NMDAR-dependent somatic potentiation of synaptic inputs is correlated with ß amyloid-mediated neuronal hyperactivity.

BACKGROUND: ß Amyloid (Aß)-mediated neuronal hyperactivity, a key feature of the early stage of Alzheimer's disease (AD), is recently proposed to be initiated by the suppression of glutamate reuptake. Nevertheless, the underlying mechanism by which the impaired glutamate reuptake causes neuronal hyperactivity remains unclear. Chronic suppression of the glutamate reuptake causes accumulation of ambient glutamate that could diffuse from synaptic sites at the dendrites to the soma to elevate the tonic activation of somatic N-methyl-D-aspartate receptors (NMDARs). However, less attention has been paid to the potential role of tonic activity change in extrasynaptic glutamate receptors (GluRs) located at the neuronal soma on generation of neuronal hyperactivity. METHODS: Whole-cell patch-clamp recordings were performed on CA1 pyramidal neurons in acute hippocampal slices exposed to TFB-threo-ß-benzyloxyaspartic acid (TBOA) or human Aß1-42 peptide oligomer. A series of dendritic patch-clamp recordings were made at different distances from the soma to identify the location of the changes in synaptic inputs. Moreover, single-channel recording in the cell-attached mode was performed to investigate the activity changes of single NMDARs at the soma. RESULTS: Blocking glutamate uptake with either TBOA or the human Aß1-42 peptide oligomer elicited potentiation of synaptic inputs in CA1 hippocampal neurons. Strikingly, this potentiation  specifically occurred at the soma, depending on the activation of somatic GluN2B-containing NMDARs (GluN2B-NMDARs) and accompanied by a substantial and persistent increment in the open probability of somatic NMDARs. Blocking the activity of GluN2B-NMDARs at the soma completely reversed both the TBOA-induced or the Aß1-42-induced somatic potentiation and neuronal hyperactivity. CONCLUSIONS: The somatic potentiation of synaptic inputs may represent a novel amplification mechanism that elevates cell excitability and thus contributes to neuronal hyperactivity initiated by impaired glutamate reuptake in AD.

Chronic fluoxetine reverses the effects of chronic corticosterone treatment on α2-adrenoceptors in the rat frontal cortex but not locus coeruleus.

Disruption of the hypothalamic-pituitary-adrenal axis is an established finding in patients with anxiety and/or depression. Chronic corticosterone administration in animals has been proposed as a model for the study of these stress-related disorders and the antidepressant action. Alterations of the central noradrenergic system and specifically of inhibitory α2-adrenoceptors seem to be part of the pathophysiology of depression and contribute to the antidepressant activity. The present study evaluates in male rats the effect of chronic corticosterone treatment during 35 days (16-20 mg kg-1 day-1) on the sensitivity of α2-adrenoceptors expressed in the somatodendritic and terminal noradrenergic areas locus coeruleus (LC) and prefrontal cortex (PFC), respectively. Further, the effect of chronic fluoxetine treatment (5 mg kg-1, i.p., since the 15th day) on the sensitivity of α2-adrenoceptors was examined under control conditions and in corticosterone-treated rats. The α2-adrenoceptor functionality was analysed in vitro by agonist-mediated [35S]GTPγS binding stimulation and in vivo through the modulation of noradrenaline (NA) release evaluated by dual-probe microdialysis. The concentration-effect curves of the [35S]GTPγS binding stimulation by the agonist UK14304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) demonstrated a desensitization of cortical α2-adrenoceptors induced by corticosterone (-logEC50 = 6.7 ±â€¯0.2 vs 8.2 ±â€¯0.3 in controls) that was reverted by fluoxetine treatment (-logEC50 = 7.5 ±â€¯0.3). Local administration of the α2-adrenoceptor antagonist RS79948 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) (0.1-100 µmol L-1) into the LC induced a concentration-dependent NA increase in the PFC of the control group (Emax = 191 ±â€¯30%) but non-significant effect was observed in corticosterone-treated rats (Emax = 133 ±â€¯46%), reflecting a desensitization of α2-adrenoceptors that control the firing of noradrenergic neurons. Fluoxetine treatment did not alter the corticosterone-induced desensitization in this area (Emax = 136 ±â€¯19%). No effect of fluoxetine on α2-adrenoceptor functionality was observed in control animals (Emax = 223 ±â€¯30%). In PFC, the local administration of RS79948 increased NA in controls (Emax = 226 ±â€¯27%) without effect in the corticosterone group (Emax = 115 ±â€¯26%), suggesting a corticosterone-induced desensitization of terminal α2-adrenoceptors. Fluoxetine administration prevented the desensitization induced by corticosterone in the PFC (Emax = 233 ±â€¯33%) whereas desensitized α2-adrenoceptors in control animals (Emax = -24 ±â€¯10%). These data indicate that chronic corticosterone increases noradrenergic activity by acting at different α2-adrenoceptor subpopulations. Treatment with the antidepressant fluoxetine seems to counteract these changes by acting mainly on presynaptic α2-adrenoceptors expressed in terminal areas.

NMDA Receptor-Dependent Cholinergic Modulation of Mesolimbic Dopamine Cell Bodies: Neurochemical and Behavioral Studies.

Substance abuse disorders are devastating, costly, and difficult to treat. Identifying the neurochemical mechanisms underlying reinforcement promises to provide critical information in the development of effective treatments. Several lines of evidence suggest that striatal dopamine (DA) release serves as a teaching signal in reinforcement learning, and that shifts in DA release from the primary reward to reward-predicting stimuli play a critical role in the self-administration of both natural and non-natural rewards. However, far less is known about the reinforcing effects of motivationally neutral sensory stimuli, or how these signals can facilitate self-administration behavior. Thus, we trained rats ( n = 7) to perform a visual stimulus-induced instrumental task, which involved lever pressing for activation of a stimulus light. We then microinfused vehicle (phosphate buffered saline), carbachol (acetylcholine receptor agonist), or carbachol in the presence of an N-methyl-d-aspartate (NMDA) receptor-specific drug (NMDA itself, or the antagonist, AP5) into the ventral tegmental area (VTA). This enabled us to directly evaluate how chemical modulation of dopamine cell bodies affects the instrumental behavior, as well as the nature of extracellular dopamine transients recorded in the nucleus accumbens shell (NAc shell) using fast-scan cyclic voltammetry (FSCV). Intra-VTA infusion of carbachol enhanced the magnitude and frequency of dopamine transients in the NAc shell and potentiated active lever responding without altering inactive lever responding, as compared to infusion of vehicle. Coinfusion of carbachol with AP5 abolished dopamine transients recorded in the NAc and attenuated active lever responding without altering inactive lever responding. Finally, coadministration of carbachol and NMDA into the VTA restored both lever pressing and dopaminergic signals recorded in the striatum. Together, these results suggest that acetylcholine and glutamate synergistically act at dopamine cells in the VTA to modulate VTA-NAc shell dopaminergic output, and this underlies motivation to lever press for a motivationally neutral visual stimulus.

Phospho-Regulation of Soma-to-Axon Transcytosis of Neurotrophin Receptors.

Axonal targeting of signaling receptors is essential for neuronal responses to extracellular cues. Here, we report that retrograde signaling by target-derived nerve growth factor (NGF) is necessary for soma-to-axon transcytosis of TrkA receptors in sympathetic neurons, and we define the molecular underpinnings of this positive feedback regulation that enhances neuronal sensitivity to trophic factors. Activated TrkA receptors are retrogradely transported in signaling endosomes from distal axons to cell bodies, where they are inserted on soma surfaces and promote phosphorylation of resident naive receptors, resulting in their internalization. Endocytosed TrkA receptors are then dephosphorylated by PTP1B, an ER-resident protein tyrosine phosphatase, prior to axonal transport. PTP1B inactivation prevents TrkA exit from soma and causes receptor degradation, suggesting a "gatekeeper" mechanism that ensures targeting of inactive receptors to axons to engage with ligand. In mice, PTP1B deletion reduces axonal TrkA levels and attenuates neuron survival and target innervation under limiting NGF (NGF+/-) conditions.

Beneficial immunomodulatory and neuro digestive effect in Trypanosoma cruzi infection after Lycopodium clavatum 13c treatment.

Studies show that highly diluted medications demonstrate benefits in treating infections, constituting an alternative for their treatment. The present study evaluated the effects of Lycopodium clavatum, dynamization 13c, in Wistar rats infected with T. cruzi. In this study 42 male rats were intraperitoneally inoculated with T. cruzi - Y strain and allocated into groups: IC (infected control group) and Ly (treated with L. clavatum 13c). The cytokines dosage (IFN-γ, IL-12, IL-10, IL-4), quantification and morphometry of myenteric neurons were evaluated. The treatment with L. clavatum modifies the immune response, with increase of IFN-γ on day 10 a.i. and IL-12 on day 24 a.i., decrease of IL-10 concentration on day 10 a.i. and subsequent increase of this cytokine and IL-4 on day 24 a.i., affording a bigger number of myenteric neurons compared to IC group. Thus, L. clavatum 13c promoted on rats infected with T. cruzi a beneficial immunomodulatory action reducing the pathogenic progression of digestive Chagas disease.

Synaptically driven phosphorylation of ribosomal protein S6 is differentially regulated at active synapses versus dendrites and cell bodies by MAPK and PI3K/mTOR signaling pathways.

High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR and MAPK/ERK-dependent kinases. Using phospho-specific antibodies for rpS6 at different sites (ser235/236 versus ser240/244), we show that delivery of the PI3-kinase inhibitor, wortmannin, decreased rpS6 phosphorylation throughout the somatodendritic compartment (granule cell layer, inner molecular layer, outer molecular layer), especially in granule cell bodies while sparing phosphorylation at activated synapses (middle molecular layer). In contrast, delivery of U0126, an MEK inhibitor, attenuated rpS6 phosphorylation specifically in the dendritic laminae leaving phosphorylation in the granule cell bodies intact. Delivery of the mTOR inhibitor, rapamycin, abolished activation of rpS6 phosphorylation in granule cell bodies and dendrites, whereas delivery of a selective S6K1 inhibitor, PF4708671, or RSK inhibitor, SL0101-1, attenuated rpS6 phosphorylation throughout the postsynaptic cell. These results reveal that MAPK/ERK-dependent signaling is predominately responsible for the selective induction of rpS6 phosphorylation at active synapses. In contrast, PI3-kinase/mTOR-dependent signaling induces rpS6 phosphorylation throughout the somatodendritic compartment but plays a minimal role at active synapses. Collectively, these results suggest a potential mechanism by which PI3-kinase/mTOR and MAPK/ERK pathways regulate translation at specific subcellular compartments in response to synaptic activity.

Widespread reduction of dopamine cell bodies and terminals in adult rats exposed to a low dose regimen of MDMA during adolescence.

Although MDMA (3,4-methylendioxymethamphetamine, ecstasy) neurotoxicity in serotonin neurons is largely recognized in a wide variety of species including man, neurotoxicity in dopamine (DA) neurons is thought to be species-specific. MDMA is mainly consumed by adolescents, often in conjunction with caffeine (Energy Drinks) and this association has been reported to exacerbate MDMA toxic effects. In order to model these aspects of MDMA use, vis-à-vis their impact on DA neurons, we investigated the effects of adolescent exposure to low doses of MDMA (5 mg/kg for 10 days), alone or in combination with caffeine (10 mg/kg) on neuronal and functional DA indices and on recognition memory in adult rats. MDMA reduced density of tyrosine hydroxylase (TH) positive neurons in the ventral tegmental area and in the substantia nigra pars compacta, and immunoreactivity of TH and DA transporter in the nucleus accumbens (NAc) shell and core, and caudate-putamen. This same treatment caused a reduction of basal dialysate DA in the NAc core. MDMA-pretreated rats also showed behavioral sensitization to a MDMA challenge at adulthood and potentiation of MDMA-induced increase of dialysate DA in the NAc core, but not in the NAc shell. In addition, MDMA-treated rats displayed a deficit in recognition memory. Caffeine co-administration did not affect the above outcomes. Our results show that adolescent exposure of rats to low doses of MDMA induces long-lasting and widespread reduction of DA neurons indicative of a neurotoxic effect on DA neurons and suggestive of a degeneration of the same neurons.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (ß-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons.

The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.

Folic acid deficiency increases brain cell injury via autophagy enhancement after focal cerebral ischemia.

Folic acid (FA) deficiency is not only associated with an increased risk of ischemic stroke, but also with increased oxidative DNA damage and brain injury after cerebral ischemia-reperfusion. However, the cellular and molecular mechanisms underlying FA deficiency-associated neuropathogenesis are not completely understood. In the present study, we tested the hypothesis that neuronal autophagy in focal cerebral ischemia rats may be involved in the mechanisms of FA deficiency-induced injury to neuronal cells. The results demonstrated that, accompanied by obvious neuron damage, the expression of the autophagic markers LC3 and Beclin-1, and the formation of 8-OHdG (a marker of oxidative stress to DNA) and autophagosomes were significantly increased in the brain cortex after ischemia-reperfusion. FA deficiency further induced neuronal cell death, and significantly increased the formation of autophagosomes and the expression of LC3 and Beclin-1 in NeuN-positive cell bodies after ischemia-reperfusion. The elevated level of 8-OHdG was also observed in the ischemic cortex of FA deficiency-treated animals. Conversely, the neuronal cell injury, autophagosome accumulation and the effects of LC3 and Beclin1 overexpression caused by FA deficiency were partially blocked by an autophagic inhibitor 3-methyladenine. These results suggest that FA deficiency progresses autophagic activation and aggravates the damage in rat brain cortex following focal cerebral ischemia-reperfusion. The oxidative injury may be involved in cell morphological damage and autophagy alteration caused by FA deficiency.

Differential effects in CGRPergic, nitrergic, and VIPergic myenteric innervation in diabetic rats supplemented with 2% L-glutamine.

The objective of this study was to investigate the effects of 2% L-glutamine supplementation on myenteric innervation in the ileum of diabetic rats, grouped as follows: normoglycemic (N); normoglycemic supplemented with L-glutamine (NG); diabetic (D); and diabetic supplemented with L-glutamine (DG). The ileums were subjected to immunohistochemical techniques to localize neurons immunoreactive to HuC/D protein (HuC/D-IR) and neuronal nitric oxide synthase enzyme (nNOS-IR) and to analyze varicosities immunoreactive to vasoactive intestinal polypeptide (VIP-IR) and calcitonin gene-related peptide (CGRP-IR). L-Glutamine in the DG group (i) prevented the increase in the cell body area of nNOS-IR neurons, (ii) prevented the increase in the area of VIP-IR varicosities, (iii) did not prevent the loss of HuC/D-IR and nNOS-IR neurons per ganglion, and (iv) reduced the size of CGRP-IR varicosities. L-Glutamine in the NG group reduced (i) the number of HuC/D-IR and nNOS-IR neurons per ganglion, (ii) the cell body area of nNOS-IR neurons, and (iii) the size of VIP-IR and CGRP-IR varicosities. 2% L-glutamine supplementation exerted differential neuroprotective effects in experimental diabetes neuropathy that depended on the type of neurotransmitter analyzed. However, the effects of this dose of L-glutamine on normoglycemic animals suggests there are additional actions of this beyond its antioxidant capacity.

MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains.

In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known regulator of polarized trafficking in epithelial cells, and given its presence in OLGs it was therefore of interest to investigate whether MAL played a similar role in PLP transport in OLGs, taking into account its timely expression in these cells. Our data revealed that premature expression of mCherry-MAL in oligodendrocyte progenitor cells interfered with terminal OLG differentiation, although myelin membrane formation per se was not impaired. In fact, also PLP transport to myelin membranes via the cell body plasma membrane was unaffected. However, the typical shift of PLP from TX-100-insoluble membrane domains to CHAPS-resistant, but TX-100-soluble membrane domains, seen in the absence of MAL expression, is substantially reduced upon expression of the MAL protein. Interestingly, not only in vitro, but also in developing brain a strongly diminished shift from TX-100 resistant to TX-100 soluble domains was observed. Consistently, the MAL-expression mediated annihilation of the typical membrane microdomain shift of PLP is also reflected by a loss of the characteristic surface expression profile of conformation-sensitive anti-PLP antibodies. Hence, these findings suggest that MAL is not involved in vesicular PLP trafficking to either the plasma membrane and/or the myelin membrane as such. Rather, we propose that MAL may regulate PLP's distribution into distinct membrane microdomains that allow for lateral diffusion of PLP, directly from the plasma membrane to the myelin membrane once the myelin sheath has been assembled.

Mechanism of induction of fibroblast to corneal endothelial cell.

OBJECTIVE: To explore mechanism of nduction of fibroblast to corneal endothelial cell. METHODS: Rabbit conjunctiva fibroblasts were used as feeder cells, rabbit oral mucosa epithelial cells were used as seed cells, and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium. The transformation effect was observed. RESULTS: As concentration of mitomycin C increased, cell survival rate gradually decreased, cell proliferation was obviously inhibited when concentration≥25 µg/mL; 5 days after being treated by 5 µg/mL mitomycin C, cell body was enlarged and extended without cell fusion, however after being treated by 0.5 µg/mL mitomycin C, cell body was significantly proliferated and gradually fused; after 3 weeks of culture, stratified epithelium appeared on rabbit oral mucosa epithelial cells, differentiation layers were 4-5 and were well differentiated, the morphology was similar to corneal endothelial cells; Under electron microscope, surface layer of cells were polygonal, tightly connected to another with microvilli on the border, there was hemidesmosome between basal cells and human denuded amniotic membrane. CONCLUSIONS: Fibroblast cells have the potential of multi-directional differentiation, effective induction can promote emergence of intercellular desmosomes between seed cells and emergence of epithelial surface microvilli, and differentiate to the corneal endothelial cell. However, clinical application still needs more research and safety evaluation.

Oxygen radicals elicit paralysis and collapse of spinal cord neuron growth cones upon exposure to proinflammatory cytokines.

A persistent inflammatory and oxidative stress is a hallmark of most chronic CNS pathologies (Alzheimer's (ALS)) as well as the aging CNS orchestrated by the proinflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). Loss of the integrity and plasticity of neuronal morphology and connectivity comprises an early step in neuronal degeneration and ultimate decline of cognitive function. We examined in vitro whether TNFα or IL-1ß impaired morphology and motility of growth cones in spinal cord neuron cultures. TNFα and IL-1ß paralyzed growth cone motility and induced growth cone collapse in a dose-dependent manner reflected by complete attenuation of neurite outgrowth. Scavenging reactive oxygen species (ROS) or inhibiting NADPH oxidase activity rescued loss of neuronal motility and morphology. TNFα and IL-1ß provoked rapid, NOX-mediated generation of ROS in advancing growth cones, which preceded paralysis of motility and collapse of morphology. Increases in ROS intermediates were accompanied by an aberrant, nonproductive reorganization of actin filaments. These findings suggest that NADPH oxidase serves as a pivotal source of oxidative stress in neurons and together with disruption of actin filament reorganization contributes to the progressive degeneration of neuronal morphology in the diseased or aging CNS.

Inhibiting geranylgeranylation increases neurite branching and differentially activates cofilin in cell bodies and growth cones.

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 µM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 µM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.