bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondria and Na+/Ca2+ exchange buffer glutamate-induced calcium loads in cultured cortical neurons
Authors: White RJ , Reynolds IJ
Published: April 2018

Utilizing Indo-1 microfluorimetry, we have investigated the role of mitochondria and Na+/Ca2+ exchange in buffering calcium loads induced by glutamate stimulation or depolarization of cultured rat forebrain neurons. A 15 sec pulse of 3 microM glutamate or 50 mM potassium with veratridine was followed by a 2 min wash with a solution containing either Na(+)-free buffer or the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or both. For glutamate-induced Ca2+ loads, a Na(+)-free wash delayed recovery to baseline by twofold, mitochondrial uncoupling delayed recovery by greater than fourfold, and the combined treatment essentially prevented recovery of [Ca2+]i for the duration of the wash. Although the depolarization stimulus was able to elicit a larger peak [Ca2+]i, the neurons required significantly less time to recover from depolarization- induced Ca2+ loads after identical wash manipulations, indicating a fundamental difference between calcium loads induced by glutamate as opposed to those induced by depolarization. We show evidence that the delayed recovery is not primarily the result of perturbations in intracellular pH regulation and have also demonstrated that a substantial portion of the delayed recovery is independent of Ca2+ entry during the washout phase. We conclude that glutamate and depolarization both induce Ca2+ loads whose buffering is critically dependent on functional mitochondria and secondarily reliant on Na+/Ca2+ exchange. The two systems overlap and seem to be responsible for buffering most of the glutamate-induced Ca2+ load, because manipulations that compromised both systems completely disabled the neurons' ability to recover [Ca2+]i to baseline.
The influence of heregulins on human Schwann cell proliferation
Authors: Levi AD , Bunge RP , Lofgren JA , Meima L , Hefti F , Nikolics K , Sliwkowski MX
Published: April 2018

The use of Schwann cell (SC) autotransplantation to influence neural repair in humans is dependent upon identifying mitogens that will effectively expand human Schwann cells (SCs) in culture. The recent purification and molecular cloning of glial growth factor (GGF), a potent mitogen for rat Schwann cells, has led to the recognition that a family of proteins (GGF/HRG/NDF/ARIA) are alternatively spliced products of a single gene. The heregulins (HRGs) have been characterized with respect to their influence on human breast cancer cell lines; here we examined whether the HRGs have mitogenic activity for human SCs. Using DNA synthesis assays and serial passaging of cells in culture, we demonstrate that HRG is an effective mitogen for human SCs and that, in the presence of agents that elevate cAMP, it is possible to expand these cells over multiple passages without overwhelming fibroblast contamination. One putative target for this family of proteins is p185erbB2, and EGF-like receptor tyrosine kinase that is encoded by the erbB2 protooncogene. In this report we also demonstrate that the erbB2/3/4 messages as well as the erbB2/3 receptor proteins are present within cultured human SCs. The addition of HRG to human SCs results in tyrosine phosphorylation of a 185 kDa protein. In the presence of stimulatory concentrations of HRG, a blocking monoclonal antibody (2C4) to p185erbB2 is capable of significantly inhibiting phosphorylation of a 185 kDa protein as well as the subsequent incorporation of 3H-thymidine within the human SC. These latter results implicate an important role for p185erbB2 in mediating the mitogenic response of human SCs to HRGs.
Postnatal development of GABAA receptor function in somatosensory thalamus and cortex: whole-cell voltage-clamp recordings in acutely isolated rat neurons
Authors: Oh KS , Lee CJ , Gibbs JW , Coulter DA
Published: April 2018

GABAergic inhibition synchronizes oscillatory activity in the thalamocortical system. To understand better the role of this neurotransmitter in generation of thalamocortical rhythmicity, the postnatal development of GABAergic function mediated through activation of GABAA receptors was studied in thalamus and cortex. GABA-evoked chloride currents were recorded in dissociated rat cortical and thalamic neurons during postnatal development. Kinetic fits of GABA concentration/response relationships revealed developmental and regional alterations in the potency of GABA. Early in postnatal development (p5-p8), both thalamic and cortical neurons exhibited reduced potency of GABA (27–31 microM KD). Potency increased by p18-p25 in thalamic and cortical neurons (19–22 microM KD), to a level maintained in adult thalamic neurons. Adult cortical neurons exhibited reduced potency of GABA (40 microM KD). Benzodiazepine modulation of GABAA currents was also studied. Kinetic analyses of benzodiazepine augmentation of GABAA currents were best fitted assuming two effective sites with different affinities for clonazepam. The high-affinity site (KD of 0.05–0.27 nM) showed little variation with development in cortical neurons, contributing about 16–23% potentiation at all postnatal ages. Developing thalamic neurons (p5-p25) showed similar potency and efficacy of the high-affinity benzodiazepine site to cortical neurons. High-affinity benzodiazepine effects disappeared in adult thalamic neurons. A lower-affinity benzodiazepine site (25–50 nM KD) was greater in efficacy in cortical neurons compared to thalamic neurons at all ages, with efficacy ranging from 50% to 110% in cortex and from 20% to 60% in thalamus. Knowledge of developmental and regional alterations in GABAA receptor function may aid in understanding mechanisms involved in generation and control of normal and pathological thalamocortical rhythms.
Expression of neuronal acetylcholine receptor genes in vertebrate skeletal muscle during development
Authors: Corriveau RA , Romano SJ , Conroy WG , Oliva L , Berg DK
Published: April 2018

Of the 15 nicotinic ACh receptor genes identified in vertebrates, only four (alpha 1, beta 1, gamma, and delta) have been shown to be expressed in embryonic skeletal muscle at early times. In mammalian muscle a fifth gene (epsilon) replaces the gamma gene in expression at later times. The remaining 10 nicotinic receptor genes identified to date (alpha 2-alpha 8, beta 2-beta 4) are expressed in the nervous system and are considered neuronal genes. Using RNase protection assays, we show here that four of the neuronal-type genes (alpha 4, alpha 5, alpha 7, and beta 4) are expressed in developing chick skeletal muscle. Two of them (alpha 4 and alpha 7) decline substantially in transcript abundance between embryonic days 11 and 17, as does alpha 1, while the other two (alpha 5 and beta 4) show only moderate decreases over the same time period. At embryonic day 8, alpha 7 transcripts are nearly 20% as abundant as alpha 1 transcripts. In situ hybridizations confirm the presence of alpha 7 transcripts in muscle cells both in cell culture and in embryonic tissue. No evidence was found for expression of the alpha 2, alpha 3, alpha 8, or beta 3 genes in muscle. Immunoprecipitations and immunoblot analysis using subunit-specific monoclonal antibodies reveal alpha 7 protein in muscle, and the amount of protein rises and declines with the amount of alpha 7 mRNA during development. Sucrose gradient analysis demonstrates that the alpha 7 protein is present in muscle as a species of 10S, the size expected for a nicotinic receptor. The alpha 7 species in muscle binds alpha-bungarotoxin but does not contain alpha 1 subunits, indicating that the two kinds of alpha-type gene products segregate during assembly. The results suggest that neuronal AChRs may play a role in early muscle development.
Facilitation of acetylcholine release and cognitive performance by an M(2)-muscarinic receptor antagonist in aged memory-impaired
Authors: Quirion R , Wilson A , Rowe W , Aubert I , Richard J , Doods H , Parent A , White N , Meaney MJ
Published: April 2018

Aged memory-impaired (AI) and unimpaired (AU) 24–25-month-old Long- Evans rats were used to investigate the integrity of various cholinergic markers during normal aging and to establish if alterations can possibly relate to cognitive disabilities. AI and AU rats were classified on the basis of their performance in the Morris swim maze task. Choline acetyltransferase activity (ChAT) was not differentially altered in various cortical and hippocampal areas between these two groups. Similarly, quantitative receptor autoradiography did not reveal significant differences in 3H-pirenzepine/muscarinic M1 and 3H- hemicholinium-3/high-affinity choline uptake binding sites in AI versus AU rats. In contrast, 3H-AF-DX 384/putative muscarinic M2 binding was significantly increased in certain cortical and hippocampal areas of the age-impaired animals. These increments were correlated with decreased in vivo acetylcholine (ACh) release capacity in the AI rats. Most interestingly, the muscarinic M2 antagonist BIBN-99 reversed, in a dose-dependent manner, the impaired ACh release as well as the cognitive deficits observed in the AI group. Similarly, BIBN-99 reversed scopolamine-induced amnesia in young animals. The efficacy of BIBN-99 likely relates to its antagonistic properties on negative muscarinic M2 autoreceptors that are apparently increased in the AI animals, leading to altered ACh release. Taken together, these findings strengthen the role of ACh in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic functions, such as Alzheimer's disease.
Analysis of connectivity in the cat cerebral cortex
Authors: Scannell JW , Blakemore C , Young MP
Published: April 2018

The mammalian cerebral cortex is innervated by a large number of corticocortical connections. The number of connections makes it difficult to understand the organization of the cortical network. Nonetheless, conclusions about the organization of cortical systems drawn from examining connectional data have often been made in a speculative and informal manner, unsupported by any analytic treatment. Recently, progress has been made toward more systematic ways of extracting organizing principles from data on the network of connections between cortical areas of the monkey. In this article, we extend these approaches to the cortical systems of the cat. We collated information from the neuroanatomical literature about the corticocortical connections of the cat. This collation incorporated 1139 reported corticocortical connections between 65 cortical areas. We have previously used an optimization technique (Scannell and Young, 1993) to analyze this database in order to represent the connectional organization of cortical systems in the cat. Here, we report the connectional database and analyze it in a number of further ways. First, we employed rules from Felleman and Van Essen (1991) to investigate hierarchical relations among the areas. Second, we compared quantitatively the results of the optimization method with the results of the hierarchical method. Third, we examined quantitatively whether simple connection rules, which may reflect the development and evolution of the cortex, can account for the experimentally identified corticocortical connections in the database. The results showed, first, that hierarchical rules, when applied to the cat visual system, define a largely consistent hierarchy. Second, in both auditory and visual systems, the ordering of areas by hierarchical analysis and by optimization analysis was statistically significantly related. Hence, independent analyzes concur broadly in their ordering of areas in the cortical hierarchies. Third, the majority of corticocortical connections, and much of the pattern of connectivity, were accounted for by a simple “nearest-neighbor-or-next-door-but-one” connection rule, which may suggest one of the mechanisms by which the development of cortical connectivity is controlled.
Two kinetically distinct 5-hydroxytryptamine-activated Cl- conductances at Retzius P-cell synapses of the medicinal leech
Authors: Lessmann V , Dietzel ID
Published: April 2018

The properties of serotonin (5-HT)-activated Cl- receptor/ion channel complexes in neurons of the CNS of the medicinal leech were analyzed. These channels mediate the postsynaptic response at the serotoninergic Retzius P-cell as well as Retzius-Retzius cell synapses. 5-HT-induced Cl- currents were activated by fast superfusion of transmitter on cells dissociated from embryonic leeches including histochemically identified Retzius cells. Whole-cell currents elicited by partial superfusion of the membrane and summated single-channel currents of outside-out patches showed times to peak of 18 +/- 4 msec and 10 +/- 5 msec, respectively, and desensitized with time constants of 28 +/- 3 msec and 20 +/- 11 msec. Persistence of single-channel openings in the outside- out configuration as well as lack of effect of dialysis of the whole cell with AMP-PNP or GDP-beta-S indicated that 5-HT directly gates the Cl- channels without involving second messenger cascades. In outside- out patches, two single-channel conductances of 13 pS and 32 pS were identified. While the 13 pS conductance desensitized, the 32 pS conductance activated within several tens of msec and showed no desensitization. We postulate that two subtypes of channels are coactivated by 5-HT and that the activation of the fast desensitizing channel could be responsible for the fast decaying component of the post-synaptic response. The slow conductance explains the second slower decay time constant of the postsynaptic response and could account for the tonic component sometimes observed at Retzius P-cell synapses.
Sensory neurons selectively upregulate synthesis and transport of the beta III-tubulin protein during axonal regeneration
Authors: Moskowitz PF , Oblinger MM
Published: April 2018

The effects of peripheral nerve injury on the content, synthesis, and axonal transport of the class III beta-tubulin protein in adult rat dorsal root ganglion (DRG) neurons were examined. Recent reports of selective increases in the steady-state levels of the beta III-tubulin mRNA during axonal regeneration (Moskowitz et al., 1993) led to the hypothesis that upregulated levels of expression of the beta III- tubulin isotype that alter the composition of neuronal microtubules is important for effective axonal regrowth. If this is the case, the increases in mRNA levels must be translated into increased beta III- tubulin protein levels and subsequently modify the axonal cytoskeleton via axonal transport mechanisms. The present study assessed whether or not this occurs by examining beta III-tubulin protein content in adult rat lumbar DRG neurons at different times (1–14 d) after a distal sciatic nerve crush (approximately 55 mm from the DRG) by Western blotting and immunocytochemistry with a beta III-tubulin specific monoclonal antibody. These studies showed substantial increases in beta III-tubulin content in DRG neurons, as well as in proximal regions of peripheral sensory axons (0–6 mm from the DRG), from 1–2 weeks after a distal nerve injury. Pulse labeling of DRG neurons with 35S-methionine and 35S-cysteine and immunoprecipitation of labeled beta III-tubulin indicated that the synthesis of beta III-tubulin was increased in the DRG after axotomy. Studies of axonal transport, wherein L5 DRG proteins were labeled with 35S-methionine and 35S-cysteine by microinjection, revealed that slow component b(SCb) of axonal transport conveyed more labeled tubulin moving at apparently faster rates through the intact regions of sciatic nerve axons in response to crush injury of the distal sciatic nerve. Immunoprecipitation experiments using proximal peripheral nerve segments showed that SCb in distally injured DRG neurons was enriched in the beta III-tubulin isotype. These findings demonstrate that the augmented synthesis of beta III-tubulin after axotomy alters the composition of the axonally transported cytoskeleton that moves with SCb. The increased amounts and rate of delivery of beta III-tubulin in axons of regenerating DRG neurons suggest that the altered pattern of tubulin gene expression that is initiated by axotomy impacts on the composition and organization of the axonal cytoskeleton in a manner that can facilitate axonal regrowth.
The dendritic lamellar body: a new neuronal organelle putatively associated with dendrodendritic gap junctions
Authors: De Zeeuw CI , Hertzberg EL , Mugnaini E
Published: April 2018

It is shown in rat that antiserum alpha 12B/18 specifically labels a new lamellar organelle that is exclusively located in dendritic appendages. These dendritic lamellar bodies occur in a restricted number of brain regions, which include areas like the inferior olive, area CA1 and CA3 of the hippocampus, dentate gyrus, olfactory bulb, and cerebral cortex. In these regions the neurons with lamellar bodies form dendrodendritic gap junctions. Immunoreactivity in the inferior olive is first detected between P9 and P15, which coincides with the development of gap junctions in this nucleus. In the adult inferior olive, the density of dendritic lamellar bodies is highest in the rostral medial accessory olive, the subnucleus where electrotonic coupling is most prominent. Antiserum alpha 12B/18, thus, specifically detects a new neuronal organelle that may be related to dendrodendritic gap junctions.
The neurophysiology of figure-ground segregation in primary visual cortex
Authors: Lamme VA
Published: April 2018

The activity of neurons in the primary visual cortex of the awake macaque monkey was recorded while the animals were viewing full screen arrays of either oriented line segments or moving random dots. A square patch of the screen was made to perceptually pop out as a circumscribed figure by virtue of differences between the orientation or the direction of motion of the texture elements within that patch and the surround. The animals were trained to identify the figure patches by making saccadic eye movements towards their positions. Almost every cell gave a significantly larger response to elements belonging to the figure than to similar elements belonging to the background. The figure- ground response enhancement was present along the entire extent of the patch and was absent as soon as the receptive field was outside the patch. The strength of the effect had no relation with classical receptive field properties like orientation or direction selectivity or receptive field size. The response enhancement had a latency of 30–40 msec relative to the onset of the neuronal response itself. The results show that context modulation within primary visual cortex has a highly sophisticated nature, putting the image features the cells are responding to into their fully evaluated perceptual context.
Developmental kinetics of GAD family mRNAs parallel neurogenesis in the rat spinal cord
Authors: Somogyi R , Wen X , Ma W , Barker JL
Published: April 2018

GABA (gamma-amino butyric acid), a fast-acting synaptic transmitter in the mature CNS, is synthesized from glutamate by GAD (glutamic acid decarboxylase). We have developed an ultrasensitive PCR technique to quantify the expression of GAD-related mRNAs during the development of the rat cervical spinal cord and have localized them using in situ hybridization. GAD65, GAD67, and an alternatively spliced variant of GAD67, EP10, were quantified each day from embryonic (E) day 11 through E21, and at postnatal days 0, 7, 14, and adult. GAD65 and GAD67 mRNAs were detected at E11 and increased exponentially over three orders of magnitude during embryonic development, then declined approximately threefold in the first-2 postnatal weeks. While the exponential growth phase coincided with the progressive appearance of GAD67 in situ signals in both the ventral and dorsal cord, the postnatal decline coincided with the virtual disappearance of expression in the ventral region. EP10 expression was prominent in the embryo, then declined markedly together with the mRNA encoding the neuroepithelial stem cell marker, nestin. The concerted appearance of GAD-related mRNAs paralleled transcripts encoding neuronal markers (light and heavy neurofilaments) and also closely correlated with the expression of GABA, mRNAs encoding GABAA receptor subunits, and depolarizing responses to GABA. We have used the results on GAD-related mRNA expressions to formulate a simple, minimal mathematical model that accounts for their kinetics in terms of positive and negative feedback loops.
GABAA/benzodiazepine receptors in acutely isolated hippocampal astrocytes
Authors: Fraser DD , Duffy S , Angelides KJ , Perez-Velazquez JL , Kettenmann H , MacVicar BA
Published: April 2018

The properties of GABA receptor-mediated responses were examined in noncultured astrocytes, acutely isolated from the mature rat hippocampus. Whole-cell patch clamping revealed a GABA-activated Cl- conductance that was mimicked by the GABAA receptor agonist muscimol and depressed by the GABAA antagonists bicuculline and picrotoxin. The GABAA-activated currents were potentiated by the barbiturate pentobarbital and the benzodiazepine diazepam. The benzodiazepine inverse agonist DMCM either enhanced or depressed the astrocytic GABAA- mediated responses, suggesting receptor heterogeneity with respect to pharmacologic profiles. In addition, GABA evoked an increase in [Ca2+]n measured by indo-1 fluorometry, which was depressed in the presence of verapamil or picrotoxin. A GABAA-induced depolarization, therefore, causes Ca2+ influx through voltage-gated Ca2+ channels. The expression and subcellular localization of GABAA receptors and its subunits were examined using immunohistochemical and fluorescent benzodiazepine binding techniques. Polyclonal antisera raised against the GABAA/benzodiazepine receptor, which recognizes multiple subunit isoforms, labeled receptors on the astrocytic cell body and most large processes. In contrast, antisera generated against either alpha 1 or beta 1 subunit peptides revealed immunoreactivity predominantly on a subset of processes. To determine the subcellular distribution of membrane-bound receptors, a fluorescent benzodiazepine derivative was superfused over live astrocytes and visualized with laser-scanning confocal microscopy. Specific fluorescence was distributed in discrete clusters on the cell soma and a subset of distal processes. Collectively, these data support the view that astrocytes, like neurons, express GABAA receptors and target subunit isoforms to distinct cellular localizations. Astrocytic GABAA receptors may be involved in both [Cl-]o and [pH]o homeostasis, and a GABA-evoked increase in [Ca2+]i could serve as a signal between GABAergic neurons and astrocytes.
Cyclooxygenase inhibition and the spinal release of prostaglandin E2 and amino acids evoked by paw formalin injection: a microdialysis study in unanesthetized rats
Authors: Malmberg AB , Yaksh TL
Published: April 2018

Injection of formalin into the hind paw evokes a biphasic flinching of the injured paw. Pharmacological characterization of this behavior has implicated the spinal release of excitatory amino acids (EAAs) and cyclooxygenase (COX) products. To address this hypothesis, we examined the effect of paw formalin injection on release of EAAs and prostaglandin E2-like immunoreactivity (PGE2-LI) from the spinal cord in unanesthetized rats using a dialysis probe placed in the lumbar subarachnoid space. To assess the contribution of spinal COX products, the effects of S(+)- and R(-)-ibuprofen (active and inactive COX inhibitors) were examined. Paw formalin injection evoked a biphasic spinal release of PGE2-LI with an increase above resting concentrations of 110% in the 0–10 min sample, and of 83% in the 20–30 min sample. Significantly increased release of glutamate (Glu; 110%) and aspartate (Asp; 112%) was only observed in the 0–10 min sample. Saline injection into the paw had no effect on behavior, PGE2-LI, or EAA release. Intraperitoneal administration of 10 mg/kg, but not 1 mg/kg, S(+)- ibuprofen reduced paw flinching, blocked the elevated levels of PGE2- LI, and suppressed Glu and Asp release to 50% of control. Intrathecal delivery of 10 micrograms, but not 1 microgram, S(+)-ibuprofen also suppressed formalin-induced behavior, PGE2-LI, Glu, and Asp release. R(- )-ibuprofen showed no effect on formalin-induced behaviors or spinal release. These data demonstrate that paw formalin injection produces spinal release of PGE2-LI corresponding to the biphasic behavioral response and that the evoked release is blocked by antinociceptive doses of COX inhibitors.
Somatic gene transfer of NGF to the aged brain: behavioral and morphological amelioration
Authors: Chen KS , Gage FH
Published: April 2018

Primary fibroblasts modified to secrete nerve growth factor (NGF) were implanted into the nucleus basalis magnocellularis (NBM) of aged memory impaired rats. The NGF-producing fibroblasts survived for 6 weeks following transplantation and continued expressing NGF mRNA through the duration of the experiment. A significant amelioration of the memory impairment and a significant increase in size and number of low- affinity NGF receptor (p75)-positive neurons in the basal forebrain were observed. Implantation of NGF-producing cells into normal young adult rats resulted in a transient but significant memory impairment and hypertrophy of low-affinity NGF receptor-positive neurons. These results show that naturally occurring age-related memory loss can be reversed by grafting cells engineered to secrete NGF directly to the NBM, and that either cholinergic hyper- or hypofunction may lead to cognitive impairments.
Spontaneous quantal transmitter secretion from myocytes and fibroblasts: comparison with neuronal secretion
Authors: Girod R , Popov S , Alder J , Zheng JQ , Lohof A , Poo MM
Published: April 2018

When exogenous ACh is loaded into the cytoplasm of cultured amphibian myocytes and fibroblasts, the cells undergo spontaneous quantal ACh secretion, as detected by the appearance of pulsatile membrane currents in Xenopus myocytes which are manipulated into contact with the cells. These currents resemble in many ways the miniature endplate currents (MEPCs) observed at developing neuromuscular synapses formed on these Xenopus myocytes. Analyses of the frequency, amplitude, and time course of these currents suggests similarity in the cellular mechanisms involved in the packaging and secretion of ACh quanta in fibroblasts, myocytes, and developing neurons. The size of the ACh packets released by the non-neuronal cells were found to be very similar to the size of the neuronal ACh quanta, which are thought to result from the exocytotic release of synaptic vesicles. Moreover, the kinetics with which the ACh packets are discharged from all three cell types are comparable, although the speed of secretion in non-neuronal cells is somewhat slower and more irregular. The spontaneous quantal ACh secretion from neurons and myocytes was decreased by reducing cytosolic Ca2+ level and enhanced by activation of protein kinase C with phorbol ester, but secretion from fibroblasts was unaffected by both treatments. The spontaneous secretion from fibroblasts did show some sensitivity to a rise in cytosolic Ca2+ after treatment with a Ca2+ ionophore. These observations support the hypothesis that the basic machinery for transmitter secretion operating in neurons derive from a more ubiquitous mechanism used for constitutive secretion and membrane trafficking in non-neuronal cells, and neuronal differentiation involves expression of additional unique components for the regulation of the spontaneous quantal secretion.
Pharmacological types of calcium channels and their modulation by baclofen in cerebellar granules
Authors: Amico C , Marchetti C , Nobile M , Usai C
Published: April 2018

Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the dihydropyridine nimodipine reversibly inhibited the maximum current by 25% and the dose dependence showed IC50 close to 50 nM. omega-Conotoxin GVIA (cgtx, 5 microM) and omega-agatoxin IVA (agatx, 200 nM) irreversibly inhibited the current by 17% and by 47%, respectively. The effect of nimodipine was additive with that of the toxins. The GABAB agonist (+/-)baclofen, or (- )baclofen (100 microM), reduced the calcium current by 30 +/- 5%, with a IC50 4 microM. The effect was mediated by a pertussis toxin-sensitive G-protein. In cells treated with cgtx during the experiment or preincubated with the toxin for 30 min, the effect of baclofen was significantly reduced. However, the action of baclofen was not confined to cgtx-sensitive channels: application of nimodipine or agatx resulted in a 50% reduction of the baclofen effect as well. In contrast, baclofen inhibited approximately the same amount of current both before and after the increase caused by the dihydropyridine agonist BayK 8644 and did not modify the slow BayK-induced tail current. These results indicate (1) the modulation through GABAB receptors does not clearly discriminate between pharmacologically distinct calcium channels and (2) L-type calcium channels represent an heterogeneous population in these neurons.
Overexpression of potassium channel RNA: in vivo development rescues neurons from suppression of morphological differentiation in vitro
Authors: Jones SM , Hofmann AD , Lieber JL , Ribera AB
Published: April 2018

Neuronal differentiation often proceeds differently in vitro than it does in vivo. Previous work demonstrated that overexpression of potassium channel RNA reduces the number of morphologically identifiable neurons that appear in cultures prepared from neural plate stage (17–1/2 hr) embryos (Jones and Ribera, 1994). Here, we report that morphological differentiation of neurons in situ is only slightly affected by overexpression of potassium channels. Endogenous factors appear to compensate for the effect of channel overexpression. Consistent with this view, when cultures are prepared from older neural tube embryos (22–24 hr), more neurons containing excess potassium channel RNA differentiate morphologically in vitro. Exposure in situ to a rapid intracellular calcium chelator, but not to tetrodotoxin, omega- conotoxin or a slow calcium chelator, prevents the compensation provided by extended development in vivo. Typically, RNA overexpression is limited to half of the embryo in order to provide an internal control. However, when potassium channel RNA is overexpressed throughout the embryo, few neurons differentiate morphologically in vitro, even if cultures are prepared from older neural tube embryos. Thus, recovery is possible if a minimum of 5 hr of further development in vivo is allowed under conditions in which rapid elevations of intracellular calcium are permitted and half of the nervous system has normal levels of potassium channel RNA. These results suggest that different or additional mechanisms operate in situ than in vitro to promote morphological differentiation of neurons.
The first retinal axons and their microenvironment in zebrafish: cryptic pioneers and the pretract
Authors: Burrill JD , Easter SS
Published: April 2018

The initial development of the optic tract was studied with light and electron microscopy in the zebrafish (Danio rerio). Intraocular injections of the fluorescent marker, 1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate (dil), labeled retinal axons and growth cones anterogradely, and injections of dil into the optic chiasm labeled retinal ganglion cells retrogradely. Labeled tissue was photoconverted and examined electron microscopically. The ventronasal retinal quadrant produced the first growth cones. They were the first growth cones in the optic stalk. The leading retinal growth cones, typically 4–10 in number, advanced alongside the tract of the postoptic commissure but rarely sent filopodia into it and never wrapped its axons. Instead, the retinal growth cones followed a pretract, a subpial region that was morphologically distinct from its surroundings and extended out in front of the leading growth cones, presaging the optic tract. Thus, the retinal growth cones, previously thought to be followers of preexisting axons, are actually cryptic pioneers whose proximity to the earlier axons masks their pioneering nature. We suggest that cryptic pioneers and pretracts are probably common elsewhere in the nervous system.
Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice
Authors: Kelley MW , Talreja DR , Corwin JT
Published: April 2018

This study examined the potential for hair cell regeneration in embryonic and neonatal mouse organs of Corti maintained in vitro. Small numbers of hair cells were killed by laser microbeam irradiation and the subsequent recovery processes were monitored by differential interference contrast (DIC) microscopy combined with continuous time- lapse video recordings. Replacement hair cells were observed to develop in lesion sites in embryonic cochleae and on rare occasions in neonatal cochleae. In embryonic cochleae, replacement hair cells did not arise through renewed proliferation, but instead developed from preexisting cells that changed from their normal developmental fates in response to the loss of adjacent hair cells. In cochleae established from neonates, lost hair cells usually were not replaced, but 11 apparently regenerated hair cells and a single hair cell labeled by 3H-thymidine were observed as rare responses to the creation of hair cell lesions in these organs. The results indicate that the organ of Corti can replace lost hair cells during embryonic and on rare occasions during early neonatal development. The ability of preexisting cells to change their developmental fates in response to hair cell death is consistent with the hypothesis that during embryonic development hair cells may inhibit neighboring cells from specializing as hair cells. In neonatal cultures, the rare occurrence of apparently regenerated hair cells indicates that some cells in the postembryonic organ of Corti retain response mechanisms that can lead to self-repair.
The role of heparin-binding growth-associated molecule (HB-GAM) in the postsynaptic induction in cultured muscle cells
Authors: Peng HB , Ali AA , Dai Z , Daggett DF , Raulo E , Rauvala H
Published: April 2018

The heparan sulfate proteoglycan (HSPGs) is a components of the extracellular matrix of skeletal muscle that is concentrated at the neuromuscular junction (NMJ). Recent studies have suggested that HSPG, together with its bound peptide growth factors, plays important roles in autocrine or paracrine types of regulation of cell growth and differentiation. Heparin-binding growth-associated molecule (HB-GAM; also known as pleiotrophin, or p18) is a newly discovered HSPG-bound factor that is expressed at high levels in the developing CNS and PNS. In this study, we examined the role of this factor in NMJ development by examining its relationship to the formation of ACh receptor (AChR) clusters. Using an antibody against recombinant rat brain HB-GAM, we found that this protein is present prominently on the surface of cultured Xenopus myotomal muscle cells by immunocytochemistry. It is associated with HSPGs as evidenced by the fact that heparin and heparinase treatment greatly diminished the antibody labeling. HB-GAM is concentrated at preexisting AChR hot spots as well as at those induced by polystyrene beads. In addition, this molecule is also concentrated at AChR clusters induced by spinal cord neurons in nerve- muscle cocultures. To assess its function in synaptic induction, we applied recombinant HB-GAM-coated beads to cultured muscle cells to effect its focal presentation. Over 70% of these beads induced the formation of AChR clusters as shown by fluorescent alpha-bungarotoxin labeling. Furthermore, bath application of HB-GAM inhibited the nerve- induced formation of AChR clusters. Thus, HB-GAM is an endogenous muscle-derived factor that may be a component of the molecular mechanism in postsynaptic induction.
Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth
Authors: Tuttle R , Schlaggar BL , Braisted JE , O'Leary DD
Published: April 2018

We have tested the hypothesis that maturation-dependent changes in the cortical plate affect the spatiotemporal growth patterns of developing thalamocortical and corticocortical axonal projections. Given a choice between alternating lanes of embryonic (E18–19) and neonatal (P0–1) rat cortical plate membranes, embryonic (E18–19) thalamic and cortical neurites prefer to extend on neonatal membranes. Thalamic and cortical explants do extend neurites on uniform carpets of E19 cortical plate membranes, but the outgrowth is consistently greater on uniform carpets of P1 cortical plate membranes. These experiments demonstrate a maturation-dependent enhancement in the ability of cortical plate to support neurite growth from thalamic and cortical explants. In contrast, retinal and cerebellar neurites, which do not grow into cortex in vivo, generally grew poorly on these membranes, suggesting a degree of specificity to the neurite growth response. Immunohistochemical analysis of developing cortex suggests that several extracellular matrix (ECM) and cell adhesion molecules are upregulated in cortical plate. However, immunocharacterization of membrane carpets for these same ECM and cell adhesion molecules suggests that the growth preferences of thalamic and cortical neurites in vitro are predominantly influenced by membrane-anchored, rather than ECM, molecules. Western analysis of E19 and P1 cortical plate membranes supports this conclusion, and indicates that the membrane-anchored cell adhesion molecules L1 and N-CAM are more abundant in the P1 cortical plate membrane preparation. Experiments in which cortical plate membranes were treated to remove molecules sensitive to phosphatidylinositol (PI)-specific phospholipase C demonstrate that neurite growth promoters present in E19 cortical plate membranes are predominantly PI linked, whereas those present in P1 membranes are predominantly non-PI linked. These findings indicate that the neurite growth preferences are mediated, at least in part, by an upregulation of neurite growth-promoting molecules in developing cortical plate that are not PI linked. Taken together, these findings suggest that a maturation-dependent upregulation of neurite growth-promoting molecules on cortical plate cells controls the invasion of the cortical plate by thalamocortical and corticocortical axons.
A novel, abundant sodium channel expressed in neurons and glia
Authors: Schaller KL , Krzemien DM , Yarowsky PJ , Krueger BK , Caldwell JH
Published: April 2018

A novel, voltage-gated sodium channel cDNA, designated NaCh6, has been isolated from the rat central and peripheral nervous systems. RNase protection assays showed that NaCh6 is highly expressed in the brain, and NaCh6 mRNA is as abundant or more abundant than the mRNAs for previously identified rat brain sodium channels. In situ hybridization demonstrated that a wide variety of neurons express NaCh6, including motor neurons in the brainstem and spinal cord, cerebellar granule cells, and pyramidal and granule cells of the hippocampus. RT-PCR and/or in situ hybridization showed that astrocytes and Schwann cells express NaCh6. Thus, this sodium channel is broadly distributed throughout the nervous system and is shown to be expressed in both neurons and glial cells.
Topographic organization of corticospinal projections from the frontal lobe: motor areas on the medial surface of the hemisphere
Authors: He SQ , Dum RP , Strick PL
Published: April 2018

We examined the topographic organization of corticospinal neurons in the four premotor areas on the medial wall of the hemisphere of macaques. These motor areas include the supplementary motor area (SMA) and three areas buried within the cingulate sulcus: the caudal cingulate motor area on the dorsal bank (CMAd), the caudal cingulate motor area on the ventral bank (CMAv), and the rostral cingulate motor area (CMAr). In one set of animals, we injected one fluorescent tracer into lower cervical segments of the spinal cord and another fluorescent tracer into lower lumbosacral segments to define the topographic organization of arm and leg representation within each premotor area. Similarly, in another set of animals, we injected different tracers into upper cervical and lower cervical segments to provide an indication of the topographic organization of proximal and distal arm representation within the arm representation of each premotor area. We found that all four of the premotor areas on the medial wall project to cervical and lumbosacral segments of the spinal cord. Three of these areas (SMA, CMAd, and CMAv) are like the primary motor cortex in having distinct arm and leg representations. The arm representation in each of the four motor areas on the medial wall contains separate regions that project densely to upper or to lower cervical segments. This observation suggests that each motor area contains distinct proximal and distal representations of the arm. Surprisingly, the size of the distal representation is comparable to or larger than the size of the proximal representation in each motor area. Thus, contrary to some previous hypotheses, the anatomical substrate exists for the premotor areas on the medial wall to be involved in the control of distal, as well as proximal arm movements. Our results provide a new map for guiding the exploration of the motor functions of the medial wall of the hemisphere. Furthermore, the observations of the present study support our suggestion that each of the premotor areas may be an important source of descending commands for the generation and control of movement.
Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation
Authors: Reynolds IJ , Hastings TG
Published: April 2018

Using the fluorescent dye 2′,7′-dichlorodihydrofluorescein (DCF-H2) we investigated the role of glutamate in the production of reactive oxygen species (ROS) in cultured neurons from fetal rat forebrain. The addition of an excitotoxic concentration of glutamate (100 microM) produced a generalized decrease in cellular DCF fluorescence accompanied by local areas of increased fluorescence around the margins of the cell body that could be observed within 2–4 min of glutamate exposure. Increases in fluorescence were dependent on NMDA receptor activation and Ca2+ entry and were blocked by the mitochondrial proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Additional studies suggested that the generalized decrease in fluorescence was due to intracellular acidification. These studies suggest a critical role for mitochondria in the production of ROS in association with glutamate excitotoxicity, and additionally demonstrate the feasibility of measuring the production of ROS at the level of the single cell.
Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord
Authors: Arvidsson U , Riedl M , Chakrabarti S , Lee JH , Nakano AH , Dado RJ , Loh HH , Law PY , Wessendorf MW , Elde R
Published: April 2018

Opioid receptors regulate neuronal activity by both pre- and postsynaptic mechanisms. We recently reported that the cloned delta- opioid receptor (DOR1) is primarily targeted to axons, suggesting a presynaptic role. In the present study we have studied the distribution and targeting of another opioid receptor, the mu-opioid receptor (MOR1), by raising anti-peptide antisera to the C-terminal peptide of MOR1. The specificity of the antisera was determined by analysis of transfected cells, Western blots, and immunoisolation studies. Immunohistochemistry showed that MOR1 immunoreactivity was enriched in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus, and the superficial laminae of the dorsal horn. Moreover, MOR1-expressing neurons seem to target this receptor preferentially to their somatodendritic domain as determined by double- labeling experiments with MAP2. However, discrete populations of neurons target MOR1 to their axons, including some primary afferent neurons that express DOR1. In many regions enkephalin-containing axons were complementary to MOR1, suggesting by their proximity that enkephalins may be physiologically relevant ligands for this receptor. Thus, these results provide a morphological basis for understanding pre- and postsynaptic functions mediated by MOR1.
Dopamine D2 receptor mechanisms contribute to age-related cognitive decline: the effects of quinpirole on memory and motor performance in monkeys
Authors: Arnsten AF , Cai JX , Steere JC , Goldman-Rakic PS
Published: April 2018

The D2 dopamine (DA) receptor agonist, quinpirole, was characterized in young adult monkeys, young reserpine-treated monkeys and aged monkeys to assess the contribution of DA to age-related loss of prefrontal cortical (PFC) cognitive function. Monkeys were tested on a delayed response memory task that depends on the PFC, and a fine motor task that taps the functions of the motor cortex. In young adult monkeys, low quinpirole doses impaired performance of the PFC and fine motor tasks, while higher doses improved memory performance and induced dyskinesias and “hallucinatory-like” behaviors. The pattern of the quinpirole response in reserpine-treated monkeys suggested that the impairments in delayed response and fine motor performance resulted from drug actions at D2 autoreceptors, while the improvement in delayed response performance, dyskinesias and “hallucinatory-like” behaviors resulted from actions at postsynaptic receptors. In aged monkeys, low doses of quinpirole continued to impair fine motor performance, but lost their ability to impair delayed response performance. The magnitude of cognitive improvement and the incidence of “hallucinatory- like” behaviors were also reduced in the aged animals, suggesting some loss of postsynaptic D2 receptor function. The pattern of results is consistent with the greater loss of DA from the PFC than from motor areas in aged monkey brain (Goldman-Rakic and Brown, 1981; Wenk et al., 1989), and indicates that DA depletion contributes significantly to age- related cognitive decline.
Axoplasm enriched in a protein mobilized by nerve injury induces memory- like alterations in Aplysia neurons
Authors: Ambron RT , Dulin MF , Zhang XP , Schmied R , Walters ET
Published: April 2018

Axon regeneration after injury and long-term alterations associated with learning both require protein synthesis in the neuronal cell body, but the signals that initiate these changes are largely unknown. Direct evidence that axonal injury activates molecular signals in the axon was obtained by injecting axoplasm from crushed or uncrushed nerves into somata of sensory neurons with uncrushed axons. Those injected with crush axoplasm behaved as if their axons had been crushed, exhibiting increases in both repetitive firing and spike duration, and a decrease in spike afterhyperpolarization 1 d after injection. Because similar changes occur in the same cells after learning, these data suggest that some of the long-lasting adaptive changes that occur after injury and learning may be induced by common axoplasmic signals. Since the signals in axoplasm must be conveyed to the cell soma, we have begun to test the hypothesis that at least some of these signals are proteins containing a nuclear localization signal (NLS). Axoplasmic proteins at the crush site and those that accumulated at a ligation proximal to the crush were probed with an antibody to an amino acid sequence (sp) containing a NLS that provides access to the retrograde transport/nuclear import pathway. One protein, sp97, displayed properties expected of an axonal injury signal: it responded to injury by undergoing an anterograde-to-retrograde change in movement and, when the ligation was omitted, it was transported to the cell bodies of the injured neurons.