Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P₂, but this function appears normal in Synaptojanin^RQ knock‐in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P₂, two lipids found in autophagosomal membranes. Using advanced imaging, we show that Synaptojanin^RQ mutants accumulate the PI(3)P/PI(3,5)P₂‐binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell‐derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in Synaptojanin^RQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic‐specific autophagy defects to Parkinson's disease.     

Biophysical model of synaptic plasticity dynamics

We discuss a biophysical model of synaptic plasticity that provides a unified view of the outcomes of synaptic modification protocols, including: (1) prescribed time courses of postsynaptic intracellular Ca²⁺ release, (2) postsynaptic voltage clamping with presentation of presynaptic spike trains at various frequencies, (3) direct postsynaptic response to presynaptic spike trains at various frequencies, and (4) LTP/LTD as a response to precisely timed presynaptic and postsynaptic spikes.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Pro-BDNF–induced synaptic depression and retraction at developing neuromuscular synapses

Postsynaptic cells generate positive and negative signals that retrogradely modulate presynaptic function. At developing neuromuscular synapses, prolonged stimulation of muscle cells induces sustained synaptic depression. We provide evidence that pro–brain-derived neurotrophic factor (BDNF) is a negative retrograde signal that can be converted into a positive signal by metalloproteases at the synaptic junctions. Application of pro-BDNF induces a dramatic decrease in synaptic efficacy followed by a retraction of presynaptic terminals, and these effects are mediated by presynaptic pan-neurotrophin receptor p75 (p75^NTR), the pro-BDNF receptor. A brief stimulation of myocytes expressing cleavable or uncleavable pro-BDNF elicits synaptic potentiation or depression, respectively. Extracellular application of metalloprotease inhibitors, which inhibits the cleavage of endogenous pro-BDNF, facilitates the muscle stimulation–induced synaptic depression. Inhibition of presynaptic p75^NTR or postsynaptic BDNF expression also blocks the activity-dependent synaptic depression and retraction. These results support a model in which postsynaptic secretion of a single molecule, pro-BDNF, may stabilize or eliminate presynaptic terminals depending on its proteolytic conversion at the synapses.     

Significance of the degree of synaptic Zn<Superscript>2+</Superscript> signaling in cognition

Zinc is a trace nutrient for the brain and a signal factor to serve for brain function. A portion of zinc is released from glutamatergic (zincergic) neuron terminals in the brain. Synaptic Zn²⁺ signaling is involved in synaptic plasticity such as long-term potentiaion (LTP), which is a cellular mechanism of memory. The block and/or loss of synaptic Zn²⁺ signaling in the hippocampus and amygdala with Zn²⁺ chelators affect cognition, while the role of synaptic Zn²⁺ signal is poorly understood, because zinc-binding proteins are great in number and multi-functional. Chronic zinc deficiency also affects cognition and cognitive decline induced by zinc deficiency might be associated with the increase in plasma glucocorticoid rather than the decrease in synaptic Zn²⁺ signaling. On the other hand, excess glutamatergic (zincergic) neuron activity induces excess influx of extracellular Zn²⁺ into hippocampal neurons, followed by cognitive decline. Intracellular Zn²⁺ dynamics, which is linked to presynaptic glutamate release, is critical for LTP and cognitive performance. This paper deals with insight into cognition from zinc as a nutrient and signal factor.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Vav independently regulates synaptic growth and plasticity through distinct actin-based processes

Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. Here, we show that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. We also present evidence that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca²⁺ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin–stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. We propose that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively.     

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca²⁺-ATPase (PMCA) isoforms 2 and 3, and Na⁺/Ca²⁺-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na⁺/K⁺-ATPase in controlling local Na⁺ increases derived from GlyT2 activity after neurotransmitter release.     

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

Ca²⁺ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca²⁺ in triggering vesicle fusion though the Ca²⁺ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca²⁺ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca²⁺–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca²⁺-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca²⁺ sensor for presynaptic vesicle fusion.     

Presynaptic endoplasmic reticulum and neurotransmission

Synaptic transmission relies on rapid calcium (Ca²⁺) influx into presynaptic terminal via voltage-gated Ca²⁺ channels. However, smooth ER is present in presynaptic terminals and accumulating evidence indicate that ER Ca²⁺ signaling may play a modulatory role in synaptic transmission. Most recent publication by Lindhout and colleagues (EMBO J, 38 (2019) e101345) suggested that the fragmentation state of the ER affects synaptic vesicle release. Here we discuss these results as well as several key publications that addressed a connection between ER Ca²⁺ signaling and synaptic transmission.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Presynaptic Mechanisms of Lead Neurotoxicity: Effects on Vesicular Release,Vesicle Clustering and Mitochondria Number

Childhood lead (Pb²⁺) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb²⁺ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb²⁺ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb²⁺ neurotoxicity, we show that Pb²⁺ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb²⁺ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.     

Inter-and intraganglionar synaptic connections formed by neurons of the <Emphasis Type="Italic">Helix</Emphasis> visceral ganglion

In experiments on the subpharyngeal complex of the Helix ganglia, we found an excitatory monosynaptic input to the pacemaker PPa2 neuron from an unidentified cell of the visceral ganglion and a polysynaptic inhibitory influence of another unidentified neuron of this ganglion on the PPa1 cell. In addition, we revealed three pairs of neurons synaptically connected with each other (excitatory connections) in the visceral ganglion. In the case where we used high-frequency (11 sec⁻¹) stimulation of presynaptic elements, synaptic transmission to the PPa2 neuron demonstrated the greatest efficiency and stability. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 32–36, January–February, 2007.     

Action of some surfactants on neuromuscular transmission in frogs

The effect of bile salts, saponin, and Tween-80 on miniature end-plate potentials and electrotonic potentials of frog muscle fibers was studied. During the action of bile salts in a concentration of 10^–4 g/ml the frequency of the synaptic potentials rose sharply. Their amplitude also increased. The input resistance of the muscle fiber decreased during the action of these substances. With an increase in their concentration to 10^–3 g/ml bile salts caused an initial increase in frequency of the spontaneous synaptic potentials followed by their depression and complete disappearance. Tween-80 caused no appreciable change in synaptic activity, whereas saponin inhibited it. Lowering the external calcium ion concentration by two to eight times had no influence on the stimulating effect of bile salts, but the total removal of calcium reduced it. The substances tested stimulated secretion of acetylcholine from the nerve endings, probably through changes caused in the structure of the presynaptic membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 305–310, May–June, 1976.     

Selective pressure modulation of synaptic voltage‐dependent calcium channels—involvement in HPNS mechanism

Exposure to hyperbaric pressure (HP) exceeding 100 msw (1.1 MPa) is known to cause a constellation of motor and cognitive impairments named high‐pressure neurological syndrome (HPNS), considered to be the result of synaptic transmission alteration. Long periods of repetitive HP exposure could be an occupational risk for professional deep‐sea divers. Previous studies have indicated the modulation of presynaptic Ca²⁺ currents based on synaptic activity modified by HP. We have recently demonstrated that currents in genetically identified cellular voltage‐dependent Ca²⁺ channels (VDCCs), Ca_V1.2 and Ca_V3.2 are selectively affected by HP. This work further elucidates the HPNS mechanism by examining HP effect on Ca²⁺ currents in neuronal VDCCs, Ca_V2.2 and Ca_V2.1, which are prevalent in presynaptic terminals, expressed in Xenopus oocytes. HP augmented the Ca_V2.2 current amplitude, much less so in a channel variation containing an additional modulatory subunit, and had almost no effect on the Ca_V2.1 currents. HP differentially affected the channels' kinetics. It is, therefore, suggested that HPNS signs and symptoms arise, at least in part, from pressure modulation of various VDCCs.     

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18‐1

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal‐regulated kinase (ERK) that mediates CB1R‐ and mGluR2/3‐induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock‐induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18‐1. Mimicking constitutive phosphorylation of Munc18‐1 results in a drastic decrease in synaptic transmission. ERK‐mediated phosphorylation of Munc18‐1 ultimately leads to degradation by the ubiquitin–proteasome system. Conversely, preventing ERK‐dependent Munc18‐1 phosphorylation increases synaptic strength. CB1R‐ and mGluR2/3‐induced synaptic inhibition and depolarization‐induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK‐dependent Munc18‐1 phosphorylation is blocked. Thus, ERK‐dependent Munc18‐1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.     

Brain-derived Neurotrophic Factor (BDNF)-induced Mitochondrial Motility Arrest and Presynaptic Docking Contribute to BDNF-enhanced Synaptic Transmission

Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca²⁺ buffering requirements at synapses. Brain-derived neurotrophic factor (BDNF) plays an essential role in regulating synaptic transmission and plasticity. However, whether and how BDNF can regulate mitochondrial transport and distribution are still unclear. Here, we find that in cultured hippocampal neurons, application of BDNF for 15 min decreased the percentage of moving mitochondria in axons, a process dependent on the activation of the TrkB receptor and its downstream PI3K and phospholipase-Cγ signaling pathways. Moreover, the BDNF-induced mitochondrial stopping requires the activation of transient receptor potential canonical 3 and 6 (TRPC3 and TRPC6) channels and elevated intracellular Ca²⁺ levels. The Ca²⁺ sensor Miro1 plays an important role in this process. Finally, the BDNF-induced mitochondrial stopping leads to the accumulation of more mitochondria at presynaptic sites. Mutant Miro1 lacking the ability to bind Ca²⁺ prevents BDNF-induced mitochondrial presynaptic accumulation and synaptic transmission, suggesting that Miro1-mediated mitochondrial motility is involved in BDNF-induced mitochondrial presynaptic docking and neurotransmission. Together, these data suggest that mitochondrial transport and distribution play essential roles in BDNF-mediated synaptic transmission.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000