Sequential steps in synaptic targeting of sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs.

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.     

Sequential steps of carbohydrate signaling mediate sensory afferent differentiation

Differences in carbohydrate signaling control sequential steps in synaptic growth of sensory afferents in the leech. The relevant glycans are constitutive and developmentally regulated modifications of leechCAM and Tractin (family members of NCAM and L1) that are specific to the surface of sensory afferents. A mannosidic glycosylation mediates the dynamic growth of early afferents as they explore their target region through sprouting sensory arbors rich with synaptic vesicles. Later emerging galactosidic glycosylations serve as markers for subsets of the same sensory afferents that correlate with different sensory modalities. These developmentally regulated galactose markers now oppose the function of the constitutive mannose marker. Sensory afferents gain cell-cell contact with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant vesicles are confined to sites of en passant synapse formation. The transformation of sensory afferent growth, progressing from mannose- to galactose-specific recognition, is consistent with a change from cell-matrix to cell-cell contact. While the constitutive mannosidic glycosylation promotes dynamic growth, developmentally regulated galactosidic glycosylations of the same cell adhesion molecules promote tissue stability. The persistence of both types of neutral glycans beyond embryonic age allows their function in synaptic plasticity during habituation and learning.     

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell–cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity. © 1993 John Wiley & Sons, Inc.     

Ectopic CNS projections guide peripheral neuron axons along novel pathways in leech embryos

Previous studies have indicated that the formation of stereotyped segmental nerves in leech embryos depends on the interactions between CNS projections and ingrowing afferents from peripheral neurons. Especially, CNS-ablation experiments have suggested that CNS-derived guidance cues are required for the correct navigation of several groups of peripheral sensory neurons. In order to directly test this hypothesis we have performed transplantations of CNS ganglia into ectopic sites in segments from which the resident ganglia have been removed. We find that the transplanted ganglia extend numerous axons distributed roughly equally in all directions. When these CNS projections reach and make contact with peripheral sensory axons they are used as guides for peripheral neurons to grow toward and into the ectopic ganglia even when this means following novel pathways that cross the midline and/or segmental boundaries. The peripheral sensory axons turn and grow toward the ectopic ganglia only when in physical contact with CNS axons, suggesting that diffusible chemoattractants are not a factor. These results demonstrate that the guidance cues provided by ectopic CNS projections are both necessary and sufficient to steer peripheral sensory neuron axons into the CNS.     

In vivo dynamics of CNS sensory arbor formation: a time-lapse study in the embryonic leech

In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality-specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality-specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye-filled sensory afferents as a function of time. Using 2-photon laser-scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time-lapse series of images revealed a highly dynamic and maturation-state-dependent pattern of growth. Upon entering the CNS, the growth cone-tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior-posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher-order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en-passant specializations accompanying the developmental onset of synapse formation.     

In vivo dynamics of CNS sensory arbor formation: A time‐lapse study in the embryonic leech

In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality‐specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality‐specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye‐filled sensory afferents as a function of time. Using 2‐photon laser‐scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time‐lapse series of images revealed a highly dynamic and maturation‐state‐dependent pattern of growth. Upon entering the CNS, the growth cone‐tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior‐posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher‐order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en‐passant specializations accompanying the developmental onset of synapse formation. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 41–53, 2003     

Effect of stimulus-driven pruning on the detection of spatiotemporal patterns of activity in large neural networks

Adult patterns of neuronal connectivity develop from a transient embryonic template characterized by exuberant projections to both appropriate and inappropriate target regions in a process known as synaptic pruning. Trigger signals able to induce synaptic pruning could be related to dynamic functions that depend on the timing of action potentials. We stimulated locally connected random networks of spiking neurons and observed the effect of a spike-timing-dependent synaptic plasticity (STDP)-driven pruning process on the emergence of cell assemblies. The spike trains of the simulated excitatory neurons were recorded. We searched for spatiotemporal firing patterns as potential markers of the build-up of functionally organized recurrent activity associated with spatially organized connectivity.     

Ectopic neurons and the organization of insect sensory systems

Summary The transplantation of appendages from one place to another on the body of crickets (Acheta domesticus) has been used to study the similarities and differences between the sensory systems of various ganglia. Mesothoracic legs have been transplanted to the abdomen in place of a cercus and cerci have been transplanted to thoracic leg stumps. After the ectopic sensory neurons had time to regenerate into the CNS, they were stained and their axonal arborizations examined. The results, which were concerned primarily with bristle receptors, revealed that bristle afferents on ectopic cerci arborized in ventral neuropil (the ventralmost association center) and leg afferents arborized in a ventral anterior region of the terminal abdominal ganglion. The results support the idea that each ganglion contains only a few distinct regions of neuropil (probably three), each receiving separate subsets of the afferent projection.The ectopic cerci were also shown to excite interneurons in the thoracic ganglia whose dendrites were located in the most ventral neuropil. These neurons normally respond to thoracic bristle afferents. Thus, the segregation of afferent axons has a correlate in the interneurons they excite.     

Specificity of a target cell-derived stop signal for afferent axonal growth.

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a "stop-growing signal" for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with another cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation.     

Specificity of a target cell-derived stop signal for afferent axonal growth

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a ?stop-growing signal”? for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with an other cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation. © 1992 John Wiley & Sons, Inc.     

Decabrominated diphenyl ether and methylmercury impair fetal nervous system development in mice at documented human exposure levels

The central nervous system (CNS) is extremely vulnerable to the toxic effects of environmental pollutants during development. Polybrominated diphenyl ethers (PBDEs) are persistent contaminants, increasingly present in the environment and in human tissues. Recent investigations identified a correlation between maternal exposure to PBDEs and impairment in fetal neurobehavioral development, suggesting that these contaminants pose a potential risk for children. We investigated on the potential effects of environmental decabrominated diphenyl ether (decaBDE, the fully brominated congener) on key neurodevelopmental molecules (e.g., synaptic proteins and immature neuron markers) in fetal mouse neurons. Methylmercury was used as reference neurotoxic contaminant and to evaluate its possible synergism with decaBDE. The neurotoxic effects of decaBDE and methylmercury were determined in developing cultured neurons from mouse fetal hippocampus and cerebellum. Neuron death, dendritic branching, synaptic protein expression, markers of immature neurons, and microglia activation were evaluated by immunocytochemistry. Brain samples from prenatally treated embryos were also examined for neurotoxicity signs by immunoblotting and histochemistry. DecaBDE significantly affected (down to 0.4 nM) the number of dendritic branches, and the levels of synaptic proteins and doublecortin in cultured neurons. Prenatal exposure to decaBDE decreased the synaptic proteins and increased the expression of the immature neuron and microglial markers in mouse fetuses. In conclusion, prenatal exposure to realistic (relevant for human exposure) concentrations of decaBDE induces impairment of fetal CNS development in mice, suggesting a potential risk of fetotoxicity in humans. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 23–38, 2015     

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6?weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.     

Differential fate of multipotent and lineage-restricted neural precursors following transplantation into the adult CNS

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

Corticothalamic feedback and sensory processing

Although nearly half of the synaptic input to neurons in the dorsal thalamus comes from the cerebral cortex, the role of corticothalamic projections in sensory processing remains elusive. Although sensory afferents certainly establish the basic receptive field properties of thalamic neurons, increasing evidence indicates that feedback from the cortex plays a crucial role in shaping thalamic responses. Here, we review recent work on the corticothalamic pathways associated with the visual, auditory, and somatosensory systems. Collectively, these studies demonstrate that sensory responses of thalamic neurons result from dynamic interactions between feedforward and feedback pathways.     

Central projections of a homoeotic regenerate, antennapedia, in a stick insect, Carausius morosus (Phasmida)

Homoeotic appendages provide a system for the analysis of neural path-finding in which the appendage is mismatched with its segmented ganglion. Central projections of sensory neurons from homoeotic antennapedia regenerates induced by antennal amputation in the stick insect, Carausius morosus, are described. The majority of afferent axons project to the olfactory lobe as in the normal antennal nerve, but they do not give rise to compact glomeruli. Nor does the form of the projection resemble that of leg sensory nerves in thoracic ganglia. The projection of antennapedia regenerate neurons in Carausius resembles the antennapedia mutant of Drosophila except that some primary afferents bypass the olfactory lobe and take several courses through the brain, sometimes reaching distant contralateral areas. It appears that these wandering fibers, having bypassed the olfactory lobe, tend to follow established tracts and to arborize or to deviate at circumscribed synaptic areas. The behavioral evidence for sensory input from antennapedia regenerates is equivocal.     

Surgical generation of supernumerary appendages for studying neuronal specificity in Drosophila melanogaster

The ability of sensory neurons to establish specific synaptic contacts in the central nervous system (CNS) can be studied by changing the spatial relationship between the periphery and the CNS. In contrast to the genetic displacement of appendages by homoeotic mutations, the surgical approach used in this study allows one to place homologous as well as heterologous appendages to the same site on the body surface. Using an improved technique of “surface transplantation,” we generated supernumerary appendages of any desired type in a particular abdominal position. The sensory axons originating from these grafts enter the CNS through the main abdominal nerve and arborize in the fused abdominal ganglia; many fibers extend also into thoracic centers. In the abdominal ganglia, terminals from dorsal transplants (wings and halteres) stay on the ipsilateral side, whereas terminals from ventral transplants (legs and antennae) distribute ipsi- and contralaterally. The same preference holds true for dorsal and ventral abdominal bristles, respectively, whose projection patterns served as a reference. In thoracic ganglia, axons from dorsal and ventral grafts yield completely different terminal patterns. Dorsal grafts project into the ipsilateral wing center, even in the mutant wingless, in which normal wing afferents are suppressed. In contrast, fibers from ventral grafts often extend along the thoracic midline. These data indicate that sensory axons of homologous appendages on the one hand, and their central targets on the other, share serially repeated surface markers. This may enable sensory fibers to recognize centers of homologous appendages.     

Plasticity in the nucleus tractus solitarius and its influence on lung and airway reflexes.

The nucleus tractus solitarius (NTS) is the first central nervous system (CNS) site for synaptic contact of the primary afferent fibers from the lungs and airways. The signal processing at these synapses will determine the output of the sensory information from the lungs and airways to all downstream synapses in the reflex pathways. The second-order NTS neurons bring to bear their own intrinsic and synaptic properties to temporally and spatially integrate the sensory information with inputs from local networks, higher brain regions, and circulating mediators, to orchestrate a coherent reflex output. There is growing evidence that NTS neurons share the rich repertoire of forms of plasticity demonstrated throughout the CNS. This review focuses on existing evidence for plasticity in the NTS, potential targets for plasticity in the NTS, and the impact of this plasticity on lung and airway reflexes.     

Activity-dependent morphological synaptic plasticity in an adult neurosecretory system: magnocellular oxytocin neurons of the hypothalamus.

Oxytocin and vasopressin neurons, located in the supraoptic and paraventricular nuclei of the hypothalamus, send their axons to the neurohypophysis where the neurohormones are released directly into the general circulation. Hormone release depends on the electrical activity of the neurons, which in turn is regulated by different afferent inputs. During conditions that enhance oxytocin secretion (parturition, lactation, and dehydration), these afferents undergo morphological remodelling which results in an increased number of synapses contacting oxytocin neurons. The synaptic changes are reversible with cessation of stimulation. Using quantitative analyses on immunolabelled preparations, we have established that this morphological synaptic plasticity affects both inhibitory and excitatory afferent inputs to oxytocin neurons. This review describes such synaptic modifications, their functional significance, and the cellular mechanisms that may be responsible.     

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.