Mechanisms regulating spill‐over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons

N‐Methyl‐d ‐aspartate glutamate receptors (NMDARs) contribute to neural development, plasticity and survival, but they are also linked with neurodegeneration. NMDARs at synapses are activated by coincident glutamate release and depolarization. NMDARs distal to synapses can sometimes be recruited by ‘spill‐over’ of glutamate during high‐frequency synaptic stimulation or when glutamate uptake is compromised, and this influences the shape of NMDAR‐mediated postsynaptic responses. In substantia nigra dopamine neurons, activation of NMDARs beyond the synapse during different frequencies of presynaptic stimulation has not been explored, even though excitatory afferents from the subthalamic nucleus show a range of firing frequencies, and these frequencies change in human and experimental Parkinson's disease. This study reports that high‐frequency stimulation (80 Hz/200 ms) evoked NMDAR‐excitatory postsynaptic currents (EPSCs) that were larger and longer lasting than those evoked by single stimuli at low frequency (0.1 Hz). MK‐801, which irreversibly blocked NMDAR‐EPSCs activated during 0.1‐Hz stimulation, left a proportion of NMDAR‐EPSCs that could be activated by 80‐Hz stimulation and that may represent activity of NMDARs distal to synapses. TBOA, which blocks glutamate transporters, significantly increased NMDAR‐EPSCs in response to 80‐Hz stimulation, particularly when metabotropic glutamate receptors (mGluRs) were also blocked, indicating that recruitment of NMDARs distal to synapses is regulated by glutamate transporters and mGluRs. These regulatory mechanisms may be essential in the substantia nigra for restricting glutamate diffusion from synaptic sites and keeping NMDAR‐EPSCs in dopamine neurons relatively small and fast. Failure of glutamate transporters may contribute to the declining health of dopamine neurons during pathological conditions.     

Modulation of allopregnanolone on excitatory transmitters release from single glutamatergic terminal

Neurosteroids such as allopregnanolone (Allo) are widely distributed in the brain and may modulate neuronal excitability under physiological or pathological states. Allo modulates GABA_A receptor responses, and in this study we investigated the functional effects of Allo on presynaptic GABA_A receptors on single glutamatergic nerve terminal projecting on CA3 neurons. In the present study, we measured spontaneous and evoked excitatory postsynaptic currents (sEPSCs and eEPSCs), the latter was elicited with single or paired-pulse focal electrical stimulation, using mechanically isolated ‘synaptic bouton’ preparation. Allo (10 nM) increased significantly eEPSC amplitude while decreasing the failure rate (R_f) and the paired-pulse response ratio (PPR). Conversely high concentration (100 nM) of Allo decreased eEPSC amplitude and increased R_f and PPR. Allo also increased significantly the frequency and amplitude of sEPSCs at low concentrations (10–30 nM) but at high concentration (100 nM) it had no effect on current amplitude but modestly decreased sEPSC frequency. Application of Allo at nanomolar concentrations facilitated exogenous muscimol-induced outward postsynaptic currents but had no effect on glutamate-induced inward postsynaptic currents. Our results demonstrate that Allo modulates glutamate release via presynaptic GABA_A receptors, in addition to its better characterized effects to modulate postsynaptic GABA_A responses. Both pre- and postsynaptic GABA_A receptor modulation is likely to contribute to the physiological actions of neurosteroids.     

Glutamate transporters and metabotropic receptors regulate excitatory neurotransmission in the medial entorhinal cortex of the rat

In layer III of the medial entorhinal cortex (mEC), a region that is especially prone to cell damage in Alzheimer's disease, schizophrenia and epilepsy, effects of blocking glutamate uptake on excitatory synaptic transmission were studied. Two competitive glutamate transporter antagonists, TBOA and tPDC, reduced the amplitude of pharmacologically isolated AMPAR and NMDAR mediated EPSPs/EPSCs without changing the time course of the events. This effect was mimicked by tACPD, an agonist of groups I and II metabotropic glutamate receptors (mGluRs). The competitive groups I and II mGluR antagonist MCPG blocked the depression of the EPSC amplitude induced by tACPD and also prevented the effect of either TBOA or tPDC. Furthermore, EGLU, which selectively antagonizes group II mGluRs, blocked the effect of tPDC and LY3414965, a specific group I mGluR antagonist, abolished the reduction of amplitude caused by TBOA. Additionally, application of TBOA increased the paired-pulse index, suggesting a presynaptic mechanism for the depression of EPSP/EPSC amplitude. The present data suggest that glutamate transporters and group I/II mGluRs regulate excitatory synaptic transmission in the mEC. Presynaptic mGluRs may limit excessive glutamate accumulation if uptake becomes compromised.     

Kinetic characterization of l-[H]glutamate uptake inhibition and increase oxidative damage induced by glutaric acid in striatal synaptosomes of rats

Glutaric acidemia type I (GA-I) is an inherited metabolic disease characterized by accumulation of glutaric acid (GA) and striatal degeneration. Although growing evidence suggests that excitotoxicity and oxidative stress play central role in the neuropathogenesis of this disease, mechanism underlying striatal damage in this disorder is not well established. Thus, we decided to investigate the in vitro effects of GA 10 nM (a low concentration that can be present initial development this disorder) on l-[³H]glutamate uptake and reactive oxygen species (ROS) generation in synaptosomes from striatum of rats. GA reduced l-[³H]glutamate uptake in synaptosomes from 1 up to 30 min after its addition. Furthermore, we also provided some evidence that GA competes with the glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), suggesting a possible interaction of GA with glutamate transporters on synaptosomes. Moreover, GA produced a significant decrease in the V_MAX of l-[³H]glutamate uptake, but did not affect the K_D value. Although the GA did not show oxidant activity per se, it increased the ROS generation in striatal synaptosomes. To evaluate the involvement of reactive species generation in the GA-induced l-[³H]glutamate uptake inhibition, trolox (0.3, 0.6 and 6 μM) was added on the incubation medium. Statistical analysis showed that trolox did not decrease inhibition of GA-induced l-[³H]glutamate uptake, but decreased GA-induced reactive species formation in striatal synaptosomes (1, 3, 5, 10, 15 and 30 min), suggesting that ROS generation appears to occur secondarily to glutamatergic overstimulation in this model of organic acidemia. Since GA induced DCFH oxidation increase, we evaluate the involvement of glutamate receptor antagonists in oxidative stress, showing that CNQX, but not MK-801, decreased the DCFH oxidation increase in striatal synaptosomes. Furthermore, the results presented in this report suggest that excitotoxicity elicited by low concentration of GA, could be in part by maintaining this excitatory neurotransmitter in the synaptic cleft by non-competitive inhibition of glutamate uptake. Thus the present data may explain, at least partly, initial striatal damage at birth, as evidenced by acute bilateral destruction of caudate and putamen observed in children with GA-I.     

Transporter‐mediated replacement of extracellular glutamate for GABA in the developing murine neocortex

During early development, cortical neurons migrate from their places of origin to their final destinations where they differentiate and establish synaptic connections. During corticogenesis, radially migrating cells move from deeper zone to the marginal zone, but they do not invade the latter. This “stop” function of the marginal zone is mediated by a number of factors, including glutamate and γ‐aminobutyric acid (GABA), two main neurotransmitters in the central nervous system. In the marginal zone, GABA has been shown to be released via GABA transporters (GAT)‐2/3, whereas glutamate transporters (EAATs) operate in the uptake mode. In this study, GABAergic postsynaptic currents (GPSCs) were recorded from Cajal‐Retzius cells in the marginal zone of murine neonatal neocortex using a whole‐cell patch‐clamp technique. Minimal electrical stimulation was applied to elicit evoked GPSCs using a paired‐pulse protocol. EAAT blockade with dl ‐threo‐b‐benzyloxyaspartic acid (dl ‐TBOA), a specific non‐transportable EAAT antagonist, abolishes constitutive GAT‐2/3‐mediated GABA release. In contrast to dl ‐TBOA, d ‐aspartate, an EAAT substrate, fails to block GAT‐2/3‐mediated GABA release. SNAP‐5114, a specific GAT‐2/3 antagonist, induced an elevation of intracellular sodium concentration ([Na⁺]_i) under resting conditions and in the presence of d ‐aspartate, indicating that GAT‐2/3 operates in reverse mode. In the presence of dl ‐TBOA, however, SNAP‐5114 elicited a [Na⁺]_i decrease, demonstrating that GAT‐2/3 operates in uptake mode. We conclude that EAATs via intracellular Na⁺ signaling and/or cell depolarization can govern the strength/direction of GAT‐mediated GABA transport.     

Ceftriaxone-mediated upregulation of the glutamate transporter GLT-1 contrasts neurotoxicity evoked by kainate in rat organotypic spinal cord cultures

Excitotoxicity is a major pathological trigger of neurodegenerative diseases like amyotrophic lateral sclerosis. This process is caused by excessive release of the transmitter glutamate that overwhelms the capacity of astroglia transporters to maintain a low extracellular level of this aminoacid and strongly stimulates neurons. Using an in vitro model of rat organotypic spinal slice culture, we explored if the excitotoxicity caused by the potent glutamate analogue kainate, widely employed as a paradigm to evoke neurotoxicity in the central nervous system, was prevented by the antibiotic ceftriaxone known to enhance glutamate transporter expression. We also tested if excitotoxicity was made worse by inhibiting glutamate uptake with dl-threo-β-benzyloxyaspartate (TBOA). These experiments were aimed at clarifying the relative contribution to neurotoxicity by kainate-activation of glutamate receptors or kainate-mediated release of glutamate. Neither ceftriaxone nor TBOA alone had adverse effects. Ceftriaxone (10 μM; 3 days) significantly decreased delayed cell death induced by kainate (100 μM; 1 h) and limited neuronal damage especially to motoneurons. This effect was associated to stronger astrocytic immunostaining of the glutamate transporter GLT-1. Conversely, pharmacological inhibition of glutamate uptake with TBOA was per se unable to induce neurotoxicity, yet it intensified cell death evoked by kainate. These data indicate that kainate-mediated glutamate release was critical to damage neurons, an effect prevented by up regulating glutamate uptake. These data suggest that modulating glutamate uptake is an important strategy to preserve neuronal networks.     

Decreased glutamate transport enhances excitability in a rat model of cortical dysplasia

Glutamate transporters function to maintain low levels of extracellular glutamate and play an important role in synaptic transmission at many synapses. Disruption of glutamate transporter function or expression can result in increased extracellular glutamate levels. Alterations in glutamate transporter expression have been reported in human epilepsy and animal seizure models. Functional electrophysiological changes that occur when transporter expression is disrupted in chronic epilepsy models have not been examined. Here, we used a freeze-induced model of cortical dysplasia to test the role of glutamate transporters in synaptic hyperexcitability. We report that inhibiting glutamate transporters with the non-selective antagonist, DL-threo-beta-benzylozyaspartic acid (TBOA) preferentially prolongs postsynaptic currents (PSCs) and decreases the threshold for evoking epileptiform activity in lesioned compared to control cortex. The effect of inhibiting uptake is mediated primarily by the glia glutamate transporter (GLT-1) since the selective antagonist dihydrokainate (DHK) mimicked the effects of TBOA. The effect of uptake inhibition is mediated by activation of N-methyl-D-aspartate (NMDA) receptors since D-(-)-2-amino-5-phosphonovaleric acid (APV) prevents TBOA-induced effects. Neurons in lesioned cortex also have a larger tonic NMDA current. These results indicate that chronic changes in glutamate transporters and NMDA receptors contribute to hyperexcitability in cortical dysplasia.     

Presynaptic and postsynaptic modulation of glutamatergic synaptic transmission by activation of α1‐ and β‐adrenoceptors in layer V pyramidal neurons of rat cerebral cortex

Adrenergic agonists have different modulatory effects on excitatory synaptic transmission depending on the receptor subtypes involved. The present study examined the loci of α₁‐ and β‐adrenoceptor agonists, which have opposite effects on excitatory neural transmission, involved in modulation of glutamatergic transmission in layer V pyramidal cells of rat cerebral cortex. Phenylephrine, an α₁‐adrenoceptor agonist, suppressed the amplitude of AMPA receptor‐mediated excitatory postsynaptic currents evoked by repetitive electrical stimulation (eEPSCs, 10 pulses at 33 Hz). The coefficient of variation (CV) of the 1st eEPSC amplitude and paired‐pulse ratio (PPR), which were sensitive to extracellular Ca²⁺ concentration, were not affected by phenylephrine. Phenylephrine suppressed miniature EPSC (mEPSC) amplitude without changing its frequency. In contrast, isoproterenol, a β‐adrenoceptor agonist, strongly increased the amplitude of the 1st eEPSC compared with that of the 2nd to 10th eEPSCs, which resulted in a decrease in PPR. Isoproterenol‐induced enhancement of eEPSC amplitude was accompanied by a decrease in CV. Isoproterenol increased the frequency of mEPSCs without significant effect on amplitude. Phenylephrine suppressed inward currents evoked by puff application of glutamate, AMPA, or NMDA, whereas isoproterenol application was not accompanied by significant changes in these inward currents. These findings suggest that phenylephrine decreases eEPSCs through postsynaptic AMPA or NMDA receptors, while the effects of isoproterenol are mediated by facilitation of glutamate release from presynaptic terminals without effect on postsynaptic glutamate receptors. These two different mechanisms of modulation of excitatory synaptic transmission may improve the “signal‐to‐noise ratio” in cerebral cortex. Synapse 63:269–281, 2009. © 2008 Wiley‐Liss, Inc.     

Differences in myocardial sympathetic degeneration and the clinical features of the subtypes of Parkinson’s disease

Parkinson’s disease (PD) can be divided into the akinetic-rigid (ART), mixed (MT), and tremor-dominant (TDT) subtypes according to the clinically dominant symptoms. We analyzed the correlations between ¹²³I-meta-iodobenzylguanidine (MIBG) uptake and the clinical features of patients with various PD subtypes. In addition, we evaluated the relationship between MIBG uptake and the severity of the cardinal motor symptoms among patients with PD subtypes. The mean Unified Parkinson’s Disease Rating Scale motor scores differed significantly among patients with different PD subtypes (± standard deviation [SD]) (ART, 34.6 ± 18.28; MT, 24.63 ± 7.78; TDT, 16.22 ± 4.15, p = 0.002), especially between the ART and TDT subtypes (p = 0.022). The mean MIBG uptake (± SD) was decreased in the TDT (1.69 ± 0.39), MT (1.35 ± 0.32), and ART (1.35 ± 0.22) subtypes (p = 0.049). The MIBG uptake values differed significantly between the ART and TDT subtypes (p = 0.02). The MIBG uptake was inversely correlated with the severity of hypokinesia in the ART subtype (r = −0.75; p = 0.01) and the MT subtype (r = −0.8; p = 0.02), but it was not correlated with the severity of any of the parkinsonian motor symptoms in the TDT subtype. These results imply that hypokinesia is strongly associated with sympathetic myocardial degeneration and that sympathetic myocardial degeneration can reflect the progression of the disease in patients with the ART and mixed MT subtypes of PD.     

Modification of Epileptiform Discharges in Neocortical Neurons Following Glutamate Uptake Inhibition

Summary:  Sodium-dependent high-affinity glutamate transporters regulate synaptic glutamate levels to maintain low ambient levels of glutamate and prevent excitotoxicity. Most studies using pharmacological inhibition of glutamate transport to examine the involvement of glutamate transporters in regulating synaptic activity have examined small synaptic currents. Using in vitro brain slices, we investigated the effects of uptake inhibition on two types of epileptiform activity, bicuculline-induced paroxysmal activity, and epileptiform responses in the freeze-lesion epilepsy model. In layer II/III pyramidal cells of the prefrontal cortex, inhibiting uptake with low concentrations of DL-threo-ß-benzyloxyaspartic acid (TBOA) (20 or 30 μM) prolonged bicuculline-induced epileptiform activity. At higher concentrations, TBOA (150 or 300 μ M ) caused a transient enhancement of epileptiform discharges that was followed by a decrease. In the freeze-lesion model, inhibiting uptake also increased the amplitude and response area of evoked activity. The prolongation of epileptiform activity exhibited by the inhibition of glutamate uptake (TBOA 20 or 30 μ M ) is attributed to an increase in the level of glutamate extracellularly during uptake blockade, resulting in sustained activation of glutamate receptors. The decrease in epileptiform activity at higher TBOA concentration could be due to glutamate receptor desensitization or loss of excitability due to a depolarization block. The present results suggest that decreases in glutamate uptake can be proconvulsant in the two models of epilepsy examined.     

Molecular evidence that cortical synaptic growth predominates during the first decade of life in humans

Theories concerning the pathology of human neurodevelopmental disorders that emerge in adolescence, such as schizophrenia, often hypothesize that there may be a failure of normal cortical synaptic loss or pruning. However, direct evidence that synaptic regression is a major developmental event in the adolescent human cortex is limited. Furthermore, developmental work in rodents suggested that synaptic regression in adolescence is not a major feature of cortical development. Thus, we set out to determine when and to what extent molecular markers of synaptic terminals [synaptophysin (SYP), SNAP-25, syntaxin1A (STX1A), and vesicle-associated membrane protein 1 (VAMP1)] are reduced during postnatal human life spanning from 1 month to 45 years (n = 69) using several different quantitative methods, microarray, qPCR and immunoblotting. We found little evidence for a consistent decrease in synaptic-related molecular markers at any time point, but instead found clear patterns of gradual increases in expression of some presynaptic markers with postnatal age (including SNAP-25, VAMP1 and complexin 1 (CPLX1) mRNAs and 6/6 presynaptic proteins evaluated). A measure of synaptic plasticity [growth-associated protein of 43 kDa (GAP-43)] was elevated in neonates, and continued robust expression throughout life. Since CPLX1 protein is enriched in inhibitory terminals we also tested if the protein product of complexin 2 (CPLX2), which is enriched in excitatory neurons, is more specifically reduced in development. In contrast to CPLX1, which showed a steady increase in both mRNA and protein levels during postnatal development (both r > 0.58, p < 0.001), CPLX2 mRNA decreased from infants to toddlers (r = −0.56, p < 0.001), while CPLX2 protein showed a steady increase until young adulthood (r = 0.55, p < 0.001). Furthermore, we found that indices of the dendrites [microtubule associated protein 2 (MAP2)] and spines (spinophilin and postsynaptic density protein of 95 kDa (PSD95)] showed some evidence of reduction over time at the mRNA level but the opposite pattern, of a developmental increase, was found for PSD95 and spinophilin protein levels. Taken together, the postnatal changes in molecular components of synapses supports the notion that growth and strengthening of synaptic elements is a major developmental event occurring in the frontal cortex throughout childhood and that maintenance of steady state levels of synapse-associated molecules may predominate during human adolescence.     

Differential effects of calcium entry blockers on pre- and postsynaptic influx of calcium in the rat hippocampus in vitro

A decrease in extracellular free Ca ([Ca²⁺]₀) in response to stimulation of Schaffer collaterals could be recorded in or near the stratum pyramidale even when synaptic transmission was completely blocked. Under the same conditions, alvear stimulation also evoked a decrease in [Ca²⁺]₀ at the same site. We attributed the former to influx of Ca²⁺ into presynaptic terminals and the latter to influx postsynaptic (pyramidal) cells. Both pre- and postsynaptic Ca²⁺ influx were completely blocked by Ni²⁺ (2.5 mM). Nifedipide (5–10 μM), verapamil (50–100 μM) and fendiline (100–200 μM) reduced the posysnaptic influx of Ca²⁺ but did not alter Ca²⁺ loss from the extracellular space into presynaptic terminals. The calcium channel activators, BAY-K 8644 and CGP 28,392, had no consistent effect on either pre- or postsynaptic influx. Occasional enhancement of both pre- and postsynaptic responses was seen. In most studies the agents were without effect and on occasions a reduction in both responses was seen. The results could indicate that Ca-channels at pre- and postsynaptic sites in CA₁ may be of different types.     

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act^E84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act^E84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act^E84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act^E84K mutants. Genetic interactions between act^E84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin–Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.     

Charcot-Marie-Tooth 1A patients with low level of impairment have a higher energy cost of walking than healthy individuals

The study aimed at quantifying the walking energy cost of a group of Charcot-Marie-Tooth 1A patients (CMT1A), with low severity of walking impairment, in comparison with healthy individuals. Oxygen uptake was measured in 8 patients (age-range 20-48 years; Barthel >90; Tinetti >20) and 8 healthy individuals, matched for age and gender, when walking on a circuit for 5-min at their self-selected speeds (“slow”, “comfortable” and “fast”). Both comfortable and fast speeds were lower in patients than in the control group (0.92 ± 0.16 vs 1.16 ± 0.22 and 1.27 ± 0.27 vs 1.61 ± 0.22 m s⁻¹, respectively; P < 0.05), whereas walking energy cost per unit of distance was higher in patients than in the control group (P < 0.05) at both “comfortable” (2.27 ± 0.35 vs 1.92 ± 0.21 J kg⁻¹ m⁻¹) and “fast” speed (3.05 ± 0.35 vs 2.37 ± 0.42 J kg⁻¹ m⁻¹). CMT1A patients, therefore, choose to walk slower but with higher metabolic cost compared to healthy individuals, despite no clinically evident walking impairment, which is likely due to altered walking patterns.     

Decreased kainate receptors in the hippocampus of apolipoprotein D knockout mice

Apolipoprotein D (ApoD) has many actions critical to maintaining mammalian CNS function. It is therefore significant that levels of ApoD have been shown to be altered in the CNS of subjects with schizophrenia, suggesting a role for ApoD in the pathophysiology of the disorder. There is also a large body of evidence that cortical and hippocampal glutamatergic, serotonergic and cholinergic systems are affected by the pathophysiology of schizophrenia. Thus, we decided to use in vitro radioligand binding and autoradiography to measure levels of ionotropic glutamate, some muscarinic and serotonin 2A receptors in the CNS of ApoD^-/- and isogenic wild-type mice. These studies revealed a 20% decrease (mean ± SEM: 104 ± 10.2 vs. 130 ± 10.4 fmol/mg ETE) in the density of kainate receptors in the CA 2–3 of the ApoD^-/- mice. In addition there was a global decrease in AMPA receptors (F_1,214 = 4.67, p < 0.05) and a global increase in muscarinic M2/M4 receptors (F_1,208 = 22.77, p < 0.0001) in the ApoD^-/- mice that did not reach significance in any single cytoarchitectural region. We conclude that glutamatergic pathways seem to be particularly affected in ApoD^-/- mice and this may contribute to the changes in learning and memory, motor tasks and orientation-based tasks observed in these animals, all of which involve glutamatergic neurotransmission.     

Glutamate transporters and retinal excitotoxicity

Glutamate appears to play a major role in several degenerative retinal disorders. However, exogenous glutamate is only weakly toxic to the retina when glutamate transporters on Müller glial cells are operational. In an ex vivo rat retinal preparation, we previously found that exogenous glutamate causes Müller cell swelling but does not trigger excitotoxic neurodegeneration unless very high concentrations that overwhelm the capacity of glutamate transporters are administered. To determine the role of glutamate transporters in Müller cell swelling and glutamate-mediated retinal degeneration, we examined the effects of DL-threo-beta-benzyloxyaspartate (TBOA), an agent that blocks glutamate transport but that unlike most available transport inhibitors is neither a substrate for transport nor a glutamate receptor agonist. We found that TBOA triggered severe retinal neurodegeneration attenuated by ionotropic glutamate receptor antagonists. TBOA-induced neuronal damage was also diminished by riluzole, an agent that inhibits endogenous glutamate release. In the presence of riluzole, to inhibit glutamate release plus TBOA to block glutamate uptake, the addition of low concentrations of exogenous glutamate triggered severe excitotoxic neuronal damage without inducing Müller cell swelling. We conclude that TBOA-sensitive glutamate transporters play an important role in regulating the neurodegenerative effects of glutamate in the rat retina.     

Magnetic resonance imaging assessment of cardiac dysfunction in δ-sarcoglycan null mice

Delta-sarcoglycan (δ-sarcoglycan) null, Scgd^−/−, mice develop cardiac and skeletal muscle histopathological alterations similar to those in humans with limb-girdle muscular dystrophy. The objective of this study was to assess the feasibility of using MRI to investigate cardiac dysfunction in Scgd^−/− mice. Cardiac MRI of 8 month old Scgd^−/− and wild type (WT) mice was performed. Compared to WT, Scgd^−/− mice had significantly lower LV ejection fraction (44 ± 5% vs. 66 ± 4%, p = 0.014), lower RV ejection fraction (25 ± 2% vs. 51 ± 3%, p < 0.001) lower myocardial circumferential strain, (15.0 ± 0.3% vs. 16.9 ± 0.3%, p = 0.007) and RV dilatation (54 ± 3 μL vs. 40 ± 3 μL, p = 0.007). The regional circumferential strain also demonstrated significant temporal dyssynchrony between opposing regions of the Scgd^−/− LV. Our results demonstrate severe cardiac dysfunction in Scgd^−/− mice at 8 months. The study identifies a set of non-invasive markers that could be used to study efficacy of novel therapeutic agents in dystrophic mice.     

Impaired motor cortex plasticity in patients with Noonan syndrome

Objective

Noonan syndrome (NS; OMIM 163950) is a developmental disorder caused by activating mutations in various components of the RAS-MAPK pathway. Recent in vitro studies demonstrated impairment of synaptic plasticity caused by RAS-MAPK pathway hyperactivity. Induction of synaptic plasticity critically depends on the level of attention. We therefore studied the induction of synaptic plasticity in patients with NS and healthy volunteers under different conditions of attention using transcranial magnetic stimulation.

Methods

We investigated 10 patients with NS and healthy controls (HC) using paired associative stimulation (PAS) with different attention levels (unspecific, visual and electrical attention control). Changes in motor evoked potential (MEP) amplitudes were assessed immediately after as well as 30 and 60 min after PAS.

Results

We demonstrated that MEP amplitudes of healthy controls significantly increased from 1.00 ± 0.17 to 1.74 ± 0.50 mV (p = 0.001), which was not seen in patients with Noonan-Syndrome (0.88 ± 0.09 to 1.10 ± 0.48 mV, p = 0.148) and there was a significant difference between both groups (p = 0.003) when using an unspecific attention control. Under specific electrical attention control, MEP amplitudes decreased significantly in patients with NS, whereas a visual attention focus diminished synaptic plasticity in healthy controls.

Conclusion

Our study provides evidence that synaptic plasticity is impaired in patients with NS, which is probably a consequence of constitutive activity of the RAS-MAPK pathway. The induction of synaptic plasticity in these patients critically depends on attention.

Significance

This is the first study that indicates reduced synaptic plasticity in patients with a RAS-pathway disorder. Our results may have direct implications for learning and memory strategies in patients with a RAS-pathway disorder.     

Rapid oscillatory activity in delta brushes of premature and term neonatal EEG

We compared frequency and power of neonatal EEG delta brush rapid oscillatory activity (ROA) using multiple band frequency analysis (MBFA) in three groups; pre-term (PT, post-conceptional age 33–35.6 weeks, n = 5); full-term (FT, 39.4–40.6 weeks, n = 5) and pre-term or full-term with phenobarbital exposure (PB, n = 5). Mean number of delta brushes analyzed was 29.4 (range 26–47) in PT, 20.8 (14–33) in FT and 20 (7–37) in PB. Mean frequency ± standard deviation (s.d.) was 16.9 ± 2.1 Hz (range 15–20 Hz) in PT, 17.3 ± 1.9 Hz (15–20 Hz) in FT and 16.1 ± 1.6 Hz (14–19 Hz) in PB. Mean power ± s.d. was 22.9 ± 6.2 μV² (range 16–39 μV²) in PT, 11.9 ± 4.1 μV² (7–19 μV²) in FT and 17.1 ± 6.2 μV² (9–26 μV²) in PB. Power was significantly higher in PT than FT (p < 0.005). Power after merging PB into respective PT (PT′, n = 8) and FT (FT′, n = 7) groups, remained significantly higher in PT′ (mean ± s.d. 21.8 ± 7.4 μV²) than FT′ (11.4 ± 3.6 μV²) (p < 0.05). We characterise ROA in delta brushes in maturing neonates using MBFA, which may provide additional information for assessing future seizure recurrence and epilepsy risk.     

Comparative effects of pentobarbital on spontaneous and evoked transmitter release from inhibitory and excitatory nerve terminals in rat CA3 neurons

Pentobarbital (PB) modulates GABA_A receptor-mediated postsynaptic responses through various mechanisms, and can directly activate the channel at higher doses. These channels exist both pre- and postsynaptically, and on the soma outside the synapse. PB also inhibits voltage-dependent Na⁺ and Ca²⁺ channels to decrease excitatory synaptic transmission. Just how these different sites of action combine to contribute to the overall effects of PB on inhibitory and excitatory synaptic transmission is less clear. To compare these pre- and postsynaptic actions of PB, we used a ‘synaptic bouton’ preparation of isolated rat hippocampal CA3 pyramidal neurons where we could measure in single neurons the effects of PB on spontaneous and single bouton evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic currents (sIPSCs, sEPSCs, eIPSCs and eEPSCs), respectively. Low (sedative) concentrations (3–10 μM) of PB increased the frequency and amplitude of sIPSCs and sEPSCs, and also presynaptically increased the amplitude of both eIPSCs and eEPSCs. There was no change in current kinetics at this low concentration. At higher concentrations (30–300 μM), PB decreased the frequency, and increased the amplitude of sIPSCs, and presynaptically decreased the amplitude of eIPSCs. The current decay phase of sIPSCs and eIPSCs was increased. An increase in both frequency and amplitude was seen for sEPSCs, while the eIPSCs was also decreased by a bicuculline-sensitive presynaptic effect. The results confirm the multiple sites of action of PB on inhibitory and excitatory transmission and demonstrate that the most sensitive site of action is on transmitter release, via effects on presynaptic GABA_A receptors. At low concentrations, however, both glutamate and GABA release is similarly enhanced, making the final effects on neuronal excitability difficult to predict and dependent on the particular systems involved and/or on subtle differences in susceptibility amongst individuals. At higher concentrations, release of both transmitters is decreased, while the postsynaptic effects to increase IPSPs and decrease EPSCs would be expected to both results in reduced neuronal excitability.