Presynaptic Development Research Articles

Interaction between GRIP and Liprin-α/SYD2 Is Required for AMPA Receptor Targeting

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-α/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-α and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-α is important for postsynaptic as well as presynaptic maturation.

Gene Expression Profiling in Postmortem Rett Syndrome Brain: Differential Gene Expression and Patient Classification

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Neuroligin Expressed in Nonneuronal Cells Triggers Presynaptic Development in Contacting Axons

Most neurons form synapses exclusively with other neurons, but little is known about the molecular mechanisms mediating synaptogenesis in the central nervous system. Using an in vitro system, we demonstrate that neuroligin-1 and -2, postsynaptically localized proteins, can trigger the de novo formation of presynaptic structure. Nonneuronal cells engineered to express neuroligins induce morphological and functional presynaptic differentiation in contacting axons. This activity can be inhibited by addition of a soluble version of β-neurexin, a receptor for neuroligin. Furthermore, addition of soluble β-neurexin to a coculture of defined pre- and postsynaptic CNS neurons inhibits synaptic vesicle clustering in axons contacting target neurons. Our results suggest that neuroligins are part of the machinery employed during the formation and remodeling of CNS synapses.

Nerve growth factor collaborates with myocyte‐derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle-containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors.

Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture.

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naïve neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article.

Presynaptic differentiation induced in cultured neurons by local application of basic fibroblast growth factor

Recent studies have suggested a role for molecules residing at the muscle surface in signaling presynaptic development at the neuromuscular junction (NMJ). Since heparan sulfate-proteoglycan is a major component of the extracellular matrix of skeletal muscle, factors that are bound to this proteoglycan, such as basic fibroblast growth factor (bFGF), are in a strategic position for neuronal signaling. To test this idea, we applied bFGF to cultured Xenopus spinal cord neurons and monitored the change in intracellular Ca2+ level with fura-2 ratio imaging. In one-third of the neurons, local application of bFGF elicited a 30-140% increase in intracellular Ca2+ level. Ca(2+)-free medium or suramin abolished this change. Latex beads coated with bFGF induced clustering of synaptic vesicles at the bead-neurite contacts as evidenced by anti-synaptotagmin antibody labeling and electron microscopy. This response was also blocked by Ca(2+)-free medium and by suramin. Uncoated beads or beads coated with PDGF were ineffective. This induction was also inhibited by a tyrosine kinase inhibitor, tyrphostin RG-50864, suggesting the role of receptor tyrosine kinase activation in this process. In addition, bFGF-coated beads also induced the localization of depolarization-dependent Ca2+ influx to the bead-neurite contacts. In contrast, depolarization caused a distributed Ca2+ elevation in untreated neurites. These results suggest that local presentation of bFGF can mimic the muscle target in signaling the development of both a cytoplasmic and a membranous specialization for excitation-secretion coupling observed at the NMJ.

Retrograde regulation of presynaptic development during synaptogenesis.

Major advances are occurring in our understanding of the events leading to synapse formation. Contact between the growth cone and target tissue leads to intercellular signaling which controls both pre- and postsynaptic development of the synapse. The identity of retrograde signals that regulate presynaptic development are beginning to emerge, and the signal transduction cascades that are activated presynaptically are being characterized. Recent studies have shown that both the resting calcium level and activation of presynaptic protein kinase A are critical in the development of the presynaptic terminal. An understanding of these regulatory mechanisms is beginning to provide insight into the molecular control of synaptic specificity.

6-Hydroxydopamine treatment of neonatal rats. I. Effects on the development of the spinal cord noradrenergic system

The spinal cord contains noradrenergic (NA) pathways which descend from cell bodies in the medulla oblongata and pons to terminate at all levels in the spinal gray matter. The present studies sought to determine the patterns of postnatal development of pre- and postsynaptic elements of NA transmission in the spinal cord. Significant presynaptic development is evident at birth as reflected by substantial high-affinity uptake of norepinephrine (NE) into synaptosomes (0.65–0.90 pmol/mg protein). There is a subsequent increase in uptake on postnatal day (PND) 5, followed by a decrease in 5–10 days to essentially adult levels, starting on PND 20 (0.30–0.35 pmol/mg protein). This decrease in NE uptake occurs coincident with increases in the density of postsynaptic α 1 and β adrenergic receptors and also NE-stimulated accumulation of 3′,5′-cyclic adenosine monophosphate (cAMP). Peaks in the development of α 1 receptors (PND 10) and β receptors (PND 20) and NE-stimulated cAMP accumulation (PND 15) were also followed by decreases to adult levels. The neurotoxin 6-hydroxydopamine (6-OHDA) was administered at birth to determine the effects of denervation on the development of the spinal NA systems. At each day following 6-OHDA, synaptosomal uptake of [ 3H]NE was reduced by two-thirds compared with control values. α 1 and β adrenergic receptor binding are uniformly increased along with a parallel increase in NE-stimulated accumulation of cAMP. While uniformly increased over control, the pattern of postnatal increases and decreases in receptors and cAMP accumulation is maintained. These results suggest that developmental signals for the expression of α 1 and β receptors and adenylate cyclase activity in the postsynaptic receptor systems are maintained despite the loss of presynaptic input from descending NA pathways.

Artificial rearing alters development of the nucleus of the solitary tract

Previous studies have shown that damage induced to fungiform papillae of the anterior tongue at postnatal day 2 (P2) alters both pre- and postsynaptic development of gustatory recipient zones within the rostral nucleus of the solitary tract (NST). The present study was conducted to determine whether or not artificial rearing (AR) manipulations, which reduce normal orochemical stimulation during early postnatal development, would be sufficient to produce alterations in anatomical development of the rostral gustatory NST. Two groups of Long-Evans hooded rats were examined. One group received normal rearing with a lactating dam from birth to weaning (mother reared; MR). A second group of animals received artificial rearing via intragastric cannulae between the ages of P4 and P14, and were thereafter returned to lactating dams until the age of weaning (P21). Following weaning and maturation to adulthood (P49), the organization of gustatory afferent terminal fields in the NST was examined using fluorescent tracing procedures which permit the simultaneous visualization of gustatory afferent terminal fields arising from the seventh and ninth cranial nerves. Results show that AR manipulations between the ages of P4 and P14 produce alterations in development of gustatory afferent terminal fields in the NST that are essentially similar to those observed following early postnatal receptor damage. These results confirm previous suggestions that orochemical stimulation during a limited portion of rats' postnatal life is essential in inducing normal presynaptic development in the gustatory NST.

Perinatal exposure to methadone: how do early biochemical alterations cause neurofunctional disturbances?

Publisher Summary This chapter summarizes the progress made toward identifying the sequence of events occurring in the perinatal opiate syndrome. Development of function of a noradrenergic synapse requires a coordinated maturation of nerve terminals, their ability to synthesize, store and release transmitter, and post-synaptic ontogeny of receptors and appropriate linkages of the receptors to cellular processes (adenylate cyclase, phosphoinositide turnover, ion channels). In the rat, the majority of these events occur postnatally synthesis. The final stage of presynaptic development appears to be the arrival in the nerve terminals of synaptic vesicles, which are necessary to convert dopamine to norepinephrine, to store the norepinephrine and to release it into the synapse upon appropriate stimulation. Catecholaminergic synapses, because of their ubiquity in both the central and the peripheral nervous system, have provided some of the most useful information concerning mechanisms of neurobehavioral teratology. Vesicle maturation can be evaluated through measurements of uptake of norepinephrine into isolated vesicle preparations and is a process separable from synaptosomal uptake.

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.

Synaptogenesis in chick paravertebral sympathetic ganglia: a morphometric analysis

Synaptogenesis was studied in lumbar sympathetic ganglia of chicken by light and electron microscopic morphometric methods. At 10 days in ovo, fewer than 1% of the adult number of synapses are present. The total numbers of synapses and of synaptic vesicles per ganglion increase progressively with age; however, the majority of both are formed after 30 days after hatching. The average number of synaptic vesicles per synapse increases several fold after hatching. The number of synapses and of synaptic vesicles per ganglion increase roughly in concert with biochemical markers of presynaptic development (activity of choline acetyltransferase and levels of acetylcholine) as well as postsynaptic development (tyrosine hydroxylase; based on biochemical data reported elsewhere). The amount of acetylcholine and activity of choline acetyltransferase per synaptic vesicle at 10 days in ovo are 8 and 27 times the corresponding adult values. By 1 day after hatching, these ratios have fallen to near adult levels. These data are consistent with the early presence of cholinergic neuroblasts, as suggested by others, and suggest further that such cholinergic neuroblasts are eliminated, or their cholinergic properties suppressed, before hatching.

Maternal methadone administration: Deficit in development of alpha-noradrenergic responses in developing rat brain as assessed by norepinephrine stimulation of 33P i incorporation into phospholipids in vivo

The effects of perinatal methadone exposure on the development of noradrenergic responses in the brain were examined by assessing the ability of intracisternally administered norepinephrine to stimulate 33P i incorporation into phospholipids in vivo; the effect of norepinephrine is mediated by alpha 1-receptors juxtaposed to noradrenergic nerve terminals. Although there was no difference in basal (unstimulated) incorporation of 33P i, a deficit in norepinephrine-induced stimulation of incorporation was found throughout the preweanling period in offspring of dams treated daily with methadone beginning in midgestation. This effect was not seen when methadone was given during the postnatal period. Since perinatal methadone exposure also delays development of presynaptic catecholaminergic nerve terminals in the brain, these results support the view that perinatal exposure to methadone depresses overall central noradrenergic synaptic function; however, the effects on presynaptic development and on receptor-mediated responses appear to be separable in that they display differences in the critical age periods of sensitivity to perturbation by the drug.