The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.
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According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles. (+info)
Central peptidergic neurons are hyperactive during collateral sprouting and inhibition of activity suppresses sprouting.
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Little is known regarding the effect of chronic changes in neuronal activity on the extent of collateral sprouting by identified CNS neurons. We have investigated the relationship between activity and sprouting in oxytocin (OT) and vasopressin (VP) neurons of the hypothalamic magnocellular neurosecretory system (MNS). Uninjured MNS neurons undergo a robust collateral-sprouting response that restores the axon population of the neural lobe (NL) after a lesion of the contralateral MNS (). Simultaneously, lesioned rats develop chronic urinary hyperosmolality indicative of heightened neurosecretory activity. We therefore tested the hypothesis that sprouting MNS neurons are hyperactive by measuring changes in cell and nuclear diameters, OT and VP mRNA pools, and axonal cytochrome oxidase activity (COX). Each of these measures was significantly elevated during the period of most rapid axonal growth between 1 and 4 weeks after the lesion, confirming that both OT and VP neurons are hyperactive while undergoing collateral sprouting. In a second study the hypothesis that chronic inhibition of neuronal activity would interfere with the sprouting response was tested. Chronic hyponatremia (CH) was induced 3 d before the hypothalamic lesion and sustained for 4 weeks to suppress neurosecretory activity. CH abolished the lesion-induced increases in OT and VP mRNA pools and virtually eliminated measurable COX activity in MNS terminals. Counts of the total number of axon profiles in the NL revealed that CH also prevented axonal sprouting from occurring. These results are consistent with the hypothesis that increased neuronal activity is required for denervation-induced collateral sprouting to occur in the MNS. (+info)
Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy.
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Sensitivity to the pungent vanilloid, capsaicin, defines a subpopulation of primary sensory neurons that are mainly polymodal nociceptors. The recently cloned vanilloid receptor subtype 1 (VR1) is activated by capsaicin and noxious heat. Using combined in situ hybridization and histochemical methods, we have characterized in sensory ganglia the expression of VR1 mRNA. We show that this receptor is almost exclusively expressed by neurofilament-negative small- and medium-sized dorsal root ganglion cells. Within this population, VR1 mRNA is detected at widely varying levels in both the NGF receptor (trkA)-positive, peptide-producing cells that elicit neurogenic inflammation and the functionally less characterized glial cell line-derived neurotrophic factor-responsive cells that bind lectin Griffonia simplicifolia isolectin B4 (IB4). Cells without detectable levels of VR1 mRNA are found in both classes. A subpopulation of the IB4-binding cells that produce somatostatin has relatively low levels of VR1 mRNA. A previously uncharacterized population of very small cells that express the receptor tyrosine kinase (RET) and that do not label for trkA or IB4-binding has the highest relative levels of VR1 mRNA. The majority of small visceral sensory neurons of the nodose ganglion also express VR1 mRNA, in conjunction with the BDNF receptor trkB but not trkA. Axotomy results in the downregulation of VR1 mRNA in dorsal root ganglion cells. Our data emphasize the heterogeneity of VR1 mRNA expression by subclasses of small sensory neurons, and this may result in their differential sensitivity to chemical and noxious heat stimuli. Our results also indicate that peripherally derived trophic factors may regulate levels of VR1 mRNA. (+info)
Cannabinoid suppression of noxious heat-evoked activity in wide dynamic range neurons in the lumbar dorsal horn of the rat.
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The effects of cannabinoid agonists on noxious heat-evoked firing of 62 spinal wide dynamic range (WDR) neurons were examined in urethan-anesthetized rats (1 cell/animal). Noxious thermal stimulation was applied with a Peltier device to the receptive fields in the ipsilateral hindpaw of isolated WDR neurons. To assess the site of action, cannabinoids were administered systemically in intact and spinally transected rats and intraventricularly. Both the aminoalkylindole cannabinoid WIN55,212-2 (125 microg/kg iv) and the bicyclic cannabinoid CP55,940 (125 microg/kg iv) suppressed noxious heat-evoked activity. Responses evoked by mild pressure in nonnociceptive neurons were not altered by CP55,940 (125 microg/kg iv), consistent with previous observations with another cannabinoid agonist, WIN55,212-2. The cannabinoid induced-suppression of noxious heat-evoked activity was blocked by pretreatment with SR141716A (1 mg/kg iv), a competitive antagonist for central cannabinoid CB1 receptors. By contrast, intravenous administration of either vehicle or the receptor-inactive enantiomer WIN55,212-3 (125 microg/kg) failed to alter noxious heat-evoked activity. The suppression of noxious heat-evoked activity induced by WIN55,212-2 in the lumbar dorsal horn of intact animals was markedly attenuated in spinal rats. Moreover, intraventricular administration of WIN55,212-2 suppressed noxious heat-evoked activity in spinal WDR neurons. By contrast, both vehicle and enantiomer were inactive. These findings suggest that cannabinoids selectively modulate the activity of nociceptive neurons in the spinal dorsal horn by actions at CB1 receptors. This modulation represents a suppression of pain neurotransmission because the inhibitory effects are selective for pain-sensitive neurons and are observed with different modalities of noxious stimulation. The data also provide converging lines of evidence for a role for descending antinociceptive mechanisms in cannabinoid modulation of spinal nociceptive processing. (+info)
CNTF, not other trophic factors, promotes axonal regeneration of axotomized retinal ganglion cells in adult hamsters.
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PURPOSE: To investigate the in vivo effects of trophic factors on the axonal regeneration of axotomized retinal ganglion cells in adult hamsters. METHODS: The left optic nerve was transected intracranially or intraorbitally, and a peripheral nerve graft was apposed or sutured to the axotomized optic nerve to enhance regeneration. Trophic factors were applied intravitreally every 5 days. Animals were allowed to survive for 3 or 4 weeks. Regenerating retinal ganglion cells (RGCs) were labeled by applying the dye Fluoro-Gold to the distal end of the peripheral nerve graft 3 days before the animals were killed. RESULTS: Intravitreal application of ciliary neurotrophic factor substantially enhanced the regeneration of damaged axons into a sciatic nerve graft in both experimental conditions (intracranial and intraorbital optic nerve transections) but did not increase the survival of distally axotomized RGCs. Basic fibroblast growth factor and neurotrophins such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 failed to enhance axonal regeneration of distally axotomized RGCs. CONCLUSIONS: Neurons of the adult central nervous system can regenerate in response to trophic supply after injury, and ciliary neurotrophic factor is at least one of the trophic factors that can promote axonal regeneration of axotomized RGCs. (+info)
Neurotrophin modulation of the monosynaptic reflex after peripheral nerve transection.
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The effects of neurotrophin-3 (NT-3) and NT-4/5 on the function of axotomized group Ia afferents and motoneurons comprising the monosynaptic reflex pathway were investigated. The axotomized medial gastrocnemius (MG) nerve was provided with NT-3 or NT-4/5 for 8-35 d via an osmotic minipump attached to its central end at the time of axotomy. After this treatment, monosynaptic EPSPs were recorded intracellularly from MG or lateral gastrocnemius soleus (LGS) motoneurons in response to stimulation of the heteronymous nerve under pentobarbital anesthesia. Controls were preparations with axotomized nerves treated directly with vehicle; other axotomized controls were administered subcutaneous NT-3. Direct NT-3 administration (60 microgram/d) not only prevented the decline in EPSP amplitude from axotomized afferents (stimulate MG, record LGS) observed in axotomy controls but, after 5 weeks, led to EPSPs larger than those from intact afferents. These central changes were paralleled by recovery of group I afferent conduction velocity. Removal of NT-3 4-5 weeks after beginning treatment resulted in a decline of conduction velocity and EPSP amplitude within 1 week to values characteristic of axotomy. The increased synaptic efficacy after NT-3 treatment was associated with enhanced connectivity of single afferents to motoneurons. NT-4/5 induced modest recovery in group I afferent conduction velocity but not of the EPSPs they elicited. NT-3 or NT-4/5 had no effect on the properties of treated motoneurons or their monosynaptic EPSPs. We conclude that NT-3, and to a limited extent NT-4/5, promotes recovery of axotomized group Ia afferents but not axotomized motoneurons or the synapses on them. (+info)
Ultrastructural analysis of ectopic synaptic boutons arising from peripherally regenerated primary afferent fibers.
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The central axons of peripherally regenerated Abeta primary sensory neurons were impaled in the dorsal columns of alpha-chloralose-anesthetized cats 9-12 mo after axotomy. The adequate peripheral stimulus was determined, and the afferent fibers intracellularly stimulated while simultaneously recording the resulting cord dorsum potentials (CDPs). Fibers that successfully had reinnervated the skin responded to light tactile stimulation, and evoked CDPs that suggested dorsally located boutons were stained intracellularly with horseradish peroxidase (HRP). Two HRP-stained regenerated Abeta afferent fibers were recovered that supported large numbers of axon collaterals and swellings in laminae I, IIo, and IIi. Sections containing the ectopic collateral fibers and terminals in the superficial dorsal horn were embedded in plastic. Analyses of serial ultrathin sections revealed that ectopic projections from both regenerated fibers supported numerous synaptic boutons filled with clear round vesicles, a few large dense core vesicles (LDCVs) and several mitochondria (>3). All profiles examined in serial sections (19) formed one to three asymmetric axo-dendritic contacts. Unmyelinated portions of ectopic fibers giving rise to en passant and terminal boutons often contained numerous clear round vesicles. Several boutons (47%) received asymmetric contacts from axon terminals containing pleomorphic vesicles. These results strongly suggest that regenerated Abeta fibers activated by light tactile stimuli support functional connections in the superficial dorsal horn that have distinct ultrastructural features. In addition, the appearance of LDCVs suggests that primary sensory neurons are capable of changing their neurochemical phenotype. (+info)
Nature of the retrograde signal from injured nerves that induces interleukin-6 mRNA in neurons.
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In previous studies, interleukin-6 was shown to be synthesized in approximately one-third of lumbar dorsal root ganglion neurons during the first week after nerve transection. In present studies, interleukin-6 mRNA was found to be induced also in axotomized facial motor neurons and sympathetic neurons. The nature of the signal that induces interleukin-6 mRNA in neurons after nerve injury was analyzed. Blocking of retrograde axonal transport by injection of colchicine into an otherwise normal nerve did not induce interleukin-6 mRNA in primary sensory neurons, but injection of colchicine into the nerve stump prevented induction of interleukin-6 mRNA by nerve transection. Therefore, it was concluded that interleukin-6 is induced by an injury factor arising from the nerve stump rather than by interruption of normal retrograde trophic support from target tissues or distal nerve segments. Next, injection into the nerve of a mast cell degranulating agent was shown to stimulate interleukin-6 mRNA in sensory neurons and systemic administration of mast cell stabilizing agents to mitigate the induction of interleukin-6 mRNA in sensory neurons after nerve injury. These data implicate mast cells as one possible source of the factors that lead to induction of interleukin-6 mRNA after nerve injury. In search of a possible function of inducible interelukin-6, neuronal death after nerve transection was assessed in mice with null deletion of the interleukin-6 gene. Retrograde death of neurons in the fifth lumbar dorsal root ganglion was 45% greater in knockout than in wild-type mice. Thus, endogenous interleukin-6 contributes to the survival of axotomized neurons. (+info)