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Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivothe subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell‐based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1genes. Biocompatible poly (lactic‐co‐glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine‐loaded microparticles facilitated neuronal differentiation of SVZ‐GFP cells grafted in hippocampal slices and in in vivorodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial‐derived vehicles may represent a new tool for brain repair strategies. STEMCELLS2012; 30:773–784

The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 μg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivodelivery of proneurogenic factors and neurodegenerative disease treatment.

Overactivation of N-methyl-D-aspartate receptors is known to mediate excitotoxicity due to excessive entry of calcium, leading to the activation of several calcium-dependent enzymes. Calpains are calcium-activated proteases that appear to play a role in excitotoxic neuronal death. Several cellular proteins are substrates for these proteases, particularly the N-methyl-D-aspartate receptor. Recently, cleavage of NR2B subunits has been implicated in excitotoxic neurodegeneration in ischemia. In this work, we investigated the proteolysis by calpains of NR2B subunits of the N-methyl-D-aspartate receptor in the hippocampus of epileptic rats. Our results show that cleaved forms of NR2B subunits are formed after status epilepticus, in the same areas of the hippocampus where calpain activation was detected by immunohistochemical staining of calpain-specific spectrin breakdown products.

Valeriana officinalishave been used in traditional medicine for its sedative, hypnotic, and anticonvulsant effects. There are several eports in the literature supporting a GABAergic mechanism of action for valerian. The rationale of the present work is based on the concept that by decreasing neuronal network excitability valerian consumption may contribute to neuroprotection. The aim of our investigation was to evaluate the neuroprotective effects ofV. officinalisagainst the toxicity induced by amyloid beta peptide 25–35 [Aβ(25–35)]. Cultured rat hip-pocampal neurons were exposed to Aβ(25–35)(25 μM) for 24–48 h,after which morphological and biochemical properties were evaluated. The neuronal injury evoked by Aβ, which includes a decrease in cell educing capacity and associated neuronal degeneration, was prevented by valerian extract. Analysis of intracellular free calcium ([Ca2+]i)indicated that the neuroprotective mechanisms may involve the inhibition of excess influx of Ca2+following neuronal injury. Moreover, membrane peroxidation in rat hippocampal synaptosomes was evaluated, and our data indicate that valerian extract partially inhibited ascorbate/iron-induced peroxidation.

In the present study we assessed the neuroprotective role of aHypericum perforatumethanolic extract and obtained fractions in amyloid-β peptide (Aβ)(25–35)-induced cell death in rat cultured hippocampal neurons. Lipid peroxidation was used as a marker of oxidative stress by following the formation of TBARS in rat cortical synaptosomes, after incubation with ascorbate/Fe2+, alone or in the presence of EC97effective concentrations ofH. perforatumfractions. Induced lipid peroxidation was significantly inhibited by fractions containing flavonol glycosides, flavonol and biflavone aglycones, and by a fraction containing several phenols, mainly chlorogenic acid-type phenolics (21%,77%and 98%, respectively). Lipid peroxidation evaluated after incubation with 25 μM Aβ(25–35), was significantly inhibited byH. perforatumextract.

Based on the idea that seizures may arise from an overshoot of excitation over inhibition, all substances that may decrease glutamatergic function while having no effect or even increasing GABAergic neurotransmission are likely to be effective anticonvulsants. We now review the possible role of three such neuromodulators, kainate, adenosine, and neuropeptide Y receptors in controlling hyperexcitability and epileptogenesis. Particular emphasis is given on the robust neuromodulatory role of these three groups of receptors on the release of glutamate in the hippocampus, a main focus of epilepsy. Moreover, we also give special attention to the mechanisms of receptor activation and coupled signaling events that can be explored as attractive targets for the treatment of epilepsy and excitotoxicity. The present paper is a tribute to Arsélio Pato de Carvalho who has been the main driving force for the development of Neuroscience in Portugal, notably with a particular emphasis on the presynaptic mechanisms of modulation of neurotransmitter release.

Activation of calcium permeable glutamate receptors is likely to be important for neuronal death associated with brain trauma, stroke and neurodegenerative diseases. Cobalt uptake can be used to identify cells containing Ca2-permeable non-NMDA ionotropic glutamate receptors. However, the relative contribution of AMPA and kainate receptors, and also the role of receptor desensitization on the influx of Co2, remain to be established. We found that the selective non-desensitizing activation of AMPA receptors was efficient in promoting Co2staining. However, the selective activation of kainate receptors, even under non-desensitizing conditions, did not result in Co2staining. Taken together, our results show that non-desensitizing stimulation of AMPA, but not of kainate receptors, mediates the influx of Co2in cultured rat hippocampal neurons.

Kainate receptors are ionotropic receptors, also reported to couple to Gi/Goproteins, increasing neuronal excitability through disinhibition of neuronal circuits. We directly tested in hippocampal synaptosomes if kainate receptor‐mediated inhibition of GABA release involved a metabotropic action. The kainate analogue, domoate (3 μM), inhibited by 24% [3H]GABA‐evoked release, an effect reduced by 76% in synaptosomes pre‐treated with pertussis toxin. Protein kinase C inhibition attenuated by 82% domoate‐induced inhibition of GABA release whereas protein kinase C activation did not change kainate receptor binding. Thus, domoate inhibition of GABA release recruits Gi/Goproteins and a protein kinase C pathway.

Kainate receptors are a subtype of ionotropic glutamate receptors, permeable to cations and thus expected to have an excitatory depolarizing action on neurons. However, kainate receptor activation inhibits γ-aminobutyric acid release in the hippocampus through activation of protein kinase C in a pertussis toxin-dependent manner, suggesting a coupling of kainate receptors to G proteins. Thus, we directly investigated the G protein coupling of kainate receptors in the rat hippocampus by using a selective kainate receptor agonist, [3H](2S,4R)-4-methylglutamate ([3H]MGA). [3H]MGA bound to a single site to hippocampal membranes with a KDvalue of 32 nM and a Bmaxvalue of 1024 fmol/mg protein. This binding likely represents kainate receptors because it was displaced by domoate (Ki= 4 nM), kainate (Ki= 11 nM), and 6-cyano-7-nitroquinoxaline-2,3-dione (Ki= 1.4 μM), but not by α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (Ki> 10 μM), (RS)-α-methyl-4-phosphonophenylglycine (Ki> 10 μM), or (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (Ki> 10 μM). Guanylylimidodiphosphate (30 μM), which uncouples all G protein-coupled receptors, shifted to the right the saturation curve of [3H]MGA (KD= 133 nM). This effect was mimicked by pretreatment of hippocampal membranes with modifiers of Gi/Goproteins [30 μMN-ethylmaleimide (KD= 98 nM) or 25 μg/ml pertussis toxin (KD= 95 nM)] but not by a modifier of Gsproteins [50 μg/ml cholera toxin (KD= 32 nM)]. Treatment of solubilized hippocampal membranes with pertussis toxin (25 μg/ml) decreased [3H]MGA affinity (KD= 105–113 nM), which was recovered by reconstitution of these pretreated solubilized hippocampal membranes with Gi/Goproteins (KD= 41–76 nM). These results indicate that hippocampal kainate receptors are coupled to Gi/Goproteins.

We used hippocampal synaptosomes to study the effect of NO originating from NO donors and from the activation of the NO synthase on the Ca2+-dependent release of glutamate due to 4-aminopyridine (4-AP) depolarization. We distinguished between the effects of NO on the exocytotic and on the carrier-mediated release of glutamate, which we found to be related to an increase in cGMP content and to a reduction of the ATP/ADP ratio, respectively. The NO donor hydroxylamine, at concentrations ≤0.3 mM, inhibited the Ca2+-dependent glutamate release evoked by 4-AP, and addition of the NO donor, NOC-7, had a similar effect, which was reversed by the NO scavenger, carboxy-PTIO. Increasing the activity of NO synthase by addition ofl-arginine also led to a decrease in the Ca2+-dependent release of glutamate induced by 4-AP, and this effect was reversed by inhibiting NO synthase withNG-nitro-l-arginine. This depression of the exocytotic release of glutamate was accompanied by an increase in cGMP levels due to the stimulation of soluble guanylyl cyclase by NO, produced either by the NO donors (hydroxylamine <0.3 mM) or by the endogenous NO synthase, but no significant decrease in ATP/ADP ratio was observed. However, at concentrations ≥0.3 mM, hydroxylamine drastically increased the basal release and completely inhibited the Ca2+-dependent release of glutamate (IC50= 168 μM). At these higher levels of NO, cGMP levels dropped to about 40% of the maximal values obtained at lower concentrations, and the ATP/ADP ratio decreased to about 50% (at 0.3 mM hydroxylamine). The large increase in the basal release could be partially inhibited byl-trans-2,4-PDC, previously loaded into the synaptosomes, suggesting that the nonexocytotic basal release occurred by reversal of the glutamate carrier. Therefore, the increase in cGMP induced by NO stimulation of the guanylyl cyclase decreases the exocytotic release of glutamate, but higher NO levels reduce the ATP/ADP ratio by inhibiting mitochondrial function, which therefore causes the massive release of cytosolic glutamate through the glutamate carrier.

WE investigated the role of kainate receptor activation in modulating the influx of Ca2coupled to the exocytotic release of glutamate in rat hippocampal synaptosomal fractions (P2). In whole hippocampus synaptosomes stimulation with domoic acid increased the intracellular free Ca2concentration ([Ca2]i) in a dose-dependent manner with an EC50of 0.16 μM, whereas the EC50for kainate was 0.86 μM and for (±)-n-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) was 43.04 μM. Stimulation of the synaptosomes with 10 μM domoic acid induced Ca2-dependent release of endogenous glutamate. Also, in synaptosomes isolated from the CA3 sub-region of the hippocampus the domoic acid-induced release of glutamate was higher than that from the dentate gyrus (221.3), from the CA1 (188.1) or from the whole hippocampal synaptosomes (131.5). These results support the existence of a presynaptic kainate receptor which may control the exocytotic release of glutamate in the CA3 sub-region of the rat hippocampus.

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)‐(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)‐PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid‐induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor‐mediated neurodegeneration in the hippocampus.