bluuebunny/crossref_metadata_2025 huggingface dataset:
Three distinct axonal transport rates for tau, tubulin, and other microtubule-associated proteins: evidence for dynamic interactions of tau with microtubules in vivo
Authors: Mercken M , Fischer I , Kosik KS , Nixon RA
Published: April 2018

Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50–60 kDa tau isoforms and a second 90–95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S- methionine intravitreously and, after 6–30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2–0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1–0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature.
Connexin32 is a myelin-related protein in the PNS and CNS
Authors: Scherer SS , Deschenes SM , Xu YT , Grinspan JB , Fischbeck KH , Paul DL
Published: April 2018

We have examined the expression of a gap junction protein, connexin32 (Cx32), in Schwann cells and oligodendrocytes. In peripheral nerve, Cx32 is found in the paranodal myelin loops and Schmidt-Lanterman incisures of myelinating Schwann cells, and the levels of Cx32 protein and mRNA change in parallel with those of other myelin-related genes during development, Wallerian degeneration, and axonal regeneration. In the central nervous system, Cx32 is found in oligodendrocytes and their processes, but not in compact myelin, and the levels of Cx32 protein and mRNA increase during development in parallel with those of the other myelin genes. Thus, Cx32 is expressed as part of the myelinating phenotype of both Schwann cells and oligodendrocytes, indicating that this gap junction protein plays in important role in the biology of myelin-forming cells.
NCAM-associated polysialic acid on ciliary ganglion neurons is regulated by polysialytransferase levels and interaction with muscle
Authors: Bruses JL , Oka S , Rutishauser U
Published: April 2018

NCAM in its high polysialic acid (PSA) form is expressed on chick hindlimb motoneurons during their growth, and then decreases at about the time that synaptogenesis is completed. In order to characterize this regulation at the cell and molecular level, the present studies use the chick ciliary ganglion (CG) system, which constitutes a homogeneous and developmentally synchronized population of motoneurons that can be used for in vitro studies. Levels of PSA in the CG were evaluated both by SDS-PAGE immunoblot analysis of total NCAM and by pulse radiolabeling of newly synthesized NCAM. Up- and downregulation of PSA expression on newly synthesized NCAM in the CG was found to be closely correlated with in vivo innervation and synaptogenesis, respectively. Moreover, the downregulation observed at synaptogenesis was prevented by in vivo blockade of neuromuscular activity with alpha- bungarotoxin. The developmental regulation of PSA expression was found to coincide precisely with an increase and decrease in levels of specific polysialyltransferase activity. By contrast, the expression of the VASE exon in NCAM, which in CNS is temporally correlated with PSA downregulation, was not expressed in the CG. Cocultures of CG neurons with myotubes were used to provide direct evidence that neuron-muscle interaction can cause a specific downregulation of both neuronal PSA and polysialyltransferase activity.
G protein-coupled receptors mediate a fast excitatory postsynaptic current in CA3 pyramidal neurons in hippocampal slices
Authors: Miller LD , Petrozzino JJ , Connor JA
Published: April 2018

Synaptic activation in the presence of competitive (D,L-APV,CNQX) and noncompetitive (MK-801,GYKI-52466) ionotropic glutamate receptor antagonists induced fast (10–90% rise time of 15–30 msec) postsynaptic responses in CA3 pyramidal neurons from acute and cultured hippocampal slices. Postsynaptic currents were studied extensively in slice cultures, and displayed a linear current-voltage relationship, with a reversal potential between 0 mV and +10 mV, suggesting the activation of a nonselective cationic conductance. Inhibition of the GTPase cycle by intracellular perfusion with the nonhydrolyzable analog of GDP, GDP beta S, blocked the fast postsynaptic responses evoked in ionotropic antagonists, as well as baclofen-mediated outward K+ currents, known to be mediated by G protein-coupled GABAB receptors. Intracellular perfusion with GDP beta S did not affect the AMPA/kainate component of the synaptic currents. Irreversible activation of G proteins by intracellular perfusion with the nonhydrolyzable analog of GTP, GMP- PNP, occluded the baclofen responses, and evoked an inward current, consistent with the synaptically mediated conductance. Incubation of the slice cultures in pertussis toxin for 72 hr blocked baclofen- induced outward K+ currents, while the fast postsynaptic currents remained. The metabotropic glutamate receptor (mGluR) agonists 1S,3R- ACPD and 1S,3S-ACPD induced an inward current in the presence of the ionotropic antagonists, and occluded the fast EPSCs. The fast EPSCs were partially blocked by the mGluR antagonists L-AP3 and (+)MCPG, but there was differential antagonists sensitivity in two pathways stimulated (CA3 stratum radiatum vs CA3 stratum oriens). These data suggest that fast postsynaptic responses evoked in the presence of ionotropic glutamate receptor antagonists are mediated by G protein- coupled mGluRs linked to nonselective cationic channels.
Neurofilaments help maintain normal morphologies and support elongation of neurites in Xenopus laevis cultured embryonic spinal cord neurons
Authors: Lin W , Szaro BG
Published: April 2018

Neurofilament number and subunit composition, which are highly regulated during development, have been proposed to help regulate axonal diameter and stability. From experiments on dissociated cell cultures of Xenopus laevis embryonic spinal cord, we have obtained direct evidence that neurofilaments help maintain the structural integrity of newly developing axons. An anti-neurofilament monoclonal antibody specific for Xenopus NF-M and the cell lineage tracer, lysinated FITC-dextran, were coinjected into a single blastomere of 2- cell stage embryos. Within neurons descended from the injected blastomere, this antibody specifically confined neurofilaments to the cell body for the first two days of culture, as assayed by immunocytochemical staining with antiserum against the low molecular weight neurofilament protein XNIF. Although whole IgGs and Fab fragments both affected neurofilament distribution, the whole IgGs were more effective. For the first 9 hr of culture, neurites containing anti- NF-M developed normally. By 21 hr, they were shorter than those of sibling control neurons within the same dish, and many became morphologically abnormal. Defects included large variations in diameter, poorly defined separations between the growth cone and neurite, and more collateral branching. Despite these abnormal features, neurons containing anti-NF-M had normal distributions of alpha-tubulin immunoreactivity and phalloidin-stained F-actin. These latter observations argued that defects resulted from the absence of neurofilaments rather than from interference of the movement of other structural materials essential for axonal growth. These results support the hypothesis that neurons use neurofilaments to help maintain the characteristic shapes of axons against the increasing structural demands placed upon the elongating process.
Differential cholinergic regulation of dopamine release in the dorsal and ventral neostriatum of the rat: an in vivo microdialysis study
Authors: O'Connor WT , Drew KL , Ungerstedt U
Published: April 2018

We used in vivo microdialysis to investigate the effects of local perfusion with the AChE inhibitor neostigmine on the basal and haloperidol evoked increase in dialysate dopamine levels in the dorsolateral and fundus striata of the bilaterally implanted halothane anaesthetized rat. In the absence of neostigmine basal dopamine was consistently higher in the dorsolateral striatum compared with the fundus striati. Local perfusion with neostigmine (10 and 100 microM) increased basal dopamine in the fundus striati compared to the contralateral (control) side but not in the dorsolateral striatum. In the absence of neostigmine haloperidol (0.05–0.5 mg/kg, s.c.) increased dopamine release in both the dorsolateral and fundus striata. However, local perfusion with neostigmine (10 microM) attenuated this increase in the dorsolateral striatum at all doses of haloperidol while only the effect of the highest (0.5 mg/kg) dose of haloperidol was counteracted in the fundus striati. Both the basal and haloperidol (0.25 mg/kg) induced increase in dopamine release in the control (no neostigmine) and neostigmine (+10 microM) treated dorsolateral striata were abolished following local perfusion with tetrodotoxin (1 microM). The data demonstrate that the introduction of neostigmine into the neostriatum selectively increases basal DNA levels in the fundus striati and strongly counteracts the haloperidol evoked DA release in the dorsolateral striatum and thus provide strong evidence for a differential cholinergic regulation of striatal DA release in vivo. In addition, we demonstrate that the stimulatory and inhibitory effects of neostigmine operate independently and have a regional specificity within the neostriatum.
Targeted neocortical cell death in adult mice guides migration and differentiation of transplanted embryonic neurons
Authors: Sheen VL , Macklis JD
Published: April 2018

Local expression of cellular and molecular signals is required for normal neuronal migration and differentiation during neocortical development and during periods of plasticity in the adult brain. We have previously shown that neonatal and juvenile mice that induction of apoptotic degeneration in neocortical pyramidal neurons by targeted photolysis provides an altered environment that directs migration and differentiation of transplanted embryonic neurons. Here we employ the same paradigm in adult mice to test whether targeted photolysis induces the reexpression in the mature brain of developmental signals that control migration, differentiation and integration of embryonic neurons. We examined both the time course of migration and the morphologic and immunocytochemical differentiation of embryonic neurons transplanted into regions of targeted photolytic cell death. Pyramidal neurons in neocortical lamina II/III underwent photolytically induced apoptosis after retrograde incorporation of the photoactive chromophore chlorine e6 and transdural exposure to 674 nm near-infrared laser energy. Embryonic day 17 neocortical neurons were prelabeled with fluorescent nanospheres and the lipophilic dye PKH26, transplanted into regions of ongoing neuronal degeneration in adult mice, and examined histologically and immunocytochemically. Transplanted neurons began migration into regions of neuronal death within 3 d and differentiated into large pyramidal neurons similar to those degenerating. In contrast, neurons transplanted into intact cortex did not migrate, and they differentiate into small presumptive interneurons. Migration up to 430 microM in experimental mice was complete by 2 weeks; approximately 45% of the donor neurons migrated greater than 3 SDs beyond the mean for neurons transplanted into intact neocortex of age-matched adult hosts. Following migration, dendrites and axons of many donor neurons were properly oriented toward the pial surface and corpus callosum, indicating integration into the host parenchyma. Neurofilament and neuron-specific enolase staining further support appropriate differentiation and integration. These results indicate that signals guiding neuronal migration and differentiation in neocortex are reexpressed in adult mice well beyond the period of corticogenesis within regions of targeted photolytic cell death. Elucidating the molecular mechanisms underlying these events by comparison with adjacent unperturbed regions will contribute to efforts toward future therapeutic transplantation and control over endogenous plasticity.
mRNA expression of KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, KIF5, and cytoplasmic dynein during axonal regeneration
Authors: Takemura R , Nakata T , Okada Y , Yamazaki H , Zhang Z , Hirokawa N
Published: April 2018

Mouse brain expresses multiple kinesin superfamily proteins (KIFs), which are involved in vesicle transport. The expression of KIFs is developmentally regulated, and both the mRNA and proteins of KIF2 and KIF4 are expressed abundantly in the juvenile brain. To elucidate the role of individual kinesin superfamily motor proteins during regenerative outgrowth of axons, we examined the mRNA expression of KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 in adult mouse dorsal root ganglion cells after sciatic nerve crush. Seven to fourteen days after the nerve crush, the mRNA expression pattern of neurofilament and beta-tubulin isotypes suggested that the regenerative outgrowth of axons was active. At these stages, levels of mRNA for KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 were 50.80% of control. The levels of mRNA for KIF4, which are detected in juvenile brain but not in the adult, were under the detection limit in both control and regenerating dorsal root ganglion cells. Because mRNA of neither KIF2 nor KIF4 increased significantly, the results suggest that the gene expression of KIFs during regeneration does not recapitulate the embryonic development and support the hypothesis that different series of events take place during the regenerative and embryonic outgrowths of axons. In contrast, mRNA for cytoplasmic dynein was slightly increased, up to 140%. This is consistent with the hypothesis that retrograde transport plays critical roles in regeneration such as the transport of neurotrophic factors.
Components of glial responses to exogenous and synaptic glutamate in rat hippocampal microcultures
Authors: Mennerick S , Benz A , Zorumski CF
Published: April 2018

Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling.
Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons
Authors: Rohrbough J , Spitzer NC
Published: April 2018

Rohon-Beard (RB) spinal neurons of Xenopus larvae are depolarized by GABA. To study the mechanisms underlying this distinctive response, intracellular and patch-clamp recordings were made from RB neurons in situ. The intracellularly recorded GABA reversal potential (EREV) was near -30 mV in normal saline and was approximately 25 mV more negative in Na(+)-free saline. Whole-cell recordings from RB neurons and from neighboring dorsolateral interneurons (DLi) revealed that GABA responses of both cells were mediated by GABAA receptors. Currents elicited by GABA were mimicked by muscimol and reversibly blocked by bicuculline, and EREV shifted with changes in Cl- concentration ([Cl]) in agreement with Cl- selectivity. In perforated patch recordings, EREV for RB cells was significantly more positive than for DLi cells (-38 vs -63 mV), indicating that intact RB cells maintain higher levels of intracellular Cl-. Replacement of external Na+ or exposure to the Cl- transport inhibitor bumetanide (100 microM) shifted RB cell EREV to move negative values, consistent with Na+(-)dependent Cl cotransport contributing to higher internal [Cl]. In contrast, these treatments did not change DLi cell EREV. The results indicate that a Na+(-)dependent Cl- transport mechanism underlies GABAA receptor-mediated depolarizing Cl- conductances in RB neurons. Thus, both inhibitory and excitatory GABA responses appear to be present during the same developmental period in vivo. GABA may stimulate Ca2+ influx in RB neurons because the intracellular GABA EREV is above the threshold for low voltage- activated Ca2+ channels.
Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons
Authors: von Gersdorff H , Matthews G
Published: April 2018

Giant synaptic terminals (approximately 10 micrometer diameter) of bipolar neurons from goldfish retina were used to directly investigate calcium-dependent inactivation of presynaptic calcium current. During sustained depolarization, calcium current was initially constant for a period lasting up to several hundred milliseconds and then it declined exponentially. The duration of the initial delay was shorter and the rate of inactivation was faster with larger calcium current. The fastest time constant of inactivation (in the range of 2–5 sec) was observed under weak calcium buffering conditions. Inactivation was attenuated when external Ca2+ was replaced with Ba2+ and when terminals were dialyzed with high concentrations of internal BAPTA. Elevation of intracellular calcium concentration ([Ca2+]i) by application of the calcium ionophore ionomycin or by dialysis with pipette solutions containing buffered elevated [Ca2+] produced inactivation of calcium current. The rate of recovery from inactivation was not determined by the recovery of [Ca2+]i to baseline after a stimulus. The results demonstrate that the presynaptic calcium current in bipolar neurons is inactivated by elevated [Ca2+]i, but the inactivation is approximately 100-fold slower than previously described calcium-dependent inactivation in other types of cells.
Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H1 receptors and cyclic nucleotides
Authors: Hatton GI , Yang QZ
Published: April 2018

Activating direct olfactory (glutamatergic) inputs to supraoptic nucleus (SON) neurons increases interneuronal coupling in slices from lactating but from not virgin or male rats. Studied here were influences on coupling of another monosynaptic input to SON, the histaminergic tuberomammillary nucleus (TM) projection, activation of which selectively excites phasically firing (putative vasopressin) cells. Effects of TM stimulation and its possible downstream consequences on Lucifer yellow (LY) dye coupling among putative vasopressin cells were determined in male rat SONs. In unstimulated slices, 12 LY injections (1 cell/SON) yielded eight single and four pairs of coupled neurons. In slices in which TM was stimulated for 10 min at 10 Hz, 13 injections yielded 4 single and 28 coupled cells, with groups of 2 to 4 cells coupled to the injected neuron, a threefold increase in the number of coupled cells per injection (p < 0.02). Bathing slices in medium containing 10 microM pyrilamine (H1 antagonist) blocked this stimulation-induced coupling increase, suggesting mediation by activation of guanylate cyclase-cGMP to which H1 receptors often are linked . Bathing slices in medium containing 0.5– 1 mM 8-bromo-cGMP yielded results similar to those of TM stimulation, a 2.5-fold increase over control in the number of coupled cells per injection. Effects of TM stimulation on coupling also were blocked by bathing slices in a guanylate cyclase inhibitor (10 microM LY83583). In contrast to cGMP, 1 mM 8-bromo-cAMP significantly reduced coupling. We conclude that synaptically released histamine increases coupling via cGMP-dependent mechanisms.
Rise in intracellular calcium via a nongenomic effect of allopregnanolone in fetal rat hypothalamic neurons
Authors: Dayanithi G , Tapia-Arancibia L
Published: April 2018

This study examines the early effects of 3 alpha-hydroxy-5 alpha- pregnan-20-one (allopregnanolone on cytosolic free calcium concentration ([Ca2+]i in primary cultures of fetal rat hypothalamic neurons. Microspectrofluorimetry of fluorescent Ca2+(-)sensitive indicator Fura-2 was used to quantify these changes. Allopregnanolone (1 pM to 100 nM) increased [Ca2+]i within 2–3 sec, in a dose dependent manner, with an EC50 of 10 +/- 4 nM. The stimulatory effect of allopregnanolone was attributable principally to a Ca2+ influx, as shown by the strong inhibition of external Ca2+ removal or by the calcium channel blocker nifedipine. The effect was stereospecific because the allopregnanolone isomer 3 beta-hydroxy-5 alpha-pregnan-20- one had no effect on [Ca2+]i. Among two other steroids examined, progesterone had no effect on [Ca2+]i, but 17 beta-estradiol evoked a rise in [Ca2+]i, although to a lesser extent than allopregnanolone. The allopregnanolone-induced [Ca2+]i rise was inhibited by picrotoxin and bicuculline but was unaffected by tetrodotoxin or by pretreatment of neurons with pertussis toxin. These results are consistent with a membrane site of action for allopregnanolone associated with GABAA receptors, leading to rapid changes in [Ca2+]i in fetal rat hypothalamic neurons.
Localization of L-type Ca2+ channels at perisynaptic glial cells of the frog neuromuscular junction
Authors: Robitaille R , Bourque MJ , Vandaele S
Published: April 2018

The presence of L-type Ca2+ channels at the frog neuromuscular junction (nmj) was studied by monitoring changes in intracellular Ca2+ evoked in presynaptic terminals and perisynaptic Schwann cells (PSCs) and by studying the distribution of Ca2+ channels using a monoclonal antibody directed against the alpha 2/delta subunit of L channels. L-type Ca2+ channel agonist and antagonist had no effect on resting level of fluorescence and nerve-evoked Ca2+ responses in presynaptic terminals. However, depolarization of PSCs induced by KCl (25 mM) produced entry of Ca2+, which was prevented by L-type Ca2+ channel blockers, in (+)R Bay K 8644 of nimodipine. Labeling of Ca2+ channels revealed an intracellular epitope with an irregular and spotty distribution along the endplate. Similar results were obtained with a fluorescent phenylalkylamine [(-)DM-BODIPY-PAA], a blocker of L-type Ca2+ channels. Ca2+ channel labeling remained in absence of nerve terminals but was absent after mechanical removal of nerve terminals and PSCs. Most Ca2+ channel spots were distributed in between bands of cholinergic receptors labeled with alpha-bungarotoxin-TRITC. Cross sections of motor endplates revealed that labeling of Ca2+ channels was found only at the level of the synaptic cleft and not all around the PSCs. We conclude that L-type Ca2+ channels are located in perisynaptic glial cells in an appropriate location to sense depolarization induced by neurotransmitters and thus may support possible roles of glial cells on synaptic function.
Properties of the endosomal-lysosomal system in the human central nervous system: disturbances mark most neurons in populations at risk to degenerate in Alzheimer's disease
Authors: Cataldo AM , Hamilton DJ , Barnett JL , Paskevich PA , Nixon RA
Published: April 2018

Specific antibodies and cytochemical markers combined with several imaging and morphometric techniques were used to characterize the endosomal-lysosomal system in mature neurons of the normal human central nervous system and to quantitate changes in its function in Alzheimer's disease. Compartments containing cathespin D (Cat D) and other acid hydrolases included a major subpopulation of mature lysosomes lacking mannose-6-phosphate receptors (MPR) and smaller populations of late endosomes (MPR-positive) and lipofuscin granules (MPR-negative). Antibodies to the pro-isoform of Cat D decorated perinuclear vacuolar compartments corresponding to late endosomes. Neurons and glia contained lysosomes with differing complements of acid hydrolases, implying different processing capabilities. Endosome/lysosome number per unit volume of cytoplasm was relatively well conserved within populations of normal neurons. By contrast, in morphometric analyses of Alzheimer's disease brains, 80–93% of pyramidal cells in the prefrontal cortex (laminae III or V) and hippocampus (CA2, CA3) displayed two- to eightfold higher numbers of hydrolase-positive vacuolar compartments than did corresponding cell populations in age-matched normal brains. Only 5–10% of cerebellar Purkinje cells, a less vulnerable population, showed the same statistically significant elevations. Most affected in these brain regions and in subcortical areas seemed otherwise normal by conventional histological staining and ultrastructural inspection. That both lysosomal and pro-Cat D- and MPR-positive endosomal compartments increased in number demonstrates that the endosomal-lysosomal system is activated markedly in vulnerable neuronal populations of Alzheimer's disease brains and implies that endocytosis or autophagy or both are accelerated persistently at an early stage of cellular compromise, greatly surpassing the degree of activity associated with normal aging. Early activation of the endosomal-lysosomal system represents a biological event potentially linking major etiological factors in Alzheimer's disease, including defective membrane proteins, apolipoprotein E function, and altered amyloid precursor protein processing.
AMPA receptor activation is rapidly toxic to cortical astrocytes when desensitization is blocked
Authors: David JC , Yamada KA , Bagwe MR , Goldberg MP
Published: April 2018

Although cultured astrocytes express functional glutamate receptors, they are generally resistant to excitotoxic cell death. We explored the role of receptor desensitization in glutamate-mediated astrocyte injury. In cultures of type 1 astrocytes from mouse neocortex, brief application of AMPA evoked small, rapidly desensitizing inward currents, whereas kainate evoked small, sustained currents. Neither agonist increased cytosolic calcium, and astrocyte toxicity occurred only after 24 hr exposure to high (500–1000 microM) concentrations of kainate but not to AMPA or glutamate. Cyclothiazide, a drug that selectively blocks AMPA receptor desensitization, greatly potentiated AMPA- or kainate-gated currents and intracellular calcium elevation. Coapplication of 10–100 microM cyclothiazide with glutamate, AMPA, or kainate produced widespread astrocyte cell death within 2 hr or application. The enhancement of toxicity by cyclothiazide, which alone was not toxic, was concentration-dependent for each of the tested agonists (EC50 30–100 microM) and was blocked by further addition of the selective AMPA/kainate antagonist 2,3-dioxo-6-nitro-7- sulfamoylbenzo(f)quinoxaline (NBQX). NMDA caused no injury even in the presence of cyclothiazide. Cyclothiazide-enhanced injury varied with the age of astrocyte cultures; the maximal effect occurred at approximately 2 weeks in vitro, and little death was seen after 4 weeks. Type 1 astrocytes express AMPA-type glutamate receptors that are unmasked by reducing their desensitization with cyclothiazide. Although overactivation of AMPA receptors can be rapidly lethal to astrocytes, rapid desensitization normally limits this toxicity. The extent of AMPA receptor desensitization may be an important determinant of glial vulnerability to excitotoxic insults.
Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study
Authors: Bohlhalter S , Weinmann O , Mohler H , Fritschy JM
Published: April 2018

To assess the significance of GABAA-receptor heterogeneity, which is based on a family of at least 15 subunits, the cellular localization and subunit composition of GABAA-receptor subtypes were analyzed immunohistochemically in the rat spinal cord. The distribution of subunits alpha 1, alpha 2, alpha 3, alpha 5, beta 2,3, and gamma 2 was investigated with subunit-specific antibodies, and their colocalization within individual neurons was visualized by double-immunofluorescence staining. The results reveal a widespread expression of the subunits, alpha 3, beta 2,3, and gamma 2 in the spinal cord, whereas the three other alpha subunits displayed a more restricted, lamina-specific distribution. The alpha 1 and alpha 5 subunits were most abundant in the intermediate zone, whereas the alpha 2 subunit was predominant in the superficial layers of the dorsal horn and in somatic and preganglionic motoneurons. From colocalization studies, seven subunit combinations could be identified (alpha 3/beta 2,3/gamma 2; alpha 2/beta 2,3/gamma 2; alpha 1/beta 2,3/gamma 2; alpha 5/beta 2,3/gamma 2; alpha 1/alpha 5/beta 2,3/gamma 2; alpha 2/gamma 2; alpha 2/alpha 5/gamma 2) that correspond presumably to distinct receptor subtypes. Although most neurons expressed the subunit triplet alpha x/beta 2,3/gamma 2, the beta 2,3 subunits could not be detected in motoneurons that may thus possess “atypical” receptor subtypes (alpha 2/gamma 2 and alpha 2/alpha 5/gamma 2). ON the subcellular level, aggregates of immunoreactivity, suggestive of postsynaptic GABAA receptors, typically were seen on the surface of neuronal somata and proximal dendrites. In addition, an intense diffuse staining was observed in laminae I--III for the subunits alpha 2, alpha 3, beta 2,3, and gamma 2, presumably localized on primary afferent terminals. The localization of GABAA- receptor subtypes in distinct laminar compartments of the spinal cord suggests that GABAA-receptor heterogeneity is of relevance for the modulation of sensory inputs, nociception, and motor control at segmental levels.
The synaptic drive from the spinal locomotor network to motoneurons in the newborn rat
Authors: Cazalets JR , Borde M , Clarac F
Published: April 2018

The nature of the synaptic drive from the locomotor spinal network onto the motoneurons was studied in the newborn rat. For this purpose, an in vitro isolated spinal cord preparation of newborn rat was used. The recording chamber was partitioned with Vaseline walls to separate the L1/L2 lumbar segments, in which the spinal locomotor network is located, from the motoneurons in the lower lumbar segments. Locomoter- like activity was induced by bath-applying a mixture of serotonin and NMDA to segments L1/L2. In this way, the synaptic activity could be modified at the lower lumbar level without affecting the motor pattern. The drive elicited onto the motoneurons during sequences of locomoter- like activity, which was monitored by performing intracellular recordings, consisting of an inhibitory component followed by an excitatory component. The inhibitory synaptic volley was reversed at a membrane potential of --60 mV with K acetate electrodes, whereas it was shifted toward positive values with KCl electrodes. The glycinergic blocker strychnine, bath-applied to segments L3/L5, blocked the inhibitory drive without affecting the rhythmic activity, whereas it disrupted the locomoter-like activity when bath-applied to segments L1/L2. The inhibitory part of the drive was more sensitive than the excitatory part to changes in the membrane potential. The excitatory phase was mixed and consisted of an NMDA and a non-NMDA component, which were sensitive to 2-amino-5-phosphonovaleric acid and 6-cyano-7- nitroquinoxaline-2,3-dione, respectively. It was concluded that the locomotor network located in segments L1/L2 sends a biphasic projection to the various groups of motoneurons located along the lumbar spinal cord.
A dendritic GABAA-mediated IPSP regulates facilitation of NMDA-mediated responses to burst stimulation of afferent fibers in piriform cortex
Authors: Kanter ED , Kapur A , Haberly LB
Published: April 2018

Studies in a number of cortical systems have shown that the NMDA component of the EPSP is strongly regulated by GABAA-mediated inhibition. The present study explored the possibility that specificity in inhibitory circuitry could allow such regulation to occur during normal function without increasing the propensity for epileptiform bursting, which occurs with indiscriminate GABAA blockade. Specifically, the hypothesis was tested that a dendritic GABAA-mediated IPSP is present which strongly modulates the NMDA component and can be activated independently of the somatic IPSP. The experiments were performed on slices of piriform cortex in which the NMDA component of the EPSP was pharmacologically isolated by bath-applied 6,7- dinitroquinoxaline-2,3-dione. A facilitation of NMDA responses to burst stimulation of afferent fibers is described, which required GABAA blockade and served as an assay for the presence of a functionally significant GABAA input. When bicuculline was applied focally in the somatic region, the feedback IPSP was blocked with little or no increase in the NMDA component of the response to burst stimulation of afferent fibers. In contrast, when bicuculline was applied focally in the dendritic region, the NMDA-mediated response to burst stimulation was facilitated with minimal effect on the somatic IPSP, confirming the hypothesis.
Amyloidogenic processing of the human amyloid precursor protein in primary cultures of rat hippocampal neurons
Authors: Simons M , de Strooper B , Multhaup G , Tienari PJ , Dotti CG , Beyreuther K
Published: April 2018

The aim of this study was to investigate the proteolytic processing of the amyloid precursor protein (APP) in polarized primary cultures of hippocampal neurons. We have used the Semliki Forest virus (SFV) vector to express human APP695 in hippocampal neurons, sympathetic ganglia, and glial cells. The latter two cells secrete little or no APP, whereas hippocampal neurons secrete two forms of APP695, which differ in sialic acid content and in their kinetic appearance in the culture medium. In addition, rat hippocampal neurons expressing human APP produced significant amounts of the 4 kDa peptide beta A4. After 3 hr of metabolic labeling, the relative amount of beta A4 peptide to total cellular APP was 5.3%. Fibroblasts expressing APP695 using the same SFV vector mainly produced a related 3 kDa p3 peptide, a nonamyloidogenic fragment. Remarkably, the hippocampal neurons also produced significant amounts of beta A4-containing C-terminal fragments (10–12 kDa) intracellularly. Radiosequencing showed that these fragments were created at a previously described beta-secretase cleavage site and at a cleavage site 12 residues from the N terminus of the beta A4 domain (Thr584 of APP695), which we named delta-cleavage. Based on the observation that mature hippocampal neurons produce two potentially amyloidogenic fragments and secrete substantial amounts of beta A4 when expressing human APP, our results strengthen the hypothesis that neurons play a central role in the process of beta A4 deposition in cases of Alzheimer's disease and in aged primates.
Proneuropeptide Y processing in large dense-core vesicles: manipulation of prohormone convertase expression in sympathetic neurons using adenoviruses
Authors: Paquet L , Massie B , Mains RE
Published: April 2018

The efficient delivery of foreign genes into postmitotic cells is becoming very important for studies of nervous system functions. Cultured sympathetic neurons synthesize neuropeptide Y (NPY) in addition to catecholamines, providing an experimental model for studying neuronal peptide biosynthesis. In this work, we have studied the biosynthetic processing of NPY in primary cultures of rat superior cervical ganglion (SCG) neurons. NPY activation is complex, requiring sequential actions of a prohormone convertase (PC), carboxypeptidase H, and peptidylglycine alpha-amidating mono-oxygenase. Northern analyses established that SCG neurons in the animal contain mRNAs for both PC1 and PC2, and simultaneous immunocytochemistry for NPY and PC1 or PC2 established a 1:1 correspondence between NPY and PC2 expression in two thirds of the neurons that express NPY, both in the animal and in tissue culture. Biosynthetic studies on proneuropeptide Y (pro-NPY) processing to mature NPY established a close similarity to the rates seen in endocrine cells expressing PC2 and established clear differences between the patterns in SCG neurons and in endocrine cells expressing PC1. Recombinant adenoviruses were used to increase the level of PC1 in the cultured neurons from negligible to a level comparable with the level of PC1 in the anterior pituitary, and pro-NPY processing was markedly accelerated. When the viruses were used to lower the endogenous PC2 levels, using an antisense construct, pro-NPY processing was retarded. Taken together, these results support a major role for PC2 as the pro-NPY converting enzyme, and they establish the cultured SCG neurons as a model to study neuronal peptide biosynthesis.
The possible role of hydrogen sulfide as an endogenous neuromodulator
Authors: Abe K , Kimura H
Published: April 2018

Hydrogen sulfide (H2S), which is well known as a toxic gas, is produced endogenously from L-cysteine in mammalian tissues. H2S is present at relatively high levels in the brain, suggesting that it has a physiological function. Two other gases, nitric oxide and carbon monoxide, are also endogenously produced and have been proposed as neuronal messengers in the brain. In this work we show the following: (1) an H2S-producing enzyme, cystathionine beta-synthase (CBS), is highly expressed in the hippocampus; (2) CBS inhibitors hydroxylamine and amino-oxyacetate suppress the production of brain H2S; and (3) a CBS activator, S-adenosyl-L-methionine, enhances H2S production, indicating that CBS contributes to the production of endogenous H2S. We also show that physiological concentrations of H2S selectively enhance NMDA receptor-mediated responses and facilitate the induction of hippocampal long-term potentiation. These observations suggest that endogenous H2S functions as a neuromodulator in the brain.
Multiple voltage-dependent mechanisms potentiate calcium channel activity in hippocampal neurons
Authors: Kavalali ET , Plummer MR
Published: April 2018

Neuronal voltage-gated calcium channels provide a pathway for calcium influx that is required for processes ranging from intracellular signaling to alterations in cellular excitability. In hippocampal neurons, we have characterized a subtype of dihydropyridine-sensitive L- type calcium channels (Lp channel) that shows multiple kinds of voltage- dependent potentiation of its activity. One type of potentiation is elicited by low-voltage stimuli (-10 mV) and can be seen in dual-pulse protocols in which a transient hyperpolarization is interposed between conditioning and test pulses. The second type of potentiation is elicited by much higher voltages (+60 mV) and is selectively deactivated at hyperpolarized voltages. We have compared these types of potentiation in the Lp channel, the “standard” L-type channel, and the cardiac L-type channel. Our results show that the high-voltage potentiation is common to all three channel types. The low-voltage form of potentiation, however, is unique to the Lp channel. Thus, the Lp channel shows two kinds of potentiation that differ in their voltage dependence and rate of decay. Therefore, calcium channel plasticity in the hippocampus has a variety of forms distinguished by their stimulus requirements and duration.
Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient
Authors: Zheng JQ , Wan JJ , Poo MM
Published: April 2018

Pathfinding of growing neurites depends on turning of the growth cone in response to extracellular cues. Motile filopodia of the growth cone are known to be critical for mediating contact-dependent guidance of the growth cone. However, whether filopodia also play an essential role in growth cone turning response induced by a diffusible chemotropic substance is unclear. Growth cones of cultured Xenopus spinal neurons exhibited chemotropic turning responses in a gradient of glutamate within a limited range of concentrations. This turning response depends on the activation of the NMDA subtype of glutamate receptors and requires the presence of extracellular Ca2+. Time-lapse differential interference contrast microscopy with quantitative analysis of filopodia dynamics showed a close correlation between an increased number of filopodia on the side of the growth cone facing the glutamate source and the turning. Such filopodia asymmetry was observed within minutes after the onset of the glutamate gradient, before any detectable turning of the growth cone. In Ca(2+)-free medium, no filopodia asymmetry was induced by the glutamate gradient, and no growth cone turning was observed. Furthermore, elimination of filopodia with a low concentration of cytochalasin B completely abolished the turning response without substantially affecting neurite extension. Thus, filopodia may be required for chemotropic guidance of the growth cone, and an asymmetry in filopodia distribution may be an early cellular event responsible for determining the direction the growth cone advances.
Specificity in the axonal connections of layer VI neurons in tree shrew striate cortex: evidence for distinct granular and supragranular systems
Authors: Usrey WM , Fitzpatrick D
Published: April 2018

Pyramidal neurons in layer VI of striate cortex are the source of descending projections to the lateral geniculate nucleus (LGN) as well as a major source of axon terminals in the layers of striate cortex that receive LGN projections. This study examines how the connections of layer VI neurons are arranged with respect to the functionally distinct classes of neurons that compose their cortical and subcortical targets. By placing injections of biocytin into layer VI of tree shrew striate cortex, we identified two sublayers that differ in their intracortical and thalamic connections. Neurons in the upper part of layer VI, layer VIa, terminate in cortical layer IV, whereas those in the lower part of layer VI, layer VIb, terminate throughout the supragranular layers, layers I-III. The selectivity of layer VI subdivisions for the granular and supragranular layers is also evident in their descending projections. Neurons in layer VIa terminate preferentially in the LGN layers that supply layer IV (LGN layers 1, 2, 4, and 5), whereas neurons in layer VIb terminate in the LGN layers that supply layers I-III (LGN layers 3 and 6) and in the pulvinar nucleus. Additional subclasses of layer VIa neurons were identified based on the restriction of their terminal fields to narrow subtiers within layer IV. By influencing the activity of distinct populations of cortical neurons and the thalamic neurons that supply them, layer VI neurons could exert a powerful influence on the flow of activity in functionally distinct cortical circuits.
Cocaine alters cerebral metabolism within the ventral striatum and limbic cortex of monkeys
Authors: Lyons D , Friedman DP , Nader MA , Porrino LJ
Published: April 2018

The functional consequences of acute cocaine administration in nonhuman primates were assessed using the quantitative 2-[14C]deoxyglucose method. Local rates of cerebral metabolism were determined after an intravenous infusion of 1.0 mg/kg cocaine or vehicle in six awake cynomolgus monkeys (Macaca fascicularis) trained to sit calmly in a primate chair. Cocaine administration decreased glucose utilization in a discrete set of structures that included both cortical and subcortical portions of the limbic system. Glucose metabolism in the core and shell of the nucleus accumbens was decreased markedly, and smaller decrements were observed in the caudate and anterior putamen. In addition, cocaine administration produced significant decreases in limbic cortex. Metabolism was decreased in orbitofrontal cortex (areas 11, 12o, 13, 13a, 13b), portions of the gyrus rectus including area 25, entorhinal cortex, and parts of the hippocampal formation. The cortical regions in which functional activity was altered provide dense projections to the nucleus accumbens, and the decreased activity in these projections may be responsible in part of the large alterations in functional activity within the ventral striatum. Decreased metabolism also was evident in the anterior nuclear group of the thalamus, raphe nuclei, and locus ceruleus. The acute cerebral metabolic effects of cocaine in the conscious macaque, therefore, were contained primarily within a set of interconnected limbic regions, including ventral prefrontal cortex, medial temporal regions, the ventral striatal complex, and anterior thalamus. The decreased rates of glucose metabolism reported here resemble decrements found using positron emission tomography in humans. In the rat, by contrast, metabolic activity increased and changes were focused in subcortical regions. The present results represent an important expansion of the neural circuitry on which cocaine acts in the monkey as compared with the rat, and this in turn implies that cocaine affects a broader spectra of behaviors in primates than in rodents.
Antagonists of cyclic nucleotide-gated channels and molecular mapping of their site of action
Authors: Kramer RH , Tibbs GR
Published: April 2018

Activation of photoreceptor and olfactory cyclic nucleotide-gated (CNG) channels involves distinct ligand-binding and channel-gating reactions. To dissociate binding from gating, we identified the first competitive antagonists of CNG channels: specific phosphorothioate derivatives of cAMP and cGMP. We also identified membrane-permeant forms of these molecules that are antagonists and that will be useful for elucidating physiological roles for CNG channels in intact cells. The photoreceptor and olfactory CNG channels determine which of the phosphorothioate derivatives are agonists and which are antagonists based on different structural features of the ligand. The photoreceptor channel uses the nature of the purine ring (adenine vs guanine), whereas the olfactory channel uses the isomeric position of the thiophosphate S atom (Rp vs Sp). Interestingly, the same ligand, Rp-cGMPS, has opposite effects on the two channels, activating the photoreceptor channel and antagonizing the olfactory channel. Because Rp-cGMPS binds to both channels but activates only one, the channels must differ in a protein region that couples binding to gating. Chimeric photoreceptor and olfactory CNG channels reveal that the cytoplasmic C-terminal domain determines whether bound ligand activates the channel successfully. Hence, the C terminus contains not only the cyclic nucleotide-binding site, but also a region that couples ligand binding to channel gating.