bluuebunny/crossref_metadata_2025 huggingface dataset:
Silent Synapses in the Developing Rat Visual Cortex: Evidence for Postsynaptic Expression of Synaptic Plasticity
Authors: Rumpel Simon , Hatt Hanns , Gottmann Kurt
Published: April 2018

In the developing visual cortex activity-dependent refinement of synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP). The recently described conversion of so-called silent synapses to functional ones might underlie some forms of LTP. Using whole-cell recording and minimal stimulation procedures in immature pyramidal neurons, we demonstrate here the existence of functionally silent synapses, i.e., glutamatergic synapses that show only NMDA receptor-mediated transmission, in the neonatal rat visual cortex. The incidence of silent synapses strongly decreased during early postnatal development. After pairing presynaptic stimulation with postsynaptic depolarization, silent synapses were converted to functional ones in an LTP-like manner, as indicated by the long-lasting induction of AMPA receptor-mediated synaptic transmission. This conversion was dependent on the activation of NMDA receptors during the pairing protocol.

The selective activation of NMDA receptors at silent synapses could be explained presynaptically by assuming a lower glutamate concentration compared with functional ones. However, we found no differences in glutamate concentration-dependent properties of NMDA receptor-mediated PSCs, suggesting that synaptic glutamate concentration is similar in silent and functional synapses. Our results thus support a postsynaptic mechanism underlying silent synapses, i.e., that they do not contain functional AMPA receptors. Synaptic plasticity at silent synapses might be expressed postsynaptically by modification of nonfunctional AMPA receptors or rapid membrane insertion of AMPA receptors. This conversion of silent synapses to functional ones might play a major role in activity-dependent synaptic refinement during development of the visual cortex.
The Major Cell Populations of the Mouse Retina
Authors: Jeon Chang-Jin , Strettoi Enrica , Masland Richard H.
Published: April 2018

We report a quantitative analysis of the major populations of cells present in the retina of the C57 mouse. Rod and cone photoreceptors were counted using differential interference contrast microscopy in retinal whole mounts. Horizontal, bipolar, amacrine, and Müller cells were identified in serial section electron micrographs assembled into serial montages. Ganglion cells and displaced amacrine cells were counted by subtracting the number of axons in the optic nerve, learned from electron microscopy, from the total neurons of the ganglion cell layer. The results provide a base of reference for future work on genetically altered animals and put into perspective certain recent studies. Comparable data are now available for the retinas of the rabbit and the monkey. With the exception of the monkey fovea, the inner nuclear layers of the three species contain populations of cells that are, overall, quite similar. This contradicts the previous belief that the retinas of lower mammals are “amacrine-dominated”, and therefore more complex, than those of higher mammals.
Intradermal Injection of Capsaicin in Humans Produces Degeneration and Subsequent Reinnervation of Epidermal Nerve Fibers: Correlation with Sensory Function
Authors: Simone Donald A. , Nolano Maria , Johnson Timothy , Wendelschafer-Crabb Gwen , Kennedy William R.
Published: April 2018

The ability of capsaicin to excite and subsequently to desensitize a select group of small sensory neurons has made it a useful tool to study their function. For this reason, application of capsaicin to the skin has been used for a variety of painful syndromes. We examined whether intradermal injection of capsaicin produced morphological changes in cutaneous nerve fibers that would account for its analgesic properties by comparing cutaneous innervation in capsaicin-treated skin with psychophysical measures of sensation. At various times after capsaicin injection, nerve fibers were visualized immunohistochemically in skin biopsies and were quantified. In normal skin the epidermis is heavily innervated by nerve fibers immunoreactive for protein gene product (PGP) 9.5, whereas fibers immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) are typically associated with blood vessels. There was nearly complete degeneration of epidermal nerve fibers and the subepidermal neural plexus in capsaicin-treated skin, as indicated by the loss of immunoreactivity for PGP 9.5 and CGRP. The effect of capsaicin on dermal nerve fibers immunoreactive for SP was less obvious. Capsaicin decreased sensitivity to pain produced by sharp mechanical stimuli and nearly eliminated heat-evoked pain within the injected area. Limited reinnervation of the epidermis and partial return of sensation occurred 3 weeks after treatment; reinnervation of the epidermis was ∼25% of normal, and sensation improved to 50–75% of normal. These data show that sensory dysfunction after capsaicin application to the skin results from rapid degeneration of intracutaneous nerve fibers.
Presynaptic Calcium/Calmodulin-Dependent Protein Kinase II Regulates Habituation of a Simple Reflex in AdultDrosophila
Authors: Jin Ping , Griffith Leslie C. , Murphey R. K.
Published: April 2018

On repetitive stimulation, the strength of a reflex controlling leg position inDrosophiladecreased, and this response decrement conformed to the parametric features of habituation. To study the presynaptic function of CaMKII in this nonassociative form of learning, we used a P[Gal4] insertion line to target the expression of mutant forms of CaMKII to the sensory neurons controlling the reflex. Targeted expression of a calcium-independent CaMKII construct (T287D) in the sensory neurons eliminated habituation. Targeted expression of a mutant CaMKII incapable of achieving calcium independence (T287A) reduced the initial reflex response, but a strong facilitation then occurred, and this eliminated most of the habituation. Finally, when a CaMKII inhibitory peptide (ala) was expressed in sensory neurons, the initial response was reduced, followed by facilitation. These results suggest that basal CaMKII levels in the presynaptic neurons set the response level and dynamics of the entire neural circuit.
Changes in Medial Prefrontal Cortical Dopamine Levels Associated with Response-Contingent Food Reward: An Electrochemical Study in Rat
Authors: Richardson Nicole R. , Gratton Alain
Published: April 2018

Voltammetry was used to monitor in rats changes in medial prefrontal cortex (PFC) dopamine (DA) levels associated with response-contingent presentation of a condensed milk reward. During two initial training sessions, minor DA signal fluctuations were seen when animals consumed a standard 30 sec (0.2 ml) meal earned on a continuous reinforcement schedule. There was no evidence of experience-dependent changes in these fluctuations. Under delayed reinforcement conditions, lever-presses were followed by DA signal increases that were time-locked to the delay duration, and these were followed by signal decreases when animals eventually received the reward. Such decreases became more pronounced when the standard rate of milk delivery was tripled, but were attenuated when milk delivery was reduced to half the usual rate. Withholding earned milk resulted in signal increases. In contrast, DA signal increases were observed during milk consumption when the standard meal duration was unexpectedly shortened to 15 sec or lengthened to 60 or 90 sec. Orderly changes in DA signal were also observed under partial reinforcement conditions. Unreinforced responses were associated with DA signal decreases, whereas transient increases were seen during the 30 sec meal that followed reinforced responses. These findings indicate that response-contingent reward presentation elicits synchronous changes in PFC DA transmission. They also suggest that the DA input to PFC is activated when rewards are presented under conditions that deviate from those that the animals had come to expect, particularly so when the temporal structure of learned associations is altered.
Heterologous Expression of the Kv3.1 Potassium Channel Eliminates Spike Broadening and the Induction of a Depolarizing Afterpotential in the Peptidergic Bag Cell Neurons
Authors: Whim Matthew D. , Kaczmarek Leonard K.
Published: April 2018

The bag cell neurons ofAplysiaare a cluster of cells that control egg laying behavior. After brief synaptic stimulation, they depolarize and fire spontaneously for up to 30 min. During the first few seconds of this afterdischarge, the action potentials of the bag cell neurons undergo pronounced broadening. Single bag cell neurons in culture also show spike broadening in response to repeated depolarizations. This broadening is frequency-dependent and associated with the induction of a depolarizing afterpotential lasting minutes. In some neurons the depolarizing afterpotential is sufficient to trigger spontaneous firing. To test the possibility that spike broadening during stimulation is required to trigger the depolarizing afterpotential, we eliminated frequency-dependent broadening by heterologous expression of the Kv3.1 potassium channel. This channel has rapid activation and deactivation kinetics and no use-dependent inactivation. Expression of Kv3.1 prevented spike broadening and also eliminated the depolarizing afterpotential. Measurements of the integral of calcium current during voltage commands, which simulated the action potentials of the control neurons and those expressing Kv3.1, indicate that spike broadening produces up to a fivefold increase in calcium entry. Manipulations that limit calcium entry during action potentials or chelation of intracellular calcium using BAPTA AM prevented the induction of the depolarizing afterpotential. We conclude that spike broadening is essential for the induction of the depolarizing afterpotential probably by regulating calcium influx and suggest that one of the physiological roles of spike broadening may be to regulate long-term changes in neuronal excitability.
SVOP, an Evolutionarily Conserved Synaptic Vesicle Protein, Suggests Novel Transport Functions of Synaptic Vesicles
Authors: Janz Roger , Hofmann Kay , Südhof Thomas C.
Published: April 2018

We describe a novel synaptic vesicle protein called SVOP that is distantly related to the synaptic vesicle proteins SV2A, SV2B, and SV2C (20–22% sequence identity). Both SVOP and SV2 contain 12 transmembrane regions. However, SV2 is highly glycosylated, whereas SVOP is not. Databank searches revealed that closely related homologs of SVOP are present inCaenorhabditis elegansandDrosophila(48% sequence identity), suggesting that SVOP is evolutionarily ancient. In contrast, no invertebrate orthologs of SV2 were detected. The sequences of SVOP and SV2 exhibit homology with transport proteins, in particular with mammalian organic cation and anion transporters. SVOP and SV2 are more distantly related to eukaryotic and bacterial phosphate, sugar, and organic acid transporters. SVOP is expressed at detectable levels only in brain and endocrine cells where it is primarily localized to synaptic vesicles and microvesicles. SVOP is present in all brain regions, with particularly high levels in large pyramidal neurons of the cerebral cortex. Immunocytochemical staining of adjacent rat brain sections for SVOP and SV2 demonstrated that SVOP and SV2 are probably coexpressed in most neurons. Although the functions of SV2 and SVOP remain obscure, the evolutionary conservation of SVOP, its hydrophobic nature, and its homology to transporters strongly support a role in the uptake of a novel, as yet unidentified component of synaptic vesicles. Thus synaptic vesicles contain two classes of abundant proteins with 12 transmembrane regions that are related to transporters, nonglycosylated SVOP and highly glycosylated SV2, suggesting that the transport functions of synaptic vesicles may be more complex than currently envisioned.
Specific Agrin Isoforms Induce cAMP Response Element Binding Protein Phosphorylation in Hippocampal Neurons
Authors: Ji Ru-Rong , Böse Christian M. , Lesuisse Christian , Qiu Dike , Huang Justin C. , Zhang Qin , Rupp Fabio
Published: April 2018

The synaptic basal lamina protein agrin is essential for the formation of neuromuscular junctions. Agrin mediates the postsynaptic clustering of acetylcholine receptors and regulates transcription in muscles. Agrin expression is not restricted to motor neurons but can be demonstrated throughout the CNS. The functional significance of agrin expression in neurons other than motor neurons is unknown. To test whether agrin triggers responses in neurons that lead to the activation of transcription factors, we have analyzed phosphorylation of the transcriptional regulatory site serine 133 of the transcription factor CREB (cAMP response element binding protein) in primary hippocampal neurons. Our results indicate that the neuronal (Ag4,8), but not the non-neuronal (Ag0,0), isoform of agrin induces CREB phosphorylation in hippocampal neurons. The kinetics of agrin- and BDNF-induced CREB phosphorylation are similar: peak levels are reached in minutes and are strongly reduced 2 hr later. Neuronal responses to agrin require extracellular calcium, and, in contrast to tyrosine kinase inhibitors, the specific inhibition of protein kinase A (PKA) does not affect agrin-evoked CREB phosphorylation. Our results show that hippocampal neurons specifically respond to neuronal agrin in a Ca2+-dependent manner and via the activation of tyrosine kinases.
Morphology of Single Geniculocortical Afferents and Functional Recovery of the Visual Cortex after Reverse Monocular Deprivation in the Kitten
Authors: Antonini Antonella , Gillespie Deda C. , Crair Michael C. , Stryker Michael P.
Published: April 2018

To investigate the possible anatomical basis for the functional recovery of visual cortical responses after reverse monocular deprivation, we have studied the morphology of single geniculocortical afferents to area 17. In kittens reverse-sutured for 10 d after an initial week of monocular deprivation, single-unit and intrinsic signal optical recordings confirmed that the effects of the initial deprivation were largely reversed. Responses through the originally nondeprived (OND) eye were drastically diminished, but remained much more selective for orientation than after an initial monocular deprivation (Crair et al., 1997). Responses through the originally deprived (OD) eye recovered completely. Geniculocortical afferent arbors in layer IV of area 17 were filled by iontophoresis ofPhaseoluslectin into lamina A of the lateral geniculate nucleus (LGN) and were serially reconstructed. Arbors serving both the OD and the OND eye were analyzed. The plastic changes of both OD and OND arbors were evaluated by comparison with arbors reconstructed in normal animals and in animals studied after an equivalent initial period of deprivation (Antonini and Stryker, 1996). These analyses demonstrate that closure of the OND eye caused a substantial shrinkage of the arbors serving that eye. Moreover, reopening the OD eye induced regrowth only in some arbors, whereas others appeared to be largely unaffected and continued to have the characteristics of deprived arbors. Quantitatively, the initial and the second deprivation caused similar proportional changes in total arbor length and numbers of branches, whereas several other features were more severely affected by the initial deprivation.
Chemotopic, Combinatorial, and Noncombinatorial Odorant Representations in the Olfactory Bulb Revealed Using a Voltage-Sensitive Axon Tracer
Authors: Friedrich Rainer W. , Korsching Sigrun I.
Published: April 2018

Odor information is first represented in the brain by patterns of input activity across the glomeruli of the olfactory bulb (OB). To examine how odorants are represented at this stage of olfactory processing, we labeled anterogradely the axons of olfactory receptor neurons with the voltage-sensitive dye Di8-ANEPPQ in zebrafish. The activity induced by diverse natural odorants in afferent axons and across the array of glomeruli was then recorded optically. The results show that certain subregions of the OB are preferentially activated by defined chemical odorant classes. Within these subregions, “ordinary” odorants (amino acids, bile acids, and nucleotides) induce overlapping activity patterns involving multiple glomeruli, indicating that they are represented by combinatorial activity patterns. In contrast, two putative pheromone components (prostaglandin F2αand 17α,20β-dihydroxy-4-pregnene-3-one-20-sulfate) each induce a single focus of activity, at least one of which comes from a single, highly specific and sensitive glomerulus. These results indicate that the OB is organized into functional subregions processing classes of odorants. Furthermore, they suggest that individual odorants can be represented by “combinatorial” or “noncombinatorial” (focal) activity patterns and that the latter may serve to process odorants triggering distinct responses such as that of pheromones.
Neurons Containing Hypocretin (Orexin) Project to Multiple Neuronal Systems
Authors: Peyron Christelle , Tighe Devin K. , van den Pol Anthony N. , de Lecea Luis , Heller H. Craig , Sutcliffe J. Gregor , Kilduff Thomas S.
Published: April 2018

The novel neuropeptides called hypocretins (orexins) have recently been identified as being localized exclusively in cell bodies in a subregion of the tuberal part of the hypothalamus. The structure of the hypocretins, their accumulation in vesicles of axon terminals, and their excitatory effect on cultured hypothalamic neurons suggest that the hypocretins function in intercellular communication. To characterize these peptides further and to help understand what physiological functions they may serve, we undertook an immunohistochemical study to examine the distribution of preprohypocretin-immunoreactive neurons and fibers in the rat brain. Preprohypocretin-positive neurons were found in the perifornical nucleus and in the dorsal and lateral hypothalamic areas. These cells were distinct from those that express melanin-concentrating hormone. Although they represent a restricted group of cells, their projections were widely distributed in the brain. We observed labeled fibers throughout the hypothalamus. The densest extrahypothalamic projection was found in the locus coeruleus. Fibers were also seen in the septal nuclei, the bed nucleus of the stria terminalis, the paraventricular and reuniens nuclei of the thalamus, the zona incerta, the subthalamic nucleus, the central gray, the substantia nigra, the raphe nuclei, the parabrachial area, the medullary reticular formation, and the nucleus of the solitary tract. Less prominent projections were found in cortical regions, central and anterior amygdaloid nuclei, and the olfactory bulb. These results suggest that hypocretins are likely to have a role in physiological functions in addition to food intake such as regulation of blood pressure, the neuroendocrine system, body temperature, and the sleep–waking cycle.
Red, Green, and Red-Green Hybrid Pigments in the Human Retina: Correlations between Deduced Protein Sequences and Psychophysically Measured Spectral Sensitivities
Authors: Sharpe Lindsay T. , Stockman Andrew , Jägle Herbert , Knau Holger , Klausen Gert , Reitner Andreas , Nathans Jeremy
Published: April 2018

To analyze the human red, green, and red-green hybrid cone pigmentsin vivo, we studied 41 male dichromats, each of whose X chromosome carries only a single visual pigment gene (single-gene dichromats). This simplified arrangement avoids the difficulties of complex opsin gene arrays and overlapping cone spectral sensitivities present in trichromats and of multiple genes encoding identical or nearly identical cone pigments in many dichromats. It thus allows for a straightforward correlation between each observer’s spectral sensitivity measured at the cornea and the amino acid sequence of his visual pigment. For each of the 41 single-gene dichromats we determined the amino acid sequences of the X-linked cone pigment as deduced from its gene sequence. To correlate these sequences with spectral sensitivitiesin vivo, we determined the Rayleigh matches to different red/green ratios for 29 single-gene dichromats and measured psychophysically the spectral sensitivity of the remaining green (middle wavelength) or red (long wavelength) cones in 37 single-gene dichromats. Cone spectral sensitivity maxima obtained from subjects with identical visual pigment amino acid sequences show up to a ∼3 nm variation from subject to subject, presumably because of a combination of inexact (or no) corrections for variation in preretinal absorption, variation in photopigment optical density, optical effects within the photoreceptor, and measurement error. This variation implies that spectral sensitivities must be averaged over multiple subjects with the same genotype to obtain representative values for a given pigment. The principal results of this study are that (1) ∼54% of the single-gene protanopes (and ∼19% of all protanopes) possess any one of several 5′red-3′green hybrid genes that encode anomalous pigments and that would be predicted to produce protanomaly if present in anomalous trichromats; (2) the alanine/serine polymorphism at position 180 in the red pigment gene produces a spectral shift of ∼2.7 nm; (3) for each exon the set of amino acids normally associated with the red pigment produces spectral shifts to longer wavelengths, and the set of amino acids normally associated with the green pigment produces spectral shifts to shorter wavelengths; and (4) changes in exons 2, 3, 4, and 5 from green to red are associated with average spectral shifts to long wavelengths of ∼1 nm (range, −0.5 to 2.5 nm), ∼3.3 nm (range, −0.5 to 7 nm), ∼2.8 nm (range, −0.5 to 6 nm), and ∼24.9 nm (range, 22.2–27.6 nm).
Dopamine Modulates the Susceptibility of Striatal Neurons to 3-Nitropropionic Acid in the Rat Model of Huntington’s Disease
Authors: Reynolds David S. , Carter Rebecca J. , Morton A. Jennifer
Published: April 2018

Huntington’s disease (HD) is a progressive neurodegenerative disorder characterized by chorea, psychiatric disturbances, and dementia. The striatum is the primary site of neuronal loss in HD; however, neither the mechanism of neurodegeneration nor the underlying cause of the selectivity for the striatum is understood. Chronic systemic injection of 3-nitropropionic acid (3-NP) into rats induces bilateral striatal lesions with many neuropathological features of HD and is widely used as a model of HD. In this study we examine the role striatal dopamine plays in 3-NP-induced striatal toxicity.

The effect of elevated striatal dopamine levels on 3-NP toxicity was examined by using acute administration of methamphetamine. After 7 d of 3-NP treatment, a single low dose of methamphetamine markedly increased the frequency of striatal lesion formation. This effect was mediated via dopamine receptors because it could be blocked by the administration of dopamine receptor antagonists. The effect of decreased striatal dopamine on 3-NP toxicity was examined by lesioning the nigrostriatal dopamine input to one striatum 7 d before 3-NP treatment was started. Removal of the dopamine input protected the denervated striatum from the neurotoxic effects of systemic 3-NP but did not prevent the formation of lesions in the intact striatum. Thus the formation of 3-NP lesions is critically dependent on an intact dopamine input.

Our data show that dopamine plays an important role in the formation of 3-NP lesions. We suggest that modulation of the dopaminergic system should be reevaluated as a potential drug target in the treatment for HD.
Mating-Related Stimulation Induces Phosphorylation of Dopamine- and Cyclic AMP-Regulated Phosphoprotein-32 in Progestin Receptor-Containing Areas in the Female Rat Brain
Authors: Meredith John M. , Moffatt Christopher A. , Auger Anthony P. , Snyder Gretchen L. , Greengard Paul , Blaustein Jeffrey D.
Published: April 2018

Vaginal–cervical stimulation induces a number of physiological and behavioral events, including the facilitation of mating behavior. Although the facilitation of one component of mating behavior, lordosis, by vaginal–cervical stimulation does not require the presence of progesterone, it appears to be mediated by neural progestin receptors. Abundant evidence suggests that dopamine may play a role in the neural circuitry activated by vaginal–cervical stimulation, including the mating-induced release of dopamine in progestin receptor-containing areas of the brain, changes in the activational state of progestin receptors because of dopamine D1receptor stimulation, facilitation of lordosis by D1receptor stimulation in estradiol-primed rats via progesterone-independent events, and D1agonist-induced neuronal responses in progestin receptor-containing areas and cells. We tested the hypothesis that vaginal–cervical stimulation induces phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32;Mr= 32,000), a protein phosphorylated predominantly in response to the stimulation of D1receptors. At 9 d after ovariectomy, female rats were injected subcutaneously with a behaviorally effective dose of estradiol benzoate. At 48 hr later they received vaginal–cervical or control (perineal) stimulation, and they were perfused 1 hr later. Vaginal–cervical stimulation increased the number of cells expressing pDARPP-32 immunoreactivity by 92% in the medial preoptic nucleus, 134% in the caudal ventromedial hypothalamic nucleus, 123% in the posterodorsal medial amygdala, and 103% in the bed nucleus of the stria terminalis. These results suggest that some of the neuronal effects of vaginal–cervical stimulation, and perhaps other social or environmental stimuli, are mediated by phosphorylation of DARPP-32, perhaps via stimulation of D1receptors, within progestin receptor-containing areas.
Enhancement of Neurotransmitter Release Induced by Brain-Derived Neurotrophic Factor in Cultured Hippocampal Neurons
Authors: Li Yong-Xin , Zhang Yinong , Lester Henry A. , Schuman Erin M. , Davidson Norman
Published: April 2018

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF also can induce rapid changes in synaptic efficacy. We have investigated the mechanism(s) of these synaptic effects on cultured embryonic hippocampal neurons. In the presence of the GABAAreceptor antagonist, picrotoxin, the application of BDNF (100 ng/ml) for 1–5 min increased the amplitude of evoked synaptic currents by 48 ± 9% in 10 of 15 pairs of neurons and increased the frequency of EPSC bursts to 205 ± 20% of the control levels. There was no detectable effect of BDNF on various measures of electrical excitability, including the resting membrane potential, input resistance, action potential threshold, and action potential amplitude. In addition, BDNF did not change the postsynaptic currents induced by the exogenous application of glutamate. BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 ± 46.8% of control frequency), however, without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by the removal of extracellular calcium ([Ca2+]o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was dependent on [Ca2+]o; it was blocked by K252a and by thapsigargin, but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca2+]cthat requires the release of Ca2+from IP3-gated stores.
Nerve Growth Factor-Dependent Activation of NF-κB Contributes to Survival of Sympathetic Neurons
Authors: Maggirwar Sanjay B. , Sarmiere Patrick D. , Dewhurst Stephen , Freeman Robert S.
Published: April 2018

Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. In this report, we show that nerve growth factor (NGF) activates the transcription factors NF-κB and AP-1 in cultured sympathetic neurons. Activated NF-κB complexes were shown to consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-κB activation was found to occur independently ofde novoprotein synthesis but in a manner that required the action of the proteasome complex. Treatment with the NF-κB inhibitory peptide SN50 in the continuous presence of NGF resulted in dose-dependent induction of cell death. Under the conditions used, SN50 was shown to selectively inhibit NF-κB activation but not the activation of other cellular transcription factors such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibited morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing were accelerated relative to death induced by NGF withdrawal. Finally, experiments were conducted to test directly whether NF-κB could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-κB (c-Rel) resulted in enhanced neuronal survival after withdrawal of NGF, whereas cells that were transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain underwent cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-κB/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons.
Neurite Outgrowth Stimulated by Neural Cell Adhesion Molecules Requires Growth-Associated Protein-43 (GAP-43) Function and Is Associated with GAP-43 Phosphorylation in Growth Cones
Authors: Meiri Karina F. , Saffell Jane L. , Walsh Frank S. , Doherty Patrick
Published: April 2018

The mechanisms whereby cell adhesion molecules (CAMs) promote axonal growth and synaptic plasticity are poorly understood. Here we show that the neurite outgrowth stimulated by NCAM-mediated fibroblast growth factor (FGF) receptor activation in cerebellar granule cells is associated with increased GAP-43 phosphorylation on serine-41. In contrast, neither NCAM nor FGF was able to stimulate neurite outgrowth in similar neurons from mice in which the GAP-43 gene had been deleted by homologous recombination. Integrin-mediated neurite outgrowth was unaffected by GAP-43 deletion. Both neurite outgrowth and rapid phosphorylation of GAP-43 in isolated growth cones required the first three Ig domains of a NCAM-Fc chimera and were stimulated maximally at 5 μg/ml (∼50 nm). Likewise, GAP-43 phosphorylation in isolated growth cones also was stimulated by an L1-Fc chimera. Both neurite outgrowth and NCAM-stimulated GAP-43 phosphorylation were inhibited by antibodies to the FGF receptor and a diacylglycerol lipase inhibitor (RHC80267) that blocks the production of arachidonic acid in response to activation of the FGF receptor. Direct activation of the FGF receptor and the arachidonic acid cascade with either basic FGF or melittin also resulted in increased GAP-43 phosphorylation. These data suggest that the stimulation of neurite outgrowth by NCAM requires GAP-43 function and that GAP-43 phosphorylation in isolated growth cones occurs via an FGF receptor-dependent increase in arachidonic acid.
UNC-55, an Orphan Nuclear Hormone Receptor, Orchestrates Synaptic Specificity among Two Classes of Motor Neurons inCaenorhabditis elegans
Authors: Zhou H. Mimi , Walthall W. W.
Published: April 2018

Loss of UNC-55 function in the nematodeCaenorhabditis eleganscauses one motor neuron class, the ventral D (VD) motor neurons, to adopt the synaptic pattern of another motor neuron class, the dorsal D (DD) motor neurons. Here we show thatunc-55encodes a member of the nuclear hormone receptor gene family that is similar to the vertebrate chicken ovalbumin upstream promoter transcription factors. Although the VD and DD motor neuron classes arise from different lineages at different developmental stages, they share a number of structural and functional features that appear to be the product of identical genetic programs. UNC-55 is expressed in the VD but not the DD motor neurons to modify this genetic program and to create the synaptic pattern that distinguishes the two motor neuron classes from one another.
The Chemokine Growth-Regulated Oncogene-α Promotes Spinal Cord Oligodendrocyte Precursor Proliferation
Authors: Robinson Shenandoah , Tani Marie , Strieter Robert M. , Ransohoff Richard M. , Miller Robert H.
Published: April 2018

Chemokines, (chemotactic cytokines) are a family of regulatory molecules involved in modulating inflammatory responses. Here we demonstrate that the chemokine growth-regulated oncogene-α (GRO-α) is a potent promoter of oligodendrocyte precursor proliferation. The proliferative response of immature spinal cord oligodendrocyte precursors to their major mitogen, platelet derived growth factor (PDGF), is dramatically enhanced by GRO-α present in spinal cord conditioned medium. One source of GRO-α is a subset of spinal cord astrocytes. Cultures of astrocytes contain GRO-α mRNA and protein and secrete biologically active concentrations of GRO-α. In postnatal spinal cord white matter the location of GRO-α-immunoreactive cells is developmentally regulated: GRO-α+ cells first appear in ventral and later in dorsal spinal cord white matter. These results suggest that localized proliferation of oligodendrocytes is mediated by synergy between PDGF and GRO-α.
Expression Pattern and Neurotrophic Role of the c-fmsProto-Oncogene M-CSF Receptor in Rodent Purkinje Cells
Authors: Murase Shin-ichi , Hayashi Yokichi
Published: April 2018

To investigate whether the c-fmsproto-oncogene plays a role in the CNS, we examined its expression in mouse brain. We found that c-fms-positive Purkinje cells first appeared in caudal cerebellum at postnatal day 0 (P0) arranged in a parasagittal manner, and most Purkinje cells gradually became positive by P6. This differential expression was not seen from P7 to adulthood, and the parasagittal pattern until P5 was different from those of L7, zebrins, and the integrin β1 subunit. No neuronal expression of c-fmswas found in the other brain regions examined. In both reeler and weaver mutant mice in the adult stage, all Purkinje cells were positive for c-fmsas in the wild-type controls; however, the parasagittal bands of c-fms-positive Purkinje cells were observed even in the adult staggerer mutant. To check the neurotrophic effect of macrophage colony-stimulating factor (M-CSF), we immunostained cerebella derived from osteopetrotic mutant mice, that is, those devoid of active M-CSF. We found that the number of calbindin-positive Purkinje cells in a given cerebellum began to decrease substantially during the initial 4–5 weeks of the postnatal period. In addition, cultured Purkinje cells were dependent on M-CSF for their survival. These data suggest that expression of the c-fmsgene is intrinsically programmed in the Purkinje cells and never affected by the afferent synaptic input and that neuronal survival of Purkinje cells is dependent on M-CSF after weaning. Therefore, c-fmsis considered to be a new developmental marker for Purkinje cells.
The Role of Nitric Oxide and NMDA Receptors in the Development of Motor Neuron Dendrites
Authors: Inglis Fiona M. , Furia Fran , Zuckerman Katharine E. , Strittmatter Stephen M. , Kalb Robert G.
Published: April 2018

Nitric oxide (NO) has been implicated in the establishment of precise synaptic connectivity throughout the neuroaxis in several species. To determine the contribution of NO to NMDA receptor-dependent dendritic growth in motor neurons, we administered the NMDA antagonist MK-801 to wild-type mice and neuronal nitric oxide synthase (nNOS) knock-out mice between postnatal days 7 and 14. Compared to saline-treated wild-type animals the number of dendritic bifurcations was significantly reduced in nNOS knock-out animals and MK-801-treated wild-type animals. There was no significant difference in dendritic bifurcation between MK-801-treated wild-type, MK-801-treated nNOS knock-out, and saline-treated nNOS knock-out animals, suggesting that nNOS knock-out and NMDA receptor block had similar effects. The path of the longest dendrite and the number of primary dendrites was the same in all treatment groups, indicating an effect specific to bifurcation. Sholl analysis revealed that differences in bifurcation numbers occurred between 160 and 320 μm from the cell body, the distance at which second, third, and fourth order dendrites are most prevalent. Dendrite order analyses confirmed a significant reduction in numbers, but not lengths, of third and fourth order dendrites in nNOS knock-out and drug-treatment groups. Finally, immunohistochemical examination of the developing spinal cord indicated that NMDA receptors and nNOS are colocalized within interneurons surrounding the motor neuron pool. These results support the view that at least part of NMDA receptor-dependent arborization of motor neuron dendrites is mediated by the local production of NO within the developing spinal cord.
Regeneration of Adult Rat Corticospinal Axons Induced by Transplanted Olfactory Ensheathing Cells
Authors: Li Ying , Field Pauline M. , Raisman Geoffrey
Published: April 2018

Precisely localized focal stereotaxic electrolytic lesions were made in the corticospinal tract at the level of the first to second cervical segments in the adult rat. This consistently destroyed all central nervous tissue elements (axons, astrocytes, oligodendrocytes, microglia, and microvessels) in a highly circumscribed area.

In a group of these rats immediately after lesioning, a suspension of cultured adult olfactory ensheathing cells was transplanted into the lesion site. Within the first week after transplantation, the cut corticospinal axons (identified by anterograde transport of biotin dextran) extended caudally along the axis of the corticospinal tract as single, fine, minimally branched sprouts that ended in a simple tip, often preceded by a small varicosity. By 3 weeks, the regenerating axons, ensheathed by P0-positive peripheral myelin had accumulated into parallel bundles, which now extended across the full length of the lesioned area and reentered the caudal part of the host corticospinal tract.

The transplants contained two main types of cells: (1) p75-expressing S cells, which later formed typical peripheral one-to-one myelin sheaths around individual ensheathed axons, and (2) fibronectin-expressing A cells, which aggregated into tubular sheaths enclosing bundles of myelinated axons. The point of reentry of the axons into the central nervous territory of the caudal host corticospinal tract was marked by the resumption of oligodendrocytic myelination. Thus the effect of the transplant was to form a “patch” of peripheral-type tissue across which the cut central axons regenerated and then continued to grow along their original central pathway.
Mechanisms of Action and Targets of Nitric Oxide in the Oculomotor System
Authors: Moreno-López Bernardo , Estrada Carmen , Escudero Miguel
Published: April 2018

Nitric oxide (NO) production by neurons in the prepositus hypoglossi (PH) nucleus is necessary for the normal performance of eye movements in alert animals. In this study, the mechanism(s) of action of NO in the oculomotor system has been investigated. Spontaneous and vestibularly induced eye movements were recorded in alert cats before and after microinjections in the PH nucleus of drugs affecting the NO–cGMP pathway. The cellular sources and targets of NO were also studied by immunohistochemical detection of neuronal NO synthase (NOS) and NO-sensitive guanylyl cyclase, respectively. Injections of NOS inhibitors produced alterations of eye velocity, but not of eye position, for both spontaneous and vestibularly induced eye movements, suggesting that NO produced by PH neurons is involved in the processing of velocity signals but not in the eye position generation. The effect of neuronal NO is probably exerted on a rich cGMP-producing neuropil dorsal to the nitrergic somas in the PH nucleus. On the other hand, local injections of NO donors or 8-Br-cGMP produced alterations of eye velocity during both spontaneous eye movements and vestibulo-ocular reflex (VOR), as well as changes in eye position generation exclusively during spontaneous eye movements. The target of this additional effect of exogenous NO is probably a well defined group of NO-sensitive cGMP-producing neurons located between the PH and the medial vestibular nuclei. These cells could be involved in the generation of eye position signals during spontaneous eye movements but not during the VOR.
Glutamate Inhibits GABA Excitatory Activity in Developing Neurons
Authors: van den Pol Anthony N. , Gao Xiao-Bing , Patrylo Peter R. , Ghosh Prabhat K. , Obrietan Karl
Published: April 2018

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonistl-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neuronsin vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.
Capsaicin Binds to the Intracellular Domain of the Capsaicin-Activated Ion Channel
Authors: Jung Jooyoung , Hwang Sun Wook , Kwak Jiyeon , Lee Soon-Youl , Kang Chang-Joong , Kim Won Bae , Kim Donghee , Oh Uhtaek
Published: April 2018

Capsaicin (CAP) excites small sensory neurons, causing pain, neurogenic inflammation, and other visceral reflexes. These effects have been proposed to be the result of CAP activation of a nonselective cation current. It is generally assumed that CAP binds to an extracellular domain of the membrane receptor. However, the exact binding site is not known because of the lipophilic nature of CAP. To determine whether the binding domain is extracellular or intracellular, we tested the effect of a synthetic water-soluble CAP analog, DA-5018·HCl, on current activation. CAP activated the 45 pS (at −60 mV) nonselective cation channel from either side of the membrane. However, DA-5018·HCl, which had a greater potency and efficacy than CAP, activated the channels only from the cytosolic side of the patch membrane in a capsazepine, a CAP receptor antagonist, reversible manner. When applied extracellularly, DA-5018·HCl did not, but CAP did, activate whole-cell currents in sensory neurons, as well as in oocytes expressing vanilloid receptor 1, a recently cloned CAP receptor. Hydrogen ions, reported as a possible endogenous activator of cation current, failed to elicit any current when acidic medium (pH 5.0–6.0) was applied intracellularly, indicating that H+does not mediate the CAP effect. These results indicate that CAP and its analog bind to the cytosolic domain of the CAP receptor and suggest that an endogenous CAP-like substance other than H+may be present in the cell.
Agonist-Induced Changes in Substituted Cysteine Accessibility Reveal Dynamic Extracellular Structure of M3–M4 Loop of Glutamate Receptor GluR6
Authors: Basiry Shahin S. , Mendoza Paul , Lee Peter D. , Raymond Lynn A.
Published: April 2018

Recent evidence suggests that the transmembrane topology of ionotropic glutamate receptors differs from other members of the ligand-gated ion channel superfamily. However, the structure of the segment linking membrane domains M3 and M4 (the M3–M4 loop) remains controversial. Although various data indicate that this loop is extracellular, other results suggest that serine residues in this segment are sites of phosphorylation and channel modulation by intracellular protein kinases. To reconcile these data, we hypothesized that the M3–M4 loop structure is dynamic and, more specifically, that the portion containing putative phosphorylation sites may be translocated across the membrane to the cytoplasmic side during agonist binding. To test this hypothesis, we mutated Ser 684, a putative cAMP-dependent protein kinase site in the kainate-type glutamate receptor GluR6, to Cys. Results of biochemical and electrophysiological experiments are consistent with Cys 684 being accessible, in the unliganded state, from the extracellular side to modification by a Cys-specific biotinylating reagent followed by streptavidin (SA). Interestingly, our data suggest that this residue becomes inaccessible to the extracellular biotinylating reagent during agonist binding. However, we find it unlikely that Cys 684 undergoes membrane translocation, because the addition of SA to Cys-biotinylated GluR6(S684C) has no effect on peak glutamate-evoked current and only a small effect on macroscopic desensitization. We conclude that residue 684 in GluR6 is extracellular in the receptor-channel’s closed, unliganded state and does not cross the membrane after agonist binding. However, an agonist-induced conformational change in the receptor substantially alters accessibility of position 684 to the extracellular environment.
Generation of Neuronal Intranuclear Inclusions by Polyglutamine-GFP: Analysis of Inclusion Clearance and Toxicity as a Function of Polyglutamine Length
Authors: Moulder Krista L. , Onodera Osamu , Burke James R. , Strittmatter Warren J. , Johnson Eugene M.
Published: April 2018

Recent evidence suggests that, in huntingtin and many other proteins, polyglutamine repeats are a toxic stimulus in neurodegenerative diseases. To investigate the mechanism by which these repeats may be toxic, we transfected primary rat cerebellar granule neurons with polyglutamine-green fluorescent protein (GFP) fusion constructs containing 19 (Q19-GFP), 35 (Q35-GFP), 56 (Q56-GFP), or 80 (Q80-GFP) glutamine residues. All constructs, except Q19-GFP, aggregated within the nuclei of transfected cells in a length- and time-dependent manner. Although Q35-GFP expression led to the development of several small aggregates per cell, these aggregates were cleared or degraded, and the cells remained viable. In contrast, Q80-GFP expression resulted in one or two large aggregates and induced cell death. Caspase activation was observed after Q80-GFP aggregation, but inhibition of caspases with Boc-aspartyl(OMe)-fluoromethylketone (BAF) only served to delay, not prevent, toxicity. In addition, aggregation and toxicity were not affected by other modulators of neuronal cell death such as genetic deletion of the proapoptoticbcl-2family memberbaxor addition of the protein synthesis inhibitor cycloheximide. Lastly, nuclear condensation did not occur as part of the toxicity. These data suggest that polyglutamine-GFP expression is toxic to primary neurons but that the death is distinct from classical apoptosis.