Formation Of Chemical Synapses Research Articles

NMDA Receptors Contribute to Postnatal Developmental Downregulation of Connexin Expressions in the Rat Locus Coeruleus

It has been shown that during postnatal development, gap junctions decrease. This phenomenon overlaps with the major period of chemical synapse formation and increased synaptic activity during postnatal weeks 1–3. In current study, the expression of connexin 36 (Cx36), Cx32, and Cx43 proteins in locus coeruleus (LC) was examined using Western blot analysis in (postnatal day) PND14 and PND28 following blockade of NMDA receptors in rats to evaluate the role of these receptors in the expression of Cxs. MK801, as an NMDA receptor antagonist, was injected subcutaneously from the first day of birth until 14 or 28 days depending on the experimental groups. The control group received a saline injection in the same volume. Expression of Cxs declined in PND28 compared to PND14 in LC neurons. Also, we found that the expression of Cx36, Cx32, and Cx43 augmented in the rats’ LC in PND14 and PND28 following the blockade of NMDA receptors. The present results suggest that the activity of NMDA receptors may contribute to the downregulation of Cx36, Cx32, and Cx43 in LC during postnatal development.

Neuronal Glycoprotein M6a: An Emerging Molecule in Chemical Synapse Formation and Dysfunction.

The cellular and molecular mechanisms underlying neuropsychiatric and neurodevelopmental disorders show that most of them can be categorized as synaptopathies—or damage of synaptic function and plasticity. Synaptic formation and maintenance are orchestrated by protein complexes that are in turn regulated in space and time during neuronal development allowing synaptic plasticity. However, the exact mechanisms by which these processes are managed remain unknown. Large-scale genomic and proteomic projects led to the discovery of new molecules and their associated variants as disease risk factors. Neuronal glycoprotein M6a, encoded by the GPM6A gene is emerging as one of these molecules. M6a has been involved in neuron development and synapse formation and plasticity, and was also recently proposed as a gene-target in various neuropsychiatric disorders where it could also be used as a biomarker. In this review, we provide an overview of the structure and molecular mechanisms by which glycoprotein M6a participates in synapse formation and maintenance. We also review evidence collected from patients carrying mutations in the GPM6A gene; animal models, and in vitro studies that together emphasize the relevance of M6a, particularly in synapses and in neurological conditions.

Asymmetry of an Intracellular Scaffold at Vertebrate Electrical Synapses

Neuronal synaptic connections are either chemical orelectrical, and these two types of synapses work together to dynamically define neural circuit function [1]. Although we know a great deal about the molecules that support chemical synapse formation andfunction, we know little about the macromolecular complexes that regulate electrical synapses. Electrical synapses are created by gap junction (GJ) channels that provide direct ionic communication between neurons [2]. Although they are often molecularly and functionally symmetric, recent work has found that pre- and postsynaptic neurons can contribute different GJ-forming proteins, creating molecularly asymmetric channels that are correlated with functional asymmetry at the synapse [3, 4]. Associated with the GJs are structures observed by electron microscopy termed the electrical synapse density (ESD) [5]. The ESD has been suggested to be critical for the formation and function of the electrical synapse, yet the biochemical makeup of these structures is poorly understood. Here we find that electrical synapse formation invivo requires an intracellular scaffold called Tight Junction Protein 1b (Tjp1b). Tjp1b is localized to the electrical synapse, where it is required for the stabilization of the GJs and for electrical synapse function. Strikingly, we find that Tjp1b protein localizes and functions asymmetrically, exclusively on the postsynaptic side of the synapse. Our findings support a novel model of electrical synapse molecular asymmetry at the level of an intracellular scaffold that is required for building the electrical synapse. We propose that such ESD asymmetries could be used by all nervous systems to support molecular and functional asymmetries at electrical synapses.

Precise inhibitory microcircuit assembly of developmentally related neocortical interneurons in clusters

GABA-ergic interneurons provide diverse inhibitions that are essential for the operation of neuronal circuits in the neocortex. However, the mechanisms that control the functional organization of neocortical interneurons remain largely unknown. Here we show that developmental origins influence fine-scale synapse formation and microcircuit assembly of neocortical interneurons. Spatially clustered neocortical interneurons originating from low-titre retrovirus-infected radial glial progenitors in the embryonic medial ganglionic eminence and preoptic area preferentially develop electrical, but not chemical, synapses with each other. This lineage-related electrical coupling forms predominantly between the same interneuron subtype over an extended postnatal period and across a range of distances, and promotes action potential generation and synchronous firing. Interestingly, this selective electrical coupling relates to a coordinated inhibitory chemical synapse formation between sparsely labelled interneurons in clusters and the same nearby excitatory neurons. These results suggest a link between the lineage relationship of neocortical interneurons and their precise functional organization.

Electrical coupling regulates layer 1 interneuron microcircuit formation in the neocortex.

The coexistence of electrical and chemical synapses among interneurons is essential for interneuron function in the neocortex. However, it remains largely unclear whether electrical coupling between interneurons influences chemical synapse formation and microcircuit assembly during development. Here, we show that electrical and GABAergic chemical connections robustly develop between interneurons in neocortical layer 1 over a similar time course. Electrical coupling promotes action potential generation and synchronous firing between layer 1 interneurons. Furthermore, electrically coupled interneurons exhibit strong GABA-A receptor-mediated synchronous synaptic activity. Disruption of electrical coupling leads to a loss of bidirectional, but not unidirectional, GABAergic connections. Moreover, a reduction in electrical coupling induces an increase in excitatory synaptic inputs to layer 1 interneurons. Together, these findings strongly suggest that electrical coupling between neocortical interneurons plays a critical role in regulating chemical synapse development and precise formation of circuits.

Neurobeachin Is Required Postsynaptically for Electrical and Chemical Synapse Formation

Neural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses, transmission between neurons is mediated by neurotransmitters, whereas at electrical synapses, direct ionic and metabolic coupling occur via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood, and whether they share mechanisms of formation with chemical synapses is not clear. Here, using a forward genetic screen in zebrafish, we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain-containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wild-type, we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization. Our data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis.

Neuronal somata and extrasomal compartments play distinct roles during synapse formation between Lymnaea neurons.

Proper synapse formation is pivotal for all nervous system functions. However, the precise mechanisms remain elusive. Moreover, compared with the neuromuscular junction, steps regulating the synaptogenic program at central cholinergic synapses remain poorly defined. In this study, we identified different roles of neuronal compartments (somal vs extrasomal) in chemical and electrical synaptogenesis. Specifically, the electrically synapsed Lymnaea pedal dorsal A cluster neurons were used to study electrical synapses, whereas chemical synaptic partners, visceral dorsal 4 (presynaptic, cholinergic), and left pedal dorsal 1 (LPeD1; postsynaptic) were explored for chemical synapse formation. Neurons were cultured in a soma-soma or soma-axon configuration and synapses explored electrophysiologically. We provide the first direct evidence that electrical synapses develop in a soma-soma, but not soma-axon (removal of soma) configuration, indicating the requirement of gene transcription regulation in the somata of both synaptic partners. In addition, the soma-soma electrical coupling was contingent upon trophic factors present in Lymnaea brain-conditioned medium. Further, we demonstrate that chemical (cholinergic) synapses between soma-soma and soma-axon pairs were indistinguishable, with both exhibiting a high degree of contact site and target cell type specificity. We also provide direct evidence that presynaptic cell contact-mediated, clustering of postsynaptic cholinergic receptors at the synaptic site requires transmitter-receptor interaction, receptor internalization, and a protein kinase C-dependent lateral migration toward the contact site. This study provides novel insights into synaptogenesis between central neurons revealing both distinct and synergistic roles of cell-cell signaling and extrinsic trophic factors in executing the synaptogenic program.

Nitric Oxide Synthesis and cGMP Production Is Important for Neurite Growth and Synapse Remodeling after Axotomy

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways.

Gap Junction Expression Is Required for Normal Chemical Synapse Formation

Electrical and chemical synapses provide two distinct modes of direct communication between neurons, and the embryonic development of the two is typically not simultaneous. Instead, in both vertebrates and invertebrates, gap junction-based electrical synapses arise before chemical synaptogenesis, and the early circuits composed of gap junction-based electrical synapses resemble those produced later by chemical synapses. This developmental sequence from electrical to chemical synapses has led to the hypothesis that, in developing neuronal circuits, electrical junctions are necessary forerunners of chemical synapses. Up to now, it has been difficult to test this hypothesis directly, but we can identify individual neurons in the leech nervous system from before the time when synapses are first forming, so we could test the hypothesis. Using RNA interference, we transiently reduced gap junction expression in individual identified neurons during the 2-4 d when chemical synapses normally form. We found that the expected chemical synapses failed to form on schedule, and they were still missing months later when the nervous system was fully mature. We conclude that the formation of gap junctions between leech neurons is a necessary step in the formation of chemical synaptic junctions, confirming the predicted relation between electrical synapses and chemical synaptogenesis.

Mec-15 Encodes an F-Box Protein Required for Touch Receptor Neuron Mechanosensation, Synapse Formation and Development

Selective protein degradation is a key regulator of neuronal development and synaptogenesis. Complexes that target proteins for degradation often contain F-box proteins. Here we characterize MEC-15, an F-box protein with WD repeats, which is required for the development and function of Caenorhabditis elegans touch receptor neurons (TRNs). Mutations in mec-15 produce defects in TRN touch sensitivity, chemical synapse formation, and cell-body morphology. All mec-15 mutant phenotypes are enhanced by mutations in a MAP kinase pathway composed of the MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAPK PMK-3. A mutation of the rpm-1 gene, which encodes an E3 ubiquitin ligase that negatively regulates this pathway to promote synaptogenesis, suppresses only the mec-15 cell-body defect. Thus, MEC-15 acts in parallel with RPM-1, implicating a second protein degradation pathway in TRN development. In addition, all mec-15 phenotypes can be dominantly suppressed by mutations in mec-7, which encodes a beta-tubulin, and dominantly enhanced by mutations in mec-12, which encodes an alpha-tubulin. Since mec-15 phenotypes depend on the relative levels of these tubulins, MEC-15 may target proteins whose function is affected by these levels.

Transient electrical coupling regulates formation of neuronal networks

Electrical synapses are abundant before and during developmental windows of intense chemical synapse formation, and might therefore contribute to the establishment of neuronal networks. Transient electrical coupling develops and is then eliminated between regenerating Helisoma motoneurons 110 and 19 during a period of 48–72 h in vivo and in vitro following nerve injury. An inverse relationship exists between electrical coupling and chemical synaptic transmission at these synapses, such that the decline in electrical coupling is coincident with the emergence of cholinergic synaptic transmission. In this study, we have generated two- and three-cell neuronal networks to test whether predicted synaptogenic capabilities were affected by previous synaptic interactions. Electrophysiological analyses demonstrated that synapses formed in three-cell neuronal networks were not those predicted based on synaptogenic outcomes in two-cell networks. Thus, new electrical and chemical synapse formation within a neuronal network is dependent on existing connectivity of that network. In addition, new contacts formed with established networks have little impact on these existing connections. These results suggest that network-dependent mechanisms, particularly those mediated by gap junctional coupling, regulate synapse formation within simple neural networks.

The C. elegans gap junction protein UNC-7 is required for chemical synapse formation

The C. elegans gap junction protein UNC-7 is required for chemical synapse formation

Disruption and Recovery of Patterned Retinal Activity in the Absence of Acetylcholine

Many developing neural circuits generate synchronized bursting activity among neighboring neurons, a pattern thought to be important for sculpting precise neural connectivity. Network output remains relatively constant as the cellular and synaptic components of these immature circuits change during development, suggesting the presence of homeostatic mechanisms. In the retina, spontaneous waves of activity are present even before chemical synapse formation, needing gap junctions to propagate. However, as synaptogenesis proceeds, retinal waves become dependent on cholinergic neurotransmission, no longer requiring gap junctions. Later still in development, waves are driven by glutamatergic rather than cholinergic synapses. Here, we asked how retinal activity evolves in the absence of cholinergic transmission by using a conditional mutant in which the gene encoding choline acetyltransferase (ChAT), the sole synthetic enzyme for acetylcholine (ACh), was deleted from large retinal regions. ChAT-negative regions lacked retinal waves for the first few days after birth, but by postnatal day 5 (P5), ACh-independent waves propagated across these regions. Pharmacological analysis of the waves in ChAT knock-out regions revealed a requirement for gap junctions but not glutamate, suggesting that patterned activity may have emerged via restoration of previous gap-junctional networks. Similarly, in P5 wild-type retinas, spontaneous activity recovered after a few hours in nicotinic receptor antagonists, often as local patches of coactive cells but not waves. The rapid recovery of rhythmic spontaneous activity in the presence of cholinergic antagonists and the eventual emergence of waves in ChAT knock-out regions suggest that homeostatic mechanisms regulate retinal output during development.

Lymnaea stagnalis and the development of neuroelectronic technologies.

The recent development of techniques for stimulating and recording from individual neurons grown on semiconductor chips has ushered in a new era in the field of neuroelectronics. Using this approach to construct complex neural circuits on silicon from individual neurons will require improvements at the neuron/semiconductor interface and advances in controlling synaptogenesis. Although devices incorporating vertebrate neurons may be an ultimate goal, initial investigations using neurons from the pond snail Lymnaea stagnalis have distinct advantages. Simple two-cell networks connected by electrical synapses have already been reconstructed on semiconductor chips. Furthermore, considerable progress has been made in controlling the processes that underlie chemical synapse formation in Lymnaea. Studies of Lymnaea neural networks on silicon chips will lead to a deeper understanding of the long-term dynamics of simple neural circuits and may provide the basis for reliable interfaces for new neuroprosthetic devices.

Developmental maturation of synaptic vesicle cycling as a distinctive feature of central glutamatergic synapses

The formation of chemical synapses in the mammalian brain involves complex pre- and postsynaptic differentiation processes. Presynaptically, the progressive accumulation of synaptic vesicles is a hallmark of synapse maturation in the neocortex [J Neurocytol 12 (1983b) 697]. In this study, we analyzed the functional consequences of presynaptic vesicle-pool maturation at central glutamatergic and GABAergic synapses. Using ( N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) staining of recycling synaptic vesicles, we demonstrate a pronounced developmental increase in presynaptic vesicle accumulation during differentiation of neocortical neurons in culture. Using electrophysiological methods to study functional synaptic maturation, we found an improved recovery from hypertonic solution-induced depletion. As supported by the FM1-43 staining results, this change is most likely caused by a developmental increase in the number of reserve-pool vesicles. In addition, assuming a rapid reuse of freshly recycled vesicles, a developmental maturation of the endocytosis process may also contribute. The observed presynaptic maturation process occurred selectively at glutamatergic synapses, while GABAergic synapses did not show similar developmental alterations. Furthermore, we used high-frequency stimulation (HFS) of glutamatergic and GABAergic synapses to reveal the physiological consequences of reserve-pool maturation. As expected, recovery from HFS-induced depletion was incomplete at immature glutamatergic synapses and strongly improved during synapse maturation. Again, GABAergic synapses did not show similar developmental changes. Taken together, our study characterizes the functional consequences of a pronounced accumulation of reserve-pool vesicles occurring selectively at glutamatergic synapses.

Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system.

Gap junctions (GJs) are composed of proteins from two distinct families. In vertebrates, GJs are composed of connexins; a connexin hexamer on one cell lines up with a hexamer on an apposing cell to form the intercellular channel. In invertebrates, GJs are composed of an unrelated protein family, the innexins. Different connexins have distinct properties that make them largely non-interchangeable in the animal. Innexins are also a large family with high sequence homology, and some functional differences have been reported. The biological implication of innexin differences, such as their ability to substitute for one another in the animal, has not been explored. Recently, we showed that GJ proteins are necessary for the development of normal neural transmission in the Drosophila visual system. Mutations in either of two Drosophila GJ genes (innexins), shakB and ogre, lead to a loss of transients in the electroretinogram (ERG), which is indicative of a failure of the lamina to respond to retinal cell depolarization. Ogre is required presynaptically and shakB(N) postsynaptically. Both act during development. Here we ask if innexins are interchangeable in their role of promoting normal neural development in flies. Specifically, we tested several innexins for their ability to rescue shakB(2) and ogre mutant ERGs and found that, by and large, innexins are not interchangeable. We mapped the protein regions required for this specificity by making molecular chimeras between shakB(N) and ogre and testing their ability to rescue both mutants. Each chimera rescued either shakB or ogre but never both. Sequences in the first half of each protein are necessary for functional specificity. Potentially crucial residues include a small number in the intracellular loop as well as a short stretch just N-terminal to the second transmembrane domain. Temporary GJs, possibly between the retina and lamina, may play a role in final target selection and/or chemical synapse formation in the Drosophila visual system. In that case, specificity in GJ formation or function could contribute, directly or indirectly, to chemical synaptic specificity by regulating which neurons couple and what signals they exchange. Cells may couple only if their innexins can mate with each other. The partially overlapping expression patterns of several innexins make this 'mix and match' model of GJ formation a possibility.

Gap junction proteins expressed during development are required for adult neural function in the Drosophila optic lamina.

We provide evidence that gap junction proteins, expressed during development, are necessary for the formation of normally functioning connections in the Drosophila optic lamina. Flies with mutations in the gap junction genes (innexins), shakingB, and ogre have normal photoreceptor potentials but a defective response of the postsynaptic cells in the optic lamina. This is indicated by a reduction in, or absence of, transients in the electroretinogram. Ogre is required in the presynaptic retinal photoreceptors. ShakingB(N) is, at a minimum, required in postsynaptic lamina neurons. Transgenic expression of the appropriate innexins during pupal development (but not later) rescues connection defects. Transient gap junctions have been observed to precede chemical synapse formation and have been hypothesized to play a role in connectivity and synaptogenesis; however, no causal role has been demonstrated. Here we show that developmental gap junction genes can be required for normally functioning neural connections to form.

Gap Junction Proteins Expressed during Development Are Required for Adult Neural Function in theDrosophilaOptic Lamina

We provide evidence that gap junction proteins, expressed during development, are necessary for the formation of normally functioning connections in the <i>Drosophila</i> optic lamina. Flies with mutations in the gap junction genes (<i>innexin</i>s),<i>shakingB</i>, and <i>ogre</i> have normal photoreceptor potentials but a defective response of the postsynaptic cells in the optic lamina. This is indicated by a reduction in, or absence of, transients in the electroretinogram. Ogre is required in the presynaptic retinal photoreceptors. ShakingB(N) is, at a minimum, required in postsynaptic lamina neurons. Transgenic expression of the appropriate innexins during pupal development (but not later) rescues connection defects. Transient gap junctions have been observed to precede chemical synapse formation and have been hypothesized to play a role in connectivity and synaptogenesis; however, no causal role has been demonstrated. Here we show that developmental gap junction genes can be required for normally functioning neural connections to form.

Trophic and contact conditions modulate synapse formation between identified neurons.

We tested the ability of an identified interneuron from the mollusk, Lymnaea stagnalis, to reestablish appropriate synapses in vitro. In the CNS, the giant dopaminergic neuron, designated as right pedal dorsal one (RPeD1), makes an excitatory, chemical synapse with a pair of essentially identical postsynaptic cells known as visceral dorsal two and three (VD2/3). When the somata of the pre- and postsynaptic neurons were juxtaposed and cultured in vitro in defined medium, i.e. , a soma-soma synapse, only an inappropriate electrical synapse was observed. The postsynaptic cell still responded to applied dopamine, the presynaptic transmitter, indicating that the lack of chemical synapse formation was not due to lack of dopamine receptors. When the somata were cultured apart in conditioned medium (medium previously incubated with Lymnaea CNS, thereby deriving trophic factors), the cells exhibited overlapping neurite outgrowth that resulted in an appropriate excitatory, chemical synapse from RPeD1 to VD2/3. On the other hand, when the cell pair was cultured in a soma-soma configuration, but in conditioned medium, a mixed chemical-electrical synapse was observed. Because conditioned medium could partially overcome the limitations of the soma-soma configuration and initiate chemical synapse formation, this data suggests that conditioned medium contains a factor(s) that supports synaptogenesis.

Cholinesterases precede "ON-OFF" channel dichotomy in the embryonic chick retina before onset of synaptogenesis.

Retinal physiology is balanced by the interplay between an ON and an OFF channel. The formation of this wiring dichotomy is not understood. Two neuropil sublayers of the inner plexiform layer (IPL) represent levels of cholinergic synaptic circuitry. These two sublayers are better segregated in avians than in most vertebrates; in the chick they are called a and d and participate in the OFF and ON channel, respectively. Both levels can be detected easily in the mature retina by choline acetyltransferase (ChAT) or by acetylcholinesterase (AChE); however, the usefulness of these enzymes as developmental markers is restricted, since ChAT is detected too late, while AChE labels not only cholinergic neuropil. To establish that individual AChE+ cells indeed project into cholinergic subbands a or d (and thus are involved in either channel), in the first part of this study we used the AChE-specific monoclonal antibody 1A2 plus confocal microscopy. We show that at embryonic stage E17 two AChE+ cell types of the inner nuclear layer project into the cholinergic subband a, and another one projects into d. Moreover, two others project into noncholinergic subbands, b and c, which develop only from E14 onwards. In contrast to immunocytochemistry, sensitive AChE histochemistry allows us to trace back the establishment of subbands a and d before E10, with the first AChE subband appearing along with IPL differentiation at E7. The establishment of AChE subbands is preceded by butyrylcholinesterase subbands; with differentiation following a central-peripheral gradient. Since synapses do not develop before E12 in the chick, we conclude that retinal ON-OFF dichotomy is laid down long before the formation of chemical synapses.