bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies reveal novel aspects of the biochemistry and organization of olfactory neurons following unilateral olfactory bulbectomy
Authors: Hempstead JL , Morgan JI
Published: March 2018

Following unilateral olfactory bulbectomy in rats the ipsilateral olfactory neuroepithelium undergoes degeneration. Subsequently, the receptor neuron complement of the tissue is restored by the proliferation and differentiation of immature neuroblasts. However, as noted by other workers, in the absence of a target organ the dynamics of neuron regeneration is altered such that there is an overall reduction in the number of cells positive for the olfactory marker protein when cellular equilibrium is re-established. Immunocytochemical staining of the olfactory epithelium of unilaterally bulbectomized rats with a series of anti-neuronal monoclonal antibodies reveals an attenuation of binding of some antibodies to the neurons of the ipsilateral epithelium. In contrast, other anti-neuronal monoclonal antibodies show no difference in staining intensity when ipsilateral and control contralateral epithelia are compared. These data suggest that the expression of some neuronal antigens is subject to control by the target olfactory bulb, whereas others are independent of such putative regulation. Besides altering the expression of some antigenic determinants, olfactory bulbectomy also results in certain organizational changes in epithelium. First, bulbectomy produces an increase in the incidence of a cell type that appears to span the neuroepithelium. Although the morphology of these cells is more akin to a sustentacular cell than to a receptor neuron, they are not immunoreactive with antibodies to sustentacular cells. The cells are stained, however, by an anti-neuronal antibody, NEU-9. The second aspect of altered organization is the appearance of novel olfactory marker protein-positive structures in the olfactory mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)
Quantitative in vivo receptor binding. II. Autoradiographic imaging of muscarinic cholinergic receptors
Authors: Frey KA , Ehrenkaufer RL , Agranoff BW
Published: March 2018

The in vivo distribution of [3H]scopolamine in rat brain following establishment of constant, saturating arterial tracer concentrations was examined with the use of quantitative autoradiography. The equilibrium drug distribution, studied 240 min after initiation of tracer infusion, was highly correlated with the regional density of muscarinic receptor sites determined in vitro in the same animals by autoradiographic analysis of [3H]quinuclidinyl benzilate binding. Brain regions of highest receptor density were generally correlated with known terminal fields of cholinergic neurons, and they demonstrated a protracted time course of in vivo labeling. An exception was noted in the basal pons, where a receptor population of high density without documented cholinergic innervation was rapidly labeled. It is suggested that synaptic muscarinic receptors are labeled slowly, as a consequence of either restricted tracer accessibility or competition between tracer and endogenous acetylcholine for available binding sites, and that the pontine receptors may be functionally distinct from those in other brain regions. The in vivo equilibrium binding technique used in the present study results in regional tissue radioligand concentrations directly proportional to receptor density and may, thus, provide a basis for receptor imaging in the human brain by means of positron emission tomography.
gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]- t-butylbicyclophosphorothionate binding to cerebellar granule cells
Authors: Gallo V , Wise BC , Vaccarino F , Guidotti A
Published: March 2018

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma- aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel- modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3- carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)
The regional distribution and the chemical, chromatographic, and immunologic characterization of motilin brain peptides: the evidence for a difference between brain and intestinal motilin-immunoreactive peptides
Authors: Beinfeld MC , Korchak DM
Published: March 2018

Motilin-like immunoreactive peptides (MLIPs) have been detected in the brain by radioimmunoassay (RIA), (Yanaihara, C., H. Sato, N. Yanaihara, S. Naruse, W. G. Forssman, V. Helmstaedter, T. Fujita, K. Yamaguchi, and K. Abe (1977) Adv. Exp. Biol. Med. 106: 269–283; Chey, W. Y., R. Escoffery, F. Roth, T. M. Chang, and H. Yajima (1980) Regul. Pept. Suppl. 1: 519; O'Donohue, T. L., M. C. Beinfeld, W. Y. Chey, T. M. Chang, G. Nilaver, E. A. Zimmerman, H. Yajima, H. Adachi, M. Roth, R. P. McDevitt, and D. M. Jacobowitz (1981) Peptides 2: 467–477). Previous studies (O'Donohue et al., 1981) demonstrated that MLIPs in rat brain probably differ chemically from porcine intestinal motilin (PIM), the first motilin peptide isolated. The possibility that this rat-pig difference represents a species difference was not examined in the previous study (O'Donohue et al., 1981), neither was the question of the cross-species distribution of MLIP. This study was initiated to examine brain MLIP distribution by RIA in three additional species: cow, pig, and guinea pig. The question of rat-pig species differences was addressed by characterizing MLIP in the brains of these species in comparison to PIM. By RIA, MLIPs were widely distributed in the brains of all species examined. MLIP concentration was highest in rat brain and lowest in pig brain. Some motilin antisera consistently detected less or no MLIPs in some brain regions of all species. Rat pituitary, pineal gland, and retina had substantially higher MLIP concentrations than did brain. MLIPs were abundant throughout the rat gastrointestinal tract and in some other peripheral organs.(ABSTRACT TRUNCATED AT 250 WORDS)
Two different mechanisms of calcium spike modulation by dopamine
Authors: Paupardin-Tritsch D , Colombaioni L , Deterre P , Gerschenfeld HM
Published: March 2018

Dopamine (10 to 50 microM) modulates in two different ways the duration of the Ca2+-dependent action potential recorded in the cell body of identified neurons of the snail Helix aspersa. In some neurons (cells E13 and F1) dopamine increases the amplitude of their Ca2+-dependent spike plateau by decreasing the S-current (Klein, M., J.S. Camardo, and E. R. Kandel (1982) Proc. Natl. Acad. Sci. U.S.A. 79: 5713–5717), a K+ current controlled by cyclic AMP. In another neuron (cell D2), dopamine decreases the Ca2+-dependent plateau of the somatic action potential by evoking a decrease in Ca2+-current resulting from a decrease in Ca2+ conductance. Both modulatory effects could be observed in the same single neuron in which dopamine induces decreases of both the Ca2+ conductance and cyclic AMP-dependent K+ conductance. Nevertheless, in these cells (such as cell F5) dopamine only evokes a decrease of the amplitude of the Ca2+ spike plateau. Since the modulation of the duration of the Ca2+ action potential recorded in the neuronal soma has been shown to constitute a good model of events taking place at synaptic endings, it is suggested that these modulatory mechanisms evoked by dopamine may be involved in processes of presynaptic facilitation and inhibition.
Decreased cerebellar 3',5'-cyclic guanosine monophosphate levels and insensitivity to harmaline in the genetically dystonic rat (dt)
Authors: Lorden JF , Oltmans GA , McKeon TW , Lutes J , Beales M
Published: March 2018

The dystonic rat (dt) is an autosomal recessive mutant displaying a complex motor syndrome that includes sustained axial twisting movements. The syndrome is correlated with increased glutamic acid decarboxylase activity in the deep cerebellar nuclei and increased cerebellar norepinephrine levels in comparison with phenotypically normal littermates. Biochemical, behavioral, and anatomical techniques were used to investigate the possibility that the abnormalities noted in the cerebellum of the dt rat were indicative of altered function of the major projection neurons of the cerebellar cortex, the Purkinje cells. Phenotypically normal rats showed tremor in response to harmaline, a drug that acts on the inferior olive to produce bursting in the climbing fiber pathway. Dystonic rats were insensitive to the effects of harmaline but did respond to oxotremorine. Levels of the cyclic nucleotide 3′,5′-cyclic guanosine monophosphate, a biochemical marker for Purkinje cells, increased in response to harmaline in normal rats but were significantly lower in dystonic rats under both basal and harmaline-stimulated conditions. Purkinje cell soma size was reduced in the dystonic rats but no other morphological correlates of the behavioral or biochemical deficits were noted. Taken together with other observations on this mutant, the results suggest an impairment in the cerebellum or in its connections with lower brainstem and spinal cord sites.
Abnormal development of the retinogeniculate projection in Siamese cats
Authors: Kliot M , Shatz CJ
Published: March 2018

In the visual system of Siamese cats, the lateral geniculate nucleus (LGN) receives an abnormally large projection from the contralateral eye and a correspondingly reduced projection from the ipsilateral eye. In order to determine how this abnormal pattern of retinal input arises, the prenatal development of the retinogeniculate projection was studied in Siamese cats using the anterograde transport of intraocularly injected [3H]leucine and horseradish peroxidase. Labeled axons from the ipsilateral eye can be detected in the optic tract by embryonic day 30 (E30; gestation is 65 days), several days later than found in normally pigmented animals. The ipsilateral projection is not only apparently delayed but also is reduced in size as compared with normal animals, and this reduction persists throughout development, indicating that the Siamese mutation acts to misdirect growing optic axons to the contralateral side of the brain as originally proposed (Guillery, R. W. (1969) Brain Res. 14: 739–741). The effect of an altered retinal projection on the ingrowth and segregation of retinal fibers to the LGN was also examined. In Siamese fetuses, not until E41 can significant label be seen within the ipsilateral LGN as compared to E35 in normally pigmented fetuses. As in normal animals, in Siamese fetuses, also, the labeled retinogeniculate afferents from the two eyes initially overlap within regions of the LGN before segregating into layers. However, measurements of the area occupied by labeled afferents from the ipsilateral and contralateral eyes indicate that maximum overlap of the two sets of afferents, although close to normal in amount, does not occur until about E51--again several days later than in normally pigmented animals (E47). The time course of segregation is also altered in Siamese cats. The onset of segregation, as signaled by the removal of contralateral eye afferents from territory destined for the ipsilateral eye and by the restriction of ipsilateral eye afferents, does not occur until about E51 in Siamese cats as compared with E47 in normally pigmented animals. Despite this delay in onset, the final segregation of the two sets of afferents in Siamese cats reaches adult-like levels at about the normal time. Thus, the misrouting of axons at the optic chiasm in Siamese cats not only alters the final pattern of innervation from the two eyes within the LGN, but also delays the onset and shortens the total duration of segregation itself.
Calcium/phosphatidylserine/diacylglycerol-dependent protein phosphorylation in the Aplysia nervous system
Authors: DeRiemer SA , Greengard P , Kaczmarek LK
Published: March 2018

It has been shown that intracellular injection of protein kinase C (calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), purified from mammalian brain, or application of the tumor-promoting phorbol diester, 12-O-tetradecanoyl-13-phorbol acetate (TPA), leads to an enhancement of calcium currents in the bag cell neurons of Aplysia. We now present evidence of an endogenous enzyme in bag cell neurons which is activated by TPA and which has properties similar to those of mammalian protein kinase C. Calcium/phosphatidylserine/diacylglycerol- dependent protein kinase activity was found in both cytosolic and particulate fractions prepared from isolated clusters of bag cell neurons. This endogenous enzyme phosphorylated an 87,000-dalton protein from bovine brain, which appears to be a specific substrate for protein kinase C, as well as several substrates present in cytosolic fractions prepared from isolated bag cell clusters. Similar results were obtained using preparations made from pooled head ganglia from Aplysia. The pharmacological properties of the calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity in the Aplysia nervous system were similar to those of protein kinase C from mammalian tissues. Thus, the same group of endogenous substrate proteins were phosphorylated when diacylglycerol was replaced by TPA in cytosolic fractions prepared from isolated bag cell clusters. Non-tumor-promoting phorbols (4-alpha-phorbol, 4-alpha-phorbol-12,13- didecanoate, and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate) did not stimulate protein phosphorylation in these preparations. Phosphorylation by the Aplysia calcium/phosphatidylserine/diacylglycerol-dependent protein kinase was inhibited by polymixin B sulfate, by calmodulin, and by the “calmodulin antagonists” trifluoperazine, calmidazolium and W7.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of a component in chick ciliary ganglia that cross- reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor
Authors: Smith MA , Stollberg J , Lindstrom JM , Berg DK
Published: March 2018

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the “main immunogenic region” of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross- reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.
Neural, mechanical, and geometric factors subserving arm posture in humans
Authors: Mussa-Ivaldi FA , Hogan N , Bizzi E
Published: March 2018

When the hand is displaced from an equilibrium posture by an external disturbance, a force is generated to restore the original position. We developed a new experimental method to measure and represent the field of elastic forces associated with posture of the hand in the horizontal plane. While subjects maintained a given posture, small displacements of the hand along different directions were delivered by torque motors. The hand was held in the displaced positions and, at that time, we measured the corresponding restoring forces before the onset of any voluntary reaction. The stiffness in the vicinity of the hand equilibrium position was estimated by analyzing the force and displacement vectors. We chose to represent the stiffness both numerically, as a matrix, and graphically, as an ellipse characterized by three parameters: magnitude (the area), shape (the ratio of axis) and orientation (direction of the major axis). The latter representation captures the main geometrical features of the elastic force field associated with posture. We also evaluated the conservative and nonconservative components of this elastic force field. We found that the former were much larger than the latter and concluded that the behavior of the neuromuscular system of the multiarticular arm is predominantly spring-like. Our data indicated that the shape and orientation of the stiffness were invariant over subjects and over time. We also investigated the ability of our subjects to produce voluntary and adaptive changes in the stiffness. Our findings indicated that, when a disturbance acting along a fixed and predictable direction was imposed, the magnitude of the stiffness was increased but only minor changes in shape and orientation occurred. Taken together, all of these experiments represent a step toward the understanding of the interactions between geometrical and neural factors involved in maintaining hand posture and its interactions with the environment.
Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells
Authors: Vale RD , Ignatius MJ , Shooter EM
Published: March 2018

Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors (10% of those found in intact cells) are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor, since greater than 90% of cell surface glycoprotein receptors for WGA become associated with Triton X-100-insoluble material at lectin concentrations greater than 33 micrograms/ml. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin.
Neural regulation of the circadian vasopressin rhythm in cerebrospinal fluid: a pre-eminent role for the suprachiasmatic nuclei
Authors: Schwartz WJ , Reppert SM
Published: March 2018

The neuroanatomical system responsible for the generation and expression of the circadian vasopressin rhythm in cerebrospinal fluid (CSF) is investigated. CSF was serially withdrawn in individual, unanesthetized, unrestrained rats after neuroendocrine extirpations or stereotaxic brain lesions were made; the peptide was assayed using a sensitive and specific radioimmunoassay. Hypophysectomy or pinealectomy did not eliminate vasopressin from CSF; both day and night-time peptide levels in hypophysectomized rats were elevated above control levels. Complete lesions of the suprachiasmatic nuclei (SCN) abolished both the rhythm and in most cases the measurable level of peptide in CSF. Neither lesions of the paraventricular nuclei nor hypothalamic knife cuts interrupting most neural efferents from the SCN had this effect; in these cases, vasopressin rhythms persisted with diminished amplitude. Our results suggest that the circadian CSF vasopressin rhythm is produced by a neural system topographically separate from the classical magnocellular hypothalamo-neurohypophyseal system for the secretion of peptide into blood. The SCN are an important component of this new system and are necessary for the generation of the CSF rhythm.
Reduced metabolic response of the aged rat brain to haloperidol
Authors: Pizzolato G , Soncrant TT , Holloway HW , Rapoport SI
Published: March 2018

Local cerebral glucose utilization (LCGU) was determined in 49 brain regions of 3-, 12-, 24-, and 33-month-old awake Fischer-344 rats, at 30 to 120 min after administration of the dopaminergic antagonist haloperidol (HAL) at 1 mg/kg (i.p.). The quantitative autoradiographic [14C]-2-deoxyglucose technique was employed. In 3-month rats, HAL produced statistically significant (p less than 0.05) reductions in LCGU in 63% of the brain regions examined, including those having dopaminergic as well as non-dopaminergic function. With increasing age, LCGU was progressively less affected by HAL. Significant declines in LCGU occurred in 21 to 23% of the observed regions in 12- and 24-month rats, and in 10% of the 49 regions in 33-month-old rats. The peak effects of HAL on LCGU occurred later in the 33-month- than in the 3- month-old rats (90 and 60 min after HAL, respectively). The behavioral effect (catalepsy) of HAL was less in old than in young rats. Age- related differences in the cerebral metabolic and behavioral responsivities of rats to HAL are not due to differences in brain concentrations of HAL, as higher concentrations are obtained in older animals (Kapetanovic, I.M., D.J. Sweeney, and S.I. Rapoport (1982) J. Pharmacol. Exp. Ther. 221: 434–438). The differences correlate with known age-dependent structural and biochemical deficits of the central dopaminergic system and indicate reduced functioning of that system in the rat brain after 12 months of age.
Immunological uniqueness of human monoamine oxidases A and B: new evidence from studies with monoclonal antibodies to human monoamine oxidase A
Authors: Kochersperger LM , Waguespack A , Patterson JC , Hsieh CC , Weyler W , Salach JI , Denney RM
Published: March 2018

Monoamine oxidase (EC 1.4.3.4; MAO) is the primary enzyme responsible for the intraneuronal degradation of biogenic amines in the central nervous system. An understanding of the physiological significance of the functional and regulatory differences between the two forms of the enzyme, MAOs A and B, would be facilitated by the availability of antibodies specific for the two forms of the enzyme. We previously isolated and characterized a monoclonal antibody (MAO B-1C2, previously designated MAO-1C2) which binds human MAO B but not A. We describe here four new monoclonal antibodies (designated MAO A-3C9, A-4F10, A-7B10, and A-7E10) which were elicited to highly purified MAO A from human placenta and which, in the presence of antimouse IgG and Staphylococcus aureus, immunoprecipitate greater than 90% of the catalytically active purified MAO A. MAO A-3C9 appears to have a lower affinity for purified MAO A than the other three antibodies and does not immunoprecipitate either MAO A or MAO B from human platelets or from Triton X-100 extracts of human placental and liver mitochondria. MAO A-4F10, A-7B10, and A-7E10 immunoprecipitate catalytically active MAO A from Triton X-100 extracts of human placental and liver mitochondria, but not catalytically active MAO B from either pletelets or from Triton X-100 extracts of human liver mitochondria. Collectively, these anti-MAO monoclonal antibodies reveal unique epitopes on human MAO A not shared by MAO B, and at least one epitope on MAO B not shared by MAO A. These immunochemical differences support the hypothesis that MAO A and MAO B are different proteins, presumably isozymes.
The characterization of the specific binding of [3H]-N- acetylaspartylglutamate to rat brain membranes
Authors: Koller KJ , Coyle JT
Published: March 2018

[3H]-N-Acetylaspartylglutamate (NAAG) bound saturably and reversibly to crude synaptosomal rat brain membranes. Optimal binding occurred in Tris-HCl buffer, pH 7.2, at 37 degrees C using previously frozen, preincubated membranes. Saturation experiments revealed an apparent KD of 383 +/- 33 nM and a Bmax of 31 +/- 2 pmol/mg of protein. [3H]NAAG specific binding was displaceable by serine-o-sulfate, quisqualate, ibotenate, and glutamate with K1's in the nanomolar range, whereas the amino-phosphono analogues displaced [3H] NAAG in the micromolar range (APB greater than APV greater than APH). No specific binding was found in peripheral tissues. Within the central nervous system, the thalamus exhibited the greatest amount of binding, whereas binding was lowest in cortex. Calcium ions enhanced the specific binding, whereas sodium ions caused a concentration-dependent inhibition. These results suggest that [3H]NAAG labels an acidic amino acid receptor site designated “A-4,” which recognizes the antagonist, 2-amino-4-phosphonobutyric acid, and that this receptor may mediate the neurophysiologic effects of endogenous NAAG.
On the mechanism by which phenytoin blocks post-tetanic potentiation at the frog neuromuscular junction
Authors: Selzer ME , David G , Yaari Y
Published: March 2018

Post-tetanic potentiation (PTP) was elicited at the frog sartorius and cutaneous pectoris neuromuscular junctions. A 30-sec, 30-Hz tetanus produced a 2- to 3-fold post-tetanic increase in endplate potential (EPP). In surface-recorded responses this PTP decayed in a double exponential way with time constants of 12.7 sec +/- 2.4 (SEM) and 146.8 sec +/- 36.6. In acute experiments 0.2 to 0.8 mM phenytoin (5,5- diphenylhydantoin, DPH) dramatically and reversibly reduced the early component. The late component was also reduced, although to a lesser extent and often not reversibly. DPH reduced PTP even when there was no failure of the EPP during the tetanus. Thus, the DPH effect did not require a complete block of the presynaptic action potential. At longer exposures and higher DPH concentrations EPP failures did develop, and this was associated with a more profound suppression of PTP. PTP was also elicited in tetrodotoxin (TTX)-containing solutions using electronic stimulation of nerve terminals to elicit transmitter release. This PTP had a much shorter duration (about 30 sec) than that seen in normal Ringer's solution and was followed by depression of EPP amplitudes. Thus, sodium entry into nerve terminals enables a mechanism which greatly prolongs PTP. DPH had no effect on PTP in TTX. These results, together with others in the literature, suggest that the reduction of PTP by DPH involves a graded reduction of sodium influx into nerve terminals during high rates of axon stimulation. The development of all-or-none failures of the presynaptic action potential results in even greater suppression of PTP.
Distribution of N-methyl-D-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain
Authors: Monaghan DT , Cotman CW
Published: March 2018

N-methyl-D-aspartate (NMDA) is an acidic amino acid which depolarizes neurons by selectively interacting with a distinct class of excitatory amino acid receptor. Recent evidence has indicated that this receptor is a neurotransmitter receptor in the spinal cord, cerebral cortex, and hippocampus for which the endogenous ligand is likely to be L-glutamate or a structurally related compound. Using quantitative autoradiography, we have studied the anatomical distribution of the class of L- [3H]glutamate-binding sites displaced by NMDA, which appear to correspond to NMDA receptors. The CA1 region of the hippocampus contains the highest density of sites. In general, telencephalic regions have high levels of binding sites. The cerebral cortex shows significant density variations among the differing layers and regions, with the highest levels found in the frontal cortex layers I to III. Within the basal ganglia, the highest levels are found in the nucleus accumbens, intermediate levels are found in the caudate/putamen, and very low levels are found in the globus pallidus. Thalamic regions have moderate levels with variations among differing regions. Midbrain and brainstem have low levels of binding sites, but within these regions there are structures exhibiting higher levels, e.g., the nucleus of the solitary tract and the inferior olive. The distribution of NMDA sites is consistent with most, but not all, of the regions previously proposed to use glutamate as an excitatory transmitter. Thus, the distribution of NMDA-sensitive L-[3H]glutamate-binding sites suggests that the NMDA receptor represents a major, distinct subset of excitatory amino acid receptors and indicates regions in which neurotransmission may be mediated or modulated by this receptor.
Slowing of neurofilament transport and the radial growth of developing nerve fibers
Authors: Hoffman PN , Griffin JW , Gold BG , Price DL
Published: March 2018

Several lines of evidence indicate that neurofilaments are major intrinsic determinants of axonal caliber in myelinated nerve fibers, and that the delivery of neurofilaments by slow axonal transport is an important mechanism by which neurons regulate axonal caliber. To further clarify the relationship between neurofilament transport and axonal caliber, we examined transport in developing motor fibers of rat sciatic nerve. In 3-, 10-, 12-, and 20-week-old rats, lumbar motor neurons were labeled by the intraspinal injection of radioactive amino acids, and the distributions of labeled cytoskeletal proteins within the sciatic nerve were analyzed at various times afterwards using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel fluorography, and liquid scintillation spectroscopy. There was a progressive decline in the velocity of neurofilament transport with increasing distance along axons undergoing radial growth. By examining transport in different regions of the nerve in animals of the same age, we separated age-dependent reductions in velocity from those related to position along the nerve. The cross-sectional areas of these motor axons (in the L5 ventral root) increased linearly between 3 and 18 weeks of age. Quantitative electron microscopic analysis at 3 and 10 weeks of age revealed that neurofilament density was comparable in fibers of all calibers, indicating that the radial growth of these myelinated nerve fibers correlates with a proportional increase in neurofilament content. We propose that progressive reduction in the velocity of neurofilament transport along the nerve provides for radial growth during development.
The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences
Authors: Zeller NK , Behar TN , Dubois-Dalcq ME , Lazzarini RA
Published: March 2018

The developmental expression of the myelin basic protein (MBP) gene was studied in rat cultured oligodendrocytes using immunofluorescence and in situ hybridization. In newborn rat brain cultures, which contain only glial cells, large amounts of MBP-specific mRNA (as assayed by grain counts in autoradiograms) abruptly accumulated within immature oligodendrocytes 5 to 6 days postnatal. MBP always emerged 6 to 8 days after birth; thus, a week after, galactocerebroside (GC), an early oligodendrocyte marker, had appeared. The percentage of MBP mRNA and MBP-positive cells peaked at about 15 days postnatal and decreased thereafter. The time of emergence of MBP in these cultured oligodendrocytes appears to be determined at a very early stage in their development and independent of continuous neuronal influences. There is a striking correspondence between the times of appearance of MBP in cultured oligodendrocytes and those in the intact animal. Thus, primary cultures made from 5-day prenatal, newborn, and 2-day postnatal animals all express MBP at about the same developmental stage, namely, after 14, 8, and 6 days in culture, respectively. Furthermore, cultured oligodendrocytes obtained from the spinal cord express MBP before those obtained from midbrain or hemispheres, as they would in the intact animal. Thus, the developmental expression of the MBP gene occurs in a similar time frame in vitro and in vivo. In oligodendrocyte-enriched cultures, where 60% to 80% of the cells express MBP, in situ hybridization with the cDNA clone revealed MBP-specific mRNA in the cell body and sometimes in the processes of the differentiated oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes
Authors: Schwartz RD , Jackson JA , Weigert D , Skolnick P , Paul SM
Published: March 2018

Membrane chloride (Cl-) permeability was studied in a novel subcellular brain preparation, the synaptoneurosome. Using a radioactive tracer exchange technique, Cl- transport was determined by measuring 36Cl- efflux from rat cerebral cortical synaptoneurosomes. Barbiturates increased 36Cl- efflux in a dose-dependent manner with the following relative order of potency: 5-(1,3-dimethylbutyl)-5-ethyl barbituric acid ((-)-DMBB) greater than pentobarbital greater than secobarbital greater than (+)-DMBB greater than hexobarbital greater than amobarbital greater than mephobarbital. Phenobarbital and barbital were virtually inactive. A good correlation was observed between the potencies of these barbiturates in stimulating 36Cl- efflux and their anesthetic potencies in mice (r = 0.90, p less than 0.01) and their abilities to enhance [3H] diazepam binding to brain membranes (r = 0.77, p less than 0.05). The effect of pentobarbital in enhancing 36Cl- efflux was reversed by the gamma-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline. Picrotoxin and bicuculline both decreased 36Cl- efflux in the absence of pentobarbital, suggesting the presence of endogenous GABA. Incubation of synaptoneurosomes with 4,4′-di- isothiocyano- or dinitro-2,2′-disulfonic acid stilbene, inhibitors of anion transport, also decreased both basal and pentobarbital-induced 36Cl- efflux. Pentobarbital (500 microM) was most effective in inducing 36Cl- efflux in the cerebellum, hippocampus, and cortex (23.7, 23.6, and 22.5%, respectively), and was less effective in stimulating 36Cl- efflux in the striatum (15.1%) and pons-medulla (6.2%). The relative efficacy of pentobarbital in enhancing 36Cl- efflux among these various brain regions was highly correlated (r = 0.96, p 0.01) with the relative densities of [35S]-t-butylbicyclophosphorothionate-binding sites, a measure of GABA-gated Cl- channel density.(ABSTRACT TRUNCATED AT 250 WORDS)
Responsiveness of ventrobasal thalamic neurons after suppression of S1 cortex in the anesthetized rat
Authors: Yuan B , Morrow TJ , Casey KL
Published: March 2018

Corticofugal influences on the responses of ventrobasal (VB) thalamic neurons to repetitive stimuli were studied in anesthetized rats by suppressing primary somatosensory (S1) electrocortical activity with topically applied lidocaine. Effective concentrations of lidocaine were confined to S1 and immediately adjacent cortex and suppressed evoked S1 responses and corticofugal discharges. Suppression of S1 cortex reduced the average number of spikes discharged by 83 VB neurons in response to each of 25 electrical somatic stimuli delivered at frequencies ranging from 1 to 50 Hz. Of 20 units studied both before and after S1 suppression, 14 (70%) showed a similar reduced response to repetitive stimuli. Cortical suppression produced no consistent changes in spontaneous activity, somatic stimulus threshold, response latency, or size of receptive field. There was no significant difference in the effect of cortical suppression on the responsiveness of 8 VB neurons to repetitive medial lemniscal, as compared to somatic, stimuli. We conclude that, in the anesthetized rat, S1 corticofugal activity facilitates somato-sensory transmission to VB neurons and that this facilitation is mediated, at least in part, by corticothalamic neurons.
Laminin and optic nerve regeneration in the goldfish
Authors: Hopkins JM , Ford-Holevinski TS , McCoy JP , Agranoff BW
Published: March 2018

Previous work from our laboratory had shown that goldfish retinal fragments explanted onto a polylysine substratum 1 to 2 weeks following optic nerve crush exhibit a striking clockwise pattern of neuritic outgrowth. In the present study, however, when the basal lamina component laminin was used as a substratum, neurites grew out as uncurved spokes, were less fasciculated, and had an increased rate of elongation. When laminin was combined with polylysine in the substratum, the degree of fasciculation and rate of elongation were similar to those seen on laminin alone, whereas the tendency for clockwise outgrowth was even more pronounced than that observed with polylysine alone. These results suggest that regenerating neurites have an affinity for laminin. Using an antibody to murine Engelbreth-Holm- Swarm sarcoma laminin, which cross-reacted with basal lamina in goldfish tissue sections, we studied the histochemical distribution of laminin in the goldfish visual system. Immunoperoxidase staining for laminin showed a characteristic scalloped pattern of staining in cross- sections of optic nerve bundles. Following optic nerve crush, the reaction product became much more diffuse and intense, especially distal to the crush site. When the retinal ganglion cell bodies were eliminated by removing the eye, the degenerating optic nerve stump still showed the intensive staining. We interpret these results to indicate that optic nerve glia are responsible in large part for the formation of laminin. Taken together, these in vivo and in vitro findings suggest that laminin plays a role in nerve regeneration in the goldfish central nervous system.
The expression and localization of synaptic vesicle antigens at neuromuscular junctions in vitro
Authors: Bixby JL , Reichardt LF
Published: March 2018

To examine the biochemical differentiation of presynaptic nerve terminals in vitro, we studied the expression and localization at synapses of two synaptic vesicle-specific antigens, synapsin I and protein p65. We purified these proteins from brain and raised a rabbit antiserum against each of them. Chick synapsin I had a slightly smaller molecular weight than its mammalian homologue, although the two share several properties. With the anti-p65 serum, the protein p65 could be detected consistently in presynaptic terminals at the neuromuscular junction in vivo, as had been previously shown for synapsin I. These antisera proved sufficiently sensitive to allow a study of the developmental expression of the antigens in embryonic chick brain, using protein blots. The two antigens are not coordinately regulated; protein p65 was detected substantially sooner in development that was synapsin I. Both synapsin I and protein p65 are expressed by ciliary ganglion neurons in vitro, as assessed by immunofluorescence using the affinity-purified antisera. The two antigens co-localized at all times in culture. Neurons grown alone were reliably stained only after 4 to 5 days in culture, but comparable levels of staining were found after 1 day when neurons were co-cultured with embryonic myotubes. In the co- cultures, staining was initially high in growth cones and neurites, but the brightest staining became confined to sites of nerve-muscle contact over 4 to 5 days in culture. In mature cultures, patches of bright staining for the vesicle antigens coincided with patches of acetylcholine receptors, suggesting that the antigens had become localized at synapses. The time course of this localization process suggests that it corresponds to the morphological maturation of synapses. It should be possible to exploit this system to obtain information about the molecules and processes involved in the induction of presynaptic differentiation.
The neuronal endomembrane system. I. Direct links between rough endoplasmic reticulum and the cis element of the Golgi apparatus
Authors: Lindsey JD , Ellisman MH
Published: March 2018

This is the first of three papers describing new components and structural relationships within the neuronal endomembrane system. This system includes: the nuclear envelope, rough endoplasmic reticulum, the Golgi apparatus, lysosomes, axoplasmic reticulum, and discrete cytoplasmic compartments such as vesicles, multivesicular bodies, and so on. Previous high voltage electron microscope studies of osmium- impregnated Golgi apparatus have shown that small varicose tubules often arise from the cis element. In bullfrog spinal ganglia, these tubules have been seen to extend into cytoplasmic domains occupied by rough endoplasmic reticulum (RER). This study was undertaken to determine whether varicose tubules form direct connections with RER and to measure cytoplasmic density of varicose tubules. Spinal ganglia from Rana catesbeiana were fixed by osmium impregnation and, in some cases, were then stained en bloc with lead aspartate. Sixty- to 3000-nm-thick sections of this tissue, as well as of ganglia prepared by conventional protocol, were studied using standard and high voltage electron microscopy. Quantitative analysis revealed that 9% of the varicose tubules connected with the Golgi cis element. To determine whether continuities existed between RER and any of these tubules, more than 50 tubules were fully reconstructed from images of serial sections. The “ends” of these reconstructed tubules were then studied by high magnification stereoscopy. This was facilitated by the en bloc lead staining which allowed visualization of unimpregnated RER elements along with the impregnated tubules. Small varicose tubules as well as larger smooth tubules were found to have formed confluent bridges between the cis element and RER. In addition, the varicose tubules were found to bridge widely separated elements of RER. Finally, numerous examples of varicose and smooth tubules were seen to extend from RER and from cis elements to eventually form blind endings. These findings raise the possibility that the tubules form highly dynamic transitory connections between RER and the Golgi apparatus as well as between separated elements of RER. Those between RER and Golgi apparatus are ideally positioned to play a major role in the transfer of protein or lipid components first assembled in RER to the Golgi apparatus for terminal synthetic modification.
Formation of electrical synapses between isolated, cultured Helisoma neurons requires mutual neurite elongation
Authors: Hadley RD , Bodnar DA , Kater SB
Published: March 2018

Our previous experiments have suggested the hypothesis that conjoint active neuronal outgrowth may be necessary for formation of new electrical synapses between identified neurons of adult Helisoma buccal ganglia. This growth dependence hypothesis now has been tested by examining the responses of individual pairs of neurons in isolation from the influences of the ganglionic environment. Isolated cell culture of identified neurons (neuron 5) showed that: (i) neurons growing in cell culture undergo a predictable sequence of morphological changes culminating in a stable morphological state (i.e., growth stops); (ii) contact between actively growing neurons in cell culture results in the formation of electrical connections, just as in ganglia; and (iii) when an actively growing neuron encounters a neuron that is morphologically stable, electrical connections do not form or are very weak, even though strong connections are made between pairs of actively growing neurons in the same culture. These results establish that processes closely associated with growth are required for formation of electrical synapses between these neurons.
Neurite guidance by non-neuronal cells in culture: preferential outgrowth of peripheral neurites on glial as compared to nonglial cell surfaces
Authors: Fallon JR
Published: March 2018

Growing axons in the peripheral nervous system (PNS) encounter a variety of cellular and extracellular substrates. Since it is difficult to sort out the possible contributions of these diverse components of the extracellular environment to axonal guidance in vivo, I have developed an in vitro system to study neurite outgrowth on two classes of cells which may provide as substrates for growing axons during development or regeneration: glial cells, e.g., astrocytes and Schwann cells, and nonglial cells, e.g., fibroblasts. Although neurites from sympathetic and spinal sensory ganglia explants grew onto preformed monolayers of both glial and nonglial cells, glial cells were a markedly better substrate. On the glial cells the neurites extended at a rate of 25 to 30 micron/hr and traveled singly or in fine fascicles; their growth cones displayed long filopodia and migrated on the upper surface of the monolayer cells. Conditioned media experiments suggested that neurite outgrowth on glial cell monolayers was not mediated by soluble secreted factors. These results indicate that the glial cell surface is an attractive substrate for neurite outgrowth. In contrast, on nonglial cells the rate of outgrowth was only 10 to 15 micron/hr, large neurite fascicles were common, and the growth cones migrated beneath the monolayer cells in contact with the underlying artificial substrate. This location of the growth cone, coupled with the observation that conditioned medium from these cells promoted neurite outgrowth only when bound to artificial substrates, suggests that secreted substrate-associated components may be an important determinant of neurite outgrowth on nonglial cell monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Representation of tones in noise in the responses of auditory nerve fibers in cats. I. Comparison with detection thresholds
Authors: Costalupes JA
Published: March 2018

Rate and temporal responses evoked by 1-kHz or 8-kHz tones in continuous broadband noise are described for large populations of auditory nerve fibers in anesthetized cats. The signal-to-noise ratios (S/Ns) of the tone and noise stimuli were above behavioral detection thresholds. Stimulus combinations were presented (1) over a range of moderate to high noise intensities at a constant S/N and (2) using high intensity noise and varying S/N. Responses of low (less than 1 spike/sec) and medium (1 to 19 spikes/sec) spontaneous rate (SR) fibers were compared with those of high SR (greater than 19 spikes/sec) fibers. Low and medium SR fibers with best frequencies in the region of the test tone frequency exhibited tone-evoked rate changes at all sound levels tested. High SR fibers, in contrast, exhibited much weaker tone- evoked rate changes at the lowest noise level tested. In the presence of high intensity noise, high SR fibers did not exhibit tone-evoked rate changes due to saturation by the noise. Fibers with best frequencies in the region of 1 kHz also exhibited strong phase-locking to the 1 kHz tone which increased as the tone level increased but which did not differ for the various SR groups. Results suggest that information in the rate responses of low and medium SR fibers can account for the encoding of information about tones in noise by the nervous system.