Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.

Long-term synaptic transformation of hippocampal CA1 gamma-aminobutyric acid synapses and the effect of anandamide.

Evidence is presented for a distinctive type of hippocampal synaptic modification [previously described for a molluscan gamma-aminobutyric acid (GABA) synapse after paired pre- and postsynaptic excitation]: transformation of GABA-mediated synaptic inhibition into synaptic excitation. This transformation persists with no further paired stimulation for 60 min or longer and is termed long-term transformation. Long-term transformation is shown to contribute to pairing-induced long-term potentiation but not to long-term potentiation induced by presynaptic stimulation alone. Further support for such mechanistic divergence is provided by pharmacologic effects on long-term transformation as well as these two forms of long-term potentiation by Cl- channel blockers, glutamate and GABA antagonists, as well as the endogenous cannabinoid ligand anandamide.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors are ligand-gated channels that release intracellular Ca2+ stores in response to the second messenger, IP3. We investigated the potential role of IP3 receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca2+ mobilization is required for nuclear vesicle fusion and implicated IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibited IP3-stimulated CA2+ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP3. Because nuclear vesicle fusion was inhibited by antibodies that block Ca2+ flux, but not by control and preimmune antibodies, we concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown.

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin.

Rapid endocytosis (RE) occurs immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endoocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular Ca2+; Sr2+ and Ba2+ do not substitute, although all three cations support secretion. Thus the divalent cation receptors for RE and exocytosis must be distinct molecules. RE is dependent on GTP hydrolysis; it is blocked by GTP removal or replacement with guanosine 5'-[gamma-thio]triphosphate. In the presence of GTP, multiple rounds of secretion followed by RE could be elicited from the same cell. RE requires participation of dynamin, a guanine nucleotide binding protein, as revealed by intracellular immunological antagonism of this protein. Intact microtubules may be essential, as nocodazole also blocked RE. Whereas anti-dynamin antibodies blocked RE, anti-clathrin antibodies did not, suggesting that clathrin-coated vesicles are not involved in this form of endocytosis. RE may represent the initial step in the rapid recycling of secretory granules in the chromaffin cell.

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

Identification of a nonneuronal isoform of synaptotagmin.

Synaptotagmins, which have been found exclusively in neuroendocrine or exocrine tissues, have been implicated in the regulation of secretory vesicle fusion with the plasma membrane. The present paper describes a synaptotagmin isoform (synaptotagmin-5) which exhibits 49% amino acid identity to synaptotagmin-1 and -2. Synaptotagmin-5 mRNA is expressed in rat kidney, adipose tissue, lung, and heart, as well as at higher levels in brain and PC12 cells. Antiserum specific for the synaptotagmin-5 isoform recognizes a protein of about 50 kDa which is about 6-fold more abundant in brain synaptic vesicles than in whole brain membranes.

Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation.

Somatodendritic expression of an immediate early gene is regulated by synaptic activity.

Trans-synaptic activation of gene expression is linked to long-term plastic adaptations in the nervous system. To examine the molecular program induced by synaptic activity, we have employed molecular cloning techniques to identify an immediate early gene that is rapidly induced in the brain. We here report the entire nucleotide sequence of the cDNA, which encodes an open reading frame of 396 amino acids. Within the hippocampus, constitutive expression was low. Basal levels of expression in the cortex were high but can be markedly reduced by blockade of N-methyl-D-aspartate receptors. By contrast, synaptic activity induced by convulsive seizures increased mRNA levels in neurons of the cortex and hippocampus. High-frequency stimulation of the perforant path resulted in long-term potentiation and a spatially confined dramatic increase in the level of mRNA in the granule cells of the ipsilateral dentate gyrus. Transcripts were localized to the soma and to the dendrites of the granule cells. The dendritic localization of the transcripts offers the potential for local synthesis of the protein at activated postsynaptic sites and may underlie synapse-specific modifications during long-term plastic events.