Contextual Fear Conditioning Test Research Articles

Phenotypic Insights Into Anti-IgLON5 Disease in IgLON5-Deficient Mice.

Anti-IgLON5 disease is an autoimmune neurodegenerative disorder characterized by various phenotypes, notably sleep and movement disorders and tau pathology. Although the disease is known to be associated with the neuronal cell adhesion protein IgLON5, the physiologic function of IgLON5 remains elusive. There are conflicting views on whether autoantibodies cause loss of function, activation of IgLON5, or inflammation-associated neuronal damage, ultimately leading to the disease. We generated IgLON5 knockout (-/-) mice to investigate the functions of IgLON5 and elucidate the pathomechanism of anti-IgLON5 disease. IgLON5 knockout (-/-) mice underwent behavioral tests investigating motor function, psychiatric function (notably anxiety and depression), social and exploratory behaviors, spatial learning and memory, and sensory perception. Histologic analysis was conducted to investigate tau aggregation in mice with tauopathy. IgLON5-/- mice had poorer performance in the wire hang and rotarod tests (which are tests for motor function) than wild-type mice. Moreover, IgLON5-/- mice exhibited decreased anxiety-like behavior and/or hyperactivity in behavior tests, including light/dark transition test and open field test. IgLON5-/- mice also exhibited poorer remote memory in the contextual fear conditioning test. However, neither sleeping disabilities assessed by EEG nor tau aggregation was detected in the knockout mice. These results suggest that IgLON5 is associated with activity, anxiety, motor ability, and contextual fear memory. Comparing the various phenotypes of anti-IgLON5 disease, anti-IgLON5 disease might partially be associated with loss of function of IgLON5; however, other phenotypes, such as sleep disorders and tau aggregation, can be caused by gain of function of IgLON5 and/or neuronal damage due to inflammation. Further studies are needed to elucidate the role of IgLON5 in the pathogenesis of anti-IgLON5 diseases.

Astrocytic HILPDA promotes lipid droplets generation to drive cognitive dysfunction in mice with sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is manifested as a spectrum of disturbed cerebral function ranging from mild delirium to coma. However, the pathogenesis of SAE has not been clearly elucidated. Astrocytes play important roles in maintaining the function and metabolism of the brain. Most recently, it has been demonstrated that disorders of lipid metabolism, especially lipid droplets (LDs) dyshomeostasis, are involved in a variety of neurodegenerative diseases. The aim of this study was to investigate whether LDs are involved in the underlying mechanism of SAE. The open field test, Y-maze test, and contextual fear conditioning test (CFCT) were used to test cognitive function in SAE mice. Lipidomics was utilized to investigate alterations in hippocampal lipid metabolism in SAE mice. Western blotting and immunofluorescence labeling were applied for the observation of related proteins. In the current study, we found that SAE mice showed severe cognitive dysfunction, including spatial working and contextual memory. Meanwhile, we demonstrated that lipid metabolism was widely dysregulated in the hippocampus by using lipidomic analysis. Furthermore, western blotting and immunofluorescence confirmed that LDs accumulation in hippocampal astrocytes was involved in the pathological process of cognitive dysfunction in SAE mice. We verified that LDs can be inhibited by specifically suppress hypoxia-inducible lipid droplet-associated protein (HILPDA) in astrocytes. Meanwhile, cognitive dysfunction in SAE was ameliorated by reducing A1 astrocyte activation and inhibiting presynaptic membrane transmitter release. The accumulation of astrocytic lipid droplets plays a crucial role in the pathological process of SAE. HILPDA is an attractive therapeutic target for lipid metabolism regulation and cognitive improvement in septic patients.

Inhibition of glutamatergic neurons in the dorsomedial periaqueductal gray alleviates excessive defensive behaviors of mice with post-traumatic stress disorder

To investigate the role of glutamatergic neurons in the dorsomedial periaqueductal grey (dmPAG) in regulating excessive defensive behaviors in mice with post-traumatic stress disorder (PTSD). Eight-week-old male C57BL/6 mice were subjected to stereotactic injections of different recombinant adeno- associated viral vectors (rAAV2/9-CaMKII-mCherry, rAAV2/9-CaMKII-hM3Dq-mCherry and rAAV2/9-CaMKII-hM4Di-mCherry) into the bilateral dmPAG for chemogenetic activation or inhibition of the glutamatergic neurons, followed 2 weeks later by PTSD modeling by single prolonged stress. The looming test, response to whisker stimulation test and contextual fear conditioning (CFC) test were used to observe changes in defensive behaviors of the PTSD mice. The activity of glutamatergic neurons in the dmPAG were observed using immunofluorescence staining. Compared with the control mice, the mouse models of PTSD showed a shortened latency of flights with increased time spent in the nest, response scores of defensive behaviors and freezing time (all P<0.01). Immunofluorescence staining revealed significantly increased c-fos-positive glutamatergic neurons in the dmPAG of PTSD mice with defensive behaviors. Activation of the glutamatergic neurons in the dmPAG (in PTSD hM3Dq group) did not cause significant changes in the latency of flights or time in nest but obviously increased response scores of defensive behaviors and freezing time of the mice, whereas inhibiting the glutamatergic neurons in the dmPAG (in PTSD hM4Di group) caused the reverse changes and obviously alleviated defensive behaviors in the PTSD mice (P<0.05 or 0.01). Inhibiting the activity of glutamatergic neurons in the dmPAG can alleviate defensive behaviors in mice with PTSD.

HDAC9-mediated calmodulin deacetylation induces memory impairment in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and memory impairment. AD pathology involves protein acetylation. Previous studies have mainly focused on histone acetylation in AD, however, the roles of nonhistone acetylation in AD are less explored. The protein acetylation and expression levels were detected by western blotting and co-immunoprecipitation. The stoichiometry of acetylation was measured by home-made and site-specific antibodies against acetylated-CaM (Ac-CaM) at K22, K95, and K116. Hippocampus-dependent learning and memory were evaluated by using the Morris water maze, novel object recognition, and contextual fear conditioning tests. We showed that calmodulin (CaM) acetylation is reduced in plasma of AD patients and mice. CaM acetylation and its target Ca2+ /CaM-dependent kinase II α (CaMKIIα) activity were severely impaired in AD mouse brain. The stoichiometry showed that Ac-K22, K95-CaM acetylation were decreased in AD patients and mice. Moreover, we screened and identified that lysine deacetylase 9 (HDAC9) was the main deacetylase for CaM. In addition, HDAC9 inhibition increased CaM acetylation and CaMKIIα activity, and hippocampus-dependent memory in AD mice. HDAC9-mediated CaM deacetylation induces memory impairment in AD, HDAC9, or CaM acetylation may become potential therapeutic targets for AD.

Seizure-induced LIN28A disrupts pattern separation via aberrant hippocampal neurogenesis.

Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.

The delta opioid receptor agonist KNT-127 relieves innate anxiety-like behavior in mice by suppressing transmission from the prelimbic cortex to basolateral amygdala.

Excitatory projections from the prelimbic cortex (PL) to the basolateral nucleus of the amygdala (BLA) are implicated in the regulation of anxiety-like behaviors, and we previously demonstrated that anxiolytic-like effects of the selective delta-opioid receptor (DOP) agonist KNT-127 is involved in suppressing glutamate neurotransmission in the PL. Here, we investigated the mechanisms underlying the anxiolytic-like effect of KNT-127 in mice by combining optogenetic stimulation of the PL-BLA pathway with behavioral analyses. Four-week-old male C57BL/6J mice received bilateral administration of adeno-associated virus (AAV)2-CaMKIIa-hChR2(H134R)-enhanced yellow fluorescent protein (EYFP) into the PL to induce expression of the light-activated excitatory ionic channel ChR2. Subsequently, an optic fiber cannula connected to a wireless photo-stimulator was implanted into the BLA for optogenetic PL-BLA pathway stimulation. We evaluated innate anxiety using the elevated plus maze (EPM) and open field (OF) tests as well as learned anxiety using the contextual fear conditioning (CFC) test. Optogenetic activation of the PL-BLA pathway enhanced anxiety-like behaviors in the EPM and OF, while prior subcutaneous administration of KNT-127 (10 mg/kg) reduced this anxiogenic effect. In contrast, optogenetic activation of the PL-BLA pathway had no significant effect on conditioned fear. Our findings indicate that the PL-BLA circuit contributes to innate anxiety and that the anxiolytic-like effects of KNT-127 are mediated at least in part by suppression of PL-BLA transmission. The PL delta-opioid receptor may thus be an effective therapeutic target for anxiety disorders.

The Alzheimer multifactorial therapeutic agent E2F4DN prevents long‐term potentiation and cognitive loss and improves neuronal survival in homozygous 5xFAD mice

AbstractBackgroundDespite many decades of work, we still lack effective therapies against Alzheimer’s disease (AD), likely due to its complex etiology, which requires a multifactorial therapeutic approach. Among the variety of proposed etiological factors, synaptic failure has proof to be an early hallmarks of AD (Nat. Neurosci. 2016;19:443‐453), which precedes neuronal‐death (J. Neurosci. 2006;26:10129‐10140). We have recently shown, using both transgenic mice and gene therapy, that E2F4 is a multifactorial therapeutic target against AD (Neurotherapeutics 2021;18:2484‐2503, Mol. Neurobiol. 2022;59:3016‐3039), likely due to its homeostatic function (Int. J. Mol. Sci. 2022;23:12093). The phosphorylation of two conserved Thr residues of E2F4 is necessary to induce cognitive loss in AD (Neurotherapeutics 2021;18:2484‐2503). Therefore, we have developed a novel therapy consisting in neuronal expression of a dominant negative form of E2F4 (E2F4DN), which cannot be phosphorylated in the conserved Thr residues (Neurotherapeutics 2021;18:2484‐2503, Mol. Neurobiol. 2022;59:3016‐3039). In this work we wanted to verify whether the expression of E2F4DN could correct AD‐associated synaptic dysfunction and neuronal loss in 5xFAD mice, a known mouse model of AD.MethodWe expressed E2F4DN (intravenous administration of AAV‐E2F4DN) in 6 weeks old control and homozygous 5xFAD mice, and stereological, electrophysiological and behavioral studies were performed at 6 months. Cell counting was achieved by unbiased stereology in the CA1 hippocampal region. Cognition was analyzed through novel‐object location and contextual fear conditioning test. Synaptic function (basal synaptic transmission, presynaptic function and long‐term potentiation (LTP)) were measured by electrophysiological recordings of field excitatory postsynaptic potentials in hippocampal CA3‐CA1 synapses. Afterwards, the mechanism underlying memory recovery was evaluated analyzing protein levels of different subunits of the synaptic receptors AMPA and NMDA.ResultE2F4DN expression in neurons prevents neuronal death in the hippocampus (CA1) of homozygous 5xFAD mice, reaching similar values of cell number and CA1 volume to those observed in control mice. Moreover, E2F4DN prevents LTP loss leading to cognitive improvement, as 5xFAD mice treated with our therapy present a noticeable improvement hippocampal‐dependent memory. This effect correlates with the increase of key LTP regulators.ConclusionE2F4‐Based Gene Therapy improves neuronal survival, LTP and cognition, being a safe multifactorial approach for AD treatment.

Deletion of microRNA‐21 exacerbates multiple phenotypes associated with Alzheimer’s disease through the RECK and ADAM10 pathway in a mouse model of Aβ amyloidosis

AbstractBackgroundmicroRNAs have emerged as a new class of therapeutic targets that regulate the expression of target genes by binding their 3’‐untranslated regions. Growing evidence suggests that dysregulation of microRNAs may contribute to the onset and/or progression of various diseases. However, the functional and therapeutic implications of microRNA dysregulation in Alzheimer’s disease (AD) remain largely unclear. To gain insight into the roles of microRNAs in AD pathogenesis, we recently performed unbiased microRNA profiling with entorhinal cortices of AD and age‐matched non‐demented subjects. We found that miRNA‐21 (miR‐21) expression was markedly increased in AD patients. In addition, miR‐21 levels were increased in oligomeric amyloid‐beta (Aβ)‐treated neurons, suggesting that miR‐21 induction is directly associated with Aβ pathology. Therefore, we investigated the pathogenic/therapeutic potential of miR‐21 in AD.MethodTo investigate the role of miR‐21 in amyloid pathology in vivo, we crossed APP/PS1 mice with mir‐21−/− mice to generate APP/PS1;mir‐21+/+, APP/PS1;mir‐21+/− and, APP/PS1;mir‐21−/− mice. We evaluated the changes in the pathological features of AD, such as Aβ levels, amyloid plaques, and gliosis in these mice. Moreover, to investigate the effects of miR‐21 deletion on spatial learning and memory functions, we performed the Morris Water Maze (MWM) and Contextual fear conditioning (CFC) tests.ResultDeletion of miR‐21 increased the levels of insoluble and soluble Aβ40 and Aβ42 levels, as well as increased Aβ‐positive and X‐34‐positive plaque deposition in the cortex and hippocampus. CD45‐positive, IBA1‐positive microgliosis, and GFAP‐positive astrogliosis were also increased in the brain of APP/PS1;mir‐21+/− and APP/PS1;mir‐21−/− mice compared to APP/PS1;mir‐21+/+ mice. Mechanistic studies identified RECK as a potential target gene of miR‐21. miR‐21 deficiency upregulated the RECK protein level, leading to the inhibition of ADAM10 activity. This finding was also corroborated by the decrease in the soluble APPα level. Furthermore, we demonstrated that miR‐21 deficiency decreased spatial learning and contextual memory in MWM and CFC tests, respectively in APP/PS1 mice.ConclusionTaken together, our results demonstrated that miR‐21 may regulate multiple AD‐associated pathologies. Therefore, modulation of miR‐21 may represent a novel potential therapeutic strategy for AD.

PPARα agonist fenofibrate prevents postoperative cognitive dysfunction by enhancing fatty acid oxidation in mice.

Due to high rates of incidence and disability, postoperative cognitive dysfunction (POCD) currently receives a lot of clinical attention. Disturbance of fatty acid oxidation is a potential pathophysiological manifestation underlying POCD. Peroxisome proliferator-activated receptor α (PPARα) is a significant transcription factor of fatty acid oxidation that facilitates the transfer of fatty acids into the mitochondria for oxidation. The potential role of PPARα intervention in POCD warrants consideration. The present study is aimed to investigate whether PPARα agonist fenofibrate (FF) could protect long-term isoflurane anesthesia-induced POCD model and to explore the potential underlying function of fatty acid oxidation in the process. We established the POCD model via 6 h long-term isoflurane anesthesia in vivo with C57BL/6J mice and in vitro with N2a cells. Cells and mice were pretreated with PPARα agonist FF before anesthesia, after which fatty acid oxidation and cognitive function were assessed. The level of fatty acid oxidation-related proteins was determined using western blotting. The contextual fear conditioning test was utilized to evaluate mice's learning and memory. Our results showed that 6 h long-term isoflurane anesthesia induced contextual memory damage in mice, accompanied by decreases of fatty acid oxidation-related proteins (peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1A, and PPARα) both in the hippocampus of POCD mice and in N2a cells. In the N2a cell model, pretreatment of PPARα agonist FF led to the upregulation of fatty acid oxidation-related proteins. In vivo results showed that preconditioned FF reached similar effects. More crucially, FF has been shown to reduce cognitive damage in mice after long-term isoflurane anesthesia. Additionally, our data showed that after blocking fatty acid oxidation by Etomoxir, FF failed to protect cognitive function from long-term isoflurane anesthesia. Pretreatment of PPARα agonist FF can protect against long-term isoflurane anesthesia-induced POCD by enhancing fatty acid oxidation.

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.

Preventing Post-Traumatic Stress Disorder (PTSD) in rats with pulsed 810 nm laser transcranial phototherapy

Post-traumatic stress disorder (PTSD) is a debilitating condition that occurs following exposure to traumatic events. Current treatments, such as psychological debriefing and pharmacotherapy, often have limited efficacy and may result in unwanted side effects, making early intervention is a more desirable strategy. In this study, we investigated the efficacy of a single dose of pulsed (10 Hz) 810 nm laser-phototherapy (P-PT) as an early intervention for preventing PTSD-like comorbidities in rats induced by single inescapable electric foot shock following the single prolonged stress (SPS&S). As indicated by the results of the open filed test, elevated plus maze test, and contextual fear conditioning test, P-PT prevented the development of anxiety and freezing behaviors in rats exposed to the SPS&S. We also compared the effects of P-PT and continuous wave 810 nm laser-phototherapy (CW-PT) in preventing PTSD-like comorbidities in rats. The results revealed that P-PT was effective in preventing both freezing and anxiety behavior in stressed rats. In contrast, CW-PT only had a preventive effect on freezing behavior but not anxiety. Additionally, P-PT significantly reduced the c-fos expression in cingulate cortex area 1(Cg1) and infralimbic cortex (IL) of stressed rats, while CW-PT had no significant effects on c-fos expression. Taken together, our results demonstrate that P-PT is a highly effective strategy for preventing the occurrence of PTSD-like comorbidities in rats.

TAK1 Reduces Surgery-induced Overactivation of RIPK1 to Relieve Neuroinflammation and Cognitive Dysfunction in Aged Rats.

Postoperative cognitive dysfunction (POCD) is a common clinical complication in elderly patients, but its underlying mechanism remains unclear. Receptor-interacting protein kinase 1 (RIPK1), a key molecule mediating necroptosis and regulated by transforming growth factor β-activated kinase 1 (TAK1), was reported to be associated with cognitive impairment in several neurodegenerative diseases. This study was conducted to investigate the possible role of TAK1/RIPK1 signalling in POCD development following surgery in rats. Young (2-month-old) and old (24-month-old) Sprague-Dawley rats were subjected to splenectomy under isoflurane anaesthesia. The young rats were treated with the TAK1 inhibitor takinib or the RIPK1 inhibitor necrostatin-1 (Nec-1) before surgery, and old rats received adeno-associated virus (AAV)-TAK1 before surgery. The open field test and contextual fear conditioning test were conducted on postoperative day 3. The changes in TNF-α, pro-IL-1β, AP-1, NF-κB p65, pRIPK1, pTAK1 and TAK1 expression and astrocyte and microglia activation in the hippocampus were assessed. Old rats had low TAK1 expression and were more susceptible to surgery-induced POCD and neuroinflammation than young rats. TAK1 inhibition exacerbated surgery-induced pRIPK1 expression, neuroinflammation and cognitive dysfunction in young rats, and this effect was reversed by a RIPK1 inhibitor. Conversely, genetic TAK1 overexpression attenuated surgery-induced pRIPK1 expression, neuroinflammation and cognitive dysfunction in old rats. Ageing-related decreases in TAK1 expression may contribute to surgery-induced RIPK1 overactivation, resulting in neuroinflammation and cognitive impairment in old rats.

Lactate Improves Long-term Cognitive Impairment Induced By Repeated Neonatal Sevoflurane Exposures Through SIRT1-mediated Regulation of Adult Hippocampal Neurogenesis and Synaptic Plasticity in Male Mice.

Repeated neonatal exposures to sevoflurane induce long-term cognitive impairment that has been reported to have sex-dependent differences. Exercise promotes learning and memory by releasing lactate from the muscle. The study tested the hypothesis that lactate may improve long-term cognitive impairment induced by repeated neonatal exposures to sevoflurane through SIRT1-mediated regulation of adult hippocampal neurogenesis and synaptic plasticity. C57BL/6 mice of both genders were exposed to 3% sevoflurane for 2h daily from postnatal day 6 (P6) to P8. In the intervention experiments, mice received lactate at 1g/kg intraperitoneally once daily from P21 to P41. Behavioral tests including open field (OF), object location (OL), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function. The number of 5-Bromo-2'- deoxyuridine positive (BrdU+) cells and BrdU+/DCX+ (doublecortin) co-labeled cells, expressions of brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeletal-associated protein (Arc), early growth response 1 (Egr-1), SIRT1, PGC-1α and FNDC5, and long-term potentiation (LTP) were evaluated in the hippocampus. Repeated exposures to sevoflurane induced deficits in OL, NOR and contextual FC tests in male but not female mice. Similarly, adult hippocampal neurogenesis, synaptic plasticity-related proteins and hippocampal LTP were impaired after repeated exposures to sevoflurane in male but not female mice, which could rescue by lactate treatment. Our study suggests that repeated neonatal exposures to sevoflurane inhibit adult hippocampal neurogenesis and induce defects of synaptic plasticity in male but not female mice, which may contribute to long-term cognitive impairment. Lactate treatment rescues these abnormalities through activation of SIRT1.

Effect of 40 Hz light flicker on behaviors of adult C57BL/6J mice

40 Hz light flicker can activate multiple brain regions of wild-type mice. However, there are no systematic studies on the behavioral effects of 40 Hz light flicker on wild-type mice. Adult wild-type C57BL/6J mice were treated with 40 Hz light flicker (200 lx, 40 Hz, 1 h/day for 3 weeks) to evaluate its effects on several behaviors, including mood, locomotor activity, memory, social interaction, mechanical pain, and sense of smell. In the open field test, the elevated zero-maze test, forced swimming test, and tail suspension test, 40 Hz mice showed no anxiety and depression-like behaviors. In the rotarod test, no differences were found between the anti-fatigue ability and motor coordination ability. In memory-related tests, 40 Hz mice showed the short-term cognitive enhancement in the novel object recognition test. Interestingly, 40 Hz mice showed no enhanced the long-term memory performance in the contextual fear conditioning test, and tone-cued fear conditioning test. Besides, 40 Hz mice increased their exploration of social cues that were unfamiliar to them and differed significantly from their own experiences. In terms of sensory abilities, 40 Hz mice had unchanged pain sensitivity in the von Frey fiber test and significant enhancement in the olfactory ability in the food-seeking test. In conclusion, this 40 Hz light stimulation paradigm has high safety and can improve the specific behavioral ability, which provides a theoretical basis for the future use of 40 Hz light flicker as a disease prevention or treatment method.

Sanwei DouKou Decoction ameliorate Alzheimer disease by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway.

Sanwei DouKou decoction (SDKD) is a traditional Chinese medicine (TCM) prescription derived from the Tibetan medical book "Si Bu Yi Dian" and is clinically used for the treatment of Alzheimer's disease (AD). However, the potential mechanism of SDKD treatment for AD remains elusive. This study aims to explore the potential mechanism by which SDKD alleviates AD. Extracts of SDKD were identified with Gas chromatograph-mass spectrometer (GC-MS). 5×FAD mice were treated with SDKD for 8 weeks. The efficacy of SDKD against AD was evaluated by in-vivo experiments. Morris water maze and contextual fear conditioning tests were used to detect the learning and memory ability of mice. Hematoxylin-eosin staining (H&E) staining was used to observe the pathological changes of brain tissue. Immunohistochemistry was used to detect the positive expression of Nestin in hippocampus. In in-vitro experiments, the Cell Counting Kit 8 (CCK-8) technique was used to detect cell viability, the proliferation of neural stem cells was detected by immunofluorescence staining, the intracellular protein expression was detected by Western Blot. The results of this study suggested that SDKD may ameliorate AD. SDKD significantly shortened the escape latency of mice in the Morris water maze experiment, increased the number of times the mice crossed the target quadrant, and prolonged freezing time in the contextual fear memory experiment. SDKD also improved neuronal pathology in the hippocampus, decreased neuronal loss, and increased Nestin protein levels. Furthermore, in in-vitro experiments, SDKD could significantly increase Neural stem cells (NSCs) viability, promoted NSCs proliferation, and also effectively activated the Wnt/β-catenin signalling pathway, increased Wnt family member 3A (Wnt3a), β-catenin and CyclinD1 protein levels, activated the NSCs proliferation pathways in AD model mouse brain tissue. The present study demonstrated that sanwei doukou decoction can ameliorate AD by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway. Our observations justify the traditional use of SDKD for a treatment of AD in nervous system.

Chaperone-mediated autophagy (CMA) alleviates cognitive impairment by reducing neuronal death in sepsis-associated encephalopathy (SAE)

Sepsis-associated encephalopathy (SAE) is a common and severe complication of sepsis, which causes long-term neurological deficits, such as cognitive impairment. Despite extensive research, there is still lack of specific treatments for SAE. Chaperone-mediated autophagy (CMA), a selective type of autophagy, has been reported to be related to cognitive dysfunctions in many neurodegenerative diseases. The aim of this study was to investigate the alteration of CMA activity in the hippocampus of SAE mice and explore the neuroprotective effect of enhanced CMA. Cecal ligation and puncture (CLP) was conducted to induce SAE. In the contextual fear conditioning test, the ratio of freezing time of CLP mice significantly decreased compared with that of the mice in the Sham group, indicating cognitive impairment in SAE mice. The expression of lysosome-associated membrane protein type 2A (Lamp2a) and chaperone heat shock cognate 71 kDa protein (Hsc70), positive markers for CMA activity, decreased in hippocampal neurons of SAE mice. Although overexpression of Lamp2a in neurons via adeno-associated virus injection in the hippocampus had little effect on the mortality of septic mice, this intervention significantly alleviated the memory impairments in contextual fear conditioning test, Y-maze test and novel objective recognition test, and attenuated the neural death observed in SAE mice. We further demonstrated that the overexpression of Lamp2a in the hippocampus increased the expression of phosphorylated cyclic-AMP response element binding protein (p-CREB), brain-derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2), and suppressed the expression of cleaved caspase-3. Taken together, our study results suggested that the upregulation of CMA activity ameliorated cognitive impairments and neuron loss in SAE mice partially through the p-CREB-BDNF/Bcl-2 signaling pathways, providing a potential therapeutic target for SAE.

Cannabidiol modulates contextual fear memory consolidation in animals with experimentally induced type-1 diabetes mellitus.

In view of the neuroprotective characteristic of cannabidiol (CBD) and its beneficial action on aversive memory in non-diabetic animals, we aimed to investigate in animals with experimentally induced type-1 diabetes mellitus (T1DM) whether CBD treatment would be able to impair the contextual fear memory consolidation, its generalisation and whether the effect would be lasting. We also investigated the CBD effect on anxiety-like responses. After T1DM induction, animals received single or more prolonged treatment with CBD and were submitted to the contextual fear conditioning test. As expression of activity-regulated cytoskeletal-associated (Arc) protein is necessary for memory consolidation, we evaluated its expression in the dorsal hippocampus (DH). For evaluating anxiety-related responses, animals were submitted to the elevated plus maze test (EPMT), in which the time and number of entries in the open arms were used as anxiety index. A single injection of CBD impaired the contextual fear memory consolidation and its generalisation, which was evaluated by exposing the animal in a neutral context. This single injection was able to reduce the elevated expression of Arc in the DH from these animals. Interestingly, more prolonged treatment with CBD also impaired the persistence of context-conditioned fear memory and induced an anxiolytic-like effect, as the treated group spent more time in the open arms of the EPMT. CBD interferes with contextual fear memory and the dosage regimen of treatment seems to be important. Moreover, we cannot rule out the involvement of emotional aspects in these processes related to fear memory.

Trpc6 knockout improves behavioral dysfunction and reduces Aβ production by inhibiting CN-NFAT1 signaling in T2DM mice

As the prevalence of diabetes and health awareness increase, type 2 diabetes mellitus -associated cognitive dysfunction is receiving increasing attention. However, the pathogenesis is not entirely understood. Transient receptor potential cation channel 6 (TRPC6) is highly correlated with intracellular Ca2+ concentrations, and neuronal calcium overload is an important cause of cognitive dysfunction. In the present study, we investigated the effect and mechanism of Trpc6 knockout in high-fat diet and streptozotocin-induced T2DM mice. The body weight and fasting blood glucose were recorded during the experiment. Behavioral dysfunction was detected using the open field test (OFT), elevated plus maze (EPM), hole-board test (HBT), Morris water maze (MWM) test and contextual fear conditioning (CFC) test. Nissl and H&E staining were used to examine neuronal damage. Western blot, quantitative real-time polymerase chain reaction (q-PCR), and immunofluorescence were performed to detect amyloid beta protein (Aβ) deposition and related indicators of neurological impairments in the cerebral cortex and hippocampus. The results indicated that Trpc6 knockout inhibited body weight loss and fasting blood glucose increase, improved spontaneous activity, learning and memory dysfunction, and alleviated neuroinflammation and neuronal damage in T2DM mice. The further results demonstrated that Trpc6 knockout decreased Aβ generation and deposition, and reduced the expressions of inflammasome-related proteins in T2DM mice. In addition, Trpc6 knockout inhibited intracellular calcium overload in diabetic mice and primary cultured hippocampal neurons, which in turn suppressed CN and NFAT1 expression. These data suggest that Trpc6 knockout may inhibit the CN-NFAT1 signaling pathway by decreasing intracellular calcium overload in the brain of T2DM mice, which consequently reduce Aβ deposition and neuroinflammation, and ultimately delay the development of T2DM-associated cognitive dysfunction.

A decrease in NR2B expression mediated by DNA hypermethylation induces perioperative neurocognitive disorder in aged mice.

This study was designed to investigate the role of NR2B and the contribution of DNA methylation to NR2B expression in the pathogenesis of PND. Eighteen-month-old C57BL/6J mice were subjected to experimental laparotomy under 1.4% isoflurane anesthesia. Hippocampus-dependent learning and memory were evaluated by using the Barnes maze and contextual fear conditioning tests. The protein and mRNA expression levels of NR2B were evaluated by western blotting and qRT-PCR respectively, and the methylation of the NR2B gene was examined by using targeted bisulfite sequencing. Long-term synaptic plasticity (LTP) was measured by electrophysiology. Mice that underwent laparotomy exhibited hippocampus-dependent cognitive deficits accompanied by decreased NR2B expressions and LTP deficiency. The overexpression of NR2B in the dorsal hippocampus could improve learning and memory in mice subjected to laparotomy. In particular, the decreased NR2B expressions induced by laparotomy was attributed to the NR2B gene hypermethylation. Preoperative administration of S-adenosylmethionine (SAM) could hypomethylate the NR2B gene, upregulate NR2B expression and improve LTP, exerting a dose-dependent therapeutic effect against PND. Moreover, inhibiting NR2B abrogated the benefits of SAM pretreatment. Laparotomy cause hippocampus-dependent cognitive decline by hypermethylating the NR2B gene, allowing us to understand the pathogenesis of PND in an epigenetic landscape.

PERK-STING-RIPK3 pathway facilitates cognitive impairment by inducing neuronal necroptosis in sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a common but serious complication in septic survivors and often causes long-term cognitive impairments. The role of RIPK3-participated necroptosis in SAE remains obscured. STING is a key molecule in regulating necroptosis and apoptosis. However, there is uncertainty as to the mechanisms of STING in CLP-induced SAE. The aim of this study was to investigate whether STING is involved in the underlying mechanism of SAE. The contextual fear conditioning test (CFCT) assesses cognitive impairment. A transmission electron microscope (TEM) was used to notice the necroptosis. Western blotting and immunofluorescence labeling were applied for the observation of related proteins. The phosphorylated STING in the hippocampal neuron of SAE mice was significantly elevated. Knocking down STING inhibited necroptosis and attenuated cognitive impairment in SAE mice. Moreover, RIPK3-/- mice had less cognitive deficit in the SAE model. However, STING overexpression did not deteriorate cognitive impairment in RIPK3-/- mice with SAE, indicating that STING is upstream involved in necroptosis. Furthermore, PERK inhibition ameliorated cognitive deficits through a STING-dependent pathway in SAE mice. PERK-STING-RIPK3 pathway facilitates cognitive impairment by inducing neuronal necroptosis in the pathology of SAE, which provided a new therapeutic target in SAE treatment.