Spine Density In Hippocampal Neurons Research Articles

Saikosaponin a activates tet1/dll3/notch1 signalling and promotes hippocampal neurogenesis to improve depression-like behavior in mice

Ethnopharmacological relevanceRadix Bupleuri, also named “Chaihu” in Chinese, is a substance derived from the dry roots of Bupleurum chinense DC. [Apiaceae] and Bupleurum scorzonerifolium Willd. [Apiaceae]. Radix Bupleuri was initially recorded as a medicinal herb in Shen Nong Ben Cao Jing, the earliest monograph concerning traditional Chinese medicine (TCM). Ever since, Radix Bupleuri has been broadly used to alleviate exterior syndrome, disperse heat, modulate the liver-qi, and elevate yang-qi in TCM. Radix Bupleuri has also been utilized as an important component in Xiaoyaosan, a classical formula for relieving depression, which was originated from the famous Chinese medical book called “Tai Ping Hui Min He Ji Ju Fang” in Song Dynasty. Currently, many valuable pharmacological effects of Radix Bupleuri have been explored, such as antidepressant, neuroprotective activities, antiinflammation, anticancer, immunoregulation, etc. Former studies have illustrated that Saikosaponin A (SSa), one of the primary active components of Radix Bupleuri, possesses potential antidepressant properties. However, the underlying mechanisms still remain unknown. Aim of the studyWe used a chronic social defeat stress (CSDS) mouse model to explore the ameliorative effects and potential mechanisms of SSa in depressive disorder in vivo. Materials and methodsThe CSDS mouse model was established and mice underwent behavioral studies using assays such as the social interaction test (SIT), sucrose preference test (SPT), forced-swim test (FST), tail suspension test (TST), and open field test (OFT). Western blotting, immunofluorescence, and Golgi staining were performed to investigate signaling pathway activity, and alterations in synaptic spines in the hippocampus. To model the anticipated interaction between SSa and Tet1, molecular docking and microscale thermophoresis (MST) techniques were employed. Finally, sh-RNA Tet1 was employed for validation via lentiviral transfection in CSDS mice to confirm the requirement of Tet1 for SSA efficacy. ResultsSSa dramatically reduced depressed symptoms, boosted the expression of Tet1, Notch, DLL3, and BDNF, encouraged hippocampus development, and enhanced the dendritic spine density of hippocampal neurons. In contrast, Tet1 knockdown in CSDS mice dampened the beneficial effects of SSa on depressive symptoms. ConclusionsTherefore, our results suggest that SSa significantly activates the Tet1/Notch/DLL3 signaling pathways and promotes hippocampal neurogenesis to exert antidepressant effects in the CSDS mouse model in vivo. The present results also provide new insight into the importance of the Tet1/DLL3/Notch pathways as potential targets for novel antidepressant development.

14-3-3ζ Plays a key role in the modulation of neuroplasticity underlying the antidepressant-like effects of Zhi-Zi-Chi-Tang

BackgroundZhi-Zi-Chi-Tang (ZZCT) is an effective traditional Chinese medicinal formula. ZZCT has been used for the treatment of depression for centuries. Its clinical efficacy in relieving depression has been confirmed. However, the molecular mechanisms of ZZCT regarding neuroplasticity in the pathogenesis of depression have not yet been elucidated. PurposeThe present study aimed to examine the effects of ZZCT on neuroplasticity in mice exposed to chronic unpredictable mild stress (CUMS), and to explore the underlying molecular mechanisms. MethodsFor this purpose, a murine model of depression was established using the CUMS procedure. Following the intragastric administration of ZZCT or fluoxetine, classic behavioral experiments were performed to observe the efficacy of ZZCT as an antidepressant. Immunofluorescence was used to label and quantify microtubule-associated protein (MAP2) and postsynaptic density protein (PSD95) in the hippocampus. Golgi staining was applied to visualize the dendritic spine density of neurons in the hippocampi. Isolated hippocampal slices were prepared to induce long-term potentiation (LTP) in the CA1 area. The hippocampal protein expression levels of glycogen synthase kinase-3β (GSK-3β), p-GSK-3β (Ser9), cAMP response element binding protein (CREB), p-CREB (Ser133), brain-derived neurotrophic factor (BDNF) and 14–3–3ζ were detected using western blot analysis. The interaction of 14–3–3ζ and p-GSK-3β (Ser9) was examined using co-immunoprecipitation. LV-shRNA was used to knockdown 14–3–3ζ by an intracerebroventricular injection. ResultsZZCT (6 g/kg) and fluoxetine (20 mg/kg) alleviated depressive-like behavior, restored hippocampal MAP2+ PSD95+ intensity, and reversed the dendritic spine density of hippocampal neurons and LTP in the CA1 region of mice exposed to CUMS. Both low and high doses of ZZCT (3 and 6 g/kg) significantly promoted the binding of 14–3–3ζ to p-GSK-3β (Ser9) in the hippocampus, and ZZCT (6 g/kg) significantly promoted the phosphorylation of GSK-3β Ser9 and CREB Ser133 in the hippocampus. ZZCT (3 and 6 g/kg) upregulated hippocampal BDNF expression in mice exposed to CUMS. LV-sh14–3–3ξ reduced the antidepressant effects of ZZCT. ConclusionZZCT exerted antidepressant effects against CUMS-stimulated depressive-like behavior mice. The knockdown of 14–3–3ζ using lentivirus confirmed that 14–3–3ζ was involved in the ZZCT-mediated antidepressant effects through GSK-3β/CREB/BDNF signaling. On the whole, these results suggest that the antidepressant effects of ZZCT are attributed to restoring damage by neuroplasticity enhancement via the 14–3–3ζ/GSK-3β/CREB/BDNF signaling pathway.

Toward Antifragility: Social Defeat Stress Enhances Learning and Memory in Young Mice Via Hippocampal Synaptosome Associated Protein 25

Social adversity not only causes severe psychological diseases but also may improve people’s ability to learn and grow. However, the beneficial effects of social adversity are often ignored. In this study, we investigated whether and how social adversity affects learning and memory in a mouse social defeat stress (SDS) model. A total of 652 mice were placed in experimental groups of six to 23 mice each. SDS enhanced spatial, novelty, and fear memory with increased synaptosome associated protein 25 (SNAP-25) level and dendritic spine density in hippocampal neurons among young but not middle-aged mice. Chemogenetic inhibition of hippocampal CaMK2A+ neurons blocked SDS-induced enhancement of learning or memory. Knockdown of SNAP-25 or blockade of N-methyl-D-aspartate (NMDA) receptor subunit GluN2B in the hippocampus prevented SDS-induced learning memory enhancement in an emotion-independent manner. These findings suggest that social adversity promotes learning and memory ability in youths and provide a neurobiological foundation for biopsychological antifragility.

Facial nerve axotomy induces morphological changes in hippocampal pyramidal neurons.

Facial nerve injury in rats have been widely used to study functional and structural changes that occur in the injured motoneurons and other central nervous system structures related with sensorimotor processing. A decrease in long-term potentiation of hippocampal CA3-to-CA1 commissural synapse has recently been reported related to this peripheral injury. Additionally, it has been found increased corticosterone plasmatic levels, impairment in spatial memory consolidation, and hippocampal microglial activation in animals with facial nerve axotomy. In this work, we analyzed the neuronal morphology of hippocampal CA1 and CA3 pyramidal neurons in animals with either reversible or irreversible facial nerve injury. For this purpose, brain tissues of injured animals sacrificed at different postlesion times, were stained with the Golgi-Cox method and compared with control brains. It was found that both reversible and irreversible facial nerve injury-induced significant decreases in dendritic tree complexity, dendritic length, branch points, and spine density of hippocampal neurons. However, such changes' timing varied according to hippocampal area (CA1 vs. CA3), dendritic area (apical vs. basal), and lesion type (reversible vs. irreversible). In general, the observed changes were transient when animals had the possibility of motor recovery (reversible injury), but perdurable if the recovery from the lesion was impeded (irreversible injury). CA1 apical and CA3 basal dendritic tree morphology were more sensible to irreversible injury. It is concluded that facial nerve injury induced significant changes in hippocampal CA1 and CA3 pyramidal neurons morphology, which could be related to LTP impairments and microglial activation in the hippocampal formation, previously described.

Hippocampal semaphorin 3B improves depression-like behaviours in mice by upregulating synaptic plasticity and inhibiting neuronal apoptosis.

Depression is a global health problem, and there is a pressing need for a better understanding of its pathogenesis. Semaphorin 3B (Sema 3B) is an important axon guidance molecule that is primarily expressed in neurons and contributes to synaptic plasticity. Our previous studies using a high-throughput microarray assay suggested that Sema 3B expression was tremendously decreased during the development of depression, but the specific role and mechanisms of Sema 3B in depression are still unknown. Herein, we report that levels of Sema 3B protein are decreased in the hippocampus and serum of chronic mild stress (CMS)-treated mice. Increasing the levels of Sema 3B, either by injecting AAV-Sema 3B into the hippocampus or by injecting recombinant Sema 3B protein into the lateral ventricles, alleviated CMS-induced depression-like behaviours and enhanced the resistance to acute stress by increasing dendritic spine density in hippocampal neurons. In contrast, interfering with the function of Sema 3B by injecting anti-Sema 3B antibody into the lateral ventricles decreased the resistance to acute stress. In vitro, corticosterone (CORT) treatment decreased the survival rate and protein levels of Sema 3B and synapse-associated proteins in HT22 cells. Overexpression of Sema 3B improved the decreased survival rate caused by CORT by inhibiting apoptosis and increasing levels of synaptic-associated proteins, and knockdown of Sema 3B reduces the cellular resistance to CORT and the levels of synapse-associated proteins. These findings represent the first evidence for the neuroprotective mechanism of Sema 3B against stresses, suggesting that Sema 3B could be a promising novel target for the prevention and treatment of depression.

Seasonal differences in the morphology and spine density of hippocampal neurons in wild ground squirrels.

Seasonally reproducing small mammals often undergo changes in brain anatomy throughout the year. Much of the research on this seasonal neuroplasticity has focused on changes in hippocampus volume and neurogenesis, with relatively little attention paid to neuronal morphology. Here, we test for sex, season and sex-season interaction effects on hippocampal neuron morphology and dendritic spine density in a seasonally reproducing rodent: Richardson's ground squirrel (Urocitellus richardsonii). We quantified the morphology and spine densities of Golgi-stained pyramidal neurons and granule cells in the hippocampus and tested for differences between sexes and seasons with generalized linear models. Although we found no significant sex differences or sex-season interaction effects on any of our morphological measurements, there were significant differences in neuron morphology and spine density between breeding and non-breeding seasons. In the non-breeding season, ground squirrels had CA1 neurons with longer basal dendrites with more branches than in the breeding season. Non-breeding season animals also had higher apical and basal dendrite spine density in CA1 and CA3 neurons than breeding-season animals. Conversely, the spine densities of CA1 somata and granule cells were higher in breeding than in non-breeding season. These differences in neuron morphology and spine density between breeding and non-breeding seasons likely arise from a combination of activity levels, stress hormones, and photoperiod. Although the functional implications of seasonal changes in hippocampal neuron morphology and spine density are uncertain, our data suggest that ground squirrels may be a good model for understanding seasonal neuroplasticity in mammals.

The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism.

RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we present an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species.

Impaired Cognitive Function and Hippocampal Changes Following Chronic Diazepam Treatment in Middle-Aged Mice.

Background: Gamma-aminobutyric acid (GABA) type A receptors are positively allosterically modulated by benzodiazepine binding, leading to a potentiated response to GABA. Diazepam (DZP, a benzodiazepine) is widely prescribed for anxiety, epileptic discharge, and insomnia, and is also used as a muscle relaxant and anti-convulsant. However, some adverse effects – such as tolerance, dependence, withdrawal effects, and impairments in cognition and learning – are elicited by the long-term use of DZP. Clinical studies have reported that chronic DZP treatment increases the risk of dementia in older adults. Furthermore, several studies have reported that chronic DZP administration may affect neuronal activity in the hippocampus, dendritic spine structure, and cognitive performance. However, the effects of chronic DZP administration on cognitive function in aged mice is not yet completely understood.Methods: A behavioral test, immunohistochemical analysis of neurogenic and apoptotic markers, dendritic spine density analysis, and long-term potentiation (LTP) assay of the hippocampal CA1 and CA3 were performed in both young (8 weeks old) and middle-aged (12 months old) mice to investigate the effects of chronic DZP administration on cognitive function. The chronic intraperitoneal administration of DZP was performed by implanting an osmotic minipump. To assess spatial learning and memory ability, the Morris water maze test was performed. Dendritic spines were visualized using Lucifer yellow injection into the soma of hippocampal neurons, and spine density was analyzed. Moreover, the effects of exercise on DZP-induced changes in spine density and LTP in the hippocampus were assessed.Results: Learning performance was impaired by chronic DZP administration in middle-aged mice but not in young mice. LTP was attenuated by DZP administration in the CA1 of young mice and the CA3 of middle-aged mice. The spine density of hippocampal neurons was decreased by chronic DZP administration in the CA1 of both young and middle-aged mice as well as in the CA3 of middle-aged mice. Neither neurogenesis nor apoptosis in the hippocampus was affected by chronic DZP administration.Conclusion: The results of this study suggest that the effects of chronic DZP are different between young and middle-aged mice. The chronic DZP-induced memory retrieval performance impairment in middle-aged mice can likely be attributed to decreased LTP and dendritic spine density in hippocampal neurons in the CA3. Notably, prophylactic exercise suppressed the adverse effects of chronic DZP on LTP and spine maintenance in middle-aged mice.

Prenatal exposure to koumine results in cognitive deficits and increased anxiety-like behavior in mice offspring

Koumine (KM) is a major alkaloid monomer in the traditional Chinese medicine herb Gelsemium elegans Benth that has exhibited therapeutic potential in clinical applications. However, the pharmacological toxicological mechanism of this drug has not been fully explored. The purpose of this study was to evaluate the impacts of KM administration at a therapeutic dose in offspring. On gestational day 0, mice were injected with KM once daily for 4 consecutive days. Male and female offspring were subjected to behavioral tests and neuropathological analyses from postnatal day 60. Prenatal KM exposure resulted in cognitive and memory impairments in the Morris water maze, Y-maze test, and novel object recognition test. The open field test and elevated plus maze test indicated that prenatal KM exposure induced anxiety-like behavior in offspring. Electrophysiological experiments demonstrated that KM exposure inhibited hippocampal long-term potentiation. Immunostaining for neurogenesis markers DCX and BrdU demonstrated that KM suppressed adult neurogenesis in the subgranular zone of the dentate gyrus. In addition, prenatal KM exposure induced a significant reduction in dendritic spine density in hippocampal neurons. Synaptic formation-related proteins were decreased in the KM group based on western blot. No sex differences in the effects of KM were observed. Collectively, our results indicate that prenatal KA exposure has detrimental neural effects on offspring. This study provides a preliminary preclinical toxicological assessment of the safety of KM use during pregnancy.

Langat virus infection affects hippocampal neuron morphology and function in mice without disease signs

BackgroundTick-borne encephalitis virus (TBEV) is an important human pathogen that can cause the serious illness tick-borne encephalitis (TBE). Patients with clinical symptoms can suffer from severe meningoencephalitis with sequelae that include cognitive disorders and paralysis. While less than 30% of patients with clinical symptoms develop meningoencephalitis, the number of seropositive individuals in some regions indicates a much higher prevalence of TBEV infections, either with no or subclinical symptoms. The functional relevance of these subclinical TBEV infections and their influence on brain functions, such as learning and memory, has not been investigated so far.MethodsTo compare the effect of low and high viral replication in the brain, wildtype and Irf-7−/− mice were infected with Langat virus (LGTV), which belongs to the TBEV-serogroup. The viral burden was analyzed in the olfactory bulb and the hippocampus. Open field, elevated plus maze, and Morris water maze experiments were performed to determine the impact on anxiety-like behavior, learning, and memory formation. Spine density of hippocampal neurons and activation of microglia and astrocytes were analyzed.ResultsIn contrast to susceptible Irf-7−/− mice, wildtype mice showed no disease signs upon LGTV infection. Detection of viral RNA in the olfactory bulb revealed CNS infections in wildtype and Irf-7−/− mice. Very low levels of viral replication were detectable in the hippocampus of wildtype mice. Although wildtype mice develop no disease signs, they showed reduced anxiety-like behavior and impaired memory formation, whereas Irf-7−/− mice were not affected. This impairment was associated with a significant decrease in spine density of neurons in the hippocampal CA1 region of wildtype mice. Microglia activation and astrogliosis were detected in the hippocampus.ConclusionIn this study, we demonstrate that subclinical infections by viruses from the TBEV-serogroup affected anxiety-like behavior. Virus replication in the olfactory bulb induced far-reaching effects on hippocampal neuron morphology and impaired hippocampus-dependent learning and memory formation.

Acute Pannexin 1 Blockade Mitigates Early Synaptic Plasticity Defects in a Mouse Model of Alzheimer's Disease.

Synaptic loss induced by soluble oligomeric forms of the amyloid β peptide (sAβos) is one of the earliest events in Alzheimer’s disease (AD) and is thought to be the major cause of the cognitive deficits. These abnormalities rely on defects in synaptic plasticity, a series of events manifested as activity-dependent modifications in synaptic structure and function. It has been reported that pannexin 1 (Panx1), a nonselective channel implicated in cell communication and intracellular signaling, modulates the induction of excitatory synaptic plasticity under physiological contexts and contributes to neuronal death under inflammatory conditions. Here, we decided to study the involvement of Panx1 in functional and structural defects observed in excitatory synapses of the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (Tg) mice, an animal model of AD. We found an age-dependent increase in the Panx1 expression that correlates with increased Aβ levels in hippocampal tissue from Tg mice. Congruently, we also observed an exacerbated Panx1 activity upon basal conditions and in response to glutamate receptor activation. The acute inhibition of Panx1 activity with the drug probenecid (PBN) did not change neurodegenerative parameters such as amyloid deposition or astrogliosis, but it significantly reduced excitatory synaptic defects in the AD model by normalizing long-term potentiation (LTP) and depression and improving dendritic arborization and spine density in hippocampal neurons of the Tg mice. These results suggest a major contribution of Panx1 in the early mechanisms leading to the synaptopathy in AD. Indeed, PBN induced a reduction in the activation of p38 mitogen-activated protein kinase (MAPK), a kinase widely implicated in the early neurotoxic signaling in AD. Our data strongly suggest that an enhanced expression and activation of Panx1 channels contribute to the Aβ-induced cascades leading to synaptic dysfunction in AD.

The endocannabinoid system regulates the moderate exercise-induced enhancement of learning and memory in mice.

Exercise has been reported to enhance cognitive functions via mechanism(s) yet to be fully understood. The endogenous cannabinoid system (ECS) is involved in regulating cognitive function, including learning and memory. This system may also be involved in enhancing learning and memory after exercise. The objective of this study is to explore whether and how ECS participates in the enhancement of learning and memory after exercise. In this study, a treadmill exercise training model was established. Wild-type C57BL/6J mice and those deficient in the cannabinoid receptor 1 (CB1R) coding gene, Cnr1, specifically in the glutamatergic neurons, γ-aminobutyric acid (GABA) neurons or glial cells were randomly grouped for 4 weeks' moderate treadmill exercise. The Morris water maze was used to evaluate the spatial learning and memory abilities of mice in each group. The expression of brain-derived neurotrophic factor (BDNF) and CB1R in hippocampus was detected by western blot. The dendritic spine density of pyramidal cells in the hippocampal CA1 region was analyzed by quantitative Golgi staining. This study consisted of eight single-factor inter-subject designs, and each batch of experiments was divided into two groups. Corresponding experimental items and data analysis were carried out according to the experimental objectives. CB1R antagonist administration or CB1R knockout in glutamatergic neurons eliminated the effect of exercise on learning and memory, and counteracted exercise-elicited upregulation of BDNF in the hippocampus; CB1R-specific knockout on GABAergic neurons and glial cells did not affect the moderate exercise-induced enhancement of learning and memory. In addition, the results of Golgi staining showed that exercise increased dendritic spine density in hippocampal neurons, which was abolished by specific CB1R depletion in glutamatergic neurons. The ECS, particularly CB1R signaling in glutamatergic neurons, mediates the enhancement of learning and memory by exercise, which involves increased BDNF production and dendritic spine density.

Pyk2 Signaling through Graf1 and RhoA GTPase Is Required for Amyloid-β Oligomer-Triggered Synapse Loss.

The intracellular tyrosine kinase Pyk2 (PTK2B) is related to focal adhesion kinase and localizes to postsynaptic sites in brain. Pyk2 genetic variation contributes to late onset Alzheimer's disease (AD) risk. We recently observed that Pyk2 is required for synapse loss and for learning deficits in a transgenic mouse model of AD. Here, we explore the cellular and biochemical basis for the action of Pyk2 tyrosine kinase in amyloid-β oligomer (Aβo)-induced dendritic spine loss. Overexpression of Pyk2 reduces dendritic spine density of hippocampal neurons by a kinase-dependent mechanism. Biochemical isolation of Pyk2-interacting proteins from brain identifies Graf1c, a RhoA GTPase-activating protein inhibited by Pyk2. Aβo-induced reductions in dendritic spine motility and chronic spine loss require both Pyk2 kinase and RhoA activation. Thus, Pyk2 functions at postsynaptic sites to modulate F-actin control by RhoA and regulate synapse maintenance of relevance to AD risk.SIGNIFICANCE STATEMENT Genetic variation at the Pyk2 locus is a risk for Alzheimer's disease. We have observed that Pyk2 is required for AD transgenic synapse loss and memory dysfunction. However, the cellular and biochemical basis for Pyk2 function related to AD is not defined. Here, we show that brain Pyk2 interacts with the RhoGAP protein Graf1 to alter dendritic spine stability via RhoA GTPase. Amyloid-β oligomer-induced dendritic spine loss requires the Pyk2/Graf1 pathway.

Adolescence benzo[a]pyrene treatment induces learning and memory impairment and anxiolytic like behavioral response altering neuronal morphology of hippocampus in adult male Wistar rats

Exposure to benzo[a]pyrene (B[a]P), a prototype of polycyclic aromatic hydrocarbons (PAHs) easily cross blood brain barrier (BBB) and is associated with impaired learning and memory in adult rats. However, there is no symmetric study reported on association between B[a]P exposure during the early development and hippocampal dendritic architecture causing behavioral changes like learning and memory deficit of adult rats. We investigated a fourteen day consecutive B[a]P administration, intraperitonial (i.p.), with two different doses (0.1 and 1μM) during early adolescence at PND30-44 and learning behavior assessed between PND 45-60 in adult male rats. The anxiolytic like behavioural analysis was done by LDPT. Depressive like behaviour was estimated through sucrose preference and learning and memory by T-maze. After B[a]P administration oxidative stress markers like glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), reduced (GSH) and oxidized glutathione (GSSG) were evaluated. To parallel these behavioral and antioxidant level changes to alteration in dendritic morphology, Golgi-Cox staining was performed in the hippocampus. Our study showed anxiolytic like behavioral response with significant increase in time spent in light zone and significant (p < 0.05) decrease in preference for sucrose and a reduction in percentage of spontaneous responses in T-maze test in B[a]P administered group as compared to vehicle control. B[a]P exposed male rats showed significant increase in GST activity (p < 0.05) and concentration of GSSG with a decay in GSH, GPx and GR in both the groups as compared to control. B[a]P administered rats showed significant loss in total dendritic length and number (28%) with reduced spine density (18%) in both higher and lower doses. These results suggested that B[a]P administration can be associated with an increase ROS production showing altered antioxidant defence system through glutathione biosynthesis and causing profound alterations in dendritic length and spine density of hippocampal neurons leading towards learning and memory deficits in adult rats.

Sleep and circadian abnormalities precede cognitive deficits in R521C FUS knockin rats.

Mutations in fused in sarcoma (Fus) cause familial amyotrophic lateral sclerosis (ALS) and occasionally frontotemporal dementia. Here we report the establishment and characterization of a novel knockin (KI) rat model expressing a Fus point mutation (R521C) via CRISPR/Cas9. The mutant animals developed adult-onset learning and memory behavioral deficits, with reduced spine density in hippocampal neurons. Remarkably, sleep-wake cycle and circadian abnormalities preceded the onset of cognitive deficit. RNA-seq study further demonstrated altered expression of some key sleep and circadian regulators, such as orexin/hypocretin receptor type 2 and casein kinase 1 epsilon, in the mutant rats. Therefore, we have established a rodent model expressing physiological level of a pathogenic mutant FUS, and we found cognitive impairment as a main behavioral deficit at mid age. Furthermore, we have revealed a new role of FUS in sleep and circadian regulation and demonstrated that functional change in FUS could cause sleep-wake and circadian disturbance as early symptoms.

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/−) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/− mice. We found that Cdkl5 +/− mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/− mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/− mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations

Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV–neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.

FOXO in Neural Cells and Diseases of the Nervous System.

The evolutionarily conserved FOXO family of transcription factors has emerged as a significant arbiter of neural cell fate and function in mammals. From the neural stem cell (NSC) state through mature neurons under both physiological and pathological conditions, they have been found to modulate neural cell survival, stress responses, lineage commitment, and neuronal signaling. Lineage-specific FOXO knockout mice have provided an invaluable tool for the dissection of FOXO biology in the nervous system. Within the NSC compartments of the brain, FOXOs are required for the maintenance of NSC quiescence and for the clearance of reactive oxygen species. Within mature neurons, FOXO transcriptional activity is essential for the prevention of age-dependent axonal degeneration. Acutely, FOXO3 has been found to cause axonal degeneration upon withdrawal of neurotrophic factors. In more active neural signaling, FOXO6 promotes increased dendritic spine density of hippocampal neurons and is required for the consolidation of memories. In addition to the central nervous system (CNS), FOXOs also influence the functionality of the peripheral nervous system (PNS). FOXO1 knockout within the PNS results in a reduction of sympathetic tone and decreased levels of brain-derived norepinephrine and lower energy expenditure. FOXO3 knockout mice have impaired hearing which may be due to defects in synapse localization within the ear. Given the scope of FOXO activities in both the CNS and PNS, it will be of interest to study FOXOs within the context of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis. From within the nervous system, FOXOs may also regulate important parameters such as whole-body metabolism, motor function, and catecholamine production, making FOXOs key players in physiologic homeostasis.

Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons.

Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant’s effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol) increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP) mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine–induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine’s effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats.

Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in dorsal and ventral hippocampus (VH). Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day) during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH). By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR) subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR) subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.