bluuebunny/crossref_metadata_2025 huggingface dataset:
Unilateral Lesions of the Dorsal Striatum in Rats Disrupt Responding in Egocentric Space
Authors: Brasted Peter J. , Humby Trevor , Dunnett Stephen B. , Robbins Trevor W.
Published: April 2018

Rats were trained in a specially designed, multichoice operant chamber on a visual choice reaction time task designed to assess performance on each side of the rat’s body. The task required animals to sustain a nose poke in a central hole, until a brief light stimulus was presented in either of two holes that were located on the same side of the box. Once the rats were trained to perform the task to both sides independently they received unilateral injections of quinolinic acid into the dorsal striatum.

Postoperatively, lesioned animals were impaired when performing the task on the side contralateral to the lesion. The time taken to initiate contralateral responses was increased. Contralateral responses were also exclusively biased toward the nearer of the two response locations, regardless of the location of the stimulus. This was interpreted as a specific impairment in generating responses in contralateral space. In contrast, no comparable deficit was seen when the animals performed the task on the side ipsilateral to the lesion. Additional postoperative challenges, in which response options were presented bilaterally, showed this response deficit to be defined in egocentric coordinates, with the severest response deficits for the most contralateral locations.
Altered Trafficking of Mutant Connexin32
Authors: Deschênes Suzanne M. , Walcott Jessica L. , Wexler Tamara L. , Scherer Steven S. , Fischbeck Kenneth H.
Published: April 2018

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot–Marie–Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting in a potentially toxic cytoplasmic accumulation of Cx32 in these cells. These results and evidence of cytoplasmic accumulation of other mutated myelin proteins suggest that diseases affecting myelinating cells may share a common pathophysiology.
Transplanted Oligodendrocyte Progenitor Cells Expressing a Dominant-Negative FGF Receptor Transgene Fail to MigrateIn Vivo
Authors: Osterhout Donna J. , Ebner Sylvie , Xu Jingsong , Ornitz David M. , Zazanis George A. , McKinnon Randall D.
Published: April 2018

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factorsin vitro. The requirement and roles for individual factorsin vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte developmentin vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitorsin vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migrationin vivoand illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS developmentin vivo.
Mice Deficient in the α7 Neuronal Nicotinic Acetylcholine Receptor Lack α-Bungarotoxin Binding Sites and Hippocampal Fast Nicotinic Currents
Authors: Orr-Urtreger Avi , Göldner Finn M. , Saeki Mayuko , Lorenzo Isabel , Goldberg Leah , De Biasi Mariella , Dani John A. , Patrick James W. , Beaudet Arthur L.
Published: April 2018

The α7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding α-bungarotoxin (α-BGT). A null mutation for the α7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]α-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the α-BGT binding sites are equivalent to the α7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the α7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene productin vivo.
Apoptosis-Suppressor Genebcl-2Expression after Traumatic Brain Injury in Rats
Authors: Clark Robert S. B. , Chen Jun , Watkins Simon C. , Kochanek Patrick M. , Chen Minzhi , Stetler R. Anne , Loeffert J. Eric , Graham Steven H.
Published: April 2018

Neuronal death after experimental traumatic brain injury (TBI) has features of both apoptosis and necrosis. Neurons in the peritrauma cortex, hippocampus, and dentate gyrus are particularly vulnerable. The apoptosis-suppressor genebcl-2is induced in brain after ischemia and epilepsy-induced injury and may serve to regulate neuronal death. We studied expression ofbcl-2mRNA and protein after experimental TBI in rats. To determine whether bcl-2 protein expression occurred in cells with evidence of apoptosis, triple-labeling studies were performed using (1) antibody against bcl-2, (2) bis-benzimide dye to examine gross nuclear morphology, and (3) terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) to assess for DNA fragmentation. At 6 and 24 hr,bcl-2mRNA was induced in ipsilateral peritrauma cortex, hippocampus, and dentate gyrus. By 72 hr the increase inbcl-2mRNA was detected only in cortex. bcl-2 protein was induced at 8, 24, 72, and 168 hr in ipsilateral cortex and hippocampus. Cells expressing bcl-2 protein included neurons in the peritrauma cortex, hippocampus, hilus, and dentate gyrus. The gross nuclear morphology of neurons expressing bcl-2 appeared normal. Furthermore, biochemical evidence of DNA fragmentation, in a pattern characteristic of either apoptosis or necrosis, was seldom seen in neurons expressing bcl-2 protein (bcl-2 colocalized with TUNEL in 0–2% of TUNEL-positive cells observed). These data suggest that bcl-2 may play an important role in the regulation of neuronal death after TBI, and they support a role forbcl-2as an inducible neuroprotective gene.
Partial Hippocampal Kindling Decreases Efficacy of Presynaptic GABABAutoreceptors in CA1
Authors: Wu Chiping , Leung L. Stan
Published: April 2018

The effect of partial hippocampal kindling, a model of temporal lobe seizure, on monosynaptic inhibition mediated by GABA was studied. Kindled rats were given 15 nonconvulsive hippocampal afterdischarges, and control rats were given low frequency or no stimulations. At 1–2 d after kindling, paired-pulse depression (PPD) of the IPSCs recorded in CA1 neuronsin vitrowas significantly smaller in kindled as compared with control rats. The difference in PPD persisted for at least 21 d after kindling. The decrease in PPD of the IPSCs after partial hippocampal kindling was likely caused by a reduced GABA autoinhibition after downregulation of presynaptic GABABreceptors. The GABABantagonist CGP35348 (1 mm) suppressed PPD of the IPSCs more strongly in control than in kindled rats. Direct activation of the presynaptic GABABreceptors by baclofen suppressed the monosynaptic IPSCs significantly more in control than in kindled rats. The decay rate of a single-pulse IPSC was faster in kindled than in control rats on day 1 or day 21 after partial kindling. The difference in IPSC decay between kindled and control rats was found with or without a GABABreceptor antagonist. The low efficacy of the presynaptic GABABreceptors in kindled rats may provide compensatory stabilization of the postsynaptic membrane against further seizures or plasticity.
Detectability Index Measures of Binaural Masking Level Difference Across Populations of Inferior Colliculus Neurons
Authors: Jiang Dan , McAlpine David , Palmer Alan R.
Published: April 2018

In everyday life we continually need to detect signals against a background of interfering noise (the “cocktail party effect”): a task that is much easier to accomplish using two ears. The binaural masking level difference (BMLD) measures the ability of listeners to use a difference in binaural attributes to segregate sound sources and thus improve their discriminability against interfering noises. By computing the detectability of tones from rate-versus-level functions in the presence of a suprathreshold noise, we previously demonstrated that individual low-frequency delay-sensitive neurons in the inferior colliculus are able to show BMLDs. Here we consider the responses of a population of such neurons when the noise level is held constant (as conventionally in psychophysical paradigms). We have sampled the responses of 121 units in the inferior colliculi of five guinea pigs to identical noise and 500 Hz tones at both ears (NoSo) and to identical noise but with the 500 Hz tone at one ear inverted (NoSπ). The result suggests that the neurons subserving detection of So tones in No (identical noise at the two ears) noise are those neurons with best frequencies (BFs) close to 500 Hz that respond to So tones with an increase in their discharge rate from that attributable to the noise. The detection of the inverted (Sπ) signal is also attributable to neurons with BFs close to 500 Hz. However, among these neurons, the presence of the Sπ tone was indicated by an increased discharge rate in some neurons and by a decreased discharge rate in others.
Differential Brainstem Fos-Like Immunoreactivity after Laryngeal-Induced Coughing and Its Reduction by Codeine
Authors: Gestreau Christian , Bianchi Armand Louis , Grélot Laurent
Published: April 2018

We used the expression of the immediate-early gene c-fos, a marker of neuronal activation, to localize brainstem neuronal populations functionally related to fictive cough (FC). In decerebrate, paralyzed, and ventilated cats, the level of Fos-like immunoreactivity (FLI) was examined in five groups of animals: (1) controls, sham-operated unstimulated animals; (2) coughing cats, including both animals in which FC was elicited by unilateral electrical stimulation of the superior laryngeal nerve (SLN) and (3) those in which FC was elicited by bilateral SLN stimulation; (4) stimulated–treated cats, in which bilateral SLN stimulation was applied after selective blockade of FC by codeine; and (5) codeine controls, sham-operated unstimulated cats subjected to administration of codeine. Fifteen brainstem structures were compared for numbers of labeled cells. Because codeine selectively blocks FC, brainstem nuclei activated specifically during FC were identified as regions showing increased FLI after FC and significant reductions in FLI after FC suppression by codeine in stimulated–treated cats. In coughing animals, we observed a selective immunoreactivity in the interstitial and ventrolateral subdivisions of the nucleus of the tractus solitarius, the medial part of the lateral tegmental field, the internal division of the lateral reticular nucleus, the nucleus retroambiguus, the para-ambigual region, the retrofacial nucleus, and the medial parabrachial nucleus. FLI in all these nuclei was significantly reduced in stimulated–treated cats. Our results are consistent with the involvement of neurons overlapping the main brainstem respiratory-related regions as well as the lateral tegmental field and the lateral reticular nucleus in the neural processing of laryngeal-induced FC.
Double Dissociation between the Involvement of the Bed Nucleus of the Stria Terminalis and the Central Nucleus of the Amygdala in Startle Increases Produced by Conditioned versus Unconditioned Fear
Authors: Walker David L. , Davis Michael
Published: April 2018

The amplitude of the acoustic startle response is reliably enhanced when elicited in the presence of bright light (light-enhanced startle) or in the presence of cues previously paired with shock (fear-potentiated startle). Light-enhanced startle appears to reflect an unconditioned response to an anxiogenic stimulus, whereas fear-potentiated startle reflects a conditioned response to a fear-eliciting stimulus. We examine the involvement of the basolateral nucleus of the amygdala, the central nucleus of the amygdala, and the bed nucleus of the stria terminalis in both phenomena. Immediately before light-enhanced or fear-potentiated startle testing, rats received intracranial infusions of the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)-quinoxaline (3 μg) or PBS. Infusions into the central nucleus of the amygdala blocked fear-potentiated but not light-enhanced startle, and infusions into the bed nucleus of the stria terminalis blocked light-enhanced but not fear-potentiated startle. Infusions into the basolateral amygdala disrupted both phenomena. These findings indicate that the neuroanatomical substrates of fear-potentiated and light-enhanced startle, and perhaps more generally of conditioned and unconditioned fear, may be anatomically dissociated.
CaM Kinase II and Visual Input Modulate Memory Formation in the Neuronal Circuit Controlling Courtship Conditioning
Authors: Joiner Mei-ling A. , Griffith Leslie C.
Published: April 2018

InDrosophila,calcium/calmodulin-dependent protein kinase II (CaM kinase) has been shown to be important in the expression of both learning and memory for the associative behavior courtship conditioning. In this study we examine the role of visual input in producing this behavior and the effects of modifying visual input on CaM kinase-dependent memory formation. Inhibition of CaM kinase blocked apparent learning regardless of visual input. Visual input selectively affected the memory phase of courtship conditioning: normal visual input masked the memory effects of inhibition of CaM kinase resulting in generation of memory without apparent learning, whereas disruption of visual input revealed the CaM kinase-dependence of memory. Visual input was found to be important only during the training period, which implies that vision and CaM kinase are interacting in the formation rather than the retrieval of memory. Our results suggest a model for courtship conditioning in which multiple sensory inputs are integrated at a CaM-kinase-dependent neuronal switch to modulate courtship behavior.
Isoform Specificity in the Relationship of Actin to Dendritic Spines
Authors: Kaech Stefanie , Fischer Maria , Doll Thierry , Matus Andrew
Published: April 2018

Dendritic spines contain high concentrations of actin, but neither the isoforms involved nor the mechanism of accumulation is known.In situhybridization with specific probes established that β- and γ-cytoplasmic actins are selectively expressed at high levels by spine-bearing neurons. Transfecting cultured hippocampal neurons with epitope-tagged actin isoforms showed that cytoplasmic β- and γ-cytoplasmic actins are correctly targeted to spines, whereas α-cardiac muscle actin, which is normally absent from neurons, formed aggregates in dendrites. The transfected actin cDNAs contained only coding domains, suggesting that spine targeting involves amino acid sequences in the proteins, an interpretation supported by experiments with chimeric cDNAs in which C-terminal actin sequences were found to be determinative in spine targeting. By contrast to actin, microtubule components, including tubulin and MAP2, were restricted to the dendritic shaft domain. The close association of cytoplasmic actins with spines together with their general involvement in cell surface motility further supports the idea that actin motility-based changes in spine shape may contribute to synaptic plasticity.
BDNF and NT-4/5 Prevent Atrophy of Rat Rubrospinal Neurons after Cervical Axotomy, Stimulate GAP-43 and Tα1-Tubulin mRNA Expression, and Promote Axonal Regeneration
Authors: Kobayashi Nao R. , Fan Da-Peng , Giehl Klaus M. , Bedard Annie M. , Wiegand Stanley J. , Tetzlaff Wolfram
Published: April 2018

Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Tα1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors.In situhybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Tα1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and Tα1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and Tα1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.
Experience-Dependent Modifications in MAP2 Phosphorylation in Rat Olfactory Bulb
Authors: Philpot Benjamin D. , Lim Jae H. , Halpain Shelley , Brunjes Peter C.
Published: April 2018

Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein, enriched in dendrites and cell bodies, that helps determine dendritic shape. MAP2 regulates microtubule stability in a phosphorylation-dependent manner. The present study used immunocytochemistry with phosphoepitope-specific and phosphorylation state-independent antibodies to examine experience-dependent changes in MAP2 expression during postnatal development of the olfactory bulb. Our results demonstrate that immunoreactivity reflecting total MAP2 expression reaches a maximal level by postnatal day 20 (P20). The degree of staining for phosphoindependent forms of MAP2 is relatively unaffected by blocking odorant passage to one half the nasal epithelium via unilateral naris closure, a manipulation that attenuates physiological activity in the bulb. However, olfactory restriction from P1 dramatically reduces immunoreactivity for antibody AP18, which recognizes MAP2 only when phosphorylated on Ser136. Quantification of staining in the granule cell layer indicates that the greatest difference (64%) between control and experimental bulbs occurs after occlusion from P1 to P30 compared with animals deprived from P1 to P10 or P1 to P20. The shift in MAP2 phosphorylation occurs even when deprivation is delayed until P30, after the sensitive period for experience-dependent changes in bulb volume. Thus, the degree of the phosphorylation shift depends on the duration but not the time of onset of naris closure.

Because staining for phosphorylation-independent forms of MAP2 is unchanged by naris closure, the total amount of the protein per unit area is probably not significantly altered. However, the large reductions of AP18-immunoreactivity in the bulb after olfactory restriction suggest that there is an activity-dependent stimulation of MAP2 phosphorylation.
The Origin, Location, and Projections of the Embryonic Abdominal Motorneurons ofDrosophila
Authors: Landgraf Matthias , Bossing Torsten , Technau Gerd M. , Bate Michael
Published: April 2018

We have used a retrograde labeling technique to identify motorneurons for each of the 30 body wall muscles of an abdominal hemisegment in the late stage 16Drosophilaembryo. Each motorneuron has a characteristic cell body position, dendritic arborization, and axonal projection. In addition, we have determined the neuroblasts of origin for most of the motorneurons we describe. Some organizational principles for the neuromuscular system have become apparent: (1) There is no obvious topographic relationship between the cell body positions of motorneurons and the position or orientation of the muscles they innervate; (2) motorneurons that innervate muscles of similar position and orientation are often clustered and have overlapping dendritic trees; (3) morphologically similar motorneurons are generally derived from a common neuroblast and innervate operationally related muscles; and (4) neuroblasts can give rise to more than one morphological type of motorneuron.
Lobular Patterns of Cerebellar Activation in Verbal Working-Memory and Finger-Tapping Tasks as Revealed by Functional MRI
Authors: Desmond John E. , Gabrieli John D. E. , Wagner Anthony D. , Ginier Bruce L. , Glover Gary H.
Published: April 2018

The lobular distributions of functional activation of the cerebellum during verbal working-memory and finger movement tasks were investigated using functional magnetic resonance imaging (fMRI). Relative to a rest control, finger tapping of the right hand produced ipsilateral-increased activation in HIV/HV [Roman numeral designations based on Larsell’s (Larsell and Jansen, 1972) nomenclature] and HVI and weaker activation in HVIII that was stronger on the ipsilateral side. For a working-memory task, subjects were asked to remember six (high load) or one (low load) visually presented letters across a brief delay. To assess the motoric aspects of rehearsal in the absence of working memory, we asked the subjects to repeatedly read subvocally six or one letters at a rate that approximated the internally generated rehearsal of working memory (motoric rehearsal task). For both tasks, bilateral regions of the superior cerebellar hemispheres (left superior HVIIA and right HVI) and portions of posterior vermis (VI and superior VIIA) exhibited increased activation during high relative to low load conditions. In contrast, the right inferior cerebellar hemisphere (HVIIB) exhibited this load effect only during the working-memory task. We hypothesize that HVI and superior HVIIA activation represents input from the articulatory control system of working memory from the frontal lobes and that HVIIB activation is derived from the phonological store in temporal and parietal regions. From these inputs, the cerebellum could compute the discrepancy between actual and intended phonological rehearsal and use this information to update a feedforward command to the frontal lobes, thereby facilitating the phonological loop.
Regulation of Ca2+-Dependent K+Channel Expression in Rat Cerebellum during Postnatal Development
Authors: Muller Yunhua Li , Reitstetter Raven , Yool Andrea J.
Published: April 2018

Potassium channels govern duration and frequency of excitable membrane events and may regulate signals that are important in neuronal development. This study assesses the developmental expression of the large conductance Ca2+-dependent K+channelin vivoandin vitroin rat cerebellum.In vivo, transcript levels for the Ca2+-dependent K+channel (KCa) were shown by Northern analysis to increase during development, whereas transcript levels for the voltage-gated K+channel Kv3.1, a delayed rectifier (KD), remained relatively constant. A comparable pattern was demonstrated by expression inXenopusoocytes of poly(A)-enriched RNA isolated from postnatal rat cerebella. In cerebellar cultures, increased external K+provided a simple manipulation of cell excitability that influenced KCatranscript levels during development. With low external K+(5.3 mm), the levels of KCachannel transcript (assessed by semiquantitative PCR) remained constant throughout development. However, in culture medium that supported significant dendritic outgrowth (10 mmextracellular K+), an upregulation of KCatranscript level was observed similar to that seenin vivo. Tetraethylammonium (TEA; 1 mm) similarly enhanced KCaexpression, suggesting that depolarizing stimuli increased KCaexpression. The stimulatory effects of increased K+or TEA on KCaexpression required extracellular Ca2+and were abolished in low external calcium (0.1 mm, buffered with EGTA), although morphological development and survival were not impaired. The regulation of KCachannel expression by depolarization and Ca2+entry provides evidence of a logical feedback mechanism governing Ca2+signals that may be significant in cerebellar development.
Potassium Channel Distribution, Clustering, and Function in Remyelinating Rat Axons
Authors: Rasband Matthew N. , Trimmer James S. , Schwarz Thomas L. , Levinson S. Rock , Ellisman Mark H. , Schachner Melitta , Shrager Peter
Published: April 2018

The K+channel α-subunits Kv1.1 and Kv1.2 and the cytoplasmic β-subunit Kvβ2 were detected by immunofluorescence microscopy and found to be colocalized at juxtaparanodes in normal adult rat sciatic nerve. After demyelination by intraneural injection of lysolecithin, and during remyelination, the subcellular distributions of Kv1.1, Kv1.2, and Kvβ2 were reorganized. At 6 d postinjection (dpi), axons were stripped of myelin, and K+channels were found to be dispersed across zones that extended into both nodal and internodal regions; a few days later they were undetectable. By 10 dpi, remyelination was underway, but Kv1.1 immunoreactivity was absent at newly forming nodes of Ranvier. By 14 dpi, K+channels were detected but were in the nodal gap between Schwann cells. By 19 dpi, most new nodes had Kv1.1, Kv1.2, and Kvβ2, which precisely colocalized. However, this nodal distribution was transient. By 24 dpi, the majority of K+channels was clustered within paranodal regions of remyelinated axons, leaving a gap that overlapped with Na+channel immunoreactivity. Inhibition of Schwann cell proliferation delayed both remyelination and the development of the K+channel distributions described. Conduction studies indicate that neither 4-aminopyridine (4-AP) nor tetraethylammonium alters normal nerve conduction. However, during remyelination, 4-AP profoundly increased both compound action potential amplitude and duration. The level of this effect matched closely the nodal presence of these voltage-dependent K+channels. Our results suggest that K+channels may have a significant effect on conduction during remyelination and that Schwann cells are important in K+channel redistribution and clustering.
The Relation of Exocytosis and Rapid Endocytosis to Calcium Entry Evoked by Short Repetitive Depolarizing Pulses in Rat Melanotropic Cells
Authors: Mansvelder Huibert D. , Kits Karel S.
Published: April 2018

Melanotropic cells release predocked, large, dense-cored vesicles containing α-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only ∼3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 μmrelative to 50 μm, but exocytosis was strongly depressed at 400 μm, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 μmEGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of ∼116 msec at 33°C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.
Amphiphysin I Antisense Oligonucleotides Inhibit Neurite Outgrowth in Cultured Hippocampal Neurons
Authors: Mundigl Olaf , Ochoa Gian-Carlo , David Carol , Slepnev Vladimir I. , Kabanov Alexander , De Camilli Pietro
Published: April 2018

Amphiphysin I is an SH3 domain-containing neuronal protein, enriched in axon terminals, which was reported to act as a physiological binding partner for dynamin I in synaptic vesicle endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I, have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a role in actin dynamics and cell polarity by testing the effect of amphiphysin I suppression on neurite outgrowth. Freshly plated hippocampal neurons were exposed to antisense oligonucleotides via a new delivery system based on a polycationic amphipathic polymer, PS980. Western blot analysis revealed that amphiphysin I levels steadily increased with neuronal differentiation, whereas in antisense-treated cultures amphiphysin I levels were reduced to ∼10% of control levels at 48 hr. Concomitantly, a collapse of growth cones and a severe inhibition of neurite outgrowth and axon formation were observed. A similar effect was observed previously after dynamin I suppression in the same culture system (Torre et al., 1994). We also have found that amphiphysin I and dynamin I colocalize in developing neurons at all developmental stages and that a pool of both proteins is colocalized with actin patches at the leading edge of growth cones. Our findings suggest a conserved role of the amphiphysin protein family in the dynamics of the cortical cell cytoskeleton and provide new evidence for a close functional link between amphiphysin I and dynamin I.
Activation Kinetics of AMPA Receptor Channels Reveal the Number of Functional Agonist Binding Sites
Authors: Clements John D. , Feltz Anne , Sahara Yoshinori , Westbrook Gary L.
Published: April 2018

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, seeEdmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.
Phosphorylation of Mammalian Olfactory Cyclic Nucleotide-Gated Channels Increases Ligand Sensitivity
Authors: Müller Frank , Bönigk Wolfgang , Sesti Federico , Frings Stephan
Published: April 2018

In vertebrate olfactory sensory neurons, odorant receptors couple the sensory signal to the synthesis of the second messenger cAMP. Cyclic nucleotide-gated (CNG) channels are activated by binding of cAMP and conduct a depolarizing receptor current that leads to electrical excitation of the neuron. The sensitivity of olfactory CNG channels for cAMP can be significantly reduced by binding of calmodulin to a regulatory domain that resides within the N-terminus of the α-subunit of the channel. This regulatory domain also contains a consensus phosphorylation sequence for protein kinase C (PKC). We have investigated the effect of channel phosphorylation by PKC and found that phosphorylation increases ligand sensitivity without counteracting modulation of the channel by calmodulin. We have identified the amino acid residue that is phosphorylated by PKC and have localized three isoforms of PKC in olfactory sensory cilia. The results of this study provide information about the control of ligand sensitivity in olfactory CNG channels by an intrinsic regulatory domain, representing both a calmodulin-binding site and a substrate for PKC.
Laminin Directs Growth Cone Navigation via Two Temporally and Functionally Distinct Calcium Signals
Authors: Kuhn Thomas B. , Williams Cheri V. , Dou Ping , Kater S. B.
Published: April 2018

During development, growth cones navigate to their targets via numerous interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisions are poorly understood. We have examined the role of intracellular Ca2+in laminin (LN)-mediated growth cone navigationin vitro, using chick dorsal root ganglion neurons. Subsequent to contacting LN-coated beads with filopodia, growth cones displayed a series of stereotypic changes in behavior, including turning toward LN-coated beads and a phase of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turning required an influx of extracellular Ca2+, likely in filopodia with LN contact, and activation of calmodulin (CaM). Surprisingly, fluorescent Ca2+imaging revealed no LN-induced rise in intracellular Ca2+in filopodia attached to their parent growth cone. However, isolation of filopodia by laser-assisted transection unmasked a rapid, LN-specific rise in intracellular Ca2+(+73 ± 11 nm). Additionally, a second, sustained rise in intracellular Ca2+(+62 ± 8 nm) occurred in growth cones, with a distinct delay 28 ± 3 min after growth cone filopodia contacted LN-coated beads. This delayed, sustained Ca2+signal paralleled the phase of increased rates of outgrowth, and both events were sensitive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) with 2 μmKN-62. We propose that LN-mediated growth cone guidance can be attributed, in part, to two temporally and functionally distinct Ca2+signals linked by a signaling cascade composed of CaM and CaM-kinase II.
Isoform-Specific Effect of Apolipoprotein E on Cell Survival and β-Amyloid-Induced Toxicity in Rat Hippocampal Pyramidal Neuronal Cultures
Authors: Jordán Joaquı́n , Galindo Marı́a F. , Miller Richard J. , Reardon Catherine A. , Getz Godfrey S. , LaDu Mary Jo
Published: April 2018

Although the genetic link between the ε4 allele of apolipoprotein E (apoE) and Alzheimer’s disease is well established, the isoform-specific activity of apoE underlying this correlation remains unclear. To determine whether apoE influences the neurotoxic actions of β-amyloid (Aβ), we examined the effect of native preparations of apoE3 and E4 on Aβ-induced toxicity in primary cultures of rat hippocampal pyramidal neurons. The source of apoE was conditioned medium from HEK-293 cells stably transfected with human apoE3 or E4 cDNA. ApoE4 (10 μg/ml) alone was toxic to the cultures, whereas apoE3 had no effect. ApoE3 treatment prevented the toxicity induced by 10 μmAβ(1–40) or Aβ(25–35). The apoE3 protective effect appears to be specific to Aβ-induced toxicity, because apoE3 did not protect against the cytotoxicity produced by NMDA or staurosporine, nor did apoE3 affect the increase in intracellular calcium induced by either NMDA or KCl. ApoE3 had no effect on the toxicity produced by Aβ in the presence of receptor-associated protein, an inhibitor of apoE receptors, particularly the LDL-receptor-related protein. Interaction with apoE receptors may not mediate the toxic actions of apoE4, because receptor-associated protein did not affect apoE4-induced neurotoxicity. Consistent with our previous biochemical experiments, analysis of the culture medium revealed that SDS-stable apoE3:Aβ complex is present in greater abundance than apoE4:Aβ complex. Thus, the protection from Aβ-induced neurotoxicity afforded by apoE3 treatment may result from clearance of the peptide by apoE3:Aβ complex formation and uptake by apoE receptors.
Comparison of Neurite Outgrowth Induced by Intact and Injured Sciatic Nerves: A Confocal and Functional Analysis
Authors: Agius Eric , Cochard Philippe
Published: April 2018

Mechanisms regulating axon growth in the peripheral nervous system have been studied by means of anin vitrobioassay, the tissue section culture, in which regenerating neurons are grown on substrata made up of tissue sections. Sections from intact and degenerated sciatic nerves proved to be different in their ability to support neurite outgrowth of embryonic chick sensory neurons from both qualitative and quantitative points of view. On denervated nerve sections, the total length of neurites elaborated per neuron was almost twice that found on intact nerve sections. In addition, confocal microscopy revealed a striking difference between intact and denervated nerve substrata: on denervated nerve sections, neurites grew inside the internal structures of endoneurial Schwann cell tubes, within the underlying tissue sections, whereas on intact nerve sections neurites extended along endoneurial basal laminae but never entered Schwann cell tubes. Perturbation experiments were used to analyze some of the molecular determinants that control neurite outgrowth in this system. Antibodies directed against the β1-integrin subunit inhibited neurite extension on both normal and degenerated rat sciatic nerve tissue. Strikingly, however, differential inhibition was observed using antibodies directed against extracellular matrix molecules. Anti-laminin-2 (merosin) antibodies drastically reduced both the percentage of growing neurons and the total length of neurites on denervated nerve sections, but they did not modify these parameters on sections of normal nerve. Taken together, these results suggest that laminin-2/merosin promotes neurite outgrowth in peripheral nerve environments but only after Wallerian degeneration, which is when axons are allowed to extend within endoneurial tubes.
Peripheral Target Regulation of the Development and Survival of Spinal Sensory and Motor Neurons in the Chick Embryo
Authors: Calderó Jordi , Prevette David , Mei Xun , Oakley Robert A. , Li Ling , Milligan Carol , Houenou Lucien , Burek Michael , Oppenheim Ronald W.
Published: April 2018

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40–50% (90% total), only ∼25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidantN-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin β1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.
Region-Specific Phosphorylation of Rabphilin in Mossy Fiber Nerve Terminals of the Hippocampus
Authors: Lonart György , Südhof Thomas C.
Published: April 2018

In mossy fiber synapses of the CA3 region of the hippocampus, long-term potentiation (LTP) is induced presynaptically by activation of cAMP-dependent protein kinase A (PKA). Rab3A is a synaptic vesicle protein that regulates vesicle fusion and is essential for mossy fiber LTP. Rab3A probably acts via two effector proteins, rabphilin and RIM, of which rabphilin is anin vitrosubstrate for PKA. To test if rabphilin is phosphorylated in nerve terminals and if its PKA-dependent phosphorylation correlates with the PKA-dependent induction of LTP in mossy fiber terminals, we have studied the phosphorylation of rabphilin in synaptosomes isolated from the CA1 and CA3 regions of the hippocampus. Rabphilin was phosphorylated in both CA1 and CA3 synaptosomes. However, when we treated the CA1 and CA3 synaptosomes with forskolin (an agent that enhances PKA activity) or induced Ca2+influx into synaptosomes with high K+, rabphilin phosphorylation was increased selectively in mossy fiber CA3 synaptosomes, but not in CA1 synaptosomes. In contrast, the phosphorylation of synapsin, studied as a control for the specificity of the region-specific phosphorylation of rabphilin, was augmented similarly by both treatments in CA1 and CA3 synaptosomes. These results reveal that the phosphorylation states of two synaptic substrates for PKA and CaM KII, rabphilin and synapsin, are regulated differentially in a region-specific manner, an unexpected finding because rabphilin and synapsin are similarly present in CA1 and CA3 synaptosomes and are colocalized on the same synaptic vesicles. The region-specific phosphorylation of rabphilin agrees well with the restricted induction of LTP by presynaptic PKA activation in mossy fiber, but not CA1, nerve terminals.
Intracellular Calcium Regulates Agrin-Induced Acetylcholine Receptor Clustering
Authors: Megeath Laura J. , Fallon Justin R.
Published: April 2018

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR β-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.