Electrical synapse formation disrupts calcium-dependent exocytosis, but not vesicle mobilization

Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission.     

Dopamine induces sign reversal at mixed chemical-electrical synapses

A mixed chemical/electrical synapse can generate variable output when the strength of each synaptic component is modulated. At mixed synapses of the lobster pyloric network, the chemical component is inhibitory. Without neuromodulation, the chemical component is weak or absent and the electrical component often dominates. Dopamine reverses the sign of these mixed synaptic interactions by a reduction in the strength of electrical coupling and an enhancement of chemical inhibition, including activation of silent chemical synapses. Sign reversal at mixed synapses by neuromodulators may contribute to functional rewiring of neural networks.     

Electrical synapses, a personal perspective (or history)

Gap junctions are the morphological substrate of one class of electrical synapse. This memoir records the author's involvement in the development of our knowledge of the physiology and ultrastructure of electrical synapses. The answer to whether neurotransmission is electrical or chemical is either. One lesson is that Occam's razor sometimes cut too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray [M.V.L. Bennett, Nicked by Occam's razor: unitarianism in the investigation of synaptic transmission, Biol. Bull. 168 (1985) 159–167]. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. The new molecular, cellular and physiological techniques will clarify where gap junctions and electrical coupling do and do not occur and permit experimental manipulation with high specificity.     

Neuronal sandwiches: a method for rapid and controlled initiation of synapses

Synapse formation is a fast, dynamic process that involves the assembly of many molecules following axodendritic contact. Neuronal cultures are often used to study the insertion of fluorescently tagged pre- and postsynaptic molecules in vitro. However, this task still remains challenging, since the time-point and location of newly forming synapses are largely unpredictable and rely on random contact events. We developed a technique that controls the time-point of interaction between axons and dendrites, and thus the onset of synapse formation. Dissociated hippocampal neurons were cultivated on two different coverslips, allowing for the separate outgrowth of axonal networks and of neurons with sparsely innervated dendrites. Pre- and postsynaptic partners were brought in contact as coverslips were merged. Time-lapse imaging showed clustering of GFP/PSD-95 in postsynaptic neurons within 1-3h, indicating the rapid formation of new synaptic sites. Localization of DsRed, as a control protein, remained unchanged. Imaging of neuronal activity using calcium sensitive dyes revealed that in a number of cases neurons of the pre- and postsynaptic layer were synchronously active, suggesting the functionality of newly formed synapses across layers. Therefore, our new method is a valuable tool to control synapse formation and for investigating the temporal role of signaling molecules during this process.     

Aminergic modulation of graded synaptic transmission in the lobster stomatogastric ganglion

Graded chemical synaptic transmission is important for establishing the motor patterns produced by the pyloric central pattern generator (CPG) circuit of the lobster stomatogastric ganglion (Raper, 1979; Anderson and Barker, 1981; Graubard et al., 1983). We examined the modulatory effects of the amines dopamine (DA), serotonin (5-HT), and octopamine (Oct) on graded synaptic transmission at all the central chemical synapses made by the pyloric dilator (PD) neuron onto its follower cells, using synaptic input-output curves measured from cell somata. DA strongly reduced the graded synaptic strength at all the PD synapses. DA reduction of chemical synaptic strength from PD onto the inferior cardiac (IC) neuron could change the sign of synaptic interaction between these 2 cells from inhibitory to excitatory by uncovering a weak electrical connection. 5-HT had weaker and more variable effects, reducing graded synaptic strength from the PD onto the lateral pyloric and pyloric neurons and enhancing the weak synapse from the PD to the IC cell. Oct strongly enhanced the graded synaptic strength at all the PD central synapses. Oct enhancement of graded synaptic strength between the PD and IC cells could also change the sign of the interaction: weak, excitatory electrical coupling, which was sometimes dominant before Oct, was masked by the enhanced chemical inhibitory interaction during Oct application. Measurements of electrical coupling between 2 PD cells and between 2 postsynaptic cells suggest that Oct does not change the input resistance of these cells and may act directly at the PD synapses. The effects of DA and 5-HT are most easily explained by their general reductions in pre- and postsynaptic input resistance. DA, 5-HT, and Oct each produce a distinct pyloric motor pattern (Flamm and Harris-Warrick, 1986a). These amine-induced motor patterns may be explained by the unique actions of each amine on the intrinsic membrane properties of different pyloric CPG neurons (Flamm and Harris-Warrick, 1986b) and by modulation of graded synaptic transmission between the pyloric neurons.     

Synaptic integration of newly generated neurons in rat dissociated hippocampal cultures

In the dentate gyrus of the hippocampus new neurons are born from precursor cells throughout development and into adulthood. These newborn neurons hold significant potential for self-repair of brain damage caused by neurodegenerative disease. However, the mechanism by which newborn neurons integrate into the brain is not understood due to a lack of knowledge of the molecular and functional characteristics of the synapses formed by newborn neurons. Here we report that dissociated hippocampal cultures continue to produce new granule cells in vitro that fire action potentials and become synaptically integrated into the existing network of mature hippocampal neurons. Quantification of the expression of synaptic proteins at newborn and mature granule cell synapses revealed synapse development onto newborn neurons occurs sequentially with initial synaptic contacts evident from 6 days after cell birth. These data also showed that the dendrites of newborn neurons have a high density of Piccolo and Bassoon puncta on them and therefore have a high potential to be integrated into the neuronal network through new synaptic connections. Electrophysiological recordings from newborn neurons reveal these synapses are functional within 10 days of cell birth. GABAergic input synapses were found to mature faster in newborn neurons than glutamatergic synapses where sequential recruitment of postsynaptic glutamate receptors occurred. Group I metabotropic glutamate receptors (mGluR1/5) were present at higher levels compared with ionotropic glutamate receptors (NMDA and AMPA receptors), suggesting that metabotropic and ionotropic receptors play differential roles at glutamatergic synapses in the integration and the maturation of newborn neurons. These data show that dissociated hippocampal cultures can provide a useful model system in which to study the integration of newborn neurons into existing neuronal circuits to increase our understanding of how the function of newborn neuron synapses could contribute to restoring damaged neuronal networks.     

Postnatal development of the cerebellar cortex in the rat. 3. Maturation of the components of the granular layer

The migration of granule cells and the maturation of the various elements of the granular layer were studied in the cerebellar cortex of rats aged 0, 3, 5, 7, 10, 12, 15, 21 and 30 days with histological, histochemical, autoradiographic and electron microscopic techniques. The bulk of the granule cells are formed during the second week, but due to the time required for their migration and the lag in the formation of dendrites, few glomerular synapses are formed with mossy fibers before the beginning of the third week and the process is still in progress at 30 days, long after the dissolution of the external germinal layer. The maturation of Golgi cells is a protracted process. Their axons synapse with granule cell dendrites as soon as the glomeruli begin to mature. Evidence was obtained that mossy fibers synapse with the dendrites of Golgi cells. Towards the end of the second week the Lugaro cells are formed and synapses appear on their somata during the third week. Among these synapses the recurrent collaterals of Purkinje cell axons were identified. The Lugaro cells may be the primary targets of the infra and supraganglionic plexuses formed by these collaterals. In conclusion it was suggested that there are three major, successive stages in the neurogenesis of the cerebellar cortex, the morphogenic, synaptogenic and gliogenic. However, in the large Purkinje cell the synaptic maturation of one region (the soma) may begin before the morphogenic and synaptogenic maturation of the entire cell (the dendrites) is completed.     

Synaptogenesis and changes in synaptic morphology related to acquisition of a new behavior

Systematic testosterone treatment induces adult female canaries to develop male-like song. This same treatment induces a doubling in size of the forebrain nucleus robustus archistriatalis (RA), known to be involved in song control, and a 51% increase in the number of synapses formed on RA neurons. In central RA, the number of synaptic vesicles per synapse increases as do several measures of synaptic size. Housing in spring-like conditions is also associated with larger synapses and more vesicles per synapse than housing in fall-like conditions. We suggest that formation of new synapses on existing neurons leading to or associated with modifications in synaptic morphology is important for acquisition of a new behavior. We also suggest that maximal behavioral and anatomical effects are associated with testosterone given under spring-like conditions.     

Digital detection and analysis of branching and cell contacts in neural cell cultures

Changes in human/animal behaviour and the involved neural functions are characterized by structural alterations in the brain circuitry. These changes comprise the formation of new synapses and the elimination of existing synapses aside from the modulation of connecting properties within other ones. The mechanisms of neuronal branching and cell contacting regulate and prepare for the processes of synaptic formation. In this study, we present a set of methods to detect, describe and analyse the dynamics attributed to the process of cell contacting in cell cultures in vitro. This involves the dynamics of branching and seeking for synaptic partners. The proposed technique formally distinguishes between the actual formed synapses and the potential synaptic sites, i.e. where cell contacts are likely. The study investigates the dynamic behaviour of these potential synaptic sites within the process of seeking for contacts. The introduced tools use morphological image processing algorithms to automatically detect the sites of interest. Results indicate that the introduced tools can reliably describe experimentally observed branching and seeking for contacts dynamics. Being straightforward in terms of implementation and analysis, our framework represents a solid method for studying the neural preparation phases of synaptic formation via cell contacting in random networks using standard phase contrast microscopy.     

Ephrin regulation of synapse formation, function and plasticity

Synapses enable the transmission of information within neural circuits and allow the brain to change in response to experience. During the last decade numerous proteins that can induce synapse formation have been identified. Many of these synaptic inducers rely on trans-synaptic cell-cell interactions to generate functional contacts. Moreover, evidence now suggests that the same proteins that function early in development to regulate synapse formation may help to maintain and/or regulate the function and plasticity of mature synapses. One set of receptors and ligands that appear to impact both the development and the mature function of synapses are Eph receptors (erythropoietin-producing human hepatocellular carcinoma cell line) and their surface associated ligands, ephrins (Eph family receptor interacting proteins). Ephs can initiate new synaptic contacts, recruit and stabilize glutamate receptors at nascent synapses and regulate dendritic spine morphology. Recent evidence demonstrates that ephrin ligands also play major roles at synapses. Activation of ephrins by Eph receptors can induce synapse formation and spine morphogenesis, whereas in the mature nervous system ephrin signaling modulates synaptic function and long-term changes in synaptic strength. In this review we will summarize the recent progress in understanding the role of ephrins in presynaptic and postsynaptic differentiation, and synapse development, function and plasticity.     

Long-term plasticity at GABAergic and glycinergic synapses: mechanisms and functional significance

Activity-dependent long-term changes in synaptic efficacy are thought to be important in learning, memory formation, neuronal development and pathological states of neuronal excitability in the CNS. For the past two decades, numerous studies have investigated long-term changes in synaptic efficacy at excitatory glutamatergic synapses. Although inhibitory synapses are essential for proper functioning of the neuronal network, attention has focused only recently on describing and characterizing plasticity at these types of synapse. Not surprisingly, different forms of plasticity at GABAergic, and the closely related glycinergic, synapses have been reported in several regions of the brain. Here we review these different forms of plasticity and focus on their possible roles in developing and adult neuronal networks.     

Synapse formation in the mouse olfactory bulb Quantitative studies

A quantitative study of synapse formation in the mouse olfactory bulb has been carried out using serial sections. Volumetric synaptic density as well as absolute number of synapses per olfactory bulb for eight distinct synaptic types have been determined at 15 different ages, from the beginning of synapse formation at embryonic day 14 (E14) to postnatal day 44 (P44). Synapses are first found in appreciable numbers at E15 when both axo-dendritic and a few dendrodendritic synapses occur in the presumptive glomerular layer. Initial synapse formation correlates closely with the reorientation of mitral cells from a primitive tangential to a defintive radial direction. Synapse formation by mitral cell dendrites occurs after mitral cell axons have grown into the future olfactory cortical areas, either simultaneous with or before synapse formation by these axons. Virtually all synaptic types detected in adults have been found on the day of birth, consistent with the idea that olfaction is an important sensory modality for newborn mice. Volumetric density of a given synaptic type generally increases 50–100 times during development while the absolute number increases about 1,000 times. Synapses in glomeruli develop more precociously than those in the external plexiform and internal granular layers, which correlates well with the time of origin and differentiation of the principal postsynaptic elements of these two divisions (mitral cells and internal granule cells) Correlation of the time of synapse formation of various synaptic types with their putative excitatory or inhibitory role determined in adult studies suggests that excitatory synapses generally form somewhat earlier, although throughout nearly all of synaptic development, both excitatory and inhibitory synapses are present. Reciprocal dendro-dendritic synapses in the external plexiform layer appear to have a special mode of formation. It is suggested that a granule-to-mitral dendro-dendritic synapse only forms next to an already existing mitral-to-granule synapse on the same gemmule.     

Astrocyte‐mediated synapse remodeling in the pathological brain

Astrocytes, a major type of glia, reciprocally influence synaptic transmission and connectivity, forming the “tripartite synapses”. Astrocytic metabotropic glutamate receptor (mGluR)‐mediated Ca²⁺ waves and release of gliotransmitters or synaptogenic molecules mediate this neuron‐glia interaction in the developing brain, but this signaling has been challenged for adult brain. However, cumulative evidence has suggested that mature astrocytes exhibit re‐awakening of such immature phenotype in the pathological adult brain. This phenotypic change in astrocytes in response to injury may induce neural circuit and synapse plasticity. In this review article, we summarize astrocyte‐mediated synapse remodeling during physiological development, discuss re‐emergence of immature astrocytic signaling in adult pathological brain, and finally highlight its contribution to significant modification of synaptic connections correlating with functional progress of brain pathology.     

Multiple cell adhesion molecules shaping a complex nicotinic synapse on neurons

Neuroligin, SynCAM, and L1-CAM are cell adhesion molecules with synaptogenic roles in glutamatergic pathways. We show here that SynCAM is expressed in the chick ciliary ganglion, embedded in a nicotinic pathway, and, as shown previously for neuroligin and L1-CAM, acts transcellularly to promote synaptic maturation on the neurons in culture. Moreover, we show that electroporation of chick embryos with dominant negative constructs disrupting any of the three molecules in vivo reduces the total amount of presynaptic SV2 overlaying the neurons expressing the constructs. Only disruption of L1-CAM and neuroligin, however, reduces the number of SV2 puncta specifically overlaying nicotinic receptor clusters. Disrupting L1-CAM and neuroligin together produces no additional decrement, indicating that they act on the same subset of synapses. SynCAM may affect synaptic maturation rather than synapse formation. The results indicate that individual neurons can express multiple synaptogenic molecules with different effects on the same class of nicotinic synapses.     

The morphological and physiological properties of a regenerating synapse in the C.N.S. of the leech.

Regeneration of an electrical synapse between particular interneurons in the medicinal leech was traced physiologically and morphologically using intracellular recording the horseradish peroxidase (HRP) injection. The synapse between S-cell interneurons lies in the connective midway between segmental ganglia, so crushing near one ganglion severs only one S-cell's axon. The severed distal stump remains connected to the adjacent uninjured S-cell and continues for weeks to conduct impulses. The injured cell regenerates, while its uninjured "target" neuron in the next ganglion does not grow. After the sprouts of the regenerating neuron cross the crush, one or a few branches grow along the surviving distal stump toward the original synapse. After about one month when the region of original synapse has been reached, regenerating neurons form electrical junctions and stop growing. Thereafter electrical coupling improves in stages. Within two months the regenerated neuron attains full caliber, the stump degenerates and function is normal. In some instances within days or weeks of crushing, the regenerating neuron forms a basket of synapses upon its severed distal stump and then continues growing to synapse with the target. When this occurs, electrical coupling and subsequent impulse transmission between S-cells rapidly resumes. These experiments indicated that the regenerating neuron is guided to its proper synaptic target by recognizing and following its severed distal stump. Sometimes the distal stump itself becomes an intermediate synaptic target.     

Matching of pre- and postsynaptic specializations during synaptogenesis.

Formation of chemical synapses in the central nervous system is a highly regulated, multistep process that requires bidirectional communication across the synaptic cleft. Neurotransmitter receptors, scaffolding proteins, and signaling molecules need to be concentrated in the postsynaptic density, a specialized membrane microdomain apposed to the active zone of presynaptic terminals, where transmitter release occurs. This precise, synapse-specific matching implicates that sorting and targeting mechanisms exist for the molecular constituents of different types of synapses to ensure correct formation of neuronal circuits in the brain. There is considerable evidence from in vitro and in vivo studies that neurotransmitter signaling is not required for proper sorting during synapse formation, whereas active neurotransmission is essential for long-term synapse maintenance. Here, the authors review recent studies on the role of cell adhesion molecules in synaptogenesis and on possible mechanisms ensuring correct matching of pre- and postsynaptic sites. They discuss the role of neurotransmitter receptors and scaffolding proteins in these processes, focusing on fundamental differences between synapse formation during development and synapse maintenance and plasticity in adulthood.     

Dual constraints on synapse formation and regression in schizophrenia: neuregulin, neuroligin, dysbindin, DISC1, MuSK and agrin

During adolescence there is a loss of approximately 30% of the synapses formed in the cortex during childhood. Comprehensive studies of the visual cortex show that this loss of synapses does not occur as a consequence of less appropriate projections being eliminated in favour of more appropriate ones. Rather it seems that synapses with low efficacy for transmission are eliminated in favour of those with higher efficacy. The loss of low-efficacy synapses is known, on theoretical grounds, to enhance the function of neural networks, but large synapse losses lead to failure of network function. In the dorsolateral prefrontal cortex (DLPC) of those suffering from schizophrenia the number of synapses is relatively very low, approximately 60% lower than that observed in normal childhood. It is not known if this is due to an additional loss over that during normal adolescence or whether it results from a failure to form a normal complement of synapses during childhood. The first study of synapse loss in the mammalian nervous system was made on the neuromuscular junction at Sydney University in 1974. Since then this junction has provided principal insights into the molecular basis of synapse formation and regression, so providing a paradigm for investigations of these phenomena in the DLPC. For example the molecules muscle-specific receptor tyrosine kinase (MuSK), agrin and neuregulin have been identified and their critical roles in the formation and maintenance of synapses elucidated. Loss of function of MuSK or agrin leads to failure of neuromuscular synapse formation as well as a loss of approximately 30% of excitatory synapses in the cortex. Similar synapse loss occurs on failure of neuregulin in vitro and of neuroligin in vivo. It is suggested that three important questions need to be answered: first, over what development period are the synapse numbers in DLPC of subjects with schizophrenia lower than normal; second, what are the relative importance of MuSK/agrin, neuregulin/ErB and neurexin/neuroligin in synapse formation and regression in the DLPC; and third, to what extent have these molecules gone awry in schizophrenia.     

Imaging of experience-dependent structural plasticity in the mouse neocortex in vivo

The functionality of adult neocortical circuits can be altered by novel experiences or learning. This functional plasticity appears to rely on changes in the strength of neuronal connections that were established during development. Here we will describe some of our studies in which we have addressed whether structural changes, including the remodeling of axons and dendrites with synapse formation and elimination, could underlie experience-dependent plasticity in the adult neocortex. Using 2-photon laser-scanning microscopes and transgenic mice expressing GFP in a subset of pyramidal cells, we have observed that a small subset of dendritic spines continuously appear and disappear on a daily basis, whereas the majority of spines persists for months. Axonal boutons from different neuronal classes displayed similar behavior, although the extent of remodeling varied. Under baseline conditions, new spines in the barrel cortex were mostly transient and rarely survived for more than a week. However, when every other whisker was trimmed, the generation and loss of persistent spines was enhanced. Ultrastructural reconstruction of previously imaged spines and boutons showed that new spines slowly form synapses. New spines persisting for a few days always had synapses, whereas very young spines often lacked synapses. New synapses were predominantly found on large, multi-synapse boutons, suggesting that spine growth is followed by synapse formation, preferentially on existing boutons. Altogether our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons and promotes the formation of new synapses. These synaptic changes likely underlie experience-dependent functional remodeling of specific neocortical circuits.     

Quantification of synapse turnover in cell culture

The on and off rates of synaptogenesis can be quantitated using a cell culture system in which embryonic retinal cells form functional synapses with striated muscle cells. Quantification showed that synapse formation and termination can occur simultaneously in culture. Quantification also revealed that some retina-muscle synapses are transient, terminating within 8 h, while other synapses are much more stable. Relatively stable and transient synaptic pairs could be identified prospectively based on the strength of evoked transmission across the retina-muscle synapse.     

Role of activity in the selection of new electrical synapses between adult Helisoma neurons

Our aim was to determine whether neural activity in the form of sodium-dependent action potentials play a role in the formation, maintenance and specificity of electrical synapses between regenerating neurons. We axotomized buccal neurons of the mollusc, Helisoma trivolvis, and placed ganglia into organ culture in the absence or presence of tetrodotoxin (TTX), a specific sodium channel blocker. Electrical coupling was measured using intracellular microelectrodes positioned within the soma of identified neurons. Neurite outgrowth was assessed by epifluorescence microscopy after filling neurons by iontophoresis with Lucifer yellow. Previous studies found that two days after axotomy transient electrical synapses form between heterologous neurons (e.g. buccal neurons 4 and 5). Five days after axotomy these transient connections disappeared and a new electrical synapse was stabilized between the paired buccal neurons 5. To determine whether blocking neural activity with TTX affected the specificity and formation of new electrical synapses, we examined electrical coupling between the heterologous neurons 4 and 5 two days after axotomy, and the paired buccal neurons 5 five days after axotomy. Our electrophysiological recordings indicated that different neurons in the buccal ganglion varied in their sensitivity to TTX (i.e. sensitivity of buccal neurons 19 greater than 5 greater than 4), but spontaneous activity was abolished in all 3 neurons by 2 x 10(-5) M TTX. Furthermore, the inhibitory effects of TTX occurred within seconds of superfusion and persisted for at least 6 days. Inhibition of activity by TTX could be reversed after superfusion with normal saline. Neurite outgrowth from axotomized neurons was not appreciably altered in the presence of TTX. Furthermore, no differences in the incidence of electrical coupling or the coupling resistance were detected between neurons 4 and 5 two days after axotomy and organ culture in the presence of TTX. However, electrical coupling between the symmetrically paired neurons 5 was elevated in the presence of TTX after 5 days. We conclude from these results that neural activity in the form of sodium-dependent action potentials does not play an important role in the formation or breaking of transient electrical synapses during neuronal regeneration in the mollusc Helisoma trivolvis.