How Are Synapses Born? A Functional and Molecular View of the Role of the Wnt Signaling Pathway.

Synaptic transmission is a dynamic process that requires precise regulation. Early in life, we must be able to forge appropriate connections (add and remove) to control our behavior. Neurons must recognize appropriate targets, and external soluble factors that activate specific signaling cascades provide the regulation needed to achieve this goal. Wnt signaling has been implicated in several forms of synaptic plasticity, including functional and structural changes associated with brain development. The analysis of synapses from an electrophysiological perspective allows us to characterize the functional role of cellular signaling pathways involved in brain development. The application of quantal theory to principles of developmental plasticity offers the possibility of dissecting the function of structural changes associated with the birth of new synapses as well as the maturation of immature silent synapses. Here, we focus on electrophysiological and molecular evidence that the Wnt signaling pathway regulates glutamatergic synaptic transmission, specifically N-methyl-d-aspartate receptors (NMDARs), to control the birth of new synapses. We also focus on the role of Wnts in the conversion of silent synapses into functional synapses.

Gamma-Decanolactone Alters the Expression of GluN2B, A1 Receptors, and COX-2 and Reduces DNA Damage in the PTZ-Induced Seizure Model After Subchronic Treatment in Mice.

Gamma-decanolactone (GD) has been shown to reduce epileptic behavior in different models, inflammatory decreasing, oxidative stress, and genotoxic parameters. This study assessed the GD effect on the pentylenetetrazole (PTZ) model after acute and subchronic treatment. We evaluated the expression of the inflammatory marker cyclooxygenase-2 (COX-2), GluN2B, a subunit of the NMDA glutamate receptor, adenosine A1 receptor, and GD genotoxicity and mutagenicity. Male and female mice were treated with GD (300 mg/kg) for 12 days. On the tenth day, they were tested in the Hot Plate test. On the thirteenth day, all animals received PTZ (90 mg/kg), and epileptic behavior PTZ-induced was observed for 30 min. Pregabalin (PGB) (30 mg/kg) was used as a positive control. Samples of the hippocampus and blood were collected for Western Blotting analyses and Comet Assay and bone marrow to the Micronucleus test. Only the acute treatment of GD reduced the seizure occurrence and increased the latency to the first stage 3 seizures. Males treated with GD for 12 days demonstrated a significant increase in the expression of the GluN2B receptor and a decrease in the COX-2 expression. Acute and subchronic treatment with GD and PGB reduced the DNA damage produced by PTZ in males and females. There is no increase in the micronucleus frequency in bone marrow after subchronic treatment. This study suggests that GD, after 12 days, could not reduce PTZ-induced seizures, but it has been shown to protect against DNA damage, reduce COX-2 and increase GluN2B expression.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway.

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Reduced Expression of Hippocampal GluN2A-NMDAR Increases Seizure Susceptibility and Causes Deficits in Contextual Memory.

N-methyl-D-aspartate receptors are heterotetramers composed of two GluN1 obligatory subunits and two regulatory subunits. In cognitive-related brain structures, GluN2A and GluN2B are the most abundant regulatory subunits, and their expression is subjected to tight regulation. During development, GluN2B expression is characteristic of immature synapses, whereas GluN2A is present in mature ones. This change in expression induces a shift in GluN2A/GluN2B ratio known as developmental switch. Moreover, modifications in this relationship have been associated with learning and memory, as well as different pathologies. In this work, we used a specific shRNA to induce a reduction in GluN2A expression after the developmental switch, both in vitro in primary cultured hippocampal neurons and in vivo in adult male Wistar rats. After in vitro characterization, we performed a cognitive profile and evaluated seizure susceptibility in vivo. Our in vitro results showed that the decrease in the expression of GluN2A changes GluN2A/GluN2B ratio without altering the expression of other regulatory subunits. Moreover, rats expressing the anti-GluN2A shRNA in vivo displayed an impaired contextual fear-conditioning memory. In addition, these animals showed increased seizure susceptibility, in terms of both time and intensity, which led us to conclude that deregulation in GluN2A expression at the hippocampus is associated with seizure susceptibility and learning-memory mechanisms.

Glutamate transporter-1 link astrocytes with heightened aggressive behavior induced by steroid abuse in male CF1 mice.

The astrocytic glutamate transporter GLT-1 performs glutamate uptake thereby mediating NMDAr responses in neurons. Ceftriaxone (CEF) upregulates astrocytic GLT-1 expression/activity, which could counteract excessive glutamate levels and aggressive behavior induced by anabolic synthetic steroids such as nandrolone decanoate (ND). Here, adult male CF-1 mice were allocated to oil (VEH), ND, CEF, and ND/CEF groups. Mice were subcutaneously (s.c.) injected with ND (15 mg/kg) or VEH for 19 days, and received intraperitoneal (i.p.) injections of CEF (200 mg/kg) or saline for 5 days. The ND/CEF group received ND for 19 days plus coadministration of CEF in the last 5 days. On the 19th day, the aggressive phenotypes were evaluated through the resident-intruder test. After 24 h, cerebrospinal fluid was collected to measure glutamate levels, and the pre-frontal cortex was used to assess GLT-1, pGluN2BTyr1472, and pGluN2ATyr1246 by Western blot. Synaptosomes from the left brain hemisphere was used to evaluate mitochondrial function including complex II-succinate dehydrogenase (SDH), Ca2+ handling, membrane potential (ΔÑ°m), and H2O2 production. ND decreased the latency for the first attack and increased the number of attacks by the resident mice against the intruder, mechanistically associated with an increase in glutamate levels and pGluN2BTyr1472 but not pGluN2ATyr1244, and GLT-1 downregulation. The abnormalities in mitochondrial Ca2+ influx, SDH, ΔÑ°m, and H2O2 implies in deficient energy support to the synaptic machinery. The ND/CEF group displayed a decreased aggressive behavior, normalization of glutamate and pGluN2BTyr1472levels, and mitochondrial function at synaptic terminals. In conclusion, the pharmacological modulation of GLT-1 highlights its relevance as an astrocytic target against highly impulsive and aggressive phenotypes.

Neuronal surface P antigen (NSPA) modulates postsynaptic NMDAR stability through ubiquitination of tyrosine phosphatase PTPMEG.

BACKGROUND: Cognitive dysfunction (CD) is common among patients with the autoimmune disease systemic lupus erythematosus (SLE). Anti-ribosomal P autoantibodies associate with this dysfunction and have neuropathogenic effects that are mediated by cross-reacting with neuronal surface P antigen (NSPA) protein. Elucidating the function of NSPA can then reveal CD pathogenic mechanisms and treatment opportunities. In the brain, NSPA somehow contributes to glutamatergic NMDA receptor (NMDAR) activity in synaptic plasticity and memory. Here we analyze the consequences of NSPA absence in KO mice considering its structural features shared with E3 ubiquitin ligases and the crucial role of ubiquitination in synaptic plasticity. RESULTS: Electrophysiological studies revealed a decreased long-term potentiation in CA3-CA1 and medial perforant pathway-dentate gyrus (MPP-DG) hippocampal circuits, reflecting glutamatergic synaptic plasticity impairment in NSPA-KO mice. The hippocampal dentate gyrus of these mice showed a lower number of Arc-positive cells indicative of decreased synaptic activity and also showed proliferation defects of neural progenitors underlying less adult neurogenesis. All this translates into poor spatial and recognition memory when NSPA is absent. A cell-based assay demonstrated ubiquitination of NSPA as a property of RBR-type E3 ligases, while biochemical analysis of synaptic regions disclosed the tyrosine phosphatase PTPMEG as a potential substrate. Mice lacking NSPA have increased levels of PTPMEG due to its reduced ubiquitination and proteasomal degradation, which correlated with lower levels of GluN2A and GluN2B NMDAR subunits only at postsynaptic densities (PSDs), indicating selective trafficking of these proteins out of PSDs. As both GluN2A and GluN2B interact with PTPMEG, tyrosine (Tyr) dephosphorylation likely drives their endocytic removal from the PSD. Actually, immunoblot analysis showed reduced phosphorylation of the GluN2B endocytic signal Tyr1472 in NSPA-KO mice. CONCLUSIONS: NSPA contributes to hippocampal plasticity and memory processes ensuring appropriate levels of adult neurogenesis and PSD-located NMDAR. PTPMEG qualifies as NSPA ubiquitination substrate that regulates Tyr phosphorylation-dependent NMDAR stability at PSDs. The NSPA/PTPMEG pathway emerges as a new regulator of glutamatergic transmission and plasticity and may provide mechanistic clues and therapeutic opportunities for anti-P-mediated pathogenicity in SLE, a still unmet need.

Zika Virus Promotes Neuronal Cell Death in a Non-Cell Autonomous Manner by Triggering the Release of Neurotoxic Factors.

Zika virus (ZIKV) has recently caused a worldwide outbreak of infections associated with severe neurological complications, including microcephaly in infants born from infected mothers. ZIKV exhibits high neurotropism and promotes neuroinflammation and neuronal cell death. We have recently demonstrated that N-methyl-d-aspartate receptor (NMDAR) blockade by memantine prevents ZIKV-induced neuronal cell death. Here, we show that ZIKV induces apoptosis in a non-cell autonomous manner, triggering cell death of uninfected neurons by releasing cytotoxic factors. Neuronal cultures infected with ZIKV exhibit increased levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and glutamate. Moreover, infected neurons exhibit increased expression of GluN2B and augmented intracellular Ca2+ concentration. Blockade of GluN2B-containing NMDAR by ifenprodil normalizes Ca2+ levels and rescues neuronal cell death. Notably, TNF-α and IL-1ß blockade decreases ZIKV-induced Ca2+ flux through GluN2B-containing NMDARs and reduces neuronal cell death, indicating that these cytokines might contribute to NMDAR sensitization and neurotoxicity. In addition, ZIKV-infected cultures treated with ifenprodil exhibits increased activation of the neuroprotective pathway including extracellular signal-regulated kinase and cAMP response element-binding protein, which may underlie ifenprodil-mediated neuroprotection. Together, our data shed some light on the neurotoxic mechanisms triggered by ZIKV and begin to elucidate how GluN2B-containing NMDAR blockade can prevent neurotoxicity.

Stress-induced resistance to the fear memory labilization/reconsolidation process. Involvement of the basolateral amygdala complex.

Consolidated memories can enter into a labile state after reactivation followed by a restabilization process defined as reconsolidation. This process can be interfered with Midazolam (MDZ), a positive allosteric modulator of the GABA-A receptor. The present study has evaluated the influence of prior stress on MDZ's interfering effect. We also assessed the influence of both systemic and intra-basolateral amygdala (BLA) infusion of d-cycloserine (DCS), a partial agonist of the NMDA receptors, on the MDZ effect in previously stressed rats. Furthermore, we analyzed the effect of stress on the expression of Zif-268 and the GluN2B sites, two molecular markers of the labilization/reconsolidation process, following reactivation. The results revealed that prior stress resulted into a memory trace that was insensitive to the MDZ impairing effect. Both systemic and intra-BLA DCS administration previous to reactivation restored MDZ's disruptive effect on memory reconsolidation in stressed animals. Further, reactivation enhanced Zif-268 expression in the BLA in control unstressed rats, whereas no elevation was observed in stressed animals. In agreement with the behavioral findings, DCS restored the increased level of Zif-268 expression in the BLA in stressed animals. Moreover, memory reactivation in unstressed animals elevated GluN2B expression in the BLA, thus suggesting that this effect is involved in memory destabilization, whereas stressed animals did not reveal any changes. These findings are consistent with resistance to the MDZ effect in these rats, indicating that stress exposure prevents the onset of destabilization following reactivation. In summary, prior stress limited both the occurrence of the reactivation-induced destabilization and restabilization.

Effects of a Flavonoid-Rich Fraction on the Acquisition and Extinction of Fear Memory: Pharmacological and Molecular Approaches.

The effects of flavonoids have been correlated with their ability to modulate the glutamatergic, serotoninergic, and GABAergic neurotransmission; the major targets of these substances are N-methyl-D-aspartic acid receptor (NMDARs), serotonin type1A receptor (5-HT1ARs), and the gamma-aminobutyric acid type A receptors (GABAARs). Several studies showed that these receptors are involved in the acquisition and extinction of fear memory. This study assessed the effects of treatment prior to conditioning with a flavonoid-rich fraction from the stem bark of Erythrina falcata (FfB) on the acquisition and extinction of the conditioned suppression following pharmacological manipulations and on gene expression in the dorsal hippocampus (DH). Adult male Wistar rats were treated before conditioned fear with FfB, vehicle, an agonist or antagonist of the 5-HT1AR, GABAARs or the GluN2B-NMDAR or one of these antagonists before FfB treatment. The effects of these treatments on fear memory retrieval, extinction training and extinction retrieval were evaluated at 48, 72, and 98 h after conditioning, respectively. We found that activation of GABAARs and inactivation of GluN2B-NMDARs play important roles in the acquisition of lick response suppression. FfB reversed the effect of blocking GluN2B-NMDARs on the conditioned fear and induced the spontaneous recovery. Blocking the 5-HT1AR and the GluN2B-NMDAR before FfB treatment seemed to be associated with weakening of the spontaneous recovery. Expression of analysis of DH samples via qPCR showed that FfB treatment resulted in the overexpression of Htr1a, Grin2a, Gabra5, and Erk2 after the retention test and of Htr1a and Erk2 after the extinction retention test. Moreover, blocking the 5-HT1ARs and the GluN2B-NMDARs before FfB treatment resulted in reduced Htr1a and Grin2b expression after the retention test, but played a distinct role in Grin2a and Erk2 expression, according session evaluated. We show for the first time that the serotoninergic and glutamatergic receptors are important targets for the effect of FfB on the conditioned fear and spontaneous recovery, in which the ERK signaling pathway appears to be modulated. Further, these results provide important information regarding the role of the DH in conditioned suppression. Taken together, our data suggest that FfB represents a potential therapy for preventing or treating memory impairments.

GluN2B N-methyl-D-aspartic acid receptor subunit mediates atorvastatin-Induced neuroprotection after focal cerebral ischemia.

Statins are potent cholesterol biosynthesis inhibitors that exert protective effects in humans and in experimental models of stroke. The mechanisms involved in these protective actions are not completely understood. This study evaluates whether atorvastatin (ATV) treatment affects the GluN1 and GluN2B subunits of the N-methyl-D-aspartic acid receptor in the somatosensory cerebral cortex at short and long periods following ischemia. Sham and ischemic male Wistar rats received 10 mg/kg of ATV or placebo by gavage every 24 hr for 3 consecutive days. The first dose was administered 6 hr after ischemia-reperfusion or the sham operation. ATV treatment resulted in faster recovery of neurological scores than placebo, prevented the appearance of pyknotic neurons, and restored microtubule-associated protein 2 and neuronal nuclei staining to control values in the somatosensory cerebral cortex and the hippocampus at 72 hr and 15 days postischemia. Furthermore, ATV prevented spatial learning and memory deficits caused by cerebral ischemia. Cerebral ischemia reduced the number of GluN1/PSD-95 and GluN2B/PSD-95 colocalization clusters in cortical pyramidal neurons and reduced the levels of brain-derived neurotrophic factor (BDNF) in the cerebral cortex. These effects of the ischemic insult were prevented by ATV, which also induced GluN2B/PSD-95 colocalization in neuronal processes and an association of GluN2B with TrkB. The GluN2B pharmacological inhibitor ifenprodil prevented the increase in BDNF levels and the motor and cognitive function recovery caused by ATV in ischemic rats. These findings indicate that GluN2B is involved in the neuroprotective mechanism elicited by ATV to promote motor and cognitive recovery after focal cerebral ischemia.