Effect of capsaicin on neuropeptides in areas of termination of primary sensory neurones

Capsaicin stimulates chemosensitive peripheral pain receptors, and neonatal administration produces degeneration of a population of primary afferent fibres. It has been shown previously that the effects of capsaicin are accompanied by the loss of substance P from areas of primary afferent termination and that enkephalin is not depleted from such areas. However, a number of other peptides are thought to be contained in sensory fibre systems and so we have used immunohistochemistry to examine the effect of capsaicin on the distribution of five different peptides in the substantia gelatinosa of the spinal trigeminal nucleus and spinal cord. Neonatal capsaicin treatment produces a depletion of somatostatin and cholecystokinin immunofluorescence in addition to substance P, but enkephalin and neurotensin immunofluorescence are not depleted. The implications of this result for theories of peptide involvement in nociceptive mechanisms are discussed.     

Effects of semiconductor substrate and glia-free culture on the development of voltage-dependent currents in rat striatal neurones

An essential requirement for successful long-term coupling between neuronal assemblies and semiconductor devices is that the neurones must be able to fully develop their electrogenic repertoire when growing on semiconductor (silicon) substrates. While it has for some time been known that neurones may be cultured on silicon wafers insulated with SiO2 and Si3N4, an electrophysiological characterisation of their development under such conditions is lacking. The development of voltage-dependent membrane currents, especially of the rapid sodium inward current underlying the action potential, is of particular importance because the conductance change during the action potential determines the quality of cell-semiconductor coupling. We have cultured rat striatal neurones on either glass coverslips or silicon wafers insulated with SiO2 and Si3N4 using both serum-containing and serum-free media. We here report evidence that not only serum-free culture media but also growth on semiconductor surfaces may negatively affect the development of voltage-dependent currents in neurones. Furthermore, using surface-charge measurements with the atomic force microscope, we demonstrate a reduced negativity of the semiconductor surface compared to glass. The reduced surface charge may affect cellular development through an effect on the binding and/or orientation of extracellular matrix proteins, such as laminin. Our findings therefore suggest that semiconductor substrates are not entirely equivalent to glass in terms of their effects on neuronal cell growth and differentiation.     

Depolarization-activated K+ currents of the bushy neurones of the rat cochlear nucleus in a thin brain slice preparation

Depolarization-activated outward currents of bushy neurones of 6-14-day-old Wistar rats have been investigated in a brain slice preparation. Under current-clamp, the cells produced a single action potential at the beginning of suprathreshold depolarizing current steps. On voltage-clamp depolarizations, the cells produced a mixed outward K+ current that included a component with rapid activation and rapid inactivation, little TEA+ sensitivity, a half-inactivation voltage of -77 +/- 2 mV (T = 25 degrees C; n = 7; Mean +/- S.E.M.) and single-exponential recovery from inactivation (taurecovery= 12 +/- 1 ms at -100 mV; n=3). This transient component was identified as an A-type K+ current. Bushy cells developed a high-threshold TEA-sensitive K+ current that exhibited less prominent inactivation. These characteristics suggested that this current was associated with the activation of delayed rectifier K+ channels. Bushy neurones also possessed a low-threshold outward K+ current that showed partial inactivation and high 4-aminopyridine sensitivity. Part of this current component was blocked by 200 nmol/l dendrotoxin-I. Application of 100 micromol/l 4-aminopyridine changed the firing behaviour of the bushy neurones from the primary-like pattern to a much less rapidly adapting one, suggesting that the low-threshold current might have important roles in maintaining the physiological function of the cells.     

Effect of ciguatoxin 3C on voltage-gated Na+ and K+ currents in mouse taste cells

The marine dinoflagellate Gambierdiscus toxicus produces highly lipophilic, polycyclic ether toxins that cause a seafood poisoning called ciguatera. Ciguatoxins (CTXs) and gambierol represent the two major causative agents of ciguatera intoxication, which include taste alterations (dysgeusiae). However, information on the mode of action of ciguatera toxins in taste cells is scarce. Here, we have studied the effect of synthetic CTX3C (a CTX congener) on mouse taste cells. By using the patch-clamp technique to monitor membrane ion currents, we found that CTX3C markedly affected the operation of voltage-gated Na(+) channels but was ineffective on voltage-gated K(+) channels. This result was the exact opposite of what we obtained earlier with gambierol, which inhibits K(+) channels but not Na(+) channels. Thus, CTXs and gambierol affect with high potency the operation of separate classes of voltage-gated ion channels in taste cells. Our data suggest that taste disturbances reported in ciguatera poisoning might be due to the ability of ciguatera toxins to interfere with ion channels in taste buds.     

Effect of superoxide derived from lucifer yellow CH on voltage-gated currents of mouse taste bud cells

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.     

Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture

Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.     

Effect of melittin on ion currents in heart cell membranes

It has been shown on auricle fibres of the frog that neurotoxin from bee poison melittin suppresses the ionic currents entering the cell through calcium and sodium channels of the membrane, increases the background potassium current, suppresses phasic and tonic contraction of the fibres. Toxin modifies the kinetics of calcium channels, but does not affect activation and desensitization of beta adrenoreceptors. Effects of melittin are not decreased when adding the inhibitor of phospholipase A2 indomethacin. The results show that melittin directly affects the protein components of the membrane-ionic channels, probably binding with them.     

Maturation involves suppression of voltage-gated currents in the frog oocyte

Voltage- and time-dependent currents having slow kinetics have been studied in plasma membranes of immature oocytes of the european frog, Rana esculenta. IK, corresponding to an outward flow of K+, is activated at potentials more positive than about -40 mV, and subserves outward rectification; Iir, corresponding to an outward flow of Cl-, is activated at potentials more negative than about -80 mV and subserves inward rectification. Such currents can act as negative feedback mechanisms in the control of membrane potential in the immature oocyte and limit to a somewhat restricted range its possible deviations from resting values. Besides IK, membrane depolarizations to potentials more positive than about +30 mV are capable of activating INa, corresponding to outflow of Na+. By contrast, the frog mature egg-cell has a single voltage- and time-dependent current, IM, activated at potentials more positive than +30 mV, with properties similar to INa. The disappearance of IK and Iir along with remarkable reduction in leakage lowers impedance in the egg membrane. It seems reasonable to suggest that the observed changes in membrane permeability reflect changes which have taken place along the maturation process and are of importance for successful fertilization.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Synaptic and voltage-gated currents in interplexiform cells of the tiger salamander retina

We have correlated the membrane properties and synaptic inputs of interplexiform cells (IPCs) with their morphology using whole-cell patch-clamp and Lucifer yellow staining in retinal slices. Three morphological types were identified: (a) a bistratified IPC with descending processes ramifying in both sublaminas a and b of the inner plexiform layer (IPL), and an ascending process that branched in the outer plexiform layer (OPL) and originated from the soma, (b) another bistratified IPC with descending processes ramifying in both sublaminas a and b, and an ascending process that branched in the OPL and originated directly from IPC processes in the IPL, and (c) a monostratified IPC with a descending process ramifying over large lateral extents within the most distal stratum of the IPL, and sending an ascending process to the OPL with little branching. Similar voltage- gated currents were measured in all three types including: (a) a transient inward sodium current, (b) an outward potassium current, and (c) an L-type calcium current. All cells generated multiple spikes with frequency increasing monotonically with the magnitude of injected current. The IPCs that send their descending processes into both sublaminas of the IPL (bistratified) receive excitatory synaptic inputs at both light ON and OFF that decay with a time constant of approximately 1.3 s. Slowly decaying excitation at both ON and OFF suggests that bistratified IPCs may spike continuously in the presence of a dynamic visual environment.     

Effect of chemical ablation of myenteric neurones on intestinal cell proliferation

The duodenum or descending colon of male Wistar rats (average weight 60 g) was treated by a serosal application of a 0.2% solution of benzalkonium chloride (BAC) for 30 min. Control animals were treated with 0.9% (physiological) saline. The rats were allocated to four groups: Group DC (N = 8) in which the duodenum was treated with physiological saline; Group DB (N = 8) in which the duodenum was treated with BAC; Group CC (N = 7) in which the descending colon was treated with physiological saline and Group CB (N = 7) in which the descending colon was treated with BAC. After treatment, the animals were followed up for 5 months. At the end of the experiment, the animals were injected intraperitoneally with vincristine sulphate before sacrifice. Three segments were removed from the duodenum and descending colon for neuronal counting, catecholamine and serotonin measurements and morphokinetic studies of the epithelium. The following results were obtained: (1) there was a significant reduction in neurone number in the myenteric plexus of segments treated with BAC; (2) in the denervated intestinal segments, catecholamine levels were unchanged whereas serotonin levels were increased; (3) epithelial hyperplasia was observed in the denervated duodenum and descending colon; and (4) crypt cell production rate in the duodenum was similar in groups DC and DB but was significantly increased in the descending colon in group CB as compared with controls (CC). The present findings indicate that selective myenteric neuronal denervation caused by benzalkonium chloride plays a causative role in the hyperplasia and crypt cell production rate of the intestinal epithelium (duodenum and descending colon). These changes are probably induced by functional imbalance by the surviving neuronal elements in the gut, implicating neurotransmitters such as acetylcholine, noradrenaline, serotonin, somatostatin and vasoactive intestinal peptide.     

Comparative characteristics of activity in grafted and intact neocortex in slice preparations

Background single unit activity and mixed single and multiunit responses to electrical stimulation of rat's embryonic cortex grafted into the somatosensory cortex of adult rats were investigated in a slice preparation. The relative number of neurons exhibiting background activity in the grafts was higher than in slices of intact cortex (23 and 6%, respectively), however, the portion of neurons responding to electrical stimulation of areas of neighboring neocortex in the recipient was lower than in intact cortex. The latent periods of population responses in the grafts were consistently longer than in those in intact neocortex (19.4±5 and 5.8±1.1 msec, respectively), although the extreme values coincided. The duration of population responses in the grafts was approximately an order of magnitude longer. The data presented suggests the existence of local connections between the graft and the brain of the recipient and shows weakness of inhibitory processes in the grafts. A functional integration of graft and brain in recipients with craniocerebral trauma was demonstrated.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino, Moscow. Institute of Neurosurgery, Ministry of Public Health of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 273–280, May–June, 1991.     

The morphological consequences of neonatal treatment with capsaicin on primary afferent neurones in adult rats

A comparison was made using our work and that reported in the literature of the losses of myelinated and unmyelinated fibres in a variety of nerves and also of losses of nerve cells in dorsal root ganglia, after treatment of neonatal rats with capsaicin. In L3 and L4 dorsal roots 85-93% of unmyelinated fibres and 9-33% of myelinated fibres were lost after 50-100 mg/kg capsaicin neonatally. In rats treated with 85 mg/kg capsaicin, percentage losses of unmyelinated (89%) and myelinated (36%) fibres of L4 dorsal roots were remarkably similar to the calculated losses of small dark (92%) and large light (34%) neurones respectively in these ganglia. Studies with monoclonal antibody RT97 which labels the large light neurones only, confirmed that some RT97 negative cells (i.e. small dark neurones) remain after capsaicin treatment. At present no evidence exists to suggest that the cell death of small dark neurones or C fibres after neonatal capsaicin treatment is completely selective for subgroups of these neurones, either in relation to sensory modality, or in relation to immunocytochemical cell markers and peptide content. However much more data is required to establish whether this cell death is really nonselective as regards immunocytochemical markers.     

Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations.

Glial progenitors colonize the CNS widely in the perinatal period, but the pathways and mechanisms of migration are not well understood. We investigated the migration of progenitors from the neonatal rat forebrain subventricular zone (SVZ) by labeling them in vivo with a retrovirus encoding green fluorescent protein and visualizing movements by time lapse microscopy in slices. Cells within the dorsolateral SVZ moved in an undirected fashion but migrated radially and tangentially after emigration into white matter, cortex, and striatum. Cells in the striatal SVZ migrated parallel to the ventricular surface. During migration, elongation of the leading process and nuclear translocation were independent or linked. Orthogonal turning involved either cessation of cell body movement and formation of a new leading process or continuous cell body movement and bending of the leading process.     

Transmission and scanning electron microscope preparations of the same cell culture.

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3,393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.