bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective activation of Ca(2+)-activated PKCs in Aplysia neurons by 5- HT
Authors: Sossin WS , Schwartz JH
Published: March 2018

Ca(2+)-activated and Ca(2+)-independent protein kinase Cs (PKCs) are present in the nervous system of the marine mollusk Aplysia californica (Kruger et al., 1991). Sensitizing stimuli or application of the facilitatory transmitter 5-HT to intact isolated ganglia produces the presynaptic facilitation of sensory-to-motor neuron synapses that underlies behavioral sensitization, which is a simple form of learning. Activation of PKC can also produce this presynaptic facilitation (Braha et al., 1990). To determine which type of PKC is activated, we developed a sensitive and selective assay to measure both Ca(2+)- activated and Ca(2+)-independent PKC activities in crude supernatant and membrane fractions of nervous tissue. This assay is based on the specific binding of the Ca(2+)-activated PKCs to phosphatidylserine vesicles in the presence of Ca2+ and makes use of a novel synthetic peptide with sequences conforming to phylogenetically conserved pseudosubstrate regions of the Ca(2+)-independent kinases. We provide evidence that the presynaptic facilitation is produced by a Ca(2+)- activated isoform: application of 5-HT increases the amount of the Ca(2+)-activated PKC activity associated with the membrane. Under these conditions, no increase in Ca(2+)-independent kinase activity is seen.
Development of topographic order in the mammalian retinocollicular projection
Authors: Simon DK , O'Leary DD
Published: March 2018

We have used the anterograde axon tracer 1,1′-dioctodecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate (Dil) to characterize the development of topographic order in the rat retinocollicular projection. Retinal axons were labeled by Dil injections covering 0.15– 2% of peripheral temporal, nasal, superior, or inferior retina, or more central retina, in rats ranging in age from embryonic day 20 to postnatal day (P) 19. At P11-P12 and later, such injections label retinal axons that form overlapping arbors restricted to a topographically correct terminal zone covering about 1% of the superior colliculus (SC) area. At perinatal ages, though, axons labeled from each retinal site are distributed in the SC over much of its medial- lateral axis and extend caudally well beyond the rostral-caudal location of their correct terminal zone; some continue caudally into the inferior colliculus. Axons typically form side branches and often arborize at topographically incorrect positions throughout the SC; however, they appear to branch preferentially in a region that includes, but is much larger than, their correct terminal zone. The mature, retinotopically ordered projection emerges during an early postnatal remodeling period through the rapid remodeling of the early, diffuse projection. This process involves the large-scale removal of axons, axon segments, branches, and arbors from topographically inappropriate positions concurrently with a dramatic increase in branching and arborization at topographically correct locations. Quantitative measurements show that elimination of aberrant branches without loss of the primary axons contributes substantially to the development of order. By P6, fewer mistargeted axons persist, but those that do persist tend to branch or arborize more extensively in topographically inappropriate regions. By P8, the labeling patterns begin to approximate those seen at maturity. Further refinement leads to an adultlike topographic ordering of axonal arborizations by P11- P12. At maturity, some axons take very indirect routes to reach their correct terminal zone. However, such trajectory changes typically correct only small positional inaccuracies, indicating that axons and axon segments that make larger targeting errors do not survive the remodeling phase. Previous retrograde labeling studies indicate that some retinal axons make topographic targeting errors (O'Leary et al., 1986; Yhip and Kirby, 1990), but none have suggested the degree of diffuseness revealed by anterograde labeling with Dil. Our findings show that directed axon growth is inadequate as a mechanism to develop the topographic ordering of retinal axons in the rat SC. Rather, mechanisms that control the removal of mistargeted axons and promote the arborization of correctly positioned axons are critical for the development of retinotopic order.(ABSTRACT TRUNCATED AT 400 WORDS)
Bipolar cells specific for blue cones in the macaque retina
Authors: Kouyama N , Marshak DW
Published: March 2018

A distinct subpopulation of bipolar cells in macaque monkey retina was labeled with antisera that recognize glycine-extended cholecystokinin precursors. The labeled bipolar cells were found throughout the retina and had dendrites contacting a subpopulation of cone pedicles and axons ramifying in the fifth stratum of the inner plexiform layer. Several lines of evidence indicate that the labeled bipolar cells are a single type despite some variations in their morphology. First, the density of perikarya and their diameters varied continuously as a function of eccentricity. Second, the positions of perikarya within the inner nuclear layer and the level at which the axons branched in the inner plexiform layer were constant at all eccentricities. Bipolar cells with similar morphology have been described previously as “blue cone bipolar cells” (Mariani, 1984b), but there was no direct evidence that this was the case. In this study, we show by light microscopy that labeled bipolar cells have dendrites ending exclusively upon presumptive blue cones labeled by Procion black dye. All blue cones were contacted by labeled bipolar cells, and virtually all bipolar cells contacted blue cones, the only exceptions being in regions where blue cones had been lost. Approximately 20% more labeled bipolar cells than blue cones were found at every eccentricity; thus, connections between blue cones and labeled bipolar cells were not strictly one to one. The mean number of cones presynaptic to each bipolar cell was 1.2, and the mean number of bipolar cells postsynaptic to each cone was 1.8. By an electron microscopic study of labeled bipolar cell dendrites, we determined that they became central elements of ribbon synapses in blue cones. Some of their ribbon synapses were unusual: in one type, a single, large labeled dendrite was postsynaptic to two or more ribbons, while in the other type, ribbons had two or more central elements. The presence of these invaginating contacts and the axonal terminals in the proximal inner plexiform layer suggest that the labeled bipolar cells depolarize to short-wavelength stimuli and function to relay information from blue cones to the inner plexiform layer. There were also other, unlabeled bipolar cell dendrites that received inputs from blue cones at basal junctions and triad-associated flat contacts, which suggests that there are additional types of bipolar cells conveying information from short- wavelength cones in the primate retina.
Cellular analogs of visual cortical epigenesis. I. Plasticity of orientation selectivity
Authors: Fregnac Y , Shulz D , Thorpe S , Bienenstock E
Published: March 2018

A differential pairing procedure was applied in vivo to individual neurons in the primary visual cortex of anesthetized paralyzed cats, in order to produce changes in their relative orientation preference. While we recorded from a single cell, its visual response to a light bar was driven iontophoretically to a “high” level when stimulating with an initially nonpreferred orientation (S+), and alternately reduced to a “low” level when stimulating with the preferred orientation (S). This associative procedure was devised to test the possible role of neuronal coactivity in controlling the plasticity of orientation selectivity. Among 87 cells tested, 35 (40%) showed significant long-lasting changes, either in the relative orientation preference for the two “paired” stimuli S+ and S-, in the global orientation tuning profile, or in both. Measurements of relative orientation preference demonstrated significant effects in 27 cells (31%), all in favor of the positively reinforced orientation (S+). Modifications of orientation selectivity (studied over the entire orientation spectrum in 45 of the conditioned cells) usually consisted (21 out of 25 modified cells) of a competitive reorganization of the orientation tuning curve: the preferred orientation shifted toward S+, and a loss of relative visual responsiveness was observed for orientations close to the negatively reinforced orientation (S-). The largest changes were found in deprived kittens at the peak of the critical period, although the probability of inducing a significant change studied during the first year of postnatal life was independent of age. These functional modifications demonstrated at the cellular level are analogous to those induced by a global manipulation of the visual environment, when only a restricted spectrum of orientations is experienced during the critical period. Our results support the hypothesis that covariance levels between pre- and postsynaptic activity determine the sign and the amplitude of the modification of efficacy of cortical synapses.
Does lineage determine the dopamine phenotype in the tadpole hypothalamus?: A quantitative analysis
Authors: Huang S , Moody SA
Published: March 2018

The ancestry of dopaminergic (DA) neurons in the Xenopus laevis hypothalamus was investigated by combining intracellular lineage dye injections of 16- and 32-cell blastomeres with the immunofluorescent detection of tyrosine hydroxylase at tadpole stages. At these stages, DA neurons in the hypothalamus comprise a discrete nucleus that contains from 22 to 45 cells on each side [mean = 32.6 +/- 6.6 (SD)]. The DA nucleus descends from only four of the 16-cell blastomeres. The two dorsal midline blastomeres (D1.1) are the major progenitors, and in all embryos studied they contributed to the DA nucleus. The two dorsal lateral blastomeres (D1.2) contribute to the DA nucleus in only about half of the embryos. Thus, the DA nucleus descends only from a discrete group of progenitors, and the participation of some of the progenitors in the DA lineage is only probabilistic. The number of DA neurons generated by the same blastomere varied greatly in different animals. This variation in cell number correlated with the degree of coherence and the density of the clone in the hypothalamus, rather than with clonal ancestry. Bilateral deletion of the major 32-cell progenitor (D1.1.1) resulted in a nearly complete restitution of the DA nucleus in 74% of the embryos that successfully completed gastrulation and neurulation. In the rest, the hypothalamus was smaller than normal or missing, and the DA nucleus was significantly reduced in size or absent. These results show that the DA nucleus can be restored after its normal lineage is deleted, but complete regulation is not always accomplished. Several blastomere progenitors dramatically altered their contribution to the DA nucleus after D1.1.1 ablation, including two blastomeres that normally do not contribute to the DA lineage. Thus, the fate to produce DA neurons is not determined at cleavage stages.
Endogenous NGF and nerve impulses regulate the collateral sprouting of sensory axons in the skin of the adult rat
Authors: Diamond J , Holmes M , Coughlin M
Published: March 2018

We have investigated the co-involvement of endogenous NGF and impulses in the collateral sprouting of cutaneous sensory nerves in adult rats, specifically the A delta-axons involved in mechanonociception and the C- fibers that mediate heat nociception. Their collateral sprouting was measured by the progressive expansion, respectively, of the behaviorally defined “pinch” and “heat” fields into surrounding denervated skin (the light-touch A alpha-fibers do not sprout in adult mammals). The expansions of such “isolated” fields were totally prevented in animals injected daily with anti-NGF serum, but developed normally after treatment was discontinued. Light microscopic and EM examination of the skin confirmed that the effect of the anti-NGF treatment was attributable to its prevention of collateral sprouting. Initiation of treatment would also rapidly halt sprouting already in progress. Finally, intradermal injections of purified NGF protein would not only increase the rate of nociceptive fiber sprouting, but also evoke sprouting de novo within normally innervated skin (again, A alpha- axons were unaffected). We conclude that the collateral sprouting of intact nociceptive nerves following partial denervation of skin is entirely dependent on endogenous NGF. The observed latency of this sprouting was 10–12 d; we estimate, however, that at least 2 d of field expansion is required for its reliable detection. Thus, about 8–10 d are required for NGF levels in the skin to rise to effective levels, and for the neurons to respond and initiate sprouting. From indirect findings, the NGF component of this sprouting latency appears to be about 2 d. In accord with earlier findings, the remaining “initiation time” was reduced by 5–6 d if the neurons were briefly excited, even 2 d prior to the isolation of their fields. Unexpectedly, this phenomenon of “precocious sprouting” requires that endogenous NGF be available; the sprouting latency reverted to normal values when the conditioning impulses were evoked during a 2 d anti-NGF “umbrella.” In contrast to the impulse-sensitive neuronal mechanisms involved in the initiation of sprouting, those underlying the sprouting rate were unaffected by nerve activity and were entirely dependent on the level of endogenous NGF. We suggest that interactions like that revealed in these studies between a sprouting agent and impulses that seem to prime the neuron's response to it contribute to plasticity within the nervous system.
Involvement of ventromedial medulla "multimodal, multireceptive" neurons in opiate spinal descending control system: a single-unit study of the effect of morphine in the awake, freely moving rat
Authors: Martin G , Montagne-Clavel J , Oliveras JL
Published: March 2018

In the present work, we have studied the effects of systemic morphine on the electrophysiological properties of ventromedial medulla (VMM) neurons in the awake, freely moving rat. By means of a chronically implanted single-unit recording device, a drug delivery catheter, and the use of controlled innocuous and noxious cutaneous stimuli, we were able to study precisely the spontaneous and evoked VMM neuronal activities. We have particularly focused our attention upon the VMM “multimodal, multireceptive” units, excited by non-noxious and noxious stimuli (VMM MULT ON), which we have already determined as the neuronal class potentially involved in nociceptive processes at VMM level. We found that morphine (3 mg/kg, i.v.) does not affect the spontaneous activity of these neurons whereas their responses to noxious heat are strongly attenuated (70%), over a prolonged period (about 2 hr) associated with an increase in the response latency. This action of morphine appears to be pharmacologically specific since it is dose dependent to some extent, and is reversed by 0.3 mg/kg of naloxone. In parallel with this pharmacological specificity, we have also demonstrated a preferential physiological effect since the response of the VMM MULT ON neurons to light touch application is not affected by morphine. This specificity is emphasized by the fact that morphine does not modify the activity of the other VMM neuronal groups such as the units unresponsive to any kind of peripheral stimuli, and does not reveal “new” neuronal classes such as those we have found in previous studies after barbiturate administration. The differential effect upon the noxious versus innocuous inputs of these units produced by the opiate reinforces their participation in nociceptive processing since similar effects have been reported in well-known nociceptive somatosensory structures such as the dorsal horn of the spinal cord. Furthermore, although the precise mechanisms of action have not yet been determined, the spinal projection of the VMM MULT ON neurons, previously demonstrated by our group, suggests their involvement in an opiate descending spinal control system of nociception. Although speculative, one can imagine either a direct facilitatory MULT ON spinal effect being attenuated by morphine (disfacilitation), or a morphine-induced disinhibition of inhibitory GABAergic neurons acting upon the MULT ON neurons.
Analysis of cis-regulatory elements in the 5' flanking region of the Drosophila melanogaster choline acetyltransferase gene
Authors: Kitamoto T , Ikeda K , Salvaterra PM
Published: March 2018

We have analyzed the cis-regulatory elements in the 5′ flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6). DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation. A 7.4 kb 5′ flanking sequence directed beta- galactosidase expression in the adult optic lobes and other well- defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein. In contrast, the proximal 3.3 kb and 1.2 kb of 5′ flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct. Our results indicate that both qualitative and quantitative regulatory elements are present in the 5′ flanking DNA and that these elements distinguish various subsets of cholinergic neurons. We have also fused the same 5′ flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies. Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature- dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild- type ChAT expression to overcome these mutant phenotypes.
The role of premotor interneurons in phase-dependent modulation of a cutaneous reflex during swimming in Xenopus laevis embryos
Authors: Sillar KT , Roberts A
Published: March 2018

Phase-dependent reflex modulation during fictive “swimming” in Xenopus laevis embryos has been examined with intracellular recordings from rhythmically active spinal neurons. (1) At rest, cutaneous trunk or tail skin stimulation evokes EPSPs in motoneurons and premotor excitatory and inhibitory interneurons of the opposite motor system. During swimming, these EPSPs can only be evoked during the depolarized phase of activity and can then produce extra action potentials that lead to phase-dependent reflexes in ventral roots. On the stimulated side, IPSPs are evoked in rhythmic neurons that can block centrally generated action potentials if the stimulus coincides with the inhibited phase of the swimming cycle. This inhibition suppresses ventral root discharge in a phase-dependent manner. (2) The presence of premotor interneurons in the crossed reflex pathway suggests two parallel routes for cutaneous excitation to reach the motoneurons, one direct and the other indirect through excitatory premotor interneurons. During swimming, the crossed excitation through both routes is gated by the rhythm-generating circuit to allow summation in motoneurons only during the depolarized phase of the swim cycle. (3) Following phase- dependent reflexes, the frequency of swimming is raised for several cycles, a phenomenon that requires sensory activation of premotor rhythm-generating interneurons. The results provide evidence on the role of identified premotor spinal interneurons in phase-dependent reflex modulation.
A circuit for coding interaural time differences in the chick brainstem
Authors: Overholt EM , Rubel EW , Hyson RL
Published: March 2018

Third-order auditory neurons in the avian nucleus laminaris (NL) are the first to receive binaural input. In the chick, NL consists of a monolayer of neurons with polarized dendritic arbors oriented dorsally and ventrally. Afferents from second-order neurons in the ipsilateral nucleus magnocellularis (NM) innervate the dorsal dendrites of NL neurons, distributing processes of approximately equal length to NL neurons along an isofrequency band (roughly caudomedial to rostrolateral). Afferents from the contralateral NM innervate the ventral dendrites of NL neurons, distributing collateral branches sequentially as they proceed from caudomedial to rostrolateral along the isofrequency band of neurons. This innervation pattern could be the basis of a “delay line” circuit, as postulated in models of neural networks mediating sound localization. We examined this circuit by analyzing evoked field potentials using a brain slice preparation containing both NL and NM. The results were consistent with the previous anatomical findings. When the ipsilateral auditory nerve or ipsilateral NM was stimulated, there was no consistent variation in the latency of postsynaptic field potentials across the medial-to-lateral extent of NL. In contrast, when the contralateral NM or NM axons in the crossed dorsal cochlear tract were stimulated, a linear increase in the latency of postsynaptic potentials was observed from medial to lateral positions in NL. When stimulation amplitudes for both the ipsilateral and contralateral inputs were adjusted so as to produce little or no postsynaptic field potential, simultaneous bilateral stimulation evoked a pronounced response. Thus, NL neurons can act as “coincidence detectors.” The amplitude of the postsynaptic response was dependent on the relative timing of stimulation of the two inputs. The optimal time difference changed systematically across the medial-to-lateral extent of NL. This system of delay lines and coincidence detectors could provide a mechanism for converting interaural time differences into a “place map” within NL.
Internal models of limb geometry in the control of hand compliance
Authors: Lacquaniti F , Borghese NA , Carrozzo M
Published: March 2018

The aim of this article is to describe the role of some neural mechanisms in the adaptive control of limb compliance during preplanned mechanical interaction with objects. We studied the EMG responses and the kinematic responses evoked by pseudorandom perturbations continuously applied by means of a torque motor before and during a catching task. The temporal changes of these responses were studied by means of an identification technique for time-varying systems. We found a transient reversal of EMG stretch reflex responses centered on the time of ball impact on the hand; this reversal results in a transient coactivation of antagonist muscles at both the elbow and the wrist. The kinematic responses describe the relation between torque input and position output. Thus, they provide a global measure of limb compliance. The changes in limb compliance during catching were quantified by computing error criteria either in the Cartesian coordinates of the hand or in the angular coordinates of the elbow and wrist joints. We found that only the hand compliance in Cartesian coordinates is consistently minimized around impact, in coincidence with the transient reversal of the stretch reflex responses. By contrast, the error criteria expressed in the angular coordinates of the joints have a variable time course and are not minimized around impact. It is known that hand compliance depends on both the pattern of muscle activities and the geometrical configuration of the limb. Therefore, the lack of consistent correlation between the changes in hand compliance and the changes in the geometrical configuration of the limb during catching indicates that the gating of the stretch reflex responses around impact time is based on an internal model of limb geometry.
Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture
Authors: Liu DW , Westerfield M
Published: March 2018

Previous culture studies have demonstrated that muscle cells autonomously express and cluster ACh receptors (AChRs) and that contact by neurites induces a reorganization of these clusters. We studied these phenomena in zebrafish embryos where the same cells could be examined in vivo and in vitro, and where contacts between cells could be viewed repeatedly. Receptor clusters first appeared when the pioneer growth cones emerged from the spinal cord, were always associated with labeled branches, and developed normally in the presence of neuromuscular transmission blockers. When motoneurons were removed, the muscles failed to cluster receptors. In contrast, muscle cells grown in cell culture uncontacted by nerves clustered AChRs. Our results suggest that clustering of AChRs in living embryos is induced by the presence of neurites and does not occur in the absence of neuronal contact. We suggest that conditions in cell culture, which differ from those in the intact embryo, induce clusters on isolated muscle cells. Moreover, our results demonstrate that receptors cluster without binding transmitter and in the absence of neuronal activity.
On the frequency limit and phase of outer hair cell motility: effects of the membrane filter
Authors: Santos-Sacchi J
Published: March 2018

Whole-cell voltage clamp and displacement-measuring photodiode techniques were used to study electrophysiological and mechanical properties of the guinea pig outer hair cell (OHC). OHCs demonstrate a voltage-mechanical response (V-M) function that can be fit by a two state Boltzmann relation, where the cell normally rests near the hyperpolarizing saturation region (-70 to -90 mV). The voltage at half- maximal length change (Vh) is depolarized relative to the resting potential, and this ensures that for symmetrical sinusoidal voltage stimulation about the resting potential, AC and DC mechanical responses will be generated. Analysis of OHC motility using pure tone voltage bursts from 11 to 3200 Hz demonstrates both AC and DC mechanical responses. By exploiting the frequency-dependent current-voltage phase separation that is characteristic of an RC-dominated system under voltage clamp, it is demonstrated that OHC motility follows the phase of AC transmembrane voltage and not that of current. For voltage stimulation across frequencies in the acoustic range, the motility cutoff frequency corresponds to the cutoff frequency of the imposed transmembrane voltage. Frequency cutoffs approaching 1 kHz have been measured but are clamp time constant limited. These observations are congruent with the voltage dependency hypothesis of OHC motility. In addition, the DC component of the mechanical response is shown to be frequency independent, but to decrease in magnitude disproportionately compared to the AC component as the magnitude of the driving voltage decreases. This is predicted from the form of the V-M function, whose level dependent DC nonlinearity is a consequence of the resting potential being displaced from Vh. The net effect is that the mechanical DC: AC ratio approaches zero for small AC voltages. Taken together, these findings question the ability of the OHC mechanical response to influence organ of Corti micromechanics at high acoustic frequencies where a tuned amplification of basilar membrane motion is hypothesized.
Quantitative enzyme radioautography with 3H-Ro 41-1049 and 3H-Ro 19- 6327 in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain
Authors: Saura J , Kettler R , Da Prada M , Richards JG
Published: March 2018

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. Since the cellular localization of the isoenzymes in the CNS and peripheral organs determines to a large extent which substrate has access to which isoenzyme, knowledge of their tissue distribution and cellular localization is essential. Here we describe how reversible and selective inhibitors of MAO-A and MAO-B [Ro 41–1049 and Ro 19–6327 (lazabemide), respectively] can be used, as tritiated radioligands, to map the distribution and abundance of the enzymes in microscopic regions of the rat CNS and peripheral organs, and human brain by quantitative enzyme radioautography. The in vitro binding characteristics of both radiolabeled inhibitors revealed them to be selective, high-affinity ligands for the respective enzymes. KD and Bmax values for 3H-Ro 41–1049 in rat cerebral cortex were 10.7 nM and 7.38 pmol/mg protein, respectively, and for 3H-Ro 19–6327 were 18.4 nM and 3.45 pmol/mg protein, respectively. In accordance with their potencies as enzyme inhibitors, binding to MAO-A and MAO-B was competitively inhibited by clorgyline (IC50 = 1.4 nM) and L-deprenyl (selegiline; IC50 = 8.0 nM), respectively. The capacities of various rat and human tissues to bind the radioligands correlated extremely well with their corresponding enzyme activities. As revealed by the respective binding assays, the distribution and abundance of MAO-A and MAO-B in the tissues investigated differed markedly. MAO-A was most abundant in the locus coeruleus, paraventricular thalamus, bed nucleus of the stria terminalis, median habenular nucleus, ventromedial hypothalamus, raphe nuclei, solitary tract nucleus, inferior olives, interpeduncular nucleus, claustrum, and numerous peripheral tissues, including liver, vas deferens, heart, superior cervical ganglion, and exocrine and endocrine pancreas. In contrast, MAO-B was most abundant in the ependyma, circumventricular organs, olfactory nerve layer, periventricular hypothalamus, cingulum, hippocampal formation, raphe nuclei, paraventricular thalamus, mammillary nuclei, cerebellar Bergmann glia cells, liver, posterior pituitary, renal tubules, and endocrine pancreas. The cellular localization of the isoenzymes in both rat and human brain differs markedly and does not reflect the distribution of the presumed natural substrates, for example, absence of MAO-A in serotoninergic neurons. Indeed, the present evidence suggests that, whereas MAO-A is found in noradrenergic and adrenergic neurons, MAO-B occurs in astrocytes, serotoninergic neurons, as well as ventricular cells, including most circumventricular organs. The physiological roles of the enzymes are discussed in the light of these findings, some of which were unexpected.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression of the H-ras oncogene induces potassium conductance and neuron-specific potassium channel mRNAs in the AtT20 cell line
Authors: Hemmick LM , Perney TM , Flamm RE , Kaczmarek LK , Birnberg NC
Published: March 2018

Expression of the EJ-ras oncogene in the AtT20 cell line results in several changes in their properties that correspond to a switch of these anterior pituitary-derived cells to a more neuronlike phenotype. The width of action potentials following transfection with ras is reduced 20-fold from over 200 msec in control AtT20 cells to less than 10 msec in ras-transfected cells. This is associated with a two- to threefold increase in the density of voltage-dependent potassium currents. In addition, the rate of inactivation of these currents is decreased approximately twofold in ras-transfected cells. At least part of the change in potassium current may be due to differential expression of potassium channel mRNAs. In the ras-transfected cells, mRNA species were detected using a probe for the voltage-dependent potassium channels, Kv4, a species that appears to be uniquely expressed in the nervous system, and NGK2, an alternatively spliced product transcribed from the same gene. These mRNAs are not detected in control AtT20 cells. The results suggest that the ras protein modulates the phenotype of excitable cells by influencing the expression of specific potassium channels and thereby altering the density and types of channels in the plasma membrane.
Analysis of in vitro glucose utilization in a circadian pacemaker model
Authors: Newman GC , Hospod FE , Patlak CS , Moore RY
Published: March 2018

An in vitro glucose utilization method, based upon 14C-2-deoxyglucose kinetics in brain slices, has been used to study circadian rhythms in hypothalamic slices containing the suprachiasmatic nucleus (SCN). Spontaneous SCN metabolic activity in vitro is similar to that observed in vivo with higher metabolic rates in subjective daytime and lower rates during subjective night. However, in vitro SCN metabolic activity during late subjective day is above that seen when glucose utilization is measured in vivo, suggesting that an inhibitory influence normally active in vivo is lost during slice isolation. Incubation of slices containing SCN in the presence of TTX exposes a TTX-insensitive component of metabolic activity in early subjective day, supporting prior suggestions that glucose utilization by the circadian oscillator continues in the absence of Na(+)-dependent action potentials. Studies with high Mg2+ concentrations are consistent with the hypothesis that most metabolic activity above the basal level observed with the glucose utilization method is related to synaptic activity. Pharmacological studies of the SCN brain slice model with radiotracers offer potential for analysis of both circadian rhythmicity and neural regulation.
Fibroblast growth factor stimulates photoreceptor differentiation in vitro
Authors: Hicks D , Courtois Y
Published: March 2018

Dissociated newborn rat retinal cells were maintained in monolayer culture for periods of up to 11 d. When grown in the absence of exogenous growth factors, 1–2% of the total neuronal population expressed opsin (the photopigment that is specific for maturing photoreceptors). Addition of a single dose of 10 ng/ml basic fibroblast growth factor (bFGF) to the culture medium induced an average increase of sixfold in the numbers of neurons expressing opsin. This supplementation had little effect on the total number of differentiated neurons or of glial cells when measured at the same time points. Furthermore, another specific class of retinal neurons, the amacrine cells, showed no changes following exposure to this growth factor. Two other growth factors known to exert neurotrophic effects, epidermal and nerve growth factor, were without effect. The effect of bFGF was dose dependent, with highly significant differences being observed with as little as 100 pg/ml, and with 700 pg/ml eliciting half-maximal stimulation; maximal effects were observed at 10 ng/ml. Induction of opsin expression by low concentrations of bFGF was blocked completely by an antiserum directed specifically against bFGF, but not by preimmune serum immunoglobulins. This increase in the number of photoreceptors expressing opsin following exposure to bFGF could have been due to either increased cell survival, increased proliferation of progenitor cells, or increased differentiation of immature photoreceptors. There was no increase in overall cell survival under the experimental conditions used, and double labeling immunocytochemistry combined with autoradiographic analysis of 3H- thymidine uptake showed that proliferation of neuronal precursors was not enhanced by the addition of bFGF. In contrast to these observations, cultures established from older (postnatal day 3) retina revealed large numbers of opsin-expressing photoreceptors in all culture plates, with or without added growth factors. This reduction in the stimulatory effects of bFGF with increasing postnatal age is consistent with the period of sensitivity being limited to the cycling of neuronal precursors. It is possible that a bFGF-like molecule is secreted by neighboring cells such as the retinal pigmented epithelium, to participate in retinal development and differentiation. To our understanding, this molecule is the first protein identified to influence specifically the differentiation of photoreceptor cells.
Central and peripheral neurite outgrowth differs in preference for heparin-binding versus integrin-binding sequences
Authors: Haugen PK , McCarthy JB , Roche KF , Furcht LT , Letourneau PC
Published: March 2018

Neurons of the CNS and PNS differ in their response to fibronectin (FN) and proteolytic fragments of FN. The 33 kDa C-terminal cell and heparin- binding fragment of FN, in particular, is a strong promoter of CNS neurite outgrowth. To define further the neurite-promoting activity of the 33 kDa fragment, and to investigate further the differences between PNS and CNS responses to FN and the 33 kDa fragment, we contrasted neurite outgrowth by CNS and PNS neurons on three synthetic peptides representing sequences from this fragment of FN: two heparin-binding peptides, FN-C/H I and FN-C/H II (McCarthy et al., 1990), and an integrin-binding peptide, CS1 (Humphries et al., 1987). Spinal cord (SC) neurons, from the CNS, differed from dorsal root ganglion (DRG) neurons, from the PNS, with respect to substratum preference for heparin-binding versus integrin-binding peptides. SC neurite outgrowth was greatest on the heparin-binding peptide FN-C/H II, while DRG neurite outgrowth was greatest on the a4 beta 1 integrin-binding peptide CS1. To test whether the difference in substratum preference was due to differences in the molecular mechanism by which SC and DRG neurons interact with the 33 kDa fragment of FN, anti-beta 1 integrin antibodies and/or soluble heparin were added to the cultures as potential inhibitors of integrin-mediated or proteoglycan-mediated interactions with FN. SC neurite outgrowth was much more sensitive to the effect of heparin than anti-beta 1 integrin, indicating SC neurite outgrowth may involve predominantly a heparin-sensitive mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Voltage-activated calcium currents in identified neurons from a hydrozoan jellyfish, Polyorchis penicillatus
Authors: Przysiezniak J , Spencer AN
Published: March 2018

Calcium currents were studied in isolated “swim motor neurons” from the jellyfish Polyorchis penicillatus, using the tight-seal, whole-cell, voltage-clamp technique. Two high-voltage-activated (HVA) currents were observed. The transient current, HVA-t, activated rapidly (time to peak, 4 msec), inactivated with two time constants (26 msec, 187 msec) in a positive voltage range (Vi = -23 mV), and was larger when carried by calcium than by barium ions. The sustained current, HVA-s, inactivated slowly or not at all, even at very positive voltages and had the same amplitude whether carried by Ca2+ or Ba2+. It is likely that the two HVA current components arise from distinct channel populations, because the ionic selectivity of calcium channels is not known to depend on their inactivation kinetics. A third current appeared to activate at very positive voltages, and at a slower rate than did HVA-t. It is likely to be an artifact of inhomogeneous space clamping. A low-voltage-activated, cadmium-insensitive calcium current may also be present. Calcium currents in this primitive, multicellular animal have properties similar to calcium currents in other phyla; however, they do not fit neatly into the “T, N, L” classification scheme of vertebrate calcium currents.
Differential properties of tetrodotoxin-sensitive and tetrodotoxin- resistant sodium channels in rat dorsal root ganglion neurons
Authors: Roy ML , Narahashi T
Published: March 2018

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) sodium channel currents were analyzed in acutely dissociated dorsal root ganglion (DRG) neurons isolated from 3–12-d-old and adult rats. Currents were recorded using the whole-cell patch-clamp technique. TTX-R current was more likely to be present in younger animals (3–7 d), whereas TTX-S current was more common in older animals (7–10 d), although TTX-R current was recorded from adult rat DRG neurons. The TTX-R and TTX-S currents differed in their steady-state inactivation, with 50% inactivation voltage at -40 +/- 5 mV (n = 10) for TTX-R currents and -70 +/- 4 mV (n = 10) for TTX- S currents. These current types also differed in their activation kinetics, with 50% activation values of -15 +/- 5 mV (n = 5) for TTX-R currents and -26 +/- 6 mV (n = 5) for TTX-S currents. The interactions of TTX-R and TTX-S channels with various pharmacological agents and divalent cations were studied. The Kd values for TTX-S and TTX-R currents were estimated to be 0.3 nM and 100 microM for TTX, 0.5 nM and 10 microM for saxitoxin, and 50 microM and 200 microM for lidocaine, respectively. TTX-S channels did not exhibit a marked use-dependent block by lidocaine, whereas lidocaine significantly decreased TTX-R current in a use-dependent manner at frequencies ranging from 1 to 33.3 Hz. Several external divalent cations exerted different effects on these current types.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of a novel synapse-specific protein. I. Developmental expression and cellular localization of the F1-20 protein and mRNA
Authors: Sousa R , Tannery NH , Zhou S , Lafer EM
Published: March 2018

A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse- specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1– 20. Immunohistochemical and biochemical analyses revealed that the F1– 20 protein is synapse associated. Western blot analyses revealed that the F1–20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1–20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1–20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1– 20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1–20 protein by immunohistochemistry, and of the F1–20 mRNA by in situ hybridization. We found that the F1–20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1–20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1–20 protein are found in the cortically organized regions of the brain. The highest levels of F1–20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1–20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1–20 mRNA between different nuclear groups.
Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein
Authors: Zhou S , Sousa R , Tannery NH , Lafer EM
Published: March 2018

The F1–20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1–20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1–20 protein. We found two distinct isoforms of F1–20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1–20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3′ acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1–20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1–20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1–20 protein. We then determined biochemically that the F1–20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1–20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1–20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1–20 protein is phosphorylated in vivo.
Excitatory amino acid regulation of intracellular Ca2+ in isolated catfish cone horizontal cells measured under voltage- and concentration- clamp conditions
Authors: Linn CP , Christensen BN
Published: March 2018

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5- phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1- amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10–200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)- sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine- induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.