bluuebunny/crossref_metadata_2025 huggingface dataset:
Tau Is Enriched on Dynamic Microtubules in the Distal Region of Growing Axons
Authors: Black Mark M. , Slaughter Theresa , Moshiach Simon , Obrocka Maria , Fischer Itzhak
Published: April 2018

It is widely held that tau determines the stability of microtubules in growing axons, although direct evidence supporting this hypothesis is lacking. Previous studies have shown that the microtubule polymer in the distal axon and growth cone is the most dynamic of growing axons; it turns over more rapidly and is more sensitive to microtubule depolymerizing drugs than the polymer situated proximally. We reasoned that if the stability of axonal microtubules is directly related to their content of tau, then the polymer in the distal axon should have less tau than the polymer in the proximal axon. We tested this proposition by measuring the relative tau content of microtubules along growing axons of cultured sympathetic neurons immunostained for tau and tubulin. Our results show that the tau content of microtubules varies along the axon, but in the opposite way predicted. Specifically, the relative tau content of microtubules increases progressively along the axon to reach a peak near the growth cone that is severalfold greater than that observed proximally. Thus, tau is most enriched on the most dynamic polymer of the axon. We also show that the gradient in tau content of microtubules does not generate corresponding gradients in the extent of tubulin assembly or in the sensitivity of axonal microtubules to nocodazole. On the basis of these findings, we propose that tau in growing axons has functions other than promoting microtubule assembly and stability and that key sites for these functions are the distal axon and growth cone.
17β-Estradiol Potentiates Kainate-Induced Currents via Activation of the cAMP Cascade
Authors: Gu Qin , Moss Robert L.
Published: April 2018

Evidence for nongenomic actions of steroids is now coming from a variety of fields of steroid research. Mechanisms of steroid action are being studied with regard to the membrane receptors and the activation of second messengers. The present study investigated the mechanism for the rapid effect of estrogen on acutely dissociated hippocampal CA1 neurons by using the whole-cell, voltage-clamp recording. Under the perforated patch configuration, 17β-estradiol potentiated kainate-induced currents in 38% of tested neurons. The potentiation was stereospecific, rapid in onset, and reversible after the removal of the steroid. Dose–response curves show that the potentiation by 17β-estradiol was evident at a concentration as low as 10 nmand saturated at 10 μm. 17β-Estradiol did not affect the kinetics (i.e., affinity and cooperativity) and reversal potential of kainate-induced currents. This suggests that the potentiation did not result from direct interaction with kainate receptors nor the activation of ion channels other than kainate receptor-channels. The potentiation by 17β-estradiol was similar to the enhancement of kainate-induced currents evoked by 8-bromo-cAMP, and was modulated by an inhibitor of phosphodiesterase (IBMX). The estrogen potentiation was blocked by a specific blocker of PKA (Rp-cAMPS). Under standard recording configuration, the effect was significantly affected by intracellular perfusing with GDP-β-S or GTP-γ-S. The data suggest that the potentiation of kainate-induced currents by 17β-estradiol was likely a G-protein(s) coupled, cAMP-dependent phosphorylation event. By involvement of this nongenomic mechanism, estrogen may play a role in the modulation of excitatory synaptic transmission in the hippocampus.
Developmental Changes of Inhibitory Synaptic Currents in Cerebellar Granule Neurons: Role of GABAAReceptor α6 Subunit
Authors: Tia Sutthichai , Wang Jin Feng , Kotchabhakdi Naiphinich , Vicini Stefano
Published: April 2018

Eye opening and increased motor activity after the second postnatal week in rats imply an extensive development of motor control and coordination. We show a parallel developmental change in spontaneous IPSC (sIPSC) kinetics in cerebellar granule neurons. sIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from 7–30 postnatal day old rats. Early in development, sIPSCs had slow decay kinetics whereas in older rats faster decaying sIPSCs were found in larger proportion. Currents elicited by 1 mmGABA pulses (GABACs) in nucleated patches excised from cerebellar granule neurons revealed that GABACs kinetics better approximate sIPSC decay in young but not in more developed rats. The expression of α6 subunit of GABAAreceptors, unique in cerebellar granule neurons, has been shown to increase during development. Therefore, we took advantage of the recently reported selective inhibition of GABAAreceptors by furosemide to characterize the relative contribution of α6 subunits to native receptors in inhibitory synapses of cerebellar granule neurons. Although furosemide inhibition of sIPSCs amplitude was highly variable among distinct granule cells, it increased during development. At the same time, furosemide failed to inhibit sIPSCs recorded from Purkinje neurons. From the comparison of furosemide inhibition and kinetics of sIPSCs with GABACs recorded from mammalian HEK293 cells transfected with combinations of α1 and α6 GABAAreceptor subunits together with β2γ2 subunits, we propose that an increased α6 subunit contribution in the molecular assembly of postsynaptic receptors in cerebellar glomeruli is responsible for the developmental changes observed.
Endogenous Substance P Mediates Cold Water Stress-Induced Increase in Interleukin-6 Secretion from Peritoneal Macrophages
Authors: Zhu Goafa F. , Chancellor-Freeland Cheryl , Berman Ari S. , Kage Reinhard , Leeman Susan E. , Beller David I. , Black Paul H.
Published: April 2018

Previous studies from this laboratory had shown that exposure of mice to cold water stress leads to an increase in the secretion of interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) from their peritoneal macrophages. We now report that the secretion of IL-6 from peritoneal macrophages is also increased after cold water stress and that the peptide substance P (SP) participates in this stress-induced response. The stress paradigm involved subjecting male C57BL/6J mice to 5 min swim tests in 10 ± 2°C water twice daily for 4 d. Cold water stress augments the lipopolysaccharide-induced IL-6 secretion from peritoneal macrophages, elevates immunoreactive SP (iSP) in the peritoneal wash fluid, and reduces iSP in certain peritoneum-containing tissues or organs (i.e., diaphragm, abdominal wall, ileum, and rectum). The 10 d stress time course studies indicate that increased IL-6 secretion is positively related to elevated iSP in the peritoneal wash fluid and inversely related to reduced iSP in certain peritoneum-containing tissues. Pretreatment with capsaicin, which depletes SP in the sensory nerve endings, eliminates stress-control differences in the peritoneal wash fluid and in certain peritoneal tissues. Moreover, RP67,580, a specific SP antagonist, eliminates the cold water stress-induced augmentation of IL-6 secretion from peritoneal macrophages. These results suggest that cold water stress promotes the release of SP from peritoneal tissues into the peritoneal cavity, where it participates in the cold water stress-induced macrophage functional alterations.
Null Mutation of c-fosImpairs Structural and Functional Plasticities in the Kindling Model of Epilepsy
Authors: Watanabe Yoshinori , Johnson Randall S. , Butler Linda S. , Binder Devin K. , Spiegelman Bruce M. , Papaioannou Virginia E. , McNamara James O.
Published: April 2018

It has been suggested that expression of the immediate early gene c-foslinks fleeting changes in neuronal activity to lasting modifications of neuronal structure and function in the mammalian nervous system. To test this idea, we examined behavioral and electrophysiological indices of kindling development and kindling-induced sprouting of hippocampal granule cell axons in wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice carrying a null mutation of c-fos. The rate of kindling development was significantly attenuated in −/− compared with +/+ mice, as evidenced by both electrophysiological and behavioral measures. Kindling-induced granule cell axon sprouting as measured by the Timm stain was also attenuated in homozygous null mutants compared with +/+ mice, with an intermediate effect in +/− mice. The impairment of kindling-induced axonal sprouting in the null mutants could not be attributed to either detectable loss of dentate hilar neurons or reduced activation of the dentate granule cells by seizures. Instead, our data are consistent with the hypothesis that the null mutation of c-fosattenuates a pathological activity-determined functional plasticity (kindling development) as well as a structural plasticity (mossy fiber reorganization). We favor the hypothesis that this “fos-less phenotype” is attributable to impaired seizure-induced transcriptional activation of one or more growth-related genes.
Muscarinic Activation of a Voltage-Dependent Cation Nonselective Current in Rat Association Cortex
Authors: Haj-Dahmane Samir , Andrade Rodrigo
Published: April 2018

The ionic mechanism underlying the acetylcholine-induced depolarization of layer V pyramidal neurons of rat prefrontal cortex was examined using whole-cell recording inin vitrorat brain slices. Consistent with previous results, pressure application of acetylcholine to layer V pyramidal neurons elicited a strong depolarization. Pharmacological analysis of this response indicated that it was mediated by the stimulation of muscarinic receptors as it was mimicked by muscarinic agonists, but not by nicotine, and was blocked by atropine.

The inward current responsible for the depolarization resulted from the activation of a voltage-dependent, cation nonselective current. Thus, the amplitude of the current was critically dependent on extracellular sodium concentration but not on extracellular potassium or chloride concentration. Examination of theI–Vrelationship for the muscarinic current using voltage clamp revealed that the current reversed near −15 mV and exhibited a strong voltage dependence, turning off rapidly in the subthreshold range. The voltage dependence of the current led to the appearance of a current associated with a conductance decrease when examined using steady-state voltage- or current-clamp measurements. This might have led to earlier misidentification of this response as mediated by a decrease in potassium conductance.

These results question the traditional interpretation that muscarinic depolarization in cortex is mediated by a decrease in potassium conductance. They indicate that the fundamental mechanism responsible for muscarinic depolarization in prefrontal cortex involves the activation of a voltage-dependent, cation nonselective current. This current might represent a previously unsuspected mechanism capable of mediating slow depolarization in the central nervous system.
Layer-Specific Properties of the Transient K Current (IA) in Piriform Cortex
Authors: Banks Matthew I. , Haberly Lewis B. , Jackson Meyer B.
Published: April 2018

Piriform cortex in the rat is highly susceptible to induction of epileptiform activity. Experimentsin vivoandin vitroindicate that this activity originates in endopiriform nucleus (EN). In slices, EN neurons are more excitable than layer II (LII) pyramidal cells, with more positive resting potentials and lower spike thresholds. We investigated potassium currents in EN and LII to evaluate their contribution to these differences in excitability. Whole-cell currents were recorded from identified cells in brain slices. A rapidly inactivating outward current (IA) had distinct properties in LII (IA,LII) versus EN (IA,EN). The peak amplitude ofIA,ENwas 45% smaller thanIA,LII, and the kinetics of activation and inactivation was significantly slower forIA,EN. The midpoint of steady-state inactivation was hyperpolarized by 10 mV forIA,ENversusIA,LII, whereas activation was similar in the two cell groups. Other voltage-dependent potassium currents were indistinguishable between EN and LII. Simulations using a compartmental model of LII cells argue that different cellular distributions ofIAchannels in EN versus LII cells cannot account for these differences. Thus, at least some of the differences are intrinsic to the channels themselves. Current-clamp simulations suggest that the differences betweenIA,LIIandIA,ENcan account for the observed difference in resting potentials between the two cell groups. Simulations show that this difference in resting potential leads to longer first spike latencies in response to depolarizing stimuli. Thus, these differences in the properties ofIAcould make EN more susceptible to induction and expression of epileptiform activity.
Biosynthesis of an Endogenous Cannabinoid Precursor in Neurons and its Control by Calcium and cAMP
Authors: Cadas Hugues , Gaillet Sylvie , Beltramo Massimiliano , Venance Laurent , Piomelli Daniele
Published: April 2018

Understanding the mechanisms involved in the biogenesis ofN-arachidonoylethanolamine (anandamide) andNpalmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) andN-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid,N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca2+-activated D-type phosphodiesterase activity. We report here that thede novobiosynthesis of NAPE is enhanced in a Ca2+-dependent manner when rat cortical neurons are stimulated with the Ca2+-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronalN-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimeticN-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.
Schwann Cell Apoptosis during Normal Development and after Axonal Degeneration Induced by Neurotoxins in the Chick Embryo
Authors: Ciutat Dolors , Calderó Jordi , Oppenheim Ronald W. , Esquerda Josep E.
Published: April 2018

In the present work, we show that chick embryo Schwann cells die by apoptosis both during normal development and after axonal degeneration induced by neurotoxin treatment. Schwann cell apoptosis during development takes place during a period roughly coincidental with normally occurring motoneuron death. Administration of NMDA to chick embryos on embryonic day 7 induces extensive excitotoxic motoneuronal damage in the spinal cord without any apparent effects on neurons in the dorsal root ganglia (DRG). The death of Schwann cells inventralnerve roots after NMDA treatment causes degenerative changes that display ultrastructural features of apoptosis and exhibitin situdetectable DNA fragmentation. By contrast, NMDA treatment does not increase the death of Schwann cells indorsalnerve roots.In situdetection of DNA fragmentation in combination with the avian Schwann cell marker 1E8 antibody demonstrates that dying cells in ventral nerve roots are in the Schwann cell lineage. Administration of cycloheximide does not prevent the toxic effects of NMDA on motoneurons, but dramatically reduces the number of pyknotic Schwann cells and DNA fragmentation profiles in the ventral nerve roots.In ovoadministration of various tissue extracts (muscle, brain, and spinal cord) from the chick embryo or of the motoneuron conditioned medium fails to prevent Schwann cell apoptosis in NMDA-treated embryos. Intramuscular administration of the snake toxin β-bungarotoxin produces a massive death of both lateral motor column motoneurons and DRG neurons, resulting in a substantial increase in the number of pyknotic Schwann cells in both ventral and dorsal nerve roots. It is concluded that during development, axonal-derived trophic signals are involved in the regulation of Schwann cell survival in peripheral nerves.
Expression of NGF and NT3 mRNAs in Hippocampal Interneurons Innervated by the GABAergic Septohippocampal Pathway
Authors: Rocamora Nativitat , Pascual Marta , Acsàdy Laszlo , de Lecea Luı́s , Freund Tamàs F. , Soriano Eduardo
Published: April 2018

We usedin situhybridization for the detection of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3) mRNAs combined with immunocytochemistry against the calcium-binding proteins parvalbumin (PARV), calbindin 28k (CALB), and calretinin (CALR) to determine the expression of neurotrophins in functionally distinct subsets of hippocampal interneurons. Most PARV-immunoreactive neurons in the hippocampus were NGF mRNA-positive (82%), which corresponds to 71% of NGF-positive neurons in the hippocampus proper and in the dentate gyrus (excluding granule cells). In contrast, only a subset of CALB- and CALR-immunoreactive interneurons (24% and 23%, respectively) displayed hybridization signals for NGF. Small subsets of PARV- and CALR-positive cells expressed NT3 mRNA, but we did not find hippocampal interneurons expressing BDNF mRNA. These results show that NGF and NT3 genes are differentially regulated in distinct subsets of GABAergic cells, and these interneurons are a major source of NGF production in the hippocampus.

We also addressed whether hippocampal interneurons expressing neurotrophins were targets of the GABAergic septohippocampal pathway. We developed a triple-labeling method that combines anterograde tracing of this pathway by means ofPhaseolus vulgarisleucoagglutinin injections, within situhybridization for the detection of neurotrophins, and immunocytochemistry for calcium-binding proteins. Virtually every PARV-positive neuron innervated by GABAergic septohippocampal baskets expressed NGF mRNA (86%), whereas 39–59% of CALR- and CALB-positive interneurons that were contacted by GABAergic septohippocampal axons showed NGF gene expression. A small subset of NT3 mRNA-expressing interneurons was also innervated by septohippocampal baskets. These findings show that the GABAergic septohippocampal pathway preferentially terminates on interneurons expressing NGF mRNA, suggesting that this neurotrophic factor might be involved in the specification of this connection and in its maintenance and normal function in the adult brain.
Analysis of the Globose Basal Cell Compartment in Rat Olfactory Epithelium Using GBC-1, a New Monoclonal Antibody against Globose Basal Cells
Authors: Goldstein Bradley J. , Schwob James E.
Published: April 2018

The olfactory epithelium (OE) supports ongoing neurogenesis throughout life and regenerates after experimental injury. Although evidence indicates that proliferative cells within the population of globose (light) basal cells (GBCs) give rise to new neurons, little is known about the biology of GBCs. Because GBCs have been identifiable only by an absence of staining with reagents that mark other cell types in the epithelium, we undertook to isolate antibodies that specifically react against GBCs and to characterize the GBC compartment in normal and regenerating OE. Monoclonal antibodies were produced using mice immunized with regenerating rat OE, and a monoclonal antibody designated GBC-1, which reacts against GBCs of the rat OE, was isolated. In immunohistochemical analyses, antibody GBC-1 was found to label GBCs in both normal and regenerating OE as we are currently able to define them: basal cells that incorporate the mitotic tracer bromodeoxyuridine and fail to express cytokeratins or neural cell adhesion molecule. During epithelial reconstitution after direct experimental injury with methyl bromide, expression of the GBC-1 antigen overlaps to a limited extent with expression of cell-specific markers for horizontal basal cells, Bowman’s gland and sustentacular cells, and neurons. These data suggest that GBC-1 may mark multipotent cells residing in the GBC compartment, which are prominent during regeneration. However, a limited number of cells in the regenerating OE with other phenotypic characteristics of GBCs lack expression of the GBC-1 antigen. GBC-1 has revealed novel aspects of GBC biology and will be useful for studying the process of olfactory neurogenesis.
Gonadotropin-Releasing Hormone and NMDA Receptor Gene Expression and Colocalization Change during Puberty in Female Rats
Authors: Gore Andrea C. , Wu T. J. , Rosenberg Jacob J. , Roberts James L.
Published: April 2018

During development, an increase in gonadotropin-releasing hormone (GnRH) release occurs that is critical for the initiation of puberty. This increase is attributable, at least in part, to activation of the GnRH neurosecretory system by inputs from neurotransmitters, such as glutamate, acting via NMDA receptors. We examined changes in GnRH and NMDA-R1 gene expression by RNase protection assay of preoptic area-anterior hypothalamic (POA-AH) dissections of female rats undergoing normal puberty or in which precocious puberty was induced by treatment with the glutamate agonist NMA. GnRH mRNA levels increased significantly throughout normal development; this was accelerated by treatment with NMA. NMDA-R1 mRNA levels increased only between P10 and P20. The acceleration of the elevation in GnRH mRNA levels by NMDA suggests that a stimulation of GnRH gene expression may be a rate-limiting factor for the onset of puberty. This is attributable to a post-transcriptional mechanism because GnRH primary transcript levels, an index of proGnRH gene transcription, were not observed to change during puberty. Alterations in the colocalization of GnRH neurons with the NMDA-R1 subunit during puberty also were assessed immunocytochemically. The percentage of GnRH neurons that double-labeled with NMDA-R1 was 2% in prepubertal rats and 3% in pubertal rats; this increased to 19% in postpubertal rats. Taken together, these studies suggest that an increase in glutamatergic input to GnRH neurons plays a role in the increase in GnRH release and gene expression that occurs at the initiation of puberty.
Calcium-Dependent Paired-Pulse Facilitation of Miniature EPSC Frequency Accompanies Depression of EPSCs at Hippocampal Synapses in Culture
Authors: Cummings Dana D. , Wilcox Karen S. , Dichter Marc A.
Published: April 2018

Two forms of evoked neurotransmitter release at excitatory synapses between cultured hippocampal neurons have been described. After an action potential, it has been shown that transmitter initially is released synchronously, and this is followed by a period of “slow” asynchronous release. The “fast” synchronous component of release at these synapses has been found routinely to demonstrate paired-pulse and tetanic depression, whereas the short-term plasticity of asynchronous release has not been investigated. In the present experiments, we have used the whole-cell patch-clamp technique to record from pairs of neurons in a low-density hippocampal culture preparation to determine both the properties and underlying mechanisms of short-term plasticity of asynchronous release. It was found that an increase in miniature EPSC (mEPSC) frequency accompanied both single and multiple stimuli, and this mEPSC increase was facilitated during paired stimuli, even when the evoked synchronous release was depressed. In addition, both the activity-dependent depression of evoked EPSCs and facilitation of asynchronous mEPSC release were dependent on Ca accumulation in the nerve terminal. However, the Ca-dependent mechanisms underlying these two processes could be distinguished by the differential effects of two membrane-permeant calcium chelators, BAPTA-AM and EGTA-AM. Frequency-dependent depression of evoked EPSCs involves a rapid rise in intraterminal Ca, which likely triggers a process that proceeds in a Ca-independent manner, whereas the asynchronous release may be linked more directly to a sustained increase in intraterminal Ca.
Bidirectional Regulation of Protein Kinase Mζ in the Maintenance of Long-Term Potentiation and Long-Term Depression
Authors: Hrabetova Sabina , Sacktor Todd Charlton
Published: April 2018

Long-term potentiation (LTP) and long-term depression (LTD) are persistent modifications of synaptic efficacy that may contribute to information storage in the CA1 region of the hippocampus. Persistently enhanced phosphorylation has been implicated in the maintenance phase of LTP. This hypothesis is supported by our previous observation that protein kinase Mζ (PKMζ), the constitutively active catalytic fragment of a single protein kinase C isoform (PKCζ), increases in LTP maintenance. In contrast, dephosphorylation may be important in LTD maintenance, because phosphatase inhibitors reverse established LTD, in addition to blocking its induction. Because phosphorylation is determined by a balance of phosphatases and kinases, both increases in phosphatase activity and decreases in kinase activity could contribute to LTD. We now report that the reduction of protein kinase activity by H7, as well as selective inhibition of PKC by chelerythrine, mimics and occludes the maintenance phase of homosynaptic LTD in rat hippocampal slices. Conversely, saturated LTD occludes the synaptic depression caused by chelerythrine. Biochemical analysis demonstrates a decrease of PKMζ, as well as PKCs γ and ε, in LTD maintenance and a concomitant loss of constitutive PKC activity. LTD and the downregulation of PKMζ are prevented by NMDA receptor antagonists and Ca2+-dependent protease inhibitors. Both LTD and the downregulation of PKMζ are reversible by high-frequency afferent stimulation. Our findings indicate that the molecular mechanisms of LTP and LTD maintenance are inversely related through the bidirectional regulation of PKC.
Traffic of Dynamin within IndividualDrosophilaSynaptic Boutons Relative to Compartment-Specific Markers
Authors: Estes Patricia S. , Roos Jack , van der Bliek Alexander , Kelly Regis B. , Krishnan K. S. , Ramaswami Mani
Published: April 2018

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identifiedDrosophilaneuromuscular synapse that several of these compartments—synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton—labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers andDrosophila shibirets1mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. Inshits1nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show “hot spots” of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.
Ca2+-Permeable AMPA/Kainate and NMDA Channels: High Rate of Ca2+Influx Underlies Potent Induction of Injury
Authors: Lu You Ming , Yin Hong Zhen , Chiang Juna , Weiss John H.
Published: April 2018

Neurodegeneration may occur secondary to glutamate-triggered Ca2+influx through any of three routes: NMDA channels, voltage-sensitive Ca2+channels (VSCC), and Ca2+-permeable AMPA/kainate channels (Ca-A/K). This study aims to examine Ca2+ion dynamics in the generation of excitotoxic injury by correlating the relative amounts of45Ca2+that flow into cortical neurons through each of these routes over a 10 min epoch (“10 min Ca2+loads;” a measure of influx rate), with resultant levels of intracellular free Ca2+([Ca2+]i) and subsequent injury. Neurons possessing Ca-A/K make up a small subset (∼13%) of cortical neurons in culture, which can be identified by a histochemical stain based on kainate-stimulated Co2+uptake (Co2+(+) neurons) and which are unusually vulnerable to AMPA/kainate receptor-mediated injury. Initial studies using brief kainate exposures (to selectively destroy Co2+(+) neurons) along with kainate-triggered45Ca2+influx measurements suggested that kainate causes rapid Ca2+influx into Co2+(+) neurons (comparable to that caused by NMDA). Influx through both Ca-A/K and NMDA channels increased proportionately with extracellular Ca2+, suggesting that these channels have high Ca2+permeability.

When cultures were subjected to exposures that gave similar 10 min Ca2+loads through different routes, comparable levels of injury were observed, suggesting that net intracellular Ca2+accumulation is a critical determinant of injury. However, the relationship between [Ca2+]iand influx was less direct: although exposures that gave the lowest or highest 10 min Ca2+loads showed correspondingly lower or higher mean [Ca2+]iresponses, there appears to be a wide range of exposures over which individual neuronal differences and sequestration/buffering mechanisms obscure [Ca2+]ias a reflection of influx rate.
Rapid, Active Hair Bundle Movements in Hair Cells from the Bullfrog’s Sacculus
Authors: Benser Michael E. , Marquis Robert E. , Hudspeth A. J.
Published: April 2018

Hair bundles, the mechanically sensitive organelles of hair cells in the auditory and vestibular systems, are elastic structures that are deflected by sound or acceleration. To examine rapid mechanical events associated with mechanoelectrical transduction, we stimulated individual hair bundles with flexible glass fibers and measured their responses with a temporal resolution of 400 μsec. When a hair bundle from the bullfrog’s sacculus was abruptly deflected in the positive direction, the bundle’s motion in the direction of stimulation was interrupted within the initial few milliseconds by an active movement, or twitch. This response was biphasic, with an initial component in the direction of the stimulus and a second component in the opposite direction. The amplitude and duration of the twitch depended on the bundle’s initial position and the size and rise time of the stimulus; the twitch was largest over the range of bundle deflections in which transduction was most sensitive. Under displacement clamp conditions, in which a hair bundle’s position was changed and then held constant with negative feedback, the twitch manifested itself as a biphasic force exerted by the bundle. Some hair bundles produced twitches in response to negatively directed stimuli, exhibited stimulus-evoked damped oscillations, or twitched spontaneously. The hair bundle’s ability to perform work against an external load and to oscillate in response to stimulation indicates that the bundle could supply feedback for mechanical amplification in vertebrate auditory organs.
Retinoic Acid Stimulates α-CAMKII Gene Expression in PC12 Cells at a Distinct Transcription Initiation Site
Authors: Chen Jing , Kelly Paul T.
Published: April 2018

The promoter region of the α-subunit of the calcium/calmodulin-dependent protein kinase II (α-CaMKII) gene was inserted into a β-galactosidase (β-gal) reporter plasmid, and β-gal activities were examined in neuroblastoma (NB2a) and pheochromocytoma (PC12) cells after transient or stable transfections. The α-CaMKII promoter was 12- to 45-fold more active in NB2a compared with PC12 cells after transient or stable transfections. All-transretinoic acid (RA) stimulated reporter gene expression at both protein and mRNA levels in transfected PC12 cells. RA increased the level of endogenous α-CaMKII mRNA in untransfected PC12 cells by 4.4-fold. The transcription initiation site(s) (TIS) of the α-CaMKII gene in PC12 cells and rat brain was examined by RNase protection assays (RPA) and reverse transcriptase PCRs. The TIS for the α-CaMKII/β-gal reporter gene in transfected PC12 cells was indistinguishable from the TIS+1in rat hippocampus. In contrast, the only detectable TIS for the α-CaMKII gene in untransfected PC12 cells was located near the ATG translation start codon, 147 nucleotides 3′ to TIS+1in hippocampus. This unusual TIS was also the predominant TIS in rat cerebellum. These results suggest that the α-CaMKII promoter may contain sequences that respond directly or indirectly to RA. In addition, the unusual TIS of the α-CaMKII gene in PC12 cells and rat cerebellum may contribute to the very low expression of this gene compared with that in hippocampus.
Laser Ablation ofDrosophilaEmbryonic Motoneurons Causes Ectopic Innervation of Target Muscle Fibers
Authors: Chang Te Ning , Keshishian Haig
Published: April 2018

We have tested the effects of neuromuscular denervation inDrosophilaby laser-ablating the RP motoneurons in intact embryos before synaptogenesis. We examined the consequences of this ablation on local synaptic connectivity in both 1st and 3rd instar larvae. We find that the partial or complete loss of native innervation correlates with the appearance of alternate inputs from neighboring motor endings and axons. These collateral inputs are found at ectopic sites on the denervated target muscle fibers. The foreign motor endings are electrophysiologically functional and are observed on the denervated muscle fibers by the 1st instar larval stage. Our data are consistent with the existence of a local signal from the target environment, which is regulated by innervation and influences synaptic connectivity. Our results show that, despite the stereotypy ofDrosophilaneuromuscular connections, denervation can induce local changes in connectivity in wild-typeDrosophila, suggesting that mechanisms of synaptic plasticity may also be involved in normalDrosophilaneuromuscular development.
Interkinetic and Migratory Behavior of a Cohort of Neocortical Neurons Arising in the Early Embryonic Murine Cerebral Wall
Authors: Takahashi Takao , Nowakowski Richard S. , Caviness Jr. Verne S.
Published: April 2018

Neocortical neuronogenesis occurs in the pseudostratified ventricular epithelium (PVE) where nuclei of proliferative cells undergo interkinetic nuclear movement. A fraction of daughter cells exits the cell cycle as neurons (the quiescent, or Q, fraction), whereas a complementary fraction remains in the cell cycle (the proliferative, or P, fraction). By means of sequential thymidine and bromodeoxyuridine injections in mouse on embryonic day 14, we have monitored the proliferative and postmitotic migratory behaviors of 1 and 2 hr cohorts of PVE cells defined by the injection protocols. Soon after mitosis, the Q fraction partitions into a rapidly exiting (up to 50 μm/hr) subpopulation (Qr) and a more slowly exiting (6 μm/hr) subpopulation (Qs).QrandQsare separated as two distributions on exit from the ventricular zone with an interpeak distance of ∼40 μm. Cells inQrandQsmigrate through the intermediate zone with no significant change in the interpeak distance, suggesting that they migrate at approximately the same velocities. The rate of migration increases with ascent through the intermediate zone (average 2–6.4 μm/hr) slowing only transiently on entry into the developing cortex. Within the cortex,QrandQsmerge to form a single distribution most concentrated over layer V.
p53-Independent Cyclin G Expression in a Group of Mature Neurons and Its Enhanced Expression during Nerve Regeneration
Authors: Morita Naonori , Kiryu Sumiko , Kiyama and Hiroshi
Published: April 2018

An increase in cyclin G expression after nerve injury was demonstrated by differential display PCR, carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the rat. The nerve injury dramatically upregulated the expression of cyclin G mRNA in the motoneurons during the early phase of the nerve regeneration process, suggesting an involvement of cyclin G in the early stage of nerve regeneration. In brain,in situhybridization studies also demonstrated cyclin G expression in a restricted group of matured neurons, particularly in the telencephalon and the thalamus. This constitutive expression in mature neurons suggests that cyclin G may have a function different from other members of the cyclin group. In addition, although cyclin G has been shown to be a transcription target of p53, the upregulation of cyclin G in injured motoneurons, as well as the expression in the adult rat brain, was not affected in the p53-deficient mouse. These data suggest that the expression of cyclin G, at least in the nervous system, is not regulated by p53 predominantly, and that there may be alternative regulatory factors or pathways for cyclin G expression.
Activation of a Metabotropic Excitatory Amino Acid Receptor Potentiates Spike-Driven Calcium Increases in Neurons of the Dorsolateral Septum
Authors: Zheng Fang , Gallagher Joel P. , Connor John A.
Published: April 2018

(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), an agonist for metabotropic glutamate receptors (mGluRs), causes depolarization and burst firing in rat dorsolateral septal nucleus (DLSN) neurons and results in long-term potentiation (LTP) at DLSN synapses. In the present study, we investigated whether these actions of 1S,3R-ACPD are attributable to the release of calcium from an inositol triphosphate-sensitive store after activation of mGluRs coupled to phospholipase C. Our data demonstrated that the ACPD-induced depolarization was associated with a small but significant decrease, not an increase, in [Ca2+]i; however, changes of [Ca2+]iduring ACPD-induced bursting were up to seven times larger than those produced by regular firing. Depletion of internal calcium stores by thapsigargin or ryanodine had a small to insignificant effect on the maximum changes of [Ca2+]iassociated with ACPD-induced bursting. Thus, elevation of [Ca2+]iduring firing by 1S,3R-ACPD is likely attributable to enhancement of calcium influx through voltage-gated channels and not to calcium release from internal stores. ACPD-induced burst firing elevated somatic and dendritic calcium levels up to 3 and 6 μm, respectively. Such an increase may be the underlying mechanism for ACPD-induced LTP as well as ACPD-induced acute cell death in rat DLSN.
A Family of Rat CRMP Genes Is Differentially Expressed in the Nervous System
Authors: Wang Li-Hsien , Strittmatter Stephen M.
Published: April 2018

Members of the collapsin/semaphorin family play an important role in creating the complex pattern of neuronal connectivity. Inhibition of growth cone motility by chick collapsin is mediated by the intraneuronal protein CRMP-62. We have now isolated four rat sequences that are highly related to chick CRMP-62. All four genes are expressed exclusively in the nervous system and primarily during development. Rat CRMP-2/TOAD-64 is most closely related to chick CRMP-62 and is the most widely expressed CRMP within the nervous system. Rat CRMP-1 and CRMP-4/rUlip are expressed during discrete periods of neuronal development and are not found in the adult nervous system. Rat CRMP-3 has a distinct distribution, being expressed transiently in developing spinal cord and selectively in the postnatal cerebellum. The differential expression of these genes suggests that CRMPs may transduce signals from different semaphorins and that semaphorins may regulate the plasticity of the adult nervous system.
TrkA, But Not TrkC, Receptors Are Essential for Survival of Sympathetic NeuronsIn Vivo
Authors: Fagan Anne M. , Zhang Hong , Landis Story , Smeyne Richard J. , Silos-Santiago Inmaculada , Barbacid Mariano
Published: April 2018

Neurotrophins and their signaling receptors, the Trk family of protein tyrosine kinases, play a major role in the development of the mammalian nervous system. To determine the precise stages that require Trk receptor signaling during development of the sympathetic system, we have analyzed the superior cervical ganglion (SCG) of embryonic and postnatal mice defective for each of the known Trk receptors. Transcripts encoding TrkC are detected in early sympathetic development, before the coalescence of the SCG.trkA expression appears at E13.5, becoming robust from E15.5 onward. In contrast,trkC expression decreases significantly after E15.5 and remains detectable only in a small subpopulation of cells. No significanttrkB expression could be detected in the SCG at any developmental stage. Ablation of TrkA receptors does not affect neurogenesis, expression of neuronal markers, or initial axonal growth. However, these receptors are absolutely necessary for the survival of sympathetic neurons after E15.5 and for proper innervation of their distal targets. In contrast, mice defective for either TrkC or TrkB tyrosine kinase receptors do not display detectable defects in their SCGs. These results illustrate the differential roles of the Trk family of receptors during SCG development and define a critical role for TrkA signaling in the survival, but not differentiation, of SCG neurons. Moreover, these observations raise the possibility that at least some SCG neurons become neurotrophin-dependent before complete target innervation.
Specific Induction of Protein Kinase Cδ Subspecies after Transient Middle Cerebral Artery Occlusion in the Rat Brain: Inhibition by MK-801
Authors: Miettinen Susanna , Roivainen Reina , Keinänen Riitta , Hökfelt Tomas , Koistinaho Jari
Published: April 2018

Protein kinase C (PKC) consists of a family of closely related Ca2+/phospholipid-dependent phosphotransferase isozymes, most of which are present in the brain and are differentially activated by second messengers. Calcium-dependent PKC activity may cause neuronal degeneration after ischemic insult. PKC is also involved in trophic-factor signaling, indicating that activity of some PKC subspecies may be beneficial to the injured brain. Therefore, we screened long-term changes in the expression of multiple PKC subspecies after focal brain ischemia. Middle cerebral artery occlusion was produced by using an intraluminal suture for 30 min or 90 min. Inin situhybridization experiments, mRNA levels of PKCα, -β, -γ, -δ, -ε and -ζ were decreased in the infarct core 4 hr after ischemia and were lost completely 12 hr after ischemia. In areas surrounding the core, PKCδ mRNA was specifically induced 4, 12, and 24 hr after ischemia in the cortex. At 3 and 7 d, the core and a rim around it showed increased mRNA levels of PKCδ. No other subspecies were induced. At 2 d, immunoblotting demonstrated increased levels of PKCδ protein in the perifocal tissue, and immunocytochemistry revealed an increased number of PKCδ-positive neurons in the perifocal cortex. In the core, PKCδ-positive macrophages and endothelial cells were seen. Pretreatment with MK-801, an NMDA antagonist, inhibited cortical PKCδ mRNA induction. The data show that focal brain ischemia induces PKCδ mRNA and protein but not other PKC subspecies through the activation of NMDA receptors and that the upregulation lasts for several days in neurons of the perifocal zone.
Optic Flow Processing in Monkey STS: A Theoretical and Experimental Approach
Authors: Lappe Markus , Bremmer Frank , Pekel Martin , Thiele Alexander , Hoffmann Klaus-Peter
Published: April 2018

How does the brain process visual information about self-motion? In monkey cortex, the analysis of visual motion is performed by successive areas specialized in different aspects of motion processing. Whereas neurons in the middle temporal (MT) area are direction-selective for local motion, neurons in the medial superior temporal (MST) area respond to motion patterns. A neural network model attempts to link these properties to the psychophysics of human heading detection from optic flow. It proposes that populations of neurons represent specific directions of heading. We quantitatively compared single-unit recordings in area MST with single-neuron simulations in this model. Predictions were derived from simulations and subsequently tested in recorded neurons. Neuronal activities depended on the position of the singular point in the optic flow. Best responses to opposing motions occurred for opposite locations of the singular point in the visual field. Excitation by one type of motion is paired with inhibition by the opposite motion. Activity maxima often occur for peripheral singular points. The averaged recorded shape of the response modulations is sigmoidal, which is in agreement with model predictions. We also tested whether the activity of the neuronal population in MST can represent the directions of heading in our stimuli. A simple least-mean-square minimization could retrieve the direction of heading from the neuronal activities with a precision of 4.3°. Our results show good agreement between the proposed model and the neuronal responses in area MST and further support the hypothesis that area MST is involved in visual navigation.
δ Subunit Inhibits Neurosteroid Modulation of GABAAReceptors
Authors: Zhu Wei Jian , Wang Jian Feng , Krueger Karl E. , Vicini Stefano
Published: April 2018

Neurosteroid modulation of GABAAreceptors has been observed with all subunit combinations investigated; however, hetero-oligomeric GABAAreceptors containing δ subunits were not studied previously. We describe the effect of δ subunit expression on 3α,21-dihydroxy-5α-pregnan-20-1 (THDOC)-induced potentiation of GABA-gated currents in transfected HEK 293 cells and in cerebellar granule cellsin vitro. THDOC (100 nm) significantly potentiated GABA-gated currents in cells transfected with combinations of α1, α6, β3, and γ2 subunit cDNAs, whereas cotransfection of δ subunit cDNA inhibited this potentiation. In contrast, the direct Cl−channel activation by THDOC at higher concentrations (1–10 μm) was not significantly dependent on δ subunit cotransfection. These results suggest that the presence of the δ subunit inhibits GABAAreceptor modulation but not the direct activation by neurosteroids. Cotransfection with δ subunit also affected the negative allosteric modulation by pregnenolone sulfate. THDOC potentiation of GABA-gated currents was greater in cerebellar granule cell cultures at 4 din vitro(DIV) compared with those at 14 DIV. Single-cell reverse transcription-PCR analysis of the mRNAs expressed in cultured cerebellar granule cells shows that an increased number of granule cells at 14 DIV express δ subunit mRNAs as compared with 4 DIV granule cells. The presence of δ subunit mRNAs detected in individual cells correlated well with the lack of sensitivity to THDOC. These results suggest that developmental expression of GABAAreceptor δ subunits may play an important role in determining the region-specific neurosteroid-induced modification of fast inhibitory synaptic function.