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.

Multiple forms of endocytosis in bovine adrenal chromaffin cells.

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent "compensatory" retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of approximately 6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed "excess" retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of approximately 248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.

Ultrastructural organization of bovine chromaffin cell cortex-analysis by cryofixation and morphometry of aspects pertinent to exocytosis.

We have analyzed ultrathin sections from isolated bovine chromaffin cells grown on plastic support, after fast freezing, by quantitative electron microscopy. We determined the size and intracellular distribution of dense core vesicles (DVs or chromaffin granules) and of clear vesicles (CVs). The average diameter of DVs is 356 nm, and that of CVs varies between 35-195 nm (average 90 nm). DVs appear randomly packed inside cells. When the distance of the center of DVs to the cell membrane (CM) is analyzed, DV density is found to decrease as the CM is approached. According to Monte Carlo simulations performed on the basis of the measured size distribution of DVs, this decay can be assigned to a "wall effect." Any cortical barrier, regardless of its function, seems to not impose a restriction to a random cortical DV packing pattern. The number of DVs closely approaching the CM (docked DVs) is estimated to be between 364 and 629 (average 496), i.e., 0.45 to 0.78 DVs/micron2 CM. Deprivation of Ca2+, priming by increasing [Ca2+]i, or depolarization by high [K+]e for 10 s (the effect of which was controlled electrophysiologically and predicted to change the number of readily releasable granules [RRGs]) does not significantly change the number of peripheral DVs. The reason may be that (a) structural docking implies only in part functional docking (capability of immediate release), and (b) exocytosis is rapidly followed by endocytosis and replenishment of the pool of docked DVs. Whereas the potential contribution of DVs to CM area increase by immediate release can be estimated at 19-33%, that of CVs is expected to be in the range of 5.6-8.0%.

A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells.

Intense stimuli, such as trains of depolarizing pulses or the caffeine-induced release of calcium from intracellular stores, readily depress the secretory response in neuroendocrine cells. Secretory responses are restored by rest periods of minutes in duration. This recovery was accelerated when the concentration of cytosolic calcium was moderately increased and probably resulted from calcium-dependent replenishment of a pool of release-ready granules. Continuously increased concentrations of calcium led the over-filling of such a pool. Subsequently, secretory responses to stronger calcium stimuli were augmented. Hormone-induced calcium transients with a plateau phase of increased concentration of calcium may enhance the secretory response in this way.

IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels.

Lambert-Eaton syndrome, an autoimmune disorder frequently associated with small-cell carcinoma of the lung, is characterized by impaired evoked release of acetylcholine from the motor nerve terminal. Immunoglobulin G (IgG) antibodies from patients with the syndrome, applied to bovine adrenal chromaffin cells, reduced the voltage-dependent calcium channel currents by about 40 percent. When calcium was administered directly into the cytoplasm, however, the IgG-treated cells exhibited normal exocytotic secretion, as assayed by membrane capacitance measurement. Measurement with the fluorescent calcium indicator fura-2 indicated that the IgG treatment reduced potassium-stimulated increase in free intracellular calcium concentration. The pathogenic IgG modified neither kinetics of calcium channel activation nor elementary channel activity, suggesting that a reduction in the number of functional calcium channels underlies the IgG-induced effect. Therefore, Lambert-Eaton syndrome IgG reacts with voltage-dependent calcium channels and blocks their function, a phenomenon that can account for the presynaptic impairment characteristic of this disorder.

Multiple calcium-dependent processes related to secretion in bovine chromaffin cells.

We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient--slow secretion for up to 1 min due to displacement of Ca2+ by cytoplasmic Mg2+. Flash photolysis elicited 100 microM [Ca2+]i steps that evoked intense secretion, lasting a few seconds. In cells experiencing a loading transient, [Ca2+]i steps evoked an especially fast secretion. A persistent, slow secretion often followed these fast phases. Distinct kinetic components may reflect secretion from pools that are differentially capable of release. Both secretion and movement of vesicles between pools appear to be [Ca2+]i sensitive. Later [Ca2+]i steps sometimes evoked a rapid capacitance decrease, indicating a fast, [Ca2+]i-dependent phase of endocytosis.

Ca(2+)-dependent exocytosis in the somata of dorsal root ganglion neurons.

Using capacitance measurements and the single-cell immunoblot assay to study secretion in dorsal root ganglion neurons, we found that the somata underwent robust exocytosis upon depolarization and released substance P, in response to KCl stimulation. The parallel changes between capacitance responses and intracellular Ca2+ concentration ([Ca2+]i) at different membrane potentials and the inhibition of exocytosis by Ca2+ chelators suggest that soma release is Ca(2+)-dependent. We also assessed the level of Ca2+ required for exocytosis by raising the average [Ca2+]i with the Ca2+ ionophore, ionomycin. Capacitance changes were triggered by cytosolic Ca2+ > 0.6 microM; the [Ca2+]i at the release sites during depolarizations was estimated to be 3-10 microM. These Ca2+ levels are similar to those obtained from neuroendocrine cells, but are at least 10 times lower than those required for transmitter release from nerve terminals.

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.

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.

Low mobility of the Ca2+ buffers in axons of cultured Aplysia neurons.

Cellular Ca2+ buffers determine amplitude and diffusional spread of neuronal Ca2+ signals. Fixed Ca2+ buffers tend to retard the signal and to lower the apparent diffusion coefficient (D(app)) of Ca2+, whereas mobile buffers contribute to Ca2+ redistribution. To estimate the impact of the expression of specific Ca2+-binding proteins or the errors in Ca2+ measurement introduced by indicator dyes, the diffusion coefficient De and the Ca2+-binding ratio kappa(e) of endogenous Ca2+ buffers must be known. In this study, we obtain upper bounds to these quantities (De < 16 microm2/s; kappa(e) < 60) for axoplasm of metacerebral cells of Aplysia california. Due to these very low values, even minute concentrations of indicator dyes will interfere with the spatiotemporal pattern of Ca2+ signals and will conceal changes in the expression of specific Ca2+-binding proteins, which in the native neuron are expected to have significant effects on Ca2+ signals.

Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules.

We have used membrane capacitance measurements to assay Ca2+-triggered exocytosis in single bovine adrenal chromatin cells. Brief application of phorbol ester (PMA) enhances depolarization-evoked exocytosis severalfold while actually decreasing the Ca2+ current. Ca2+ metabolism is unchanged. Three different protocols were used to show that PMA increases the size of the readily releasable pool of secretory granules. PMA treatment leads to a large increase in amplitude, but little change in the time course of the exocytic burst that results from rapid elevation of [Ca2+]i upon photolysis of DMI-Nitrophen. Thus, PKC appears to affect a late step in secretion but not the Ca2+ sensitivity of the final step.

Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse.

The size of a pool of readily releasable vesicles at a giant brainstem synapse, the calyx of Held, was probed with three independent approaches. Using simultaneous pre- and postsynaptic whole-cell recordings, two forms of presynaptic Ca2+ stimuli were applied in rapid succession: uncaging of Ca2+ by flash photolysis and the opening of voltage-gated Ca2+ channels. The ensuing transmitter release showed a nearly complete cross-inhibition between the two stimuli, indicating the depletion of a limited pool of about 700 transmitter quanta. The pool size was confirmed in experiments using enhanced extracellular Ca2+ concentrations, as well as short, high-frequency stimulus trains. The results reveal a surprisingly large pool of functionally available vesicles, of which a fraction of about 0.2 is released by a single presynaptic action potential under physiological conditions.

Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells.

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.

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.

Fractional contribution of calcium to the cation current through glutamate receptor channels.

The Ca2+ fraction of the ion current flowing through glutamatergic NMDA and AMPA/kainate receptor channels was determined in forebrain neurons of the medial septum. The neurons were overloaded with the Ca2+ indicator dye fura-2 (1 mM) via the recording patch pipettes. This approach allowed the direct determination of the Ca2+ influx from changes in the Ca(2+)-sensitive fura-2 fluorescence. We found that, at negative membrane potentials and at an extracellular free Ca2+ concentration of 1.6 mM, the Ca2+ fraction of the current through the NMDA receptor channels is only 6.8%, about 2-fold lower than previously estimated from reversal potential measurements. Interestingly, a quite high fractional Ca2+ current of 1.4% was determined for the linearly conducting AMPA/kainate receptor channels found in these neurons.

Mechanisms determining the time course of secretion in neuroendocrine cells.

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.

Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release.

Munc13-1, a mammalian homolog of C. elegans unc-13p, is thought to be involved in the regulation of synaptic transmission. We now demonstrate that Munc13-1 is a presynaptic high-affinity phorbol ester and diacylglycerol receptor with ligand affinities similar to those of protein kinase C. Munc13-1 associates with the plasma membrane in response to phorbol ester binding and acts as a phorbol ester-dependent enhancer of transmitter release when overexpressed presynaptically in the Xenopus neuromuscular junction. These observations establish Munc13-1 as a novel presynaptic target of the diacylglycerol second messenger pathway that acts in parallel with protein kinase C to regulate neurotransmitter secretion.

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.

Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity.

The secretion of synaptic and other vesicles is a complex process involving multiple steps. Many molecular components of the secretory apparatus have been identified, but how they relate to the different stages of vesicle release is not clear. We examined this issue in adrenal chromaffin cells, where capacitance measurements and amperometry allow us to measure vesicle fusion and hormone release simultaneously. Using flash photolysis of caged intracellular calcium to induce exocytosis, we observed three distinct kinetic components to vesicle fusion, of which only two are related to catecholamine release. Intracellular dialysis with botulinum neurotoxin E, D or C1 or tetanus-toxin light chains abolishes the catecholamine-related components, but leaves the third component untouched. Botulinum neurotoxin A, which removes nine amino acids from the carboxy(C)-terminal end of SNAP-25, does not eliminate catecholamine release completely, but slows down both catecholamine-related components. Thus we assign a dual role to SNAP-25 and suggest that its nine C-terminal amino acids are directly involved in coupling the calcium sensor to the final step in exocytosis.

The patch-clamp technique in the study of secretion.

One of the basic cellular functions of virtually every cell type is the exocytotic release of molecules synthesized, stored and packaged into intracellular vesicles or granules. Over decades much effort has been concentrated on elucidating the chain of events leading to exocytosis. Unfortunately, the nature of the process that ultimately induces membrane fusion is not known, nor has it been established definitively whether or not the final steps in the secretory cascade are identical in different cells. Although the fusion between vesicle and plasma membrane has been neatly documented by electron micrographs, it was only recently that the technique of time-resolved membrane capacitance measurement has provided a more detailed insight into mechanistic aspects of exocytosis, both in terms of the fusion event and the steps involved in stimulus-secretion coupling.