Engineered synaptic tools reveal localized cAMP signaling in synapse assembly

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

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.     

Electron microscope observations on synaptic vesicles in synapses of the retinal rods and cones

The submicroscopic organization of the rod and cone synapses of the albino rabbit has been investigated with the use of the electron microscope. The most common rod synapse consists of an enlarged expansion of the rod fiber (the so called spherule) into which the dendritic postsynaptic fiber of the bipolar cell penetrates and digitates. The membrane surrounding the terminal consists of a double layer, the external of which is interpreted as belonging to the intervening glial cells. The synaptic membrane has a pre- and a postsynaptic layer with a total thickness of 180 to 300 A. The presynaptic layer is frequently denser and is intimately associated with the adjacent synaptic vesicles. The synaptic membrane shows processes constituted by foldings of the presynaptic layer. The entire spherule is filled with synaptic vesicles varying in diameter between 200 and 650 A with a mean of 386 A. In addition, the spherule contains a few large vacuoles near the rod fiber, interpreted as endoplasmic reticulum, and a matrix in which with high resolution a fine filamentous material can be observed. The postsynaptic fiber is homogeneous and usually does not show synaptic vesicles. In animals maintained in complete darkness for 24 hours vesicles appear to accumulate near the synaptic membrane and its processes. After 9 days there is a sharp decrease in size of the synaptic vesicles. A special rod synapse in which the dendritic postsynaptic expansion penetrates directly into the rod cell body has been identified. In line with Cajal's classification this type of synapse could be considered as a somatodendritic one. The cone synapse has a much larger terminal with a more complex relationship with the postsynaptic fiber. However, the same components recognized in the rod synapse can be observed. In animals maintained for 9 days in complete darkness there is also a considerable diminution in size of the synaptic vesicles.     

Action of thiamine on different synapses

Thiamine at a concentration of 1×10^–14 to 1×10^–4 M facilitated neuromuscular transmission at the glutaminergic synapse of the crayfish adapter, manifesting as increased amplitude and quantal content of excitatory postsynaptic potentials and raised frequency of miniature excitatory postsynaptic potentials. Thiamine augmented spontaneous electrical activity and the amplitude of synaptic potentials in the longitudinal muscle of guinea pig taenia coli. It was found from studying the effects of thiamine on the membrane potential of rat brain synaptosomes that its presynaptic action is brought about by depolarization of the nerve terminal membrane. Interaction between thiamine and the nerve endings was described by a Hill coefficient of 0.22–0.30, indicating that it has several binding sites within the structure of the receptor concerned.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 621–629, September–October, 1986.     

A new hypothesis on the mechanism by which temporary connections are formed

A new hypothesis is proposed on the mechanism by which temporary connections are formed. It is postulated that depolarization of the oligodendrocyte membrane serves as signal to its myelinizing function, and this process requires that there be, at the moment of oligodendrocyte depolarization, physicochemical "trace" of a preceding presynaptic excitation of the terminal in the intercellular clefts between its processes and these terminals. Contact of the presynaptic terminal with the glial process and enclosure by the myelin sheath create favorable conditions for the electrotonic propagation of the action potential, and the nerve impulse thus releases a larger amount of transmitter; the cerebro-cortical synapse changes from "potential" to "actual."Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 1, No. 2, pp. 130–137, September–October, 1969.     

Physical simulation of neuronal memory as a mosaic of chemoreceptor and electroreceptor elements of the somato-dendritic membrane

A model of neuronal memory in which recording and reading out are based on scanning of the somato-dendritic membrane is analyzed. The stepwise displacement of excesses of potential relief created by superposition of local postsynaptic potentials on the soma-to-dendritic membrane is used as the scanner. Associative, diagnostic, and dependable (distributive) properties of such a memory, similar to the properties of holograms, are demonstrated.M. I. Kalinin Leningrad Polytechnical Institute. Translated from Neirofiziologiya, Vol. 6, No. 1, pp. 90–98, January–February, 1974.     

Effects of α-latrotoxin on synaptic transmission between identified neurons in the snail central nervous system

The effects of -latrotoxin — a component of black widow spider venom — on an identified monosynaptic peptidergic synapse were investigated in the central nervous system ofHelix pomatia. Extracellular and intracellular application of the toxin was found to increase and block postsynaptic current respectively. Current induced by intracellular injection of cAMP which imitates postsynaptic current was inhibited, irrespective of the mode of toxin application. It was concluded on the basis of the experiments performed that -latrotoxin manifests its reinforcing effect, acting on the transmitter release system in the presynaptic neuron. It can also induce an inhibitory effect, however, operating on the postsynaptic cell direct — an effect which may be mediated by the cAMP system.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. A. A. Bogomolets Medical Institute of Biochemistry, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 430–437, July–August, 1992.     

Effects of some agonists of excitatory amino acids on synaptic transmission in the skate electroreceptors (ampullae of Lorenzini)

The effects on synaptic transmission of blockers of amino acids at postsynaptic receptors were investigated by means of external perfusion of the basal membrane of electroreceptor cells in ampullae of Lorenzini and by recording of spike activity from individual nerve fibers in the skateRaja clavata: glutamic acid diethyl ester (DEE-GLU), glutamic acid dimethyl ester (DME-GLU), D--aminoadipic acid (DAA), kynurenic acid (KYN), and cis-2,3-piperidine dicarboxylic acid (PDA). The most effective blocker was found to be DEE-GLU, producing reversible blockade of postsynaptic amino acid response. It was found that DAA did not change synaptic transmission in the electroreceptors, while PDA largely affected response produced by applying L-aspartic acid (L-ASP); KYN affected response produced by L-glutamic acid (L-GLU). It is deduced that L-GLU and L-ASP are the most effective agonists at the afferent synapse of ampullae of Lorenzini and that their excitatory effect is produced by activating quisqualate receptors.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 4, July–August, 1988, pp. 457–463.     

Selective targeting of glutamate receptors in neurons

Glutamate receptors mediate the majority of excitatory responses in the central nervous system (CNS). Neurons express multiple subtypes and subunits of glutamate receptors, which are differentially distributed at pre- and postsynaptic sites. This allows the cell to respond differentially depending on the subunit composition of receptors at the postsynaptic membrane. The process by which receptors are targeted selectively to the appropriate synapse is poorly understood. Evidence exists that targeting of glutamate receptors to the different neuronal compartments is regulated at multiple levels involving a general targeting step; a local step where receptor-containing organelles are moved to the synapse; and a step where the receptors are stabilized at the synapse, which may involve interaction with an anchoring protein.     

Transsynaptic Coordination of Synaptic Growth,Function, and Stability by the L1-Type CAM Neuroglian

The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg–Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.     

Auerbach's plexus of mammals and man: Electron microscopic identification of three different types of neuronal processes in myenteric ganglia of the large intestine from rhesus monkeys,guinea-pigs and man

Summary Ganglia from Auerbach's plexus of the large intestine (caecum, appendix vermiformis, colon transversum and rectum) in man, rhesus monkey and guinea-pig are composed of nerve cells and their processes, typical Schwann cells and a vast neuropil. The neuropil consists of dendrites and axons of intrinsic nerve cell perikarya and axons of extrinsic neurons. Axonal profiles in large nerve fibre bundles are of uniform size and appearance, embedded in infoldings of Schwann cell cytoplasm and contain occasional large granular vesicles, mitochondria and neurotubules. Preterminal axons widen into vesicle filled varicosities, some of which establish synaptic contact with intrinsic nerve cell bodies.At least three different types of neuronal processes can be distinguished in the myenteric neuropil according to the size, appearance and commutual proportion of vesicles present in axonal varicosities, and their ability to accumulate exogenous 5- and 6-hydroxydopamine and 5-hydroxydopa: 1. Axonal enlargements containing a major population of small electron lucent synaptic vesicles (350–600 Å in diameter) together with a small number of membrane-bound, opaque granules (800–1,100 Å). These profiles have been identified as cholinergic axons. The boutons establish synaptic contacts with dendritic processes of intrinsic nerve cell bodies; membrane specializations are found at the preand postsynaptic sites. 2. Axonal beads of sometimes very large diameter, containing an approximately equal amount of large granular vesicles (850–1,600 Å) and small, electron lucent or faintly opaque vesicles (400–600 Å). The granular core of the large vesicles is of medium electron density and may either fill the entire vesicle or is separated from the limiting membrane by a more or less clear interspace. The fibres probably belong to intrinsic neurons, and because of the similarity of the large, membrane-bound vesicles with neurosecretory elementary granules, they have been designated p-type fibres (polypeptide fibres). The granular core of the vesicles in these fibres becomes more electron dense after treatment with 5-OH-dopa. The accumulation of an amine precursor analogue in combination with a possible storage of a polypeptide substance (or an ATP-like substance) resembles the situation in several diffusely distributed endocrine cell systems. 3. Varicosities of axons equipped with small (400–600 Å) empty or sometimes granular vesicles, medium sized (500–900 Å) vesicles with highly electron dense cores and occasional large (900–1,300 Å) granular vesicles. Pretreatment with 5-OH-dopamine increases the electron density in almost all medium-sized granular vesicles and some of the large granular vesicles; an osmiophilic core develops in some small vesicles. 6-hydroxydopamine results in degenerative changes in the varicosities of this type of neurons. Concomitantly, both catecholamine analogues markedly reduce neuronal noradrenaline in the large intestine, as demonstrated by fluorescence histochemistry and in fluorimetric determinations. The ultrastructural features of these varicosities and their reaction to 5- and 6-OH-dopamine indicate that they belong to adrenergic, sympathetic nerves. No membrane specializations could be detected at sites of close contact of the adrenergic boutons with dendrites and cell bodies of intrinsic nerve cells.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by a grant from Albert Pahlsson's Foundation, Sweden. The work was carried out within a research organization sponsored by the Swedish Medical Research Council (projects No. B70-14X-1007-05B, B70-14X-712-05, and B70-14X-56-06).     

Microglial MERTK eliminates phosphatidylserine‐displaying inhibitory post‐synapses

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat‐me" signal that initiates glia‐mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal‐specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well‐known "eat‐me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post‐synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post‐synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post‐synapse elimination. Moreover, we found that phosphatidylserine is used for microglia‐mediated pruning of inhibitory post‐synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat‐me" signal for inhibitory post‐synapse elimination.     

Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction

The Akt family of serine‐threonine kinases integrates a myriad of signals governing cell proliferation, apoptosis, glucose metabolism, and cytoskeletal organization. Akt affects neuronal morphology and function, influencing dendrite growth and the expression of ion channels. Akt is also an integral element of PI3Kinase‐target of rapamycin (TOR)‐Rheb signaling, a pathway that affects synapse assembly in both vertebrates and Drosophila. Our recent findings demonstrated that disruption of this pathway in Drosophila is responsible for a number of neurodevelopmental deficits that may also affect phenotypes associated with tuberous sclerosis complex, a disorder resulting from mutations compromising the TSC1/TSC2 complex, an inhibitor of TOR (Dimitroff et al., 2012). Therefore, we examined the role of Akt in the assembly and physiological function of the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse that displays developmental and activity‐dependent plasticity. The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR). Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t‐SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA. Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration. © 2013 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 73: 723–743, 2013     

Degranulation enhances presynaptic membrane packing,which protects NK cells from perforin-mediated autolysis

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.

Natural killer cells mediate largely unidirectional potent cytotoxicity against diseased cells while sparing themselves. The authors show that the NK cell membrane contains and focuses lipids of high density which shield against self-destruction, and a similar densely packed postsynaptic membrane is responsible for the perforin resistance and NK cell-mediated killing evasion of an aggressive breast cancer cell line.     

Pharmacological study of inhibited frog olfactory bulb glomerular input produced by a puff of air on the olfactory mucosa

The effects of applying 4-aminopyridine (10^–2 M), aminooxyacetic acid (AOAA — 10^–4–10^–3 M), -alanine (10^–3–10^–2 M), and bicuculline (10^–5, 10^–4 M) to the intact frog olfactory bulb were investigated. Having measured inhibition of orthodromic potential postsynaptic components produced either by a puff of air on the olfactory mucosa (OB input inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition), it was found that 4-aminopyridine greatly intensified postsynaptic inhibition but strongly reduced that of OB input; inhibition of the latter was raised by AOAA or bicuculline and decreased by -alanine. These substances failed to exert any consistent, clear-cut effects on postsynaptic inhibition. Findings would support the hypothesis that OB input inhibition produced by a puff of air on the olfactory mucosa could occur as a result of GABA release from glial cells and subsequent binding of GABA to presynaptic GABA_B-receptors in glomeruli.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 12–20, January–February, 1987.     

Dissecting tripartite synapses with STED microscopy

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.     

ELECTRON MICROSCOPY OF SYNAPTIC STRUCTURE OF OCTOPUS BRAIN

The well known type of synapse between a presynaptic process containing vesicles and a "clear" postsynaptic process can be commonly observed in the various lobes of the brain of Octopus. The presynaptic vesicles are aggregated near regions of the synaptic membranes which show specialisation and asymmetric "thickening" indicating functional polarisation, and here chemical transmission is presumed to take place. In addition, in the vertical lobe a very interesting serial arrangement of synaptic contacts occurs. Presynaptic bags, formed from varicosities of fibres from the superior frontal lobe, contact the trunks of amacrine cells in the manner just described. The trunks, however, although apparently postsynaptic are themselves packed with synaptic vesicles. The trunks, in turn, make "presynaptic" contacts with clear spinous processes of other neurons of yet undetermined origin. Typical polarised membrane specialisations occur at the contact regions. The trunk vesicles aggregated closest to the contact regions have a shell of particles round their walls. At present, there is no way of telling whether the membrane conductance to the various ions is differently affected at either of the transmission sites, and, if an inhibitory mechanism is involved, whether it is of the presynaptic or postsynaptic variety.     

Role of non-quantum acetylcholine secretion in neural control of the membrane potential in skeletal muscle fibers of rats

Experiments on muscle fibers of the rat diaphragm (in vitro denervation) showed that their three-hour incubation in the cultural medium results in an 8-mV drop in the resting membrane potential (RMP). Addition of 5·10^–8 M carbacholine to the cultural medium, mimicing the effect of non-quantum acetylcholine, delayed depolarization of the denervated muscle. The effect of carbacholine could not be eliminated byd-tubocurarine (5·10^–6 M), a postsynaptic acetylcholine receptor blocker, and by ouabain (1·10^–4 M), and inhibitor of Na⁺, K⁺-ATPase of the membrane. At the same time, the effect could be completely eliminated by Mg²⁺ ions (5·10^–3 M), which blocked Ca²⁺ channels of the membrane, by N-nitroarginine (1·10^–4 M), which inhibited the enzyme NO-synthase, and by hemoglobin (2·10^–5 M), which inactivated the extracellular NO molecules. It is concluded that the released non-quantum acetylcholine can contribute to neural control of RMP of cross-striated muscle fibers via the Ca²⁺-dependent activation of NO synthesis in the sarcoplasm. The NO molecules can play the role of a retrograde signal indicative of the normal functioning of the neuromuscular synapse. The impairment of this link caused by a denervation-induced cessation of the non-quantum secretion can serve as a signal triggering the early changes in the muscle membrane following nerve transection.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 67–71, January–February, 1995.