Effects of Serotonin on Primary Afferents in the Frog Spinal Cord

We studied, on isolated preparations of the frog spinal cord, the effects of serotonin in different concentrations on the amplitude-temporal parameters of action potentials (AP) in primary afferent fibers, on the potentials reflecting depolarization of primary afferents (DPA), and on the properties of the membrane of these fibers. It was demonstrated that in a part of the dorsal root afferent fibers serotonin caused a drop in the AP amplitude (by 15-20%) and an increase in the AP duration (by 8-13%). Serotonin also significantly (by 70-90%) decreased the amplitude of DPA induced by stimulation of a neighboring dorsal root and noticeably reduced the input membrane resistance of afferent fibers. Serotonin-induced modulation of the AP parameters in the afferents and suppression of DPA under the influence of this amine are postulated as possible factors involved in the central control of afferentation.     

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 Frog Rana ridibunda

Transport of Na⁺ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope ^{22 22}Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na⁺ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na⁺ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of ^{22 22}Na accumulation and intracellular Na⁺ concentration. These changes of the Na⁺ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na⁺/H⁺ exchanger. Hence, PMA produced activation of Na⁺/H⁺ exchange in frog erythrocytes. The unidirectional Na⁺ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na⁺ influx (IC₅₀) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.     

Involvement of 5-HT1 and 5-HT5A Receptors in Modulation of Synaptic Transmission and Intrinsic Membrane Properties of Spinal Motoneurons of the Frog (Rana ridibunda)

Journal of Evolutionary Biochemistry and Physiology - The endogenous monoamine serotonin (5-HT) is involved in the modulation of motor output in vertebrates by interacting with different types of...     

The Distribution of Primary Nitric Oxide Synthase- and Parvalbumin- Immunoreactive Afferents in the Dorsal Funiculus of the Lumbosacral Spinal Cord in a Dog

1. The aim of the present study was to examine the distribution of unmyelinated, small-diameter myelinated neuronal nitric oxide synthase immunoreactive (nNOS-IR) axons and large-diameter myelinated neuronal nitric oxide synthase and parvalbumin-immunoreactive (PV-IR) axons in the dorsal funiculus (DF) of sacral (S1–S3) and lumbar (L1–L7) segments of the dog. 2. nNOS and PV immunohistochemical methods were used to demonstrate the presence of nNOS-IR and PV-IR in the large-diameter myelinated, presumed to be proprioceptive, axons in the DF along the lumbosacral segments. 3. Fiber size and density of nNOS-IR and PV-IR axons were used to compartmentalize the DF into five compartments (CI–CV). The first compartment (CI) localized in the lateralmost part of the DF, containing both unmyelinated and small-diameter myelinated nNOS-IR axons, is homologous with the dorsolateral fasciculus, or Lissauer tract. The second compartment (CII) having similar fiber organization as CI is situated more medially in sacral segments. Rostrally, in lower lumbar segments, CII moves more medially, and at upper lumbar level, CII reaches the dorsomedial angle of the DF and fuses with axons of CIV. CIII is the largest in the DF and the only one containing large-diameter myelinated nNOS-IR and PV-IR axons. The largest nNOS-IR and PV-IR axons of CIII (8.0–9.2 μm in diameter), presumed to be stem Ia proprioceptive afferents, are located in the deep portion of the DF close to the dorsal and dorsomedial border of the dorsal horn. The CIV compartment varies in shape, appearing first as a small triangular area in S3 and S2 segments, homologous with the Philippe–Gombault triangle. Beginning at S1 level, CIV acquires a more elongated shape and is seen throughout the lumbar segments as a narrow band of fibers extending just below the dorsal median septum in approximately upper two-thirds of the DF. The CV is located in the basal part of the DF. In general, CV is poor in nNOS-IR fibers; among them solitary PV-IR fibers are seen. 4. The analysis of the control material and the degeneration of the large- and medium-caliber nNOS-IR fibers after unilateral L7 and S1 dorsal rhizotomy confirmed that large-caliber nNOS-IR and and PV-IR axons, presumed to be proprioceptive Ia axons, and their ascending and descending collaterals are present in large number in the DF of the lumbosacral intumescence. However, in the DF of the upper lumbar segments, the decrease in the number of nNOS-IR and PV-IR fibers is quite evident.     

Dipeptide Antagonists of Amino Acid-Induced and Synaptic Excitation in the Frog Spinal Cord

Abstract: The dipeptide γ-d -glutamylglycine (γDGG) antagonizes amino acidinduced depolarization and synaptic excitation in the isolated hemisected spinal cord of the frog. In general, the effects of this compound resembled those of the structurally similar d -α-aminosuberate (DαAS) in being more effective against N-methyl-d -aspartate (NMDA)-induced responses than against responses induced by other excitatory amino acids. However γDGG appeared to be more effective than DαAS in depressing kainate-induced responses. Similar, though weaker, effects were produced by the L isomer of the dipeptide (αLGG), a natural brain constituent.     

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.     

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.     

A Study of the Mechanism of Internalisation of Tetanus Toxin by Primary Mouse Spinal Cord Cultures

The fate of tetanus toxin bound to neuronal cells at 0 degree C was followed using an anti-toxin 125I-protein A assay. About 50% of surface-bound toxin disappeared within 5 min of warming cells to 37 degrees C. Experiments with 125I-toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilizing cells with Triton X-100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin-gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin-gold particles bound to the cell body at 4 degrees C showed that it was concentrated in coated pits. After 5 min at 37 degrees C, toxin-gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti-toxins, and protein A-gold. Toxin-gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and intraaxonal uncoated vesicles, respectively.     

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.     

Characterization of the cDNA encoding proopiomelanocortin in the frog Rana ridibunda

In the amphibian pars intermedia, secretion of proopiomelanocortin (POMC)-derived peptides is controlled by multiple factors including classical neurotransmitters and neuropeptides. To pursue questions concerning the regulation of POMC gene expression in Rana ridibunda, we have isolated and characterized a full-length cDNA for frog POMC. A cDNA clone isolated from a frog pituitary library contains an open-reading frame of 780-bp that predicts a 260 amino acid POMC protein. The structure of frog POMC demonstrates considerable amino acid sequence similarity with POMC from other species. In particular, the sequence of alpha-melanotropin (alpha-MSH) is identical in frog and all mammalian species studied so far, while adrenocorticotropin (ACTH) and beta-endorphin exhibit 79% and 84% homology with their human counterpart. Frog POMC contains only one potential asparagine-linked N-glycosylation signal (Asn-Ser-Thr) within the gamma-MSH domain. The alpha-MSH sequence is C-terminally flanked by the Gly-Lys-Lys amidation signal while the joining peptide is not amidate.     

Interspecific Social Interactions and Breeding Success of the Frog Rana latastei: A Field Study

Interspecific reproductive interference can affect fitness‐related breeding performance, thus influencing fitness and distribution of populations. Laboratory studies demonstrated the social interference of Rana dalmatina males on R. latastei breeding females: the presence of heterospecific males reduced the percentage of viable embryos in R. latastei eggs. Here, we tested if the negative effects of R. dalmatina males on R. latastei reproductive success occur in field conditions. We compared the percentage of viable embryos of eggs laid in field conditions from populations where R. latastei breeds alone with the percentage of viable embryos of populations where R. latastei cohabits with R. dalmatina. We did not find any significant difference in percentage of viable embryos between R. latastei populations syntopic and allotopic with R. dalmatina, nor a relationship between the relative abundance of heterospecifics and reproductive success. In natural conditions, the presence of heterospecific males does not seem to interfere with the reproductive success of R. latastei. The experimental procedure may influence the interaction among individuals. Therefore, we suggest to validate on natural populations the results of experiments dealing with complex interactions.     

炎性小体在诱导脊髓损伤神经炎症中的作用

脊髓损伤的治疗与康复一直是医学领域的重大难题,尤其是在改善损伤的神经功能方面进展甚微。继发性损伤是造成脊髓损伤后神经功能障碍的主要原因,炎症反应是继发性损伤阶段最重要的病理过程。急性期通过抑制神经炎症来减轻继发性损伤被认为可减轻神经功能损害而达到神经保护作用。炎性小体是一类蛋白质复合体,由模式识别受体中的NLRs家族和PHYIN家族的受体蛋白质作为主要框架组装并命名,常见的炎性小体包括NLRP1、NLRP3、NLRC4(IPAF)、AIM2等。在感染或受到损伤刺激时,炎性小体在细胞质内组装,并激活促炎症蛋白酶胱天蛋白酶1(caspase-1),活化的胱天蛋白酶1一方面促进促炎症细胞因子IL-1β和IL-18的前体成熟和分泌,另一方面介导细胞焦亡。细胞焦亡以细胞肿胀破裂并释放细胞内容物为特征,是在炎症和应激的病理条件下诱导的程序性细胞死亡方式。促炎症细胞因子和焦亡释放的胞内物质都可作为促炎信号引发炎症反应。近期发现,炎性小体通过诱导促炎因子释放以及介导细胞焦亡等途径, 参与激活脊髓损伤后的炎症级联反应,加重继发性神经炎症。靶向抑制炎性小体的激活可减轻炎症反应,促进神经细胞存活,达到神经保护作用。因此,炎性小体有望成为脊髓损伤治疗的新靶点。本文拟从炎性小体的结构及其在脊髓损伤中的作用、激活机制和治疗前景进行综述,以期为后续研究提供思路。     

The autonomic innervation of the liver and gallbladder of Rana ridibunda

1--The innervation of the liver and gallbladder of Rana ridibunda has been studied by the following methods: (a) demonstration of cholinesterase activity; (b) FIF method for catecholamines; (c) immunohistochemistry for VIP and (d) electron microscopy. 2--The hepatocytes are arranged in regular rows of hepatic cords, very little connective tissue is distributed in the parenchyma, the innervation being restricted to the big branches of blood vessels. 3--Well defined cholinergic and adrenergic plexuses surround the hepatic arteries, portal veins and biliary ducts. The VIPergic innervation is scarce in the liver but a richly branched plexus spreads in the wall of the gallbladder. 4--Cholinesterase-positive cells are widely distributed accompanying the nerve trunks of the gallbladder. The innervation distribution is prominent in the portion of the gallbladder next to the hepatic hilus. 5--A population of melanin-storing cells besides free melanin granules are present in the liver parenchyma and are prominent in the gallbladder where the melanocytes are disposed in close contact with blood vessels and nerve structures. We have observed that the number of these visceral melanocytes considerably increases in winter, particularly in the liver.