200 μg/mL SXNI inhibited apoptosis of hippocampal neurons by regulating the mRNA and protein expressions of apoptotic molecules Bax, Bcl-2 and Caspase-3., Conclusions: CIRI can induce hippocampal neuronal apoptosis, which is inhibited by SXNI via regulating Bax/Bcl-2 and blocking Caspase-3 activation. Therefore, SXNI may be a promising treatment strategy to improve the prognosis of ischemic stroke., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C 1 domain, a Ca 2+ /PIP 2 -binding C 2 B domain, a MUN domain and a C 2 C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C 1 C 2 B region with the plasma membrane: (i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and (ii) another involving the DAG-Ca 2+ -PIP 2 -binding face that is predicted to result in a slanted orientation and facilitate release. Here, we have tested this model and investigated the role of the C 1 C 2 B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca 2+ -independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C 2 B domain Ca 2+ -binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca 2+ -evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C 1 -C 2 B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity., Competing Interests: MC, BQ, TT, JX, LS, JR, CR No competing interests declared, (© 2021, Camacho et al.)

Glutamatergic neurotransmission is a widespread form of synaptic excitation in the mammalian brain. The development of genetically encoded fluorescent glutamate sensors allows monitoring synaptic signaling in living brain tissue in real time. Here, we describe single- and two-photon imaging of synaptically evoked glutamatergic population signals in acute hippocampal slices expressing the fluorescent glutamate sensor SF-iGluSnFR.A184S in CA1 or CA3 pyramidal neurons. The protocol can be readily used to study defective synaptic glutamate signaling in mouse models of neuropsychiatric disorders, such as Alzheimer disease. For complete details on the use and execution of this protocol, please refer to Zott et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson's Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD., (© 2021. The Author(s).)

The regulation of adult neural stem or progenitor cell (aNSC) proliferation and differentiation as an interplay of cell-intrinsic and local environmental cues remains in part unclear, impeding their role in putative regenerative therapies. aNSCs with all major properties of NSCs in vitro have been identified in a variety of brain regions beyond the classic neurogenic niches, including the caudal periventricular regions (PVRs) of the midbrain, though active neurogenesis is either limited or merely absent in these regions. To elucidate cell-intrinsic properties of aNSCs from various PVRs, we here examined the proliferation and early differentiation capacity of murine aNSCs from non-neurogenic midbrain PVRs (PVR MB ) compared to aNSCs from the neurogenic ventricular-subventricular zone (PVR V-SVZ ) 7 days after transplantation into the permissive pro-neurogenic niche of the dentate gyrus (DG) of the hippocampus in mice. An initial in vitro characterization of the transplants displayed very similar characteristics of both aNSC grafts after in vitro expansion with equal capacities of terminal differentiation into astrocytes and Tuj1 + neurons. Upon the allogenic transplantation of the respective aNSCs into the DG, PVR MB grafts showed a significantly lower graft survival and proliferative capacity compared to PVR V-SVZ transplants, whereby the latter are exclusively capable of generating new neurons. Although these differences might be-in part-related to the transplantation procedure and the short-term study design, our data strongly imply important cell-intrinsic differences between aNSCs from neurogenic compared to non-neurogenic PVRs with respect to their neurogenic potential and/or their sensitivity to neurogenic cues.

Loss-of-function mutations in endosomal Na + /H + exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome. Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to overacidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite overacidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae , we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, β-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence, we also find a prominent increase in fusion between endosomal multivesicular bodies and the plasma membrane, indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to overacidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease. SIGNIFICANCE STATEMENT Loss-of-function mutations in the endosomal Na + /H + exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurologic disorder. Loss of NHE6 has been shown to overacidify endosomes; however, endosome trafficking mechanisms have been understudied, and the mechanisms leading to neurodegeneration are largely unknown. In NHE6-null mouse neurons in vitro , we find worsening lysosome function with days in culture. Notably, pH-dependent lysosome enzymes, such as cathepsin D, have reduced activity in lysosomes yet increased, precocious activity in endosomes in NHE6-null neurons. Further, endosomes show reduced fusion to lysosomes, and increased fusion to the plasma membrane with increased exosome release. This study identifies new mechanisms involving defective endosome maturation and trafficking that impair lysosome function in Christianson syndrome, likely contributing to neurodegeneration., (Copyright © 2021 Pescosolido et al.)

Perineuronal nets (PNNs) are emerging as critical regulators of memory-related neuronal processes. However, their exact contribution depends on type of memory, consolidation stage, or brain region, and remains to be fully investigated. We describe here a protocol to evaluate the importance of PNNs in the dorsal hippocampus in different stages of aversive memories using a mouse model. The protocol provides detailed instructions for surgical implantation of hippocampal cannulas, drug infusion, contextual fear conditioning procedures, and immunohistochemistry for PNN visualization. For complete details on the use and execution of this protocol, please refer to Jovasevic et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Tauopathies, including Alzheimer's disease (AD), are manifested by the deposition of well-characterized amyloid aggregates of Tau protein in the brain. However, it is rather unlikely that these aggregates constitute the major form of Tau responsible for neurodegenerative changes. Currently, it is postulated that the intermediates termed as soluble oligomers, assembled on the amyloidogenic pathway, are the most neurotoxic form of Tau. However, Tau oligomers reported so far represent a population of poorly characterized, heterogeneous and unstable assemblies. In this study, to obtain the oligomers, we employed the aggregation-prone K18 fragment of Tau protein with deletion of Lys280 (K18Δ280) linked to a hereditary tauopathy. We have described a new procedure of inducing aggregation of mutated K18 which leads either to the formation of nontoxic amyloid fibrils or neurotoxic globular oligomers, depending on its phosphorylation status. We demonstrate that PKA-phosphorylated K18Δ280 oligomers are toxic to hippocampal neurons, which is manifested by loss of dendritic spines and neurites, and impairment of cell-membrane integrity leading to cell death. We also show that N1, the soluble N-terminal fragment of prion protein (PrP), protects neurons from the oligomers-induced cytotoxicity. Our findings support the hypothesis on the neurotoxicity of Tau oligomers and neuroprotective role of PrP-derived fragments in AD and other tauopathies. These observations could be useful in the development of therapeutic strategies for these diseases., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Accurate navigation to a desired goal requires consecutive estimates of spatial relationships between the current position and future destination throughout the journey. Although neurons in the hippocampal formation can represent the position of an animal as well as its nearby trajectories 1-7 , their role in determining the destination of the animal has been questioned 8,9 . It is, thus, unclear whether the brain can possess a precise estimate of target location during active environmental exploration. Here we describe neurons in the rat orbitofrontal cortex (OFC) that form spatial representations persistently pointing to the subsequent goal destination of an animal throughout navigation. This destination coding emerges before the onset of navigation, without direct sensory access to a distal goal, and even predicts the incorrect destination of an animal at the beginning of an error trial. Goal representations in the OFC are maintained by destination-specific neural ensemble dynamics, and their brief perturbation at the onset of a journey led to a navigational error. These findings suggest that the OFC is part of the internal goal map of the brain, enabling animals to navigate precisely to a chosen destination that is beyond the range of sensory perception., (© 2021. The Author(s).)

Spatial memories that can last a lifetime are thought to be encoded during 'online' periods of exploration and subsequently consolidated into stable cognitive maps through their 'offline' reactivation. However, the mechanisms and computational principles by which offline reactivation stabilize long-lasting spatial representations remain poorly understood. Here, we employed simultaneous fast calcium imaging and electrophysiology to track hippocampal place cells over 2 weeks of online spatial reward learning behavior and offline resting. We describe that recruitment to persistent network-level offline reactivation of spatial experiences in mice predicts the future representational stability of place cells days in advance of their online reinstatement. Moreover, while representations of reward-adjacent locations are generally more stable across days, offline-reactivation-related stability is, conversely, most prominent for reward-distal locations. Thus, while occurring on the tens of milliseconds timescale, offline reactivation is uniquely associated with the stability of multiday representations that counterbalance the overall reward-adjacency bias, thereby predicting the stabilization of cognitive maps that comprehensively reflect entire underlying spatial contexts. These findings suggest that post-learning offline-related memory consolidation plays a complimentary and computationally distinct role in learning compared to online encoding., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/discs large/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo., (© 2021. The Author(s).)

Purpose: Childhood obesity increases risk for neural dysfunctions causing learning and memory deficits. The objective of the study is to identify the effects of high fat diet-induced obesity in postnatal period on serum lipids, memory and neural cell survival in hippocampus and compare the role of choline and DHA or environmental enrichment in attenuating the alterations., Materials and Methods: 21 day postnatal male Sprague Dawley rats were assigned as Normal control [NC] fed normal chow diet, Obesity-induced [OB] fed high fat diet, Obesity-induced fed choline & DHA [OB + CHO + DHA], Obesity-induced environmental enrichment [OB + EE] [ n  = 8/group]. Memory was assessed using radial arm maze. Subsequently blood was collected for serum lipid analysis and rats were euthanized. 5 µm hippocampal sections were processed for cresyl-violet stain. Surviving neural cells were counted using 100 µm scale., Results: Memory errors were significantly higher [ p  < 0.001, 0.01] in OB compared to same in NC rats. Mean number of surviving neural cells in hippocampus of OB was significantly lesser [ p  < 0.01] compared to same in NC. Interventions in OB + CHO + DHA and OB + EE significantly attenuated [ p  < 0.01] memory errors and number of surviving neural cells in hippocampus [CA1, CA3 and DG] compared to same in OB. Moreover, hippocampal neural cell survival was found to be inversely related to serum lipid profile in OB group and was attenuated in OB + CHO + DHA and OB + EE rats., Conclusions: High fat diet-induced postnatal obesity in rats causes CA1/CA3 hippocampal neuro-degeneration and memory deficits. Supplementation of choline and DHA in obese rats attenuates these deficits.

Objective: To investigate the effects of Moringa leaves on the cognitive dysfunction and apoptosis of hippocampal neurons in diabetic rats induced by streptozotocin (STZ). Methods: Fifty male SD rats were selected, and 10 rats were randomly selected as the control group. The other 40 rats were treated with STZ at the dose of 25 mg/kg by intraperitoneal injection. The 40 diabetic rats were randomly divided into model group, Moringa oleifera low-dose, medium-dose and high-dose group. The rats in Moringa oleifera groups were treated with Moringa oleifera at the doses of 2.0, 4.0 and 8.0 g/kg by gavage, the control group and model group were treated with the same amount of normal saline once a day, for 8 weeks. Morris water maze test was used to evaluate the learning and memory ability of rats. Pathological changes of hippocampal neurons and expressions of Bax, caspase-3 and bcl-2 protein in each group were observed by the sections were stained with HE staining and immunohistochemistry. Enzyme linked immunosorbent assay (ELISA) was used to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat. Results: compared with the control group, the blood glucose of the model group was increased significantly ( P ＜0.01), and the blood insulin level was decreased significantly ( P ＜0.05); compared with the model group, the blood glucose values of Moringa oleifera groups were decreased significantly ( P ＜0.05, P ＜0.01), and the blood insulin levels of middle and high dose Moringa oleifera group were increased significantly ( P ＜0.05, P ＜0.01). There was no significant difference in FBG and INS among the three groups ( P ＞0.05). In Morris water maze test, compared with the model group, the latency of Moringa oleifera groups was significantly shorter ( P ＜0.05); the residence time in target quadrant of Moringa oleifera groups with different doses was significantly prolonged ( P ＜0.05). Compared with the model group, the contents of TNF - α, IL-6 and protein expression in low, medium and high dose groups of Moringa oleifera were decreased significantly ( P ＜0.05). HE staining and immunohistochemical staining results showed that Moringa oleifera medium dose group was positive, brown yellow, fine granular, compared with the model group. The number of neuronal apoptosis was significantly reduced in the middle dose group (53.21±7.19, P ＜0.01); the protein expressions of Bax, caspase-3 and the ratio of Bax/Bcl-2 in hippocampus were significantly decreased in the middle dose group ( P ＜0.05). Conclusion: The mechanisms of Moringa leaves on the cognitive dysfunction and apoptosis of hippocampal neurons may be related to regulating the protein expressions of Bax, Bcl-2 and Caspase-3, reducing the contents of inflammatory factors TNF-α and IL-6.

Microglial activation and neuroinflammation induced by amyloid β (Aβ) play pivotal roles in Alzheimer's disease (AD) pathogenesis. Astragaloside IV (AS-IV) is one of the major active compounds of the traditional Chinese medicine Astmgali Radix. It has been reported that AS-IV could protect against Aβ-induced neuroinflammation and cognitive impairment, but the underlying mechanisms need to be further clarified. In this study, the therapeutic effects of AS-IV were investigated in an oligomeric Aβ (oAβ) induced AD mice model. The effects of AS-IV on microglial activation, neuronal damage and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were further studied. Different doses of AS-IV were administered intragastrically once a day after intracerebroventricularly oAβ injection. Results of behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test revealed that AS-IV administration could significantly ameliorate oAβ-induced cognitive impairment in a dose dependent manner. Enzyme linked immunosorbent assay (ELISA) results showed that increased levels of reactive oxygen species (ROS), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 in hippocampal tissues induced by oAβ injection were remarkably inhibited after AS-IV treatment. OAβ induced microglial activation and neuronal damage was significantly suppressed in AS-IV-treated mice brain, observed in immunohistochemistry results. Furthermore, oAβ upregulated protein expression of NADPH oxidase subunits gp91phox, p47phox, p22phox and p67phox were remarkably reduced by AS-IV in Western blotting assay. These results revealed that AS-IV could ameliorate oAβ-induced cognitive impairment, neuroinflammation and neuronal damage, which were possibly mediated by inhibition of microglial activation and down-regulation of NADPH oxidase protein expression. Our findings provide new insights of AS-IV for the treatment of neuroinflammation related diseases such as AD.

Three major pillars of hippocampal function are spatial navigation 1 , Hebbian synaptic plasticity 2 and spatial selectivity 3 . The hippocampus is also implicated in episodic memory 4 , but the precise link between these four functions is missing. Here we report the multiplexed selectivity of dorsal CA1 neurons while rats performed a virtual navigation task using only distal visual cues 5 , similar to the standard water maze test of spatial memory 1 . Neural responses primarily encoded path distance from the start point and the head angle of rats, with a weak allocentric spatial component similar to that in primates but substantially weaker than in rodents in the real world. Often, the same cells multiplexed and encoded path distance, angle and allocentric position in a sequence, thus encoding a journey-specific episode. The strength of neural activity and tuning strongly correlated with performance, with a temporal relationship indicating neural responses influencing behaviour and vice versa. Consistent with computational models of associative and causal Hebbian learning 6,7 , neural responses showed increasing clustering 8 and became better predictors of behaviourally relevant variables, with the average neurometric curves exceeding and converging to psychometric curves. Thus, hippocampal neurons multiplex and exhibit highly plastic, task- and experience-dependent tuning to path-centric and allocentric variables to form episodic sequences supporting navigation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

As the relationship among diet, brain aging and memory is complex, it provides ample opportunity for research in multiple directions including behaviour, epigenetics and neuroplasticity. Nutritional deficiencies together with genetic and environmental factors are the major cause of many age-associated pathologies including memory loss. A compromised vitamin B 12 -folate status in older people is highly prevalent worldwide. Researchers have established a close association between the adequate level of B 12 -folate and the maintenance of cognitive brain functions. One of the main reasons for age-associated memory loss is downregulation of neuronal immediate early genes (nIEGs). Therefore, we hypothesize here that vitamin B 12 -folic acid supplementation in old mice can improve memory by altering the expression status of nIEGs. To check this, 72-week-old male Swiss albino mice were orally administered with 2 μg of vitamin B 12 and 22 μg of folic acid/mouse/day for eight weeks. Such supplementation improved recognition memory in old and altered the expression of nIEGs. The expression of nIEGs was further found to be regulated by changes in DNA methylation at their promoter regions and CREB phosphorylation (pCREB). In addition, Golgi-Cox staining showed significant improvement in dendritic length, number of branching points and spine density of hippocampal CA1 pyramidal neurons by B 12 -folic acid supplementation. Taken together, these findings suggest that vitamin B 12 -folic acid supplementation regulates nIEGs expression and improves dendritic arborization of hippocampal neurons and memory in old male mice., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gastrodin, which is extracted from the Chinese herbal medicine Gastrodia elata Blume, can ameliorate neurogenesis after cerebral ischemia. However, it's possible underlying mechanisms remain still elusive. PDE9-cGMP-PKG signaling pathway is involved in the proliferation of neural stem cells (NSCs) after cerebral ischemia. In this study, we investigated whether the beneficial effect of gastrodin on hippocampal neurogenesis after cerebral ischemia is correlated with the PDE9-cGMP-PKG signaling pathway. Bilateral common carotid artery occlusion (BCCAO) in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cultured hippocampal NSCs were used to mimic brain ischemic injury. The Morris water maze (MWM) test was executed to detect spatial learning and memory. Proliferation, differentiation, and mature neurons were examined using immunofluorescence. The survival and proliferation of NSCs were assessed by CCK-8 assay and BrdU immunofluorescence staining, respectively. ELISA and western blot were used to detect the level of the PDE9-cGMP-PKG signaling pathway. In BCCAO mice, administering gastrodin (50 and 100 mg/kg) for 14 d restored cognitive behaviors; meanwhile, neurogenesis in hippocampus was stimulated, and PDE9 was inhibited and cGMP-PKG was activated by gastrodin. Consistent with the results, administering gastrodin (from 0.01-1 μmol/L) for 48 h dose-dependently ameliorated the cell viability and promoted greatly the proliferation in primary hippocampal NSCs exposed to OGD/R. Gastrodin further decreased PDE9 activity and up-regulated cGMP-PKG level. KT5823, a PKG inhibitor, markedly abrogated the protective effects of gastrodin on OGD/R-injured NSCs, accompanied by the down-regulation of PKG protein expression, but had no effects on PDE9 activity and cGMP level. Gastrodin could accelerate hippocampal neurogenesis after cerebral ischemia, which is mediated, at least partly, by PDE9-cGMP-PKG signaling pathway., (Copyright © 2021. Published by Elsevier Ltd.)

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Multiple sclerosis (MS) is characterized by myelin sheath injury. A disintegrin and metalloprotease-17 (ADAM17), a disintegrin and metalloproteinase, is essential in regulating oligodendrocyte (OL) regeneration and remyelination under demyelinating conditions. iRhom1, a highly conserved inactive protease that belongs to the rhomboid family, is one of key regulators for ADAM17 maturation. However, it is unknown whether iRhom1 also plays a role in central neuron system myelination under demyelinating conditions like MS. In this study, we investigated the function of iRhom1/ADAM17 in cognitive capability in MS by establishing the mice with iRhom1 overexpression in the hippocampus., (© 2021 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

The generation of new neurons in the adult brain is a currently accepted phenomenon. Over the past few decades, the subventricular zone and the hippocampal dentate gyrus have been described as the two main neurogenic niches. Neurogenic niches generate new neurons through an asymmetric division process involving several developmental steps. This process occurs throughout life in several species, including humans. These new neurons possess unique properties that contribute to the local circuitry. Despite several efforts, no other neurogenic zones have been observed in many years; the lack of observation is probably due to technical issues. However, in recent years, more brain niches have been described, once again breaking the current paradigms. Currently, a debate in the scientific community about new neurogenic areas of the brain, namely, human adult neurogenesis, is ongoing. Thus, several open questions regarding new neurogenic niches, as well as this phenomenon in adult humans, their functional relevance, and their mechanisms, remain to be answered. In this review, we discuss the literature and provide a compressive overview of the known neurogenic zones, traditional zones, and newly described zones. Additionally, we will review the regulatory roles of some molecular mechanisms, such as miRNAs, neurotrophic factors, and neurotrophins. We also join the debate on human adult neurogenesis, and we will identify similarities and differences in the literature and summarize the knowledge regarding these interesting topics.

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp ® , Equicel ® and Equitamp ® , have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B 4 staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel ® led to a neutral pH, Tabotamp ® (pH 2.8) and Equitamp ® (pH 4.8) caused a significant reduction of pH ( p < 0.001). Equicel ® and Tabotamp ® increased cytotoxicity significantly in several cell lines ( p < 0.01). On OHSC, Tabotamp ® and Equicel ® led to a stronger and deeper damage to the neuronal tissue than Equitamp ® or gauze ( p < 0.01). Equicel ® increased strongly the number of microglia cells after 24 h ( p < 0.001). Microglia cells were not detectable after Tabotamp ® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel ® , Tabotamp ® or Equitamp ® for specific applications in distinct clinical settings depending on their localization or tissue properties.

Oxidative stress-mediated neuronal damage is associated with the pathogenesis and development of neurodegenerative diseases. Chrysanthemum indicum has antioxidant properties. However, the neuroprotective effects and the cellular mechanism of C. indicum ethanol extract (CIE) against oxidative damage in hippocampal neuronal cells have not been clearly elucidated. Therefore, this study investigated whether CIE has protective effects against hydrogen peroxide (H 2 O 2 )-induced oxidative toxicity in HT22 cells. CIE pretreatment significantly improved neuronal cell viability. Moreover, the formation of intracellular reactive oxygen species and apoptotic bodies, and mitochondrial depolarization were significantly reduced in HT22 cells with H 2 O 2 -induced oxidative toxicity. Furthermore, CIE increased the phosphorylation of tropomyosin-related kinase receptor B (TrkB), protein kinase B (Akt), cAMP response element-binding protein, the expression of brain-derived neurotrophic factor, antioxidant enzymes, and the nuclear translocation of nuclear factor erythroid 2-related factor 2 by activating the TrkB/Akt signaling pathway. In contrast, the addition of K252a, a TrkB inhibitor, or MK-2206, an Akt-selective inhibitor, reduced the neuroprotective and antioxidant effects of CIE. Taken together; CIE exhibits neuroprotective and antioxidant effects against oxidative damage. Therefore, it can be a potential agent for treating oxidative stress-related neurodegenerative diseases.

Dendritic spine enlargement by synaptic activation is thought to increase synaptic efficacy underlying learning and memory. This process requires forces generated by actin polymerization and actin-adhesion coupling (clutch coupling). Here, we describe a protocol to monitor actin filament retrograde flow and actin polymerization within spines using a standard epi-fluorescence microscope. In combination with chemical long-term potentiation, this protocol allows us to quantify clutch coupling efficiency and actin polymerization rate, which are essential variables for generating forces for activity-dependent spine enlargement. For complete details on the use and execution of this protocol, please refer to Kastian et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The selection of goal-directed behaviors is supported by neural circuits located within the frontal cortex. Frontal cortical afferents arise from multiple brain areas, yet the cell-type-specific targeting of these inputs is unclear. Here, we use monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting distinct classes of local inhibitory interneurons and excitatory projection neurons in mouse infralimbic frontal cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide receive a large proportion of inputs from the hippocampus, while interneurons expressing neuron-derived neurotrophic factor receive a large proportion of inputs from thalamic regions. A similar dichotomy is present among the four different excitatory projection neurons. These results show a prominent bias among long-range hippocampal and thalamic afferent systems in their targeting to specific sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs., (© 2021. The Author(s).)

Stem cells have the potential as a regenerative therapy for cerebral ischemia by improving functional outcomes. However, cell transplantation has some limitations, including a low rate of the grafted cell survival. There is still a major challenge of promoting the harmonious symbiosis between grafted cells and the host. Acupuncture can effectively improve the functional outcome after cerebral ischemia. The present study evaluated the therapeutic effects and explored the mechanism of combined medial ganglionic eminence (MGE) neural progenitors differentiated from human embryonic stem cells (hESCs) with electroacupuncture (EA) in a bilateral common carotid artery occlusion (2VO) rat model. The results showed that EA could promote the survival of the grafted MGE neural progenitors differentiated from hESCs and alleviate learning and memory impairment in rats with cerebral ischemia. This may have partially resulted from inhibited expression of TNF- α and IL-1 β and increased vascular endothelial growth factor (VEGF) expression and blood vessel density in the hippocampus. Our findings indicated that EA could promote the survival of the grafted MGE neural progenitors and enhance transplantation therapy's efficacy by promoting angiogenesis and inhibiting inflammation., Competing Interests: The authors have declared no conflict of interest., (Copyright © 2021 Juan Li et al.)

The medial entorhinal cortex (MEC)-hippocampal network plays a key role in the processing, storage, and recall of spatial information. However, how the spatial code provided by MEC inputs relates to spatial representations generated by principal cell assemblies within hippocampal subfields remains enigmatic. To investigate this coding relationship, we employed two-photon calcium imaging in mice navigating through dissimilar virtual environments. Imaging large MEC bouton populations revealed spatially tuned activity patterns. MEC inputs drastically changed their preferred spatial field locations between environments, whereas hippocampal cells showed lower levels of place field reconfiguration. Decoding analysis indicated that higher place field reliability and larger context-dependent activity-rate differences allow low numbers of principal cells, particularly in the DG and CA1, to provide information about location and context more accurately and rapidly than MEC inputs. Thus, conversion of dynamic MEC inputs into stable spatial hippocampal maps may enable fast encoding and efficient recall of spatio-contextual information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

GABA can depolarize immature neurons close to the action potential (AP) threshold in development and adult neurogenesis. Nevertheless, GABAergic synapses effectively inhibit AP firing in newborn granule cells of the adult hippocampus as early as two weeks post-mitosis. The underlying mechanisms are largely unclear. Here, we analyze GABAergic inputs in newborn hippocampal granule cells mediated by soma-targeting parvalbumin and dendrite-targeting somatostatin interneurons. Surprisingly, both interneuron subtypes activate α5-subunit-containing GABA A receptors (α5-GABA A Rs) in young neurons, showing a nonlinear voltage dependence with increasing conductance around the AP threshold. By contrast, in mature cells, parvalbumin interneurons mediate linear GABAergic synaptic currents lacking α5-subunits, while somatostatin interneurons continue to target nonlinear α5-GABA A Rs. Computational modeling shows that the voltage-dependent amplification of α5-GABA A R opening in young neurons is crucial for inhibition of AP firing to generate balanced and sparse firing activity, even with depolarized GABA reversal potential., Competing Interests: Declaration of interests M.-C.H. is an employee at F. Hoffmann-La Roche. M.L., J.M.S., and J.B. declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Membrane lipids and their metabolism have key functions in neurotransmission. Here we provide a quantitative lipid inventory of mouse and rat synaptic junctions. To this end, we developed a multiomics extraction and analysis workflow to probe the interplay of proteins and lipids in synaptic signal transduction from the same sample. Based on this workflow, we generate hypotheses about novel mechanisms underlying complex changes in synaptic connectivity elicited by environmental stimuli. As a proof of principle, this approach reveals that in mice exposed to an enriched environment, reduced endocannabinoid synthesis and signaling is linked to increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in a subset of Cannabinoid-receptor 1 positive synapses. This mechanism regulates synaptic strength in an input-specific manner. Thus, we establish a compartment-specific multiomics workflow that is suitable to extract information from complex lipid and protein networks involved in synaptic function and plasticity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Angelica sinensis (Oliv.) Diels (ASD), commonly known as Dang Gui, is a popular Chinese herb that has long been used to treat ischemic stroke. However, the effects of ASD in chronic cerebral ischemia and its underlying mechanisms still remain unclear., Aim of the Study: This study aimed to determine the effects of the ASD extract on hippocampal neuronal survival at 28 d after transient global cerebral ischemia (GCI) and to investigate the precise mechanisms underlying the p38 mitogen-activated protein kinase (MAPK)-related signaling pathway's involvement in hippocampal neurogenesis., Materials and Methods: Rats underwent 25 min of four-vessel occlusion. The ASD extract was intragastrically administered at doses of 0.25 g/kg (ASD-0.25 g), 0.5 g/kg (ASD-0.5 g), 1 g/kg (ASD-1 g), 1 g/kg after dimethyl sulfoxide administration (D + ASD-1 g), or 1 g/kg after SB203580 (a p38 MAPK inhibitor) administration (SB + ASD-1 g) at 1, 3, 7, 10, 14, 17, 21, and 24 d after transient GCI., Results: ASD-0.5 g, ASD-1 g, and D + ASD-1 g treatments had the following effects: upregulation of bromodeoxyuridine (BrdU) and Ki67 expression, and BrdU/neuronal nuclei (NeuN) and Ki67/nestin co-expression in the hippocampal dentate gyrus (DG); upregulation of microtubule-associated protein 2/NeuN co-expression, and NeuN and glial fibrillary acidic protein (GFAP) expression, and downregulation of tumor necrosis factor-α/GFAP co-expression in the hippocampal CA1 region; upregulation of phospho-p38 MAPK (p-p38 MAPK), phospho-cAMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor A (VEGF-A) expression in the hippocampus. SB + ASD-1 g treatment abrogated the effects of ASD-1 g on the expression of these proteins., Conclusions: ASD-0.5 g and ASD-1 g treatments promotes neuronal survival by enhancing hippocampal neurogenesis. The effects of the ASD extract on astrocyte-associated hippocampal neurogenesis and dendritic growth are caused by the activation of p38 MAPK-mediated CREB/BDNF, GDNF, and VEGF-A signaling pathways in the hippocampus at 28 d after transient GCI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present study aims to investigate the protective effects of N -(3-methoxybenzyl)-(9 Z ,12 Z ,15 Z )-octadecatrienamide (M 18:3) on corticosterone-induced neurotoxicity. A neurotoxic model was established by subcutaneous injection of corticosterone (40 mg per kg bw) for 21 days. Depressive behaviors (the percentage of sucrose consumption, the immobility time in the forced swimming test, and the total distance in the open field test) were observed. The levels of the brain-derived neurotrophic factor, the contents of tumor necrosis factor-α and interleukin-6, and the numbers of positive cells of doublecortin and bromodeoxyuridine in the hippocampus were measured. The density of hippocampal neurons was calculated. The morphological changes of hippocampal neurons (the density of dendritic spines, the dendritic length, and the area and volume of dendritic cell bodies) were observed. The expression levels of synaptophysin, synapsin I, and postsynaptic density protein 95 were measured. Behavioral experiments showed that M 18:3 (5 and 25 mg per kg bw) could remarkably improve the depressive behaviors. The enzyme-linked immunosorbent assay showed that M 18:3 could considerably reduce hippocampal neuroinflammation and increase hippocampal neurotrophy. Nissl staining showed that M 18:3 could remarkably improve the corticosterone-induced decrease in the hippocampal neuron density. Immunofluorescence analysis showed that M 18:3 could considerably promote hippocampal neurogenesis. Golgi staining showed that M 18:3 could remarkably improve the corticosterone-induced changes in the hippocampal dendritic structure. Western blotting showed that M 18:3 could considerably increase the expression levels of synaptic-structure-related proteins in the hippocampus. In conclusion, the protective effects of M 18:3 may be attributed to the anti-inflammatory, neurotrophic, and synaptic protection properties.

Physiological brain aging is characterized by gradual, substantial changes in cognitive ability, accompanied by chronic activation of the neural immune system. This form of inflammation, termed inflammaging, in the central nervous system is primarily enacted through microglia, the resident immune cells. The endocannabinoid system, and particularly the cannabinoid receptor 2 (CB 2 R), is a major regulator of the activity of microglia and is upregulated under inflammatory conditions. Here, we elucidated the role of the CB 2 R in physiological brain aging. We used CB 2 R -/- mice of progressive ages in a behavioral test battery to assess social and spatial learning and memory. This was followed by detailed immunohistochemical analysis of microglial activity and morphology, and of the expression of pro-inflammatory cytokines in the hippocampus. CB 2 R deletion decreased social memory in young mice, but did not affect spatial memory. In fact, old CB 2 R -/- mice had a slightly improved social memory, whereas in WT mice we detected an age-related cognitive decline. On a cellular level, CB 2 R deletion increased lipofuscin accumulation in microglia, but not in neurons. CB 2 R -/- microglia showed an increase of activity markers Iba1 and CD68, and minor upregulation in tnfa and il6 expression and downregulation of ccl2 with age. This was accompanied by a change in morphology as CB 2 R -/- microglia had smaller somas and lower polarity, with increased branching, cell volume, and tree length. We present that CB 2 Rs are involved in cognition and age-induced microglial activity, but may also be important for microglial activation itself.

With rates of psychiatric illnesses such as depression continuing to rise, additional preclinical models are needed to facilitate translational neuroscience research. In the current study, the raccoon (Procyon lotor) was investigated due to its similarities with primate brains, including comparable proportional neuronal densities, cortical magnification of the forepaw area, and cortical gyrification. Specifically, we report on the cytoarchitectural characteristics of raccoons profiled as high, intermediate, or low solvers in a multiaccess problem-solving task. Isotropic fractionation indicated that high-solvers had significantly more cells in the hippocampus (HC) than the other solving groups; further, a nonsignificant trend suggested that this increase in cell profile density was due to increased nonneuronal (e.g., glial) cells. Group differences were not observed in the cellular density of the somatosensory cortex. Thionin-based staining confirmed the presence of von Economo neurons (VENs) in the frontoinsular cortex, although no impact of solving ability on VEN cell profile density levels was observed. Elongated fusiform cells were quantified in the HC dentate gyrus where high-solvers were observed to have higher levels of this cell type than the other solving groups. In sum, the current findings suggest that varying cytoarchitectural phenotypes contribute to cognitive flexibility. Additional research is necessary to determine the translational value of cytoarchitectural distribution patterns on adaptive behavioral outcomes associated with cognitive performance and mental health., (© 2021 Wiley Periodicals LLC.)