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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The patterning of synaptic connections during development is thought to be influenced by the correlation of neuronal impulse activity. N-methyl-D-aspartate (NMDA) receptors have been implicated in the reorganization of thalamocortical afferents in the visual system. The topographic mapping of the periphery of sensory systems onto the somatosensory cortex in the whisker-barrel field of rodents has served as another important paradigm in the study of extrinsic influences on synaptic rearrangements. In a search for the molecular cues that may contribute to synaptic plasticity, we have investigated the distribution of the glia-derived extracellular matrix glycoprotein tenascin-C, which is highly expressed during the formation of the barrel field map around birth and delineates the boundaries between barrel fields after segregation of afferent inputs. Here we show that systemic and local application of NMDA receptor antagonists at postnatal day 2 inhibited the down-regulation of tenascin mRNA and protein by postnatal day 6 and prevented the appearance of tenascin-positive barrel field boundaries. Furthermore, barrels were not distinguishable by Nissl staining, and segregation of thalamocortical afferents as monitored by anterograde Dil tracing and acetylcholinesterase histochemistry was not complete. These observations indicate that expression of tenascin-C and segregation of afferent inputs are modified by NMDA receptor-dependent neuronal activity.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have made reasonably comprehensive measurements of action potential activity in the Aplysia californica abdominal ganglion to determine the amount of feedback the central nervous system (CNS) receives from a movement which it initiates. Voltage-sensitive dye measurements of action potential activity of cells in the ganglion were made during the gill-withdrawal reflex elicited by siphon stimulation. We compared recordings in two situations which differed dramatically in the amount the gill moved. In the control sea water, the gill withdrawal was normal; in low-Ca2+, high-Mg2+ sea water, the gill movement was blocked. Both the timing and the number of spikes of the individual neurons were similar in the two situations. Histograms of the summed spike activity versus time and histograms of the number of active neurons versus time in the two conditions were also similar. Finally, two numerical measures of trial-to-trial differences, a paired t-test and a measure we named fractional similarity, did not indicate larger differences between two trials in the different sea waters than two trials in the same sea water. Feedback from sensory neurons activated by the gill movement itself does not make a large contribution to the spike activity in the abdominal ganglion. Apparently the Aplysia CNS issues the command for the withdrawal and does not make adjustments for the magnitude of the actual withdrawal. It may not even receive the information necessary for such adjustments to be made. A second motivation for these experiments was to test whether removing the feedback might simplify the neuronal activity that occurs during the gill-withdrawal reflex. This did not occur.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Apoptosis and mitosis are often thought to share certain morphological similarities and therefore to be regulated by similar sets of enzymes. In this study, the Golgi apparatus and nuclear lamina were examined in PC12 cells and rat superior cervical ganglion neurons undergoing apoptosis in response to withdrawal of nerve growth factor or addition of staurosporine. We found that the Golgi apparatus disperses during apoptosis, without obvious degradation, in a manner similar to that occurring in mitosis. In contrast, the nuclear lamina did not become completely solubilized during apoptosis, as occurs in mitosis, but remained as a distinct structure around the nucleus, although some degradation of nuclear lamins was seen. To assess the integrity of the nuclear envelope, fluorescent probes were introduced into the cytoplasm of live and dying cells. High molecular weight tracers were still excluded from the nuclei of apoptotic cells, demonstrating the continued existence of a functional nuclear barrier. These data suggest, therefore, that cell death is unlikely to occur simply as a result of inappropriate activation of cell cycle enzymes.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: α2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic α2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10μM nicotine, or by adding Ca2+ to otherwise Ca2+-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker ω-conotoxin GVIA and application of the α2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α2-adrenergic effects. Our results indicate that the α2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Acetylcholine-evoked currents were investigated using the conventional whole-cell patch-clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine-evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic-like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non-selective cation current, and could be reversibly blocked by d-tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near EK and displaying a bell shape peaking between -40 and -30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca2+ and nearby Ca2+-activated K+ channels at the synaptic pole of OHCs.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: This study demonstrates the localization and regulation of a novel neuropeptide of 33 amino acids, secretoneurin (SN), in the rat superior cervical ganglion. Gel filtration chromatography of ganglion proteins followed by a specific radioimmunoassay revealed that SN is the predominant cleavage product of secretogranin II, a member of the chromogranin/secretogranin protein family, in adult ganglia. SN was detected within the majority of nerve endings surrounding postganglionic neurons that were identified by the presence of synaptophysin and, in part, colocalized leu-encephalin. Applying immuno-electronmicroscopy, SN was localized to large dense core vesicles of neuronal and small intensely fluorescent (SIF) cells. In situ hybridization revealed the presence of secretogranin II mRNA in postganglionic neurons and, to a lesser extent, in SIF cells. One week after transection of the postganglionic branches SN levels were not significantly altered; however, a decrease of secretogranin II mRNA was observed in postganglionic neurons but not in SIF cells. After decentralization of the ganglion, SN-immunoreactive nerve terminals disappeared and intraganglionic SN levels were reduced by 70%, indicating the preganglionic origin of SN-positive nerve fibres and varicosities. Secretogranin II mRNA was slightly reduced under this condition. Combined axotomy and decentralization further diminished intraganglionic secretogranin II mRNA, although peptide levels increased significantly above control values under these conditions. Double-labelling immunofluorescence with antibodies against the somatodendritic marker microtubule-associated protein 2 (MAP2) revealed that the increase in SN immunoreactivity was due to an accumulation of SN in axonal processes of postganglionic neurons. SN immunoreactivity was also detected in dissociated neonatal superior cervical ganglion cultures and increased significantly upon treatment with nerve growth factor, the survival and differentiation factor of sympathetic neurons during perinatal development. Co-culture with non-neuronal cells or addition of leukaemia inhibitory factor, a cytokine known to stimulate synthesis of various peptides after nerve transection, did not influence SN immunoreactivity. Therefore, since no fixed relationship between SN and any of the known neuropeptides or neurotransmitters expressed in sympathetic neurons was observed, the expression of this novel peptide appears to be independently regulated.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 μM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 × 105 cells/cm2) or increasing the volume by an identical 4-fold factor reduced cell death from 90-95% to 20-30%. Addition of a conditioned medium derived from high-density to low-density cultures or to high-volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate-sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1-7, 3T3 and CHP 100. Glutamate-sensitizing activity was not mimicked by triiodo-L-thyronine, insulin-like growth factor-I (IGF-I), truncated IGF-I, GPE [a tripeptide (gly-pro-glu) derived from IGF-I], brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor-α. However, IGF-I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate-sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triodoi-L-thyronine and BDNF did not support the production of glutamate-sensitizing activity. Glutamate-sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N-Ω-nitro-L-arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate-sensitizing activity.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The localization of gp130, the signal transducing receptor component used in common for interleukin (IL)-6, IL-11, ciliary neurotrophic factor (CNTF), LIF and OSM, in the rat brain was demonstrated by immunohistochemistry using an antibody specific to gpl30. The gp130 immunoreactivity was observed in both glial and neuronal cells. Two distinct neuronal staining patterns were observed. The first showed neuropil staining, observed mainly in telencephalic structures including the hippocampus, cerebral cortex and caudate-putamen. The second pattern was observed on the cytoplasmic membrane of neuronal somata and was found primarily in the lower brainstem, in the large neurons of the reticular formation, and in spinal and cranial motor neurons. Electron-microscopic analysis revealed that both types of gpl30 immunoreactivity were primarily associated with the cytoplasmic membrane and were not localized exactly at synaptic sites. Further, gpl30 immunoreactivity was also observed in the oligodendrocytes and subependymal zone. These widespread but characteristic patterns of gp130 immunoreactivity overlap well with those of IL-6 receptor and CNTF alpha chains, suggesting a role of cytokines and growth factors such as IL-6 and CNTF via gp130 in certain specific regions of the brain.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have investigated the properties of antidromically identified lamina I neurons in the rat dorsal horn (in vivo) after neonatal administration of antibody to nerve growth factor (anti-NGF). Treatment from postnatal day (P) 2 to P9 yielded normal lamina I cell physiology; most cells responded to mechanical nociception and the remainder had a wide dynamic range (WDR). Extending anti-NGF treatment to P14 reduced the proportion of cells responding to mechanical nociception, increased the proportion of WDR cells, and caused the emergence of cells not driven by cutaneous inputs. Both nociceptive-specific and WDR cells had larger receptive fields, suggestive of enhanced central action of the remaining nociceptive afferents. These findings cannot be explained by direct action of anti-NGF on spinal cord neurons since both P2-9 and P2-14 treatments should have had similar effects given the time course of development of the blood-brain barrier. The results are discussed in terms of previous findings indicating normal numbers of D-hairs and high-threshold mechanoreceptors (HTMRs) after anti-NGF treatment from P2 to P9, but a decline in the number of HTMRs and an increase in the number of D-hairs after treatment from P2 to P14. It is suggested that the reduction in nociceptive neurons and the appearance of neurons not driven by cutaneous stimulation in lamina I results from the reduction in HTMR input. However, D-hair input to lamina I did not increase despite the larger number of these afferents, suggesting that their central action was regulated to maintain appropriate modality relationships between periphery and centre.
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