Dendritic Spine Morphology Research Articles

Role of extracellular vesicles in early synaptic dysfunction in AD

AbstractBackgroundSynaptic dysfunction is an early mechanism in Alzheimer’s Disease (AD) which involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. We hypothesised that large extracellular vesicles (EVs) released by microglia exposed to and carrying Aβ42 (Aβ‐EVs) may be responsible for these early events in AD.MethodCombining optical manipulation with time lapse imaging we placed single Aβ‐EVs and ctrl‐EVs (released by microglia not exposed to Aβ) on axons or dendrites of RFP‐positive cultured neuron to test both the dynamics of their interaction with neurons and their effects on the synapse. Then, EVs were stereotaxically injected into the mouse entorhinal cortex (EC), one of the most vulnerable regions in AD, and long‐term potentiation (LTP) was measured in the EC and in its main target region, the dentate gyrus of the hippocampus (DG), through field potential extracellular recordings in cortico‐hippocampal brain slices. Finally, taking advantage of high‐resolution accurate‐mass spectrometry SWATH™‐MS, we investigated the molecular differences between ctrl‐EVs and Aβ‐EVs.ResultAβ‐EVs were able to move along the axonal surface more efficiently than ctrl‐EVs and predominantly in an anterograde direction. They locally altered dendritic spine morphology in vitro and impaired LTP in vivo. One hour after injection in the EC, LTP was impaired in the EC of brains injected with Aβ‐EVs but not ctrl‐EVs. However, 24h after Aβ‐EV injection, LTP was impaired also in the DG, indicating a spreading of synaptic dysfunction between the two connected regions. Results were reproduced using large EVs released by microglia exposed to naturally secreted Aβ oligomers, whereas neither Aβ alone nor inflammatory EVs devoid of Aβ were able to propagate LTP impairment. Importantly, when Aβ‐EV motility was limited by annexin‐V coating, no propagation of LTP deficit occurred along the EC‐DG circuit. Proteomic analysis provided insights into the possible molecular mechanisms underneath Aβ‐EV extracellular motion at the neuronal surface.ConclusionOur data provide evidence of the involvement of microglial EVs in early synaptic dysfunction, through a new mechanism ‐ extracellular motion at the neuronal surface ‐ not implying EV internalization, paving the way for novel therapeutic strategies.

A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology.

The Rac1 guanine exchange factor Kalirin-7 is a key regulator of dendritic spine morphology, LTP and dendritic arborization. Kalirin-7 dysfunction and genetic variation has been extensively linked to various neurodevelopmental and neurodegenerative disorders. Here we characterize a Kalirin-7 missense mutation, glu1577lys (E1577K), identified in a patient with severe developmental delay. The E1577K point mutation is located within the catalytic domain of Kalirin-7, and results in a robust reduction in Kalirin-7 Rac1 Guanosine exchange factor activity. In contrast to wild type Kalirin-7, the E1577K mutant failed to drive dendritic arborization, spine density, NMDAr targeting to, and activity within, spines. Together these results indicate that reduced Rac1-GEF activity as result of E1577K mutation impairs neuroarchitecture, connectivity and NMDAr activity, and is a likely contributor to impaired neurodevelopment in a patient with developmental delay.

Changes in the Neuronal Architecture of the Hippocampus in a 6-Hydroxydopamine-Lesioned Rat Model of Parkinson Disease.

Parkinson disease (PD) is a progressive neurodegenerative disorder in which dopaminergic (DAergic) systems are destroyed (particularly in the nigrostriatal system), causing both motor and nonmotor symptoms. Hippocampal neuroplasticity is altered in PD animal models, resulting in nonmotor dysfunctions. However, little is known about the precise mechanism underlying the hippocampal dysfunctions in PD. Striatal 6-hydroxydopamine (6-OHDA) infusions were performed unilaterally in adult Sprague Dawley rats. Both motor and nonmotor symptoms alongside the expression of tyrosine hydroxylase (TH) in the substantia nigra and striatum were confirmed in 6-OHDA-lesioned rats. The neuronal architecture in the hippocampus was analyzed by Golgi staining. During the 7-8 weeks after infusion, the 6-OHDA-lesioned rats exhibited motor and nonmotor dysfunctions (especially anxiety/depression-like behaviors). Rats with unilateral 6-OHDA infusion displayed reduced TH+ immunoreactivity in the ipsilateral nigrostriatal pathway of the brain. Golgi staining revealed that striatal 6-OHDA infusion significantly decreased the dendritic complexity (i.e., number of crossing dendrites, total dendritic length, and branch points) in the ipsilateral hippocampal conus ammonis 1 (CA1) apical/basal and dentate gyrus (DG) subregions. Additionally, the dendritic spine density and morphology were significantly altered in the CA1 apical/basal and DG subregions following striatal 6-OHDA infusion. However, alteration of microglial and astrocytic distributions did not occur in the hippocampus following striatal 6-OHDA infusion. The present study provides anatomical evidence that the structural plasticity in the hippocampus is altered in the late phase following striatal 6-OHDA infusion in rats, possibly as a result of the prolonged suppression of the DAergic system, and independent of neuroinflammation.

Activation of a novel α2AAR-spinophilin-cofilin axis determines the effect of α2 adrenergic drugs on fear memory reconsolidation.

Posttraumatic stress disorder (PTSD) after the pandemic has emerged as a major neuropsychiatric component of post-acute COVID-19 syndrome, yet the current pharmacotherapy for PTSD is limited. The use of adrenergic drugs to treat PTSD has been suggested; however, it is hindered by conflicting clinical results and a lack of mechanistic understanding of drug actions. Our studies, using both genetically modified mice and human induced pluripotent stem cell-derived neurons, reveal a novel α2A adrenergic receptor (α2AAR)-spinophilin-cofilin axis in the hippocampus that is critical for regulation of contextual fear memory reconsolidation. In addition, we have found that two α2 ligands, clonidine and guanfacine, exhibit differential abilities in activating this signaling axis to disrupt fear memory reconsolidation. Stimulation of α2AAR with clonidine, but not guanfacine, promotes the interaction of the actin binding protein cofilin with the receptor and with the dendritic spine scaffolding protein spinophilin to induce cofilin activation at the synapse. Spinophilin-dependent regulation of cofilin is required for clonidine-induced disruption of contextual fear memory reconsolidation. Our results inform the interpretation of differential clinical observations of these two drugs on PTSD and suggest that clonidine could provide immediate treatment for PTSD symptoms related to the current pandemic. Furthermore, our study indicates that modulation of dendritic spine morphology may represent an effective strategy for the development of new pharmacotherapies for PTSD.

Phosphatidylserine exposure modulates adhesion GPCR BAI1 (ADGRB1) signaling activity

Brain-specific angiogenesis inhibitor 1 (BAI1; also called ADGRB1 or B1) is an adhesion G protein-coupled receptor known from studies on macrophages to bind to phosphatidylserine (PS) on apoptotic cells via its N-terminal thrombospondin repeats. A separate body of work has shown that B1 regulates postsynaptic function and dendritic spine morphology via signaling pathways involving Rac and Rho. However, it is unknown if PS binding by B1 has any effect on the receptor's signaling activity. To shed light on this subject, we studied G protein-dependent signaling by B1 in the absence and presence of coexpression with the PS flippase ATP11A in human embryonic kidney 293T cells. ATP11A expression reduced the amount of PS exposed extracellularly and also strikingly reduced the signaling activity of coexpressed full-length B1 but not a truncated version of the receptor lacking the thrombospondin repeats. Further experiments with an inactive mutant of ATP11A showed that the PS flippase function of ATP11A was required for modulation of B1 signaling. In coimmunoprecipitation experiments, we made the surprising finding that ATP11A not only modulates B1 signaling but also forms complexes with B1. Parallel studies in which PS in the outer leaflet was reduced by an independent method, deletion of the gene encoding the endogenous lipid scramblase anoctamin 6 (ANO6), revealed that this manipulation also markedly reduced B1 signaling. These findings demonstrate that B1 signaling is modulated by PS exposure and suggest a model in which B1 serves as a PS sensor at synapses and in other cellular contexts.

Extract of Mallotus oppositifolius (Geiseler) Müll. Arg. increased prefrontal cortex dendritic spine density and serotonin and attenuated para-chlorophenylalanine-aggravated aggressive and depressive behaviors in mice.

Background/Aim: Depression-related aggression is linked to serotonin (5-HT) and dendritic spine alterations. Although Mallotus oppositifolius extract (MOE) has potential for reducing this effect, its specific role remains uncertain. Herein, we evaluated this potential and associated alterations in the brain. Methods: A standard resident-intruder model of para-chlorophenylalanine (pCPA)-induced depression-associated aggression in male ICR mice was used. The resident mice received pCPA (300mg/kg, i. p.) for 3 consecutive days while saline-treated mice served as negative control. The pCPA aggressive mice were subsequently treated orally with either MOE (30, 100, 300mg/kg), fluoxetine (20mg/kg), tryptophan (20mg/kg) or saline (untreated pCPA group) for 28days. Locomotor activity was assessed using open field test. Serotonin (5-HT) levels in mice brain and phytochemical fingerprint of MOE were determined by high performance liquid chromatography (HPLC) while gas chromatography-mass spectrometry (GC-MS) was used to identify constituents of MOE. Dendritic spine density and morphology were evaluated using Golgi-Cox staining technique and analyzed with ImageJ and Reconstruct software. Results: Administration of pCPA induced aggressive behavior in mice, evidenced by increased attack behaviors (increased number and duration of attacks), which positively correlated with squeaking and tail rattling. MOE treatment significantly reduced these characteristics of aggression in comparison with vehicle (non-aggressive) and untreated pCPA groups (p < 0.001), and also reduced social exploration behavior. Although the behavioral effects of MOE were comparable to those of fluoxetine and tryptophan, these effects were quicker compared to fluoxetine and tryptophan. Additionally, MOE also markedly increased 5-HT concentration and dendritic spine density in the prefrontal cortex relative to vehicle and untreated pCPA groups (p < 0.05). Interestingly, these behavioral effects were produced without compromising locomotor activity. GC-MS analysis of the MOE identified 17 known compounds from different chemical classes with anti-inflammatory, antioxidant, neuroprotective and antidepressant activities, which may have contributed to its anti-aggressive effect. Conclusion: MOE decreased depression-associated aggressive behavior in mice via increased 5-HT concentration and dendritic spine density in the prefrontal cortex. The MOE-mediated effects were faster than those of fluoxetine and tryptophan. Our finding suggests that MOE may have clinical promise in decreasing aggressive and depressive behaviors.

Inka2, a novel Pak4 inhibitor, regulates actin dynamics in neuronal development.

The actin filament is a fundamental part of the cytoskeleton defining cell morphology and regulating various physiological processes, including filopodia formation and dendritic spinogenesis of neurons. Serine/threonine-protein kinase Pak4, an essential effector, links Rho GTPases to control actin polymerization. Previously, we identified the Inka2 gene, a novel mammalian protein exhibiting sequence similarity to Inka1, which serves as a possible inhibitor for Pak4. Although Inka2 is dominantly expressed in the nervous system and involved in focal-adhesion dynamics, its molecular role remains unclear. Here, we found that Inka2-iBox directly binds to Pak4 catalytic domain to suppress actin polymerization. Inka2 promoted actin depolymerization and inhibited the formation of cellular protrusion caused by Pak4 activation. We further generated the conditional knockout mice of the Inka2 gene. The beta-galactosidase reporter indicated the preferential Inka2 expression in the dorsal forebrain neurons. Cortical pyramidal neurons of Inka2-/- mice exhibited decreased density and aberrant morphology of dendritic spines with marked activation/phosphorylation of downstream molecules of Pak4 signal cascade, including LIMK and Cofilin. These results uncovered the unexpected function of endogenous Pak4 inhibitor in neurons. Unlike Inka1, Inka2 is a critical mediator for actin reorganization required for dendritic spine development.

Functional interdependence of the actin regulators CAP1 and cofilin1 in control of dendritic spine morphology

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.

Diverging Effects of Adolescent Ethanol Exposure on Tripartite Synaptic Development across Prefrontal Cortex Subregions.

Adolescence is a developmental period that encompasses, but is not limited to, puberty and continues into early adulthood. During this period, maturation and refinement are observed across brain regions such as the prefrontal cortex (PFC), which is critical for cognitive function. Adolescence is also a time when excessive alcohol consumption in the form of binge drinking peaks, increasing the risk of long-term cognitive deficits and the risk of developing an alcohol use disorder later in life. Animal models have revealed that adolescent ethanol (EtOH) exposure results in protracted disruption of neuronal function and performance on PFC-dependent tasks that require higher-order decision-making. However, the role of astrocytes in EtOH-induced disruption of prefrontal cortex-dependent function has yet to be elucidated. Astrocytes have complex morphologies with an extensive network of peripheral astrocyte processes (PAPs) that ensheathe pre- and postsynaptic terminals to form the ‘tripartite synapse.’ At the tripartite synapse, astrocytes play several critical roles, including synaptic maintenance, dendritic spine maturation, and neurotransmitter clearance through proximity-dependent interactions. Here, we investigate the effects of adolescent binge EtOH exposure on astrocyte morphology, PAP-synaptic proximity, synaptic stabilization proteins, and dendritic spine morphology in subregions of the PFC that are important in the emergence of higher cognitive function. We found that adolescent binge EtOH exposure resulted in subregion specific changes in astrocyte morphology and astrocyte-neuronal interactions. While this did not correspond to a loss of astrocytes, synapses, or dendritic spines, there was a corresponding region-specific and EtOH-dependent shift in dendritic spine phenotype. Lastly, we found that changes in astrocyte-neuronal interactions were not a consequence of changes in the expression of key synaptic structural proteins neurexin, neuroligin 1, or neuroligin 3. These data demonstrate that adolescent EtOH exposure results in enduring effects on neuron-glia interactions that persist into adulthood in a subregion-specific PFC manner, suggesting selective vulnerability. Further work is necessary to understand the functional and behavioral implications.

Sevoflurane exposure may cause dysplasia of dendritic spines and result in fine motor dysfunction in developing mouse through the PI3K/AKT/mTOR pathway.

Sevoflurane has become one of the most widely used volatile anesthetics in pediatric surgery. However, sevoflurane exposure may interfere with dendritic development and synaptogenesis, resulting in brain function impairment. The PI3K/AKT/mTOR pathway plays an important role in dendritic development and synaptic plasticity. Here we investigated whether sevoflurane exposure would affect the morphological proportions of dendritic spines in developing mouse and explored the role of the change of plasticity of dendritic spines in sevoflurane-induced neurodevelopmental toxicity. The related signaling pathway was also examined. C57BL/6 mice at postnatal day (PND) 7 were exposed to 2% sevoflurane for 3 h. The PI3k/AKT/mTOR agonist IGF-1 or the mTOR phosphorylation inhibitor KU0063794 was intraperitoneally injected 30 min before sevoflurane or O2 exposure at PND7. Hippocampi were harvested 6 h after sevoflurane exposure. Western blotting was applied to measure the protein expression of PI3K/AKT/mTOR pathway phosphorylation. At PND14, brains from all groups were harvested for Golgi staining, and the morphology of dendritic spines of hippocampal neurons was observed by an oil immersion lens. When the mice grew to adolescence (PND48), fine motor function was measured by the Beam walking test. Here we showed that exposure to 2% sevoflurane for 3 h decreased the proportion of thin dendritic spines and increased the proportion of mushroom dendritic spines, but not changed the density of the dendritic spines. Sevoflurane exposure also suppressed the phosphorylation of the PI3K/AKT/mTOR pathway in immature mice hippocampi, and eventually led to long-term fine motor dysfunction. Meanwhile, IGF-1 pretreatment could rescue and KU0063794 pretreatment could aggravate the impairment induced by sevoflurane. In conclusion, sevoflurane exposure may cause a change of proportions of the types of dendritic spines through impacting the phosphorylation expression of the PI3K/AKT/mTOR pathway, and eventually led to long-term fine motor dysfunction in developing mouse.

Kainate receptors GluK1 and GluK2 differentially regulate synapse morphology.

The regulation of dendritic spine morphology is a critical aspect of neuronal network refinement during development and modulation of neurotransmission. Previous studies revealed that glutamatergic transmission plays a central role in synapse development. AMPA receptors and NMDA receptors regulate spine morphology in an activity dependent manner. However, whether and how Kainate receptors (KARs) regulate synapse development remains poorly understood. In this study, we found that GluK1 and GluK2 may play distinct roles in synapse development. In primary cultured hippocampal neurons, we found overexpression of the calcium-permeable GluK2(Q) receptor variant increased spine length and spine head area compared to overexpression of the calcium-impermeable GluK2(R) variant or EGFP transfected, control neurons, indicating that Q/R editing may play a role in GluK2 regulation of synapse development. Intriguingly, neurons transfected with GluK1(Q) showed decreased spine length and spine head area, while the density of dendritic spines was increased, suggesting that GluK1(Q) and GluK2(Q) have different effects on synaptic development. Swapping the critical domains between GluK2 and GluK1 demonstrated the N-terminal domain (NTD) is responsible for the different effects of GluK1 and GluK2. In conclusion, Kainate receptors GluK1 and GluK2 have distinct roles in regulating spine morphology and development, a process likely relying on the NTD.

Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner

Morphologically dynamic dendritic spines are the major sites of neuronal plasticity in the brain; however, the molecular mechanisms underlying their morphological dynamics have not been fully elucidated. Phldb2 is a protein that contains two predicted coiled-coil domains and the pleckstrin homology domain, whose binding is highly sensitive to PIP3. We have previously demonstrated that Phldb2 regulates synaptic plasticity, glutamate receptor trafficking, and PSD-95 turnover. Drebrin is one of the most abundant neuron-specific F-actin-binding proteins that are pivotal for synaptic morphology and plasticity. We observed that Phldb2 bound to drebrin A (adult-type drebrin), but not to drebrin E (embryonic-type drebrin). In the absence of Phldb2, the subcellular localization of drebrin A in the hippocampal spines and its distribution in the hippocampus were altered. Immature spines, such as the filopodium type, increased relatively in the CA1 regions of the hippocampus, whereas mushroom spines, a typical mature type, decreased in Phldb2-/- mice. Phldb2 suppressed the formation of an abnormal filopodium structure induced by drebrin A overexpression. Taken together, these findings demonstrate that Phldb2 is pivotal for dendritic spine morphology and possibly for synaptic plasticity in mature animals by regulating drebrin A localization.

Changes in dendritic complexity and spine morphology following BCG immunization in APP/PS1 mice

ABSTRACT Bacillus Calmette – Guerin (BCG) is an immune regulator that can enhance hippocampal synaptic plasticity in rats; however, it is unclear whether it can improve synaptic function in a mouse model with Alzheimer’s disease (AD). We hypothesized that BCG plays a protective role in AD mice and investigated its effect on dendritic morphology. The results obtained show that BCG immunization significantly increases dendritic complexity, as indicated by the increased number of dendritic intersections and branch points, as well as the increase in the fractal dimension. Furthermore, the number of primary neurites and dendritic length also increased following BCG immunization, which increased the number of spines and promoted maturation. IFN-γ and IL-4 levels increased, while TNF-α levels decreased following BCG immunization; expression levels of p-JAK2, P-STAT3, SYN, and PSD-95 also increased. Therefore, this study demonstrates that BCG immunization in APP/PS1 mice mitigated hippocampal dendritic spine pathology, especially after the third round of immunization. This effect could possibly be attributed to; changes in dendritic arborization and spine morphology or increases in SYN and PSD-95 expression levels. It could also be related to mechanisms of BCG-induced increases in IFN-γ or IL-4/JAK2/STAT3 levels.

Leveraging interindividual variability of regulatory activity for refining genetic regulation of gene expression in schizophrenia

Schizophrenia is a polygenic psychiatric disorder with limited understanding about the mechanistic changes in gene expression regulation. To elucidate on this, we integrate interindividual variability of regulatory activity (ChIP-sequencing for H3K27ac histone mark) with gene expression and genotype data captured from the prefrontal cortex of 272 cases and controls. By measuring interindividual correlation among proximal chromatin peaks, we show that regulatory element activity is structured into 10,936 and 10,376 cis-regulatory domains in cases and controls, respectively. The schizophrenia-specific cis-regulatory domains are enriched for fetal-specific (p = 0.0014, OR = 1.52) and depleted of adult-specific regulatory activity (p = 3.04 × 10−50, OR = 0.57) and are enriched for SCZ heritability (p = 0.001). By studying the interplay among genetic variants, gene expression, and cis-regulatory domains, we ascertain that changes in coordinated regulatory activity tag alterations in gene expression levels (p = 3.43 × 10−5, OR = 1.65), unveil case-specific QTL effects, and identify regulatory machinery changes for genes affecting synaptic function and dendritic spine morphology in schizophrenia. Altogether, we show that accounting for coordinated regulatory activity provides a novel mechanistic approach to reduce the search space for unveiling genetically perturbed regulation of gene expression in schizophrenia.

Changes in Dendritic Spine Morphology and Density of Granule Cells in the Olfactory Bulb of Anguilla anguilla (L., 1758): A Possible Way to Understand Orientation and Migratory Behavior.

Simple SummaryThe olfactory bulb can process odour cues through granular cells (GCs) and dendritic spines, changing their synaptic plasticity properties and their morphology. The GCs’ dendritic spines density and morphology were analysed in Anguilla anguilla, considering the olfaction as a driver involved in fish orientation and migration. For the head and neck morphology, spines were classified as mushroom, long thin, stubby, and filopodia. Spines’ density decreased from juvenile migrants to no-migrant stages and increased in the adult migrants. Spines’ density was comparable between glass and silver eels as an adaptation to migration, while at non-migrating phases, spines’ density decreased. For its phylogenetic Elopomorph attribution and its complex life cycle, A. anguilla could be recommended as a model species to study the development of dendritic spines in GCs of the olfactory bulb. Considering the role of olfaction in the orientation and migration of A. anguilla, the modification of environmental stimuli (ocean alterations and climate change) could represent contributing factors that threaten this critically endangered species.Olfaction could represent a pivotal process involved in fish orientation and migration. The olfactory bulb can manage olfactive signals at the granular cell (GC) and dendritic spine levels for their synaptic plasticity properties and changing their morphology and structural stability after environmental odour cues. The GCs’ dendritic spine density and morphology were analysed across the life stages of the catadromous Anguilla anguilla. According to the head and neck morphology, spines were classified as mushroom (M), long thin (LT), stubby (S), and filopodia (F). Total spines’ density decreased from juvenile migrants to no-migrant stages, to increase again in the adult migrant stage. Mean spines’ density was comparable between glass and silver eels as an adaptation to migration. At non-migrating phases, spines’ density decreased for M and LT, while M, LT, and S density increased in silver eels. A great dendritic spine development was found in the two migratory phases, regressing in trophic phases, but that could be recreated in adults, tracing the migratory memory of the routes travelled in juvenile phases. For its phylogenetic Elopomorph attribution and its complex life cycle, A. anguilla could be recommended as a model species to study the development of dendritic spines in GCs of the olfactory bulb as an index of synaptic plasticity involved in the modulation of olfactory stimuli. If olfaction is involved in the orientation and migration of A. anguilla and if eels possess a memory, these processes could be influenced by the modification of environmental stimuli (ocean alterations and rapid climate change) contributing to threatening this critically endangered species.

Cranial irradiation induces cognitive decline associated with altered dendritic spine morphology in the young rat hippocampus.

Therapeutic irradiation is commonly used to treat brain cancers but can induce cognitive dysfunction, especially in children. The mechanism is unknown but likely involves alterations in dendritic spine number and structure. To explore the impact of radiation exposure on the alteration of dendritic spine morphology in the hippocampus of young brains, 21-day-old Sprague-Dawley rats received cranial irradiation (10Gy), and changes in spine density and morphology in dentate gyrus (DG) granules and CA1 pyramidal neurons were detected 1 and 3months later by using Golgi staining. Moreover, we analyzed synapse-associated proteins within dendritic spines after irradiation. Our data showed that cognitive deficits were detected in young rats at both time points postirradiation, accompanied by morphological changes in dendritic spines. Our results revealed significant reductions in spine density in the DG at both 1month (40.58%) and 3months (28.92%) postirradiation. However, there was a decrease in spine density only at 1month (33.29%) postirradiation in the basal dendrites of CA1 neurons and no significant changes in the apical dendrites of CA1 neurons at either time point. Notably, among our findings were the significant dynamic changes in spine morphology that persisted 3months following cranial irradiation. Meanwhile, we found that depletion of the synapse-associated proteins PSD95 and Drebrin coincided with alterations in dendritic spines. These data suggest that the decreased levels of PSD95 and Drebrin after ionizing radiation may cause changes in synaptic plasticity by affecting the morphological structure of dendritic spines, blocking the functional connectivity pathways of the brain and leading to cognitive impairment. Although the mechanism involved is unclear, understanding how ionizing radiation affects young brain hippocampal tissue may be useful to gain new mechanistic insights into radiation-induced cognitive dysfunction.

Early Changes in Transcriptomic Profiles in Synaptodendrosomes Reveal Aberrant Synaptic Functions in Alzheimer's Disease.

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative disorders characterized by the progressive decline of cognitive functions, and is closely associated with the dysfunction of synapses, which comprise the basic structure that mediates the communication between neurons. Although the protein architecture and machinery for protein translation at synapses are extensively studied, the impact that local changes in the mRNA reservoir have on AD progression is largely unknown. Here, we investigated the changes in transcriptomic profiles in the synaptodendrosomes purified from the cortices of AD mice at ages 3 and 6 months, a stage when early signatures of synaptic dysfunction are revealed. The transcriptomic profiles of synaptodendrosomes showed a greater number of localized differentially expressed genes (DEGs) in 6-month-old AD mice compared with mice 3 months of age. Gene Ontology (GO) analysis showed that these DEGs are majorly enriched in mitochondrial biogenesis and metabolic activity. More specifically, we further identified three representative DEGs in mitochondrial and metabolic pathways—Prnp, Cst3, and Cox6c—that regulate the dendritic spine density and morphology in neurons. Taken together, this study provides insights into the transcriptomic changes in synaptodendrosomes during AD progression, which may facilitate the development of intervention strategies targeting local translation to ameliorate the pathological progression of AD.

Effects of membrane androgen receptor binding on synaptic plasticity in primary hippocampal neurons

Androgens play an important role in the regulation of hippocampal synaptic plasticity. While the classical molecular mechanism of androgen's genomic activity is their binding to intracellular androgen receptors (iARs), they can also induce rapid non-genomic effects through specific membrane androgen receptors (mARs). In this study, we aimed to localize and characterize these mARs in primary rat hippocampal neurons. Specific punctate fluorescent signals on the cell surface, observed by testosterone-fetal bovine serum albumin conjugated fluorescein isothiocyanate (T-BSA-FITC), indicated the presence of mARs in hippocampal neurons. T-BSA-FITC binding to the cell membrane was incompletely blocked by the iAR-antagonist flutamide, and mAR binding site was competitively bound by free testosterone (T). Most neurons expressing androgen membrane binding sites are glutamatergic (excitatory), although several are γ-aminobutyric acid (GABA)ergic (inhibitory). Confocal microscopy and live-cell imaging techniques were used to observe the real-time rapid effects of androgens on hippocampal dendritic spine morphology. Immunofluorescence cell staining was used to observe their effects on the postsynaptic density protein 95 (PSD95) and synapsin (SYN) synaptic markers. While androgens did not cause a short-term increase in dendritic spine density of rat primary hippocampal neurons, they promoted the transformation of dendritic spines from thin to mushroom, promoted dendritic spine maturation, increased dendritic spine surface area, and rapidly increased PSD95 and SYN expression in the primary hippocampal neurons. Hippocampal synaptosomes were prepared using the Optiprep and Percoll density gradient two-step centrifuge methods, and the gene expression profiles of the synaptosomes and hippocampus were compared using a gene chip; PSD95 mRNA expression was detected by reverse transcription-polymerase chain reaction. Several mRNAs were detected at the synaptic site, including PSD95. Finally, the Venus-PSD95 plasmid was constructed and transfected into HT22 cells, which is a mouse hippocampal neuronal cell line. The real-time effect of androgen on synaptic protein PSD95 was observed by fluorescence recovery after photobleaching experiments, which involved the translation process of PSD95 mRNA. In conclusion, our findings increased our understanding of how androgens exert the neuroprotective mechanisms on synaptic plasticity.

Swimming exercise prevents hippocampal dendritic spine changes and memory loss caused by aging: An application of a new semi-automated spine analysis software

Dendritic spines are small, ratchet-like protrusions on neuronal dendrites that form synapses for receiving neuronal messages. Dendritic spine morphology is associated with synapse function. If neurons degrade or are damaged, the spine morphology of neurons changes. Given that most commercially available spine analysis software is expensive and complex, this study investigated a semi-automated spine analysis software, CTSpine, and used previously published data to verify the accuracy of the analysis results of this software. We also applied CTSpine to understand whether aging causes alterations in the hippocampal spine morphology and whether physical exercise can impede dendritic spine changes in 20 male Sprague Dawley rats. The spines of pyramidal cells in the hippocampal Cornu Ammonis 1 (CA1) region in the aging group were more enriched in filopodium type pattern than those in the control group, whereas the spines of the exercised aging group showed a similar pattern to that of the control. No significant changes were observed in neuronal dendritic spines in other hippocampal regions. However, long-term hippocampal memory was considerably decreased in the aging group, which was reversed to some extent in the exercised aging group. CTSpine, a self-developed semi-automatic spine analysis software, showed results similar to those noted in published data and can be effectively applied to the study of dendritic patterns, including neurodevelopment and disease.

Comparison of Golgi-Cox and Intracellular Loading of Lucifer Yellow for Dendritic Spine Density and Morphology Analysis in the Mouse Brain

Dendritic spines are small protrusions on dendrites that serve as the postsynaptic site of the majority of excitatory synapses. These structures are important for normal synaptic transmission, and alterations in their density and morphology have been documented in various disease states. Over 130 years ago, Ramón y Cajal used Golgi-stained tissue sections to study dendritic morphology. Despite the array of technological advances, including iontophoretic microinjection of Lucifer yellow (LY) fluorescent dye, Golgi staining continues to be one of the most popular approaches to visualize dendritic spines. Here, we compared dendritic spine density and morphology among pyramidal neurons in layers 2/3 of the mouse medial prefrontal cortex (mPFC) and pyramidal neurons in hippocampal CA1 using three-dimensional digital reconstructions of (1) brightfield microscopy z-stacks of Golgi-impregnated dendrites and (2) confocal microscopy z-stacks of LY-filled dendrites. Analysis of spine density revealed that the LY microinjection approach enabled detection of approximately three times as many spines as the Golgi staining approach in both brain regions. Spine volume measurements were larger using Golgi staining compared to LY microinjection in both mPFC and CA1. Spine length was mostly comparable between techniques in both regions. In the mPFC, head diameter was similar for Golgi staining and LY microinjection. However, in CA1, head diameter was approximately 50% smaller on LY-filled dendrites compared to Golgi staining. These results indicate that Golgi staining and LY microinjection yield different spine density and morphology measurements, with Golgi staining failing to detect dendritic spines and overestimating spine size.