Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum.

Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution, but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that selectively traces monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically evoked Ca(2+) transients with GCaMP3 and FFN200 release simultaneously, we found that only a small fraction of dopamine boutons that exhibited Ca(2+) influx engaged in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally 'silent' dopamine vesicle clusters and represents, to the best of our knowledge, the first report suggestive of presynaptically silent neuromodulatory synapses.

Regulation of TrkB receptor translocation to lipid rafts by adenosine A(2A) receptors and its functional implications for BDNF-induced regulation of synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 µM) did not modify the effects of the A(2A) receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 µM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors RpcAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain.

We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system.

Mechanisms of dopamine quantal size regulation.

The study of dopamine (DA) quantal size, or the amount of transmitter released per vesicle fusion event, has been enabled by subsecond resolution amperometric recordings. These methods, together with other electrophysiology techniques, novel optical approaches and classical molecular biology and biochemistry methodologies, have advanced our understanding of quantal size regulation in dopaminergic and other catecholaminergic systems. The presynaptic mechanisms that determine DA quantal size regulate two features: the amount of transmitter stored in each vesicle and the fraction of vesicular contents that are released per fusion event. The amount of vesicular DA is dependent on DA synthesis, DA vesicular loading and storage and on DA reuptake from the extracellular space upon exocytosis. The mode of vesicle fusion and the related fusion pore dynamics control the fraction of DA released per fusion event. We will summarize current understanding on the regulation of these steps by endogenous and exogenous factors, including drugs of abuse and DA itself.

Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation.

Brain-derived neurotrophic factor (BDNF) signaling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signaling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansylcadaverine (100 microm) did not modify the effects of the A(2A) receptor agonists but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 microm forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22), and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts induced by activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Surto de pitiose cutânea em bovinos / Outbreak of cutaneous pythiosis in cattle

Setenta e seis bovinos, sem raça definida, jovens, de ambos os sexos, apresentaram lesões cutâneas multifocais nodulares, ulceradas e crostosas nas faces medial e lateral dos membros anteriores e posteriores, região ventral do pescoço, esterno e cauda. A doença ocorreu no verão e as lesões foram observadas nas regiões do corpo que ficavam grande tempo em contato com a água em canais de irrigação. Histologicamente observaram-se múltiplos granulomas e piogranulomas contendo escassas imagens negativas de hifas na área central, as quais foram melhor evidenciadas através da técnica de metenamina nitrato de prata de Grocott. O diagnóstico etiológico definitivo foi baseado na técnica de imuno-histoquímica com anticorpo policlonal anti-Pythium insidiosum. Adicionalmente, foi realizado o teste de ELISA indireto. Surtos de pitiose cutânea bovina são incomuns e, particularmente neste relato, todos os animais afetados tiveram cura espontânea das lesões dentro de duas a três semanas.(AU)

Seventy-six young mixed breed cattle of both sexes, presented multifocal ulcerated nodular cutaneous lesions localized in the medial and lateral aspects of fore and hindlimbs, ventral neck, sternum, and tail. The disease occurred during summer and lesions were observed on areas of the body which were in contact with water of irrigation channels for long periods. Histologically, there were multiple granulomas and pyogranulomas with few negative profiles of hyphae, which were better visualized throughout Grocott methenamine silver stain. Definitive etiologic diagnosis was based on immuno-histochemistry with anti-Pythium insidiosum polyclonal antibody. Additionally, an indirect ELISA test was performed. Bovine cutaneous pythiosis outbreaks are uncommon and, particularly as occurred in the cattle of this report, all affected animals had spontaneous healing within two to three weeks.(AU)

The tyrosine kinase Fyn determines the localization of TrkB receptors in lipid rafts.

Localization of Trk neurotrophin receptors is an important factor in directing cellular communication in developing and mature neurons. One potential site of action is in lipid raft membrane microdomains. Although Trk receptors have been localized to lipid rafts, little is known about how these neurotrophin receptors are directed there or how localization to these membrane microdomains regulates Trk signaling. Here, we report that the TrkB brain-derived neurotrophic factor (BDNF) receptor specifically localized to intracellular lipid rafts in cortical and hippocampal membranes in response to BDNF and that this process was critically dependent on the tyrosine kinase Fyn. BDNF-induced TrkB accumulation at lipid rafts was prevented by blocking the internalization of TrkB. BDNF stimulation also resulted in the association between endogenous TrkB and Fyn. Moreover, in neurons derived from Fyn knock-out mice, the translocation of TrkB to lipid rafts in response to BDNF was compromised, whereas the corticohippocampal region of Fyn mutants displayed lower amounts of TrkB in lipid rafts in vivo. In support of a role for lipid rafts in neurotrophin signaling, inhibiting TrkB translocation to lipid rafts, either by using Fyn knock-out neurons or lipid raft-disturbing agents, prevented the full activation of TrkB and of downstream phospholipase C-gamma. These results indicate that the lipid raft localization of TrkB receptors is regulated by Fyn and represents an important factor in determining the outcome of BDNF signaling in neurons.

BDNF regulates the expression and traffic of NMDA receptors in cultured hippocampal neurons.

The neurotrophin BDNF regulates the activity-dependent modifications of synaptic strength in the CNS. Physiological and biochemical evidences implicate the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present study, we investigated the effect of BDNF on the expression and plasma membrane abundance of NMDA receptor subunits in cultured hippocampal neurons. Acute stimulation of hippocampal neurons with BDNF differentially upregulated the protein levels of the NR1, NR2A and NR2B NMDA receptor subunits, by a mechanism sensitive to transcription and translation inhibitors. Accordingly, BDNF also increased the mRNA levels for NR1, NR2A and NR2B subunits. The neurotrophin NT3 also upregulated the protein levels of NR2A and NR2B subunits, but was without effect on the NR1 subunit. The amount of NR1, NR2A and NR2B proteins associated with the plasma membrane of hippocampal neurons was differentially increased by BDNF stimulation for 30 min or 24 h. The rapid upregulation of plasma membrane-associated NMDA receptor subunits was correlated with an increase in NMDA receptor activity. The results indicate that BDNF increases the abundance of NMDA receptors and their delivery to the plasma membrane, thereby upregulating receptor activity in cultured hippocampal neurons.

Brain-derived neurotrophic factor regulates the expression and synaptic delivery of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity in the hippocampus, but the mechanisms involved are not fully understood. The neurotrophin couples synaptic activation to changes in gene expression underlying long term potentiation and short term plasticity. Here we show that BDNF acutely up-regulates GluR1, GluR2, and GluR3 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits in 7-day in vitro cultured hippocampal neurons. The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a tropomyosin-related [corrected] kinase (Trk) inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors [corrected] The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a Trk inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors. Accordingly, BDNF increased the mRNA levels for GluR1 and GluR2 subunits. Biotinylation studies showed that stimulation with BDNF for 30 min selectively increased the amount of GluR1 associated with the plasma membrane, and this effect was abrogated by emetine. Under the same conditions, BDNF induced GluR1 phosphorylation on Ser-831 through activation of protein kinase C and Ca(2+)-calmodulin-dependent protein kinase II. Chelation of endogenous extracellular BDNF with TrkB-IgG selectively decreased GluR1 protein levels in 14-day in vitro cultures of hippocampal neurons. Moreover, BDNF promoted synaptic delivery of homomeric GluR1 AMPA receptors in cultured organotypic slices, by a mechanism independent of NMDA receptor activation. Taken together, the results indicate that BDNF up-regulates the protein levels of AMPA receptor subunits in hippocampal neurons and induces the delivery of AMPA receptors to the synapse.

Trkb receptors modulation of glutamate release is limited to a subset of nerve terminals in the adult rat hippocampus.

Brain-derived neurotrophic factor (BDNF) modulates glutamatergic excitatory transmission in hippocampal primary cultures by acting at a presynaptic locus. Although it has been suggested that BDNF also modulates adult hippocampus glutamatergic transmission, this remains a matter of controversy. To clarify a putative role for this neurotrophin in the modulation of glutamate release we applied exogenous BDNF to isolated adult rat hippocampal nerve terminals. BDNF, at 100 ng/ml, potentiated by 25% the K(+)-evoked release of [(3)H]glutamate from hippocampal synaptosomes. The small effect of BDNF on [(3)H]glutamate release correlated with a modest increase in phospholipase Cgamma (PLCgamma) phosphorylation, and with the lack of effect of BDNF on extracellular-signal regulated kinase (ERK) and Akt phosphorylation. Immunocytochemistry studies demonstrated that only about one-third of glutamatergic synaptosomes were positive for TrkB immunoreactivity. Furthermore, biotinylation and subsynaptic fractionation studies showed that only one-fourth of total full-length TrkB was present at the plasma membrane, evenly distributed between the presynaptic active zone and the postsynaptic density. These results indicate that BDNF modulates synaptic transmission presynaptically in a small subset of hippocampal glutamatergic synapses that contain TrkB and that express the receptor on the plasma membrane.

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation.

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects.

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events. Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.