Modulations of high‐voltage activated Ca2+ channels in the central neurones of Spodoptera exigua by chlorantraniliprole

The modulation of voltage‐gated calcium channels by chlorantraniliprole in the central neurones isolated from third‐instar larvae of Spodoptera exigua is studied by the whole‐cell patch‐clamp technique. The current of calcium in the third‐instar larvae of S. exigua is identified as a high‐voltage activated Ca²⁺ current. During the 10‐min recording, the current–voltage relationship curves of whole‐cell calcium channels are shifted in a negative direction by 10 mV compared with the control group. The fact that the gravity rundown of calcium current in the treated group is more apparent than in the control group demonstrates that the open channels are constantly inactivated. In addition, chlorantraniliprole inhibits the recorded calcium currents in a concentration‐dependent manner, which is irreversible on washout.     

Matrix-mediated gene transfer to brain cortex and dorsal root ganglion neurones by retrograde axonal transport after dorsal column lesion

BACKGROUND: In previous studies, we showed that the immobilisation of DNAs encoding basic fibroblast growth factor, neurotrophin-3 and brain-derived neurotrophic factor in a gene-activated matrix (GAM) promotes sustained survival of axotomised retinal ganglion cells after optic nerve injury. Here, we evaluated if the immobilisation of DNAs in a GAM could be an effective approach to deliver genes to axotomised dorsal root ganglion (DRG) neurones after spinal cord injury and if the matrix component of the GAM would modulate the deposition of a dense scar at the injury site. METHODS: We evaluated the expression of the thymidine kinase (TK) reporter gene in brain cortex and DRG after a bilateral T8 dorsal column (DC) lesion using PCR, RT-PCR and in situ hybridisation analyses. Collagen-based GAMs were implanted at the lesion site and the cellular response to the GAM was assessed using cell-specific markers. RESULTS: At 1 week post-injury, PCR analyses confirmed that DNATK was retrogradely transported from the DC lesion where the GAM was implanted to the brain cortex and to caudal DRG neurones, and RT-PCR analyses showed expression of mRNATK. At 7 weeks post-injury, DNATK was still be detected in the GAM and DRG. In situ hybridisation localised DNATK and mRNATK within fibroblasts, glia, endothelial and inflammatory cells invading the GAM and in DRG neurones. Interestingly, the presence of a GAM also reduced secondary cavitation and scar deposition at the lesion site. CONCLUSIONS: These results establish that GAMs act as bridging scaffolds in DC lesions limiting cavitation and scarring and delivering genes both locally to injury-reactive cells and distally to the cerebral cortex and to DRG neuronal somata through retrograde axonal transport.     

Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions

The current status of insect neuronal cultures is discussed and their contribution to our understanding of the insect nervous system is explored. Neuronal cultures have been developed from a wide range of insect species and from all developmental stages. These have been used to study the morphological development of insect neurones and some of the extrinsic factors that affect this process. In addition, they have been used to investigate the physiology of sodium, potassium and calcium channels and the pharmacology of acetylcholine and GABA receptors. Insect neurones have also been grown in culture with muscle and glial cells to study cell interactions.     

Neurotrophic and neurotoxic effects of nitric oxide on fetal midbrain cultures

There is evidence suggesting that nitric oxide (NO) may play an important role in dopamine (DA) cell death. Thus, the aim of this study was to investigate the effects of NO on apoptosis and functionality of DA neurones and glial cells. The experiments were carried out in neuronal-enriched midbrain cultures treated with the NO donor diethylamine-nitric oxide complexed sodium (DEA-NO). DEA-NO, at doses of 25 and 50 microM, exerted neurotrophic effects on dopamine cells, increasing the number of tyrosine hydroxylase positive (TH(+)) cells, TH(+) neurite processes, DA levels and [(3)H]DA uptake. A dose of 25 microM DEA-NO protected DA cells from apoptosis. In addition, it induced de novo TH synthesis and increased intracellular reduced glutathione (GSH) levels, indicating a possible neuroprotective role for GSH. However, in doses ranging from 200 to 400 microM, DEA-NO decreased TH(+) cells, DA levels, [(3)H]DA uptake and the number of mature oligodendrocytes (O1(+) cells). No changes in either the amount or morphology of astrocytes and glial progenitors were detected. A dose- and time-dependent increase in apoptotic cells in the DEA-NO-treated culture was also observed, with a concomitant increase in the proapoptotic Bax protein levels and a reduction in the ratio between Bcl-xL and Bcl-xS proteins. In addition, DEA-NO induced a dose- and time-dependent increase in necrotic cells. 1H-[1,2,4]oxadiazolo[4, 3a]quinoxaline-1-one (ODQ, 0.5 microM), a selective guanylate cyclase inhibitor, did not revert the NO-induced effect on [(3)H]DA uptake. Glia-conditioned medium, obtained from fetal midbrain astrocyte cultures, totally protected neuronal-enriched midbrain cultures from NO-induced apoptosis and rescued [(3)H]DA uptake and TH(+) cell number. In conclusion, our results show that low NO concentrations have neurotrophic effects on DA cells via a cGMP-independent mechanism that may implicate up-regulation of GSH. On the other hand, higher levels of NO induce cell death in both dopamine neurones and mature oligodendrocytes that is totally reverted by soluble factors released from glia.     

Sensory pathways in amphioxus larvae II. Dorsal tracts and translumenal cells

Serial and interval EM series were used to examine the dorsal nerve tracts in the anterior nerve cord of a 12.5 day larva of Branchiostoma floridae. Fibres within the tracts derive from peripheral sensory cells and a class of intramedullary sensory neurones known as dorsal (Retzius) bipolar cells. Both form repeated synapses of similar type, apparently with the same targets. The synapses occur at points where, at intervals, the tracts expand to form large synaptic zones. The target dendrites, which form complex tangles, belong chiefly to dorsal translumenal cells, a class of neurone distinguished by their apical processes. The latter range from short extensions of the cell apex that contact the opposite side of the cord via junctions, but go no further, to elongate processes with slender branches that project to the contralateral dorsal tract. The morphology indicates that translumenal cells play the same role in amphioxus as internuncial neurones in vertebrate spinal cord. Their axons can be ipsilateral or contralateral; some synapse with motoneurones directly while others innervate other interneurones, including other translumenal cells. From the circuitry, the cells appear to be chiefly involved in integrating sensory input from peripheral mechanoreceptors. This could include acting as a filter that amplifies some input patterns over others, or that normalizes input, so that CNS circuits are not overloaded as new sensory cells differentiate during development. The functional importance of the translumenal system to the organism is reflected in a massive increase in size and cell numbers during the larval phase. The anterior, brain-like integrative centres of the cerebral vesicle, in contrast, are initially small and change very little.     

Effects of brain-derived neurotrophic factor (BDNF) on glial cells and serotonergic neurones during development

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain-derived neurotrophic factor (BDNF) and S100beta, an astrocyte-specific Ca(2+) binding protein. Here, we used BDNF-deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF-/- mice, the number of serotonin-immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5-hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP-immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100beta. Accordingly, S100beta was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia-free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100beta. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100beta and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100beta in astrocytes and the production MBP in oligodendrocytes.     

Neuropeptides and the central regulation of body temperature during fever and hibernation

Neuropeptides, acting on structures within the central nervous system influence body temperature. Non-opioid peptides induce hypothermia usually, while opioid peptides are mostly hyperthermic. Neuropeptides exert their effect only when injected into specific brain areas.

Hypo- Or hyperthermic effect of neuropeptides may be either due to changes in threshold body temperatures for induction of thermoregulatory effectors or due to changes in hypothalamic thermosensitivity.

At the cellular level the opioid peptides also act differently than the non-opioid peptides. The opioid peptides mostly inhibit spontaneous neuronal firing, while the non-opioid peptides usually stimulate it. Neuropeptides exert their influence on all neurones in the hypothalamus, independently on their temperature characteristics.

Neuropeptides may play a role in the regulation of body temperature under stressful conditions and during fever or hibernation, in particular. Some neuropeptides, namely AVP, -MSH and ACTH, act as natural antipyretic substances by lowering the threshold for cold thermogenesis.

Neuropeptides also modulate food intake, reproduction and many other functions which are substantially changed during hibernation. There appears to be a correlation between the effect of peptides on the control of food intake and on the control of body temperature. Opioid peptides, which increase food intake, induce hyperthermia, while non-opioid peptides, which are appetite inhibiting, induce hypothermia. The exact role o neuropeptides in the regulation of body temperature, food intake and gonadal activity of hibernators remains unclear, however.     

Characterization, by Size, Density, Osmotic Fragility, and Immunoaffinity, of Acetylcholine- and Vasoactive Intestinal Polypeptide-Containing Storage Particles from Myenteric Neurones of the Guinea-Pig

When cytoplasmic extracts of guinea-pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg2+-activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle-exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense-cored, enkephalin- and noradrenaline-containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor-product relationship with the classic synaptic vesicles containing the small-molecular-mass transmitters and found in the same nerve terminals.     

Impact of ethanol and acetaldehyde on DNA and cell viability of cultured neurones

Ethanol consumption has long been associated with brain damage. However, the mechanism underlying this deleterious effect remains unclear. Among different hypotheses, acetaldehyde is regarded by certain authors as playing a major role in the expression of ethanol toxicity, but there are still some uncertainties about the exact nature of its implication. We therefore tried to characterize the profile of the alterations of neuronal viability and DNA integrity obtained after either a direct exposure to ethanol or to acetaldehyde. Ethanol at concentrations within the range of blood alcohol levels in intoxicated humans (100 mmol/L) induced DNA alterations without any apparent effect on cell viability. Acetaldehyde ( 1000 mol/L) can also induce DNA alterations but with a different profile of the DNA cellular alterations. The comparison between the distributions of the comet tail DNA indicated that ethanol induced strong breaks (tail DNA 60 a.u.) generation whereas acetaldehyde rather induced lower breaks (20tail DNA 50 a.u.) formation but affecting a greater number of neurones. Acetaldehyde had thus a different genotoxic potential which may suggest a different mode of action or a different cellular target. Furthermore, when a single 100 mmol/L ethanol exposure did not lead to any loss of cell viability, the addition of an inhibitor of aldehyde dehydrogenase was followed by a significant loss in viability. In contrast, the inhibition of catalase, which suppresses acetaldehyde synthesis, led to no reduced viability in the same exposure conditions. ROS also reduced viability, but this was observed only after both cytochrome P450 stimulation and catalase inhibition. These combined results could suggest that acetaldehyde may play a significant role in the expression of ethanol toxicity in brain.