Quantification of astrocytic synaptic pruning in mouse hippocampal slices in response to ex vivo Aβ treatment via colocalization analysis with C1q

Objective To evaluate the role of mammalian target of rapamycin(mTOR) in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Methods Twenty-four healthy adult male Sprague-Dawley rats, weighing 180-240 g, were divided into 4 groups(n=6 each) by using a random number table method: control group(group C), inflammatory pain group(group IP), dimethyl sulfoxide(DMSO) group and mTOR inhibitor rapamycin group(group R). Inflammatory pain model was established by subcutaneous injection of 50 μl bee venom into the plantar surface of the left hindpaw. The equal volume of normal saline was subcutaneously injected into the plantar surface of the left hindpaw in group C. In group DMSO, 2% DMSO was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. In group R, rapamycin was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. Mechanical paw withdrawal threshold(MWT) and thermal paw withdrawal latency(TWL) were measured at 2 h after establishing the model. The rats were sacrificed after measurement of the pain threshold, and hippocampi were removed to prepare hippocampal slices. Hippocampal CA1 region and dentate gyrus(DG region) were located with an inverted microscope. Planar microelectrode array technique was used to record the number of channels and the standardized amplitude of evoked effective field excitatory postsynaptic potentials(fEPSPs)(fEPSPs amplitude>20% of the baseline value) at different stimulus intensities. Results Compared with group C, MWT was significantly decreased, TWL was shortened, the number of effective fEPSP channels at different stimulus intensities was increased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was increased in group IP(P 0.05). Compared with group IP, MWT was significantly increased, TWL was prolonged, the number of effective fEPSP channels at different stimulus intensities was decreased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was decreased in group R(P 0.05). Conclusion mTOR is involved in the changes in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Key words: Receptor-interacting protein serine-threonine kinases; Hippocampus; Entorhinal cortex; Pain; Inflammation; Neuronal plasticity

Objective For the hippocampal CA1 and CA3 regions,the possible relationship between structural plasticity of synaptic interface structure and expression of synaptophysin and their functional roles were explored in anxiety-behavioral rats.Methods The escalating doses of morphine and the elevated plus maze were applied to validate anxiety-like behavior in rats.Both electron microscopy and immunohistochemitry were applied to detect parameters,including structural plasticity of synaptic interface structure and expression of synaptophysin(P38),in the hippocampal fields CA1 and CA3 in control group,morphine-withdrawal group and cured group(n=6).Results (1)Anxiety-like behavioral symptoms were observed in the rats suffering from the escalating morphine doses(t,least significant difference test,P＜0.01 or P＜0.05).(2)Compared with control group and cured group,higher values of postsynaptic density (10.7±0.9)nm,length of postsynaptic thickening(45±4)nm,widths in synaptic interface structure on junctions(3.80±0.30)nm and curvature of the cleft region(1.37±0.12)nm were notably observed in hippocampal CA1 region in anxious rats(P＜0.01 or P＜0.05).Similarly,higher scores of postsynaptic density(12.9±1.1)nm,length of postsynaptic thickening(53±8)nm,widths in synaptic interface structure on junctions(3.81±0.59)nm and curvature of the cleft region(1.39±0.30)nm were detected in hippocampal field CA3 in anxious rats(P＜0.01 or P＜0.05).(3)Higher accumulated levels of synaptophysin were found in the hippocampal CA1 and CA3 regions in anxiety-behavioral rats(0.42±0.06and 0.43±0.05).Conclusion Our results suggested that structural plasticity of synaptic interface structure and expression of synaptophysin in the hippocampus could be contributed to development of drug addiction in rats. Key words: Morphine; Substance withdrawal syndrome; Anxiety; Hippocampus; Synapses; Synaptophysin

Event Abstract Back to Event Cholinergic control of GABA release by carbachol at perisomatic inhibitory synapses in the CA3 region of mouse hippocampus G. G. Szabó1, N. Holderith1, A. I. Gulyás1, T. F. Freund1 and N. Hájos1* 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary Action potential generation of hippocampal pyramidal neurons is controlled by perisomatic inhibition, originating from fast-spiking basket (FSBC)- and axo-axonic cells (AACs) as well as from regular spiking basket cells (RSBCs). These neurons play a role in feedforward and feedback inhibition and are tuned by cholinergic receptor activation. We aimed to clarify the impact of cholinergic receptor activation on synaptic communication between these perisomatic inhibitory neurons (PINs) and pyramidal cells (PCs). By performing paired recordings between the three types of PINs and PCs in mouse hippocampal slices, we investigated the release properties and their sensitivity to the cholinergic receptor agonist carbachol (CCh). Synaptic currents originating from the distinct types of PINs had different properties recorded in CA3 PCs. Analyzes of the decay kinetics suggested that at AAC-PC pairs synaptic cross-talk may occur between adjacent release sites. RSBCs showed robust asynchronous release, whereas the other two types of PINs did not. CCh decreased the GABA release significantly from all the three types of PINs to a different extent and via different receptor activation. In addition, CCh significantly reduced the short-term depression of synaptic transmission at FSBC- and AAC-PC pairs.These findings suggest that the contribution of different types of PINs to feedforward and feedback inhibition might be distinct that could be altered by cholinergic receptor activation. Keywords: Neurophysiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Neurophysiology Citation: Szabó GG, Holderith N, Gulyás AI, Freund TF and Hájos N (2011). Cholinergic control of GABA release by carbachol at perisomatic inhibitory synapses in the CA3 region of mouse hippocampus. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. N. Hájos, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, hajos@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Google G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Google Scholar G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos PubMed G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Protective effect of S14G-humanin against beta-amyloid induced LTP inhibition in mouse hippocampal slices

Deregulation of NMDA-receptor function and down-stream signaling in APP[V717I] transgenic mice

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-microns-thick sections, 3 microns distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 x 10(3)/mm3; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 x 10(3)/mm3, 272.4 x 10(3)/mm3, and 1.9 x 10(3)/mm3, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 x 10(3)/mm3 and 1.1 x 10(3)/mm3, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV+ neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA+ and/or PV+ neurons was 2.5 x 10(3)/mm3 and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 x 10(3)/mm3, 6.2 x 10(3)/mm3, and 5.4 x 10(3)/mm3, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.

Testosterone, acting through its androgenic metabolite 5alpha-dihydrotestosterone (DHT), can increase dendritic spine density in the CA1 region of the male rat hippocampus. The mechanisms mediating this increase in spines are presently unknown. In female rats, estrogen (E) has been shown to increase spine density, which is in part mediated by increases in N-methyl-d-aspartate (NMDA) receptors in the CA1 region and cholinergic forebrain inputs to the hippocampus. Whether similar mechanisms are responsible for the DHT-induced increase in spines in the male remains to be determined. In the first experiment, we used [(3)H]glutamate NMDA receptor binding autoradiography to assess whether DHT-treated males had higher NMDA receptor levels in the CA1 region of the hippocampus, compared with oil-treated males. In the second set of experiments, we used choline acetyltransferase (ChAT) in situ hybridization and immunohistochemistry to assess whether DHT could affect ChAT cell number in the forebrain. We also investigated the effect of DHT on hemicholinium-3-sensitive choline transporter levels in the CA1 region of the male hippocampus. We found that DHT significantly increased NMDA receptor binding in the CA1 region of males but had no effect on ChAT cell number in the forebrain or hemicholinium-3-sensitive choline transporter protein levels in the CA1 region. These data indicate that, similar to E-induced spinogenesis in females, DHT-induced increases in spine formation in males may require increases in NMDA receptors. However, unlike E-treated females, these data suggest that DHT does not influence cholinergic inputs to the hippocampus.

The role of estrogen (E) in promoting learning and memory in females has been well studied in both rodent and primate models. In female rats, E increases dendritic spine number, synaptogenesis, and synaptic proteins in the CA1 region of the hippocampus, an area of the brain that mediates learning and memory. In the present study, we used radioimmunocytochemistry to examine whether E and progesterone were capable of modulating the levels of pre- and postsynaptic proteins in the CA1 region of the female nonhuman primate hippocampus. It was found that E increased syntaxin, synaptophysin (presynaptic), and spinophilin (postsynaptic) levels in the stratum oriens and radiatum of the CA1 region, whereas combined E and progesterone treatment decreased these synaptic proteins to the levels found in untreated, spayed controls. Furthermore, progesterone treatment alone significantly increased synaptophysin levels in the stratum oriens and radiatum of the CA1 region. The levels of these synaptic proteins were unaltered by hormone treatment in the dentate gyrus, suggesting that this steroid-induced plasticity is hippocampal region specific. As these synaptic proteins are important components of the synaptic apparatus and reliable markers of synaptogenesis, it appears that E-induced increases in cognitive function of higher order mammals may be mediated in part by the effect of E on hippocampal synaptogenesis and synaptic plasticity.

Seizures develop in 80% of patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti-NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti-NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures. We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti-NMDAR encephalitis or polyclonal rabbit anti-NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2-week treatment was assessed with video-electroencephalography. We assessed memory, anxiety-related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole-cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures. Prolonged exposure to rabbit anti-NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n=4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n=12), polyclonal rabbit IgG (n=7), albumin (n=3), and normal human IgG (n=3). We did not observe memory deficits, anxiety-related behavior, or motor impairment measured at 2weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice. Our findings indicate that autoantibodies can induce seizures in anti-NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

Alzheimer's disease (AD) is a neurodegenerative disorder with the histopathological hallmark of extracellular accumulation of amyloid-β (Aβ) peptide in brain senile plaques. Though many studies have shown the neural toxicity from various forms of Aβ peptides, the subcellular mechanisms of Aβ peptide are still not well understood, partially due to the technical challenges of isolating axons or dendrites from the cell body for localized investigation. In this study, the subcellular toxicity and localization of Aβ peptides are investigated by utilizing a microfluidic compartmentalized device, which combines physical restriction and chemotactic guidance to enable the isolation of axons and dendrites for localized pharmacological studies. It is found that Aβ peptides induced neuronal death is mostly resulted from Aβ treatment at cell body or axonal processes, but not at dendritic neurites. Simply applying Aβ to axons alone induces significant hyperactive spiking activity. Dynamic transport of Aβ aggregates is only observed between axon terminal and cell body. In addition to differential cellular uptake, more Aβ-peptide secretion is detected significantly from axons than from dendritic side. These results clearly demonstrate the existence of a localized mechanism in Aβ-induced neurotoxicity, and can potentially benefit the development of new therapeutic strategies for AD.

To investigate whether Passiflora actinia hydroalcoholic extract and its major constituent, isovitexin, protect mice hippocampal brain slices from glutamate-induced neurotoxicity. Neuroprotective effect of the extract against glutamate-induced excitotoxicity (10 mm) was evaluated through cell viability of hippocampal slices. The extract or its flavonoids were directly applied to hippocampal slices and then subjected to glutamate-induced toxicity. Alternatively, hippocampal slices from extract-treated mice were also subjected to the same toxicity protocol. Mice supplementation with the extract protected hippocampal slices from in-vitro neurotoxicity. When directly applied to hippocampal slices, the extract showed a higher neuroprotective potential than a commercial dry extract of Passiflora incarnata, which was related to P. actinia extract which had higher isovitexin and total flavonoid content expressed as isovitexin. Isovitexin, but not apigenin, induced a similar neuroprotective response when applied alone, at a concentration equivalent to that found in the extract. This study highlights new neuropharmacological activity of the Passiflora genus, suggesting that it can act as modulator of the glutamatergic system. The search for improved pharmacotherapies with novel mechanisms of action has been shown of great importance for the treatment of resistant neurological and psychiatric disorders.

Regional distribution of ethanolamine plasmalogen in the hippocampal CA1 and CA3 regions and cerebral cortex of the gerbil

Microglia maintain brain homeostasis via iC3b-mediated synaptic pruning. The Arp2/3 complex has been implicated in iC3b-mediated macrophage phagocytosis, but it is unclear whether it is similarly required in microglia in the CNS. We examined the question of CR3-dependent clearance of iC3b in microglia using a combination of in vitro and in situ physical confinement studies. Arp2/3 inhibition decreased iC3b phagocytosis and cell motility in vitro. Furthermore, microglia-like cells remove immobilized iC3b from the substrate in an Arp2/3-dependent fashion, in a process reminiscent of trogocytic synaptic pruning. We also used a novel approach to immobilize an iC3b gradient onto a substrate and demonstrate Arp2/3-dependent haptotactic migration toward increasing iC3b concentrations. While Arp2/3-deficient microglia robustly respond to ATP via chemotaxis within mouse hippocampal slices, they demonstrate a persistent inability to stably interact with iC3b-coated beads. The present study establishes new approaches to systematically interrogate molecular pathways relevant to synaptic pruning, advances the understanding of iC3b phagocytosis as a haptotactic response, and confirms that the Arp2/3-dependent haptotactic response is important for microglia function in the CNS microenvironment.

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.

Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β(2) adrenergic receptor (β(2)AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β(2)AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β(2)AR in fibroblasts and endogenous β(2)AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β(2)AR into lysosome, and prolonged Aβ treatment causes β(2)AR degradation. The Aβ-induced β(2)AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β(2)AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β(2)AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β(2)AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β(2)AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β(2)AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities.