Activating effect of substance P on nerve tissue in culture

The effect of substance P on explant development was investigated in organotypic cultures of rat sympathetic ganglia and spinal cord. The pattern of evolution, cellular composition, and dimensions of the growth zone were evaluated on the basis ofin vivo observations. It was found that this peptide exercises a significant growth-promoting effect at a concentrations of 10^–5–10^–12 M for sympathetic ganglia and 10^–5–10^–14 M for spinal cord culture. The growth zone of sympathetic ganglia measured 1.3–1.6 times the control level by the 14th day of culture at all effective concentrations. The area of outgrowth of spinal cord explants increased 2.0–5.2 fold by the sixth day of culture and peak response was recorded at concentrations of 10^–5 and 10^–12 M. This effect resembled response to opioid peptides [1, 3]. The likely physiological significance of regulatory peptides for the processes of nerve tissue development and regeneration is discussed in the light of these findings, together with the part played by the nociceptive/antinociceptive system in processes of histogenesis and repair.Institute of Experimental Cardiology, All-Union Cardiologic Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 610–615, September–October, 1986.     

Influence of opioid peptides on nerve tissue in vitro

The influence of opioid peptides (gamma- and beta-endorphins, leu- and met-enkephalins, as well as certain synthetic analogs of enkephalin) was investigated on organotypic cultures of rat spinal and sympathetic ganglia. The cellular composition and size of the zone of growth were evaluated on the basis of intravital observations and an analysis of the specimen obtained using the method of impregnation, according to Holmes and the detection of catecholamines with glyoxylic acid. It was established that under the action of all the investigated substances that possess high affinity for opiate receptors, growth of the neurites from an explant was enhanced, and the number of glial and fibroblastoid cells in the growth zone was increased. The effect was observed most distinctly on a model of sympathetic ganglia. The tested compounds exhibited a significant growth-stimulating effect in the range of concentrations 10^–8–10^–14 M. The maximum size of the growth zone of the explants of the sympathetic ganglia in the case of a mean effective concentration of the peptides 10^–10 M by the third to fifth day of culturing was approximately 2–2.5 times this value in the control. The reaction was similar to the response of the nerve cells to nerve growth factor, used as a standard. Thus, the opioid peptides exhibit a pronounced growth effect on the structures of the nerve tissue under conditions of culture. It is suggested that this group of compounds, together with its currently well-known functions, may play a definite role in processes of the development and regenera-of nerve tissue.Institute of Experimental Cardiology, All-Union Cardiologic Science Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 550–557, July–August, 1985.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Effect of hydrocortisone on immunohistochemically demonstrable phenylethanolamine-N-methyltransferase in cultures of embryonic and postnatal superior cervical ganglia

Summary Pre- and postnatal superior cervical ganglia of the rat were cultured in Rose chambers for 1–7 days with or without hydrocortisone. Phenylethanolamine-N-methyltransferase (PNMT) was demonstrated by indirect immunofluorescence technique. In cultures without added hydrocortisone, no cells or fibres showed PNMT-immunoreactivity, without regard to the time in culture or the developmental stage at the time of explantation. The first PNMT-immunoreactive cells in hydrocortisone-containing cultures appeared 3 days after the explantation of E14 ganglia, or 1 day after the explantation of E15 ganglia, i.e. at the developmental stage E16–E17. The cultures of neither E14 nor E15 ganglia showed marked fibre growth from the PNMT-immunoreactive cell bodies. On the other hand, in the hydrocortisone-containing cultures of newborn or postnatal rats, there was extensive nerve fibre formation from the PNMT-immunoreactive cells in the course of the culture. PNMT-immunoreactive cells did not appear in hydrocortisone-containing cultures of ganglia taken from rats older than 17 postnatal days.     

Effects of opioid peptides and naloxone on tissue from the central and peripheral nervous system in culture

A study was made of the effects of opioid peptides (leu-enkephalin and dalargin AE-1, its synthetic analog) and of naloxone, an opiate receptor blocker, on organotypic cultures of spinal cord and spinal ganglia cells. The cellular composition and size of explant outgrowth was estimated according to in vitam morphological observations. It was found that all the opioid peptides tested at concentrations of 10^–9-10^–10M exercise a clear-cut growth-promoting effect on cultures from the spinal cord as well as those from the peripheral nervous system [4, 5]. Naloxone at a concentration of 10^–5-10^–6 M does not block peptide action, but itself stimulates growth. It was also proved that opioid peptides act as trophic factors for spinal ganglia nerve cells, increasing their survival in culture. Endorphins can thus serve as growth factors for tissues of the peripheral as well as the central nervous system. The likely processes lying at the root of the growth-promoting and trophic effects of endorphins on nerve tissue are discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 227–233, March–April, 1986.     

Spectral analysis of EEG in normal rats and in rats genetically predisposed to seizures

Spectral and visual analyses were performed on the EEG of the motor and visual cortex, hippocampus, caudate nucleus, and intralaminary thalamic nuclei in two strains of rats; animals were maintained in a state of "awake immobility." It was found that KM rats, genetically predisposed to audiogenic fits, differed from the Wistar strain not subject to this genetic predisposition in that mean relative intensity of theta rhythm diminished and high amplitude slow irregular hippocampal activity intensified in the neocortex, as did generalized spindling. Susceptibility to seizure was reduced in KM rats as a result of protracted and graded increasing camphor administration to match the level of mean EEG spectral density changes characteristic of the Wistar strain. The part which brainstem reticular formation mechanisms may play in raising susceptibility to seizures is discussed, together with the EEG pattern characteristic of this condition.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 171–179, March–April, 1987.     

Long-term action of camphor on "audio-sensitive" rats: Electrophysiological study and mathematical modeling of properties of neuronal networks

Krushinskii-Molodkina (KM) strain rats genetically predisposed to audiogenic convulsive reaction were given repeated camphor injections in gradually increasing doses (starting at the minimum threshold level required for seizures to occur) over a 4–5 month period. Animals were able to tolerate camphor at doses 3/2–3 times convulsion threshold level without seizure occurring once habituation to the action of this convulsant had been developed. At the same time, the cortical motor zone of strain KM rats acquired properties typical of an epileptic focus: spontaneous epileptiform firing peaks were noted in the background electrical activity of this zone. A decline in the parameter reflecting efficacy of the mechanisms underlying recurrent inhibition emerged in the cortical motor zone of strain KM rats receiving camphor from calculating the parameters of neuronal network from spectra of summated potentials (using the model of a neuronal network). It is suggested that the development of compensatory processes making it possible to avoid generalized seizure following administration of camphor in large doses is associated with intensification of inhibitory caudate function and attenuated hippocampal excitation.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 193–200, March–April, 1990.     

Ultrastructural characteristics of synaptogenesis in monolayer cultures of spinal cord

Stages of formation of different types of synapse between cells of the dissociated spinal cord and spinal ganglia of 12–14 day mouse embryos, in monolayer cultures, were studied electron-microscopically. The participation of cones of growth in the formation of different junctions between structures of the monolayer was traced. It was shown that the appearance of synaptic vesicles in the growing axon precedes the onset of membrane specialization in the region of contact between axon and target cell. Ultrastructural characteristics of axo-dendritic, axo-somatic, and axo-axonal synapses formed during growth are given. On the 24th day of culture structural complexes of axonal glomerulus type, incorporating axo-axonal and axo-dendritic synapses, were discovered. It is suggested that desmosomes participate in the formation of both chemical synapses and synapses of gap junction type.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 336–343, May–June, 1984.     

Modulation of the inhibiting effect of long-chained acyl-CoA on oxidative phosphorylation of liver mitochondria in rats of various lines by Z-protein

The effect of short-term starvation on the energy state of the adenine nucleotide system in the livers of rats of Wistar and Wag strains was studied. In fed rats, (4 hours after food withdrawal) both strains had the same liver content of long-chain acyl-CoAs. At the same time, in the livers of Wistar rats the phosphate potential values were much higher than in the livers of Wag rats (1.82 +/- 0.18 and 0.64 +/- 0.08, respectively), thus suggesting a strong inhibition of oxidative phosphorylation in the livers of Wag rats. Based on a comparison of the states and levels of Z-protein in the livers of fed and starved Wistar and Wag rats, it was proposed that an excess of long-chain acyl-CoAs over the Z-protein content in the liver is a necessary prerequisite for the development of inhibition of oxidative phosphorylation. Thus, the Z-protein may be considered as a possible physiological modulator of the inhibitory effect of acyl-CoAs on the energy state of the adenine nucleotide system in the liver in vivo.     

Effect of hydrocortisone on the ultrastructure of the small,intensely fluorescent,granule-containing cells in cultures of sympathetic ganglia of newborn rats

Summary Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with or without hydrocortisone. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde solution and thereafter in 1% osmium tetroxide. Hydrocortisone (10 mg/l) caused a great increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants, and the fluorescence intensity of these cells was also increased. The SIF cells corresponded to small, granule-containing (SGC) cells in the electronmicros copic preparations, and in addition to an increase in their number there was also an increase in the size and number of granular vesicles in the presence of hydrocortisone. In control cultures the granular vesicles were round (about 100 nm in diameter) or elongated (40–150 nm in cross section and 150–250 nm in length); both types of vesicles contained electron dense cores. In hydrocortisone-containing cultures round granular vesicles up to 200 nm in diameter were also observed; the cores of these vesicles were of variable electron density. It is concluded that in tissue culture, hydrocortisone causes an increased formation of catecholamine-containing granular vesicles in SIF-SGC cells and their precursors and an increase in the number of these cells.This work was supported by grants from the National Heart Foundation, the Australian Research Grants Committee and the Sigrid Juselius Foundation.University of Melbourne Senior Research Fellow, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.Holder of a grant from the National Health and Medical Research Council of Australia.Sunshine Foundation and Rowden White Research Fellow in the University of Melbourne, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.     

Regeneration of pedal ganglion neurons in the sea butterfly Clione limacina

In the pedal ganglia ofClione limacina the growth of neurites is traced in motoneurons after transection of the wing nerve and in interneurons after transection of the pedal commissure. Neurons were stained intracellularly with Lucifer yellow. In the motoneurons the neurites growing from the transected end of the axon and from the neuron soma spread to all nerve trunks departing from the ipsi- and contralateral ganglia. For nerve transection in the intact mollusk, wing movements were restored 10 days after the operation. In the interneurons the growing neurites branched within the pedal ganglion or spread to the cerebral ganglia, but they never reached the periphery.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 449–455, July–August, 1985.     

Conducting pathways of the dog solar plexus

Conducting pathways of the dog solar plexus were studied by recording action potentials from its nerves. The splanchnic nerves are composed of two groups of fast-conducting afferent A fibers (with conduction velocities of 12–15 and 25–56 m/sec), slowly conducting afferent C fibers (0.4–2.0 m/sec), and preganglionic B and C fibers (1.0–12.0 m/sec). Afferent A and C fibers from peripheral nerves run without interruption through the ganglia of the solar plexus, splanchnic nerves, and sympathetic chain and they enter the spinal cord in the composition of the dorsal roots. Cell bodies of A fibers are located in the spinal ganglia, those of the C fibers below the ganglia of the solar plexus, evidently in the walls of the internal organs. Peripheral nerves contain A fibers only with very low conduction velocities (13–20 m/sec) and no fast-conducting A fibers (25–56 m/sec) were found. Preganglionic fibers terminate synaptically on neurons of the ganglia of the solar plexus whose axons run in the peripheral nerves to the internal organs. Synaptic pathways run from some peripheral nerves of the solar plexus into others through its ganglia; in all probability these pathways participate in peripheral reflex arcs.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 76–83, January–February, 1976.     

Effects of hormones on axonal transport rate at the ventral root of the rat spinal cord

Male Wistar rats aged between 8 and 12 months were injected with 7–8 µl of aqueous L-leucine-¹⁴C (specific activity: 12543 megaBq/mM) in the region of the ventral horn at the level of segments L_5,6 of the spinal cord. Radioactivity was investigated in 3 mm segments of the ventral roots concerned within 1 h in all series of experiments. Estradiol dipropionate, testosterone propionate, insulin, and small doses of thyroxine were found to accelerate axonal transport of labeled material, while hydrocortisone, large doses of thyroxine, as well as castration and thyroidectomy delayed this process. It was thus concluded that axonal transport is under clear-cut hormonal control.Institute of Gerontology, Academy of Medical Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 459–464, July–August, 1990.     

Characterization of a pyridine-degrading branched Gram-positive bacterium isolated from the anoxic zone of an oil shale column

Summary From the anoxic zone of an oil shale leachate column three pyridine-degrading bacterial strains were isolated. Two strains were Gram-negative facultative anaerobic rods and one strain was a branched Gram-positive bacterium. The branched Gram-positive strain had the best pyridine-degrading ability. This organism was aerobic, non-motile, catalase positive, oxidase negative, and had no flagellum. The G+C content of the DNA was 66.5 mol%. The major menaquinone was MK-8(H₂). The main cellular fatty acids were saturated and monounsaturated straight chains. This organism contained mycolic acid, meso-diaminopimelic acid, arabinogalactan and glycolyl residues in the cell wall. Due to morphological, physiological and chemotaxonomic characteristics this strain was placed in the genus Rhodococcus. The optimum culture conditions were as follows: temperature 32° C, pH 8.0 and 0.1% v/v of pyridine as sole carbon, energy and nitrogen source. Utilization of pyridine by a batch fermentor culture of Rhodococcus sp. was characterized by a specific growth rate of 0.13 h^–1, growth yield of 0.61 mg cell·mg pyridine^–1 and a doubling time of 5.3 h^–1. Offprint requests to: S.-T. Lee     

Involvement of IAA in the interaction betweenAzospirillum brasilense andPanicum miliaceum roots

The possible involvement of IAA in the effect thatAzospirillum brasilense has on the elongation and morphology ofPanicum miliaceum roots was examined by comparing in a Petri dish system the effects of inoculation with a wild strain (Cd) with those of an IAA-overproducing mutant (FT-326). Both bacterial strains produced IAA in culture in the absence of tryptophan. At the stationary growth phase, production of IAA by FT-326 wasca. 12 times greater than that of Cd. When inoculation was made with bacterial concentrations higher than, 10⁶ colony forming units ml^–1 (CFU ml^–1), both strains inhibited root elongation to the same extent. At lower concentrations Cd enhanced elongation, by 15–20%, while FT-326 was ineffective. Both strains promoted root-hair development, and root-hairs were produced nearer the root tip the higher the bacterial concentration (e. g. root elongation region was reduced). Effects of FT-326 on root-hair development were greater than those of Cd. Acidified ether extracts of Cd and FT-326 cultures had inhibitory or promoting effects on root elongation depending on the dilution applied. At low dilutions, extracts from FT-326 were more inhibitory for elongation than those from Cd. At higher dilutions root elongation was promoted, but FT-326 extracts had to be more diluted than those from Cd. Dilutions that promoted root elongation contained supra-optimal concentrations of IAA, 1–3 orders of magnitude higher than those required for optimal enhancement by synthetic IAA. It is suggested that the bacteria produce in culture an IAA-antagonist or growth inhibitor that decreases the effectiveness of IAA action. The large variability reported for the effects ofAzospirillum on root elongation could be the result of the opposite effects on root elongation of IAA and other compounds, produced by the bacteria.     

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

Summary Many mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex     

Filamentous growth ofPseudomonas aeruginosa

Summary The growth of two strains ofPseudomonas aeruginosa in stirred batch cultures was monitored by optical density, DNA concentration, and acridine orange direct cell count measurements. Growth of adherent bacteria in pure culture was also observed on suspended glass discs by light and scanning electron microscopy. Strain MUCOID produced significant numbers of filamentous cells in broth culture and in the adherent population, while strain PAO 381 did not produce elongated cells. Filamentous growth of MUCOID could be prevented by the addition of 5 × 10^–2 M Mg²⁺. However, the addition of 0.66 mM EDTA caused an increased proportion of the population (>50%) of MUCOID cells to become filamentous in broth culture. The results are discussed and related to theories regarding bacterial plasticity, and filamentation of normally bacillary cells.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.