bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow axonal transport in acrylamide neuropathy: different abnormalities produced by single-dose and continuous administration
Authors: Gold BG , Griffin JW , Price DL
Published: March 2018

Alterations in axonal caliber and neurofilament content have been associated with altered neurofilament transport in several models of neurofibrillary degeneration. Acrylamide intoxication provides a prototype of distal axonal degeneration, the most frequent pattern of axonal pathology in human and experimental neurotoxic injury. Neurofibrillary changes are a variable and often minor aspect of the early pathological changes observed in acrylamide intoxication, and previous studies of slow axonal transport have produced conflicting results. In this study, we have correlated slow axonal transport, specifically neurofilament transport, with structural changes in the sciatic nerve complex of rats exposed to acrylamide. To study direct toxic effects of acrylamide, young rats were given a single dose of acrylamide (75 mg/kg, i.p.). A second group received daily injections of acrylamide at a lower dose (30 mg/kg, i.p.) in order to study animals with established acrylamide neuropathy. The slow component of axonal transport was labeled by intraspinal injections of [35S] methionine. Transport of individual slow component polypeptides was compared to profiles obtained from age-matched controls. Similarly intoxicated rats were perfused for morphometric and morphological studies. Results demonstrate that two different abnormalities of the slow component of axonal transport arise at different stages during the development of experimental acrylamide neuropathy. Both patterns of altered transport have structural correlates which reflect the changes in neurofilament transport. Following a single high dose, there was a modest retardation of the leading edge of the slow component. At this time, neurofilaments accumulated in proximal axons with formation of axonal swellings. During chronic administration, when distal axonal degeneration was present, the proportion of neurofilaments in the slow component was markedly reduced, and there was prominent loss of caliber in proximal axons. We suggest that these early changes represent a direct toxic effect of acrylamide on slow transport, whereas the later changes reflect reordering of slow transport as a neuronal response to toxin-induced axonal injury. This latter effect is of sufficient magnitude to obscure the acrylamide-induced retardation of slow transport.
Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus
Authors: Takaki M , Branchek T , Tamir H , Gershon MD
Published: March 2018

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5- hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP- DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5- hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.
Isolation and characterization of an endogenous C-terminal fragment of the alpha-neo-endorphin/dynorphin precursor from bovine caudate nucleus
Authors: Evans CJ , Barchas JD , Esch FS , Bohlen P , Weber E
Published: March 2018

Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245–248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C- fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.
Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures
Authors: Katz DM , Kimelberg HK
Published: March 2018

Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5- hydroxytryptamine; [3H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [3H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [3H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [3H]-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+- dependent component of [3H]-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. We estimated that this system enabled the cells to concentrate [3H]-5-HT up to 44- fold at an external [3H]-5-HT concentration of 10(-7) M. Inhibition of [3H]-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of [3H]-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on [3H]-5-HT uptake.
The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. I. Immunological distinction between the two toxin-binding sites of the receptor
Authors: Gu Y , Silberstein L , Hall ZW
Published: March 2018

We have examined the effect of a serum from a patient with myasthenia gravis on the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptors (AChRs) of a mouse muscle cell line, C2. After a 2-hr incubation, antibodies in the serum reduced toxin binding to C2 myotubes to a maximal extent of approximately 50%. The degradation of surface AChRs could account for the loss of only 5% of sites during the incubation; the remainder, therefore, must have been lost by blockage of binding. To investigate whether the antibodies blocked specifically one of the two toxin-binding sites that each AChR possesses, we used an analysis based on that of Sine, S. W., and P. Taylor, [1981) J. Biol. Chem. 255: 10144–10156). Although the two sites could not be distinguished by their rates of binding of alpha-BuTx, d-tubocurarine (dTC) inhibition of the initial rate of toxin binding revealed that the sites had affinities for dTC that differed by approximately 30-fold. Incubation with the myasthenic antibodies reduced the number of high affinity dTC sites, without affecting those of low affinity. We conclude that the two toxin-binding sites of the AChR are immunologically distinct.
Loss of supernumerary axons during neuronal morphogenesis
Authors: Heathcote RD , Sargent PB
Published: March 2018

The morphogenesis of individual neurons was investigated in the cardiac ganglion of the frog. Intracellular injection of horseradish peroxidase shows that mature neurons lack dendrites and have a single axon. Early in development, more than half of the neurons are multipolar and have as many as four processes emanating from their cell body. The most likely mechanism for the developmental transformation of larval neurons is that the supernumerary processes are pruned from the cell body. Supernumerary processes in larval neurons have features characteristic of axons. The processes of larval neurons can be highly branched and extend throughout the target with distinctive varicosities along the length of each process. Electron microscopy shows that all processes of individually injected cells contain clusters of vesicles apposed to active zones. Thus, the larval cardiac ganglion neuron is capable of extending more than one axon from its cell body.
Molecular weight determinations of soluble and membrane-bound fractions of choline O-acetyltransferase in rat brain
Authors: Badamchian M , Carroll PT
Published: March 2018

Three fractions of choline O-acetyltransferase (ChAT) (EC 2.3.1.6) were solubilized from a nerve ending fraction of rat forebrain using three sequential washes of an increasingly chaotrophic nature (100 mM sodium phosphate, pH 7.4; 500 mM NaCl; 2% Triton DN-65) as previously described (Benishin, C.G., and P.T. Carroll (1983) J. Neurochem. 41: 1030–1039). The molecular weights of the soluble (NaP) and membrane- bound fractions (NaCl and 2% Triton DN-65) of ChAT, following partial purification, were determined using either gel filtration on Sephadex G- 200, G-100 Superfine, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by “Western blotting” and immunochemical visualization of ChAT with four different anti-ChAT monoclonal antibodies (Ab8, Ab9, 4D7, and 1E6). Results obtained with gel filtration indicated that the NaP- and Triton DN-65-solubilized fractions of ChAT had molecular weights in the range of 73,000 to 78,000, whereas the NaCl-solubilized fraction of ChAT had a molecular weight in the range of 230,000 to 240,000. Results obtained with SDS- PAGE and Western blotting indicated that all three fractions of ChAT were composed of the same nonidentical subunits.
Cholecystokinin potentiates dopamine-mediated behaviors: evidence for modulation specific to a site of coexistence
Authors: Crawley JN , Stivers JA , Blumstein LK , Paul SM
Published: March 2018

Cholecystokinin coexists with dopamine in mesolimbic neurons in mammalian brain. When injected directly into the nucleus accumbens, cholecystokinin (CCK) potentiated dopamine (DA)-induced hyperlocomotion and apomorphine-induced stereotypy. These effects were not mimicked by nonsulfated CCK, but were blocked by proglumide, a putative CCK antagonist, as well as by antisera raised against sulfated CCK. CCK alone had no effect on locomotion or sterotypy, indicating that this peptide acts primarily as a modulator of DA-mediated behaviors in the mesolimbic pathway. In addition, CCK did not potentiate DA-induced hyperlocomotion or apomorphine-induced stereotypy when injected into the caudate nucleus, where CCK and DA are localized in separate neurons in rats. Facilitation of DA-mediated behaviors by CCK may represent a functional interaction specific to the neuromodulator-neurotransmitter coexistence phenomenon.
Differential synapse formation and neurite outgrowth at two branches of the metacerebral cell of Aplysia in dissociated cell culture
Authors: Schacher S
Published: March 2018

The metacerebral cell (MCC) of Aplysia californica was isolated with its bifurcate axon from the cerebral ganglion and maintained in vitro under three conditions: (a) with no targets, (b) with identified buccal ganglion neurons B1 or B2 placed near the stump of the large diameter cerebral-buccal connective (CBC) branch, and (c) with B1 or B2 placed near the stump of the small diameter posterior lip nerve (PLN) branch. After 5 days in culture, the two branches differed significantly in the formation of chemical connections and in the extent of neurite outgrowth. Chemical connections characteristic of MCC-B1(B2) connections in vivo were observed in more than 90% of the cultures in which the buccal neuron was contacted by neurites emerging from the CBC branch, but in only 20% of the cultures in which the buccal neuron was contacted by neurites extending from the PLN branch. Neurite outgrowth from the CBC stump was always greater than growth from the PLN and was not affected significantly by the presence of a buccal neuron target at either branch. In contrast, neurite outgrowth from the PLN decreased significantly when the target was contacted by neurites from the CBC branch. These results suggest that two branches of a single neuron can differ in their capacities to form chemical connections. In addition, the two branches show differential growth as a result of target interaction at one of the branches. This simple in vitro system may therefore be useful in exploring the ways in which individual neurons control neurite extension from different branches as they seek to form chemical connections with their targets.
Physiological and morphological analysis of synaptic transmission between leech motor neurons
Authors: Granzow B , Friesen WO , Kristan WB
Published: March 2018

In the leech Hirudo medicinalis inhibitory motor neurons to longitudinal muscles make central inhibitory connections with excitatory motor neurons to the same muscles. We have used a variety of physiological and morphological methods to characterize these inhibitory connections. The efficacy of the transmission between the inhibitors and the excitors was measured by using three intracellular electrodes, two in the inhibitor (one for injecting current and one for measuring voltage) and a third electrode in the excitor for measuring the resultant voltage changes. We have determined that delta Vpre/delta Vpost, or what we have called the transmission coefficient, is X = 0.51, as measured in the somata of the two cells. Evidence which we have obtained leads us to propose that these inhibitory connections between motor neurons are probably monosynaptic. The synaptic latency is consistent with a monosynaptic connection. In addition, a double- labeling technique, whereby one neuron was filled with Lucifer Yellow and the other with horseradish peroxidase (HRP), was used to determine the anatomical relationship between inhibitors and excitors in whole mounts. This revealed varicosities on the processes of inhibitor motor neurons which appear to make contact with processes of excitor motor neurons. A second double-labeling technique, whereby one neuron was filled with HRP and the other with an electron-dense particulate marker, revealed adjacent processes between an inhibitor and an excitor in electron microscopic thin sections which could be the sites of synaptic contact between the neurons. The connections appear to be mediated largely by graded transmitter release from the inhibitory motor neurons. Three different methods were used to demonstrate that synaptic transmission remained in the absence of impulses in the inhibitory motor neurons. These included eliminating the impulse- supporting portion of the motor neuron by pinching off its axon, abolishing impulses by replacing Na+ with Tris in the medium bathing the nerve cord, and increasing the threshold for impulse production by raising the Mg2+ and Ca2+ concentrations in the medium bathing the nerve cord.
An immunohistochemical and quantitative examination of dorsal root ganglion neuronal subpopulations
Authors: Price J
Published: March 2018

Sensory neurons of adult rat lumbar dorsal root ganglia were labeled in cryostat sections with antisera against tyrosine hydroxylase (TH), substance P (SP), and somatostatin (SOM), and with a monoclonal antibody (RT97) that labels the 145- and 200-kilodalton (kd) subunits of neurofilaments. These neurons were also histochemically stained for fluoride-resistant acid phosphatase (FRAP), and the size and distribution of each population were determined. In addition, the double-label immunoperoxidase technique of Sternberger and Joseph (Sternberger, L.A., and S.A. Joseph (1979) J. Histochem. Cytochem. 27: 1424–1429) was employed to determine whether these antibodies labeled distinct or overlapping populations of neurons. The results indicate that the dopaminergic (TH+) cells constitute a separate population from the SP+ and SOM+ neurons and that the size distributions of the SP+, SOM+, TH+, and FRAP+ cells are all different despite the fact that all of these subpopulations are part of the “small dark” subpopulation as indicated by their size and by the fact that they are RT97-. RT97 is a putative marker for the “large light” population (Anderton, B., H.B. Coakham, J. A. Garson, A.A. Harper, and S.N. Lawson (1982) J. Physiol. (Lond.) 334: 97–98P). Furthermore, the distribution data indicate that all of the “small dark” cell subpopulations are not evenly distributed within the ganglion, and the staining with RT97 and with another antibody which recognizes the 68-kd neurofilament subunit indicates heterogeneity among the “large light” population. These results are discussed in terms of the significance of the “small dark”-“large light” classification.
Specific labeling of rat brain substance P receptor with [3H]physalaemin
Authors: Mohini P , Bahouth SW , Brundish DE , Musacchio JM
Published: March 2018

The binding of [3H]physalaemin [( 3H]PHY) to rat brain membranes is specific, saturable and reversible in the presence of monovalent cations and peptidase inhibitors. Monovalent cations increase the binding of [3H]PHY in an ionic strength (mu)-dependent manner with an optimal effect at mu higher than 0.3. Addition of 2.5 mM MnCl2 results in a 2-fold increase in the affinity (KD) and a 40% increase in the maximal receptor density (Bmax). Scatchard analysis under these conditions indicates the existence of a single population of noninteracting sites with KD of 3.6 nM and a Bmax of 76 fmol/mg of protein. Substance P (SP) and physalaemin are equipotent in inhibiting the binding of [3H]PHY, whereas the potency of SP(2–11), SP(3–11), and SP(4–11) decreased in inverse proportion to their length. The relative affinity of the different tachykinins, SP, and SP fragments in competing with [3H]PHY correlates with their potency to stimulate several bioassay systems, indicating that [3H]PHY labels a physiologically relevant binding site that correspond to the SP-P tachykinin receptor. Guanine nucleotides completely abolish the increase in the binding of [3H]PHY produced by 2.5 mM MnCl2, but in its absence, the nucleotides reduce binding only by 15%. Guanine nucleotides reduce binding to the same level regardless of the presence or absence of the divalent cation. Regional distribution studies confirm that the density of SP receptors is maximal in the olfactory bulb, followed by the hypothalamus, striatum, hippocampus, cortex, and cerebellum.
Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells
Authors: Hefti F , Hartikka J , Frick W
Published: March 2018

Administration of gangliosides has been reported to stimulate regeneration of motoneurons and of central dopaminergic and cholinergic neurons. To shed light on the mechanism by which gangliosides mediate the effects on cholinergic neurons, we studied their actions on cultures of cells dissociated from the septal area of fetal rat brains. These cultures contain cholinergic neurons, which, in vivo, give rise to the cholinergic septo-hippocampal pathway. Gangliosides produced prominent changes in the morphological appearance of the cultures. In contrast to control cultures, which contained many process-bearing cells and a confluent layer of flat cells, there were no flat cells in cultures grown in the presence of gangliosides (0.2 to 0.8 mg/ml of medium). Using immunocytochemical visualization of the astrocytic marker glial fibrillary acid protein, it was shown that all astrocytes in cultures grown in the presence of gangliosides exhibited the morphology of process-bearing cells, whereas in control cultures astrocytes represented the majority of the flat cells. Furthermore, gangliosides attenuated astrocytic proliferation. The effects of gangliosides apparently were not mediated by cAMP, since they could be differentiated from actions of forskolin, an activator of adenylate cyclase. Astrocytic growth and morphology were affected by ganglioside mixtures of various sources and composition and also by the pure gangliosides GM1 and GD1a, whereas lipid and carbohydrate components of gangliosides were ineffective. In contrast to the prominent effects on astrocytes, gangliosides failed to significantly alter survival or fiber growth of cholinergic neurons.
The ultrastructural basis for synaptic transmission between primary muscle afferents and neurons in Clarke's column of the cat
Authors: Walmsley B , Wieniawa-Narkiewicz E , Nicol MJ
Published: March 2018

The synaptic connection between primary muscle afferents and dorsal spinocerebellar tract (DSCT) neurons has been studied in an attempt to reveal some of the mechanisms underlying excitatory transmission in the mammalian central nervous system. Previous electrophysiological experiments have shown that the excitatory postsynaptic potentials (EPSPs) evoked DSCT neurons by impulses in a single muscle afferent fluctuate in amplitude. These fluctuations occur between discrete amplitudes which are separated by quantal increments. Two alternative hypotheses relate such a quantal increment to all-or-nothing transmitter release from either (1) an entire synaptic bouton or (2) an individual transmitter release site, given that a bouton may contain multiple release sites. The present study was undertaken primarily to gain ultrastructural evidence on these proposals. Electrodes filled with horseradish peroxidase (HRP) were used to label single identified group Ia afferent fibers and DSCT neurons in the lumbar spinal cord of anesthetized cats. HRP-labeled Ia synaptic boutons, and the contacts formed between HRP-labeled Ia boutons and the dendrites of a DSCT neuron labeled intracellularly with HRP, were examined in serial sections under the electron microscope. Group Ia boutons were found to contain multiple synaptic specializations, as evidenced by pre- and postsynaptic thickenings and presynaptic clusters of vesicles. Careful examination of a bouton in serial sections revealed each specialization as a separate structure. These observations support the proposal that synaptic transmission between group I muscle afferents and DSCT neurons occurs with discrete all-or-nothing EPSPs associated with transmitter release sites, rather than boutons per se.
Membrane physiology of retinal glial (Muller) cells
Authors: Newman EA
Published: March 2018

Electrophysiological techniques were used to determine the ion selectivity properties and the spatial distribution of the membrane conductance of amphibian Muller cells. Membrane potential changes recorded during ion substitution experiments in frog (Rana pipiens) retinal slices demonstrated that the Muller cell K+:Na+ membrane permeability ratio is approximately 490:1 and that cell Cl- permeability is extremely low. In frog retinal slices, Muller cell input resistance was 8.5 megohms when measured in the inner plexiform layer and 4.8 megohms when measured in the optic fiber layer. Intact, enzymatically dissociated salamander (Ambystoma tigrinum) cells had an input resistance of 7.9 megohms, whereas cells lacking their endfoot process (removed by surgical microdissection or by shearing force) had a resistance of 152 megohms. Pressure ejection of a 100 mM K+ solution near the proximal surface of the endfeet of dissociated salamander cells produced depolarizations 7 times greater than did ejections near the lateral face of the endfoot and 24 to 50 times greater than did ejections near other cell regions. Similar K+ ejection results were obtained from Muller cells in salamander and frog retinal slices. Taken together, these results demonstrate that in both the frog and the salamander, approximately 95% of the total membrane conductance of Muller cells is localized in the cell's endfoot process. In salamander, the specific membrane resistance of the endfoot membrane was estimated to be 32 ohm X cm2 whereas the specific resistance of the remainder of the cell was 7300 ohm X cm2. This remarkably nonuniform conductance distribution has important consequences for theories concerning K+ regulation in the retina and for mechanisms underlying electroretinogram generation.
Kinematic features of unrestrained vertical arm movements
Authors: Atkeson CG , Hollerbach JM
Published: March 2018

Unrestrained human arm trajectories between point targets have been investigated using a three-dimensional tracking apparatus, the Selspot system. Movements were executed between different points in a vertical plane under varying conditions of speed and hand-held load. In contrast to past results which emphasized the straightness of hand paths, movement regions were discovered in which the hand paths were curved. All movements, whether curved or straight, showed an invariant tangential velocity profile when normalized for speed and distance. The velocity profile invariance with speed and load is interpreted in terms of simplification of the underlying arm dynamics, extending the results of Hollerbach and Flash (Hollerbach, J. M., and T. Flash (1982) Biol. Cybern. 44: 67–77).
Opiate- and alpha 2-adrenoceptor-induced hyperpolarizations of locus ceruleus neurons in brain slices: reversal by cyclic adenosine 3':5'- monophosphate analogues
Authors: Andrade R , Aghajanian GK
Published: March 2018

The purpose of this study was to investigate the ionic and second messenger mechanisms underlying the hyperpolarizations induced by the selective alpha 2-adrenoceptor agonist clonidine and the opiate agonist morphine in the locus ceruleus. Intracellular recordings were carried out in rat brain slices, and drugs at known concentrations were administered in the perfusate. The cyclic adenosine 3′:5′-monophosphate (cAMP) analogues 8-bromo-cAMP and dibutyryl cAMP, while not altering basal activity, reversed the hyperpolarizations induced by clonidine or morphine. In contrast, administration of the parent compound adenosine failed to affect these responses. These results are consistent with previous biochemical studies suggesting that alpha 2-adrenergic and opiate agonists might signal their actions by reducing intracellular cAMP levels. Under manual voltage clamp, both clonidine and morphine elicited outward currents. The algebraic sum of the individual currents elicited by morphine and clonidine significantly exceeded the actual current elicited by their co-administration. This nonadditivity, as well as the observation that cAMP analogues reverse the morphine- and clonidine-induced hyperpolarizations, suggests that these compounds hyperpolarize locus ceruleus neurons through a shared ionic mechanism the activation of which might be signaled by a decrease in intracellular cAMP.
Effects of body weight, adrenal status, and estrogen priming on hypothalamic progestin receptors in male and female rats
Authors: Etgen AM
Published: March 2018

Some laboratories have reported sex differences in the effect of estrogen priming on neural progestin receptor concentration, whereas others find no such differences. The present studies sought to identify experimental variables which might differentially influence the measurable level of hypothalamic cytosol progestin receptors in adult male and female rats. The influence of three variables, body weight, adrenal status, and estrogen priming regimen, was evaluated. Treatment with low doses of estrogen (2 or 8 micrograms of estradiol benzoate (EB) 48 hr before sacrifice) was slightly but not significantly more effective in elevating hypothalamic progestin receptor content in age- matched females than in males despite the 40% greater body weights of males. When the same doses of EB were administered per 200 gm of body weight to animals with and without adrenal glands, both basal and estrogen-induced levels of hypothalamic progestin receptor were significantly higher in adrenalectomized/gonadectomized rats than in rats which were gonadectomized only, regardless of sex. There was no significant effect of sex or any interaction between sex, adrenal status, or estrogen priming dose. The only significant sex difference in brain progestin receptors occurred when weight-matched males and females received multiple injections of higher priming doses of estrogen (three daily injections of 10 micrograms of EB). Under these conditions, females showed up to 2-fold higher levels of hypothalamic progestin receptors than males, regardless of adrenal status. These data suggest that sex differences may exist in the neural progestin receptor systems of male and female rats, but the relationship of these differences to sex differences in neuroendocrine function or behavior is not clear.
Formation of neuronal pathways in the imaginal discs of Drosophila melanogaster
Authors: Jan YN , Ghysen A , Christoph I , Barbel S , Jan LY
Published: March 2018

We have followed the formation of neuronal pathways in different imaginal discs of Drosophila. The pattern is highly reproducible for a given disc type but distinct for each type of discs: in leg discs, several neurons are present before metamorphosis and provide two major pathways that are joined by later neurons; in the wing and haltere discs, a few pairs of neurons appear after the onset of metamorphosis and pioneer the major pathways; in antenna discs, no pioneers are detected before massive neuronal differentiation begins. The mechanisms used for axonal guidance seem common to all discs, and the differences between discs can be accounted for simply by differences in the arrangement and birth time of pioneer neurons. Different subsets of pioneer neurons are deleted by mutations such as scute and engrailed.
Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nervous system
Authors: Macagno ER , Muller KJ , DeRiemer SA
Published: March 2018

In studies of axonal regeneration, it has been difficult to determine (a) whether growth along the normal pathway is important for restoration of connections with previous targets and (b) whether the new synapses resemble the old in strength and location. To address these problems at the level of individual nerve cells, we have studied touch (T) sensory neurons in the leech after their axons have been severed and we have confirmed that their axons regenerate electrical connections with some of their usual synaptic targets in the central nervous system. Injections of horseradish peroxidase and Lucifer Yellow dye into separate T cells in unoperated animals showed that T cell axons typically run close to one another within single ganglia or from ganglion to ganglion. Knowledge of one T cell's arborizations thus revealed the groundplan of others in the same ganglia and the sites of apparent contact with its synaptic targets. For regenerating axons, those sprouts that encountered the normal pathway (as marked by homologous axons) grew preferentially along it. Despite the striking coincidence of old and new pathways, regenerated branching patterns within the ganglionic neuropils were usually incomplete and sometimes had atypical branches. Synaptic connections with normal targets (other T cells as well as S and C cells) were abnormally weak physiologically. The numbers of apparent contacts seen with the light microscope were also lower than normal. In addition, the strength of the synaptic potentials, normalized to the number of contacts (calculated as microvolts per contact), was generally smaller in the regenerated connections than in the controls, and smallest at earliest times, during the first 6 weeks following injury. It thus appears to be characteristic of T cell regeneration that axon regrowth is aided by the recognition of specific pathways and that successful regeneration, as assayed anatomically and physiologically, occurs frequently but usually incompletely.
Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor
Authors: Boulter J , Luyten W , Evans K , Mason P , Ballivet M , Goldman D , Stengelin S , Martin G , Heinemann S , Patrick J
Published: March 2018

The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size- fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5′-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3′-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.
Muscle denervation increases the levels of two mRNAs coding for the acetylcholine receptor alpha-subunit
Authors: Goldman D , Boulter J , Heinemann S , Patrick J
Published: March 2018

The mRNA coding for the alpha-subunit of the acetylcholine receptor was studied in mouse leg and rat diaphragm muscle. We find that denervation of rat diaphragm results in a 7-fold increase in mRNA coding for the alpha-subunit, whereas denervation of mouse leg muscle results in approximately a 50-fold increase in alpha-subunit-specific mRNA. The relationship of the mRNAs purified from innervated and denervated muscle was investigated by SI nuclease mapping. Two mRNA species were found in both innervated and denervated muscle which differ in their 3′- untranslated region. The levels of both these mRNA species increase upon denervation of mouse leg muscle.
Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants
Authors: Wu CF , Haugland FN
Published: March 2018

The body wall muscles in Drosophila larvae are suitable for voltage clamp analysis of changes in membrane excitability caused by mutations. Both inward and outward ionic currents are present in these muscle fibers. The inward current is mediated by voltage-dependent Ca2+ channels. In Ca2+-free saline, the inward current is eliminated. The remaining outward K+ currents consist of two distinct components, an early transient IA and a delayed steady IK, which are separable by differences in the rate and voltage dependence of activation and inactivation. The steady-state and kinetic properties of the activation and inactivation processes of these two currents are analyzed. The results provide a basis for quantitative analysis of altered membrane currents in behavioral mutants of Drosophila. Previous studies indicate that mutations in the Shaker (Sh) locus alter excitability in both nerve and muscle in Drosophila. Our results support the idea that the channels mediating IA are molecularly distinct from those mediating IK. All Sh mutations studied specifically affect IA without changing the properties of the calcium current and IK. In certain alleles (ShKS133, Sh102, and ShM) IA is eliminated, permitting detailed studies of IK in isolation of IA. Studies of the alleles that do not eliminate IA provide additional information of the channels. In one such allele, Sh5, voltage dependence of IA activation is shifted to more positive potentials. This is accompanied by a less pronounced shift in the voltage dependence of inactivation. These results suggest that Sh5 mutation affects the voltage-sensitive mechanism of both activation and inactivation processes and that these two processes are not controlled by independent parts of the channel. Furthermore, the differential effects of these alleles on different excitable membranes imply that other genes take part in the control of IA. The effects of Sh5 on muscle depend on developmental stage. In larval muscle, Sh5 reduces the amplitude of IA because of the shift in the current-voltage (I-V) relation. In contrast, in adult Sh5 muscles, IA is reported to be normal in amplitude but shows abnormally rapid inactivation (Salkoff, L., and R. Wyman (1981) Nature 293: 228–230). A different allele, ShrK0120, causes a clear defect in nerve excitability, but analysis of IA in ShrK0120 larval muscle reveals I-V relations, inactivation, and recovery from inactivation similar to those seen in normal fibers. We suggest a possible mechanism of combinations of multiple interacting genes participating in the control of potassium channels to account for the presence of a variety of potassium channels in different excitable membranes.
Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin
Authors: Davis GE , Manthorpe M , Engvall E , Varon S
Published: March 2018

Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons. During the purification of this factor, the neurite-promoting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbent assay. The purified factor has an immunoreactivity per milligram of protein similar to that of purified rat yolk sac tumor laminin. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (kD) and two minor ones at about 130 and 35 kD. The 200-kD band comigrates with the 200-kD band of purified rat laminin. After SDS- PAGE under non-reducing conditions, the rat schwannoma factor and rat laminin both exhibit a band in the 900-kD range with the schwannoma factor band migrating slightly faster than the laminin one. The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the schwannoma factor and laminin are stained by immunoblotting with antisera raised against rat and human laminin and against a partially purified preparation of the schwannoma factor. On immunoblots the 400-kD band of laminin (a band not seen in the schwannoma factor preparation) also stains with all three antisera. When the antibodies from each of the three antisera are immobilized on protein A-agarose beads, the beads will completely remove from solution the neurite- promoting activities of both the schwannoma factor and laminin. Antibodies raised against rat laminin fail to block the neurite- promoting activity of the purified schwannoma factor but totally block that of rat laminin. In contrast, antibodies raised against the schwannoma factor will block the neurite-promoting activities of both the schwannoma factor and laminin. By rotary shadowing electron microscopy the schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse laminin. In addition, the schwannoma factor preparation contains images resembling proteoglycans.
gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture
Authors: McEachern AE , Margiotta JF , Berg DK
Published: March 2018

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma- aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.
Human spinal cord neurons in dissociated monolayer cultures: morphological, biochemical, and electrophysiological properties
Authors: Kato AC , Touzeau G , Bertrand D , Bader CR
Published: March 2018

The preparation of dissociated monolayer cultures from embryonic human spinal cord is described. Optimal survival was achieved with embryonic tissue between the eighth and ninth week. The neurons survive for as long as 7 weeks in culture and they grow in a standard tissue culture medium which contains 13% decomplemented human serum. The neurons have been identified by indirect immunofluorescence techniques using antibodies to tetanus toxin and neurofilament protein. Our biochemical studies demonstrate the presence of cholinergic and GABAergic neurons. Cholinergic neurons develop in culture and are more numerous in the cultures prepared from the anterior part of the spinal cord as compared to those from the posterior part. Therefore, it is possible that a large part of the cholinergic neurons derive from the motoneuron pool. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin (TTX). Voltage clamp experiments combined with the use of pharmacological blocking agents revealed the presence of several voltage- and time-dependent currents: a sodium current sensitive to TTX, a potassium current made up of two components, sensitive to tetraethylammonium and 4-aminopyridine, and a calcium current sensitive to cobalt. From a biochemical and electrophysiological point of view the properties of human spinal cord neurons in culture closely resemble the properties of spinal cord neurons from other species.
Neurotrophic activity in the adult rat hippocampal formation: regional distribution and increase after septal lesion
Authors: Collins F , Crutcher KA
Published: March 2018

Conditioned medium prepared from slices of the rat hippocampal formation contains an agent that shares the following properties with nerve growth factor (NGF): it promotes neurite growth from embryonic sympathetic ganglia in vitro; and it is inhibited by affinity-purified antibody against mouse submaxillary gland 2.5 S NGF. The NGF-like growth-promoting activity is regionally distributed within the hippocampal formation: the activity is consistently higher in the dentate gyrus-CA3 region than in the CA1 region. Furthermore, the level of activity is significantly increased within 1 week after a medial septal lesion, and, in the dentate-CA3 region, the increased level of activity is maintained for at least 4 weeks after the lesion. Control lesions that fail to interrupt the septohippocampal innervation, but cause equally extensive damage to nearby regions of the central nervous system, do not cause increased levels of activity in the hippocampal formation. These results provide substantial evidence linking the NGF- like agent in hippocampal conditioned medium to the sprouting of sympathetic axons into the dentate gyrus-CA3 region of the hippocampal formation after a medial septal lesion in vivo.