Hippocampal Synaptosomes Research Articles

Chronic corticosterone administration alters synaptic mitochondrial function within the hippocampus of C57Bl/6NTac mice

Chronic activation of the hypothalamic-pituitary-adrenal axis increases circulating corticosterone levels, causing a host of downstream behavioral, molecular, and metabolic changes. Here, we assess the effects of chronic exogenous CORT administration on changes in behavior and mitochondrial respiration in hippocampal synaptosomes of male and female mice. Adult male (n = 15) and female (n = 17) C57Bl/6NTac mice were given 35ug/mL CORT or vehicle dissolved in their drinking water for 21 consecutive days. Chronic CORT increased piloerection in males only. Although volume of CORT-containing water consumed was similar between males and females, circulating plasma and fecal corticosterone levels were only elevated in CORT-exposed males. Behavioral effects of CORT were evident in the Y-maze such that CORT caused a decrease in direct revisits in both sexes. There was no observed presentation of anxiety-like behavior following chronic CORT administration. Functional hippocampal synaptosomes were analyzed for mitochondrial respiration using Agilent's Cell Mito Stress test. Chronic CORT caused a decrease in synaptic mitochondria basal respiration, maximal respiration, proton leak, and ATP production in both sexes. Despite only observing an effect of chronic CORT on corticosterone concentrations in fecal and blood samples of males, chronic CORT induced marked changes in hippocampal synaptic mitochondrial function of both sexes. These data highlight the importance of considering effects of stress hormone exposure on neural function even in the absence of measurable peripheral elevations in females.

Premature cognitive decline in a mouse model of tuberous sclerosis.

Little is known about the influence of (impaired) neurodevelopment on cognitive aging. We here used a mouse model for tuberous sclerosis (TS) carrying a heterozygous deletion of the Tsc2 gene. Loss of Tsc2 function leads to mTOR hyperactivity in mice and patients. In a longitudinal behavioral analysis, we found premature decline of hippocampus-based cognitive functions together with a significant reduction of immediate early gene (IEG) expression. While we did not detect any morphological changes of hippocampal projections and synaptic contacts, molecular markers of neurodegeneration were increased and the mTOR signaling cascade was downregulated in hippocampal synaptosomes. Injection of IGF2, a molecule that induces mTOR signaling, could fully rescue cognitive impairment and IEG expression in aging Tsc2+/- animals. This data suggests that TS is an exhausting disease that causes erosion of the mTOR pathway over time and IGF2 is a promising avenue for treating age-related degeneration in mTORopathies.

Temporal-Specific Sex and Injury-Dependent Changes on Neurogranin-Associated Synaptic Signaling After Controlled Cortical Impact in Rats.

Extensive effort has been made to study the role of synaptic deficits in cognitive impairment after traumatic brain injury (TBI). Neurogranin (Ng) is a calcium-sensitive calmodulin (CaM)-binding protein essential for Ca2+/CaM-dependent kinase II (CaMKII) autophosphorylation which subsequently modulates synaptic plasticity. Given the loss of Ng expression after injury, additional research is warranted to discern changes in hippocampal post-synaptic signaling after TBI. Under isoflurane anesthesia, adult, male and female Sprague-Dawley rats received a sham/control or controlled cortical impact (CCI) injury. Ipsilateral hippocampal synaptosomes were isolated at 24h and 1, 2, and 4weeks post-injury, and western blot was used to evaluate protein expression of Ng-associated signaling proteins. Non-parametric Mann-Whitney tests were used to determine significance of injury for each sex at each time point. There were significant changes in the hippocampal synaptic expression of Ng and associated synaptic proteins such as phosphorylated Ng, CaMKII, and CaM up to 4weeks post-CCI, demonstrating TBI alters hippocampal post-synaptic signaling. This study furthers our understanding of mechanisms of cognitive dysfunction within the synapse sub-acutely after TBI.

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress

BackgroundEarly life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. MethodsMale Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. ResultsWe report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LimitationsThe study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. ConclusionDouble-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Daily fluctuations in blood glucose with normal aging are inversely related to hippocampal synaptic mitochondrial proteins

Defective brain glucose utilization is a hallmark of Alzheimer’s disease (AD) while Type II diabetes and elevated blood glucose escalate the risk for AD in later life. Isolating contributions of normal aging from coincident metabolic or brain diseases could lead to refined approaches to manage specific health risks and optimize treatments targeted to susceptible older individuals. We evaluated metabolic, neuroendocrine, and neurobiological differences between young adult (6 months) and aged (24 months) male rats. Compared to young adults, blood glucose was significantly greater in aged rats at the start of the dark phase of the day but not during the light phase. When challenged with physical restraint, a potent stressor, aged rats effected no change in blood glucose whereas blood glucose increased in young adults. Tissues were evaluated for markers of oxidative phosphorylation (OXPHOS), neuronal glucose transport, and synapses. Outright differences in protein levels between age groups were not evident, but circadian blood glucose was inversely related to OXPHOS proteins in hippocampal synaptosomes, independent of age. The neuronal glucose transporter, GLUT3, was positively associated with circadian blood glucose in young adults whereas aged rats tended to show the opposite trend. Our data demonstrate aging increases daily fluctuations in blood glucose and, at the level of individual differences, negatively associates with proteins related to synaptic OXPHOS. Our findings imply that glucose dyshomeostasis may exacerbate metabolic aspects of synaptic dysfunction that contribute to risk for age-related brain disorders.

Early-life stress and amyloidosis in mice share pathogenic pathways involving synaptic mitochondria and lipid metabolism.

Early-life stress (ES) increases the risk for Alzheimer's disease (AD). We and others have shown that ES aggravates amyloid-beta (Aβ) pathology and promotes cognitive dysfunction in APP/PS1 mice, but underlying mechanisms remain unclear. We studied how ES affects the hippocampal synaptic proteome in wild-type (WT) and APP/PS1 mice at early and late pathological stages, and validated hits using electron microscopy and immunofluorescence. The hippocampal synaptosomes of both ES-exposed WT and early-stage APP/PS1 mice showed a relative decrease in actin dynamics-related proteins and a relative increase in mitochondrial proteins. ES had minimal effects on older WT mice, while strongly affecting the synaptic proteome of advanced stage APP/PS1 mice, particularly the expression of astrocytic and mitochondrial proteins. Our data show that ES and amyloidosis share pathogenic pathways involving synaptic mitochondrial dysfunction and lipid metabolism, which may underlie the observed impact of ES on the trajectory of AD.

Interactions between Glycine and Glutamate through Activation of Their Transporters in Hippocampal Nerve Terminals.

Evidence supports the pathophysiological relevance of crosstalk between the neurotransmitters Glycine and Glutamate and their close interactions; some reports even support the possibility of Glycine-Glutamate cotransmission in central nervous system (CNS) areas, including the hippocampus. Functional studies with isolated nerve terminals (synaptosomes) permit us to study transporter-mediated interactions between neurotransmitters that lead to the regulation of transmitter release. Our main aims here were: (i) to investigate release-regulating, transporter-mediated interactions between Glycine and Glutamate in hippocampal nerve terminals and (ii) to determine the coexistence of transporters for Glycine and Glutamate in these terminals. Purified synaptosomes, analyzed at the ultrastructural level via electron microscopy, were used as the experimental model. Mouse hippocampal synaptosomes were prelabeled with [3H]D-Aspartate or [3H]Glycine; the release of radiolabeled tracers was monitored with the superfusion technique. The main findings were that (i) exogenous Glycine stimulated [3H]D-Aspartate release, partly by activation of GlyT1 and in part, unusually, through GlyT2 transporters and that (ii) D-Aspartate stimulated [3H]glycine release by a process that was sensitive to Glutamate transporter blockers. Based on the features of the experimental model used, it is suggested that functional transporters for Glutamate and Glycine coexist in a small subset of hippocampal nerve terminals, a condition that may also be compatible with cotransmission; glycinergic and glutamatergic transporters exhibit different functions and mediate interactions between the neurotransmitters. It is hoped that increased information on Glutamate-Glycine interactions in different areas, including the hippocampus, will contribute to a better knowledge of drugs acting at "glycinergic" targets, currently under study in relation with different CNS pathologies.

Early life stress lastingly alters the function and AMPA-receptor composition of glutamatergic synapses in the hippocampus of male mice.

Early postnatal life is a sensitive period of development that shapes brain structure and function later in life. Exposure to stress during this critical time window can alter brain development and may enhance the susceptibility to psychopathology and neurodegenerative disorders later in life. The developmental effects of early life stress (ELS) on synaptic function are not fully understood, but could provide mechanistic insights into how ELS modifies later brain function and disease risk. We here assessed the effects of ELS on synaptic function and composition in the hippocampus of male mice. Mice were subjected to ELS by housing dams and pups with limited bedding and nesting material from postnatal days (P) 2-9. Synaptic strength was measured in terms of miniature excitatory postsynaptic currents (mEPSCs) in the hippocampal dentate gyrus at three different developmental stages: the early postnatal phase (P9), preadolescence (P21, at weaning) and adulthood at 3 months of age (3MO). Hippocampal synaptosome fractions were isolated from P9 and 3MO tissue and analyzed for protein content to assess postsynaptic composition. Finally, dendritic spine density was assessed in the DG at 3MO. At P9, ELS increased mEPSC frequency and amplitude. In parallel, synaptic composition was altered as PSD-95, GluA3 and GluN2B content were significantly decreased. The increased mEPSC frequency was sustained up to 3MO, at which age, GluA3 content was significantly increased. No differences were found in dendritic spine density. These findings highlight how ELS affects the development of hippocampal synapses, which could provide valuable insight into mechanisms how ELS alters brain function later in life.

Increased hippocampal cannabinoid 1 receptor expression is associated with protection from severe seizures in pregnant mice with reduced uterine perfusion pressure.

Eclampsia, new-onset seizures in pregnancy, can complicate preeclampsia, a hypertensive pregnancy disorder. The mechanisms contributing to increased risk of seizures in preeclampsia are not fully known. One mechanism could be abnormal endocannabinoid system (ECS) activity and impaired neuromodulation. Indeed, increased placental cannabinoid receptor 1 (CB1R) expression and reduced serum anandamide, a CB1R ligand, have been reported in preeclampsia patients. We hypothesized that reduced uterine perfusion pressure (RUPP), used to mimic preeclampsia, leads to changes in hippocampal CB1R expression, and that manipulating CB1R activity will change seizure severity in RUPP mice. Pregnant mice underwent sham or RUPP surgery on gestational day (GD)13.5. On GD18.5, mice received: no drug treatment, pentylenetetrazol (PTZ, 40 mg/kg), Rimonabant (10 mg/kg) + PTZ, or 2-AG (1 mg/kg) + PTZ. Behaviors were video recorded (15 min for Rimonabant and 2-AG, followed by 30 min for PTZ), and the hippocampus was harvested. The expression of CB1R and ECS proteins was measured in hippocampal homogenates, synaptosomes, and cytosol. Hippocampal CB1R increased in homogenates and cytosolic fraction, and was unchanged in synaptosomes of RUPP mice. Increased CB1R colocalization on glutamate-releasing neurons within hippocampal CA1 was observed in RUPP mice. Rimonabant modestly increased seizure scores over time in RUPP mice. PTZ after rimonabant pretreatment increased seizure scores and duration, while reducing latency in sham mice, with little to no change in RUPP mice. Furthermore, RUPP mice had lower seizure scores over time than sham following CB1R blockade and activation. These data suggest that RUPP modifies CB1R activity prior to seizure induction, which protects mice from worse seizure outcomes.

Melatonin Yaşlı Sıçanlarda Postoperatif Kognitif Disfonksiyonu İyileştirir: Oksidatif Stres, PSD95 ve Ca2+/Kalmodulin Bağımlı Protein Kinazın Önemi

Aim: Postoperative cognitive dysfunction (POCD) is usually seen in the aged patients in postoperative
 period. In the present study, we aimed to investigate the effects of melatonin on cognitive and synaptic
 dysfunction and oxidative stress in POCD model constructed by abdominal surgery in aged rats.
 Material and Methods: Twenty months old male Wistar rats were randomly allocated into the control
 group, melatonin treated-control group, surgery group and melatonin treated-surgery group. The novel
 object recognition test (NORT) was used to assess the postoperative cognitive ability of rats at the
 end of the experiments. The contents of malondialdehyde (MDA) and reduced glutathione (GSH) were
 evaluated in the synaptosomes of hippocampus by spectrophotometric methods. The protein levels of
 calcium-calmodulin dependent protein kinase II (CaMKII) and postsynaptic density protein 95 (PSD95)
 were measured by using ELISA. .
 Results: Animals in surgery group showed significant an impairment of novel object recognition memory
 in NORT. Surgery group also exhibited increased content of MDA (21.05±3.96) and decreased levels
 of GSH (5.86±0.26) and PSD95 (156.04±2.56) in the hippocampus (respectively p=0.012, p

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.

Rapamycin Affects the Hippocampal SNARE Complex to Alleviate Cognitive Dysfunction Induced by Surgery in Aged Rats.

Delayed neurocognitive recovery (dNCR) is a common complication that occurs post-surgery, especially in elderly individuals. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex plays an essential role in various membrane fusion events, such as synaptic vesicle exocytosis and autophagosome-lysosome fusion. Although SNARE complex dysfunction has been observed in several neurodegenerative disorders, the causal link between SNARE-mediated membrane fusion and dNCR remains unclear. We previously demonstrated that surgical stimuli caused cognitive impairment in aged rats by inducing α-synuclein accumulation, inhibiting autophagy, and disrupting neurotransmitter release in hippocampal synaptosomes. Here, we evaluated the effects of propofol anesthesia plus surgery on learning and memory and investigated levels of SNARE proteins and chaperones in hippocampal synaptosomes. Aged rats that received propofol anesthesia and surgery exhibited learning and memory impairments in a Morris water maze test and decreased levels of synaptosome-associated protein 25, synaptobrevin/vesicle-associated membrane protein 2, and syntaxin 1. Levels of SNARE chaperones, including mammalian uncoordinated-18, complexins 1 and 2, cysteine string protein-α, and N-ethylmaleimide-sensitive factor, were all significantly decreased following anesthesia with surgical stress. However, the synaptic vesicle marker synaptophysin was unaffected. The autophagy-enhancer rapamycin attenuated structural and functional disturbances of the SNARE complex and ameliorated disrupted neurotransmitter release. Our results indicate that perturbations of SNARE proteins in hippocampal synaptosomes may underlie the occurrence of dNCR. Moreover, the protective effect of rapamycin may partially occur through recovery of SNARE structural and functional abnormalities. Our findings provide insight into the molecular mechanisms underlying dNCR.

Feedback facilitation by adenosine A2A receptors of ATP release from mouse hippocampal nerve terminals

The adenosine modulation system is mostly composed by inhibitory A1 receptors (A1R) and the less abundant facilitatory A2A receptors (A2AR), the latter selectively engaged at high frequency stimulation associated with synaptic plasticity processes in the hippocampus. A2AR are activated by adenosine originated from extracellular ATP through ecto-5’-nucleotidase or CD73-mediated catabolism. Using hippocampal synaptosomes, we now investigated how adenosine receptors modulate the synaptic release of ATP. The A2AR agonist CGS21680 (10-100 nM) enhanced the K+-evoked release of ATP, whereas both SCH58261 and the CD73 inhibitor α,β-methylene ADP (100 μM) decreased ATP release; all these effects were abolished in forebrain A2AR knockout mice. The A1R agonist CPA (10-100 nM) inhibited ATP release, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects. The presence of SCH58261 potentiated CPA-mediated ATP release and uncovered a facilitatory effect of DPCPX. Overall, these findings indicate that ATP release is predominantly controlled by A2AR, which are involved in an apparent feedback loop of A2AR-mediated increased ATP release together with dampening of A1R-mediated inhibition. This study is a tribute to María Teresa Miras-Portugal.

Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress.

Increased ATP release and its extracellular catabolism through CD73 (ecto-5'-nucleotidase) lead to the overactivation of adenosine A2A receptors (A2AR), which occurs in different brain disorders. A2AR blockade blunts mood and memory dysfunction caused by repeated stress, but it is unknown if increased ATP release coupled to CD73-mediated formation of extracellular adenosine is responsible for A2AR overactivation upon repeated stress. This was now investigated in adult rats subject to repeated stress for 14 consecutive days. Frontocortical and hippocampal synaptosomes from stressed rats displayed an increased release of ATP upon depolarization, coupled to an increased density of vesicular nucleotide transporters and of CD73. The continuous intracerebroventricular delivery of the CD73 inhibitor α,β-methylene ADP (AOPCP, 100 μM) during restraint stress attenuated mood and memory dysfunction. Slice electrophysiological recordings showed that restraint stress decreased long-term potentiation both in prefrontocortical layer II/III-layer V synapses and in hippocampal Schaffer fibers-CA1 pyramid synapses, which was prevented by AOPCP, an effect occluded by adenosine deaminase and by the A2AR antagonist SCH58261. These results indicate that increased synaptic ATP release coupled to CD73-mediated formation of extracellular adenosine contributes to mood and memory dysfunction triggered by repeated restraint stress. This prompts considering interventions decreasing ATP release and CD73 activity as novel strategies to mitigate the burden of repeated stress.

L-proline transporter inhibitor (LQFM215) promotes neuroprotection in ischemic stroke.

L-proline transporter (PROT/SLC6A7) is closely associated with glutamatergic neurotransmission, where L-proline modulates the NMDA receptor (NMDAR) function. NMDAR-mediated excitotoxicity is a primary cause of neuronal death following stroke, which is triggered by the uncontrolled release of glutamate during the ischemic process. After ischemic stroke, L-proline levels show a reduction in the plasma, but high circulating levels of this molecule indicate good functional recovery. This work aimed to produce new PROT inhibitors and explore their effects on ischemic stroke. Initially, we built a three-dimensional model of the PROT protein and run a molecular docking with the newly designed compounds (LQFM215, LQFM216, and LQFM217). Then, we synthesized new PROT inhibitors by molecular hybridization, and proline uptake was measured inex vivoandin vivomodels. The behavioral characterization of the treated mice was performed by the open-field test, elevated plus-maze, Y-maze, and forced swimming test. We used the permanent middle cerebral artery occlusion (MCAO) model to study the ischemic stroke damage and analyzed the motor impairment with limb clasping or cylinder tests. LQFM215 inhibited proline uptake in hippocampal synaptosomes, and the LQFM215 treatment reduced proline levels in the mouse hippocampus. LQFM215 reduced the locomotor and exploratory activity in mice and did not show any anxiety-related or working memory impairments. In the MCAO model, LQFM215 pre-treatment and treatment reduced the infarcted area and reduced motor impairments in the cylinder test and limb clasping. This dataset suggests that the new compounds inhibit cerebral L-proline uptake and that LQFM215 promotes neuroprotection and neuro-repair in the acute ischemic stroke model.

Synapse-specific changes in Arc and BDNF in rat hippocampus following chronic temporal lobe epilepsy

Expression of immediate early genes (IEGs) in the brain is important for synaptic plasticity, and probably also in neurodegenerative conditions. To understand the cellular mechanisms of the underlying neuropathophysiological processes in epilepsy, we need to pinpoint changes in concentration of synaptic plasticity-related proteins at subsynaptic levels. In this study, we examined changes in synaptic expression of Activity-regulated cytoskeleton-associated (Arc) and Brai Derived Neurotrophic Factor (BDNF) in a rat model of kainate-induced temporal lobe epilepsy (TLE). Western blotting showed reduced concentrations of Arc and increased concentrations of BDNF in hippocampal synaptosomes in chronic TLE rats. Then, using quantitative electron microscopy, we found corresponding changes in subsynaptic regions in the hippocampus. Specifically, we detected significant reductions in the concentrations of Arc in the presynaptic terminal of Schaffer collateral glutamatergic synapses in the stratum radiatum of the CA1 area in TLE, as well as in their adjacent postsynaptic spines. In CA3, there was a significant reduction of Arc only in the presynaptic terminal cytoplasm. Conversely, in CA3, there was a significant increase in the expression of BDNF in the presynaptic terminal, but not in the postsynaptic spine. Significant increase in BDNF concentration in the CA1 postsynaptic density was also obtained. We hypothesize that the observed changes in Arc and BDNF may contribute to both cognitive impairment and increased excitotoxic vulnerability in chronic epilepsy.

Ubiquitin‐proteasome system dysfunction induced by Alzheimer's disease‐linked Ab oligomers

AbstractBackgroundBrain accumulation of the amyloid‐β peptide (Aβ) is a pathological hallmark of Alzheimer’s disease (AD). Considerable evidence indicates that soluble Ab oligomers (AβOs) cause synapse failure/loss and cognitive decline in AD. Evidence further indicates that the ubiquitin‐proteasome system (UPS) is inhibited in AD brains, likely leading to impaired synaptic plasticity and memory. However, the mechanisms underlying UPS dysfunction in AD remain to be elucidatedMethodHere, we investigated proteasome activity in primary hippocampal cultures and in isolated hippocampal synaptosomes from mice that received an intracerebroventricular (i.c.v.) infusion of AβOs. We further investigated the possible protective actions of UCH‐L1, an enzyme that enhances proteasome‐mediated degradation of target proteins, against the impact of AβOs on hippocampal neurons and memory.ResultExposure to AβOs resulted in decreased proteasome activity in primary hippocampal cultures and in isolated hippocampal synaptosomes. Synaptosomes isolated from the hippocampi of AβO‐infused mice were also inhibited compared to hippocampal synaptosomes from control, vehicle‐infused mice. Treatment with exogenous recombinant UCH‐L1 prevented oxidative stress in AβO‐exposed hippocampal neurons and memory deficit in AβO‐infused mice.ConclusionThese results indicate that AβOs inhibit the proteasome, notably at synapses, and suggest that boosting proteasomal proteolysis prevents AβO‐induced neuronal damage and memory deficit.

Complement C3 Lowering in Adult Inducible Conditional Knockout Mice: Long‐Lasting Effects

AbstractBackgroundComplement C3, a central component of the complement cascade, participates in synaptic elimination during brain development and is elevated in the brain with aging and Alzheimer’s disease (AD). Germline C3 knockout (KO) protects against age‐ and AD‐related hippocampal synapse loss and functional decline (Shi Q et al., J Neurosci 2015; Shi Q et al., Sci Transl Med 2017). We crossed C3fl/fl mice with Rosa26‐Cre‐ERT2 mice to generate global C3 inducible conditional KO mice (C3iKO). Previously, we reported that 5 daily TAM treatments in C3iKO mice at 4‐5 mo of age led to sustained C3 reductions of ∼95% in serum and ∼80% in brain, and protected mice against age‐related cognitive decline at 16‐17 months. In addition, TAM treatment at 3‐4 mo of age protected long‐term potentiation (LTP) in hippocampal slices exposed to toxic Aβ dimers (S26C) obtained at 7‐8 mo of age. Here, we extend our analyses to include changes in complement, cytokine and ApoE expression in these same C3iKO mice and provide confirmatory results in a second cohort of mice.MethodqRT‐PCR was used to quantify brain gene expression in the first cohort. In the second cohort, C3iKO, C3fl/fl and C3KO mice were treated with TAM or corn oil (CO) at 7‐8 mo of age. Cognitive testing commenced at 17‐18 mo and included the Spatial Novelty Y Maze (SPNY), Novel Object Recognition (NOR) and Displaced Object Recognition (DOR). Mice were euthanized and hippocampal synaptosomes isolated for Western blot analysis.ResultOne‐year post‐treatment, C1qa, C1qb, C1qc, C3, CD11b, CD18, IL‐1β, IFNα, IFNβ, and APOE mRNAs were reduced, and TGFβ and TGFβR1 mRNAs were elevated in brains of TAM‐treated C3iKO and C3KO mice compared to CO‐treated C3iKO mice. In the second cohort, C3 protein levels were reduced ∼92% in serum and ∼77% in hippocampal synaptosomes in TAM‐treated C3iKO mice. TAM‐treated C3iKO mice and both C3KO groups showed significantly better performance in SNYM, LOR and DOR tests compared to controls, consistent with our first study. Pre‐ and post‐synaptic markers were elevated in TAM‐treated vs. CO‐treated C3iKO hippocampal synaptosomes.ConclusionGlobal C3 lowering in adulthood has long‐lasting effects on immune signaling and hippocampal function. (NIH RF1 AG060057‐CAL)

Learning differentially increases the synaptic levels of AMPA receptor subunits (GluA1‐GluA4) in the rodent hippocampus: a single‐synapse approach

AbstractBackgroundDecades of experimental work support the idea that synapses are the anatomical substrate for experience‐dependent plasticity in the brain. Changes in the synaptic strength underlie learning and memory via the accumulation of glutamate AMPA receptors (AMPAR) at the surface of excitatory synapses. AMPAR are tetrameric receptors constituted by multiple combination of four subunits: GluA1 GluA2, GluA3, and GluA4. However, the relative contribution of each AMPAR subunit on learning and memory is relatively unexplored.MethodHere, we introduce Fluorescence Analysis of Single‐Synapse Potentiation induced by Learning (FASS‐PiL), a flow cytometry‐based method to quantify surface levels of all four GluA‐AMPAR subunits, in parallel, in isolated synaptosomes after a learning episode in rodents. We evaluated surface levels of all four AMPAR subunits in synaptosomes of mice trained in a learning protocol commonly used for studying episodic memory in the hippocampus (e.g., Object Location Memory (OLM) and Object Recognition Memory (ORM) tasks). Briefly, after exploring novel objects in a novel environment for 10 min, mice were returned to their home cage for 60 min. After hippocampus dissection, synaptosome isolation, and immunostaining, samples were analyzed via flow cytometry.ResultWe first demonstrated that FASS‐PiL is a simple and sensitive method to track all four GluAs at the synaptosome surface (each GluA subunit was paired with the presynaptic marker Neurexin‐1beta to focus on synaptosome particles containing both pre‐ and postsynaptic compartments). Notably, by profiling hundreds of events, our data showed that the OLM training increases the number of hippocampal synaptosomes expressing high levels of GluA1 and GluA2, but not GluA3 and GluA4 (vs control animals).ConclusionOur results indicate that plasticity‐related mechanisms underlying learning are AMPA‐subunit‐specific in hippocampal synapses. Our approach could provide the basis for protocols to study behavioral‐relevant mechanisms of plasticity directly at the synapse, with single‐event resolution.

Early time course of oxidative stress in hippocampal synaptosomes and cognitive loss following impaired insulin signaling in rats: Development of sporadic Alzheimer’s disease

Oxidative stress, caused by impaired insulin signaling, plays a pivotal role in the pathogenesis of sporadic Alzheimer’s disease (sAD). We investigated the oxidative stress parameters in the synaptosomes prepared from the hippocampus tissue in order to identify their potential role in sAD development in intraperitoneal (IP) and intracerebroventricular (ICV) streptozotocin (STZ) injections models of insulin signaling impairment. Rats were harvested 1, 3, or 6 weeks post treatment. Spatial learning and memory, several antioxidants and oxidative stress markers were analyzed. Results showed a significant deficit in learning and memory in rats injected with STZ through IP and ICV routes. Glutathione, glutathione/oxidized glutathione, glutathione S-transferase, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase(SOD)-total, Zn/Cu(SOD), Mn/Fe(SOD) are significantly decreased in IP-STZ and ICV-STZ groups at 1, 3, and 6 weeks after STZ injection. Oxidized glutathione, thiobarbituric acid reactive species, glucose 6-Phosphate dehydrogenase, protein carbonyls, 4-Hydroxynonenal, and 3-Nitrotyrosine are significantly increased in IP-STZ and ICV-STZ groups at 1,3, and 6 weeks after STZ injection. Changes in oxidative stress parameters in ICV-STZ groups are greater than IP-STZ groups. STZ treatment induced cognitive impairments by 3-W and 6-W, and it was significantly correlated with the extent of oxidative damage. In conclusion, STZ administration through ICV route is deleterious in causing early synaptosomal oxidative damage that exacerbated with time and correlated with cognitive impairments. Our data implicate the involvement of oxidative stress as an early feature of sAD and provide insights into the behavioral and biochemical changes over the course of disease development.