Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation

Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. In a Drosophila genetic screen for synaptogenesis mutants, we identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. MTG is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix-dPak-Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development.     

Extracellular calcium depletion as a mechanism of short-term synaptic depression

Recent experiments have demonstrated that normal neural activity can cause significant decrements in external calcium levels, and that these decrements mediate a form of short-term synaptic depression. These findings raise the possibility that certain forms of short-term synaptic depression at glutamatergic synapses throughout the mammalian CNS may be influenced by similar changes in external calcium. We use a computational model of the extracellular space, combined with experimental data on calcium consumption, to show that such short-term depression can be accounted for by changes in calcium just outside active synapses, provided that external calcium diffusion is restricted. Remarkably, the model suggests the novel possibility that synapses may possess private pools of external calcium that enforce some forms of short-term depression in a synapse-specific manner.     

NMDA-Receptor-dependent synaptic activation of voltage-dependent calcium channels in basolateral amygdala

Afferent stimulation of pyramidal cells in the basolateral amygdala produced mixed excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors during whole cell current-clamp recordings. In the presence of GABA(A) receptor blockade, the mixed EPSPs recruited a large "all-or-none" depolarizing event. This recruited event was voltage dependent and had a distinct activation threshold. An analogous phenomenon elicited by exogenous glutamate in the presence of tetrodotoxin (TTX) was blocked by Cd(2+), suggesting that the event was a Ca(2+) spike. Selective glutamatergic blockade revealed that these Ca(2+) spikes were recruited readily by single afferent stimulus pulses that elicited NMDA EPSPs. In contrast, non-NMDA EPSPs induced by single stimuli failed to elicit the Ca(2+) spike even at maximal stimulus intensities although these non-NMDA EPSPs depolarized the soma more effectively than mixed EPSPs. Elongation of non-NMDA EPSPs by cyclothiazide or brief trains of stimulation were also unable to elicit the Ca(2+) spike. Blockade of K(+) channels with intracellular Cs(+) enabled single non-NMDA EPSPs to activate the Ca(2+) spike. The finding that voltage-dependent calcium channels are activated preferentially by NMDA-receptor-mediated EPSPs provides a mechanism for NMDA-receptor-dependent plasticity independent of Ca(2+) influx through the NMDA receptor.     

Differential discrimination of fast and slow synaptic waveforms by two low-voltage-activated calcium channels

Electrophysiological analysis of human embryonic kidney 293 cells stably expressing recombinant channels was used to compare how the biophysical properties of the low-voltage-activated Ca(2+) channels encoded by the alpha(1G) (Ca(V)3.1) or alpha(1I) (Ca(V)3.3) subunits shape their responses to excitatory synaptic potentials. In medium containing 2 mM extracellular Ca(2+) standard current-voltage relationships demonstrated both channel types to be clearly low-voltage activated with significant slowly activating current responses being observed at -66 mV. At all test potentials examined, activation of Ca(V)3.3 was substantially slower than that of Ca(V)3.1. To probe how these different T-type channels might respond to excitatory postsynaptic potentials (EPSPs), mock EPSPs with different kinetic profiles were created from the sum of exponentials. These waveforms were then used as command templates in voltage-clamp experiments. Ca(V)3.1-mediated channels responded effectively to both rapidly decaying mock EPSPs and slowly decaying EPSPs. In contrast, Ca(V)3.3-mediated channels were poorly gated by rapidly decaying EPSPs but were effectively activated by the more prolonged synaptic response. When activated with mock EPSPs Ca(V)3.3-mediated currents were more resistant to steady-state depolarisation of the pre-stimulus holding potential. Ca(V)3.3 currents were also more resistant to repetitive application of prolonged EPSPs, which caused substantial inactivation of Ca(V)3.1-mediated currents. The addition of a single mock action potential to the peak of a rapidly decaying EPSP voltage-clamp template greatly enhanced the currents produced by either Ca(V)3.1 or Ca(V)3.3-expressing cells. This facilitatory effect was considerably greater for Ca(V)3.3-mediated channels. From these data we suggest that the slow activation kinetics of Ca(V)3.3-mediated T-type channels enable them to respond selectively to either slow or suprathreshold synaptic potentials.     

Presynaptic inhibition in the crayfish CNS: pathways and synaptic mechanisms

I studied the pathways that produce primary afferent depolarization (PAD) and presynaptic inhibition during crayfish escape behavior. Simultaneous intracellular recordings were obtained from interneurons and primary afferent axons in the neuropil of the sixth abdominal ganglion. In several experiments, a sucrose-gap recording of PAD accompanied the intracellular impalements. I have identified PAD-producing inhibitory interneurons (PADIs) that are fired by a single impulse in the lateral (LG) or medial (MG) giant, escape-command axons; the PADIs appear to be directly responsible for presynaptic inhibition of primary afferent input to identified mechanosensory interneurons. PADI spikes, elicited by injection of depolarizing current, produced unitary PAD with constant short latency (mean = 0.97 +/- 0.12 SD ms). The unitary PADs were capable of following PADI impulses one for one at frequencies greater than 100 Hz, and the amplitude of unitary PAD was increased by injection of chloride into the afferent terminals. Therefore, the PADIs appear to directly produce an increase in chloride conductance in the primary afferent terminals. Intracellular injections of Lucifer yellow or horseradish peroxidase (HRP) revealed three morphological types of PADI. Their axonal branches and terminals are bilateral and overlap extensively with the innervation fields of all 10 sensory roots of the sixth ganglion. The three morphological types of PADI were physiologically indistinguishable. In several cases, the impaled PADI was shown to produce unitary PAD in more than one afferent of a given root as well as in afferents of adjacent roots. Therefore, the PADIs appear to diverge widely and contact many afferents in all of the sixth-ganglion sensory roots. Stimulation, caudal to the fifth ganglion, of an MG that had been interrupted rostral to the fifth ganglion produced no PAD in sixth-ganglion afferents. Also, stimulation of an MG or an LG in a surgically isolated sixth abdominal ganglion failed to produce PAD. Therefore, the pathway between the MGs and PADIs is activated exclusively within the rostral abdominal ganglia. Direct stimulation in the second and third abdominal ganglia of the segmental giants (SGs) produced a polysynaptic, suprathreshold response in the PADIs. This response was compound and was not due to the activity of the identified corollary discharge interneurons, CDI-2 and CDI-3, that are fired by the SGs. Therefore, the primary input to the PADIs must come from other, unidentified CDIs that are driven by the SGs. PADIs were not fired by shocks to the sensory portions of any peripheral roots even though these shocks produced PAD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Possible role of calcium ions,calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens

The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.     

Presynaptic and interactive peptidergic modulation of reticulospinal synaptic inputs in the lamprey

The modulatory effects of neuropeptides on descending inputs to the spinal cord have been examined by making paired recordings from reticulospinal axons and spinal neurons in the lamprey. Four peptides were examined; peptide YY (PYY) and cholecystokinin (CCK), which are contained in brain stem reticulospinal neurons, and calcitonin-gene-related peptide (CGRP) and neuropeptide Y (NPY), which are contained in primary afferents and sensory interneurons, respectively. Each of the peptides reduced the amplitude of monosynaptic reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The modulation appeared to be presynaptic, because postsynaptic input resistance and membrane potential, the amplitude of the electrical component of the EPSP, postsynaptic responses to glutamate, and spontaneous miniature EPSP amplitudes were unaffected. In addition, none of the peptides affected the pattern of N-methyl-D-aspartate (NMDA)-evoked locomotor activity in the isolated spinal cord. Potential interactions between the peptides were also examined. The "brain stem peptides" CCK and PYY had additive inhibitory effects on reticulospinal inputs, as did the "sensory peptides" CGRP and NPY. Brain stem peptides also had additive inhibitory effects when applied with sensory peptides. However, sensory peptides increased or failed to affect the amplitude of reticulospinal inputs in the presence of the brain stem peptides. These interactive effects also appear to be mediated presynaptically. The functional consequence of the peptidergic modulation was investigated by examining spinal ventral root responses elicited by brain stem stimulation. CCK and CGRP both reduced ventral root responses, although in interaction both increased the response. These results thus suggest that neuropeptides presynaptically influence the descending activation of spinal locomotor networks, and that they can have additive or novel interactive effects depending on the peptides examined and the order of their application.     

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.     

Sodium conductance in calcium channels of guinea-pig ventricular cells induced by removal of external calcium ions

An inward current characterized by a slow inactivation, was induced when the extracellular Ca^2– concentration was reduced by EGTA. It was suppressed by replacing external Na^– with Tris⁺ or by D-600, increased by epinephrine, and was not affected by TTX. These findings suggest that this current is carried by Na⁺ ions through the Ca channels. The Na current decreased in amplitude as the concentration of external divalent cations was elevated. Blocking the Na current by divalent cations could be approximated by a bimolecular interaction between divalent cation and channel, with a dissociation constant of 1.2 M for Ca²⁺ and 60 M for Mg²⁺. Single channel currents were recorded in the cell-attached configuration. With a pipette solution of pCa=7.5 or pCa>8, the single channel I-V relationship was linear and the slope conductance was 70–75 pS. For 40 mV depolarizations from the resting potential, unitary currents were smaller at pCa=6 than at pCa=7.5. However, single channel events, which were observed after the repolarizing step to the resting potential, were much the same amplitude. The open time histogram was fitted with a single exponential having a time constant of 1.9 ms at around –40 mV (pCa>8, with 5 M Bay K 8644 in the bath solution), which was decreased with increasing the Ca²⁺ concentration in the pipette solution. Noise power spectra of patch currents at pCa=6 revealed a high-frequency component at around 1500 Hz. These results suggest that Ca binding to the sites with a high affinity for Ca²⁺ blocks the Na conductance in Ca channels. Reduction of the unitary current at higher concentrations of Ca²⁺ might be attributed to a rapid block by Ca²⁺.     

Age-related changes in the levels of voltage-dependent calcium channels and other synaptic proteins in rat brain cortices

Neurotransmitter release from synapses is one of the most important interneuronal signaling in the nervous system. We previously reported that aging decreases depolarization-induced acetylcholine release in rat brain synaptosomes. To investigate the mechanisms underlying the age-related decrements of neurotransmission, we determined the levels of the alpha1 subunit proteins of voltage-dependent calcium channels (VDCCs) and three synaptic proteins that relate to exocytotic processes using synaptosomes prepared from cerebral cortices of young (6-month-old) and aged (27-month-old) rats. Immunoblotting analyses revealed that the protein levels of alpha1A (P/Q-type) and alpha1B (N-type) subunits in aged rats were 38% and 43% lower than the levels of young rats, respectively, but the levels of the alpha1C (L-type) subunit were not different between young and aged. On the contrary, the levels of synaptotagmin-1, synaptophysin and syntaxin were not significantly different between the two age groups in the synaptosomal preparations. These results suggest that synaptic density does not change much in the cerebral cortex in normal aging, and that the reduction of P/Q-type and N-type VDCCs, both of which participate in neurotransmitter release, is one of the causes for the decrease of neurotransmission at aged synapses.     

Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients

The cellular mechanisms responsible for large miniature currents in some brain synapses remain undefined. In Purkinje cells, we found that large-amplitude miniature inhibitory postsynaptic currents (mIPSCs) were inhibited by ryanodine or by long-term removal of extracellular Ca2+. Two-photon Ca2+ imaging revealed random, ryanodine-sensitive intracellular Ca2+ transients, spatially constrained at putative presynaptic terminals. At high concentration, ryanodine decreased action-potential-evoked rises in intracellular Ca2+. Immuno-localization showed ryanodine receptors in these terminals. Our data suggest that large mIPSCs are multivesicular events regulated by Ca2+ release from ryanodine-sensitive presynaptic Ca2+ stores.