Intracellular and extracellular labeling of neurons in fresh slice preparations combined with multiple tract tracing

We describe methods for studying axo-dendritic projections, one of the forms of neural connection involved in the complex circuits of the central nervous system, including brainstem auditory pathways. This form of neural connection is often difficult to visualize by conventional tract tracing techniques. Retrogradely identified cells were filled intracellularly with a mixture of fluorescent Lucifer yellow and nonfluorescent HRP in live slice preparations to reveal the detailed morphological features of these cells with special attention to the distal dendrite that may receive projections from suspected or known input axons. Extracellular or intracellular labeling of cells with axons that project to the distal dendrite of the identified cells was accomplished in the same live slice preparation. Using a live slice rather than a fixed slice allows accurate, visually controlled placement of anterograde tracer, which requires living axons for transport, into the source of input to the identified cells within the slice. Live slices also permit one to characterize the identified cells electrophysiologically. Intracellular labeling of cells in a potential source of local input to the identified cells also provides conclusive information concerning with connections of the cells involved.     

Pathfinding, target recognition, and synapse formation of single regenerating fibers in the adult grasshopper Schistocerca gregaria

After lesion of the peripheral tympanal nerve of the adult locust (Schistocerca gregaria), sensory axons regenerate into their original target areas. We examined the individual behavior of single regenerating auditory afferents during pathway and target selection by intracellularly recording and labeling them at different times postlesion. During axotomy, spontaneous activity is not increased in either the distal or proximal part of the cells. Stimulus response properties of lesioned cells with or without regenerating axons are not influenced. Surprisingly, only 55% of sensory neurons regenerate through the lesion site and often give rise to more than one axonal fiber. Within the central nervous system, 70% of regenerated axons consistently follow an incorrect pathway to reach the correct target region. Often, one of two processes formed by a cell chooses the correct pathway, and the other the incorrect one. In the target region, regenerated axons reconstitute somatotopically ordered projections and form synapses that resemble those of intact fibers in number and structure. The regeneration process does not induce a detectable expression of antigens that are known to be expressed during neural development in these neurons. Our study clearly demonstrates that precise synaptic regeneration is possible in adult animals within a completely differentiated central nervous system, although pathfinding and formation of arborizations are disturbed in a particular and probably system-related manner. The results strongly suggest that accurate pathfinding is unlikely to be a decisive factor in target area recognition and synaptogenesis.     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

Fine structure of the chemoreceptor sensillum in Limulus

Each chemoreceptor sensillum of Limulus polyphemus consists of 6–15 bipolar neurosensory cells with distal processes confined within a single cuticular tubule as they extend to the outside environment. The cuticular tubule, which is enveloped by the cuticulo-tubal cell, opens proximally into a fluid-filled extracellular space through which the dendrite passes before entering the cuticular tubule. Between the neurosensory cells are one to three microvillar cells also exposed to the extracellular space. This space is enclosed by a sheath cell extending proximally from the inner opening of the cuticular tubule and enveloping the proximal portions of the dendrites, the distal portions of the microvillar cells, as well as the distal portion of some neurosensory cell bodies. Most of the remaining portions of the neurosensory cells and microvillar cells are enveloped by neuroglia. Tight junctions occur between the distal portions of the dendrites in or near the cuticular tubule. Each dendrite has a cilium-like segment located where it traverses the extracellular space with a 9 + 0 pattern of fibers. Septuplelayered junctions occur among the proximal portions of some dendrites and some neurosensory cell bodies of the same sensillum. The subjacent processes of the sensillum frequently course proximally as isolated axons before joining nerve bundles. In the chilarial and gnathobasal chemoreceptors these nerve bundles course proximally to neuropile clumps of a peripheral nerve plexus. The presence of numerous synaptic vesicles in the neuropiles suggests that chemical transmission may occur among “en passant” synapses formed by the axons. Proximally the neuropiles are joined to the central nervous system by relatively long nerves.     

Direct live monitoring of heterotypic axon-axon interactions in vitro

This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somatosensory neuron axons, but the procedure should be readily adaptable to other neuron types. The protocol is based on the rapid isolation and primary culture of genetically identified motor neurons combined with straightforward vital dye labeling and culture of dorsal root ganglion sensory neurons. Subsequently, axonal interactions are directly monitored via live fluorescence microscopy, whereas axon type identities can be unambiguously delineated throughout the experiments. Through chemical compound application or by using neurons derived from genetically engineered mice, the protocol facilitates the dissection of molecular pathways driving the axonal interactions that are crucial for neural pathway and circuit assembly. The whole procedure can be completed in 3 d.     

Convergence of multisensory inputs in Xenopus tadpole tectum

The integration of multisensory information takes place in the optic tectum where visual and auditory/mechanosensory inputs converge and regulate motor outputs. The circuits that integrate multisensory information are poorly understood. In an effort to identify the basic components of a multisensory integrative circuit, we determined the projections of the mechanosensory input from the periphery to the optic tectum and compared their distribution to the retinotectal inputs in Xenopus laevis tadpoles using dye‐labeling methods. The peripheral ganglia of the lateral line system project to the ipsilateral hindbrain and the axons representing mechanosensory inputs along the anterior/posterior body axis are mapped along the ventrodorsal axis in the axon tract in the dorsal column of the hindbrain. Hindbrain neurons project axons to the contralateral optic tectum. The neurons from anterior and posterior hindbrain regions project axons to the dorsal and ventral tectum, respectively. While the retinotectal axons project to a superficial lamina in the tectal neuropil, the hindbrain axons project to a deep neuropil layer. Calcium imaging showed that multimodal inputs converge on tectal neurons. The layer‐specific projections of the hindbrain and retinal axons suggest a functional segregation of sensory inputs to proximal and distal tectal cell dendrites, respectively. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Npn-1 contributes to axon-axon interactions that differentially control sensory and motor innervation of the limb

The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs.     

Axons of sacral preganglionic neurons in the cat: I. Origin,initial segment,and myelination

Parasympathetic preganglionic neurons in the cat sacral spinal cord innervate intraspinal neurons and pelvic target organs. Retrograde tracing studies have revealed little of the morphology of their axons including their origin, initial segments, or their myelin, due to methodological limitations. Intracellular labeling of single neurons with neurobiotin or HRP has overcome these problems. Axons were studied in 24 preganglionic neurons. In 21 neurons the axon originated as a branch of a dendrite, without a detectable axon hillock, at distances from the soma ranging from 10 to 110 μm (average 34.1 μm ). In 3 neurons the axon was derived from the soma. Initial segments, present in all cells, ranged from 15 to 40 μm (average 26.8 μm). Nearly all axons followed the initial segment with unmyelinated segments that varied between 59 to 630 μm, followed by myelin and nodes of Ranvier. Internodal distances were variable and relatively short (average 93 μm). Axonal diameters measured over the intraspinal course in 18 axons averaged 1.3 μm (range 0.6–2.4 μm) and were relatively constant compared with other neurons. Spine-like protrusions were observed on the initial segments of 12 cells. Axon collaterals originated from unmyelinated sections and nodes of Ranvier. Antidromic action potentials showing initial segment, soma-dendritic inflections, did not differentiate between soma-derived and dendrite-derived axons. The data suggest that axons originating from a dendrite are the normal structure of preganglionic neurons in the lateral sacral parasympathetic nucleus. It is proposed that the particular structure of these axons may be part of a timing mechanism that coordinates preganglionic neurons with other spinal neurons involved in target organ reflexes.     

Transcriptional regulators that differentially control dendrite and axon development

Neurons are the basic units of connectivity in the nervous system.As a signature feature,neurons form polarized structures:dendrites and axons,which integrate either sensory stimuli or inputs from upst...     

Early peripheral sensory neurons in the development of trochozoan animals

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.     

Neurons projecting from the brain to the corpora allata in orthopteroid insects: anatomy and physiology

Retrograde and orthograde labeling of neurons projecting to the corpus allatum was performed in locust, grasshopper, cricket, and cockroach species in order to identify brain neurons that may be involved in the regulation of juvenile hormone production. In the acridid grasshopper Gomphocerus rufus L., and the locusts Locusta migratoria (R.&F.) and Schistocerca gregaria Forskal, the corpora allata are innervated by two morphologically distinguishable types of brain neurons. One group of 9–13 neurons (depending on species) with somata in the pars lateralis extend axons via the nervus corporis cardiaci 2 and nervus corporis allati 1 to the ipsilateral corpus allatum, whereas two cells in each pars lateralis have bilateral projections and innervate both glands. No direct connection between the pars intercerebralis and corpus allatum has been found. In contrast, neurons with paired axons innervating both glands are not present in Periplaneta americana (L.) and Gryllus bimaculatus de Geer. Instead, two cells in each pars lateralis project only to the gland contralateral to their somata. Electrophysiological experiments on acridid grasshoppers have confirmed the existence of a direct conduction pathway between the two glands via the paired axons of four cells that have been identified by neuroanatomy. These cells are not spontaneously active under experimental conditions. Ongoing discharges in the left and right nerves are unrelated, suggesting that the corpora allata receive independent neuronal inputs from the brain.     

Specific reduction of development of the Mauthner neuron lateral dendrite after otic capsule ablation in Brachydanio rerio

During development, afferent fibers may stimulate development of postsynaptic target neurons. By surgically ablating an otic vesicle in zebrafish embryos 30 hr after fertilization we deprived the developing Mauthner (M) neuron of vestibular axonal input to its lateral dendrite. After 8 days, 14 M cells were examined by light microscopy, and in each case the size and branching of the lateral dendrite was reduced. No consistent changes were observed in shape and size of other regions of the deprived cells or in the contralateral control cells. Synapses onto five of these pairs of cells were examined by electron microscopy. Except for missing vestibular terminals on the deprived dendrites, the synaptic input to the dendrites and to other regions of the M cell was normal in distribution and pattern. These data suggest that growth-promoting or trophic effects of vestibular axons upon the M cell are localized to its lateral dendrite.     

Arhabdomeric cells of the median eye retina of scorpions

Summary Based on reconstructions from serial thin sections, arhabdomeric cells within the retina of the median eyes of the scorpion,Androctonus australis, are identified. Each retinula unit (formed by mainly five retinula cells with a fused rhabdom) is associated with one arhabdomeric cell. Extending distally from its soma which is located close to the postretina, the arhabdomeric cell bears an up to 80 m long dendrite that ends at the base of the fused rhabdom. The most noteworthy morphological feature of the dendrite is the presence, at the distal dendrite tip, of numerous finger-like or bulbous evaginations that extend into every one of the five visual cells forming a retinula unit. These and other characteristics strongly suggest that the arhabdomeric cell represents an intrinsically photoinsensitive second neuron involved in visual information processing.This study was supportet by a grant from the Deutsche Forschungs-gemeinschaft (F1 77/8).     

The neural tube origin of ventral root sheath cells in the chick embryo

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.     

Organization of bilateral spinal projections to the lateral reticular nucleus of the rat

The present study was carried out to analyze the topography of bilateral spinal projections to the lateral reticular nucleus (LRN). We used retrograde transport of fluorescent tracers Fast Blue and Diamidino Yellow to identify spinal neurons projecting to the ipsilateral and/or contralateral LRN, as well as orthograde transport of Phaseolus vulgaris leucoagglutinin to identify the LRN areas where spinoreticular axons terminate. Orthograde labeling confirmed that bilateral spinoreticular projections coming from cervical and upper-thoracic segments terminate in the magnocellular division of LRN, while those coming from the lower-thoracic, lumbar and sacral segments end in the parvocellular division of the nucleus; only a sparse spinal input has been observed in the subtrigeminal division of LRN. Retrograde labeling showed that labeled neurons were present at all spinal levels and in particular large numbers in the cervical and lumbar enlargements. Retrogradely single-labeled cells were located, with contralateral predominance, in all segments of the spinal cord, within laminae IV, V, VI, VIII, and X, whereas in laminae III and VII labeled neurons were mainly observed ipsilaterally. Furthermore, a small fraction of double-labeled cells (7.4%) was observed throughout the spinal cord, mainly in laminae III, IV, VII and VIII.     

Fine structure and primary sensory projections of sensilla on the maxillary palp of Drosophila melanogaster Meigen (Diptera : Drosophilidae)

Three types of hairs were identified on the maxillary palp of Drosophila melanogaster Meigen (Diptera : Drosophilidae): (i) single-walled, multiporous sensilla basiconica, which constitute 75% of the innervated hairs; (ii) thick walled non-porous sensilla trichodea, which make up the remaining 25% of the innervated hairs; and (iii) numerous spinules, which are un-innervated. These sensilla basiconica uniformly contain 2 bipolar sense cells, whereas sensilla trichodea have a single dendrite with a tubular body at the base of each hair. A majority of the sensilla basiconica is located on the distal half of the dorsal surface, whereas sensilla trichodea are positioned on the tip and entire ventrolateral ridge of the palp. Approximately 125 axons of the sense cells join to form a single nerve. The structure of sensilla basiconica and sensilla trichodea suggests that they are olfactory and mechanosensory respectively. The contact chemoreceptors (gustatory sensilla) are conspicuously absent on the maxillary palp.Golgi silver impregnations and cobalt fills show that the primary sensory fibres from sensilla trichodea and sensilla basiconica on the maxillary palp project in the posterior suboesophageal ganglion (SOG) and the antennal lobe respectively. A single fibre projects separately either in the SOG or in the antennal lobe. In the antennal lobe, the input received from sensilla basiconica is usually bilateral and at least 5 glomeruli are innervated symmetrically on either side from both the palps.This study suggests that the sensory neurons are capable of making selective projections in the specific regions of the brain. Accordingly, the fibres from a sensillum project to the brain with respect to their functions and the individual glomeruli represent functional units of the brain, receiving inputs in a characteristic combination.     

Neuroanatomical technique for studying long axonal projections in the central nervous system: combined axonal staining and pre-labeling in parasagittal gerbil brain slices

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The polarity of axon growth in the wings of Drosophila melanogaster

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.