Early Stages Of Synapse Formation Research Articles

Brain inflammation induces post-synaptic changes during early synapse formation in adult-born hippocampal neurons

An inflammatory reaction in the brain is primarily characterized by activation of parenchymal microglial cells. Microglia regulate several aspects of adult neurogenesis, i.e. the continuous production of new neurons in the adult brain. Hippocampal neurogenesis is thought to be important for memory formation, but its role in brain diseases is not clear. We have previously shown that brain inflammation modulates the functional integration of newly formed hippocampal neurons. Here, we explored whether there is a defined time period during synaptic development when new neurons are susceptible to brain inflammation. Newly formed hippocampal neurons, born in an intact environment in the adult mouse brain, were exposed to lipopolysaccharide (LPS)-induced inflammation during either early or late phases of excitatory and inhibitory synaptogenesis. We used intra-hippocampal injections of GFP-retroviral vector (RV-GFP) to label the new neurons and ipsilateral LPS injection at either 1 or 4weeks post-RV-GFP injection. A single intra-hippocampal LPS injection induced an inflammatory response for at least 3weeks, including an acute transient pro-inflammatory cytokine release as well as a sub-acute and sustained change in microglial morphology. The general cytoarchitecture of the hippocampal dentate gyrus, including granule cell layer (GCL) volume, and astrocytic glial fibrillary acidic protein expression was not different compared to vehicle controls, and no Fluoro-Jade-positive cell death was observed. New neurons encountering this inflammatory environment exhibited no changes in their gross morphology. However, when inflammation occurred during early stages of synapse formation, we found a region-specific increase in the number of thin dendritic spines and post-synaptic density-95 (PSD-95) cluster formation on spines, suggesting an enhanced excitatory synaptic connectivity in the newborn neurons. No changes were observed in the expression of N-cadherin, an adhesion molecule primarily associated with excitatory synapses. At the inhibitory synapses, alterations due to inflammation were also evident during early but not later stages of synaptic development. Gephyrin, an inhibitory scaffolding protein, was down-regulated in the somatic region, while the adhesion molecules neuroligin-2 (NL-2) and neurofascin were increased in the somatic region and/or on the dendrites. The GABAA receptor-α2 subunit (GABAAR-α2) was increased, while pre/peri-synaptic GABA clustering remained unaltered. The disproportional changes in post-synaptic adhesion molecules and GABAA receptor compared to scaffolding protein expression at the inhibitory synapses during brain inflammation are likely to cause an imbalance in GABAergic transmission. These changes were specific for the newborn neurons and were not observed when estimating the overall expression of gephyrin, NL-2, and GABAAR-α2 in the hippocampal GCL. The expression of interleukin-1-type 1 receptor (IL-1R1) on preferentially the somatic region of new neurons, often in close apposition to NL-2 clusters, may indicate a direct interaction between brain inflammation and synaptic proteins on newborn neurons. In summary, this study provides evidence that adult-born hippocampal neurons alter their inhibitory and excitatory synaptic integration when encountering an LPS-induced brain inflammation during the initial stages of synapse formation. Changes at this critical developmental period are likely to interfere with the physiological functions of new neurons within the hippocampus.

A Comparative Quantitative Assessment of Axonal and Dendritic mRNA Transport in Maturing Hippocampal Neurons

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

Localized Acetylcholine Receptor Clustering Dynamics in Response to Microfluidic Focal Stimulation with Agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.

Synaptic precedence during synapse formation between reciprocally connected neurons involves transmitter-receptor interactions and AA metabolites.

The cellular mechanisms that determine specificity of synaptic connections between mutually connected neurons in the nervous system have not yet been fully examined in vertebrate and invertebrate species. Here we report on a novel form of synaptic interaction during early stages of synapse formation between reciprocally connected Lymnaea neurons. Specifically, using soma-soma synapses between an identified dopaminergic neuron (also known as the giant dopamine cell), right pedal dorsal 1 (RPeD1), and a FMRFamidergic neuron, visceral dorsal 4 (VD4), we demonstrate that although reciprocal inhibitory synapses re-form between the somata after 24-36 h of pairing, VD4 is, however, the first cell to establish synaptic contacts with RPeD1 (within 12-18 h). We show that VD4 "captures" RPeD1 first as a postsynaptic cell by suppressing its transmitter secretory machinery during early stages of cell-cell pairing. The VD4-induced suppression of transmitter release from RPeD1 was transient, and it required transcription and de novo protein synthesis dependent step in VD4 but not in RPeD1. The VD4-induced effects on RPeD1 were mimicked by a FMRFamide-like peptide. Perturbation of FMRFamide-activated metabolites of the arachidonic acid pathway in RPeD1 not only prevented FMRFamide-induced suppression of transmitter release from the giant dopamine cell but also shifted the synaptic balance in favor of RPeD1, thus making it the first cell to begin synaptic transmission with VD4 within 12-18 h. A single RPeD1 that had developed dopamine secretory capabilities overnight and was subsequently paired with VD4 for 12-18 h was, however, immune to VD4-induced suppression of transmitter release. Under these experimental conditions, both cells developed mutual inhibitory synapses concurrently. Taken together, our data provide evidence for novel synaptic interaction between reciprocally connected neurons and underscore the importance of transmitter-receptor interplay in regulating the timing of synapse formation in the nervous system.

CAST into the synapse

For the nervous system to function properly, neurotransmitters must be released in a regulated fashion from an active zone in the presynaptic membrane, but relatively little is known about how synaptic vesicles are directed to this zone in the first place. On page 577, Ohtsuka et al. characterize a novel protein associated with the active zone. Figure CAST (green) and Bassoon (red) colocalize at the early stages of synapse formation. The cytomatrix at the active zone (CAZ) is believed to be important in determining synaptic vesicle localization. In the new work, the authors began with traditional biochemistry, comparing crude membrane and postsynaptic density fractions from rat brain to identify proteins associated with the synapse. One novel protein was found and named CAZ-associated structural protein (CAST). Electron microscopy places CAST at the CAZ in conventional synapses. CAST associates with two known CAZ proteins, RIM1 and Munc13–1, in a ternary complex that associates with the CAZ protein bassoon, providing the first evidence that CAZ proteins form a network of protein–protein interactions in vivo. CAST appears to help determine RIM1 localization in neurons, and the authors found that CAST is expressed in the early stages of synapse formation in primary cultured neurons. The data suggest that complexes of CAZ proteins associate with vesicles during the early stages of synapse formation, and that these protein–vesicle complexes are then transported to the newly forming synapses. Fusion of the vesicles with the plasma membrane could then determine the location of the new active zone. A database search uncovered a putative orthologue of CAST in C. elegans, suggesting that the new protein is a conserved component required for CAZ formation in metazoans. If so, then targeted disruption of CAST in mice or worms should provide additional insight into the formation of this crucial structure. ▪

Cast

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Glutamate Inhibits GABA Excitatory Activity in Developing Neurons

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonist L-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neurons in vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.

Hikaru genki protein is secreted into synaptic clefts from an early stage of synapse formation in Drosophila

The development of neural circuits is regulated by a large number of factors that are localized at distinct neural sites. We report here the localization of one of these factors, hikaru genki (hig) protein, at synaptic clefts in the pupal and adult nervous systems of Drosophila. In hig mutants, unusually frequent bursting activity of the muscles and abnormal motor behavior during the adult stage suggest the misfunction of neuromuscular circuitry. Our immunohistochemical analyses revealed that hig protein, produced by neurons, is secreted from the presynaptic terminals into the spaces between the presynaptic and postsynaptic terminals. In addition, we have found that the localization of this protein in the synaptic spaces temporally correlates with its functional requirement during a critical period that occurs in the middle stage of pupal formation, a period when a number of dendrite and axon growth cones meet to form synapses. These findings indicate that hig protein functions in the formation of functional neural circuits from the early stages of synapse formation.

Tetanic stimulation and cyclic adenosine monophosphate regulate segregation of presynaptic inputs on a common postsynaptic target neuron in vitro.

Previous studies indicated that Aplysia sensory neurons (SNs) compete when reestablishing synapses with a motor cell target (L7) in vitro. The competition is characterized by a cell number-dependent decrease in the efficacy of each connection, an increase in the elimination of SN varicosities, a reduction in the formation of new SN varicosities, and the segregation of varicosities of each SN to restricted portions of the target axons. The changes do not require spike activity, since both the SNs and L7 do not fire spontaneously. Here, we examined whether adding activity to SNs during the early stages of synapse formation with stimuli known to evoke facilitatory responses in stable SN-L7 connections--tetanic stimulation or increase in intracellular cyclic adenosine monophosphate (cAMP)--would modulate the intrinsic segregatory process. Tetanic stimulation to one SN increased synapse efficacy and the number of varicosities of the stimulated SNs while reducing the functional changes by the nonstimulated SNs in the same cultures. An increase in the stability of preexisting varicosities contributed to the overall increase in varicosities evoked by tetanus. The functional changes evoked by tetanus were not expressed when the same tetanic stimulation was also given to the other SN, or when L7 was hyperpolarized during the tetanus to the SN. Raising cAMP levels in one SN increased synapse efficacy and the rate of new varicosity formation by the injected SNs without affecting the development of the connections formed by the noninjected SNs. These results suggest that different forms of presynaptic and postsynaptic activities in neurons can regulate specific aspects of the competitive process associated with the fine-tuning of connections formed by converging presynaptic inputs.

Aplysia cell adhesion molecules and serotonin regulate sensory cell- motor cell interactions during early stages of synapse formation in vitro

Long-term facilitation of sensorimotor synapses of Aplysia in culture by serotonin (5-HT) is accompanied by two changes: an increase in the number of sensory cell branches and varicosities contacting the major axons of the target motor cell L7, and a downregulation of Aplysia cell adhesion molecules (apCAM) from the surface of the presynaptic sensory cell. We tested the hypothesis that the two changes may be linked; the 5-HT-induced decrease of apCAM levels from sensory neurites may defasciculate sensory neurites from each other and make the surface of the motor axons a more attractive substrate for new growth and synapses. We used developing cultures to examine the relationship of neuritic branching, varicosity formation, and efficacy of the connections formed by sensory cells to levels of apCAM expression on the motor cell. We then determined the consequences of 5-HT applied during the early period of interaction between sensory and motor cells (day 1 or 2 in culture) on the pattern of sensory cell growth and synapse formation. We report that the number of sensory cell branches and varicosities, and the ability of sensory growth cones to fasciculate with L7 axons and form chemical connections correlate with the level of apCAM expression on different regions of L7. Early exposure to 5-HT increased the number of sensory cell branches and varicosities contacting newly regenerated distal neurites of L7 to levels that would normally occur when the sensory neurites interact with the major proximal axons of L7. Treatment with 5-HT also modulated the efficacy of the developing synaptic connections. The change in synapse efficacy was accompanied by an increase in the formation of new sensory varicosities and branches with pioneering growth cones extending on the major axons of L7. The results are consistent with the hypothesis that treatment with 5-HT modulates local differences in the expression of cell adhesion molecules on the surface of the interacting cells making motor neurites more attractive for sensory growth cones, thereby affecting new sensory neuritic growth and synapse formation.

Neuron-Muscle Contact Changes Presynaptic Resting Calcium Set-Point

The role of retrograde signaling between target cells and their presynaptic partners during early cell-cell interactions was examined in cell culture. Using time-lapse video microscopy and fura-2 calcium analysis, we followed contacts between presynaptic neurites of Helisoma motoneurons and muscle fibers dissociated from identified partners in chemical synaptogenesis. Cytosolic calcium rose dramatically at the region of cell-cell contact and subsequently increased throughout the entire neuron. Changes in the calcium set-point of presynaptic neurons were maintained even when connections with muscle targets were severed. Changes in presynaptic calcium concentration were not detected following contact with partners in electrical synapse formation. These data, taken together with the fact that calcium changes coincide with the time course of chemical synaptogenesis in these neuron-muscle cultures, suggest that calcium changes may be involved in early stages of synapse formation.

5B4-CAM expression parallels neurite outgrowth and synaptogenesis in the developing rat brain.

In order to study whether 5B4-CAM expression parallels neurite outgrowth and synaptogenesis, a monoclonal antibody, 5B4, was used, which recognizes both fetal (185-250 kD) and adult (140 kD, 180 kD) forms of the neural cell adhesion molecule (N-CAM), to identify and localize the antigen in rat tissue during developmental ages P1 through P31 and in adults between P60 and 2 years of age. A ubiquitous pattern of intense immunolabelling was detected during the earliest stages of development. 5B4-CAM expression paralleled process outgrowth and the early stages of synaptogenesis in the cerebral cortex, hippocampal formation, and cerebellum. In the adult, immunoreactivity was generally less intense, but the cerebral cortex and hippocampal and cerebellar molecular layers, all areas implicated in learning-associated plasticity, retained substantial immunoreactivity. The inner one-third of the dentate gyrus molecular layer, an area implicated in axonal sprouting and reactive synaptogenesis, was particularly intensely labelled. Evidence from this work suggests that 5B4-CAM expression may be useful in monitoring neurite outgrowth and the early stages of synapse formation during development and possibly axonal sprouting and reactive synaptogenesis in the adult.

The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study

GAP43 is a developmentally regulated phosphoprotein which is almost exclusively found in neurons. Numerous correlative studies have shown that GAP43 is expressed at high levels during neurite extension, axonal elongation and synaptogenesis. In this study we used in situ hybridization to examine GAP43 expression during late embryonic and early postnatal development. The highest relative levels of GAP43 at all stages were present in the neocortex. Levels in this and other regions peaked between postnatal days 5 and 10. These results indicate that high levels of GAP43 mRNA correlate most highly with the latter stages of axon outgrowth and with the early stages of synapse formation.

Voltage-dependent properties of electrical synapses formed between identified leech neurones in vitro.

1. The voltage-dependent properties of rectifying and non-rectifying electrical synapses formed between identified leech neurones were quantified during their regeneration in vitro. 2. Junctional conductance increased with time in culture. This was evaluated by making comparisons between cell pairs maintained in vitro for differing amounts of time, as well as by taking repeated measurements from a single cell pair at different time intervals. 3. Non-rectifying electrical synapses were formed between certain identified neurones of the same type. Thus, Leydig cells cultured with Leydig cells established non-rectifying electrical connections, as did Retzius cells, longitudinal motoneurones (L cells) and anterior pagoda (AP) cells, each paired with its own cell type. 4. Rectifying synapses developed when sensory neurones (P cells or N cells) were paired with the other neurones mentioned above that form non-rectifying connections between themselves. The cell combinations examined were L cell-P cell. Leydig cell-N cell, and AP cell-P cell. The direction of current flow across these rectifying synapses was consistently from the sensory neurone to the other cell in the pair. 5. Non-rectifying connections early in the process of synapse regeneration (1-3 days) showed non-linearities greater than those observed in established non-rectifying synapses. There was a subtle, but clear, voltage dependence even at the later stages of synapse formation (4-18 days). 6. In contrast to non-rectifying connections, rectifying synapses formed between cells at early times in culture showed less voltage dependence than those observed at later times. 7. The marked non-linearities of the non-rectifying connections at early stages in synapse formation along with the reduced voltage dependence of the rectifying connections within the same time period revealed unexpected similarities between the two. At the early stages of synapse formation, the two types of electrical synapse were essentially indistinguishable for one direction of junctional current.

Comparative aspects of hippocampal and neocortical long-term potentiation

Long-term potentiation (LTP) is a candidate for the synaptic alternations underlying memory storage in the mammalian CNS. In this chapter LTP in hippocampus and in visual neocortex are compared. Comparisons of the optimal tetanus parameters revealed that 2–3 trains of high-frequency stimulation (100–400 Hz) delivered within a brief period of time (minutes) results in maximal potentiation in hippocampal synapses. In contrast, the parameters most effective in neocortex were either low-frequency (2 Hz for 60 min) or high-frequency bursts (100 Hz, 100 ms train at 1 5 s for 10 min), both of which deliver at least an order of magnitude more afferent activation than that required for hippocampus. Hippocampal population spike potentiation averages 250% and the population excitatory postsynaptic potential (EPSP) potentiation averages 50%. Neocortical LTP also averages about 50%. The expression of LTP requires about 5 min in CA1 hippocampus, whereas about 30 min are required for expression of neocortical potentiation. Both hippocampus and visual neocortex display an enhanced potentiation early in development, with a later stabilization at lower adult levels. Centering at postnatal day 15, hippocampal CA1 displays an LTP magnitude that is over twice that seen at day 60. Neocortical responses display a similar peak at postnatal day 15 and a subsequent adult stabilization at approximately half of the day 15 maximum. Both tissues first display LTP during the early stages of synapse formation between postnatal days 6–10. The role of the NMDA receptor is implicated in aspects of both hippocampal and neocortical LTP.

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

Independent development of presynaptic specializations in olfactory cortex of the fetal rat.

Electron microscopy was used to study synaptogenesis in prepyriform cortex of fetal rat pups during early stages of synapse formation. Of special interest is the frequent occurrence of unapposed, developing synaptic specializations in axon and growth cone profiles. The location and morphology of the unapposed specializations suggests that they are presynaptic in nature. These presumably immature presynaptic specializations are found in the lateral olfactory tract and subjacent cortex. Intermediate forms between uncontacted presynaptic specializations and definitive synapses suggest a synaptogenic sequence in which initial development of an immature presynaptic specialization begins without apposition of a postsynaptic element at that location. This implies that initiation of presynaptic development is not dependent upon postsynaptic contact and also raises the question of whether synaptic contacts could be established via presynaptic induction of postsynaptic formation.

Interaction between motor axons from two different nerves reinnervating the pectoral muscle of Xenopus laevis.

1. An electrophysiological and morphological study of sprouting and regeneration of motor nerves has been performed in the dually innervated pectoral muscle of Xenopus laevis. 2. Section of one of the nerves induced axon sprouting in the intact nerve. Synapse formation by the sprouting axons was slow since the intact nerve took more than 3 months to increase its field of innervation by 70%. The rate of axon regeneration was faster than that of axon sprouting since the cut nerve reinnervated its former territory in less than 1 month. 3. At early stages of synapse formation the sprouted or regenerated endings were poorly branched but any terminal branches were, as a rule, longer than normal. Signs of degeneration and replacement of endings have been observed. 4. Low levels of transmitter release persisted for several months in newly formed endings. This depression was more pronounced at the endings formed outside the normal field of innervation of the nerve. 5. Poly-innervated muscle fibres have been observed during reinnervation by regenerated or sprouted axons. Their number decreases gradually in the months that follow the beginning of reinnervation. Synaptic efficacy was lower at poly than at mono-innervated muscle fibres. At doubly innervated spots and at separated spots on the same fibre average end-plate potential (e.p.p.) amplitude was 1/3 and 2/3 respectively of that recorded at singly innervated fibres. 6. Electrophysiological and morphological data have been compared at individual doubly innervated end-plate sites. End-plate potential amplitude was positively correlated with the degree of ending development. 7. Sprouted endings remain functional after periods of reinnervation of 30 months, although signs of regression have been observed. These are probably mediated by spontaneous degeneration of the terminals and replacement by endings from the regenerating nerve.

Storage and release of acetylcholine by a clonal cell line.

The nerve cell line PC12, in its morphologically undifferentiated state, synthesizes, stores, and secretes catecholamines and acetylcholine. At least 60% of the basal level of neurotransmitter release is due to a calcium-dependent mechanism, and the rate of secretion is enhanced by increasing external potassium. A minimum of 80% of the intracellular acetylcholine and catecholamines are stored in particulate structures. The storage site for acetylcholine is dense core vesicles that can be distinguished from those containing catecholamines on the basis of vesicle density on sucrose gradients, vesicle size, and reserpine sensitivity. These results are discussed in relation to what are thought to be the early stages of synapse formation in cell culture.