Peripheral mechanisms of itch

Detection of environmental stimuli that provoke an aversive response has been shown to involve many receptors in the periphery. Probably the least-studied of these stimuli are those that induce the perception of itch (pruritus), an often-experienced unpleasant stimulus. This review covers the ligands and their receptors which are known to cause primary sensory neuron activation and initiate itch sensation. Also covered are several itch-inducing substances which may act indirectly by activating other cell types in the periphery which then signal to primary neurons. Finally, progress in identifying candidate neurotransmitters that sensory neurons use to propagate the itch signal is discussed.     

Phosphorylation status of heat shock protein 27 influences neurite growth in adult dorsal root ganglion sensory neurons in vitro

The small heat shock protein Hsp27 influences neurite growth, potentially via phosphorylation-dependent interactions of Hsp27 with actin. To investigate the contribution of Hsp27 phosphorylation to neurite growth in adult DRG neurons, we employed hamster Hsp27 cDNA constructs (in pIRES-EGFP) with mutations in the phosphorylation sites, either mimicking constitutively phosphorylated Hsp27 (with substitution of serines 15 and/or 90 by glutamate) or preventing phosphorylation at the site (serines 15/90 replaced by alanine). Five mutant constructs were employed in this study in addition to wild-type hamster Hsp27; siRNA directed against the rat Hsp27 was used to depress endogenous Hsp27. Neurite growth was assessed in EGFP-expressing cells following immunocytochemistry and tracing of neurite growth. Hsp27 staining and phalloidin labelling were used to examine Hsp27 and actin colocalization in neurons and growth cones. Overall, our results demonstrate that the role that Hsp27 plays in neurite growth can be affected by phosphorylation, oligomerization, or a combination of both. Hsp27 constructs that are able to dimerize and/or form large oligomers [WT, Hsp27-AA, Hsp27-AE, Hsp27-Δ(5-23)] rescued siRNA-depressed neurite growth, whereas Hsp27 mutants that do not form dimers or oligomers (Hsp27-EE and Hsp27-EA) were unable to rescue the effect of the siRNA. The phalloidin labelling qualitatively showed a higher level of localization of actin with the Hsp27-AA compared with the other constructs. Although phosphorylation appears to be important in growth, the ability of Hsp27 to exist in both phospho- and nonphospho- states is likely key to its role in regulating cytoskeletal elements involved in neurite growth.     

Decrease in the number of lower affinity (type II) nerve growth factor receptors on embryonic sensory neurons does not affect fiber outgrowth

Nerve growth factor binds to two different specific receptors on responsive cells. The relationship of these two receptors is not fully understood at this time. We have studied the binding of labeled NGF to a different strain of white leghorn chicken embryo dorsal root ganglionic cells. The equilibrium dissociation constants for the two sites (K = 4.1 ± 1.8 × 10^?11M, K = 1.0 ± 0.8 × 10^?9M) are identical to those obtained previously. Also, the number of type I sites per cell (3.8 ± 1.3 × 10³) is the same as that previously determined. However, the number of type II sites per cell (1.9 ± 1.3 × 10⁴) is significantly different than that previously determined. This 2.5-fold decrease in the number of type II sites does not affect the concentration of NGF needed to obtain maximal fiber outgrowth from explanted sensory ganglia. The rate of association (1.2 ± 0.2 × 10⁷ M^?1 sec^?1 at 22°C) of labeled NGF with receptors on sensory neurons from this different strain of chickens is identical to that previously obtained. The rate of association of NGF with its receptors on sensory neurons was also determined at 4°C. This rate constant (2.1 ± 1.1 × 10⁶ M^?1 sec^?1) along with the rate constants obtained at 22° and 37°C were used to determine an activation energy for the binding of NGF to its receptors. The activation energy obtained (16.2 kcal/mole) suggests that binding is not a diffusion-controlled process.     

Recovery of C-Fiber-Induced Extravasation Following Peripheral Nerve Injury in the Rat

Peripheral nerve injury leads to substantial alterations in injured sensory neurons. These include cell death, phenotypic modifications, and regeneration. Primary sensory neurons have recently been shown not to die until a time beyond 4 months following a nerve crush or ligation and this loss is, moreover, limited to cells with unmyelinated axons, the C-fibers. The late loss of C-fibers may be due to a lack of target reinnervation during the regenerative phase. In order to investigate this, we have used a particular peripheral function, unique to C-fibers, as a measure of peripheral reinnervation: an increase in capillary permeability on antidromic activation of C-fibers, i.e., neurogenic extravasation. This was investigated in rats that had received a nerve crush injury 1 to 50 weeks earlier. Some recovery of the capacity of C-fibers to generate extravasation was detected at 8–10 weeks, which increased further at 12–14 weeks, and then plateaued at this level with no further recovery at 30 or 50 weeks. In intact and damaged sciatic nerves, Aβ-fibers never induced extravasation. These findings are compatible with the hypothesis that those C-fibers which make it back to their peripheral targets do not subsequently die and those that do not, may die.     

Influence of GM1 gangliosides on the growth of cultured rat embryonic serotonergic neurons

GM1 gangliosides were added to the medium of cultured raphe neurons enriched in the serotonergic phenotype in order to study their influence on biochemical and morphological growth parameters of serotonergic neurons. After 2 days of culture in the presence of GM1, specific uptake of serotonin measured by scintillation counting exhibited a moderate but significant increase for a GM1 concentration of 5 × 10⁻⁸M. Morphological parameters of 5-HT neurons were measured after immunocytochemical staining with specific serotonin antiserum and digitalization of immunoreactive cells. Eight parameters were studied; for concentrations of 5 × 10⁻⁸ and 10⁻⁷M of GM1, the absolute neuritic field area and the total length of the segments were significantly increased, whereas the number of neuritic segments and their mean length were not modified. We conclude that GM1 ganglioside has a significant influence on the growth of serotonergic neurons. Moreover, electron microscopy showed, on treated cultures, a dramatic increase of the number of spicules all along the neuron's process, suggesting that GM1 could act by modifying the attachment of cells to their substrate. The possible molecular mechanisms of the action of GM1 are discussed.     

Sequestration requirements for the degradation of 125I-labeled β nerve growth factor bound to embryonic sensory neurons

Nerve growth factor interacts with responsive cells by binding to cell surface membrane receptors. There are two different receptors on both embryonic sensory and sympathetic neurons, a high-affinity (type I) receptor and a lower-affinity (type II) receptor. Sequestration, which we have defined as bound nerve growth factor that becomes inaccessible to the external milieu with time, occurs through the type I receptor on both sensory and sympathetic neurons. We describe here a process subsequent to sequestration involving internalization and degradation of bound nerve growth factor and showing that bound nerve growth factor is not degraded under the following conditions: (1) low temperature, ie 4°C; (2) when a large excess of unlabeled nerve growth factor is added concomitantly with the labeled nerve growth factor and the temperature is raised from 4°C to 37°C; (3) when metabolic inhibitors sodium fluoride and dinitrophenol are added concomitantly with the labeled nerve growth factor and the temperature is raised from 4° to 37°C. On the other hand, conditions that allow bound nerve growth factor to be degraded are the following: (1) incubation of the sensory nerve cells at low temperature (ie, 4°C) only in the presence of labeled nerve growth factor, then raising the temperature to 37°C; (2) when sodium fluoride and dinitrophenol are added when the temperature is raised to 37°C; (3) when excess unlabeled nerve growth factor is added when the temperature is raised to 37°C. These studies are consistent with the idea that nerve growth factor has to bind to the cells in order to be degraded; however, binding is not sufficient for degradation to occur. Second, the bound nerve growth factor must be sequestered in order to be degraded. Third, the process of internalization of the bound nerve growth factor, unlike sequestration, is not an energy-dependent process. Thus, it seems reasonable to suggest the following steps for the interaction of nerve growth factor with responsive cells: binding to a cell surface membrane receptor, followed by sequestration of the bound nerve growth factor, and finally, internalization of the sequestered nerve growth factor.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Oral and facial representation within the medullary and upper cervical dorsal horns in the cat

Transganglionic transport of HRP was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the trigeminal nucleus caudalis and upper cervical dorsal horn of the cat. In separate animals, the superior alveolar, pterygopalatine, buccal, inferior alveolar, lingual, frontal, corneal, zygomatic, infraorbital, mental, mylohyoid, and auriculotemporal branches of the trigeminal nerve were traced in this experiment. The organization of the primary afferents innervating the oral structures is not uniform across laminae and at different rostrocaudal levels of the nucleus caudalis. The superior alveolar and pterygopalatine nerves mainly terminate in laminae I, II, and V at the level of the rostral one-third of the caudalis. By contrast, the lingual, inferior alveolar, and buccal nerve terminate in laminae I-V of, respectively, the rostral third, the entire length, and caudal two-thirds of the caudalis. In addition, the lingual, buccal, and pterygopalatine nerves terminate in the dorsal and middle parts of the interstitial islands or pockets of lamina I neuropil extending to the rostral levels parallel to the nucleus interpolaris. Mediolaterally, in laminae I, II, and V of the rostral third an extensive overlap of projections was found between the branches from each trigeminal division, and some overlap was observed between projections from the mandibular and maxillary divisions. On the other hand, the projections of primary afferents innervating the facial structures are arranged in a somatotopic fashion in rostrocaudal and mediolateral axes over the laminae (I-IV) through the nucleus caudalis and upper cervical dorsal horn. Fibers from the perioral and perinasal regions terminate most rostrally in caudalis, and fibers from progressively more posterior facial regions terminate at successively lower levels. A mediolateral somatotopic arrangement was observed, with fibers from the ventral parts of face ending in the medial regions and fibers from the progressively more dorsal parts of the face ending in successively more lateral regions of the medullary and upper cervical dorsal horns. Corneal afferent terminals are concentrated in the outer parts of lamina II at the levels of the rostral parts of the caudal two-thirds of the caudalis and the interstitial islands of lamina I. The maxillary division terminates first at the most caudal level of the caudalis, followed by the ophthalmic division descending as far as the C2 segment and the mandibular division reaching the most caudal level of the C2 segment.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development

N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ～40% of the somitic hemisegments and an ～150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection.     

Adult Rat Dorsal Root Ganglion Neurons Extend Neurites on Predegenerated But Not on Normal Peripheral Nerves In Vitro

The abilities of embryonic and adult rat sensory neurons to regenerate were compared when cultured on cryostat sections of normal and lesioned sciatic nerve tissues. Differences in neurite growth, visualized by GAP-43 immunolabelling, were most pronounced on substrata consisting of longitudinal sections of normal versus predegenerated sciatic nerve. Adult dorsal root ganglion (DRG) neurons grew only on the lesioned nerves. Neurites extended along these sections in a characteristically longitudinal orientation, and this growth was not dependent on nerve growth factor. Embryonic DRG neurons extended neurites on sections from both types of nerves. These results highlight important differences in the regenerative abilities of embryonic and adult DRG neurons when grown on physiologically appropriate substrata.     

Galectin-3 promotes neural cell adhesion and neurite growth

Galectin-3 is a member of the galectin family and belongs to a group of soluble β-galactoside-binding animal lectins. The molecule is expressed by neural and nonneural cells intra- (cytoplasm and nucleus) as well as extra-cellularly (plasma membrane and extracellular space). By using an in vitro cell-substratum adhesion assay, we have addressed the question whether galectin-3 present in the extracellular milieu may support the adhesion and/or neurite outgrowth of neural cells in a manner analogous to cell adhesion molecules. Galectin-3 was immobilized as a substratum and various cell types, N2A (neuroblastoma), PC12 (pheochromocytoma), and TSC (transformed Schwann cells) cell lines, neural cells from early postnatal mouse cerebellum, and dorsal root ganglion neurons from newborn mice were allowed to adhere to the lectin. Here we show that all cell types studied specifically adhered to galectin-3 by the following criteria: 1) the number of adherent cells was dependent on the galectin-3 concentration used for coating; 2) adhesion of cells to galectin-3, but not to collagen type I or laminin was inhibited by polyclonal antibodies to galectin-3; 3) upon addition of asialofetuin (a polyvalent carrier of terminal β-galactosides) to the cell suspension prior to the adhesion assay, cell adhesion to galectin-3 was inhibited in a dose-dependent manner; and 4) cell adhesion to galectin-3 was abolished by treatment of cells with endo-β-galactosidase. In addition, the adhesion of dorsal root ganglion neurons to galectin-3 could be inhibited by lactose. Notably, substratum-bound galectin-3 promoted the outgrowth of neurites from dorsal root ganglia explants and this neurite outgrowth promoting activity could be inhibited by polyclonal antibodies to galectin-3. J. Neurosci. Res. 54:639-654, 1998. © 1998 Wiley-Liss, Inc.     

Peripheral fields and branching patterns of buccal mechanosensory neurons in the opisthobranch mollusc,navanax inermis

A population of about 75 primary sensory cells were identified on the dorsal surface of each buccal ganglion of Navanax. Each sensory cell possesses at least one mechanosensitive field in the pharynengeal wall or lips that correlates somototopically with its position in the buccal ganglia. Many cells had additional fields that could be widely separated, requiring that their afferent processes branch. Cells were found with processes in more than one buccal nerve and with multiple processes in the same nerve. Hyperpolarization of the soma or repetitive electrical or physiological stimulation could cause failure of centripetal propagation of impulses. Impulses initiated in different branches could fail at different distances from the soma. Axon spikes that fail to invade the soma may or may not invade other branches. Axon spikes in separate branches that fail to invade the soma can summate to initiate an invading impulse. These findings suggest that integration of information from different branches may occur in a single sensory neuron.     

Activation of MrgprA3 and MrgprC11 on Bladder-Innervating Afferents Induces Peripheral and Central Hypersensitivity to Bladder Distension

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ^−/− mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ^−/− mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.     

Early pattern of neuronal differentiation in the Xenopus embryonic brainstem and spinal cord

Wholemount antibody labeling techniques and horseradish peroxidase backfilling were used to analyze the pattern of neuronal differentiation in the embryonic Xenopus central nervous system between stages 22 and 35/36. In the spinal cord, the first neurons to differentiate are the Rohon-Beard neurons; they are followed by ventral neurons with descending axons (descending interneurons, motoneurons) and lateral interneurons with commissural axons. The somata and axons of these primary neurons form dorsal, ventral, and lateral columns, respectively; the ventral and lateral columns uninterruptedly continue forward into the brainstem. The distribution and projection patterns of spinal neurons were analyzed quantitatively. Rohon-Beard neurons, commissural interneurons, and primary motoneurons vary in number from segment to segment. Thus, these neurons are not distributed in a segmental pattern. In each segment, neurons of a given type project axons whose length varies over a wide range. The numerical distribution of length of axons formed by a population of neurons of a given type was calculated and expressed as the projection profile of these neurons. For each type of neuron and spinal segment, the projection profile is different. Furthermore, the projection profiles change in a systematic way along the spinal cord. For example, the fraction of Rohon-Beard neurons with long ascending axons steadily increases if one moves towards caudal spinal levels. The findings suggest that suprasegmental cues with a graded distribution along the spinal cord determine the number and projection profile of a particular cell type in a given segment. © 1993 Wiley-Liss, Inc.     

Coculture of rat embryonic proprioceptive sensory neurons and myotubes

With the aim to study the cellular mechanisms underlying the process of muscle spindle (re)generation, dorsal root ganglia (DRG) neurons derived from 16-day rat embryos were cocultured with developing myotubes in a compartmentalized culture device. To accomplish the selective survival and neurite formation of the proprioceptive subpopulation, the neurotrophic factor, neurotrophin-3, was added to the culture medium. It appeared that the proprioceptive DRG neurons could develop specialized, Ia afferent terminal-like contacts with myotubes. However, these interactions were scarce and did not result in the induction of differentiation of the contacted myotubes into intrafusal fibers as normally occurs during in vivo development. The present coculture setup apparently lacks appropriate regulatory factors essential for the proper matching of sensory axons and intrafusal fiber precursors and the induction of a functional sensory myoneural connection. © 1996 John Wiley & Sons, Inc.     

Differential NR2A and NR2B expression between trigeminal neurons during early postnatal development

N-methyl-D-aspartate (NMDA) receptors play an important role in the production of rhythmical trigeminal motor activity resembling suckling and chewing. The developmental relationship between the expression of NMDA receptor subunits and the function of neurons comprising brainstem oral-motor circuitry is not clear. We conducted receptor immunohistochemistry studies to demonstrate the expression of NR2A and NR2B subunits in trigeminal motoneurons (Mo5) and mesencephalic trigeminal neurons (Me5) during the first 2 weeks of development. During this time period, rats begin the transition from suckling to chewing, two distinct motor behaviors. In Mo5, NR2A and NR2B immunoreactivity was observed throughout the time frame sampled. A significant increase in the NR2A:NR2B ratio occurred between P3-4 and P11 due to a reduction in the number of NR2B immunoreactive neurons. The temporal and spatial expression of NR2A and NR2B was differentially regulated between caudal and rostral regions of Me5. In contrast to Mo5, the NR2A:NR2B ratio decreased between P0-1 and P11 in caudal Me5 due to a concurrent increase in the number of NR2A and NR2B immunoreactive neurons. In rostral Me5, NR2A and NR2B immunoreactivity emerged at P3 and P11, respectively. Our data provides further insight into the molecular changes of trigeminal neurons during the transition from suckling to chewing behaviors. The differences in the NR2A:NR2B ratio between Mo5 and Me5 suggest functional differences in these neurons during NMDA-mediated neurotransmission.     

Effects of acetyl-l-carnitine on the survival of adult rat sensory neurons in primary cultures

Acetyl-L-carnitine produces a significant increase in the survival time-course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 microM acetyl-L-carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost. Patch-clamp recordings from these neurons show that superfusion with acetyl-L-carnitine (100-1000 microM) does not induce any membrane current. In addition an agonist muscarinic effect particularly concerning high-voltage activated calcium channel modulation appears to be ruled out. In conclusion our data favour the role of acetyl-L-carnitine in the trophism of sensory neurons in adult rats. In agreement with other in vivo experiments our data reinforce the hypothesis that this substance might be involved in reducing neuronal loss observed in nervous system aging.     

Chick wing innervation. II. Morphology of motor and sensory axons and their growth cones during early development

The development and distribution of neuronal projections to the developing chick wing was studied using anterograde transport of horseradish peroxidase (HRP). Small injections of HRP were made into motor or sensory neuronal populations in order to visualize individual axons and their associated growth cones. Motor growth cones were observed in different regions of the embryo at different stages, in a proximal-to-distal pattern of distribution which paralleled the process of axon outgrowth and nerve formation. Different growth cone morphologies were associated with differing regions of the developing projection. In the spinal nerves, axons destined for the limb were unbranched and terminated in simply shaped growth cones. As axons approached the developing limb and entered the plexus region, their growth cones became more complex and larger primarily because of widening, and they sometimes branched, producing processes which could extend tens of microns from a tricorne branch point on the parent axon. Both motor and sensory fibers showed similar morphological changes in the plexus region. A distinctively shaped growth cone expanded on its leading edge was observed, sequentially apparent in the distal spinal nerves, in the plexus region, in the loosely organized axonal sheets projecting to the uncleaved dorsal or ventral muscle masses, and where muscle nerves diverged from nerve trunks and within muscle nerves. It is likely that some of these are transitional growth cones preparing to branch, because complex and branched growth cones were also observed in these regions. Branched axons oriented along the anteroposterior axis were similarly observed in the plexus region and distal to the plexus when axons first projected to the limb bud. At somewhat older stages when the basic peripheral nerve branching pattern had formed, motor growth cones were observed in common nerve trunks and in individual muscle nerves, but they were no longer found in the plexus region. Branched axons were likewise restricted to these peripheral Imations. Taken together, these observations suggest that one of the ways in which axons navigate is by exploration in the form of growth cone widening, and in some cases terminal bifurcation which may produce axon branches. Selection of the most appropriately directed growth cone process and/or precocious axonal branches may be one of the ways in which axons respond to specific growth cues which guide axons into the limb bud. Alternatively, this precocious branching may be an early neurotrophic response to developing muscle and play no significant role in axon navigation. © 1995 Wiley-Liss, Inc.     

Time and dose dependencies of effects of nerve growth factor on sympathetic and sensory neurons in neonatal rats

Nerve growth factor (NGF) plays a role in the development of several components of the sympathetic and sensory nervous systems. The objectives of this study were to examine the time and dose dependencies of some of the well known effects of NGF on sympathetic ganglia and to examine qualitatively and quantitatively the recently described effects on sensory ganglia of neonatal rats. Single doses of NGF as low as 0.1 mg/kg produce increases in tyrosine hydroxylase (TOH) activity in superior cervical ganglia (SCG), and doses of 3 mg/kg produce maximal effects. Larger doses and longer treatments are required to see increases in protein content of the SCG. Larger doses are also required to affect TOH activity in the adrenal gland. Increases in TOH activity in SCG can be observed within 18 h of injection. Chronic NGF treatment for three weeks produces no change in blood pressure or heart rate in neonatal rats. Chronic administration of NGF (1 or 3 mg/kg/day) results in dose-related increases in the protein content of dorsal root ganglia (DRG). The increase in protein content of the DRG was associated with an increase in the diameter of smaller neurons (those<30 μm in diameter), but NGF caused no change in the number of neurons.     

Exuberant growth and synapse formation of olfactory sensory neuron axonal arborizations

Neural connections in the adult nervous system are established with a high degree of precision. Several examples throughout the nervous system indicate that this precision is achieved by first establishing an initial exuberant immature pattern of connectivity that is then sculpted into the adult pattern via pruning. This often emerges as an activity-dependent process. In the olfactory system, sensory axons from neurons expressing the same odorant receptor project with high precision to specific glomerular structures in the olfactory bulb. This process undergoes maturation-dependent refinements that are not fully understood. Due to technical impediments that have made it difficult to focus on single axons, it is unknown whether olfactory sensory projections are established in an exuberant fashion. Here we developed a novel technique of electroporation that allowed us to simultaneously label single olfactory sensory neuron (OSN) axonal arbors and their presynaptic specializations. Using this method we were able to incorporate plasmids into OSNs at an immature stage, thereby allowing a time-course study of axonal arbor development and synapse formation in single olfactory sensory axons. We observed that the number of branch points, the total branch length, and the number and density of presynaptic specializations peaked at postnatal day 8 and decreased afterwards. Our data demonstrate that olfactory sensory axons develop in an exuberant way, both in terms of branch growth and synaptic composition. We hypothesize that exuberant branches and synapses are eliminated to achieve the mature pattern in a process likely to be regulated by neural activity.