Charles F. Stevens

Astroglia induce neurogenesis from adult neural stem cells

During an investigation of the mechanisms through which the local environment controls the fate specification of adult neural stem cells, we discovered that adult astrocytes from hippocampus are capable of regulating neurogenesis by instructing the stem cells to adopt a neuronal fate. This role in fate specification was unexpected because, during development, neurons are generated before most of the astrocytes. Our findings, together with recent reports that astrocytes regulate synapse formation and synaptic transmission, reinforce the emerging view that astrocytes have an active regulatory role—rather than merely supportive roles traditionally assigned to them—in the mature central nervous system.

Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca(2+)-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or alpha-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca2+ triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca(2+)-independent release. We propose that synaptotagmin I is the major low affinity Ca2+ sensor mediating Ca2+ regulation of synchronous neurotransmitter release in hippocampal neurons.

Heterogeneity of Release Probability, Facilitation, and Depletion at Central Synapses

Previous studies of short-term plasticity in central nervous systems synapses have largely focused on average synaptic properties. In this study, we use recordings from putative single synaptic release sites in hippocampal slices to show that significant heterogeneity exists in facilitation and depletion among synapses. In particular, the amount of paired-pulse facilitation is inversely related to the initial release probability of the synapse. We also examined depletion at individual synapses using high frequency stimulation, and estimated the size of the readily releasable vesicle pool, which averaged 5.0 +/- 3.0 quanta (n = 13 synapses). In addition, these experiments demonstrate that the release probability at a synapse is directly correlated with the size of its readily releasable vesicle pool.

Definition of the Readily Releasable Pool of Vesicles at Hippocampal Synapses

A readily releasable pool of quanta, tentatively identified with docked synaptic vesicles, has been defined by analysis of the neurotransmitter release caused by application of hypertonic solutions. The goal of this work is to determine the relationship of this functionally defined readily releasable pool to the one drawn upon by action potential-evoked release. We find that hypertonic solutions do not act through changes in intracellular calcium. Since the release produced by action potentials and hypertonic solutions varies in parallel as the pool size is changed, we conclude that the same pool is shared by both mechanisms. This conclusion, taken together with other observations in the literature, means that the synaptic release probability depends on the size of the readily releasable pool.

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons

Neural stem cells are present both in the developing nervous system and in the adult nervous system of all mammals, including humans. Little is known, however, about the extent to which stem cells in adults can give rise to new neurons. We used immunocytochemistry, electron microscopy, fluorescence microscopy (FM imaging) and electrophysiology to demonstrate that progeny of adult rat neural stem cells, when co-cultured with primary neurons and astrocytes from neonatal hippocampus, develop into electrically active neurons and integrate into neuronal networks with functional synaptic transmission. We also found that functional neurogenesis from adult stem cells is possible in co-culture with astrocytes from neonatal and adult hippocampus. These studies show that neural stem cells derived from adult tissues, like those derived from embryonic tissues, retain the potential to differentiate into functional neurons with essential properties of mature CNS neurons.

Modified hippocampal long-term potentiation in PKCγ-mutant mice

Abstract Calcium-phospholipid-dependent protein kinase (PKC) has long been suggested to play an important role in modulating synaptic efficacy. We have created a strain of mice that lacks the γ subtype of PKC to evaluate the significance of this brain-specific PKC isozyme in synaptic plasticity. Mutant mice are viable, develop normally, and have synaptic transmission that is indistinguishable from wild-type mice. Long-term potentiation (LTP), however, is greatly diminished in mutant animals, while two other forms of synaptic plasticity, long-term depression and paired-pulse facilitation, are normal. Surprisingly, when tetanus to evoke LTP was preceded by a low frequency stimulation, mutant animals displayed apparently normal LTP. We propose that PKCγ is not part of the molecular machinery that produces LTP but is a key regulatory component.

Heterogeneous Release Properties of Visualized Individual Hippocampal Synapses

We have used endocytotic uptake of the styryl dye FM1-43 at synaptic terminals (Betz and Bewick, 1992) to study properties of individual synapses formed by axons of single hippocampal neurons in tissue culture. The distribution of values for probability of evoked transmitter release p estimated by dye uptake is continuous, with a preponderance of low p synapses and a broad spread of probabilities. We have validated this method by demonstrating that the optically estimated distribution of p at autapses in single-neuron microislands predicts, with no free parameters, the rate of blocking of NMDA responses by the noncompetitive antagonist MK-801 at the same synapses. Different synapses made by a single axon exhibited varying amounts of paired-pulse modulation; synapses with low p tended to be facilitated more than those with high p. The increment in release probability produced by increasing external calcium ion concentration also depended on a synapses initial p value. The size of the recycling pool of vesicles was strongly correlated with p as well, suggesting that synapses with higher release probabilities had more vesicles. Finally, p values of neighboring synapses were correlated, indicating local interactions in the dendrite or axon, or both.

Inactivity Produces Increases in Neurotransmitter Release and Synapse Size

When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components--active zone, postsynaptic density, and bouton--becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.

Facilitation and depression at single central synapses

Using whole-cell recording from CA1 hippocampal pyramidal neurons and minimal stimulation of Schaffer collaterals, we have studied what seem to be single synapses. Although the transmission at a putative single synapses is quite unreliable, the synapse can be made to release transmitter reliably in response to the second stimulus in a pair of stimuli that re presented in rapid succession (e.g., 50 ms separation). Statistical analysis of transsmision failures seen with such paired pulse stimulation reveals that the majority of stimulus-evoked synaptic currents (> 90%) are produced by a single synapse under the conditions of minimal stimulation, even if multiple synapses are actually present. Individual synapses appear to release either zero or one quantum; that is, a single synapse seems to have only one functional release sit at any time. After the release site has been used, approximately 20 ms is required to refill the site so that it can be used again.