Axon Synapse Research Articles

Role of Leucine-Rich Repeat Proteins in the Development and Function of Neural Circuits

The nervous system consists of an ensemble of billions of neurons interconnected in a highly specific pattern that allows proper propagation and integration of neural activities. The organization of these specific connections emerges from sequential developmental events including axon guidance, target selection, and synapse formation. These events critically rely on cell-cell recognition and communication mediated by cell-surface ligands and receptors. Recent studies have uncovered central roles for leucine-rich repeat (LRR) domain-containing proteins, not only in organizing neural connectivity from axon guidance to target selection to synapse formation, but also in various nervous system disorders. Their versatile LRR domains, in particular, serve as key sites for interactions with a wide diversity of binding partners. Here, we focus on a few exquisite examples of secreted or membrane-associated LRR proteins in Drosophila and mammals and review the mechanisms by which they regulate diverse aspects of nervous system development and function.

Coupling a dynein transport model with a model of anterograde and retrograde transport of intracellular organelles

A model of fast axonal transport of organelles that accounts for dynein transport in an inactive state toward the axonal synapse is developed. It is assumed that anterograde transport of inactive dynein in an axon is powered by kinesin motors. It is further assumed that the probability of organelle attachment to a dynein motor is directly proportional to the concentration of free dynein motors available at a particular location in the axon. The results predicted by two models (the first one is that which incorporates dynein transport and the second one is the traditional model that does not incorporate dynein transport) are compared. The obtained results suggest that the availability of dynein motors in a particular location in an axon can be a factor limiting fast axonal transport.

Age-dependent Expression of GAP-43, Netrin-1, Collapsin-1, and Neuropilin-1 in Murine Cerebellum*

GAP-43, netrin-1, collapsin-1, and neuropilin-1 have been regarded to play crucial roles in the formation of patterned neural connections. The cerebellum consists of five distinct concentric layers: white matter, internal granule layer (IGL), Purkinje cell layer (PCL), molecular layer (ML), and external granule layer (EGL) in young rodents. Cells in EGL are generated after birth. In contrast Purkinje neurons are born before birth, which receive main innervations of climbing fibers from the inferior olivary nucleus and parallel fibers from the internal granule cells. These innervations are mostly established in the first three postnatal weeks, accompanying the sprouting and maturation of Purkinje cells. The potential roles of GAP-43, netrin-1, collapsin-1 and neuropilin-1 in the postnatal development of cerebellum remain unclear. To get insights into the above issue, the expression of GAP-43, netrin-1, collapsin-1, and neuropilin-1 mRNAs and proteins were examined in the cerebellum of mice at postnatal days (P) 5, P10, P20 and adulthood. The results showed that these four molecules were expressed in different temporal and spatial patterns in the postnatal cerebellum of mice, which was in match with axonal synaptogenesis, elongation and synapse formation during postnatal development and adulthood. By using double immunohistochemistry, it was found that the Purkinje cells stained for GAP-43 were also positive for either netrin-1 or collapsin-1 at P10, and cells stained for collapsin-1 were also positive for netrin-1 or neuropilin-1. It was suggested that the four molecules are involved in the postnatal development of cerebellum.

S11-02 Activity and signalling in the development of neural morphology and locomotion

Whole animal behaviors require precise wiring of the nervous system. Yet how neuronal wiring translates into defined behaviors is not well understood. To study the interplay between nervous system development and behavior, we focus on neuronal wiring (axonal guidance and synapse formation) controlling defined motor behaviors. In genetic screens for motor axon guidance and synapse formation, we have identified a series of genes that have allowed us to dissect at the molecular and cellular level how motor axons navigate from the spinal cord to their muscle targets, and how they form the first neuromuscular synapses [1– 3]. In parallel, we have conducted genetic screens to identify genes controlling the modulation of motor behaviors. We have focused on the startle response, which is a evolutionary conserved and well-characterized motor behavior modulated by sensory stimuli. For example, the display of a sub-threshold stimulus (i.e. a stimulus too weak to elicit a response on its own) just prior to a startling stimulus, suppresses the startle response through neural processing called sensorimotor gating. Despite its importance (patients with schizophrenia have severe sensorimotor deficits) the molecular and cellular pathways underlying sensorimotor are not very well understood. We have recently established the zebrafish as a model to study sensorimotor gating, and in a genetic screen we have identified several mutants with specific defects in this process [4]. We will discuss our ongoing efforts to integrate neuronal wiring (axonal guidance and synapse formation) during development with the function of neural circuits controlling defined motor behaviors (sensorimotor gating, phototaxis).

CRMP family proteins that regulate axon guidance, synapse maturation and cell migration

CRMP family proteins that regulate axon guidance, synapse maturation and cell migration

Mitochondrial Trafficking to Synapses in Cultured Primary Cortical Neurons

Functional synapses require mitochondria to supply ATP and regulate local [Ca2+]i for neurotransmission. Mitochondria are thought to be transported to specific cellular regions of increased need such as synapses. However, little is known about how this occurs, including the spatiotemporal distribution of mitochondria relative to presynaptic and postsynaptic sites, whether mitochondria are dynamically recruited to synapses, and how synaptic activity affects these trafficking patterns. We used primary cortical neurons in culture that form synaptic connections and show spontaneous synaptic activity under normal conditions. Neurons were cotransfected with a mitochondrially targeted cyan fluorescent protein and an enhanced yellow fluorescent protein-tagged synaptophysin or postsynaptic density-95 plasmid to label presynaptic or postsynaptic structures, respectively. Fluorescence microscopy revealed longer dendritic mitochondria that occupied a greater fraction of neuronal process length than axonal mitochondria. Mitochondria were significantly more likely to be localized at synaptic sites. Although this localization was unchanged by inhibition of synaptic activity by tetrodotoxin, it increased in dendritic synapses and decreased in axonal synapses during overactivity by veratridine. Mitochondrial movement and recruitment to synapses also differed between axons and dendrites under basal conditions and when synaptic activity was altered. Additionally, we show that movement of dendritic mitochondria can be selectively impaired by glutamate and zinc. We conclude that mitochondrial trafficking to synapses is dynamic in neurons and is modulated by changes in synaptic activity. Furthermore, mitochondrial morphology and distribution may be optimized differentially to best serve the synaptic distributions in axons and dendrites. Last, selective cessation of mitochondrial movement in dendrites suggests early postsynaptic dysfunction in neuronal injury and degeneration.

Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential

Traditionally, neuronal operations in the cerebral cortex have been viewed as occurring through the interaction of synaptic potentials in the dendrite and soma, followed by the initiation of an action potential, typically in the axon. Propagation of this action potential to the synaptic terminals is widely believed to be the only form of rapid communication of information between the soma and axonal synapses, and hence to postsynaptic neurons. Here we show that the voltage fluctuations associated with dendrosomatic synaptic activity propagate significant distances along the axon, and that modest changes in the somatic membrane potential of the presynaptic neuron modulate the amplitude and duration of axonal action potentials and, through a Ca2+-dependent mechanism, the average amplitude of the postsynaptic potential evoked by these spikes. These results indicate that synaptic activity in the dendrite and soma controls not only the pattern of action potentials generated, but also the amplitude of the synaptic potentials that these action potentials initiate in local cortical circuits, resulting in synaptic transmission that is a mixture of triggered and graded (analogue) signals.

Developmental period for N‐methyl‐D‐aspartate (NMDA) receptor‐dependent synapse elimination correlated with visuotopic map refinement

During a short perinatal interval, N-methyl-D-aspartate receptor (NMDAR) function is essential to a process in which spontaneous retinal waves focus retinal axon arbors in the superficial layers of the rodent superior colliculus (sSC). Here we provide evidence that this NMDAR-dependent axonal refinement occurs through elimination of uncorrelated retinal synapses arising from disparate loci, rather than stabilization of topographically appropriate inputs. The density of synaptic release sites within fluorescently labeled retinal terminals was counted in double-labeling experiments using confocal microscopy and antibodies against synaptophysin or synapsin-1. Chronic NMDAR blockade from birth increased retinal axon synapse density at postnatal days (P) 6, 8, and 10, suggesting that NMDAR currents reduce synapse density during the refinement period. With assay at P14, after focal arborization has been established, the effect disappeared. Conversely, chronic NMDA treatment, known to induce functional synaptic depression in the sSC, decreased retinocollicular synapse density at P14, but not earlier, during the refinement period (P8). Thus during the development of retinocollicular topographic order, there is a period when NMDAR activity predominantly eliminates retinal axon synapses. We were able to extend this period by using retinal lesions to reduce synaptic density in a defined zone. Synapse density on intact retinocollicular axons sprouting into this zone was increased by NMDAR blockade, even when examined at P14. Thus, the period of NMDAR-dependent synaptic destabilization is terminated by a factor related to the density and refinement of retinal arbors.

Ciliary neurotrophic factor-immunoreactivity in olfactory sensory neurons

Ciliary neurotrophic factor (CNTF) has been implicated in processes of neuroprotection, axonal regeneration and synaptogenesis in the lesioned CNS. In the olfactory system, which is characterized by particularly robust neuroplasticity throughout life, the concentration of CNTF is high even under physiological conditions. In the present study, the cellular localization of CNTF-immunoreactivity was studied in the rat and mouse olfactory epithelium. In both species, individual olfactory sensory neurons (ONs) displayed intense CNTF-immunoreactivity. The number of CNTF-ir ONs varied interindividually in rats and was lower in mice than in rats. In olfactory epithelia of mice expressing β-galactosidase under control of the CNTF promoter, cells of the ON layer were immunoreactive for the reporter protein. CNTF-ir ONs were olfactory marker protein-positive and growth associated protein 43-negative. CNTF-ir ONs lacked apoptotic markers, and the number of specifically labeled ONs was apparently unchanged after light chemical lesioning of the epithelium, indicating that CNTF-immunoreactivity was not associated with ON death. Electron microscopy of CNTF-ir ON axons in innervated olfactory bulb glomeruli documented that they formed typical ON axonal synapses with target neurons. Three dimensional reconstructions of bulb pairs showed a striking similarity of the positions of glomeruli innervated by CNTF-ir ON axons in left and right bulbs of individual animals and interindividually. The number of innervated glomeruli differed interindividually in rats and was lower in mice than in rats. The results show that in rodents CNTF-immunoreactivity occurs in a subset of mature, functionally competent ONs. The localization of target glomeruli suggests that CNTF-immunoreactivity may be associated with the expression and/or activation of specific olfactory receptor proteins.

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.

Future developments.

Neuronal signalling cannot always be reduced to classical synaptic transmission resulting in a binary inhibition or excitation. Many types of molecules that are bioactive do not fit readily into the profile of a classical neurotransmitter. Release of bioactive substances can occur beyond the familiar axonal synapse. The actions of bioactive substances, including classical transmitters, can be modulatory, global and long term. It is erroneous to extrapolate from events at the level of the neuron or gene to wholescale functions and dysfunctions. The actions of bioactive substances have to be appreciated within the context of neuronal populations, and in turn within the holistic anatomical context of the brain. Molecular biology will contribute to this more precise understanding of the relationship between individual genes, gene mutations, molecular and cellular phenotypes, and corresponding functions and dysfunctions.

Olfactory Reciprocal Synapses: Dendritic Signaling in the CNS

Synaptic transmission between dendrites in the olfactory bulb is thought to play a major role in the processing of olfactory information. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic dendrodendritic inhibition back onto mitral dendrites. We examined the mechanisms governing reciprocal dendritic transmission in rat olfactory bulb slices. We find that NMDA receptors play a critical role in this dendrodendritic inhibition. As with axonic synapses, the dendritic release of fast neurotransmitters relies on N- and P/Q-type calcium channels. The magnitude of dendrodendritic transmission is directly proportional to dendritic calcium influx. Furthermore, recordings from pairs of mitral cells show that dendrodendritic synapses can mediate lateral inhibition independently of axonal action potentials.

Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions

We have studied the spatial deployment of synapses arising from different axons that converge on the same developing neuromuscular junctions. Labeling the competing synaptic “cartels” with different dyes in mouse muscle showed that, perinatally, each axon adds similar terminal areas, whereas later, areas occupied by the competing cartels diverged by gradual elimination of one axon's synapses and ongoing addition of synaptic area by the other. Activity-dependent labeling of synapses capable of vesicle recycling in snake muscle also revealed a gradual change in territories occupied by competing inputs, implying that an axon maintained some functional synapses even as others in its cartel were being eliminated. Thus the process of synapse elimination is gradual, with loss of one viable synapse after another, until an axon is left with no synaptic territory and withdraws.

Possible morphological substrates for GABA-mediated presynaptic inhibition in the lamprey spinal cord.

Gamma-aminobutyric acid (GABA) neurons intrinsic to the lamprey spinal cord are known to modulate synaptic transmission from interneurons active during locomotion and from mechanosensory dorsal cells. Many of these physiological effects are presynaptic. To establish the morphological substrates for these axo-axonic interactions, an ultrastructural analysis was performed with an antiserum to fixed GABA. The GABA immunoreactivity (ir) was detected by postembedding peroxidase-antiperoxidase and immunogold techniques. GABA-ir terminals were found to make appositions with unlabelled axons located in the dorsal columns and in the ventrolateral aspect of the spinal cord. In the ventrolateral part of the cord, similar appositions between different GABA-ir terminals were also observed. The immunolabelled terminals contained spherical to pleomorphic synaptic vesicles, and also glycogen granules and dense core vesicles. In some cases, the fine structure of the contacts between immunogold-labelled terminals and unlabelled axons suggested a synaptic relationship. Such a relation was found in a relatively small proportion (2-3%) of the appositions studied. These specializations were always observed in close relation to an output synapse of the postsynaptic axon. It is suggested that the axo-axonal contacts described may provide an effective modulation of the synaptic transmission from axons in the lamprey spinal cord.

In vivo observations of pre- and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions

Synaptic rearrangements in developing muscle were studied by visualizing individual neuromuscular junctions in the sternomastoid muscle of living neonatal mice as they underwent the transition from multiple to single innervation. Vital staining of ACh receptors (AChRs) with rhodamine-conjugated alpha-bungarotoxin showed that while junctions were still multiply innervated (usually by two motor axons), regions of the postsynaptic membrane within each junction became depleted of receptors. Usually, several small postsynaptic areas lost AChRs in succession. In these areas, AChRs already in the membrane rapidly disappeared compared to a low level of receptor turnover elsewhere in the junction. Moreover, there was no evidence of new AChRs being inserted into these areas. Within each postsynaptic area undergoing AChR depletion, the intensity of receptor staining decreased gradually over 1-2 d. In some junctions, it appeared that AChRs were migrating away from areas being depleted of receptors. The depletion of AChRs from some sites in combination with the spreading apart of the entire receptor-rich area due to muscle fiber growth accounts for the transformation from plaque-like to branched receptor distributions at developing neuromuscular junctions. Vital staining of presynaptic motor nerve terminals at junctions whose postsynaptic AChRs were also stained showed that motor nerve terminals were lost from the same areas that were depleted of receptors postsynaptically. Postsynaptic areas began to be depleted of AChRs before there was any obvious loss of membrane or intracellular staining in the overlying nerve terminal. Only when a single innervating axon remained at a junction did loss of motor nerve terminals and underlying AChRs largely cease. That former synaptic areas could at later times be identified as uninnervated regions within a junction indicates that synapse elimination during development leaves an indelible mark on synaptic structure. These observations suggest that the withdrawal of a motor axon from a neuromuscular junction occurs as a consequence of the stepwise elimination of all of its synapses with that muscle fiber. These results also suggest that an important aspect of synaptic competition leading to axon withdrawal is the precocious loss of AChRs beneath the nerve terminals of the axon that will be eliminated. A similar early loss of AChRs beneath one axon's synapses has been shown to occur during synapse elimination in reinnervated adult muscle (Rich and Lichtman, 1989a).(ABSTRACT TRUNCATED AT 400 WORDS)

Specificity of filiform hair afferent synapses onto giant interneurons in Periplaneta americana: Anatomy is not a sufficient determinant

The synapses between the filiform hair sensory afferents and giant interneurons (GIs) 1-6 of embryonic and first instar cockroaches, Periplaneta americana, were used to investigate the role of neuronal anatomy in determining synaptic specificity. The pattern of afferent-to-GI synapses was first determined by intracellular recording of excitatory postsynaptic potentials (EPSPs). The lateral (L) axon synapses only with GIs 3, 4, and 6, while the medial (M) axon synapses with the contralateral dendrites of all six GIs but with the ipsilateral dendrites only of GIs 1, 2, and 4. The three-dimensional anatomy of the filiform afferents and GIs was determined by injection of cobalt. There is little anatomical segregation of the filiform afferents; consequently, there is no correlation between the anatomy of the GIs and their synaptic inputs. The M axon and ipsilateral GI3 were studied in more detail by light and electron microscopy. Despite the presence of an anterior M axon branch which loops around the ipsilateral GI3 neurite at a distance of 2 microns, no synapses are formed between them. This lack of synapses is not due to the presence of physical barriers. Investigation of filiform afferents and GIs in embryonic ganglia shows that at no stage are the afferents sufficiently separated for their anatomy to be an important factor in determining the specificity of the synaptic inputs of the GIs. It was postulated that two pairs of complementary cell surface labels would be sufficient to code for this specificity, and that, in GIs 3, 5, and 6, spatial differences in the expression of these labels allow the M axon to distinguish ipsilateral dendrites from contralateral.

The developmental appearance of Thy-1 antigen in the avian nervous system.

A monoclonal antibody against chicken Thy-1 glycoprotein was utilised in an indirect binding assay and an immunohistochemical technique to investigate the developmental appearance of Thy-1 in the chicken nervous system. The largest increase of Thy-1 levels in chicken forebrain during embryonic development coincided with the major period of neuronal development and synapse formation but preceded the major period of myelination. Immunohistochemical localisation studies on sections of chicken cerebellum, retina and spinal cord during this period showed that Thy-1 first appeared in regions of tissue which contained differentiated synapses of axons; however, not all such areas were Thy-1 positive. Areas of tissue rich in actively dividing neuroblasts or postmitotic undifferentiated neurons, such as the external granular layer of the cerebellum, showed very little staining. The disappearance of Thy-1-specific staining with age observed in the white matter of sections of cerebellum and spinal cord was due to the masking effect of myelination and not to a loss of Thy-1 from neuronal membranes.

Axonal proteins of presynaptic neurons during synaptogenesis.

Changes occur in the synthesis and axonal transport of neuronal proteins in dorsal-root ganglia axons as a result of contact with cells from the spinal cord during synapse formation. Dorsal-root ganglia cells were cultured in a compartmental cel culture system that allows separate access to neuronal cell bodies and their axons. When cells from the ventral spinal cord were cultured with the dorsal-root ganglia axons, synapses were established within a few days. Metabolic labeling and two-dimensional electrophoresis revealed that four of more than 300 axonal proteins had changed in their expression by the time synapses were established. The highly selective nature of these changes suggests that the proteins involved may be important in the processes of axon growth and synapse formation and their regulation by the regional environment.

The paratrigeminal nucleus. I. Neurons and synaptic organization.

The paratrigeminal nucleus, a diffuse collection of neurons on the lateral medullary surface, lies embedded in the fibres of the restiform body, ascending spinocerebellar tract and the spinal tract of the trigeminal nerve. Its rostrocaudal extent is approximately 1500--2000 micrometer in the rat, 2800--3500 micrometer in the rhesus monkey, and 5000--6000 micrometer in the human, where it is a well-defined nucleus. In Golgi preparations the neuronal somata are generally fusiform, ranging from 8--15 micrometer in width and 15--25 micrometer in length. Electron microscope studies show that their polar dendritic processes are thick and long and they intertwine in bundles among islands of cells or myelinated fibres. The perikarya have a high nucleus to cytoplasm ratio and the scanty cytoplasm is conspicuous for its paucity of organelles and its plethora of polysomal arrays. The neuropil is complex and contains a heterogeneous population of axonal varicosities displaying markedly different axoplasmic structures. Many different types of large granular vesicle-containing axons prevail. These axons engage in axo--somatic, axo--dendritic, axo--spinous and axo--axonic synapses with the processes of the cells within the nucleus.

Reciprocal synapses between cholinergic postganglionic axon and adrenergic interneuron in the cardiac ganglion of the turtle

Electron microscopy of the cardiac ganglion from normal and bilaterally vagotomized turtles revealed the occurrence of reciprocal synapses between the postganglionic cholinergic axon and the granule-containing cell (adrenergic interneuron). In the synaptic zone polarized from cholinergic to adrenergic elements, agranular intraaxonal vesicles are concentrated toward the presynaptic membrane, but no granular vesicles of the adrenergic interneuron are gathered on the postsynaptic side. In the synaptic zone polarized from adrenergic to cholinergic elements, granular vesicles are accumulated beneath the presynaptic membrane but no vesicles are associated with the postsynaptic axolemma. The membrane thickening is asymmetrical, and larger on the presynaptic than the postsynaptic side in both cases of polarization at reciprocal synapses. The intercellular cleft at the adrenergic interneuron to cholinergic axon synapse is slightly wider than that at the synapse with opposite polarity. These findings appear to provide a basis for a disinhibition device incorporated within the inhibitory feedback system of the ganglion.