Synapse formation and function: Insights from identified leech neurons in culture

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressuresensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation. © 1995 John Wiley & Sons, Inc.     

Cell-specific contact selects transmitter responses in an identified leech neuron.

Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.     

Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.     

Mechanisms of excitatory synapse maturation by trans-synaptic organizing complexes

Synapses are specialized cell-cell adhesion contacts that mediate communication within neural networks. During development, excitatory synapses are generated by step-wise recruitment of presynaptic and postsynaptic proteins to sites of contact. Several classes of synaptic organizing complexes have been identified that function during the initial stages of synapse formation. However, mechanisms underlying the later stages of synapse development are less well understood. In recent years, molecules have been discovered that appear to play a role in synapse maturation. In this review, we highlight recent findings that have provided key insights for understanding postsynaptic maturation of developing excitatory synapses with a focus on recruitment of AMPA receptors to developing synapses.     

The differential regulation of formation of chemical and electrical connections in Helisoma

Novel chemical and electrical connections form between neurons not normally connected in the buccal ganglia of the snail Helisoma. We examined the cellular and environmental conditions required for the formation of each type of connection. Previous work in situ showed that novel electrical connections could form in response to axotomy. We have now found that axotomy can evoke the formation of novel unidirectional chemical connections between neurons B5 and B4 in addition to a novel electrical connection. The novel chemical connections display all of the normal properties of chemical synapses in Helisoma ganglia. These connections, however, are transient in nature and break 4 days following axotomy. Previous work has shown that conjoint outgrowth is required for the formation of electrical connections. In cell culture we have investigated whether conjoint outgrowth is also required for chemical synaptogenesis. Using neurons B5 and B19 we have found that when neuron pairs make contact in cell culture, under conditions of synchronous neurite extension, both electrical and chemical synapses form. However, if one neuron has ceased extension prior to contact by a growing neuron, electrical synapses never form (Hadley et al., 1983, 1985) but chemical synapses do form. Furthermore, the addition of serotonin (10(-6) M) to culture medium to inhibit neurite extension of B19, but not that of B5, selectively prevents the formation of electrical connections while permitting the formation of chemical synapses. Thus, the timing of contact in relation to the state of neurite extension can specify the type of connection a given neuron can form.     

Dynamics of postsynaptic glutamate receptor targeting

Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.     

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.     

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R? P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin. 1994 John Wiley & Sons, Inc.     

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     

Voltage gated calcium channels in molluscs: classification,Ca2+ dependent inactivation,modulation and functional roles

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorousConus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca²⁺ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca²⁺ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies,Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.     

Cell-cell interaction during synaptogenesis

1. Neuromuscular synapse formation was studied using nerve and muscle cells dissociated from Xenopus embryos and kept in culture for 1 to 3 days. Within a few minutes of manipulated contact with isolated cholinergic neurons, miniature endplate potential-like depolarizations (MEPPs) due to spontaneous release of acetylcholine (ACh) from the neurons were detected in the muscle cells. 2. Addition of an antibody to a frog neural cell adhesion molecule (anti-NCAM) into the culture medium of nerve-muscle co-cultured for 1-3 days decreased the percentage of functional nerve-muscle contacts. 3. Acute exposure to anti-NCAM (1 hour) inhibited significantly muscle cell contact-triggered ACh release from initially identified cholinergic neurons. 4. Lysed muscle cells manipulated into contact with neurons induced ACh release, whereas lysed neurons did not, suggesting the presence of specific molecules on the muscle cell membrane capable of triggering ACh release from the cholinergic neuron. 5. Transient appearance of electrical coupling was detected between neuronal soma and muscle cell, suggesting the possibility of exchange of modulators for the formation and maintenance of neuromuscular synapses. 6. Neuromuscular synaptogenesis constitutes a complex process where at least two different types of direct cell-cell interaction seem to occur: a) cell surface molecule contact (and binding) for cell recognition and triggering of ACh release, and b) transient intercytoplasmic communication between the cells for possible passage of modulatory molecules.     

Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus

Our understanding of mechanisms that regulate the differentiation of specific classes of synapses is limited. Here, we investigate the formation of synapses between hippocampal dentate gyrus (DG) neurons and their target CA3 neurons and find that DG neurons preferentially form synapses with CA3 rather than DG or CA1 neurons in culture, suggesting that specific interactions between DG and CA3 neurons drive synapse formation. Cadherin-9 is expressed selectively in DG and CA3 neurons, and downregulation of cadherin-9 in CA3 neurons leads to a selective decrease in the number and size of DG synapses onto CA3 neurons. In addition, loss of cadherin-9 from DG or CA3 neurons in vivo leads to striking defects in the formation and differentiation of the DG-CA3 mossy fiber synapse. These observations indicate that cadherin-9 bidirectionally regulates DG-CA3 synapse development and highlight the critical role of differentially expressed molecular cues in establishing specific connections in the mammalian brain.     

Synapse formation and mRNA localization in cultured Aplysia neurons

mRNA localization and regulated translation provide a means of spatially restricting gene expression within neurons during axon guidance and long-term synaptic plasticity. Here we show that synapse formation specifically alters the localization of the mRNA encoding sensorin, a peptide neurotransmitter with neurotrophin-like properties. In isolated Aplysia sensory neurons, which do not form chemical synapses, sensorin mRNA is diffusely distributed throughout distal neurites. Upon contact with a target motor neuron, sensorin mRNA rapidly concentrates at synapses. This redistribution only occurs in the presence of a target motor neuron and parallels the distribution of sensorin protein. Reduction of sensorin mRNA, but not protein, with dsRNA inhibits synapse formation. Our results indicate that synapse formation can alter mRNA localization within individual neurons. They further suggest that translation of a specific localized mRNA, encoding the neuropeptide sensorin, is required for synapse formation between sensory and motor neurons.     

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.     

Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses.     

Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Growth and synapse formation among major classes of adult salamander retinal neurons in vitro

Adult neurons, isolated from the salamander retina, were maintained in low-density cell culture and examined for synapse formation by electrophysiological and electron microscopic techniques. Morphologically identifiable rod, cone, horizontal, bipolar, and amacrine/ganglion cells survived for many months, grew processes, and formed numerous cell contacts. Intracellular recordings showed the presence of a variety of voltage- and time-dependent conductances and both electrical and chemical transmission among these cells. At the ultrastructural level, gap junctions, monad ribbon synapses, and conventional synapses, like those present in the intact retina, were observed in sibling cultures. Thus, all major classes of adult retinal neurons, in addition to ganglion cells, are able to regenerate processes and reform synapses. The regenerated synaptic contacts are functional and structurally diverse.     

Engineered synaptic tools reveal localized cAMP signaling in synapse assembly

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as “SynTAMs” for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.     

Integrating bits and pieces: synapse structure and formation in Drosophila embryos

During the development of the nervous system, numerous neurons connect to form complex networks. In order to build a functional network each neuron has to establish contacts with appropriate target cells, and at these contacts synapses of the right quality and strength have to be formed. Gaining insight into the mechanisms underlying this complex development is an important step towards a better understanding of how the nervous system is formed and behaviour generated. One model system in which synapse formation can be studied at the morphological, physiological and molecular level is that of the fruitfly Drosophila, and insights gained from Drosophila embryos are reviewed here. The first part of this review deals with the neuromuscular junction as the best-known synaptic contact in Drosophila. It describes: (1) its structure, (2) mechanisms underlying the formation of the neuromuscular cell junction and the arborisation of the presynaptic terminal, and (3) our present understanding of signal-dependent and -independent processes during synapse formation at the neuromuscular junction. The last part of this review deals with the question of how particular neurons can adopt specific synaptic properties, stating as an example the development of the neural lineage of NB7-3, which gives rise to two serotonergic neurons.