A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of Bacillus stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars l-glutamate effectively protected against the modification of the cysteines at position 269–271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.

Glutamate transporter currents in Bergmann glial cells follow the time course of extrasynaptic glutamate

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate flux. Although a complete cycle of transport may require 50–100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of l-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.

Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus

Ca2+ released from presynaptic and postsynaptic intracellular stores plays important roles in activity-dependent synaptic plasticity, including long-term depression (LTD) of synaptic strength. At Schaffer collateral–CA1 synapses in the hippocampus, presynaptic ryanodine receptor-gated stores appear to mobilize some of the Ca2+ necessary to induce LTD. Cyclic ADP-ribose (cADPR) has recently been proposed as an endogenous activator of ryanodine receptors in sea urchin eggs and several mammalian cell types. Here, we provide evidence that cADPR-mediated signaling pathways play a key role in inducing LTD. We show that biochemical production of cGMP increases cADPR concentration in hippocampal slices in vitro, and that blockade of cGMP-dependent protein kinase, cADPR receptors, or ryanodine-sensitive Ca2+ stores each prevent the induction of LTD at Schaffer collateral–CA1 synapses. A lack of effect of postsynaptic infusion of either cADPR antagonist indicates a probable presynaptic site of action.

Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-d-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Context-sensitive synaptic plasticity and temporal-to-spatial transformations in hippocampal slices

Hippocampal slices are used to show that, as a temporal input pattern of activity flows through a neuronal layer, a temporal-to-spatial transformation takes place. That is, neurons can respond selectively to the first or second of a pair of input pulses, thus transforming different temporal patterns of activity into the activity of different neurons. This is demonstrated using associative long-term potentiation of polysynaptic CA1 responses as an activity-dependent marker: by depolarizing a postsynaptic CA1 neuron exclusively with the first or second of a pair of pulses from the dentate gyrus, it is possible to “tag” different subpopulations of CA3 neurons. This technique allows sampling of a population of neurons without recording simultaneously from multiple neurons. Furthermore, it reflects a biologically plausible mechanism by which single neurons may develop selective responses to time-varying stimuli and permits the induction of context-sensitive synaptic plasticity. These experimental results support the view that networks of neurons are intrinsically able to process temporal information and that it is not necessary to invoke the existence of internal clocks or delay lines for temporal processing on the time scale of tens to hundreds of milliseconds.

Cleft palate in mice with a targeted mutation in the γ-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the β-3 GABAA receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.

Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/synaptobrevin and block of exocytosis.

Role of intrinsic synaptic circuitry in collicular sensorimotor integration

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers in vitro by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: A possible factor regulating synaptic protein synthesis

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3β, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.

Genome scan for teratogen-induced clefting susceptibility loci in the mouse: Evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate

Nonsyndromic clefting of the lip and palate in humans has a highly complex etiology, with both multiple genetic loci and exposure to teratogens influencing susceptibility. Previous studies using mouse models have examined only very small portions of the genome. Here we report the findings of a genome-wide search for susceptibility genes for teratogen-induced clefting in the AXB and BXA set of recombinant inbred mouse strains. We compare results obtained using phenytoin (which induces cleft lip) and 6-aminonicotinamide (which induces cleft palate). We use a new statistical approach based on logistic regression suitable for these categorical data to identify several chromosomal regions as possible locations of clefting susceptibility loci, and we review candidate genes located within each region. Because cleft lip and cleft palate do not frequently co-aggregate in human families and because these structures arise semi-independently during development, these disorders are usually considered to be distinct in etiology. Our data, however, implicate several of the same chromosomal regions for both forms of clefting when teratogen-induced. Furthermore, different parental strain alleles are usually associated with clefting of the lip versus that of the palate (i.e., allelic heterogeneity). Because several other chromosomal regions are associated with only one form of clefting, locus heterogeneity also appears to be involved. Our findings in this mouse model suggest several priority areas for evaluation in human epidemiological studies.

Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene Lhx8

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that Lhx8, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of Lhx8 in vivo, we generated a mutant mouse with a targeted deletion of the Lhx8 gene. Our analysis of the mutant animals revealed a crucial role for Lhx8 in palatogenesis. In Lhx8 homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in Lhx8-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in Lhx8-mutant mice suggests that Lhx8 is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.