Structuro-functional changes of bushy receptors after reoxygenation

With restarting oxygen supply to the bushy receptors of the frog urinary bladder, a gradual increase of impulse frequency in the afferent neural fibers is observed, with a successive exceed, in some cases, of the initial level and rearrangement of spike rhythmicity according to the type of "spasmodic" discharges. Certain acceleration is noted in vital staining and discoloration of the receptors. This is expressed in shortening coloration time, in increasing rate for accumulation restorative equivalents, in intensified granuloformation. Ultrastructural shifts are mainly demonstrated in normalization of the mitochondrial apparatus.     

Neurite-promoting activities for embryonic spinal neurons and their developmental changes in the chick

Spinal motoneurons may depend upon muscle-derived factors for axon outgrowth and stabilization at two principal stages of their development: during the initial invasion of the differentiating muscle masses in the embryo and during the perinatal regression of multiple innervation. Using a bioassay involving the measurement of neurite outgrowth from 4.5-day embryonic chick spinal neurons in dissociated cell culture, neurite-promoting activities were detected both in medium conditioned over embryonic chicken myotubes in vitro (embryonic muscle-conditioned medium) and in soluble extracts of chick leg muscle prepared 3-5 days after hatching (postnatal muscle extract). The molecules responsible for these two activities had physicochemical properties that distinguished them both from each other and from some other reported neurite-promoting factors. The factor in embryonic muscle-conditioned medium, although active on uncoated tissue culture wells, bound with only low affinity to tissue culture plastic under cell culture conditions. It was inactivated by incubation with trypsin, and was essentially found only in media conditioned by muscle and liver cells. The factor in PNME, on the other hand, bound to plastic culture wells and was found in extracts of a variety of tissues. Its concentration in postnatal leg muscle was developmentally regulated: the specific activity increased approximately 10-fold between hatching and Day 3 (maximum value: 3200 units/mg protein) and then fell back to nearly its original levels by Day 7. Evidence is presented that the observed effects of these two neurite-promoting factors did not result from differential survival in vitro of different cell subpopulations. Possible roles for the two active factors during motoneuron development are discussed.     

Effect of fenibut on the GABA B receptors of the spinal motor neurons

It has been established in experiments on the isolated spinal cord of 7-14-day-old rats that the GABAB-mimetic phenibut (10(-5)--10(-4) M) elicits a slow-developing depolarization of motoneurons, suppression of spontaneous activity and polysynaptic reflex discharges of motoneurons, recorded from the ventral roots. Administered under the same conditions GABA produces de- and hyperpolarization of motoneurons. The depolarization of motoneurons elicited by phenibut and GABA is not reversed by picrotoxin in contradistinction to the GABA-induced hyperpolarization of motoneurons, being associated with a direct action of the GABA-mimetics on postsynaptic GABAB receptors of motoneurons. Diazepam (10(-9)--10(-6) M) potentiates the effects of phenibut supposedly via benzodiazepine receptors bound with GABAA receptors (an independent interaction).     

Localization and interaction of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of lamprey spinal neurons.

Small volumes of N-Methyl-D-Aspartate (NMDA) and non-NMDA excitatory amino acid receptor agonists were applied to localized regions of the dendritic trees of lamprey spinal neurons along their medial-lateral axis to obtain a spatial map of glutamate receptor distribution. Voltage clamp and frequency domain methods were used to obtain quantitative kinetic data of the voltage dependent ionic channels located both on the soma and on highly branched dendritic membranes. Pressure pulses of NMDA applied to the most peripheral regions of the dendritic tree elicited large somatic impedance increases, indicating that the most peripheral dendrites are well supplied with NMDA receptors. Experiments done with kainate did not elicit somatic responses to agonist applications on peripheral dendrites. The data obtained are consistent with the hypothesis that the activation of NMDA receptors by exogenous glutamate is significantly modified by the simultaneous activation of non-NMDA receptors, which shunts the NMDA response. The non-NMDA shunting hypothesis was tested by a combined application of kainate and NMDA to mimic the action of glutamate showing that the shunting effect of non-NMDA receptor activation virtually abolished the marked voltage dependency typical of NMDA receptor activation. These data were interpreted with a compartmental neuronal model having both NMDA and non-NMDA receptors.     

Spinal ganglion neurons of rats--a model for the study of the central serotonin receptors

Application of 5-hydroxytryptamine (5-HT) (3 x 10(-5) M) on the rat lumbar dorsal ganglia (RDG) induced membrane depolarization with increased input resistance in 30% of neurons, hyperpolarization with decreased input resistance in 30% of neurons and mixed responses in 40% of neurons. Methysergide and amitriptyline (10(-6) M) blocked depolarizing but not hyperpolarizing effects of 5-HT. Propranolol (3 x 10(-6) M) was inactive in respect to both 5-HT responses. 5-HT depolarizing responses of RDG neurons were mediated by 5-HT2 receptors activation and decreased membrane potassium conductivity; 5-HT hyperpolarizing responses were mediated by 5-HT1A receptor activation and increased potassium conductivity. RDG neurons seem to be an interesting model for the investigation of central 5-HT receptor mechanism.     

Asymmetric cross-inhibition between GABAA and glycine receptors in rat spinal dorsal horn neurons

Presynaptic nerve terminals of inhibitory synapses in the dorsal horn of the spinal cord and brain stem can release both GABA and glycine, leading to coactivation of postsynaptic GABAA and glycine receptors. In the present study we have analyzed functional interactions between GABAA and glycine receptors in acutely dissociated neurons from rat sacral dorsal commissural nucleus. Although the application of GABA and glycine activates pharmacologically distinct receptors, the current induced by a simultaneous application of these two transmitters was less than the sum of currents induced by applying two transmitters separately. Sequential application of glycine and GABA revealed that the GABA-evoked current is more affected by glycine than glycine-evoked responses by GABA. Activation of glycine receptors decreased the amplitude and accelerated the rate of desensitization of GABA-induced currents. This asymmetric cross-inhibition is reversible, dependent on the agonist concentration applied, but independent of both membrane potential and intracellular calcium concentration or changes in the chloride equilibrium potential. During sequential applications, the asymmetric cross-inhibition was prevented by selective GABAA or glycine receptor antagonists, suggesting that occupation of binding sites did not suffice to induce glycine and GABAA receptors functional interaction, and receptor channel activation is required. Furthermore, inhibition of phosphatase 2B, but not phosphatase 1 or 2A, prevented GABAA receptor inhibition by glycine receptor activation, whereas inhibition of phosphorylation pathways rendered cross-talk irreversible. Taken together, our results demonstrated that there is an asymmetric cross-inhibition between glycine and GABAA receptors and that a selective modulation of the state of phosphorylation of GABAA receptor and/or mediator proteins underlies the asymmetry in the cross-inhibition.     

Metabolic, electrophysiological and structural effects of dicaine and trimecaine on tissue bushy receptors

By means of a complex technique, including electrophysiological control, vital microscopy, cytophotometry and electron microscopy, reaction of the tissue bushy receptors of the frog urinary bladder to the effect of 0.05% solution of tetracaine hydrochloride (TeH) and trimecaine hydrochloride (TrH) has been investigated. Together with fading afferent impulse activity, the level of restorative equivalents increases sharply; this demonstrates that the anaesthetics influence not only plasmolemma but cytosol of the terminal. In neuroplasm of the terminal plates and receptor fibers certain ultrastructural changes take place (rearrangement of organelles, changes in vesicles, accumulation of glycogen granules). These changes are interpreted as functional-adaptive. Simultaneously, the character of vital staining of the receptors also changes (saturation of the colour, granule formation and time of decoloration). Certain specificity is noted in reaction of the receptors to TeH and TrH. The latter alters more essentially the microstructure both at the effect during 2 and 20 min. The effect of the local anaesthetics is evidently determined not only by the membranotropic effect, but by the influence on the cytosol of the terminal.     

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.     

Comparative study of dehydrogenase and diaphorase activity in spinal ganglion neurons in vivo and during their cultivation in vitro]

Enzymatic activity was investigated in spinal ganglia of 10-day-old chick embryo and in 10-day-old cultures of these ganglia (the method of "flying glasses"). Histochemical preparations were photometrically treated with the one-wave plug-method in the device MCFB-1 (LOMO) with the diameter of the probe 8 mkm. As the result of the investigations on optic density, a definite conclusion was made about relative activity of 15 enzymes and a general scheme of neuronal metabolism was graphically presented under the conditions of cultivation, enzymatic activity of krebs cycle was demonstrated to decrease, while that of diaphorase and lactate dehydrogenase to increase. The data on topography of enzymatic distribution in neurons, satellites, lemmoncytes and fibroblasts were also presented.     

Manifestations of the mobility of sensory neurons of the spinal ganglion after stimulation of its peripheral receptors

The body of sensory neurons in the frog spinal ganglion++ possesses an active mobility. This is demonstrated as pulsation and changes in its form. The mobility is closely connected with electrogenesis of cells. When an irritation is applied (press of a needle, or application of acetylcholine on the urinary bladder receptors), the neurons of the node respond with an increasing rate of impulse activity. Simultaneously, the mobility of these neurons increases. The character and form of their mobility in response to mechano- and chemoirritation differ from each other. Possible mechanisms of connection between the form of the cell body mobility and the character (active or passive) of its electrogenesis are considered. The mobility of the neurons, together with other morphological signs (tinctorial peculiarities and some others) can serve as an indication of their functional state without application of electrodes in morphological experiments.     

Responses of rat spinal ganglion neurons mediated by activation of serotonin receptors of the third type

Application of serotonin (5-hydroxytryptamine; 5-HT) to rat dorsal root ganglion neurons under conditions in which potassium conductance was blocked by cesium ions elicited depolarizing responses followed by an increase in membrane conductance. The responses did not exhibit desensitization and were due to activation of 5-HT receptors of the third type (5-HT₃Rs), since they were insensitive to methysergide, the 5-HT₂R antagonist, but were inhibited by tropicetrone (ISC 205–930) and metoclopramide, the 5-HT₃R antagonists. The reversal potential of the 5-HT-induced depolarizing responses was –11.9 mV; their amplitude decreased following a decrease in extracellular Na⁺ concentration but remained constant after intracellular injection of GTP. The amplitude of the responses increased following elevation of intracellular cAMP concentration caused by theophylline or sodium fluoride whose potentiating effect was reduced by butamide, a protein kinase A inhibitor. Potentiation of the 5-HT-induced responses was also produced by increased intracellular Ca²⁺ concentration following either direct intracellular injections or a burst of action potentials. The potentiation could be prevented by trifluoroperazine, the calmodulin inhibitor. The 5-HT effects were also potentiated by methylfurmetide, an activator of muscarinic acetylcholine receptors. The effect of methylfurmetide was slightly decreased by trifluoroperazine and was markedly decreased by polymixin B, a protein kinase C inhibitor. The effects of 5-HT were also enhanced by ethanol.Neirofiziologiya/Neurophysiology, Vol. 25, pp. 258–263, July–August, 1993.     

Reaction of sensory neurons of the spinal ganglia to sectioning of their peripheral and central processes

Transversal ++semi-sectioning of the spinal ganglion (SG) is a good model for studying the reaction of the ganglional sensory neurons to sectioning of their peripheral and central processes. At sectioning the peripheral and central processes of the SG neurons degeneration of the neurons and their death take place. The degenerative processes are more pronounced in the neurons with the peripheral processes sectioned, and the restorative ones-with the central processes sectioned. The dynamics of the posttraumatic changes in absolute number of the neurons, profile areas of the body fields and neuronal nuclei, amount of neurons with certain signs of axonal reactions in the SG demonstrate a maximally pronounced reaction on the 7th day and beginning of restorative processes on the 15th day. They are not completed by the 180th day.     

Action of precursors of noradrenaline synthesis on membrane receptors of chick embryo spinal neurons

Effects of noradrenaline precursors on glycine and N-methyl-D-aspartate (NMDA) receptors in spinal cord neurons recently isolated from chick embryo were investigated using whole cell patch-clamp and concentration clamp techniques. Both L-alanine and L-DOPA were found to be glycine agonists capable of potentiating NMDA response, while L-tyrosine does not activate glycine but can potentiate NMDA response. Lastly, L-phenylalanine and dopamine do not interact with either glycine or NMDA receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 665–670, September–October, 1990.     

Peculiarities of dopamine receptors on the membrane of spinal cord multipolar neurons of the brook lamprey Lampetra planeri

On isolated multiporal neurons of spinal cord of amniocoete larva of the brook lamprey Lampetra planeri, by the patch-clamp method in configuration "the whole cell", a modulating effect of dopamine on potential-activated Na+ currents was studied. Application of dopamine (10 microM) was shown to produce a complex action on the sodium current amplitude. In some cases a decrease of the amplitude, on average, by 13.5 +/- 2.2% was found, while in others--an increase, on average, by 8.6 +/- 6.1%. The modulation dopamine effect was not accompanied by any changes either of the threshold of the current appearance or of resistance of neuronal cell membranes. Pharmacological analysis with use of dopamine agonist has shown that the agonist of D1-receptors (-)-SKF-38393 (10 microM) decreases the Na+ current amplitude, whereas the agonist of D2-receptors (-)-quinpirole (10 microM) can produce in different cells both an increase, by 30.7 +/- 17.0 %, and a decrease, by 13.2 +/- 3.1%, of the Na+ current amplitude. The obtained data indicate the existence of D1- and D2-receptors on the membrane of multipolar spinal neurons of the amniocoete larva of the brook lamprey. Study of action of antagonists has shown that the antagonist of D1-receptors (+)-SCH-23390 (10 microM) does not affect action of the agonist of D1-receptors (-)-SKF-38393 (10 microM); the antagonist of D2-receptors (-)-sulpiride (10 microM) blocks completely effects both of the agonist of D1-receptors (-)-SKF-38393 (10 microM) and of the agonist of D2-receptors (-)-quinpirole (10 microM). The antagonist of D1-receptors (+)-SCH-23390 (10 microM) produced no effect on action of the agonist of D1-receptors (-)-SKF-38393 (10 microM). The obtained data indicate peculiarities of dopamine receptors of Cyclostomata as compared with those in mammals.