Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus.

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.

The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.

Introduction: regulated exocytosis.

Calcium ions regulate secretory processes in several ways. Most prominently they (i) trigger the release of vesicle contents rapidly and in a highly cooperative way and they (ii) control priming steps, which prepare vesicles for release. The importance of using assays with high time resolution for separating these distinct roles is pointed out here.

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.

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image.

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal-to-noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal-to-noise ratio or optimize a newly defined 'figure of merit', which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.

Estimating transmitter release rates from postsynaptic current fluctuations.

A method is presented that allows one to estimate transmitter release rates from fluctuations of postsynaptic current records under conditions of stationary or slowly varying release. For experimental applications, we used the calyx of Held, a glutamatergic synapse, in which "residual current," i.e., current attributable to residual glutamate in the synaptic cleft, is present. For a characterization of synaptic transmission, several postsynaptic parameters, such as the mean amplitude of the miniature postsynaptic current and an apparent single channel conductance, have to be known. These were obtained by evaluating variance and two more higher moments of the current fluctuations. In agreement with Fesce et al. (1986), we found both by simulations and by analyzing experimental records that high-pass filtering of postsynaptic currents renders the estimates remarkably tolerant against nonstationarities. We also found that release rates and postsynaptic parameters can be reliably obtained when release rates are low ( approximately 10 events/msec). Furthermore, during a long-lasting stimulus, the transmitter release at the calyx of Held was found to decay to a low, stationary rate of 10 events/msec after depletion of the "releasable pool" of synaptic vesicles. This stationary release rate is compatible with the expected rate of recruitment of new vesicles to the release-ready pool of vesicles. MiniatureEPSC (mEPSC) size is estimated to be similar to the value of spontaneously occurring mEPSC under this condition.

Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis.

At the large excitatory calyx of Held synapse, the quantal size during an evoked EPSC and the number of active zones contributing to transmission are not known. We developed a nonstationary variant of EPSC fluctuation analysis to determine these quantal parameters. AMPA receptor-mediated EPSCs were recorded in slices of young (postnatal 8-10 d) rats after afferent fiber stimulation, delivered in trains to induce synaptic depression. The means and the variances of EPSC amplitudes were calculated across trains for each stimulus number. During 10 Hz trains at 2 mm Ca(2+) concentration ([Ca(2+)]), we found linear EPSC variance-mean relationships, with a slope that was in good agreement with the quantal size obtained from amplitude distributions of spontaneous miniature EPSCs. At high release probability with 10 or 15 mm [Ca(2+)], competitive antagonists were used to partially block EPSCs. Under these conditions, the EPSC variance-mean plots could be fitted with parabolas, giving estimates of quantal size and of the binomial parameter N. With the rapidly dissociating antagonist kynurenic acid, quantal sizes were larger than with a slowly dissociating antagonist, suggesting that the effective glutamate concentration was increased at high release probability. Considering the possibility of multivesicular release and moderate saturation of postsynaptic AMPA receptors, we conclude that the binomial parameter N (637 +/- 117; mean +/- SEM) represents an upper limit estimate of the number of functional active zones. We estimate that during normal synaptic transmission, the probability of vesicle fusion at single active zones is in the range of 0.25-0.4.

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I.

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca(2+)-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca(2+)-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca(2+)-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca(2+)-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.

Estimating synaptic parameters from mean, variance, and covariance in trains of synaptic responses.

Fluctuation analysis of synaptic transmission using the variance-mean approach has been restricted in the past to steady-state responses. Here we extend this method to short repetitive trains of synaptic responses, during which the response amplitudes are not stationary. We consider intervals between trains, long enough so that the system is in the same average state at the beginning of each train. This allows analysis of ensemble means and variances for each response in a train separately. Thus, modifications in synaptic efficacy during short-term plasticity can be attributed to changes in synaptic parameters. In addition, we provide practical guidelines for the analysis of the covariance between successive responses in trains. Explicit algorithms to estimate synaptic parameters are derived and tested by Monte Carlo simulations on the basis of a binomial model of synaptic transmission, allowing for quantal variability, heterogeneity in the release probability, and postsynaptic receptor saturation and desensitization. We find that the combined analysis of variance and covariance is advantageous in yielding an estimate for the number of release sites, which is independent of heterogeneity in the release probability under certain conditions. Furthermore, it allows one to calculate the apparent quantal size for each response in a sequence of stimuli.

Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse.

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.

Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.

1. In connections formed by nerve terminals of layer 2/3 pyramidal cells onto bitufted interneurones in young (postnatal day (P)14-15) rat somatosensory cortex, the efficacy and reliability of synaptic transmission were low. At these connections release was facilitated by paired-pulse stimulation (at 10 Hz). In connections formed by terminals of layer 2/3 pyramids with multipolar interneurones efficacy and reliability were high and release was depressed by paired-pulse stimulation. In both types of terminal, however, the voltage-dependent Ca2+ channels that controlled transmitter release were predominantly of the P/Q- and N-subtypes. 2. The relationship between unitary EPSP amplitude and extracellular calcium concentration ([Ca2+]o) was steeper for facilitating than for depressing terminals. Fits to a Hill equation with nH = 4 indicated that the apparent KD of the Ca2+ sensor for vesicle release was two- to threefold lower in depressing terminals than in facilitating ones. 3. Intracellular loading of pyramidal neurones with the fast and slowly acting Ca2+ buffers BAPTA and EGTA differentially reduced transmitter release in these two types of terminal. Unitary EPSPs evoked by pyramidal cell stimulation in bitufted cells were reduced by presynaptic BAPTA and EGTA with half-effective concentrations of approximately 0.1 and approximately 1 mM, respectively. Unitary EPSPs evoked in multipolar cells were reduced to one-half of control at higher concentrations of presynaptic BAPTA and EGTA (approximately 0.5 and approximately 7 mM, respectively). 4. Frequency-dependent facilitation of EPSPs in bitufted cells was abolished by EGTA at concentrations of > or = 0.2 mM, suggesting that accumulation of free Ca2+ is essential for facilitation in the terminals contacting bitufted cells. In contrast, facilitation was unaffected or even slightly increased in the terminals loaded with BAPTA in the concentration range 0.02-0.5 mM. This is attributed to partial saturation of exogenously added BAPTA. However, BAPTA at concentrations > or = 1 mM also abolished facilitation. 5. Frequency-dependent depression of EPSPs in multipolar cells was not significantly reduced by EGTA. With BAPTA, the depression decreased at concentrations > 0.5 mM, concomitant with a reduction in amplitude of the first EPSP in a train. 6. An analysis is presented that interprets the effects of EGTA and BAPTA on synaptic efficacy and its short-term modification during paired-pulse stimulation in terms of changes in [Ca2+] at the release site ([Ca2+]RS) and that infers the affinity of the Ca2+ sensor from the dependence of unitary EPSPs on [Ca2+]o. 7. The results suggest that the target cell-specific difference in release from the terminals on bitufted or multipolar cells can be explained by a longer diffusional distance between Ca2+ channels and release sites and/or lower Ca2+ channels density in the terminals that contact bitufted cells. This would lead to a lower [Ca2+] at release sites and would also explain the higher apparent K(D) of the Ca2+ sensor in facilitating terminals.

Highly nonlinear photodamage in two-photon fluorescence microscopy.

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).

Combining deconvolution and noise analysis for the estimation of transmitter release rates at the calyx of held.

The deconvolution method has been used in the past to estimate release rates of synaptic vesicles, but it cannot be applied to synapses where nonlinear interactions of quanta occur. We have extended this method to take into account a nonlinear current component resulting from the delayed clearance of glutamate from the synaptic cleft. We applied it to the calyx of Held and verified the important assumption of constant miniature EPSC (mEPSC) size by combining deconvolution with a variant of nonstationary fluctuation analysis. We found that amplitudes of mEPSCs decreased strongly after extended synaptic activity. Cyclothiazide (CTZ), an inhibitor of glutamate receptor desensitization, eliminated this reduction, suggesting that postsynaptic receptor desensitization occurs during strong synaptic activity at the calyx of Held. Constant mEPSC sizes could be obtained in the presence of CTZ and kynurenic acid (Kyn), a low-affinity blocker of AMPA-receptor channels. CTZ and Kyn prevented postsynaptic receptor desensitization and saturation and also minimized voltage-clamp errors. Therefore, we conclude that in the presence of these drugs, release rates at the calyx of Held can be reliably estimated over a wide range of conditions. Moreover, the method presented should provide a convenient way to study the kinetics of transmitter release at other synapses.

Quantitative relationship between transmitter release and calcium current at the calyx of held synapse.

A newly developed deconvolution method (Neher and Sakaba, 2001) allowed us to resolve the time course of neurotransmitter release at the calyx of Held synapse and to quantify some basic aspects of transmitter release. First, we identified a readily releasable pool (RRP) of synaptic vesicles. We found that the size of the RRP, when tested with trains of strong stimuli, was constant regardless of the exact stimulus patterns, if stimuli were confined to a time interval of approximately 60 msec. For longer-lasting stimulus patterns, recruitment of new vesicles to the RRP made a substantial contribution to the total release. Second, the cooperativity of transmitter release as a function of Ca(2+) current was estimated to be 3-4, which confirmed previous results (Borst and Sakmann, 1999; Wu et al., 1999). Third, an initial small Ca(2+) influx increased the efficiency of Ca(2+) currents in subsequent transmitter release. This type of facilitation was blocked by a high concentration of EGTA (0.5 mm). Fourth, the release rates of synaptic vesicles at this synapse turned out to be heterogeneous: once a highly Ca(2+)-sensitive population of vesicles was consumed, the remaining vesicles released at lower rates. These components of release were more clearly separated in the presence of 0.5 mm EGTA, which prevented the buildup of residual Ca(2+). Conversely, raising the extracellular Ca(2+) concentration facilitated the slower population such that its release characteristics became more similar to those of the faster population under standard conditions. Heterogeneous release probabilities are expected to support the maintenance of synaptic transmission during high-frequency stimulation.

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held.

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

Calcium signals and synaptic short-term plasticity in the central nervous system.

Plastic changes in the connectivity between neurons underly the adaptive information processing of the central nervous system. The shortest forms of such plasticity are synaptic depression and facilitation, which happen on the subsecond time scale. New techniques allow to study synaptic transmission at unprecedented resolution and to dissect its various components, such as presynaptic Ca++ currents, Ca++ signals, and transmitter stores. Precise knowledge on the components, which are altered during plastic changes, is essential for understanding these important mechanisms.

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.