Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

The development of an identified subpopulation of avian sensory neurons is regulated by interaction with the periphery

The monoclonal antibody, SN1, binds to the cell surface of a subpopulation of avian sensory neurons. Dorsal root ganglia (DRG) that innervate different peripheral fields contain significantly different proportions of SN1(+) neurons. Moreover, the developmental time of appearance of these neurons suggests that they normally express SN1 immuno-reactivity only after they have made contact with their normal peripheral targets (Marusich et al., 1986). In the present paper, we test the hypothesis that the proportion of SN1(+) neurons within DRG is regulated by interactions between the developing neurons and their peripheral targets. Thus, immature DRG explanted into a defined medium show an age-dependent increase in the proportion of cells able to become SN1(+), but fail to show the large increase in number of immunoreactive neurons that occurs in vivo. Moreover, unilateral wing bud extirpations performed at E3 (prior to wing innervation) resulted in a dramatic selective decrease in the number of SN1(+) neurons within DRG that normally innervate the wing. These results support the hypothesis that peripheral targets regulate the appearance of SN1(+) neurons.     

Reaction of sensory neurons of the spinal ganglia to sectioning of their peripheral and central processes

Transversal ++semi-sectioning of the spinal ganglion (SG) is a good model for studying the reaction of the ganglional sensory neurons to sectioning of their peripheral and central processes. At sectioning the peripheral and central processes of the SG neurons degeneration of the neurons and their death take place. The degenerative processes are more pronounced in the neurons with the peripheral processes sectioned, and the restorative ones-with the central processes sectioned. The dynamics of the posttraumatic changes in absolute number of the neurons, profile areas of the body fields and neuronal nuclei, amount of neurons with certain signs of axonal reactions in the SG demonstrate a maximally pronounced reaction on the 7th day and beginning of restorative processes on the 15th day. They are not completed by the 180th day.     

Herpes simplex virus gene expression in neurons: viral DNA synthesis is a critical regulatory event in the branch point between the lytic and latent pathways.

Herpes simplex virus establishes a latent infection in peripheral neurons. We examined viral gene expression in rat peripheral neurons in vitro and determined that viral gene expression is attenuated and delayed in these neurons compared with that in Vero cells. In addition, using pharmacologic and genetic blocks to viral DNA synthesis, we found that viral alpha and beta gene expression was upregulated by viral DNA synthesis. Although maximal gene expression in neurons requires viral DNA synthetic activity, activation of viral gene expression was seen even in the presence of herpes simplex virus DNA polymerase inhibitors, but not in the absence of the origin-binding protein. Initiation of viral DNA synthesis is apparently a key regulatory event in the balance between the lytic and latent pathways in peripheral neurons.     

Musings on the wanderer: what's new in our understanding of vago-vagal reflexes?: II. Integration of afferent signaling from the viscera by the nodose ganglia

To understand vago-vagal reflexes, one must have an appreciation of the events surrounding the encoding, integration, and central transfer of peripheral sensations by vagal afferent neurons. A large body of work has shown that vagal afferent neurons have nonuniform properties and that distinct subpopulations of neurons exist within the nodose ganglia. These sensory neurons display a considerable degree of plasticity; electrophysiological, pharmacological, and neurochemical properties have all been shown to alter after peripheral tissue injury. The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neuronal neighbors. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Neuronal organization of the left celiac ganglion in the cat

Unit responses of the isolated left celiac ganglion to stimulation of various nerves of the solar plexus were studied by intracellular microelectrode recording in cats before and after degeneration of the preganglionic fibers. The resting potential of the ganglionic neurons was ?62.2±2.9 mV and the amplitude of the spike potential 72.4±3.2 mV. The spike was followed by after-hyperpolarization with a mean amplitude of 24% of the spike amplitude and a duration of between 25 and 180 msec. A characteristic feature of the ganglion was the presence of orthodromic unit responses to stimulation of peripheral nerve fibers of the solar plexus. The higher threshold of activation of the neurons by peripheral fibers than by preganglionic fibers and the preservation of orthodromic unit responses to stimulation of peripheral nerves after degeneration of the preganglionic fibers are evidence that the peripheral reflex arc is closed in this ganglion. Neurons of the left celiac ganglion are divided into three groups. Only preganglionic fibers of the splanchnic nerve with different properties converge on the neurons of the first group (the most numerous); only afferent fibers of peripheral nerves converge on the neurons of the third group (the least numerous); both types of fibers terminate on neurons of the second group. This convergence may lie at the basis of the mechanism of the centrifugal and peripheral reflex interaction in the ganglion for coordinated visceral activity.     

Peripheral nerve extract promotes long-term survival and neurite outgrowth in cultured spinal cord neurons

The hypothesis that peripheral, skeletal muscle tissue contains a trophic factor supporting central neurons has recently been investigated in vitro by supplementing the culture medium of spinal cord neurons with muscle extracts and fractions of extract. We extended these studies asking whether or not a trophic factor is present in peripheral nerves, the connecting link between muscle and central neurons via which factors may be translocated from muscle to neurons by the retrograde transport system. Lumbar, 8-day-old chick spinal cords were dissociated into single cells and then cultured in the presence of peripheral nerve extract. Cytosine arabinoside was added to inhibit proliferation of nonneuronal cells. In the presence of nerve extract, spinal cord neurons survived for more than a month, extended numerous neurites, and showed activity of choline acetyltransferase. In the absence of extract, neurons attached and survived for a few days but then died subsequently in less than 10 days. Neurite outgrowth did not occur in the absence of extract. Withdrawal of extract from the medium of established neuronal cultures caused progressive loss of both cells and neurites. Other tissues also contained neuron supporting activity but less than that found in nerve extract. These studies indicate that peripheral nerves contain relatively high levels of spinal cord neuron-directed trophic activity, suggesting translocation of neurotrophic factor from muscle to central target neurons. The neurotrophic factor has long-term (weeks) effects, whereas short-term (days) survival is factor independent.     

Peripheral ghrelin interacts with orexin neurons in glucostatic signalling

Ghrelin interactions with glycemia in appetite control as well as the potential mechanisms involving the orexin and melanin-concentrating hormone (MCH) neurons in the orexigenic ghrelin signals were investigated by using a specific anti-ghrelin antibody (AGA). Our results confirm that peripheral ghrelin is an important signal in meal initiation and appetite. Employing immunohistochemistry techniques, we found that c-fos positive neurons in the lateral hypothalamus (LH) and perifornical area (PFA) increased after insulin or 2-deoxyglucose administration. Moreover, we have also demonstrated that peripheral ghrelin blockade by the AGA, reduces the orexigenic signal induced by insulin and 2-DG administration probably partly producing a decrease of c-fos immunoreactivity in the LH and PFA as well as a lower activation of orexin neurons. In contrast, the c-fos positive MCH neurons were not apparently affected. In summary, our findings suggest that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms by means of its role as a "hunger" signal affecting the LH and PFA areas, which may contribute to energy homeostasis through orexin neurons.     

PYY3-36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y2 receptors

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.     

Axon outgrowth along segmental nerves in the leech. I. Identification of candidate guidance cells

We studied the development of the major extraganglionic components of the germinal plate in embryos of the glossiphoniid leech Helobdella triserialis to improve our understanding of the mechanism of segmental nerve formation. We examined the outgrowth of groups of axons from ganglionic neurons into the segmental nerves, the migration of peripheral neurons and epidermal specializations to their definitive sites, and the development of circular and longitudinal muscle fibers. We visualized axons, as well as neurons and epidermal specializations, by means of fluorescent cell lineage tracers injected earlier into blastomeres and muscle fibers by means of immunofluorescence. The development of cells in all groups was found to follow a stereotyped pattern. Axons of ganglionic neurons approach some identified peripheral neurons located along the segmental nerve paths but not, in general, epidermal specializations and muscle fibers. Near the somata of a subset of peripheral neurons they approach, axons cease or interrupt their growth. These findings identify a set of candidate guidance cells for axonal outgrowth in the leech, similar to those previously described in the developing nervous system of insects.     

CaMKK-CaMK1a,a New Post-Traumatic Signalling Pathway Induced in Mouse Somatosensory Neurons

Neurons innervating peripheral tissues display complex responses to peripheral nerve injury. These include the activation and suppression of a variety of signalling pathways that together influence regenerative growth and result in more or less successful functional recovery. However, these responses can be offset by pathological consequences including neuropathic pain. Calcium signalling plays a major role in the different steps occurring after nerve damage. As part of our studies to unravel the roles of injury-induced molecular changes in dorsal root ganglia (DRG) neurons during their regeneration, we show that the calcium calmodulin kinase CaMK1a is markedly induced in mouse DRG neurons in several models of mechanical peripheral nerve injury, but not by inflammation. Intrathecal injection of NRTN or GDNF significantly prevents the post-traumatic induction of CaMK1a suggesting that interruption of target derived factors might be a starter signal in this de novo induction. Inhibition of CaMK signalling in injured DRG neurons by pharmacological means or treatment with CaMK1a siRNA resulted in decreased velocity of neurite growth in vitro. Altogether, the results suggest that CaMK1a induction is part of the intrinsic regenerative response of DRG neurons to peripheral nerve injury, and is thus a potential target for therapeutic intervention to improve peripheral nerve regeneration.     

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.     

Metamorphosis of Wing Motor System in the Silk Moth, Bombyx mori: Origin of Wing Motor Neurons

The origin of the peripheral nerve and motor neurons that innervate the adult mesothoracic dorsal longitudinal muscles (DLMs) was examined in the silk moth, Bombyx mori . The anatomical features of the peripheral nerve and motor neurons were investigated by dissection, electron microscopy, and cobalt back-fill staining at different pupal stages. These studies showed that the peripheral nerve (IIN1c) that innervates the adult DLMs originates from a branch (db branch) of the larval mesothoracic dorsal nerve that innervates the larval DLMs. During metamorphosis the larval nerve shortens or lengthens locally without change in its basic branching pattern, and the db branch moves towards the mesothoracic ganglion to become the IIN1c. All the adult DLM motor neurons are from larval ones. Nine of the 14 larval DLM motor neurons survive during metamorphosis to become adult DLM motor neurons, and 5 disappear in early pupal stages.     

Crustacean cardioactive peptide-immunoreactive neurons in the ventral nerve cord and the brain of the meal beetle Tenebrio molitor during postembryonic development

Summary By use of an antiserum against the crustacean cardioactive peptide (CCAP) several types of bilaterally symmetrical neurons have been mapped quantitatively in the ventral nerve cord and in the brain of the meal beetle, Tenebrio molitor. The general architecture of these neurons was reconstructed from peroxidase-antiperoxidase-labelled whole-mount preparations. From the subesophageal to the seventh abdominal ganglia two types of neurons show a repetitive organization of contralateral projection patterns in each neuromere. The first type has few branches in the central neuropil and a distinct peripheral projection. The second type is characterized by an elaborate central branching pattern, which includes ascending and descending processes. Some of its peripheral branches were found to supply peripheral neurohemal areas. In the protocerebrum, 10 CCAP-immunoreactive neurons occur with projections into the superior median protocerebrum and the tritocerebrum. Immunopositive neurons were mapped in larval and various pupal stages, as well as in the adult. All types of identified neurons were found to persist throughout metamorphosis maintaining their essential structural and topological characteristics. The CCAP-immunoreactive neurons of T. molitor are compared with those described for the locust. Putative structural homologies of subsets of neurons in both species are discussed.     

Distribution of serotonin-, Gamma-Aminobutyric Acid- and substance P-like immunoreactivity in the central and peripheral nervous system of Mytilus galloprovincialis

The distribution of serotonin-, GABA- and substance P-like immunoreactivity has been studied in the cerebral and visceral ganglia and in some peripheral tissues of Mytilus galloprovincialis (Moleusca, Bivalvia). Cerebral ganglia contain a developed serotonin-immunoreactive neuronal subpopulation and numerous GABA-immunoreactive neurons, whereas neurons positive for substance P are sparse. In peripheral tissues innervated by the cerebral ganglia (labial palps and oesophagus) only serotonin-immunoreactive nerve fibers were found. In the visceral ganglia, serotonin- and GABA-immunoreactive neurons are far less numerous than in the cerebral ganglia, whereas several neurons positive for substance P are scattered in all cortical zones. Serotonin-immunoreactive plexuses innervate the posterior adductor muscle and the gill filaments which contain also a developed nerve network positive for substance P. The distribution pattern of the immunoreactive elements in the ganglia and in peripheral territories indicates that GABA should exert only a central action, whereas serotonin and a substance P-like peptide are involved both in central and peripheral neurotransmission.     

Liposomes to Target Peripheral Neurons and Schwann Cells

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.     

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

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

Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth

During embryogenesis and the postnatal period, neurons and glia interact in the development and differentiation of specific populations of nerve cells. Both in the peripheral (PNS) and in the central nervous system (CNS), glial cells have been shown in various experimental conditions to constitute a favorable substrate for neural adhesion, neural polarity, shape and axonal extension, while numerous soluble molecules secreted by neurons influence the survival and differentiation of the glial cells themselves. The aim of the present work was to investigate the influence of postnatal Schwann cells (SC) on embryonic serotoninergic (5-HT) neurons of the raphe, in order to study the possible influence of the peripheral glia on the CNS neurons. Cultures of SC from sciatic nerve of postnatal rats and neurons from rat embryonic rhombencephalon were successfully established and cells were immunocytochemically characterized. The number of 5-HT neurons, and the number and length of their branches were quantified in the cultures of 5-HT neurons, in cultures added with Nerve Growth Factor (NGF) and Insulin-like Growth Factor I (IGF-I), in co-cultures with SC and in cultures added with conditioned medium obtained from SC cultures. The results indicated that SC have the capacity to promote the survival and growth of 5-HT neurons in culture, and that this activity is mediated by soluble factors. Although the precise nature and mechanism of action of the growth factor or factors produced by SC in the presence of 5-HT neurons was not identified, our results add more data on the possible activity of the peripheral glia in promoting and enhancing the survival and outgrowth of the CNS neurons.