bluuebunny/crossref_metadata_2025 huggingface dataset:
Pattern and specificity of axonal outgrowth following varying degrees of chick limb bud ablation
Authors: Tosney KW , Landmesser LT
Published: March 2018

Motoneurons grow into the chick hindlimb via consistent pathways, within which they make specific decisions leading to their correct targets. To determine which axonal guidance features dictate the position of the pathways and to examine the distribution of specific cues, we totally or partially ablated the early hindlimb bud and determined how the subsequent pattern of nerve outgrowth related to the distribution of tissue remnants. Our results suggest that local elements determine the gross anatomical pattern of outgrowth. First, determinants of individual pathways could be selectively removed without altering the pattern in other regions. Second, neurites were restricted to the plexus region at the base of the leg (within which, for unknown reasons, they proceeded posteriorly) unless distal permissive pathways or nearby target remnants were present. Finally, we found that the central region of the pelvic girdle, adjacent to the plexus region, determines the position where the major nerve trunks enter the leg. When gaps were introduced in this region of the girdle, nerves traversed the gaps and directly innervated adjacent muscle. The developing girdle is probably a nonpermissive environment for axon elongation, and axons enter the leg only where it is locally absent. Our results also support the concept that the specific cues that neurites use to reach their appropriate muscles are local. We find that neurites could make correct and specific decisions in the plexus region in the absence of all tissues distal to the pelvic girdle. This shows that the cues for these decisions are independent of the target and must reside in the local mesenchyme. In addition, when muscle remnants were present they were correctly innervated.(ABSTRACT TRUNCATED AT 250 WORDS)
Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin- immunoreactive material
Authors: Somogyi P , Hodgson AJ , Smith AD , Nunzi MG , Gorio A , Wu JY
Published: March 2018

The coexistence of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and cholecystokinin (CCK)- or somatostatin- immunoreactive material in the same neurons was studied in the hippocampus and visual cortex of the cat. One-micrometer-thick serial sections of the same neuron were reacted to reveal different antigens by the unlabeled antibody enzyme method. All CCK- and somatostatin- immunoreactive neurons in the cortex and all CCK-immunoreactive and the majority of somatostatin-immunoreactive neurons in the hippocampus that could be examined in serial sections were also immunoreactive for GABA. In neurons that were immunoreactive for GAD it was often possible to demonstrate immunoreactivity for one of the peptides as well as for GABA. GABA-immunoreactive neurons, as revealed by an antiserum to GABA, were present in all layers of the cortex and hippocampus, and their shape, size, and distribution were similar to GAD-immunoreactive neurons. All GAD-immunoreactive neurons were also positive for GABA, but the latter staining revealed additional neurons. CCK/GABA- and somatostatin/GABA-immunoreactive neurons were present mainly in layers II and upper III and in layers V and VI in the visual cortex. CCK/GABA- immunoreactive neurons were most frequently present in the strata oriens, pyramidale, and moleculare of the hippocampus and in the polymorph cell layer of the dentate gyrus. Somatostatin/GABA- immunoreactive neurons were localized mainly in the stratum oriens and in the hilus of the fascia dentata. The two peptides could not be found in the same neuron. The majority of neurons that were GABA immunoreactive did not stain for either peptide. The presence of CCK- and somatostatin-immunoreactive material in GABAergic cortical neurons raises the possibility that neuroactive peptides affect GABAergic neurotransmission.
Hypothalamic peptidyl-glycine alpha-amidating monooxygenase: preliminary characterization
Authors: Emeson RB
Published: March 2018

An enzymatic activity capable of converting mono-[125I]-D-Tyr-Val-Gly into mono-[125I]-D-Tyr-Val-NH2 was identified in a crude mitochondrial/synaptosomal preparation from rat hypothalamus. Further subcellular fractionation studies localized a majority of this enzymatic activity to fractions enriched in synaptic vesicles. The alpha-amidation activity demonstrated optimal activity at pH 7.5 to 8, was stimulated by the presence of copper ions and reduced ascorbate and required the presence of molecular oxygen. Endogenous alpha-amidation activity was inhibited by the addition of ascorbate oxidase. Kinetic analyses demonstrated Michaelis-Menten type kinetics for D-Tyr-Val-Gly as the varied substrate with the values of Km and Vmax increasing as the ascorbate concentration in the reaction increased. A variety of peptides possessing carboxyl-terminal glycine residues were potent inhibitors of the reaction, while peptides lacking a carboxyl-terminal glycine residue were not, suggesting that many glycine-extended peptides may serve as substrates in the alpha-amidation reaction. The characteristics of hypothalamic alpha-amidation activity are similar to those previously reported for the alpha-amidation activity in rat pituitary and mouse corticotropic tumor cells suggesting the presence of closely related enzymes in these tissues.
The control of firing pattern in nigral dopamine neurons: single spike firing
Authors: Grace AA , Bunney BS
Published: March 2018

Dopamine (DA) neurons have been recorded in vivo in four states of activity: hyperpolarized, nonfiring; single spike firing; burst firing; and depolarization inactivation. Nonfiring DA neurons can be made to fire by iontophoretic application of the excitatory substances glutamate and cholecystokinin, or by depolarizing current injection. Spontaneously active DA cells typically fire in a slow (3 to 8 Hz) irregular pattern. In vivo intracellular recordings revealed that this pattern is sustained by the alternation of two currents: a spontaneously occurring slow depolarization (13 +/- 3 mV amplitude, 78 +/- 40 msec duration) which brings the membrane potential of the DA cell to spike threshold (-42 mV), and an afterhyperpolarization mediated by a calcium-activated potassium conductance (IK(Ca)). The slow depolarization is a pacemaker-like conductance, with a rate of rise proportional to the membrane potential. The regular pacemaker pattern of the spontaneously occurring slow depolarization is interrupted by the IK(Ca) which appears to be triggered by calcium entry during the action potential. Thus, intracellular injection of the calcium chelator EGTA will cause DA cells to fire in a regular, pacemaker pattern. The IK(Ca) is observed after single spikes and trains of spikes with the amplitude of the afterhyperpolarization being proportional to the number of spikes in a train. Both the afterhyperpolarization and the firing accommodation observed during depolarizing current injection can be blocked by intracellular injection of the calcium chelator EGTA.
The control of firing pattern in nigral dopamine neurons: burst firing
Authors: Grace AA , Bunney BS
Published: March 2018

In addition to firing in a single spiking mode, dopamine (DA) cells have been observed to fire in a bursting pattern with consecutive spikes in a burst displaying progressively decreasing amplitude and increasing duration. In vivo intracellular recording demonstrated the bursts to typically ride on a depolarizing wave (5 to 15 mV amplitude). Although the burst-firing frequency of DA cells showed little correlation with the base line firing rate, increases in firing rate were usually associated with an increase in burst firing. Increases in burst firing could also be elicited by intracellular calcium injection and could be prevented by intracellular injection of EGTA, suggesting a calcium involvement in bursting. Blockade of potassium conductances with extracellular iontophoresis of barium or intracellular injection of tetraethylammonium bromide could also trigger an increased degree of burst firing in DA cells. These data suggest that the increased calcium influx accompanying an increased firing rate triggers burst firing, possibly by inactivating a potassium conductance. A switch from a single spiking mode to a burst-firing mode may be important in modulating striatal DA release, as shown for burst firing in other preparations.
Binding of the nicotinic cholinergic antagonist, dihydro-beta- erythroidine, to rat brain tissue
Authors: Williams M , Robinson JL
Published: March 2018

The nicotinic cholinergic antagonist, dihydro-beta-erythroidine, binds to two sites in rat cortical membranes with dissociation constants of 4 and 22 nM and respective apparent Bmax values of 52 and 164 fmol/mg of protein. Binding to the higher affinity site, defined by the use of 2 nM [3H]dihydro-beta-erythroidine, was saturable, reversible, and susceptible to protein denaturation. Binding was highest in the thalamus and lowest in the spinal cord and showed preferential enrichment in a synaptosomal subfraction of rat brain. Nicotine displaced [3H]dihydro-beta-erythroidine in a stereospecific manner, the (-)-isomer being approximately 6 times more potent than the (+)-isomer. The alkaloid nicotinic agonists, cytisine and lobeline, were potent inhibitors of binding, while acetylcholine in the presence of the cholinesterase inhibitor di-isopropylfluorophosphate was equipotent with (+)-nicotine. Binding was also inhibited by the muscarinic ligands, arecoline, atropine, and oxotremorine. The nicotinic antagonists mecamylamine, hexamethonium, and pempidine were essentially inactive in displacing [3H]dihydro-beta-erythroidine. These findings indicate that dihydro-beta-erythroidine binds to a nicotinic recognition site in rat brain which is neuromuscular, rather than ganglionic, in nature and that such binding is similar in several respects to that seen with nicotinic agonists. Whether such binding is to a nicotinic, as opposed to nicotinic cholinergic, recognition site or to a “common” nicotinic/muscarinic site is an issue that requires further study.
The effects of nerve growth factor and its antiserum on the postnatal development and survival after injury of sensory neurons in rat dorsal root ganglia
Authors: Yip HK , Rich KM , Lampe PA , Johnson EM
Published: March 2018

Nerve growth factor (NGF) is known to be essential for survival and maintenance of sympathetic ganglia and of embryonic sensory ganglia of neural crest origin. The present study examined the physiological and pharmacological roles of NGF in the postnatal development of sensory neurons in the dorsal root ganglion (DRG). In contrast to what is generally stated in the literature, administration of NGF antiserum to newborn rats for a period of 7 days resulted in a significant (approximately 20%) reduction of neuronal number in the lumbar DRG. Size spectrum analysis of surviving neurons revealed a shift toward larger sizes, presumably due to a preferential loss of small cells. The number of neurons in the L5 DRG was studied at various times after unilateral sciatic nerve crush in 1-day-old rats. Axotomy resulted in a substantial loss (40 to 50%) of neurons in the immature DRG. Administration of NGF antiserum to animals with axotomized DRG did not increase cell death when compared with the axotomized controls. However, the number of neurons in the antiserum-treated ganglia decreased by the same percentage (20%) when compared with the control serum-treated ganglia before and after axotomy. Treatment with NGF initially prevented the loss of neurons in the axotomized DRG. However, some neurons died during the first week despite continued NGF administration; and, subsequent to NGF withdrawal, neuronal number decreased to the same level as in control animals. Thus, removal of exogenous NGF resulted in the death of the sensory neurons which had been maintained.(ABSTRACT TRUNCATED AT 250 WORDS)
Dye coupling in hypothalamic slices: dependence on in vivo hydration state and osmolality of incubation medium
Authors: Cobbett P , Hatton GI
Published: March 2018

Electrotonic coupling is one mechanism which may coordinate the electrophysiological activity of a population of neurons. By measuring the incidence of dye coupling, we have investigated whether conditions that stimulate hormone secretion by hypothalamic magnocellular neuroendocrine cells affect coupling between these neurons. Neurons in the magnocellular regions of the paraventricular nucleus (PVN), in slices prepared from normally hydrated or chronically dehydrated male rats, were intracellularly injected with the fluorescent dye Lucifer Yellow CH. The dye coupling index (DCI), the ratio of the number of dye- coupled neurons to the total number of filled cells, was determined for each treatment group. The DCI for slices from dehydrated animals incubated in 310 milliosmoles/kg of medium (0.121) was significantly lower than that for slices for hydrated animals incubated in medium of the same osmolality (0.333). This decrease was reversed when slices from dehydrates were incubated in medium having an osmolality of 340 milliosmoles/kg (DCI = 0.307). There was also evidence for an interaction between slices incubated in the same chamber: the DCI in slices from dehydrated animals was significantly higher (0.475) when slices from normally hydrated rats were also present in the incubation chamber. Based on these data and on cited evidence, we suggest that the osmolality of the extracellular fluid and the local concentration of sex steroid hormones may influence dye coupling in the PVN.
Correlation between growth form and movement and their dependence on neuronal age
Authors: Argiro V , Bunge MB , Johnson MI
Published: March 2018

Neurites of superior cervical ganglion neurons from embryonic, perinatal, and adult rats extended at different rates when placed in tissue culture on similar collagen substrata. Using high resolution cinematography and a time-lapse video recording system, we concluded that these differences arise from variations in individual growth cone behavior. Growth cones of embryonic and perinatal neuronal origin exhibited high peak rates of advance and filopodial and lamellipodial excresences. Perinatal cones differed from embryonic ones in that they were somewhat larger, advanced in straighter paths, and retracted less, consequently translocating at 14 to 29 microns/hr compared with 8 to 22 microns/hr for embryonic cones (ranges of 4-hr means). The growth cones of neurons obtained from adult rats had scant cytoplasm and short branched filopodia, lacked definitive lamellipodia, and traversed the terrain at 4 to 13 microns/hr due to lack of high peak rates of advance and more time spent in stationary or minimal advance phases. Periodic pauses lasting 10 to 20 min, occurring every 20 to 90 min, interrupted the forward advance of growth cones of all ages. During pauses or slow forward movement, the growth cone displayed numerous filopodia whereas, during brief episodes when embryonic and perinatal growth cones moved at peak rates of 200 microns/hr or more, the cone periphery was predominantly lamellipodial. We conclude that the predominance of a lamellipodial or filopodial conformation correlates with the rate of growth cone advance and that age-dependent variations in neurite extension rates are related to differences in growth cone form and pattern of translocation. This is the first documentation of differing behavior of single growth cones of neurons of varying developmental ages in culture.
Inhibitors of metalloendoprotease activity prevent K+-stimulated neurotransmitter release from the retina of Xenopus laevis
Authors: Frederick JM , Hollyfield JG , Strittmatter WJ
Published: March 2018

Metalloendoprotease activity was identified in retinal homogenates using a synthetic fluorogenic metalloendoprotease substrate and specific metalloendoprotease inhibitors. The requirement of metalloendoprotease activity in neurotransmitter release was examined during the depolarization-induced release of [3H]glycine from the retina of Xenopus laevis. Neurons with high affinity uptake and calcium- dependent, K+-stimulated release of glycine have been described previously in this retina. When isolated retinas preloaded with [3H]glycine are depolarized by 22 mM K+, the usual efflux of [3H]glycine is completely abolished by the metalloendoprotease inhibitor, 1,10-phenanthroline (100 micrograms/ml). The inhibition of [3H]glycine release by 1,10-phenanthroline is dose dependent; furthermore, 1,10-phenanthroline blocks release by chelating metal and not Ca2+, since addition of equimolar calcium does not alter the inhibition. The metalloendoprotease inhibitor, carbobenzoxy (CBZ)-L- phenylalanine, also prevents release, whereas the amino acid, L- phenylalanine, has no effect. Synthetic carbobenzoxy dipeptide amides which are metalloendoprotease substrates (e.g., CBZ-Gly-Leu-amide, CBZ- Ser-Leu-amide, and CBZ-Gly-Phe-amide) also prevent [3H]glycine release in a dose-dependent and reversible manner. The synthetic dipeptide CBZ- Gly-Gly-amide, however, is not a metalloendoprotease substrate and has no effect on release. The ability of synthetic dipeptides to inhibit neurotransmitter release is amino acid specific, dose dependent, reversible, and matches their ability to interact with characterized metalloendoproteases. Depolarization-stimulated transmitter release may therefore require the activity of a metalloendoprotease.
Excitatory and inhibitory activity in the dorsal musculature of the nematode Ascaris evoked by single dorsal excitatory motonerons
Authors: Walrond JP , Stretton AO
Published: March 2018

A physiological preparation, in which identified motoneurons of the nematode Ascaris lumbricoides can be individually stimulated, was used to map the response evoked by single dorsal excitatory (DE) motoneurons in muscle cells innervated along the length of the dorsal nerve cord. As previously reported (Walrond, J. P., I. S. Kaas, A. O. W. Stretton, and J. E. Donmoyer (1985) J. Neurosci. 5: 1–8), stimulation of a DE cell produces excitatory responses in muscle cells which it directly innervates. Excitatory activity propagates along the most strongly activated region of muscle at a velocity of approximately 28 cm/sec, then relaxes into a slower velocity of approximately 12 cm/sec. When either the DE1 or DE3 neurons were stimulated, excitatory responses were also observed in muscle cells not directly innervated by the neuron. These signals propagate in the opposite direction from the fast- propagating activity at a velocity of approximately 13 cm/sec. Injection of hyperpolarizing current into muscle cells blocks this slower propagation but fails to block the faster conduction. We conclude that the fast-conducting responses result from signals propagating in the motor axon, whereas the slow responses are conducted through gap junctions which connect Ascaris muscle cells. Stimulating a single DE motoneuron also evokes hyperpolarizing muscle responses in regions adjacent to the zones of fast and slow excitation.(ABSTRACT TRUNCATED AT 250 WORDS)
Pyruvate participation in the low molecular weight trophic activity for central nervous system neurons in glia-conditioned media
Authors: Selak I , Skaper SD , Varon S
Published: March 2018

Conditioned media from glial cell cultures contain low molecular weight agents which can support survival of CNS neurons in the absence of recognized protein neuronotrophic factors. A similar support is provided to CNS neurons by selected basal media, and pyruvate is the critical medium constituent responsible for their trophic competence. Eagle's basal medium, which contains no pyruvate, acquires pyruvate when conditioned over astroglial cell cultures. Enzymatic degradation of the pyruvate in the astroglia-conditioned medium leads to corresponding losses in its low molecular weight trophic activity for CNS neurons. Quantitative correlations between pyruvate content and CNS trophic activity demonstrate that pyruvate is the main trophic ingredient of the glia-conditioned medium, and other low molecular weight substances, acquired during conditioning, reduce the pyruvate concentration required for its trophic effect. The “pyruvate-sparing” substances, as yet unidentified, are not the serine and Fe3+ which have pyruvate-sparing competence for peripheral, ciliary ganglionic neurons. These findings, together with previous observations, propose that prenatal neurons fail to generate or retain endogenous pyruvate at the levels for their survival-sustaining activities.
Cholinergic neurons of the chick ciliary ganglia express adrenergic traits in vivo and in vitro
Authors: Teitelman G , Joh TH , Grayson L , Park DH , Reis DJ , Iacovitti L
Published: March 2018

In this study, we sought to determine whether neurons of the chick embryo ciliary ganglia (CG), a parasympathetic cholinergic ganglia, can express catecholaminergic (CA) traits. To accomplish this, we used immunocytochemical techniques to examine the presence of the CA enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in CGs removed from chick embryo at day 8 of development (E8). Few neurons containing TH but not PNMT were found in the E8 CG. To examine whether CG neurons express CA enzymes in vitro, CGs removed from E8 chick embryo were dissociated and kept in culture for 3 to 12 days. In 50% of the culture dishes, some neurons contain TH or PNMT after 5 days in vitro. In an equal proportion of culture plates, CG neurons did not express the enzymes. To determine whether the proportion of CG neurons expressing TH or PNMT is increased by tissue influences, ganglion cells were co-cultured with notochord. In 90% of the co-culture experiments, most neurons present in the culture dishes stained with TH or PNMT after 5 days in vitro. To test for the presence of aromatic L-amino acid decarboxylase (AADC), another CA enzyme, cultures of CGs and CGs plus notochord were incubated with levodopa and processed for the detection of CA histofluorescence. Dopamine histofluorescence was present in all neurons after 3 days in vitro irrespective of the presence of notochord, suggesting that the expressions of TH and PNMT and that of AADC are differentially regulated. This study, therefore, demonstrates that cholinergic neurons of the CG contain CA enzymes in vivo and in vitro and that the proportion of neurons expressing CA traits during development in vitro can be increased by environmental cues such as those released by the notochord.
Sexually dimorphic regions in the medial preoptic area and the bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood
Authors: Hines M , Davis FC , Coquelin A , Goy RW , Gorski RA
Published: March 2018

Sexually dimorphic regions are described in two areas of the guinea pig brain: the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST). The volume of a darkly staining portion of the MPOA is approximately 4-fold larger in male than in female guinea pigs, and the volume of a darkly staining portion of the BNST is approximately 36% larger in male than in female animals. The sex differences in both of these areas are present in animals that have been gonadectomized as adults as well as in intact animals, suggesting that they result from differences between the sexes in the hormonal environment during early development. Both the MPOA and the BNST bind high levels of gonadal steroids early in life, during the period when functional differentiation occurs. It is possible that dramatic morphological sex differences characterize such steroid-binding areas. Furthermore, these sexually dimorphic areas may form an anatomically and functionally interrelated system. Attention to these possibilities may help elucidate more precisely the neural basis for sexually dimorphic functions, as well as the basic mechanisms underlying sexual differentiation of behavior and the brain.
Neural mechanisms of motor program switching in the mollusc Pleurobranchaea. II. Role of the ventral white cell, anterior ventral, and B3 buccal neurons
Authors: Croll RP , Kovac MP , Davis WJ
Published: March 2018

Identified buccal neurons in the mollusc Pleurobranchaea were stimulated and recorded intracellularly while recording the resultant identified motor program from buccal muscles (reduced preparation) or nerves (isolated central nervous system). Neurons studied included the ventral white cell (VWC), members of the anterior ventral (AV) population, and interneuron B3. Each of these neurons elicited the egestion motor program or its characteristic components when stimulated intracellularly. The characteristic prolonged plateau potential of the VWC was frequently associated with the egestion motor program but never with the ingestion motor program or its characteristic components. Intracellular recordings from these same neurons during spontaneous or induced buccal motor programs were consistent with the view that these neurons participate in production of the egestion motor program. The VWC discharged also during the neutral buccal rhythm, although in a different pattern from that seen during the egestion motor program, suggesting that it is multifunctional. Synaptic targets of the VWC are unknown, but synaptic influences of the AV and B3 neurons were found and are appropriate to their proposed role in egestion. This study therefore indicates that an interrelated cluster of buccal neurons is specialized to command the egestion motor program.
Embryonic development of glial cells and their junctions in the locust central nervous system
Authors: Swales LS , Lane NJ
Published: March 2018

The embryonic development of the specialized glial cells that form the perineurial blood-brain barrier in the locust CNS has been studied by freeze-fracture and tracer uptake. These cells migrate to form bracelet cell arrangements around the nervous tissues between day 4 to day 10 of embryonic differentiation which lasts 14 days in toto. A number of different kinds of intercellular junction form between the bracelet cells from day 8 to day 13 of development. These include gap junctions with features characteristic of arthropods, which seem to assemble by lateral migration of 13-nm E face intramembranous particles (IMPs), which ultimately cluster to form a large number of mature plaques of varying diameters. Less numerous are tight junctions which serve to restrict entry of exogenous molecules, including lanthanum and cationic ferritin, thereby forming the blood-brain barrier; these appear to assemble by migration of individual 8- to 10-nm P face IMPs into ridges which are found between the overlapping fingers of the perineurial bracelet cell processes. Septate junctions also mature at this stage in embryonic development by apparent assembly of IMPs into characteristic aligned rows; these may serve to slow down the entry of positively charged molecules as well as being adhesive, although anionic ferritin may leak into the CNS even after septate and tight junction formation. The observed changes in cellular associations and the formation of the blood-brain barrier coincide with the onset of mature neuronal electrical properties and spontaneous synaptic input.
Calcium- and cyclic nucleotide-dependent protein kinases and their substrates in the Aplysia nervous system
Authors: Novak-Hofer I , Lemos JR , Villermain M , Levitan IB
Published: March 2018

Homogenates of the Aplysia nervous system contain protein kinase activities sensitive to cAMP, cGMP, and Ca2+/calmodulin. The cAMP- and cGMP-dependent activities are either soluble enzymes or are only loosely bound to membranes, since they can be detected only in crude but not in washed membrane fractions, and are present in 20,000 or 100,000 X g supernatants prepared from homogenates. In contrast there are both soluble and tightly membrane-bound Ca2+/calmodulin-dependent protein kinase activities. The three activities present in supernatant fractions can be separated by chromatography on DE-cellulose, indicating that they are due to distinct enzyme species. Substrates for these enzymes were analyzed by two-dimensional gel electrophoresis. Protein phosphorylation within the identified Aplysia neuron R15 in vivo was measured by the intracellular injection of [gamma-32P]ATP. cAMP stimulates the phosphorylation of nine proteins and decreases phosphorylation of two proteins in this cell. This in vivo pattern was compared with in vitro phosphorylation measured in homogenates of whole ganglion. Most of the phosphoproteins affected by cAMP in neuron R15 in vivo are also substrates for cAMP-dependent protein kinase in vitro. Thus, the in vitro system will be a useful tool for detailed biochemical analysis of phosphoproteins which have been identified as being physiologically relevant in vivo.
Characterization of tissue-derived macromolecules affecting transmitter synthesis in rat spinal cord neurons
Authors: Kaufman LM , Barry SR , Barrett JN
Published: March 2018

Rat spinal cord cells maintained in neuron-rich cultures were exposed to extracts of skeletal muscle or to medium conditioned by non-neuronal cells. The conditioned media enhanced neuronal acetylcholine (ACh) synthesis, choline acetyltransferase activity, and protein synthesis, and decreased gamma-aminobutyric acid (GABA) synthesis. Muscle extract prepared from newborn rats produced similar enhancements but did not depress GABA synthesis. Muscle extracts prepared from normal and denervated adult rat limbs contained relatively little activity. These results suggest that different molecular factors might mediate the effects on GABA and ACh synthesis. Gel filtration of conditioned media and muscle extracts revealed that all of these activities were confined to a macromolecular fraction with an apparent Mr of 40,000. These tissue-derived factors affecting neuronal protein and transmitter synthesis are in turn distinct from a neuronal survival-promoting factor obtained from serum (Kaufman, L. M., and J. N. Barrett (1983) Science 220: 1394–1396).
Corticotropin-releasing factor desensitization of adrenocorticotropic hormone release is augmented by arginine vasopressin
Authors: Hoffman AR , Ceda G , Reisine TD
Published: March 2018

The desensitization of corticotropin-releasing factor (CRF)-stimulated ACTH release from and intracellular cyclic AMP accumulation in anterior pituitary cells was investigated in primary cultures of rat pituitary cells and in a mouse tumor cell line (AtT 20/D16–16). CRF potently stimulated ACTH secretion and cyclic AMP accumulation in both preparations. When primary cultures were preincubated with 100 nM CRF, 50% desensitization of ACTH release occurred within 4 hr while similar desensitization of cyclic AMP accumulation occurred by 1 hr. This desensitization was manifested as a reduced maximal CRF response. Concentrations of CRF as low as 1 to 10 pM induced desensitization. Pretreatment did not affect ACTH content nor did it alter the ability of epinephrine or forskolin to stimulate ACTH release. Pretreatment of AtT 20 cells, which are a homogeneous population of corticotrophs, with 100 nM CRF reduced the subsequent ability of CRF, but not of isoproterenol or of forskolin, to stimulate ACTH release and cyclic AMP formation. Preincubation of AtT 20 cells with isoproterenol did not affect the cyclic AMP or ACTH release responses induced by CRF, but did desensitize beta-adrenergic receptors. Arginine vasopressin (AVP) was a weak ACTH-releasing factor, but AVP enhanced CRF-directed ACTH release from primary cultures. When these cells were preincubated with both CRF and AVP, CRF desensitization occurred with lower concentrations of CRF than when CRF desensitization was induced by preincubating the cells with CRF alone. The ED50 for CRF-induced desensitization was 700 +/- 150 pM when the cells were exposed to CRF alone, and 20 +/- 15 pM when 1 microM AVP was added during the preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)
The regulation of synaptic strength within motor units of the frog cutaneous pectoris muscle
Authors: Trussell LO , Grinnell AD
Published: March 2018

The physiological properties of frog neuromuscular junctions may vary widely in a single muscle. In order to understand the factors that contribute to this variation, we have studied populations of synapses belonging to individual motor units of the frog cutaneous pectoris muscle. Motor units in this muscle differ widely in twitch strength. A motor axon's synaptic contacts could be found throughout the muscle, at both singly and polyneuronally innervated endplates. Indeed, over 36% of the endplates contacted by each isolated motor axon were polyneuronally innervated. Comparisons of synapses on muscle fibers in large twitch motor units with those in small twitch motor units reveal that endplate potential amplitude, transmitter release, and muscle fiber diameter are positively correlated with the strength of the motor unit contraction. Large and small twitch motor units differ more in their transmitter release than in their nerve terminal length, indicating that larger twitch motor units have a higher release per unit length of terminal. Among motor units of roughly similar twitch tension, transmitter release at endplates receiving only one axonal input is remarkably constant, independent of postsynaptic muscle fiber input resistance, or, presumably, nerve terminal size. In cases where two different motor axons contribute to a single endplate, the synaptic strength of each input is again related to properties of the contributing motoneuron, although the individual synaptic inputs are markedly reduced in strength and size relative to singly innervated endplates. Additionally, the diameter of polyneuronally innervated muscle fibers appears related to properties of both innervating motoneurons. Thus, the pre- and postsynaptic characteristics of neuromuscular junctions may be determined both by the motoneuron and by peripheral interactions between motoneurons.
Postnatal development of the phase-locked response to low frequency tones of auditory nerve fibers in the cat
Authors: Kettner RE , Feng JZ , Brugge JF
Published: March 2018

The maturation of the auditory nerve's ability to encode temporal information in an acoustic signal was studied in young kittens, 7 to 23 days old, and in adult cats by measuring the degree to which auditory nerve fiber responses are synchronized (phase locked) to low frequency tones. The major findings include the following. In 7- to 10-day-old kittens thresholds are high (around 100 dB), and secure phase locking is observed only at frequencies below about 600 Hz. The upper frequency limit for phase locking in the adult, around 4 kHz, is reached gradually toward the end of the third postnatal week, a time when thresholds also reach their adult levels. The time course of development of the phase-locked response is similar for fibers with different characteristic frequencies (CFs). At all ages studied, the maximal phase synchrony at any given low frequency is generally obtained at stimulus intensities less than 20 dB above the rate threshold and some 10 to 30 dB below the saturation firing level of the fiber. Time delays, estimated from phase-versus-frequency plots, were similar across ages, although they tended to be longer and more variable at lower CFs during the first 2 postnatal weeks. Comparing the phase-locked response of auditory nerve fibers with that of the anteroventral cochlear nucleus neurons from a previous study suggested that the upper frequency limit for phase synchrony was reached later in the nucleus than in the nerve.
Functional postnatal changes in avian brain regions responsive to retinal slip: a 2-deoxy-D-glucose study
Authors: McKenna OC , Wallman J
Published: March 2018

The postnatal development of two avian brain areas, the nucleus of the basal optic root (nBOR) and the lentiform nucleus of the mesencephalon (LM), was studied using the 2-deoxy-D-glucose (2-DG) method. Previous studies have shown that these nuclei respond to whole field visual motion, which is used for optokinetic eye movements. In the present study vertical whole field visual motion presented to one eye resulted in the accumulation of 2-DG label in the contralateral nBOR in both hatchlings and 3-week or older chicks. In hatchlings the nBOR was diffusely labeled whether upward or downward motion was used as the test stimulus, whereas in older birds the label was localized within different subdivisions of the nBOR depending on the direction of vertical motion: upward motion resulted in concentration of 2-DG label in the dorsal nBOR, whereas downward motion resulted in label in the ventral nBOR. Horizontal whole field visual motion presented to one eye resulted in 2-DG label in the contralateral LM and in the lateral portion of the contralateral nBOR in animals of both ages. In hatchlings, visual motion in both temporal-to-nasal and nasal-to- temporal directions resulted in labeling of both subdivisions of the LM, LM magnocellularis (LMmc) and LM parvocellularis. In older birds, temporal-to-nasal motion resulted in labeling of only the LMmc, whereas nasal-to-temporal motion produced labeling in both subdivisions. These results strongly suggest that the nBOR and LM continue to develop their response properties postnatally and that different functional units become separated within particular subdivisions of the nuclei. Conceivably, the functional segregation within the nBOR is due to an intrinsic reorganization, whereas functional changes in the LM may be due to the postnatal development of a projection from the telencephalon.
Adhesion of axolemmal fragments to Schwann cells: a signal- and target- specific process closely linked to axolemmal induction of Schwann cell mitosis
Authors: Sobue G , Pleasure D
Published: March 2018

Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125I- axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more.
Direct electrical stimulation promotes growth and enhances survival of aneurogenic muscles of the chick embryo
Authors: Bloom JW , Butler J , Brierley J , Cosmos E
Published: March 2018

Analyses of embryonic aneurogenic muscles indicate that several processes associated with early myogenesis in vivo proceed in the absence of peripheral nerves. However, aneurogenic muscles demonstrate impaired growth and limited survival. To investigate whether neurally mediated activity is responsible for these phenomena, aneurogenic muscles of the chick embryo were directly stimulated in vivo via implanted electrodes. Volumetric analyses of stimulated aneurogenic brachial (latissimus dorsi) muscles from Stage (St) 33 (7.5 to 8 days) through St 37 (11 days) demonstrated that growth was enhanced significantly beyond the level characteristic of unstimulated aneurogenic muscles. Moreover, for the majority of embryos, the stimulation regimen actually rescued the posterior latissimus dorsi muscle which characteristically does not survive beyond St 32 (7.5 days) in the aneurogenic state. Thus, our results implicate activity per se as an important factor necessary for the proper growth and survival of brachial muscles during early embryogenesis. The stimulation regimen, however, did not alter myosin ATPase profiles.
Cell surface Thy-1-cross-reactive glycoprotein in cultured PC12 cells: modulation by nerve growth factor and association with the cytoskeleton
Authors: Richter-Landsberg C , Greene LA , Shelanski ML
Published: March 2018

In cultured PC12 rat pheochromocytoma cells, nerve growth factor (NGF) selectively stimulated the incorporation of [3H]fucose into a glycoprotein of apparent Mr approximately 25,000, as determined by sodium dodecyl sulfate-gel electrophoresis. Neither dibutyryl cyclic adenosine 3′:5′-monophosphate nor epidermal growth factor mimicked this effect. Using gradient gels, the affected protein was resolved into two closely migrating bands. Enhancement of labeling was present by 1 to 2 days of treatment with NGF and was maximal after 4 to 7 days. Short- term extraction of PC12 cell monolayer cultures with Triton X-100 left the 25,000-dalton glycoprotein associated with the detergent-resistant cytoskeletal fraction. The Mr approximately 25,000 glycoprotein was shown to be immunologically cross-reactive with Thy-1.1 antigen by indirect immunoprecipitation with monoclonal Thy-1.1 antibodies. Anti- Thy-1.1 labeling, as demonstrated by indirect immunofluorescence, was distributed on PC12 cell bodies and along neuritic processes and remained attached to the cytoskeleton as part of the surface lamina of cells treated and untreated with NGF. The selective increase of the Mr approximately equal to 25,000 glycoprotein in NGF-treated cultures was also paralleled by increases in material immunoprecipitated from fucose- labeled cells with anti Thy-1.1 monoclonal antibodies. Immunoprecipitations with extracts of [35S]methionine-labeled cultures indicated that NGF causes an increase in synthesis, rather than merely glycosylation, of the 25,000-dalton/Thy-1-cross-reactive protein. The effect of NGF on this protein was blocked by inhibitors of RNA synthesis, suggesting involvement of a transcription-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Relationship between heterosynaptic reflex facilitation and acquisition of the nictitating membrane response in control and scopolamine- injected rabbits
Authors: Harvey JA , Gormezano I , Cool-Hauser VA
Published: March 2018

Classical conditioning of the rabbit nictitating membrane response was accomplished by presenting a 100-msec tone conditioned stimulus at intervals of 0, 100, 200, 400, and 800 msec before the presentation of a 100-msec shock unconditioned stimulus. In addition, tone-alone and shock-alone trials were interspersed during conditioning. On the first day of conditioning, during which there was no evidence of acquisition of conditioned responses to the tone conditioned stimulus, the amplitudes of the nictitating membrane response evoked on paired tone- shock trials were compared with the amplitudes obtained on shock-alone trials to provide a measure of reflex facilitation. There was a significant correlation (+0.86) in control animals between the degree of reflex facilitation and the degree of learning demonstrated at the various tone-shock intervals. Both reflex facilitation and learning were absent at the 0-msec tone-shock interval, increased at the 100- msec interval, reached a maximum at the 200-msec interval, and then declined at the longer intervals. Scopolamine (0.4 mg/kg) did not affect the amplitude of the nictitating membrane response elicited on shock-alone trials but eliminated any evidence of reflex facilitation or learning at the 100- and 800-msec intervals and significantly reduced both reflex facilitation and learning at the 200- and 400-msec intervals. The comparable effects of scopolamine on both reflex facilitation and learning were reflected by a significant correlation (+0.95) between these two measures.(ABSTRACT TRUNCATED AT 250 WORDS)
Non-quantal release of acetylcholine at a developing neuromuscular synapse in culture
Authors: Sun YA , Poo MM
Published: March 2018

Local, pulsed application of d-tubocurarine at neuromuscular synapses in embryonic Xenopus nerve-muscle culture resulted in a transient hyperpolarization of muscle membrane potential. Miniature endplate potentials (MEPPs) were abolished during the hyperpolarization and recovered after the return of resting membrane potential. The magnitude of hyperpolarization was independent of the frequency of MEPPs before curarization, and it had an average peak value of 4.3 mV in medium containing physiological levels of Ca2+. Prolonged application of curare or alpha-bungarotoxin led to sustained hyperpolarizations up to 8 mV in magnitude. Denervation produced by mechanically removing the neurite from the muscle cell also produced similar hyperpolarization, and curarization after denervation was without significant hyperpolarizing effect. Increasing the extracellular Ca2+ concentration to about 8 mM abolished the curare-induced hyperpolarization response, in sharp contrast to its effect in elevating the frequency of MEPPs. Taken together, our results indicate that innervated embryonic muscle cells were maintained at a depolarized state relative to that of uninnervated muscle cells by a steady, spontaneous release of acetylcholine (ACh) from the innervating neurite. The cellular mechanism underlying this mode of ACh release appears to be different from that of the quantal ACh release responsible for MEPPs.
Dorsal and ventral myotome motoneurons and their input during fictive locomotion in lamprey
Authors: Wallen P , Grillner S , Feldman JL , Bergelt S
Published: March 2018

Motoneurons supplying the dorsal and ventral parts of the myotome in the lamprey are shown to have different morphological characteristics; furthermore, their pattern of activation during fictive locomotion may differ considerably. Intracellular recordings from motoneurons were performed in an in vitro spinal cord-myotome preparation from segments rostral to the fins. The location of the contracting muscle fibers in the myotome could be observed directly in the dissection microscope during intracellular stimulation of the motoneuron. The motoneurons were injected with Lucifer Yellow, an intracellular dye, and were subsequently reconstructed, sometimes in both a horizontal and a transverse plane. Motoneurons supplying the ventral third of the myotome had a dense, fan-like, dendritic tree and ramifications near the midline. In contrast, motoneurons supplying the dorsal third of the myotome had a more widespread and less dense dendritic tree, with few ramifications near the midline. Some motoneurons supplying the most ventral or dorsal part of the myotome had contralateral dendrites crossing in the ventral commissure and ramifying near contralateral large, reticulospinal Muller fibers. The differences in morphology may indicate that these motoneurons receive different descending inputs. This may be related to the need for an effective control in the dorsoventral plane during righting and steering responses. During fictive locomotion elicited in the isolated spinal cord by bath-applied N-methyl-aspartate, pairs of motoneurons were recorded which subsequently were identified and characterized by intracellular injections of Lucifer Yellow.(ABSTRACT TRUNCATED AT 250 WORDS)