bluuebunny/crossref_metadata_2025 huggingface dataset:
ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine- innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus
Authors: Hemmings HC , Greengard P
Published: March 2018

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin- layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine- innervated brain regions. II. Immunocytochemical localization in rat brain
Authors: Ouimet CC , Hemmings HC , Greengard P
Published: March 2018

ARPP-21, a cAMP-regulated phosphoprotein, has been studied by immunocytochemistry to determine its cellular and regional distribution in rat brain. This study demonstrates that ARPP-21 immunoreactivity is present throughout the cytoplasm of immunoreactive neurons and that most of the immunoreactivity is associated with the basal ganglia. Within the caudatoputamen (CP), nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, and portions of the amygdaloid complex, ARPP-21 is present in neuronal somata and dendrites. In brain regions known to receive projections from these nuclei, immunoreactivity is present in puncta (presumed axons and axon terminals). These regions include the globus pallidus, ventral pallidum, entopeduncular nucleus, lateral preoptic area, and substantia nigra. Within the basal ganglia, ARPP-21 immunoreactivity is most intense in the olfactory tubercle, nucleus accumbens, medial portion of the CP, and the ventral retrochiasmatic pocket of the CP. These same areas comprise the limbic striatum, and ARPP-21 is the first substance found to be specifically enriched therein. The possibility is discussed that ARPP-21 mediates effects of multiple first messengers, including dopamine and vasoactive intestinal polypeptide, that act through cAMP.
Characterization of a partial cDNA clone for the NILE glycoprotein and identification of the encoded polypeptide domain
Authors: Prince JT , Milona N , Stallcup WB
Published: March 2018

A partial cDNA clone [2.4 kilobase (kb)] for the nerve growth factor- inducible large external (NILE) glycoprotein was selected from a lambda gt 11 expression library constructed using mRNA from PC 12 cells. A 0.2 kb subclone (pNILE-1B) was used for Northern blot analysis of NILE message present in 2 NILE-positive neuronal cell lines and 2 NILE- negative glial cell lines. pNILE-1B hybridizes with components of 6.8 and 2.0 kb in the 2 neuronal cell lines but fails to show hybridization with any components in the 2 glial cell lines. Only the 6.8 kb species would be large enough to code for the NILE polypeptide. A rabbit antiserum was prepared against the NILE-beta-galactosidase fusion protein produced by the NILE clone. This antiserum (anti-NILE-beta-gal) immunoprecipitates NILE glycoprotein from neuronal cell lines, further confirming the authenticity of the NILE cDNA clone. The epitope recognized by anti-NILE-beta-gal is contained in an 85 kDa tryptic fragment from the phosphorylated carboxy terminus of NILE. The 160 kDa tryptic fragment containing the amino terminus is not recognized by anti-NILE-beta-gal. Both immunoprecipitation and immunofluorescence experiments indicate that the anti-NILE-beta-gal epitope is not exposed on the cell surface but is accessible only after cells are treated with detergent. The cytoplasmic nature of the determinant is also indicated by its absence on a truncated, soluble form of NILE released from cells (possibly by a proteolytic mechanism) into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Nerve growth factor receptors on normal and injured sensory neurons
Authors: Verge VM , Riopelle RJ , Richardson PM
Published: March 2018

The density and binding properties of receptors for nerve growth factor (NGF) in normal and injured sensory neurons have been analyzed by quantitative radioautography following incubation of tissue sections with radioiodinated NGF. The technique is designed to study binding sites that are half-maximally saturated by picomolar concentrations of NGF: Additional sites of lower affinity have not been emphasized. In normal adult rats, approximately half of lumbar sensory neurons have high-affinity receptors for NGF. One month after the sciatic nerve is cut, the mean number of high-affinity sites on heavily labeled neurons in the fifth lumbar dorsal root ganglion falls to less than 20% of normal values because of reduced receptor density and cell volume. Neurons with high-affinity receptors are more liable to atrophy after injury than those lacking such receptors. Receptors are lost not only in the cell bodies of sensory neurons but also on their peripheral and central processes. Delayed administration of NGF to the sciatic nerve 3 weeks after it is cut restores the receptor density to normal values and partially restores the neuronal cell volume. As part of the response to axonal injury and possibly because the cell body is deprived of NGF, fewer high-affinity receptors are displayed by sensory neurons. For at least 3 weeks after nerve transection, neurons that are atrophic and depleted of NGF receptors can be resuscitated by exogenous NGF.
An EM study of the dorsal nucleus of the lateral lemniscus: inhibitory, commissural, synaptic connections between ascending auditory pathways
Authors: Oliver DL , Shneiderman A
Published: March 2018

The dorsal nucleus of the lateral lemniscus (DNLL) and its connections constitute one of the ascending auditory pathways to the inferior colliculus. One notable feature of this nucleus is the heavy commissural connections between DNLL on opposite sides of the midbrain. These commissural connections may have a significant impact on the ascending pathway. In this study, the fine structure of DNLL in the cat and its commissural connections were examined. Both anterograde and retrograde transport methods were used simultaneously at the EM level. Injections of 3H-leucine mixed with WGA-HRP were made in one DNLL. After axonal transport, EM autoradiographic methods were used to identify the anterogradely labeled axonal endings from the opposite DNLL. In the same location, retrogradely labeled neurons with crossed connections were identified with HRP histochemistry. Two types of axonal endings were found in DNLL, those with round synaptic vesicles forming asymmetrical synaptic junctions and those with pleomorphic vesicles and symmetrical synapses. Both types were equally common. However, only endings with pleomorphic vesicles were labeled after injections in the contralateral DNLL. The labeled endings from the opposite DNLL appeared to represent a homogeneous population, even though a number of variations in the 2 types of endings were found. Labeled endings were presynaptic to all parts of neurons in DNLL, but a large proportion of the synapses were on cell bodies and large dendrites. Two patterns of nuclear morphology and distribution of rough endoplasmic reticulum were identified and may represent different cell types. Examples of both cell types were observed to project to the contralateral side and received labeled synaptic endings. The major finding of this study is that the crossed connections between DNLL exhibit the morphology associated with inhibitory function. Since neurons in DNLL are thought to use GABA as a neurotransmitter, the crossed connections could provide inhibitory inputs to DNLL on each side. Since some neurons receive numerous axosomatic inputs from the contralateral DNLL and also project to the opposite side, they may participate in direct reciprocal, inhibitory connections between the nuclei. Crossed inhibitory connections in the DNLL pathway may be important in regulating the flow of ascending auditory information.
Localization of the growth-associated phosphoprotein GAP-43 (B-50, F1) in the human cerebral cortex
Authors: Benowitz LI , Perrone-Bizzozero NI , Finklestein SP , Bird ED
Published: March 2018

The growth-associated phosphoprotein GAP-43 is a component of the presynaptic membrane that has been linked to the development and functional modulation of neuronal connections. A monospecific antibody raised against rat GAP-43 was used here to study the distribution of the protein in cortical and subcortical areas of the human brain. On Western blots, the antibody recognized a synaptosomal plasma membrane protein that had an apparent molecular weight and isoelectric point similar to GAP-43 of other species. In brain tissue reacted with the antibody, the heaviest immunoreactivity was found in associative areas of the neocortex, particularly within layers 1 and 6, in the molecular layer of the dentate gyrus, the caudate putamen, and the amygdala. In contrast, primary sensory or motor regions of the cortex, portions of dorsal thalamus, and cerebellum showed only light staining. Staining was generally confined to the neuropil, which showed punctate labeling, whereas most neuronal somata and fiber bundles were unreactive. The pronounced variations in GAP-43 immunostaining among various areas of the human brain may reflect different potentials for functional and/or structural remodeling.
Corollary discharge inhibition and preservation of temporal information in a sensory nucleus of mormyrid electric fish
Authors: Bell CC , Grant K
Published: March 2018

Knollenorgan electroreceptors in mormyrid fish are concerned with electrocommunication, i.e., with detecting electric organ discharges (EODs) of other electric fish. But knollenorgan electroreceptors are also activated by the fish's own EOD. Potential interference by such self-stimulation is blocked by an inhibitory corollary discharge in the nucleus of the electrosensory lateral line lobe (NELL), the first central relay of the knollenorgan pathway. This study used intracellular recording and staining to examine the mechanism of the corollary inhibition and the specializations in anatomy and physiology that permit the accurate relaying of temporal information about the EODs of other fish. Several events are recorded inside primary knollenorgan afferents in addition to a large orthodromic action potential. The additional events include small orthodromic electronic epsps, postsynaptic action potentials, and a corollary discharge inhibitory postsynaptic potential (ipsp) associated with the EOD motor command. These additional events are also recorded in NELL cells and almost certainly originate there. Electrical coupling between afferents and cells makes it possible to observe the events inside primary afferents. The corollary discharge ipsp in the cell is associated with a conductance increase and inverts rapidly when recorded with chloride- containing electrodes, supporting a hypothesis of GABA mediation. The ipsp lasts longer in cells than in afferents. Each electrotonic excitatory postsynaptic potential (epsp) is probably caused by a single primary afferent, and any one of several epsps in a given cell seems capable of eliciting a postsynaptic spike in that cell. The epsps follow stimulation rates as high as 500/sec with minimal variability. No lateral inhibition is observed in NELL. These and other properties indicate that the knollenorgan pathway is specialized for temporal information rather than spatial or intensity information.
The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP
Authors: Prosser RA , Gillette MU
Published: March 2018

A circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus controls the daily behavioral and physiological rhythms of mammals. While the mammalian circadian system has been the focus of research for many years, very little work has been directed at understanding its underlying biochemical mechanisms. In these experiments we used the hypothalamic brain slice technique to investigate these mechanisms, focusing specifically on the intrinsic resetting properties of the circadian clock of the rat. We monitored a primary expression of the clock or pacemaker, the circadian rhythm of electrical activity of SCN neurons. This rhythm continues the oscillatory pattern seen in vivo for up to 60 hr in vitro, with an activity peak near midday that shows very little variation among SCN from different rats. The stability of the rhythm in vitro enabled us to use the time of peak activity to monitor the phase of the underlying pacemaker. Bath application of membrane-soluble cAMP analogs in 1 hr pulses induced robust advances in the phase of the rhythm that remained stable for 2 cycles. This effect depended on the phase of the pacemaker at the time of treatment: the peak was maximally advanced (4–6 hr) by treatments during the middle of the subjective day (projected from the donor's cycle); treatments during most of the subjective night and early subjective day induced no phase changes. Half-maximal phase resetting was induced at 1 x 10(-10) M concentrations of active cAMP analog.(ABSTRACT TRUNCATED AT 250 WORDS)
Gaze-dependent visual neurons in area V3A of monkey prestriate cortex
Authors: Galletti C , Battaglini PP
Published: March 2018

Extracellular recordings from single neurons of the prestriate area V3A were carried out in awake, behaving monkeys, to test the influence of the direction of gaze on cellular activity. The responsiveness to visual stimulation of about half of the studied neurons (88/187) was influenced by the animal's direction of gaze: physically identical visual stimuli delivered to identical retinotopic positions (on the receptive field) evoked different responses, depending upon the direction of gaze. Control experiments discount the possibility that the observed phenomenon was due to changes in visual background or in depth, depending on the direction in which the animal was looking. The gaze effect modulated cell excitability with different strengths for different gaze directions. The majority of these neurons were more responsive when the animal looked contralaterally with respect to the hemisphere they were recorded from. Gaze-dependent neurons seem to be segregated in restricted cortical regions, within area V3A, without mixing with non-gaze-dependent cells of the same cortical area. The most reliable differences between V3A gaze-dependent neurons and the same type of cells previously described in area 7a (Andersen and Mountcastle, 1983) concern the small receptive field size, the laterality of gaze effect, and the lack of straight-ahead facilitated or inhibited neurons in area V3A. Since the present results show that V3A gaze-dependent neurons combine information about the position of the eye in the orbit with that of a restricted retinal locus (their receptive field), we suggest that they might directly encode spatial locations of the animal's field of view in a head frame of reference. These cells might be involved in the construction of an internal map of the visual environment in which the topographical position of the objects reflects their objective position in space instead of reflecting the retinotopic position of their images. Such an objective map of the visual world might allow the stability of visual perception despite eye movement.
Rat CNS white matter, but not gray matter, is nonpermissive for neuronal cell adhesion and fiber outgrowth
Authors: Savio T , Schwab ME
Published: March 2018

In adult mammalian CNS, axons mostly fail to regenerate after injury, while in the PNS they often succeed in reaching their previous targets. Crucial differences are present in the local tissue microenvironment of CNS and PNS. To investigate the substrate properties of nervous tissue for neuronal adhesion and fiber growth, we used frozen sections of rat CNS and PNS as culture substrates for neuroblastoma cells and for sympathetic and dorsal root ganglia. The results showed that CNS white matter from adult rat spinal cord, cerebellum, forebrain, or optic nerve did not allow cell adhesion and axonal elongation. In contrast, gray matter areas, sciatic nerve, and also trout CNS white and gray matter were permissive substrates. To delineate the tissue components of white matter involved in this nonpermissive substrate effect, newborn rats were injected for 13 d with the antimitotic agent 5- azacytidine. This treatment strongly reduced the oligodendrocyte population and the myelin content of the spinal cord. The immunoreactivity for specific oligodendrocyte and astrocyte markers confirmed the selective suppression of oligodendroglia in these rats. Neuroblastoma cells plated on spinal cord sections taken from these animals were no longer exclusively localized on the gray matter but were also found on regions normally rich in myelin. A significant reduction of the white matter nonpermissive substrate effect was also obtained by the monoclonal antibody IN-1 directed against 2 defined myelin proteins with inhibitory substrate properties (Caroni and Schwab, 1988b). Our results, therefore, show that, in the adult mammalian CNS, cell adhesion and axonal elongation are prevented by white matter components, which are, at least in part, associated with oligodendrocytes and myelin.
Auditory brain stem of the ferret: some effects of rearing with a unilateral ear plug on the cochlea, cochlear nucleus, and projections to the inferior colliculus
Authors: Moore DR , Hutchings ME , King AJ , Kowalchuk NE
Published: March 2018

To examine the influence of acoustic experience on the development of the mammalian auditory brain stem, darkly pigmented ferrets were reared with a plug inserted in the right outer ear. The plugs were first inserted on postnatal day 23–34 and produced a variable, frequency- dependent attenuation of up to 60 dB. Between 3–15 months after the ear plug was begun, animals were prepared for physiological recording and injection of wheat germ agglutinin-HRP (WGA-HRP) in the left inferior colliculus (IC). The plug was removed and the condition of the right ear was assessed by pure-tone stimulation and recordings from neurons in the left IC. Neural audiograms for each animal showed a residual deficit in most cases. Following 24–60 hr survival, the animals were perfused and the right ear was examined. Brain-stem sections were reacted with tetramethylbenzidine. Outer and/or middle ear pathology was present in over half of the animals. However, the cochleas appeared to be normal and the spiral ganglion cells were normal by several quantitative criteria: number, area, and nucleolar eccentricity. The volume of each division of the cochlear nuclei (CN) and the areas of individual neurons in the anteroventral CN were the same on the right and left sides. The number of CN neurons retrogradely labeled from the left IC injection of WGA-HRP was found to be significantly increased in the left CN, relative to normal animals, when expressed as a ratio of the number labeled in the right CN. We conclude that the residual hearing loss in the previously plugged ears was predominantly or exclusively conductive. Neonatal, unilateral conductive hearing loss in the ferret does not lead to degeneration of the CN on the side of the loss, but it does lead to at least one rearrangement of auditory brain- stem connectivity. We suggest that the extent to which the brain stem is modified by early auditory deprivation is dependent on the type, degree, and symmetry of the hearing loss.
Spatial and temporal factors determining afferent fiber responses to a grating moving sinusoidally over the monkey's fingerpad
Authors: Goodwin AW , John KT , Sathian K , Darian-Smith I
Published: March 2018

Gratings of alternating grooves and ridges were moved sinusoidally across the fingerpads of anesthetized monkeys, while responses were recorded from individual slowly adapting afferents (SAs), rapidly adapting afferents (RAs), and Pacinian afferents (PCs) in the median nerve. The stimulus comprised 2 spatial variables, namely, groove width (G) and ridge width (W), and 2 temporal variables, namely, the peak speed of movement (S) and the peak temporal frequency (F) at which successive spatial cycles of the grating pass over a point in the receptive field. The responses of all 3 fiber types were determined by only 1 spatial variable, G, and only 1 temporal variable, F. Changes in W or S affected responses only if there was a concomitant change in either G or F. Responses were phase-locked to the occurrence of successive spatial cycles of the grating, and we have used the number of impulses elicited by a single spatial cycle as the fundamental measure of response. An equation of the form I = cGaexp(-b square root of F) describes the responses of all 3 fiber types. For SAs, the effect of groove width was greater (a = 2.64) than for RAs and PCs (a = 0.924 and 1.05, respectively). The reduction in response with frequency was most marked for SAs (b = 0.262), and greater for PCs (b = 0.167) than for RAs (b = 0.130). From the equation, the instantaneous response during the entire sinusoidal cycle was reconstructed as well as a second measure, the mean cyclic response. These 2 measures behaved differently with changes in the stimulus parameters. The temporal properties of the fibers, as revealed by gratings, may appear to be in conflict with those established by vibratory threshold studies; in fact, they are compatible with suprathreshold responses to vibrating probes.
Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat
Authors: Tetzlaff W , Zwiers H , Lederis K , Cassar L , Bisby MA
Published: March 2018

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Bungner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.
A pheromone-degrading aldehyde oxidase in the antennae of the moth Manduca sexta
Authors: Rybczynski R , Reagan J , Lerner MR
Published: March 2018

Antennae of the tobacco hornworm moths Manduca sexta contain an aldehyde oxidase (AOX) that oxidizes aldehydes to carboxylic acids. The enzyme, which is distinguishable from aldehyde-oxidizing activities in other tissues, is secreted into the receptor lymph that bathes the primary olfactory dendrites. First detectable about 3 d before eclosion, AOX levels increase through the first day after eclosion. This parallels the development of the antennal responsiveness to bombykal (a male attractant aldehydic pheromone produced by female M. sexta) and trans-2-hexenal (an aldehyde commonly found in leaves). The AOX is about 60% more abundant in antennae of males than in antennae of females. The antennal AOX is a dimer with Mr of 295 kDa and is capable of oxidizing a variety of aldehydes. Of all aldehydes examined, the pheromone bombykal was the best substrate with an apparent Km of 5 microM, whereas the next best substrate, benzaldehyde, had an apparent Km of 255 microM. Using kinetic parameters estimated in vitro and the assumption of first-order kinetics, the half-life of bombykal in sensilla was estimated to be about 0.6 msec. The affinity of the antennal AOX for bombykal, its location in the receptor lymph, and its pattern of developmental expression all suggest that it plays a role in modulating the sensitivity of adult M. sexta to aldehyde odors and, in particular, the sensitivity of males to the pheromone bombykal.
Early differentiation of retinal ganglion cells: an axonal protein expressed by premigratory and migrating retinal ganglion cells
Authors: McLoon SC , Barnes RB
Published: March 2018

A monoclonal antibody, RA4, was developed that recognizes retinal ganglion cell axons in the mature retina. Between embryonic days 3 and 9, the RA4 antigen was associated with cell bodies in certain regions of the retina in addition to the ganglion cell axons. The RA4-positive cells were of 3 types: an apolar cell adjacent to the ventricular surface, a bipolar cell that spanned the thickness of the retina, and a monopolar cell in the ganglion cell layer. Evidence suggests that these cells are premigratory and migrating retinal ganglion cells. The expression of the RA4 antigen is the earliest indicator of ganglion cell differentiation yet reported. The existence of RA4-positive apolar cells along the outer surface of the retina suggests that the ganglion cell phenotype is expressed as soon as the cell becomes postmitotic. Approximately 20% of the migrating ganglion cells were in pairs. The paired cells most likely arose from the terminal division of a germinal cell. One possibility suggested by these data is that a ganglion cell- specific germinal cell arises from a pluripotent germinal cell. Immunoblots and other analyses revealed the RA4 antigen to be a 140 kDa cytoplasmic protein in the retina. RA4 also recognized many long tract axons in the brain. In the brain, the RA4 epitope was observed on proteins with at least 7 different molecular weights. Evidence suggests that different cell types may express the RA4 antigen with slightly different molecular weights.
Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents
Authors: Evers J , Laser M , Sun YA , Xie ZP , Poo MM
Published: March 2018

We have studied the spontaneous and nerve-evoked synaptic currents during the initial period of nerve-muscle contact in Xenopus cell cultures. The precise timing of the contact was achieved by physically manipulating embryonic muscle cells into contact with co-cultured spinal neurons. Previous studies have shown that physical contact of the muscle membrane induces pulsatile release of acetylcholine (ACh) from the growth cone of these neurons, resulting in spontaneous synaptic currents (SSCs) in the muscle cell within seconds following the contact. In the present work, we first showed that these SSCs at the manipulated nerve-muscle contacts are similar to those observed at naturally occurring synapses. We then examined the possible cellular mechanisms responsible for the marked variation in SSC amplitude and showed that it most likely results from differences in either the amount of ACh contained in each release event or the extent of close membrane apposition near the release sites. During the first 20 min following the nerve-muscle contact, there was an increase in the frequency and mean amplitude of the SSCs. During a similar period, the evoked synaptic currents (ESCs), which were induced by suprathreshold electrical stimulation of the neuronal soma, also showed an increase in the mean amplitude and a reduction in the delay of onset following the stimulus. These postcontact changes in the efficacy of synaptic transmission may be related to an increase in the total area of close membrane apposition between the nerve and muscle cells. This was suggested by the finding that neurite-muscle adhesion increases over a similar postcontact period. The transition from low- to high-efficacy transmission during the early phase of contact may reflect the process of selective adhesion between the cells, and thus signify the formation of specific synapse. Analysis of the fluctuation in the ESC amplitude at the early nerve-muscle contact suggests that evoked release of ACh occurs as multiples of a quantal unit. However, this unit is apparently related to only a small subpopulation of SSCs of relatively high amplitudes.(ABSTRACT TRUNCATED AT 400 WORDS)
Heparin and heparan sulfate partially inhibit induction of acetylcholine receptor accumulation by nerve in Xenopus culture
Authors: Hirano Y , Kidokoro Y
Published: March 2018

It has been demonstrated that ACh receptors in Xenopus nerve-muscle cultures migrate in the membrane to the nerve contact area during junction formation (Anderson et al., 1977) and that “diffusion trapping” is the major mechanism for nerve-induced receptor accumulation (Kidokoro and Brass, 1985; Kuromi et al., 1985; Kidokoro et al., 1986). A crucial remaining question is how the nerve induces the trap for randomly diffusing ACh receptors. In this study we examined the effect of various glycosaminoglycans in the culture medium on the nerve-induced receptor accumulation and found that heparin and heparan sulfate partially inhibited nerve-induced receptor accumulation, but similar molecules, chondroitin sulfate type A and type C, did not. By chemical modification of heparin we also showed that N-sulfate residues and a large-molecular-weight molecule are essential for this inhibitory effect. Heparin did not affect ACh receptor clustering (hot-spot formation) in myocytes cultured without nerve. By changing the time and duration of heparin application, we found that heparin was effective in inhibiting nerve-induced receptor accumulation only when it was present in the culture medium during the period that neurites are actively forming contact with muscle membrane.
The role of a modulatory neuron in feeding and satiation in Aplysia: effects of lesioning of the serotonergic metacerebral cells
Authors: Rosen SC , Weiss KR , Goldstein RS , Kupfermann I
Published: March 2018

Food-induced arousal in Aplysia is characterized by a progressive increase in the speed and strength of biting responses elicited by a seaweed stimulus. Data from semi-intact and dissected preparations suggest that the identified, serotonergic, metacerebral cells (MCCs) of the cerebral ganglion contribute to food-induced arousal by enhancing the strength of buccal muscle contractions, and by modulating the output of the central pattern generator for biting movements. In order to test this hypothesis in intact, free-moving animals and to determine if the MCCs play a role in satiation of feeding, the behavior of animals that had their MCCs destroyed by intracellular injection of proteases was compared with that of B Cell-Lesion and Dye injection control animals (Experiment 1) or surgical control animals (Experiment 2). Nonfeeding behaviors such as defensive withdrawal responses, locomotion, and righting reflexes were unaffected by MCC lesioning. Also unaffected by MCC lesioning were appetitive feeding behaviors and the amount of food needed to satiate animals. Significant behavioral deficits in consummatory feeding behaviors, which remained stable for periods exceeding 10 d, were observed in the MCC-lesioned animals but not in controls. Lesioned animals exhibited a slowing of rate of repetitive biting responses by 40% of controls and had reduced magnitudes of repetitive bites, particularly at the end of a testing run of 10 consecutive bites. The deficit in bite magnitude was minimally evident in food-deprived animals (Experiment 1) but became more pronounced as animals were fed to satiation (Experiment 2). MCC- lesioned animals still exhibited a residual build-up of the rate and magnitude of biting responses at the onset of feeding behavior. This suggests that, in addition to the MCCs, there are other sources of modulation that contribute to plasticity of consummatory responses during the food-induced arousal state.
Development and death of external granular layer cells in the weaver mouse cerebellum: a quantitative study
Authors: Smeyne RJ , Goldowitz D
Published: March 2018

Previous studies have identified the cerebellar granule cell as a primary site of gene action in the weaver mutant mouse. The temporal expression of the weaver mutant granule cell phenotype has not been fully investigated. To identify early postnatal expression of the weaver mutant phenotype, we quantitated 4 parameters of cerebellar development in postnatal day 0, 2, 4, 6, and 8 +/+, wv/+, and wv/wv mice: (1) cerebellar area, (2) cells in the external granule layer (EGL), (3) number of mitotic figures in the EGL, and (4) number of pyknotic figures in the EGL. Qualitative observations suggest a generalized retardation in the development of wv/wv and wv/+ EGL cells compared with the +/+ cells. Quantitatively, the earliest detectable mutant phenotype in wv/wv and wv/+ cerebella is the increased presence of cell death in the EGL, apparent at the day of birth. Degenerating cells in the EGL, the majority of which are found in the postmitotic zone of this layer, contain abnormally clumped heterochromatin, suggestive of “nuclear” cell death. Previous hypotheses concerning the mechanism of weaver gene action have suggested that granule cells die due to their inability to migrate from the EGL. The time course, mode, and spatial organization of cell death found in the present studies lead us to suggest that an earlier event in granule cell development, such as the exit of neuroblasts from the cell cycle or axonogenesis, is affected by the weaver gene.
The earliest-generated neurons of the cat cerebral cortex: characterization by MAP2 and neurotransmitter immunohistochemistry during fetal life
Authors: Chun JJ , Shatz CJ
Published: March 2018

The earliest-generated neurons of the cat cerebral cortex have been studied here during development using a combination of 3H-thymidine birthdating with immunohistochemistry for the neuron-specific protein MAP2 or for several neuropeptides/transmitters. These neurons are the first postmitotic cells of the cortex, with birthdates during the 1- week period preceding the genesis of cells of the adult cerebral cortex (Luskin and Shatz, 1985a; Chun et al., 1987). However, they are transient and the majority disappear by adulthood (Luskin and Shatz, 1985a; Chun and Shatz, 1989). When autoradiographic birthdating is combined with MAP2 immunostaining during fetal life, the entire population of these early-generated neurons appears to be stained, resulting in labeled bands above and below the cortical plate. The band above the cortical plate (in the marginal zone) contains early- generated neurons with horizontal morphologies, while the thicker band beneath the cortical plate (within the intermediate zone) contains the somata of early-generated neurons and their elaborate processes that are frequently directed towards the ventricular surface. In view of the correspondence between the location of the early-generated neurons and the MAP2-immunostained band beneath the cortical plate, we suggest that this combined approach can be used to define accurately the subdivision of the intermediate zone known as the subplate. The early-generated neurons are also immunoreactive for GABA, neuropeptide Y (NPY), somatostatin (SRIF), and cholecystokinin (CCK) during fetal life. While GABA, NPY, and SRIF immunostaining could be detected by embryonic day 50 (E50), that for CCK was not found until E60. Moreover, there is a relationship between neuropeptide immunoreactivity and location within the cerebral wall. The marginal-zone neurons are immunoreactive only for CCK. The subplate neurons are immunoreactive for CCK, SRIF, and NPY. Most of those immunoreactive for SRIF tend to be clustered within the upper part of the subplate, while those immunoreactive for NPY tend to be located more deeply. Cells immunoreactive for GABA are more uniformly distributed throughout the cerebral wall. These observations demonstrate directly that the marginal zone and subplate contain peptide- and GABA-immunoreactive neurons that belong to the earliest- generated cell population of the cerebral cortex. The presence of these early-generated neurons, which achieve a remarkable degree of maturity during fetal life, suggests that they perform an essential, yet transient, role in the development of the cerebral cortex.
Characterization of a partial cDNA clone for the NILE glycoprotein and identification of the encoded polypeptide domain
Authors: Prince JT , Milona N , Stallcup WB
Published: March 2018

A partial cDNA clone [2.4 kilobase (kb)] for the nerve growth factor- inducible large external (NILE) glycoprotein was selected from a lambda gt11 expression library constructed using mRNA from PC 12 cells. A 0.2 kb subclone (pNILE-1B) was used for Northern blot analysis of NILE message present in 2 NILE-positive neuronal cell lines and 2 NILE- negative glial cell lines. pNILE-1B hybridizes with components of 6.8 and 2.0 kb in the 2 neuronal cell lines but fails to show hybridization with any components in the 2 glial cell lines. Only the 6.8 kb species would be large enough to code for the NILE polypeptide. A rabbit antiserum was prepared against the NILE-beta-galactosidase fusion protein produced by the NILE clone. This antiserum (anti-NILE-beta-gal) immunoprecipitates NILE glycoprotein from neuronal cell lines, further confirming the authenticity of the NILE cDNA clone. The epitope recognized by anti-NILE-beta-gal is contained in an 85 kDa tryptic fragment from the phosphorylated carboxy terminus of NILE. The 160 kDa tryptic fragment containing the amino terminus is not recognized by anti-NILE-beta-gal. Both immunoprecipitation and immunofluorescence experiments indicate that the anti-NILE-beta-gal epitope is not exposed on the cell surface but is accessible only after cells are treated with detergent. The cytoplasmic nature of the determinant is also indicated by its absence on a truncated, soluble form of NILE released from cells (possibly by a proteolytic mechanism) into the medium. This released NILE is 15–20 kDa smaller than the detergent-extracted NILE and, in addition to lacking the anti-NILE-beta-gal epitope, does not contain the cytoplasmic site(s) of phosphorylation. Nucleotide sequencing of the pNILE-1B subclone confirms the location of the anti-NILE-beta-gal epitope in the cytoplasmic domain. The clone contains an open reading frame coding for a 79 amino acid segment of the polypeptide that differs in only 2 residues from the cytoplasmic domain of the L1 glycoprotein.
Cholinergic synapses and the organization of contrast detection in the crayfish optic lobe
Authors: Pfeiffer C , Glantz RM
Published: March 2018

The actions of acetylcholine (ACh) were examined on 4 classes of multicolumnar interneurons whose dendrites lie in close proximity to the putative cholinergic transmedullary neurons described in the companion report. ACh-elicited responses in each cell type resemble visually elicited synaptic events and persist following synaptic blockade with 20 mM CoCl2. Tangential cells exhibit a hyperpolarizing response to ACh that resembles the visual response in reversal potential and dependence on extracellular chloride. The visual response is potentiated by the anticholinesterase, neostigmine (0.1 mM). Visual and carbachol-elicited responses are blocked by nicotinic ganglionic antagonists (e.g., 10(-6) M pempidine) that are 10–100 times more potent than D-tubocurarine. Medullary amacrine cells exhibit depolarizing responses to ACh (10(-6) M) and light with similar reversal potentials. The visual response is potentiated by neostigmine. Dimming fibers respond to light and ACh with a hyperpolarization that inhibits the maintained discharge. The sustaining fiber response to ACh reflects both direct responses and indirectly elicited synaptic actions. The direct action is a hyperpolarization possibly related to the visual “off-response.” It is associated with an increased conductance and a reversal potential negative to the dark potential. The off-response is abolished by curare and pempidine and potentiated by neostigmine. ACh appears to orchestrate several aspects of the dual- channel contrast detection system of the optic lobe. The actions of ACh on tangential cells, amacrine cells, and dimming fibers are all consistent with the effects of a spatially localized increment in light intensity and a corresponding local release of ACh in the retinotopic columnar array.
Identification of two protein kinases that phosphorylate the neural cell-adhesion molecule, N-CAM
Authors: Mackie K , Sorkin BC , Nairn AC , Greengard P , Edelman GM , Cunningham BA
Published: March 2018

The neural cell-adhesion molecule (N-CAM) is detected as at least 3 related polypeptides generated by alternative splicing of a single gene. In vivo the 2 larger polypeptides are phosphorylated, but the smallest polypeptide, which lacks a cytoplasmic domain, is not. We have found that the 2 larger polypeptides are phosphorylated in vivo on several common phosphorylation sites. Furthermore, the largest polypeptide has additional sites, suggesting that some phosphorylation occurs in that portion of the intracellular region unique to it. In vitro N-CAM is not a substrate for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, II, or III, protein kinase C, or casein kinase II. However, we have isolated 2 protein kinases from mammalian and avian brain that phosphorylate rodent and chicken N-CAM. On the basis of their chromatographic behavior and substrate specificity, the 2 kinases are glycogen synthase kinase 3 (GSK-3) and casein kinase I (CK I). The 2 kinases phosphorylate N-CAM rapidly, to a high stoichiometry and with a low Km for N-CAM, suggesting that the phosphorylation of N-CAM by these kinases is physiologically relevant. Both enzymes phosphorylate the 2 larger N-CAM polypeptides in vitro in the cytoplasmic domain on threonyl residues that are phosphorylated to a low level in vivo. In addition, the threonyl residues are close to seryl residues phosphorylated to a high level in vivo. Prior phosphorylation at the in vivo sites appears to be a prerequisite for phosphorylation by GSK-3 and CK I. Taken together, the results suggest that N-CAM may be physiologically phosphorylated on 2 sets of interrelated sites, one demonstrable in vivo and one in vitro. Phosphorylation on the “in vivo” sites is resistant to dephosphorylation and may be constitutive, while phosphorylation on the “in vitro” sites is much more labile.
Lesions of the amygdala that spare adjacent cortical regions do not impair memory or exacerbate the impairment following lesions of the hippocampal formation
Authors: Zola-Morgan S , Squire LR , Amaral DG
Published: March 2018

Monkeys with stereotaxic lesions of the amygdaloid complex that spared the surrounding cortex (i.e., the periamygdaloid, entorhinal, and perirhinal cortices) performed normally on the delayed nonmatching to sample task, as well as on 3 other memory tasks (object retention, concurrent discrimination, and delayed response) administered during the 1 1/2 years after surgery. These animals also performed normally on pattern discrimination and motor-skill learning, 2 tasks analogous to ones amnesic patients perform well. A second group of monkeys with conjoint lesions that included both the amygdaloid complex, as just described, and the hippocampal formation were impaired on the same 4 memory tasks. However, the severity of impairment in this group was no greater than in monkeys with lesions of the hippocampal formation alone. Thus, circumscribed bilateral lesions of the amygdala did not impair performance on 4 different memory tasks, nor did they exacerbate the memory impairment that followed hippocampal formation lesions alone. These findings suggest that one must look to structures other than the amygdala to account for the severe memory impairment that follows large lesions of the medial temporal region. One possibility is that damage to the cortical regions that surround the amygdala contributes to memory impairment.