Morphological Study of Spatial Distribution of Vestibulospinal Neurons in the Frog Rana ridibunda

In the thew frog Rana ridibunda, local microphoretic injections of horseradish peroxidase into various parts of spinal cord were used for study of trajectory of retrograde enzyme-labeled fiber systems and topography of labeled neurons in vestibulospinal nuclei, the source of vestibulospinal fibers. The vestibulospinal tracts were shown to be formed by neurons of lateral vestibular nucleus, although descending vestibular nucleus also is partially involved, while medial vestibular nucleus contributes to even lesser degree. Besides, study of spatial distribution of C- and L-vestibulospinal neurons in the frog did not confirm the presence of the definite somatotopy that is characteristic of vestibular nuclei in mammals.     

Electrophysiological Study of Spatial Distribution of Vestibulospinal Neurons in the Frog Rana ridibunda

In experiments on a perfused brain preparation of the frog Rana ridibunda, the vestibulospinal neurons were identified, based on the excitatory postsynaptic potentials (EPSP) that appeared in response to an ipsilateral stimulation of the vestibular nerve and on the antidromic activity in response to stimulation of the cervical and lumbar enlargements of the spinal cord. The cells that could be antidromically activated only by stimulation of the cervical cord were designated as C-neurons. The cells that could be antidromically activated by stimulation of the lumbar cord were designated as L-neurons. The intracellular activity was recorded in 244 neurons of the vestibular nuclear complexes, out of which 127 cells (52%) were C-neurons and 117 (48%), L-neurons. The antidromic action potentials were recorded from the cells of lateral (143 neurons, 58.6%), descending (75 neurons, 30.7%), and medial (26 neurons, 10.6%) vestibular nuclei. The axon conduction velocity was determined to amount, on average, to 10.67 m/s for C-neurons and 15.84 m/s for L-neurons. In the vestibular nuclear complex, distribution of the fast and slow C- and L-neurons was studied. This study confirmed the previously made suggestion that C- and L-neurons of the frog, as sources of vestibular fibers, are distributed separately or, more often, as small groups, which leads to a patch-like somatotopy, rather than to formation of clearly separated fields.     

Activation of Na+,H+-Exchange in Erythrocytes of the River Lamprey Lampetra fluviatilis

To study H⁺ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H⁺ efflux from the erythrocytes. There was a tenfold decrease of the H⁺ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na⁺ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H⁺ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na⁺-dependent H⁺ efflux from the cells and H⁺-dependent Na⁺ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na⁺ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H⁺-dependent Na⁺ influx and an increase of the Na⁺ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na⁺/H⁺ exchanger in erythrocytes of the river lamprey.     

Different Sensitivity of Motoneuron Synaptic Inputs in the Frog, Rana ridibunda, to Antagonists of Excitatory Amino Acids

The effects of antagonists of excitatory amino acids (AP-5, kynurenate, and CNQX) on PSP recorded intracellularly in lumbar motoneurons of a preparation of the isolated spinal cord in the frog, Rana ridibunda, in response to activation of three different synaptic inputs (stimulation of DR, RF, and VC or LC) were analyzed. It is shown that the effects of the antagonists were non-uniform in different motoneurons. Inputs of suprasegmental and sensomotor projections substantially differed from each other. A considerable amount of DC-PSP resistant to kynurenate and CNQX was found, whereas the latter regularly inhibited DR-PSP in the same cell. The disynaptic, as judged by its latency, plane-shaped component was always relatively more stable to kynurenate as compared with other components. Unlike kynurenate that inhibited the early and late components, CNQF selectively depressed the early components of DR-PSP.     

苯胺对黑斑蛙蝌蚪外周血红细胞的遗传毒性效应

为从不同遗传终点检测苯胺对黑斑蛙(Rana nigromaculata)蝌蚪红细胞的遗传毒性,将黑斑蛙蝌蚪暴露于0、3.45、17.26、34.53、69.06μg/L不同浓度的苯胺96 h后,显微镜下观察红细胞形态和数目的变化,采用微核试验测定红细胞微核率,通过彗星试验测定彗星尾长和尾距的变化。从17.26μg/L浓度组开始出现红细胞变形拉长和细胞膜破裂,且随着苯胺浓度的增加而增多。另外,各浓度组蝌蚪红细胞数目随着苯胺溶液浓度的增加而逐渐减少,且与空白对照相比差异显著(P0.01)。微核试验结果显示,各浓度处理组微核率均显著高于空白对照组(P0.05),但由于苯胺所致的红细胞破裂和Heinz小体的影响,微核率和浓度之间并未出现明显的浓度-效应关系。彗星试验结果显示,不同浓度苯胺处理组与空白对照组相比,蝌蚪红细胞尾长和尾距均显著增加(P0.05或P0.01),并与处理浓度之间存在显著的浓度-效应关系。上述结果表明,苯胺可诱发黑斑蛙蝌蚪红细胞的染色体、DNA损伤,具有较强的遗传毒性效应;苯胺最高浓度处理组69.06μg/L蝌蚪红细胞DNA损伤水平与5 mg/L环磷酰胺相近,显现明显的DNA损伤,因此建议渔业水质标准对水体中苯胺限量的规定不应高于此值。     

The autonomic innervation of Rana ridibunda intestine

1. The innervation of Rana ridibunda intestine has been studied by the following methods: (a) demonstration of cholinesterase activity; (b) FIF method for catecholamines; (c) immunohistochemistry for VIP, SP and SOM, and (d) conventional electron microscopy. 2. The intrinsic intestinal innervation is represented by cholinergic-, VIPergic-, SP- and SOM-like plexuses. The intestinal adrenergic component is of extrinsic origin. 3. The intestinal peptidergic innervation is the most developed, the large intestine being the portion where the studied peptidergic plexuses are more widely distributed. 4. Against a poorly developed cholinergic/adrenergic innervation, it seems that there is a predominant peptidergic innervation in the amphibians intestine wall. 5. Taking into account that amphibians sacral parasympathetic as well as sympathetic innervation development are limited, it could be considered that in vertebrates the intestinal peptidergic innervation is phylogenetically earlier and hence better developed.     

The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.     

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K⁺ efflux and Cl⁻ loss. When human erythrocytes were suspended in a physiological NaCl solution (pH _o = 7.4), the measured pH _i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH _i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl⁻ content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H⁺ influx with the measured unidirectional K⁺ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H⁺ influx and a K⁺ efflux in LIS solution of 108.2 ± 20.4 mmol (l _cells hr)⁻¹ and 98.7 ± 19.3 mmol (l _cells hr)⁻¹, respectively. For bovine and porcine erythrocytes, in LIS media, H⁺ influx and K⁺ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K⁺(Na⁺)/H⁺ exchanger, inhibited the K⁺ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K⁺(Na⁺)/H⁺ exchanger in the human erythrocyte membrane. Received: 22 December 1999/Revised: 10 April 2000     

Analysis of Combined Action of Antagonists of Excitatory and Inhibitory Amino Acids on Motoneuron Postsynaptic Potentials of the Frog Rana ridibunda

The effect of blockers of excitatory and inhibitory amino acid receptors on postsynaptic potentials (PSP) evoked by activation of three synaptic inputs of the lumbar motoneuron (stimulation of the dorsal root, reticular formation, ventral and lateral columns) was studied on preparation of the isolated spinal cord of the frog Rana ridibunda. It has been shown that sensitivity of PSP to antagonists differs in different motoneurons, in the same motoneuron at activation of different inputs, and in the same input in different PSP components. It has been found that many descendent (DC) PSPs resistant to kynurenate or CNQX [1] were inhibited by blockers of inhibitory receptors. In this case the early component of DC-PSP varied considerably by amplitude and changed its polarity from positive to negative on the background of a low transmembrane depolarizing current. These changes were absent under conditions of replacement of chlorine ion by sulfate in the perfusion solution or treatment of the spinal cord with a blocker of inhibitory amino acids. All this allows suggesting that these DC-PSPs or their components were inhibitory. A part of PSPs resistant to kynurenate and CNQX were also resistant to the blockers of inhibitory amino acids (strychnine, picrotoxin, and bicuculline). In some cases, as a result of treatment with convulsants, the same blockers of excitatory receptors inhibited the initially resistant PSPs.     

Ranakinin: A Novel NK1 Tachykinin Receptor Agonist Isolated with Neurokinin B from the Brain of the Frog Rana ridibunda

An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P-like immunoreactivity, measured with an antiserum directed against the COOH-terminal region of mammalian substance P and neurokinin B-like immunoreactivity, measured with an antiserum directed against the NH2-terminus of neurokinin B. The primary structure of the substance P-related peptide (ranakinin) was established as: Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin B-related peptide was established as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence is the same as that of mammalian neurokinin B. Ranakinin was equipotent with substance P and [Sar9,Met(O2)11]substance P in inhibiting the binding of 125I-Bolton-Hunter-[Sar9,Met(O2)11]substance P, a selective radioligand for the NK1 receptor, to binding sites in rat submandibular gland membranes (IC50 1.6 +/- 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.     

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda

Summary The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types are recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH (1–24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.     

Activation of Na/H exchange by thrombin in peritoneal mast cells]

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.     

A Study of the Role of GABAA- and GABAB-Receptors in Presynaptic Inhibition of Primary Afferents in the Spinal Cord of the Frog Rana ridibunda

The role of GABA_A- and GABA_B-receptors in presynaptic inhibition of primary afferent fibers was studied on an isolated preparation of the spinal cord of the frog Rana ridibunda. It is shown that the inhibitory effect of GABA on synaptic transmission from afferent fiber to motoneuron is caused by activation of both GABA_A- and GABA_B-receptors. A temporal correlation (± 5 min) was shown between the blocking action of bicuculline (a specific antagonist of GABA_A-receptors) on primary afferent fiber depolarization (PAD) and its potentiating effect on the excitatory postsynaptic potential (EPSP) at parallel intracellular recording of EPSP in motoneuron and PAD in axons of the dorsal root. As a basis of this correlation, the single GABA_A-receptor mechanism is discussed, which mediates the effect of bicuculline on PAD and EPSP. When a specific agonist of GABA_B-receptor, baclofen, and an antagonist of GABA_B-receptor, 2(OH)-saclofen, were applied, the obtained data indicated an involvement of GABA_B-receptors in inhibition of synaptic transmission from afferent fibers to the motoneuron. Analysis of parameters of the unitary synaptic responses recorded in the control experiments and of their changes under the effect of (– )-baclofen indicates that the inhibitory action caused by activation of GABA_B-receptors develops at the presynaptic level.     

Cardiac minute volume and its pattern in Rana ridibunda frogs

In frogs with an average body mass 56 g, the minute volume of the heart is equal to 4.5 ml/min X 100 g, which is approximately an order lower than in mammals with the same body mass. Pulmonary fraction constitutes 52% of the minute volume of the heart. The main bulk of systemic fraction of the minute volume of the heart (78%) passes to locomotor system and skin, whereas 19% of this volume are adressed to vegetative organs. This pattern of distribution significantly differs from that in mammals with a similar body mass, in which the vegetative and locomotor fractions are approximately equal. Differentiation in muscular blood supply was noted--there is a threefold difference in the volume of blood flow between gastrocnemius and submandibular muscles.