Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

Axons of sacral preganglionic neurons in the cat: II. Axon collaterals

Axon collaterals were identified in 21 of 24 preganglionic neurons in the lateral band of the sacral parasympathetic nucleus of the cat. Following the intracellular injection of HRP or neurobiotin the axons from 20 of these neurons were followed and 53 primary axon collaterals were found to originate from unmyelinated segments and from nodes of Ranvier. Detailed mapping done in the five best labeled cells showed bilateral axon collaterals distributions up to 25,000 μm in length with 950 varicosities and unilateral distributions up to 12,561 μm with 491 varicosities. The axon collaterals appeared to be unmyelinated, which was confirmed at EM, and were small in diameter (average 0.3 μm). Varicosities were located mostly in laminae I, V, VII, VIII and X and in the lateral funiculi. Most varicosities were not in contact with visible structures but some were seen in close apposition to Nissl stained somata and proximal dendrites. Varicosities had average minor diameters of 1.3 μm and major diameters of 2.3 μm. Most were boutons en passant while 10–20% were boutons termineaux. EM revealed axodendritic and axoaxonic synapses formed by varicosities and by the axons between varicosities. It is estimated that the most extensive of these axon collaterals systems may contact over 200 spinal neurons in multiple locations. These data lead to the conclusion that sacral preganglionic neurons have multiple functions within the spinal cord in addition to serving their target organ. As most preganglionic neurons in this location innervate the urinary bladder, it is possible that bladder preganglionic neurons have multiple functions.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Scanning electron microscope observations on the muscle innervation of Oikopleura dioica fol (appendicularia,tunicata) with notes on the arrangement of connective tissue fibres

Critical point dried and fractured appendicularia of the species Oikopleura dioica have been examined in the scanning electron microscope. The dorsal nerve cord with ganglion cells and peripheral nerve fibres could easily be observed. Thick peripheral nerve fibres leave the nerve cord as bilateral pairs at constant intervals along the tail. Most of these fibres branch from the naked nerve cord, but some evidently originate in ganglion perikarya bulging out from the nerve cord itself. These paired peripheral nerves always have elaborate end-arborizations on the medial surface of the lateral muscle cells. They are accordingly interpreted as motor axons. Some thinner peripheral nerve fibres originate at irregular intervals from both the nerve cord and the ganglion cells. Due to the numerous extracellular fibrils that connect the bilateral layers of the epidermal fins and the muscle cells to each other, these thin nerve fibres can seldom be traced to their termination. A few ones can, however, be traced ventrally between the notochord and the muscle cells and seem to end in singular bulb-like expansions. Clusters of synaptic vesicles are present in transmission electron micrographs of such nerves, and they are accordingly believed to carry efferent impulses. The extracellular fibrils are arranged in a highly ordered pattern with thick bundles crossing the gap between the structures to be interconnected and with numerous radiating insertions on the surface of the tissues.     

DIFFERENTIATION OF NEURONAL TYPES AND SYNAPSES IN MYELINATING CULTURES OF MOUSE CEREBELLUM

The Holmes silver impregnation method has made possible the recognition of multiple neuronal types and synapses in myelinating cultures of mouse cerebellum. Well stained large and medium-sized neurons are always found in small numbers near ependymal formations and are considered to be roof nuclear neurons. Neurons with poorly stained somas, abruptly demarked from intensely stained axons, are numerous and often are arranged in palisades. With prolonged maintenance in vitro these neurons develop some but not all of the features of mature Purkinje cells. A few small, densely stained, bipolar neurons, often with one process bifurcated, are found in dense regions of some cultures of newborn cerebellum. These neurons are commoner in cultures from cerebella of older mice. They closely resemble the immature granule cell in vivo. All the neuron types recognized in cultures are present in the initial explants; neurons differentiate further in vitro, but new neurons probably do not form. Synaptic boutons are found on somas and dendrites of many Purkinje cells. Two cultures contained structures resembling the basket endings which surround Purkinje cell somas in vivo. The complexity of neuronal relationships in cultures of central nervous tissue is emphasized.     

Development of "normal" dermatomes and somatotopic maps by "abnormal" populations of cutaneous neurons

During development, motor and sensory axons grow to peripheral targets with remarkable precision. Whereas much has been learned about the development of motoneuron connectivity, less is known about the regulation of cutaneous innervation. In adults, dorsal root ganglia (DRG) innervate characteristic skin regions, termed dermatomes, and their axons project somatotopically in the dorsal horn. Here, we have investigated whether cutaneous neurons are selectively matched with specific skin regions, and whether peripheral target skin influences the central connections of cutaneous neurons. To address these questions, we shifted limb buds rostrally in chick embryos prior to axon outgrowth, causing DRGs to innervate novel skin regions, and mapped the resulting dermatomes and central projections. Following limb shifts, cutaneous innervation arose from more rostral and from fewer DRGs than normal, but the overall dermatome pattern was preserved. Thus, DRGs parcel out innervation of skin in a consistent manner, with no indication of matching between skin and DRGs. Similarly, cutaneous nerves established a "normal" somatotopic map in the dorsal horn, but in more rostral segments than usual. Thus, the peripheral target skin may influence the pattern of CNS projections, but does not direct cutaneous axons to specific populations of neurons in the dorsal horn.     

Journey to the skin

The peripheral axons of vertebrate tactile somatosensory neurons travel long distances from ganglia just outside the central nervous system to the skin. Once in the skin these axons form elaborate terminals whose organization must be regionally patterned to detect and accurately localize different kinds of touch stimuli. This review describes key studies that identified choice points for somatosensory axon growth cones and the extrinsic molecular cues that function at each of those steps. While much has been learned in the past 20 years about the guidance of these axons, there is still much to be learned about how the peripheral axons of different kinds of somatosensory neurons adopt different trajectories and form specific terminal structures.     

Recurrent axon collaterals of lumbar motor neurons in turtles

A combined morphophysiological study was made of connections between motoneurons on the superfused isolated lumbar spinal cord of Testudo horsfieldi. Postsynaptic potentials of motoneurons, followed by antidromic stimulations of ventral root filaments (VR-PSPs), were recorded intracellularly. Depolarizing VP-PSPs had short latencies (1.0-1.5 mc) and amplitudes in the range of 0.3-3.0 mV. At the constant stimulus intensity, the fluctuations of amplitudes were recorded. In some motoneurons, hyperpolarizing VP-PSRs with the latencies 2.5-3.0 mc were observed. A possible structural basis of VR-PSPs was studied by the horseradish peroxidase (HRP) method. After HRP application on thin ventral root filaments the retrograde staining of motoneurons revealed recurrent axon collaterals of labeled motoneurons. Three-dimensional computer reconstructions showed one to three collaterals given off by motoneuron axons. There were up to 19 points of branching in a single collateral. In some cases the full length of collateral trees reached 4.0 mm. The collateral branches had up to 72 "en passant" and terminal axon swellings. The swellings (presumed contacting boutons) were distributed in the ventral and intermedial gray matter and in the ventromedial while matter and revealed on motoneurons and inerneurons. These data suggest the participation of the motor axon collaterals in the motoneuron--motoneuron communication in the turtle spinal cord whereas only dendro-dendritic contacts had been discussed earlier.     

Effect of reduced impulse activity on the development of identified motor terminals in Drosophila larvae

In Drosophila larvae, motoneurons show distinctive differences in the size of their synaptic boutons; that is, axon 1 has type Ib ("big" boutons) terminals and axon 2 has type Is ("small" boutons) terminals on muscle fibers 6 and 7. To determine whether axon 1 develops large boutons due to its high impulse activity, we reduced impulse activity and examined the motor terminals formed by axon 1. The number of functional Na(+) channels was reduced either with the nap(ts) mutation or by adding tetrodotoxin (TTX) to the media (0.1 microg/g). In both cases, the rate of locomotion was decreased by approximately 40%, presumably reflecting a decrease in impulse activity. Locomotor activity was restored to above wild-type (Canton-S) levels when nap(ts) was combined with a duplication of para, the Na(+)-channel gene. Lucifer yellow was injected into the axon 1 motor terminals, and we measured motor terminal area, length, the number of branches, and the number and width of synaptic boutons. Although all parameters were smaller in nap(ts) and TTX-treated larvae compared to wild-type, most of these differences were not significant when the differences in muscle fiber size were factored out. Only bouton width was significantly smaller in both different nap(ts) and TTX-treated larvae: boutons were about 20% smaller in nap(ts) and TTX-treated larvae, and 20% larger in nap(ts); Dp para(+) compared to wild-type. In addition, terminal area was significantly smaller in nap(ts) compared to wild-type. Bouton size at Ib terminals with reduced impulse activity was similar to that normally seen at Is terminals. Thus, differences in impulse activity play a major role in the differentiation of bouton size at Drosophila motor terminals.     

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.     

Cell adhesion and migration in the organization of spinal motor neurons

Spinal motor neurons are critical to the ability of animals to move and thus essential to survival. Motor neurons that project axons to distinct limb-muscle targets are topographically organized such that central nervous system position reflects the location of the muscle in the limb. The central positioning of limb-projecting motor neurons arises during development through motor neuron migration followed by a period of coalescence into discrete groupings of motor neurons which project axons to an individual muscle. These so-called motor pools are a common feature of motor organization in higher vertebrates. Recent work has highlighted the critical role for armadillo family member catenin-dependent functions of the cadherin family of cell adhesion molecules in directing the organization of motor neurons. Cadherin function appears to be important for both the motor neuron migration and coalescence phases of the emergence of motor neuron topography. Here, I review this recent work in the context of our understanding of the general development of spinal motor neurons.     

Cell adhesion and migration in the organization of spinal motor neurons

Spinal motor neurons are critical to the ability of animals to move and thus essential to survival. Motor neurons that project axons to distinct limb-muscle targets are topographically organized such that central nervous system position reflects the location of the muscle in the limb. The central positioning of limb-projecting motor neurons arises during development through motor neuron migration followed by a period of coalescence into discrete groupings of motor neurons which project axons to an individual muscle. These so-called motor pools are a common feature of motor organization in higher vertebrates. Recent work has highlighted the critical role for armadillo family member catenin-dependent functions of the cadherin family of cell adhesion molecules in directing the organization of motor neurons. Cadherin function appears to be important for both the motor neuron migration and coalescence phases of the emergence of motor neuron topography. Here, I review this recent work in the context of our understanding of the general development of spinal motor neurons.     

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive. 1994 John Wiley & Sons, Inc.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Sprouting and connectivity of embryonic leech heart excitor (HE) motor neurons in the absence of their peripheral target

The rhythmic pumping of the hearts in the medicinal leech,Hirudo medicinalis, is neurogenic and mediated by a defined circuit involving identified interneurons in a central pattern generator (CPG) and segmentally iterated motor neurons that drive the heart muscle. During early embryogenesis, presumptive heart excitor (HE) motor neurons extend many axon branches into the body wall; they later innervate the heart while retracting the supernumerary peripheral axons, and only much later in development receive synaptic input from the central pattern generator (Jellies, Kopp and Bledsoe (1992)J. Exp. Biol., 170, 71–92.)- In this study, HE motor neurons were deprived of an early interaction with the heart by surgical ablation of a circumscribed portion of body wall including the heart primordium. Anatomical and electrophysiological data were obtained using intracellular techniques to examine the hypothesis that peripheral interactions with the developing heart provide instructive cues for the final differentiation of these neurons. Target-deprived HE motor neurons continued to extend multiple axons in ventral, lateral and dorsal body wall throughout late embryonic and into postembryonic stages and they extended anomalous axons within the CNS. This resembles the early embryonic growth of HE motor neurons before heart tube differentiation. Furthermore, HE motor neurons deprived of heart contact exhibited tonic activity similar to the situation during early development before they are contacted by the CPG interneurons. In contrast, sham-operated and contralateral HE motor neurons oscillated normally. These results suggest that heart tube contact is specifically required for at least some aspects of HE development and provide a framework in which to identify cell-cell interactions that are involved in matching neurons and targets to generate behaviorally relevant neural circuits.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Motor Axon Pathfinding

Motor neurons are functionally related, but represent a diverse collection of cells that show strict preferences for specific axon pathways during embryonic development. In this article, we describe the ligands and receptors that guide motor axons as they extend toward their peripheral muscle targets. Motor neurons share similar guidance molecules with many other neuronal types, thus one challenge in the field of axon guidance has been to understand how the vast complexity of brain connections can be established with a relatively small number of factors. In the context of motor guidance, we highlight some of the temporal and spatial mechanisms used to optimize the fidelity of pathfinding and increase the functional diversity of the signaling proteins.Motor neurons residing in the brain stem and spinal cord extend axons into the periphery and are the final relay cells for locomotor commands. These cells are among the longest projection neurons in the body and their axons follow stereotypical pathways during embryogenesis to synapse with muscle and sympathetic/parasympathetic targets. Cellular studies of motor axon navigation in developing chick and zebrafish embryos have shown that motor neurons located at different rostrocaudal positions show specific preferences for axonal pathways (see Landmesser 2001; Lewis and Eisen 2003 for reviews). This early cellular research laid the foundation for molecular studies of motor axon guidance by establishing the concept that motor neurons are in fact a diverse cell population. The molecular studies covered in this article have sought to identify genetic differences between motor neurons and to characterize the signaling pathways that underlie the specificity of motor axon targeting.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

Novel peripheral motor neurons in the posterior tentacles of the snail responsible for local tentacle movements

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15–35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5–7 µm diameter) and light neurons with 12–18 (T1 type) and 18–30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus–response pathway, consisting of olfactory stimulus—local motor neuron—motor response components, to generate local lateral movements of the tentacle tip (“quiver”). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus—distal segments of central motor axons—motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.