Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells.

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.

Kinetics of both synchronous and asynchronous quantal release during trains of action potential-evoked EPSCs at the rat calyx of Held.

We studied the kinetics of transmitter release during trains of action potential (AP)-evoked excitatory postsynaptic currents (EPSCs) at the calyx of Held synapse of juvenile rats. Using a new quantitative method based on a combination of ensemble fluctuation analysis and deconvolution, we were able to analyse mean quantal size (q) and release rate (xi) continuously in a time-resolved manner. Estimates derived this way agreed well with values of q and quantal content (M) calculated for each EPSC within the train from ensemble means of peak amplitudes and their variances. Separate analysis of synchronous and asynchronous quantal release during long stimulus trains (200 ms, 100 Hz) revealed that the latter component was highly variable among different synapses but it was unequivocally identified in 18 out of 37 synapses analysed. Peak rates of asynchronous release ranged from 0.2 to 15.2 vesicles ms(-1) (ves ms(-1)) with a mean of 2.3 +/- 0.6 ves ms(-1). On average, asynchronous release accounted for less than 14% of the total number of about 3670 +/- 350 vesicles released during 200 ms trains. Following such trains, asynchronous release decayed with several time constants, the fastest one being in the order of 15 ms. The short duration of asynchronous release at the calyx of Held synapse may aid in generating brief postsynaptic depolarizations, avoiding temporal summation and preserving action potential timing during high frequency bursts.

Fine-tuning an auditory synapse for speed and fidelity: developmental changes in presynaptic waveform, EPSC kinetics, and synaptic plasticity.

Fast, precise, and sustained synaptic transmission at high frequency is thought to be crucial for the task of sound localization in the auditory brainstem. However, recordings from the calyx of Held synapse have revealed severe frequency-dependent synaptic depression, which tends to degrade the exact timing of postsynaptic spikes. Here we investigate the functional changes occurring throughout the critical period of synapse refinement from immature calyx terminal [postnatal day 5 (P5)] to after the onset of hearing (P12-P14). Surprisingly, for recordings near physiological temperature (35 degrees C), we find that P14 synapses are already able to follow extremely high input rates of up to 800 Hz. This ability stems in part from a remarkable shortening of presynaptic action potentials, which may lead to a lowering of release probability and decrease in synaptic delays during development. In addition, AMPA receptor-mediated EPSCs as well as quantal synaptic currents acquired progressively faster kinetics, although their mean amplitudes did not change significantly. NMDA receptor-mediated EPSCs, however, diminished with age, as indicated by a 50% reduction in mean amplitude and faster decay kinetics. Finally, the degree of synaptic depression was greatly attenuated with age, presumably because of a 2.5-fold or larger increase in the releasable pool of vesicles, which together with a decreasing release probability produces a fairly constant EPSC amplitude. This finely tuned orchestra of developmental changes thus simultaneously promotes speed while preventing premature vesicle pool depletion during prolonged bouts of firing. A few critical days in postnatal development can thus have a large impact on synaptic function.

Ca2+-permeable P2X receptor channels in cultured rat retinal ganglion cells.

ATP has been identified as an excitatory neurotransmitter in both the CNS and peripheral nervous system; however, little is known about the functional properties of ATP-gated channels in central neurons. Here we used a culture preparation of the postnatal rat retina to test the responsiveness of identified retinal ganglion cells (RGCs) and putative amacrines to exogenous ATP and other purinoceptor agonists. Rapidly activating ATP-induced currents (IATP) were exclusively generated in a subpopulation (approximately 65%) of RGCs. The latter were identified by Thy1.1 immunostaining, repetitive firing patterns, and activation of glutamatergic autaptic currents. None of the putative amacrine cells was ATP-sensitive. IATP could be induced with ATP, ADP, and alpha,beta-mATP but not with adenosine. It was antagonized by suramin. The current-voltage relationship of IATP showed marked inward rectification. Dose-response analysis yielded an EC50 of 14.5 microM, with a Hill coefficient of 0.9. Noise analysis of IATP suggested a mean single channel conductance of 2.3 pS. Retinal P2X purinoceptor channels exhibited a high permeability for Ca2+. PCa/PCs obtained from reversal potentials of IATP under bi-ionic conditions amounted to 2. 2 +/- 0.7. In the majority of cells, the decay of IATP was biphasic. The degree of current inactivation during the first 2 sec of agonist application was highly variable. Heterogeneity was also found with respect to the sensitivity to ADP and alpha,beta-mATP and the blocking action of suramin, suggesting expression of multiple P2X receptor subtypes. Our results indicate that activation of P2X receptor channels represents an important pathway for Ca2+ influx in postnatal RGCs.

Interaction of calcium-permeable non-N-methyl-D-aspartate receptor channels with voltage-activated potassium and calcium currents in rat retinal ganglion cells in vitro.

Calcium-permeable non-N-methyl-D-aspartate receptor channels are now characterized in much detail, but still little is known about the consequences of Ca2+ influx through these channels in specific neuron types. We are interested in the role of Ca2+-permeable non-N-methyl-D-aspartate receptor channels during differentiation of retinal ganglion cells. However, in view of the conflicting data on the relative Ca2+ permeability of non-N-methyl-D-aspartate receptor channels in these neurons, a more systematic evaluation of permeation properties of different Na+ substitutes was necessary before proceeding with the main goal of the present study evaluating the effects of non-N-methyl-D-aspartate receptor activation on repetitive firing and voltage-activated K+ and Ca2+ conductances. Retinal ganglion cells were dissociated from the rat retina on postnatal day 5. They were selected by vital anti-Thy-1 immunostaining and repetitive firing behaviour and submitted to patch-clamp recording in the whole-cell configuration. Non-N-methyl-D-aspartate receptor channels were activated by application of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. It was found that they were essentially impermeable to N-methyl-D-glucamine (P(NMDG)/P(Cs)<0.02), but not to choline (P(choline)/P(Cs)=0.24) and tetramethylammonium (P(TMA)/P(Cs)=0.23). When using N-methyl-D-glucamine as a substitute for Na+ to obtain bi-ionic conditions P(Ca)/P(Cs) varied between 0.08 to 1.40. Linear current voltage relation or little outward rectification corresponded to a low Ca2+ permeability (P(Ca)/P(Cs)=0.14). In about one third of the cells kainate-induced currents showed inward rectification and non-N-methyl-D-aspartate receptor agonists induced a substantially higher Ca2+ influx (P(Ca)/P(Cs)=0.64). Activation of non-N-methyl-D-aspartate receptors by kainate profoundly altered the repetitive discharge of retinal ganglion cells. In contrast to the continuously firing controls, cells generated only a few spikes at the beginning of a steady depolarization after kainate exposure. Among the candidates regulating the firing behaviour of retinal ganglion cells voltage-activated Ca2+ and K+ conductances were tested for their sensitivity to kainate application. It was found that even short conditioning pulses of kainate decreased the peak amplitudes of both voltage-activated K+ and voltage-activated Ca2+ currents. Only the latter effect required extracellular Ca2+ and was antagonized by increasing the intracellular Ca2+ buffering strength. Thus, suppression of calcium currents was induced by a non-N-methyl-D-aspartate receptor-mediated rise of the intracellular calcium concentration. The reduction of K+ currents did not depend on extracellular calcium and was insensitive to experimental manipulation of intracellular Ca2+ buffer strength. The interaction between Ca2+-permeable non-N-methyl-D-aspartate receptor channels and voltage-activated Ca2+ and K+ currents may represent an important regulatory mechanism to control the repetitive firing of developing retinal ganglion cells.

Synaptic current kinetics in a solely AMPA-receptor-operated glutamatergic synapse formed by rat retinal ganglion neurons.

1. Postnatal rat retinal ganglion cells (RGCs) can be maintained and identified in dissociated long-term culture. After 4-7 days in vitro they form glutamatergic synapses with other RGCs or putative amacrine cells. Here we intended to characterize the postsynaptic features of these in vitro synapses. 2. Pair patch-clamp recordings in the whole cell configuration were performed to study the properties of synaptic glutamate receptors. Immunohistochemically and physiologically identified RGCs were activated by short depolarizing voltage steps. This elicited glutamatergic excitatory postsynaptic currents (EPSCs) in coupled neurons. At room temperature, evoked EPSCs (eEPSCs) had latencies between 3 and 7 ms and amplitudes between 36.4 and 792.6 pA. 3. Postsynaptic neurons were electrotonically compact and therefore well suited for analysis of fast synaptic events. All cells were responsive to exogenous glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA). The current-voltage relationships of AMPA-activated currents were linear, whereas NMDA-induced whole cell currents displayed the typical characteristics including a negative slope conductance in the presence of Mg2+. In contrast to AMPA-activated currents, NMDA-activated currents had the usual slow onset and decay. 4. RGCs obviously failed to generate NMDA-receptor-mediated EPSCs, because all postsynaptic cells lacked a slow current component even in the absence of added Mg2+ and in the presence of glycine. Retinal eEPSCs were completely blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 5. eEPSCs as well as spontaneous EPSCs (sEPSCs) were characterized by a very rapid time course. In eEPSCs, 20-80% rise times and time constants of decay (tau DS) were on average 0.64 and 1.96 ms, respectively. eEPSCs were extremely fast, with average rise times of 0.34 ms and tau DS of 1.20 ms. The latter numbers closely correspond to the values obtained for DNQX-sensitive miniature EPSC (mEPSC) in postnatal day 5 rat RGCs in situ. 6. To clarify whether the decay of fast AMPA-receptor-mediated EPSCs of retinal neurons was determined by the onset of glutamate receptor desensitization, we compared the decay of sEPSCs with the decay of the glutamate response of excised out-side-out membrane patches. Glutamate-activated currents were elicited by a rapid superfusion device (time constant of rise = 0.7 ms). The response to 1 mM of glutamate decayed 2 to 4 times more slowly than the sEPSCs. 7. These results suggest that desensitization did not limit the rate of decay of purely AMPA-mediated EPSCs in response to ganglion cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Several types of Ca2+ channels mediate glutamatergic synaptic responses to activation of single Thy-1-immunolabeled rat retinal ganglion neurons.

A dissociated cell culture from the postnatal rat retina was established to characterize the synapses formed by retinal ganglion neurons (RGNs) in vitro. An antibody against Thy-1.1 was used to preselect putative RGNs for pair patch-clamp recording with the principal aim of identifying the released transmitter(s) and estimating the role of different types of voltage-activated Ca2+ channels in evoked transmitter release. The population of Thy-1+ neurons was heterogeneous. Staining patterns, soma-dendritic geometries and axon length displayed variations that could be related to basic electrophysiological properties, such as amplitudes of voltage-activated Na+ currents (INa(V)), action potential size and capacity for repetitive discharge. Out of 73 coupled connections, 33 pairs were glutamatergic. With no exception, these connections were formed by the axons of strongly labeled Thy-1+ neurons with large INa(V) (typically > 2 nA) and repetitive firing over a broad current range. Such neurons were classified as RGNs. Forty out of 73 coupled pairs were GABAergic. These connections were always formed by weakly stained Thy-1+ neurons with small INa(V) (typically < 2 nA) and very limited capacity for repetitive discharge. Such neurons were tentatively classified as displaced amacrine cells. Evoked EPSCs in response to RGN activation were completely blocked by low concentrations of Cd2+ or Gd3+. omega-CgTx-GVIA (5 microM) reduced EPSCs to 67 +/- 29%, omega-AgaTx-IVA (200 nM) had no effect, and nifedipine (15 microM) enhanced the evoked EPSCs. Our experiments indicate that (1) the transmitter released by RGNs is glutamate and (2) the major part of synaptic glutamate release is governed by a novel toxin-resistant Ca2+ channel. The results further suggest that the characteristic phenotype of RGNs is well maintained in dissociated cell culture. In conjunction with electrophysiological tests Thy-1+ labeling can be used for RGN identification.

In vitro development of vertebrate central synapses.

This article deals with basic determinants of synaptic efficacy during development of glutamatergic and GABAergic synaptic transmission: location and number of release sites, release probability and single cell-activated (unitary) conductances. We hypothesize that both types of neuronal connections differ in major aspects of synaptogenesis. Disregarding the fact that various test models and cell types could render diverging results, it can be observed that glutamatergic terminals display a preference for dendrites, whereas GABAergic terminals select soma locations at initial stages of development. Glutamatergic synapses are characterised by receptor accumulation in the region of terminal apposition, whereas in GABAergic synapses receptor concentration is weak, if present at all. The expression of glutamate receptors (GluRs), but not GABAA receptors is under control of interneurons. Developmental changes in glutamatergic synaptic transmission have not yet been assessed by quantal analysis. For GABAergic synapses, first results are now available from a culture preparation of the rat superior colliculus. In general terms, functional maturation seemed to lag behind the formation of structurally differentiated release sites. Compound binomial analysis revealed that during in vitro development a considerable fraction of GABAergic terminals remained in a low efficacy release state (p < 0.2). A developmental increase in synaptic strength was reached by the appearance of singular highly effective release sites. Presynaptic maturation could be manipulated by long-term drug treatment. Addition of GluR antagonists significantly increased amplitudes and decreased the coefficients of variations of evoked inhibitory postsynaptic currents. Thus, the strength of inhibitory synaptic transmission could be influenced by the status of heteronymous synaptic input.

Separation of calcium currents in retinal ganglion cells from postnatal rat.

A culture system of the postnatal rat retina was established to investigate Ca2+ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca2+ currents of RGNs and to clarify the potential use of the lanthanide Gd3+ as a selective Ca2+ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca2+ currents (ICa(LVA)) and high voltage activated Ca2+ currents (ICa(HVA)). The remaining 2/3 or RGNs in short term culture and most RGNs in long-term culture displayed only ICa(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of ICa(HVA) was irreversibly blocked by the N channel antagonist omega-CgTx (5 microM). The L channel antagonist nifedipine (10 microM) eliminated about 25% of ICa(HVA). Thus, about 1/3 of the HVA Ca2+ or Ba2+ current remained unaffected by either omega-CgTx or nifedipine. omega-AgaTx (200 nM) completely failed to block HVA Ca2+ or Ba2+ currents in RGNs. Gd3+ exerted contrasting actions on LVA and HVA Ca2+ currents. While ICa(LVA) consistently increased in the presence of Gd3+ (0.32-3.2 microM), ICa(HVA) always decreased, especially when using higher concentrations of Gd3+ (10-32 microM). The blocking action of Gd3+ was not restricted to the omega-CgTx-sensitive HVA current component, but also concerned omega-CgTx- and nifedipine-resistant components. The decay of Ca2+ currents was accelerated in the presence of Gd3+. Even in RGNs lacking ICa(LVA), application of 3.2 microM Gd3+ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from -90 to 0 mV; 10 mM [Ca2+]o; 26 degrees C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd3+.Gd3+ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd3+ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca2+ currents, that resemble in some aspects T, N and L channel currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a metabotropic glutamate agonist, trans-ACPD, on cortical epileptiform activity.

The effect of the metabotropic glutamate receptor agonist trans-1-amino-1,3,cyclopentanedicarboxylic acid (trans-ACPD) on epileptiform activity induced in rat neocortical slices by exposure to Mg(2+)-free medium was examined. Trans-ACPD dose dependently (10-200 microM) decreased the frequency of spontaneous epileptiform events whilst increasing both the duration of afterpotentials and the number of afterbursts associated with single events. This effect on afterpotentials and afterbursting was particularly pronounced in 14-17 day-old rats and was blocked by the sigma ligand ditolyguanidine (DTG) 10 microM. The putative metabotropic glutamate receptor antagonist L-AP3 did not antagonise the actions of trans-ACPD. The results suggest a role for metabotropic glutamate receptors in epilepsy, possibly in the transition from interictal to ictal activity.