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.     

Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ

Previous work with identified Helisoma neurons has characterized an array of neuroplastic responses to axotomy that include the generation of new neuritic outgrowth, the reinnervation of target organs, and the formation of new electrical synapses. These responses are not random, but rather occur in a precise, predictable manner under a variety of culture conditions. The present investigation demonstrates that specific identified neurons display similar neuroplastic "behavior" within the living animal. In response to in situ nerve crushes, neurons B4 and B5 generate new neuritic outgrowth, neuron B4 functionally reinnervates the salivary glands, and new electrical synapses form between the left and right neurons B5. The in situ paradigm employed in the present experiments made it possible to examine responses to axotomy over longer periods than in earlier studies with organ cultures. New B5R-B5L connections, previously found to be stable over the short term in culture, gradually decreased in strength in situ, and the cells effectively uncoupled by 8 weeks after axotomy. This uncoupling did not depend upon target reinnervation and occurred in the continued presence of neurites in the buccal commissure. It is suggested that the stability of new connections is related to whether the connection previously existed in the unperturbed nervous system. The similarities between the ability of identified neurons to grow and to form synaptic connections in situ and in culture suggests that neurons are endowed with a specific program of regenerative responses that can be expressed reliably in a wide variety of environmental conditions.     

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.     

Transient depletion of serotonin in the nervous system of Helisoma

The present study shows that the drug 5,7-dihydroxytryptamine (5,7-diHT) can be used reliably to deplete the neurotransmitter serotonin (5-HT) from the nervous system of the snail Helisoma. The depletion is more effective in axonal and synaptic regions (85-90%) than in the somata (55%), is reasonably specific for serotonin (dopamine is affected to a much lesser extent), and is transient, with normal levels of neurotransmitter being restored by 2 months. A physiological correlate of 5-HT depletion has been shown in that an EPSP elicited by a cerebral serotonergic neuron (C1) onto a buccal motoneuron (B19) is much smaller during depletion and also recovers with time as 5-HT regains normal concentration. Despite the severe 5-HT depletion and physiological impairment, the gross morphology of neuron C1 remains indistinguishable from controls. Serotonergic depletion is not accompanied by development of receptor supersensitivity nor by the production of serotonin in extraneuronal sources.     

Target-dependent induction of secretory capabilities in an identified motoneuron during synaptogenesis

Cholinergic neurons isolated from the buccal ganglia of Helisoma were plated into cell culture with a variety of defined target cells to study the specificity of synaptogenesis. Motoneuron B19 selectively formed chemical connections with single dissociated muscle fibers derived from its appropriate target, the supralateral radular tensor (SLT) muscle. B19 did not form such connections with novel neuronal targets. In contrast to neuron B19, cholinergic neuron B5 nonselectively formed chemical connections with novel muscle and neuronal targets. Target cells were micromanipulated into contact with presynaptic neurons to examine the latent period until the onset of functional synaptic transmission. Neuron B5 formed chemical connections within the first minutes of contact with ACh-sensitive neurons and muscle while B19 required sustained periods of muscle-specific contact to induce the acquisition of a functional excitation-secretion coupling mechanism. These different latent periods from the onset of target contact suggest that neuron B5 acquires presynaptic secretory function before target contact, while B19 must receive a specific signal(s) from its appropriate target to induce the transformation of its terminal into a secretory state.     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

Neuron-specific and state-specific differences in calcium homeostasis regulate the generation and degeneration of neuronal architecture

Many stimuli (e.g., neurotransmitters and electrical activity) regulate neuromorphogenesis by changing intracellular calcium. The ionophore A23187 was employed as a receptor-independent method to investigate neuronal calcium homeostasis. Distinctive neuron-specific (B5 versus B19) and state-specific (growing versus non-growing) differences in calcium homeostasis were observed in cultured identified Helisoma neurons. Fura-2 studies revealed that A23187 induced a transient rise in intracellular calcium in growing neurons B5 but a sustained rise in growing neurons B19. In stable-state (non-growing) cells A23187 evoked only a transient calcium rise. Both neuron-specific and state-specific differences in calcium homeostasis were dependent on extracellular sodium. Morphological studies also indicated that such differences in calcium-regulatory capacity can have profound consequences on the generation and degeneration of neuronal architecture.     

Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons.     

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron.

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiation from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22 degrees-24 degrees C when the injury was located 800 microns from the soma. Elevating the temperature to 32 degrees C or moving the lesion site to 400 or 1500 microns from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 x 10(-5) M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10(-5) M) did. The calcium channel blocker lanthanum (10(-6)-10(-4) M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10(-3)-10(-5) M), and the calcium ionophore A23187 (10(-5) M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH4Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10(-5) M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10(-5) M) had no effect. Inhibition of protein synthesis with anisomycin (2 x 10(-4) to 2 x 10(-6) M) did not significantly suppress sprouting 24 h after axotomy. Both D and L isomers of glutamate (300 microM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.     

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons.

This study focuses on the effects of K+ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K+ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K+ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K+ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K+ solution. Identified neurons differed in the magnitude of their response to K+ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K+, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K+ application evoked a brief period (5-10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K+ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca2+ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca2+ change during depolarization, reflecting their different sensitivities to depolarization.     

Interactive effects of serotonin and acetylcholine on neurite elongation

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.     

Electrically induced changes in Ca2+ in Helisoma neurons: Regional and neuron-specific differences and implications for neurite outgrowth

The present experiments addressed the questions of how electrical stimulation influenced the magnitude, time course, and regional levels of free intracellular calcium of different identified neurons. The calcium concentration in the growth cones, neurites and cell bodies of Helisoma buccal neurons B4 and B19 was measured while somata were electrically stimulated via an intracellular electrode. The findings showed that calcium levels in B4 and B19 increased monotonically with increasing stimulation frequency. However, the range of calcium levels evoked by electrical stimulation differed significantly for each type of neuron. The greater increase in calcium concentration in B4 was correlated with its longer duration action potential compared to B19. The increase in calcium concentration was much smaller in the cell bodies than in the growth cones and neurites. Extending the duration of the B19 action potential produced a sixfold increase in the change in calcium concentration at 2 Hz stimulation. Under conditions where the electrical stimulation produced a calcium concentration of < 160 nM, the elevated level of free intracellular calcium remained constant. When calcium concentration increased above 200 nM in both identified neurons, an initial peak concentration was followed by a decline to a lower concentration suggesting increased calcium buffering occurring above 200 nM. By correlating the calcium concentration data herein with growth data from a previous study, we suggest that specific calcium levels that influence neurite outgrowth may differ widely between neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 150–162, 1997.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Three-dimensional culture of leech and snail ganglia for studies of neural repair

Three-dimensional (3D) collagen gels provide a stable matrix in which isolated regenerating ganglia from leech and snail can be maintained for studies of the molecular and cellular mechanisms underlying the regenerative process. Segmental ganglia from leech, or supraoesophageal, suboesophageal or buccal ganglia from snail were maintained for up to 3 weeks in 3D matrices of mammalian Type I collagen. The collagen matrix supports the regenerative outgrowth of axon tracts as well as the migration of microglial cells, important elements in the repair process. Proteins or soluble factors or target tissue may be added to the basic collagen matrix to manipulate the environment of the regenerating tissue. We describe techniques for immunostaining of regenerating axons and microglial cells within the gel matrix in combination with staining of cell nuclei, and the use of intracellular labelling to distinguish axons of identified neurons within the regenerative outgrowth.E.J. Babington and J. Vatanparast contributed equally     

Weaver granule neurons are rescued by calcium channel antagonists and antibodies against a neurite outgrowth domain of the B2 chain of laminin

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10- 20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.     

Regulation of neuronal growth cone filopodia by nitric oxide depends on soluble guanylyl cyclase

Nitric oxide has been proposed to play an important role in neuronal development. We have previously shown that growth cones from an identified neuron, B5, in the snail Helisoma trivolvis, respond to nitric oxide (NO) donors by increasing the length of their filopodia within minutes of application (Van Wagenen and Rehder, 1999). This effect was mediated through a cGMP-induced increase in [Ca2+]i and resulted in an enlargement of the growth cone's action radius, suggesting that NO could function as a signaling molecule during neuronal pathfinding. We show here that NO functions as a specific rather than a general regulator of growth cone filopodia, because another identified neuron from the same ganglion, B19, failed to respond to NO with an increase in filopodial length. We found that, contrary to B5 neurons, B19 growth cones contained little or no soluble guanylyl cyclase (sGC) immunoreactivity, presumably preventing their response to NO. This hypothesis was supported by the finding that the sGC activator YC-1 (10 microM) had no effect on B19 filopodia but induced elongation of B5 filopodia. These results indicate that the effects of NO can be quite specific, and raise the interesting possibility that neurons could selectively tune in to NO by differentially expressing the target enzyme sGC in the appropriate cellular location during critical developmental stages. In addition, our NADPH-diaphorase staining and anti-NOS immunohistochemisty suggest that growth cones of B5 neurons, but not of B19 neurons, could be a source of NO, making NO a potential intra- and transcellular messenger.     

Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system

Exogenous serotonin has been shown to evoke a neuron-selective inhibition of neurite outgrowth and synaptogenesis in identified Helisoma neurons in vitro. We demonstrate here that serotonin is present in the embryonic nervous system of Helisoma and can act as a regulator of neuronal development in vivo. Serotonin-like immunoreactivity was first observed in neurons at an early stage of nervous system development (E20). Throughout embryogenesis, the number of serotonin-immunoreactive neurons increased in a stereotypic pattern that was unique for each type of ganglion. Strikingly, the number of serotonin-immunoreactive neurons continued to increase throughout adult life. Transient perturbation of endogenous serotonin levels during embryogenesis had profound effects on the development of specific identified neurons. Embryos treated with 5,7-dihydroxytryptamine and raised to maturity showed aberrations in neuronal morphology, neuronal dye coupling, and strength of electrical synaptic connections. These effects were restricted to neurons known to be sensitive to the growth-inhibitory effects of serotonin in vitro. These results support the hypothesis that neurotransmitters are an important class of regulatory factors during normal development of the nervous system.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Serotonin and cAMP induce excitatory modulation of a serotonergic neuron

Serotonin (5-HT) is an excitatory neurotransmitter and neuromodulator. In the Aplysia nervous system it increases excitability and induces spike broadening in sensory neurons. It is released at the synaptic terminals of the metacerebral cells (MCCs) and modulates the feeding neural circuit and buccal muscles during the aroused feeding state. We report that MCC itself is depolarized by 5-HT and becomes excitable. 5-HT induces tonic spike activity and even spike-burst activity. Conceivably, this sensitivity to its own transmitter could provide positive feedback excitation of MCC. Voltage clamp analysis of isolated cultured MCCs shows that 5-HT reduces a calcium-dependent outward current at the resting potential (-60 mV), and enhances steady state inward currents between -55 and -30 mV and between -75 and -100 mV. 8-Br-cAMP has similar effects, suggesting that cAMP mediates the 5-HT effects, in part. A transient calcium current is enhanced at voltages more positive than -40 mV. Barium and cesium selectively block the 5-HT-induced inward current between -75 and -100 mV. Substitution of N-methyl-D-glucamine for sodium and adding cobalt block this current, also indicating that it is a hyperpolarization-activated cation current. The 5-HT-induced inward current between -55 and -30 mV is also blocked by sodium substitution and added cobalt, suggesting that 5-HT increases a depolarization-activated cation current. The outward current that remains when sodium and calcium currents are blocked is reduced by 5-HT. Thus, 5-HT enhances two different cation currents and reduces potassium currents.     

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.