Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation.

Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins.

In situ measurements of external pH and optical density oscillations in Dictyostelium discoideum aggregates.

In situ measurements of extracellular pH by means of microelectrodes and in situ measurements of optical density were performed on aggregating cells of Dictyostelium discoideum. Early aggregation stage AX2 cells showed sinusoidal pH oscillations, which could be inhibited by the specific relay inhibitor caffeine, indicating that they were coupled to cAMP oscillations. Sometimes biphasic pH oscillations were found, which can be explained by the superposition of two harmonic pH oscillations. These harmonic oscillations might arise by gating of the cAMP signal; a part of the cells respond to every cAMP signal and another subpopulation to every second cAMP pulse. Late aggregation-stage cells showed complex changes of the extracellular pH, which could be inhibited by caffeine. Optical density measurements of wave propagation in aggregation streams of HG220 also revealed gating behavior. In addition to sinusoidal optical density oscillations, biphasic and still more complex oscillations were observed.

Kinetics of GABAB receptor-mediated inhibition of calcium currents and excitatory synaptic transmission in hippocampal neurons in vitro.

The time courses of the gamma-aminobutyric acid type B (GABAB) receptor-mediated inhibition of excitatory synaptic transmission and of action potential-evoked calcium currents were studied in hippocampal neurons in vitro with step-like changes of a saturating baclofen concentration. Inhibition mediated by postsynaptic GABAB receptors was excluded pharmacologically. Both presynaptic inhibition and reduction of calcium currents developed and declined exponentially with similar time constants of about 0.2 and 3 s, respectively. The close correlation of the time courses indicates that fast, G protein-mediated depression of voltage-gated calcium channels and thus direct reduction of the presynaptic calcium influx may contribute to the GABAB receptor-induced inhibition of excitatory synaptic transmission in hippocampal neurons in vitro.

Target-specific factors regulate the formation of glutamatergic transmitter release sites in cultured neocortical neurons.

Synapse formation in the mammalian CNS is thought to involve specific target recognition processes between presynaptic and postsynaptic neurons leading to the establishment of defined neuronal circuits. To study the role of target neuron-specific factors in synaptogenesis, we used cocultures of presynaptic explants and dissociated target neurons from rat neocortex, which enabled us to selectively vary the postsynaptic target neurons. Cocultures containing target neurons that were obtained early during development [embryonic day 16 (E16)] were compared to cocultures containing target neurons that were obtained at a later embryonic stage (E19). Postsynaptic currents (PSCs) were evoked in target neurons by maximal extracellular stimulation in the presynaptic explant. The mean amplitudes of AMPA and NMDA receptor-mediated PSCs were sixfold reduced in E16 target neurons, whereas the mean amplitudes of GABA(A) receptor-mediated PSCs did not differ between E16 and E19 target neurons. This reduction was in part caused by an apparently twofold reduction in mean quantal amplitude, as shown by recording AMPA receptor-mediated miniature PSCs. In addition, a reduced number of glutamatergic release sites in E16 target neurons was revealed by synapsin I immunostaining of dendritic presynaptic terminals. No differences in mean release probability were observed between E16 and E19 target neurons. Thus, the formation of glutamatergic transmitter release sites was strongly influenced by target neuron-specific factors. The formation of functional GABAergic synapses, however, was independent of the type of target neurons, suggesting specific retrograde signaling during the establishment of glutamatergic synapses.

Alzheimer's disease-related amyloid-ß induces synaptotoxicity in human iPS cell-derived neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-ß (Aß). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aß, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aß. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aß. Application of Aß for 8 days led to a reduction in the overall FM4-64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aß impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aß-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aß for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aß-induced synaptic pathomechanisms and the development of novel therapeutic approaches.

p21ras Oncogene Protein Selectively Increases Low-voltage-activated Ca2+ Current Density in Embryonic Chick Dorsal Root Ganglion Neurons.

p21ras protein resembles the alpha subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca2+ channels in vertebrate sensory neurons by p21ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca2+ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca2+ channels in chick dorsal root ganglion neurons, p21ras did not significantly affect HVA Ca2+ currents. To study the time course of p21ras action, guanosine triphosphate-preloaded p21ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 - 30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p21ras, which was found to be inactive in biological assays, enhanced LVA Ca2+ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca2+ channel by p21ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca2+ currents. The regulatory role of p21ras on LVA channels may therefore be particularly important during differentiation.

Developmental maturation of synaptic vesicle cycling as a distinctive feature of central glutamatergic synapses.

The formation of chemical synapses in the mammalian brain involves complex pre- and postsynaptic differentiation processes. Presynaptically, the progressive accumulation of synaptic vesicles is a hallmark of synapse maturation in the neocortex [J Neurocytol 12 (1983b) 697]. In this study, we analyzed the functional consequences of presynaptic vesicle-pool maturation at central glutamatergic and GABAergic synapses. Using (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) staining of recycling synaptic vesicles, we demonstrate a pronounced developmental increase in presynaptic vesicle accumulation during differentiation of neocortical neurons in culture. Using electrophysiological methods to study functional synaptic maturation, we found an improved recovery from hypertonic solution-induced depletion. As supported by the FM1-43 staining results, this change is most likely caused by a developmental increase in the number of reserve-pool vesicles. In addition, assuming a rapid reuse of freshly recycled vesicles, a developmental maturation of the endocytosis process may also contribute. The observed presynaptic maturation process occurred selectively at glutamatergic synapses, while GABAergic synapses did not show similar developmental alterations. Furthermore, we used high-frequency stimulation (HFS) of glutamatergic and GABAergic synapses to reveal the physiological consequences of reserve-pool maturation. As expected, recovery from HFS-induced depletion was incomplete at immature glutamatergic synapses and strongly improved during synapse maturation. Again, GABAergic synapses did not show similar developmental changes. Taken together, our study characterizes the functional consequences of a pronounced accumulation of reserve-pool vesicles occurring selectively at glutamatergic synapses.

Electrophysiological and morphological properties of cell types in the chick neostriatum caudolaterale.

The neostriatum caudolaterale, in the chick also referred to as dorsocaudal neostriatal complex, is a polymodal associative area in the forebrain of birds that is involved in sensorimotor integration and memory processes. We have used whole-cell patch-clamp recordings in chick brain slices to characterize the principal cell types of the neostriatum caudolaterale. Electrophysiological properties distinguished four classes of neurons. The morphological characteristics of these classes were examined by intracellular injection of Lucifer Yellow. Type I neurons characteristically fired a brief burst of action potentials. Morphologically, type I neurons had large somata and thick dendrites with many spines. Type II neurons were characterized by a repetitive firing pattern with conspicuous frequency adaptation. Type II neurons also had large somata and thick dendrites with many spines. There was no clear morphological distinction between type I and type II neurons. Type III neurons showed high-frequency firing with little accommodation and a prominent time-dependent inward rectification. They had thin, sparsely spiny dendrites and extensive local axonal arborizations. Electrophysiological and morphological properties indicated them as being interneurons. Type IV neurons had a longer action potential duration, a larger input resistance, and a longer membrane time constant than the other classes. Type IV neurons had small somata and short dendrites with few spines. The long axon collaterals of neurons in all spiny cell classes (types I, II, IV) followed similar patterns, suggesting that neurons from all these types can contribute to the projections of the neostriatum caudolaterale to sensory, limbic and motor areas. The electrophysiological and anatomical characterization of the major classes of neurons in the caudal forebrain of the chick provides a framework for the investigation of sensorimotor integration and learning at the cellular level in birds.

Fast desensitization of glutamate activated AMPA/kainate receptors in rat thalamic neurones.

An essential part of the excitatory afferent input to the thalamus is mediated by glutamate receptors of the AMPA/kainate type. In contrast to other regions of the mammalian CNS, the biophysical properties of these receptors have not been investigated in thalamic neurones. Using a fast transmitter application system we studied L-glutamate activated currents of cultured neurones in the whole cell and outside-out patch configuration. Current-voltage relationships and dose-response curves of whole cell recordings were in close correspondence to results obtained from other brain areas. Analysis of outside-out patch currents revealed two types of desensitization time constants of 3.0 and 10.2 ms, with the former close to the time constant of decay of miniature glutamatergic synaptic currents.

Presynaptic exocytosis regulates NR2A mRNA expression in cultured neocortical neurones.

N-methyl-D-aspartate (NMDA) receptors are hetero-oligomeric receptor-channel complexes composed of NR1 and NR2 subunits. Subunit composition determines the properties of NMDA receptor subtypes. However, the molecular mechanisms regulating their subunit composition are unknown. Using reverse transcription (RT)-competitive PCR we studied the expression of NR2A and NR2B mRNA in neocortical neurones differentiating in culture. We found a developmental increase in NR2A mRNA expression in relation to NR2B mRNA expression. This increase was inhibited by addition of tetanus toxin to the culture medium and by chronic pharmacological blockade of postsynaptic, ionotropic glutamate receptors. These results suggest that presynaptic exocytosis regulates NR2A mRNA expression. This mechanism might influence NMDA receptor properties and thus developmental changes in long-term synaptic plasticity.

Afferent innervation influences HVA Ca2+ current expression in cultured neocortical neurones.

Voltage-activated Ca2+ channels represent a major pathway of Ca2+ entry into neurones. The regulation of the expression of functional Ca2+ channels thus plays a central role in neuronal differentiation. To study the influence of afferent innervation on Ca2+ current expression, we compared HVA Ca2+ currents in two categories of cultured neocortical neurones that showed pronounced differences in synaptic innervation density. Neurones strongly innervated by a presynaptic explant had a two-fold greater HVA Ca2+ current density than neurones not innervated by explant fibres. Chronic blockade of synaptic activity did not affect HVA Ca2+ current density in innervated neurones. Our results thus suggest an activity-independent regulation of HVA Ca2+ current expression by afferent innervation.

Developmental regulation of subunit composition of extrasynaptic NMDA receptors in neocortical neurones.

NMDA receptors undergo drastic changes in their subunit composition during development of the mammalian neocortex. An increase in the expression of the NR2A subunit correlates with developmental changes in the properties of synaptic NMDA receptors. In this study, we investigated whether these developmental alterations are restricted to synaptic NMDA receptors or whether similar developmental changes also occur at extrasynaptic NMDA receptors. To analyse the properties of extrasynaptic receptors, glutamate-evoked ion currents mediated by extrasynaptic NMDA receptors were isolated by irreversibly blocking synaptic NMDA receptors with MK-801. Whole-cell ion currents mediated by extrasynaptic receptors showed developmental changes in their sensitivity against the NR2B subunit-specific antagonist ifenprodil similar to that of synaptic receptors. In summary, our results strongly suggest that NR2A subunit-containing NMDA receptors increasingly contribute also to extrasynaptic NMDA receptors during in vitro differentiation.

BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones.

The effects of BDNF and NT-4/5 on AMPA receptor-mediated synaptic transmission were investigated with the patch clamp technique applied to embryonic and post-natal rat hippocampal neurones, cultured in serum-free medium. Spatially restricted application of neurotrophin-containing solution on to the recorded cells was performed and evoked as well as miniature excitatory postsynaptic currents (mepscs) were monitored. In approximately 25% of neurones tested a transient augmentation of evoked synaptic currents and a transient increase in the frequency of mepscs occurred with a delay of 0.5-5 min after the onset of BDNF or NT-4/5 application. The amplitudes of the AMPA receptor mediated mepscs were unaffected, suggesting a presynaptic action of BDNF and NT-4/5.

NT-3 regulates BDNF-induced modulation of synaptic transmission in cultured hippocampal neurons.

BDNF and NT-3 can modulate the development and plasticity of central synaptic transmission. Although the expression of NT-3 and BDNF in the rodent hippocampus coincides during perinatal development, little is known about possible functional interactions between both neurotrophins in synaptic development. Here, we have investigated the effects of combined long-term application of NT-3 and BDNF on excitatory glutamatergic (mEPSC) and inhibitory GABAergic miniature synaptic currents (mIPSC) in cultured embryonic hippocampal neurons. Our results show that the BDNF-induced twofold increase in mEPSC frequency is abolished by pre-treatment with NT-3. In addition, the NT-3-induced twofold downregulation of mIPSC frequency is reversed by BDNF. Finally, the BDNF-induced increase in c-fos expression is reduced by 50% after pre-treatment with NT-3. In summary, these data suggest an NT-3 controlled modulation of BDNF signalling in differentiating hippocampal neurons.

Lesion-induced changes in NMDA receptor subunit mRNA expression in rat visual cortex.

Focal lesions in the visual cortex are well known to induce pronounced perilesional reorganization of the neuronal circuitry. Since NMDA receptors crucially control synaptic plasticity and reorganization, we studied lesion-induced changes in their subunit expression and biophysical properties. Between 8 and 10 days after focal thermolesioning, pyramidal neurones in the near surround of the lesion were studied in acute brain slices. We found a significant decrease in the ratio of NR2A and NR2B subunit mRNA as compared to neurones from sham operated animals. Interestingly, no significant differences in the properties of NMDA receptor-mediated postsynaptic currents (NMDA PSCs) were observed between lesioned and sham operated animals. Thus, the observed perilesional changes in the NR2A/NR2B mRNA ratio appear to be subthreshold to result in significant changes in the functional properties of NMDA receptors.

Synapse formation and morphological differentiation of neuron types in embryonic rat dentate gyrus explants in vitro.

Cultured explants obtained from the dentate gyrus of rat embryos (embryonic day 19-20) were used to investigate synapse formation and morphological differentiation of neuron types in the absence of extrinsic afferents. Synaptogenesis was studied by whole-cell recordings of postsynaptic currents and by ultrastructural analysis. Neurons were visualized using Lucifer Yellow filling or staining with DiI. In short-term (3-5 days) cultured explants postsynaptic currents were rarely evoked by extracellular stimulation and synapses were almost completely absent at the ultrastructural level. After 6-10 days in vitro, the incidence of evoking postsynaptic currents mediated by glutamate and GABA(A) receptors was strongly increased. At the ultrastructural level, the density of synapses increased more than 20-fold. These results demonstrate de novo formation of synapses in cultured embryonic dentate gyrus explants. Neuron types could be discriminated by their dendritic arborizations and by their electrophysiological properties. After 6-10 days in vitro, mossy-like cells exhibited 3-4 primary dendrites branching in a characteristic pattern and showed moderate spike-frequency adaptation. Application of serotonin (5-HT) to cultured explants elicited GABA(A)-receptor-mediated postsynaptic currents in mossy-like cells, indicating synaptic GABA release from local interneurons. Comparison to 5-HT evoked GABA release in mossy cells in age-matched, acute slices revealed only slight quantitative differences. In contrast to mossy cells, granule cells showing several primary dendrites originating at one cell pole were almost completely absent in cultured explants, suggesting an involvement of extrinsic afferents in the differentiation of granule cells.

The formation of glutamatergic synapses in cultured central neurons: selective increase in miniature synaptic currents.

The formation of synapses between cultured rat thalamic neurons was studied with electrophysiological and immunocytochemical methods. Thalamic neurons in culture form predominantly glutamatergic synapses. Already after 3 days in vitro glutamatergic miniature EPSCs occurred spontaneously and their frequency was strongly increased after K+ depolarization, while GABAergic mIPSCs were found after K+ depolarization at lower frequency. This demonstrates that both, excitatory glutamatergic and inhibitory GABAergic synapses were functional in close succession to initial neurite outgrowth. Synapses formed independent of spontaneous electrical activity, which was absent during the first week in culture. Spontaneous action potentials appeared during the second week and chronic action potential blockade by addition of tetrodotoxin reduced neuronal survival and the number of glutamatergic synapses per neuron. During in vitro differentiation the number of synapsin I immunoreactive presynaptic terminals and the frequency of spontaneous glutamatergic miniature EPSCs increased closely correlated, while the frequency of GABAergic mIPSCs after K+ depolarization did not increase. Thus, the continous formation of presynaptic terminals, including possible maturation of transmitter release, appeared to underlie the increase in mEPSC frequency. Analysis of miniature EPSC amplitudes at different stages in vitro revealed an increase in amplitudes, suggesting synaptic differentiation after initial establishment of functional transmission in glutamatergic synapses. This process was synapse specific as amplitudes of GABAergic mIPSCs were invariant.

Synapse formation and morphological differentiation of neuron types in embryonic rat dentate gyrus explants in vitro

Cultured explants obtained from the dentate gyrus of rat embryos (embryonic day 19-20) were used to investigate synapse formation and morphological differentiation of neuron types in the absence of extrinsic afferents. Synaptogenesis was studied by whole-cell recordings of postsynaptic currents and by ultrastructural analysis. Neurons were visualized using Lucifer Yellow filling or staining with DiI. In short-term (3-5 days) cultured explants postsynaptic currents were rarely evoked by extracellular stimulation and synapses were almost completely absent at the ultrastructural level. After 6-10 days in vitro, the incidence of evoking postsynaptic currents mediated by glutamate and GABAA receptors was strongly increased. At the ultrastructural level, the density of synapses increased more than 20-fold. These results demonstrate de novo formation of synapses in cultured embryonic dentate gyrus explants. Neuron types could be discriminated by their dendritic arborizations and by their electrophysiological properties. After 6-10 days in vitro, mossy-like cells exhibited 3-4 primary dendrites branching in a characteristic pattern and showed moderate spike-frequency adaptation. Application of serotonin (5-HT) to cultured explants elicited GABAA-receptor-mediated postsynaptic currents in mossy-like cells, indicating synaptic GABA release from local interneurons. Comparison to 5-HT evoked GABA release in mossy cells in age-matched, acute slices revealed only slight quantitative differences. In contrast to mossy cells, granule cells showing several primary dendrites originating at one cell pole were almost completely absent in cultured explants, suggesting an involvement of extrinsic afferents in the differentiation of granule cells.

Low- and high-voltage-activated Ca2+ conductances in electrically excitable growth cones of chick dorsal root ganglion neurons.

Growth cone Ca2+ currents of chick dorsal root ganglion (DRG) neurons were recorded by a patch pipette located on the cell soma. Somatic and neuritic conductances were selectively blocked either with TTX and Cd2+ or by superfusing with isotonic sucrose using a laminar flow perfusion system. DRG growth cones were electrically excitable and growth cone Ca2+ currents were similar to Ca2+ currents described in DRG somata. In particular low-voltage-activated (LVA) Ca2+ conductances were well represented contrary to previous suggestions in other cell types.

Proton-induced chloride current and voltage-activated Na+ and Ca2+ currents in embryonic neurons from the medicinal leech (Hirudo medicinalis).

Voltage-activated ionic currents and currents induced by step changes in proton concentration (pH 7.9-6.7) were studied in early embryonic neurons from Hirudo medicinalis. Ganglia were dissociated at embryonic day (E) 8-15, and the largest neurons were investigated using whole-cell patch clamp recording. All cells studied displayed voltage-activated Na+ and Ca2+ currents. Step changes in pH induced sustained currents which reversed at the equilibrium potential for Cl- and were blocked by substituting Cl- with acetate or sulfate. These currents thus differ in their ion selectivity from proton-induced currents in vertebrate neurons which are carried by Na+.