Diffusion of Ca2+ from Small Boutons en Passant into the Axon Shapes AP-Evoked Ca2+ Transients.
ABSTRACT: Not only the amplitude but also the time course of a presynaptic Ca2+ transient determine multiple aspects of synaptic transmission. In small bouton-type synapses, the mechanisms underlying the Ca2+ decay kinetics have not been fully investigated. Here, factors that shape an action-potential-evoked Ca2+ transient were quantitatively studied in synaptic boutons of neocortical layer 5 pyramidal neurons. Ca2+ transients were measured with different concentrations of fluorescent Ca2+ indicators and analyzed based on a single-compartment model. We found a small endogenous Ca2+-binding ratio (7 ± 2) and a high activity of Ca2+ transporters (0.64 ± 0.03 ms-1), both of which enable rapid clearance of Ca2+ from the boutons. However, contrary to predictions of the single-compartment model, the decay time course of the measured Ca2+ transients was biexponential and became prolonged during repetitive stimulation. Measurements of [Ca2+]i along the adjoining axon, together with an experimentally constrained model, showed that the initial fast decay of the Ca2+ transients predominantly arose from the diffusion of Ca2+ from the boutons into the axon. Therefore, for small boutons en passant, factors like terminal volume, axon diameter, and the concentration of mobile Ca2+-binding molecules are critical determinants of Ca2+ dynamics and thus Ca2+-dependent processes, including short-term synaptic plasticity.
Project description:GABAergic somatodendritic inhibition in the preBötzinger complex (preBötC), a medullary site for the generation of inspiratory rhythm, is involved in respiratory rhythmogenesis and patterning. Nevertheless, whether GABA acts distally on presynaptic terminals, evoking presynaptic inhibition is unknown. Here, we begin to address this problem by measuring presynaptic Ca<sup>2+</sup> transients in preBötC neurons, under rhythmic and non-rhythmic conditions, with two variants of genetically encoded Ca<sup>2+</sup> indicators (GECIs). Organotypic slice cultures from newborn mice, containing the preBötC, were drop-transduced with jGCaMP7s, or injected with jGCaMP7f-labeling commissural preBötC neurons. Then, Ca<sup>2+</sup> imaging combined with whole-cell patch-clamp or field stimulation was obtained from inspiratory preBötC neurons. We found that rhythmically active neurons expressed synchronized Ca<sup>2+</sup> transients in soma, proximal and distal dendritic regions, and punctate synapse-like structures. Expansion microscopy revealed morphologic characteristics of bona fide synaptic boutons of the en passant and terminal type. Under non-rhythmic conditions, we found that bath application of the GABA<sub>A</sub> receptor agonist muscimol, and local microiontophoresis of GABA, reduced action potential (AP)-evoked and field stimulus-evoked Ca<sup>2+</sup> transients in presynaptic terminals in inspiratory neurons and commissural neurons projecting to the contralateral preBötC. In addition, under rhythmic conditions, network rhythmic activity was suppressed by muscimol, while the GABA<sub>A</sub> receptor antagonist bicuculline completely re-activated spontaneous activity. These observations demonstrate that the preBötC includes neurons that show GABAergic inhibition of presynaptic Ca<sup>2+</sup> transients, and presynaptic inhibition may play a role in the network activity that underlies breathing.
Project description:The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca<sup>2+</sup> indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca<sup>2+</sup> fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f<sup>+</sup>/ChAT<sup>+</sup>) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT<sub>3</sub>Rs). Cholinergic MG neurons also display small, spontaneous Ca<sup>2+</sup> transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na<sup>+</sup> channel dependent impulses, presynaptic Ca<sup>2+</sup> channels and postsynaptic receptor function reveal that the Ca<sup>2+</sup> transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT<sub>3</sub>Rs. Electrical stimulation of axonal connectives to MG evoked Ca<sup>2+</sup> responses in the neurons that similarly depended on nAChRs or/and 5-HT<sub>3</sub>Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca<sup>2+</sup> transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca<sup>2+</sup> transients and stimulus evoked Ca<sup>2+</sup> responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT<sub>3</sub>Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca<sup>2+</sup> imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.
Project description:Purkinje cells (PC) control spike timing of neighboring PC by their recurrent axon collaterals. These synapses underlie fast cerebellar oscillations and are characterized by a strong facilitation within a time window of <20 ms during paired-pulse protocols. PC express high levels of the fast Ca(2+) buffer protein calbindin D-28k (CB). As expected from the absence of a fast Ca(2+) buffer, presynaptic action potential-evoked [Ca(2+)]i transients were previously shown to be bigger in PC boutons of young (second postnatal week) CB-/- mice, yet IPSC mean amplitudes remained unaltered in connected CB-/- PC. Since PC spine morphology is altered in adult CB-/- mice (longer necks, larger spine head volume), we summoned that morphological compensation/adaptation mechanisms might also be induced in CB-/- PC axon collaterals including boutons. In these mice, biocytin-filled PC reconstructions revealed that the number of axonal varicosities per PC axon collateral was augmented, mostly confined to the granule cell layer. Additionally, the volume of individual boutons was increased, evidenced from z-stacks of confocal images. EM analysis of PC-PC synapses revealed an enhancement in active zone (AZ) length by approximately 23%, paralleled by a higher number of docked vesicles per AZ in CB-/- boutons. Moreover, synaptic cleft width was larger in CB-/- (23.8 ± 0.43 nm) compared to wild type (21.17 ± 0.39 nm) synapses. We propose that the morphological changes, i.e., the larger bouton volume, the enhanced AZ length and the higher number of docked vesicles, in combination with the increase in synaptic cleft width likely modifies the GABA release properties at this synapse in CB-/- mice. We view these changes as adaptation/homeostatic mechanisms to likely maintain characteristics of synaptic transmission in the absence of the fast Ca(2+) buffer CB. Our study provides further evidence on the functioning of the Ca(2+) homeostasome.
Project description:Ca<sup>2+</sup> signaling in glial cells is primarily triggered by metabotropic pathways and the subsequent Ca<sup>2+</sup> release from internal Ca<sup>2+</sup> stores. However, there is upcoming evidence that various ion channels might also initiate Ca<sup>2+</sup> rises in glial cells by Ca<sup>2+</sup> influx. We investigated AMPA receptor-mediated inward currents and Ca<sup>2+</sup> transients in olfactory ensheathing cells (OECs), a specialized glial cell population in the olfactory bulb (OB), using whole-cell voltage-clamp recordings and confocal Ca<sup>2+</sup> imaging. By immunohistochemistry we showed immunoreactivity to the AMPA receptor subunits GluA1, GluA2 and GluA4 in OECs, suggesting the presence of AMPA receptors in OECs. Kainate-induced inward currents were mediated exclusively by AMPA receptors, as they were sensitive to the specific AMPA receptor antagonist, GYKI53655. Moreover, kainate-induced inward currents were reduced by the selective Ca<sup>2+</sup>-permeable AMPA receptor inhibitor, NASPM, suggesting the presence of functional Ca<sup>2+</sup>-permeable AMPA receptors in OECs. Additionally, kainate application evoked Ca<sup>2+</sup> transients in OECs which were abolished in the absence of extracellular Ca<sup>2+</sup>, indicating that Ca<sup>2+</sup> influx via Ca<sup>2+</sup>-permeable AMPA receptors contribute to kainate-induced Ca<sup>2+</sup> transients. However, kainate-induced Ca<sup>2+</sup> transients were partly reduced upon Ca<sup>2+</sup> store depletion, leading to the conclusion that Ca<sup>2+</sup> influx via AMPA receptor channels is essential to trigger Ca<sup>2+</sup> transients in OECs, whereas Ca<sup>2+</sup> release from internal stores contributes in part to the kainate-evoked Ca<sup>2+</sup> response. Endogenous glutamate release by OSN axons initiated Ca<sup>2+</sup> transients in OECs, equally mediated by metabotropic receptors (glutamatergic and purinergic) and AMPA receptors, suggesting a prominent role for AMPA receptor mediated Ca<sup>2+</sup> signaling in axon-OEC communication.
Project description:Most presynaptic terminals in the brain contain G-protein-coupled receptors that function to reduce action potential-evoked neurotransmitter release. These neuromodulatory receptors, including those for glutamate, GABA, endocannabinoids, and adenosine, exert a substantial portion of their effect by reducing evoked presynaptic Ca(2+) transients. Many axons form synapses with multiple postsynaptic neurons, but it is unclear whether presynaptic attenuation in these synapses is homogeneous, as suggested by population-level Ca(2+) imaging. We loaded Ca(2+)-sensitive dyes into cerebellar parallel fiber axons and imaged action potential-evoked Ca(2+) transients in individual presynaptic boutons with application of three different neuromodulators and found that adjacent boutons on the same axon showed striking heterogeneity in their strength of attenuation. Moreover, attenuation was predicted by bouton size or basal Ca(2+) response: smaller boutons were more sensitive to adenosine A1 agonist but less sensitive to CB1 agonist, while boutons with high basal action potential-evoked Ca(2+) transient amplitude were more sensitive to mGluR4 agonist. These results suggest that boutons within brief segment of a single parallel fiber axon can have different sensitivities toward neuromodulators and may have different capacities for both short-term and long-term plasticities.
Project description:A common comorbidity of diabetes is skeletal muscle dysfunction, which leads to compromised physical function. Previous studies of diabetes in skeletal muscle have shown alterations in excitation-contraction coupling (ECC)-the sequential link between action potentials (AP), intracellular Ca<sup>2+</sup> release, and the contractile machinery. Yet, little is known about the impact of acute elevated glucose on the temporal properties of AP-induced Ca<sup>2+</sup> transients and ionic underlying mechanisms that lead to muscle dysfunction. Here, we used high-speed confocal Ca<sup>2+</sup> imaging to investigate the temporal properties of AP-induced Ca<sup>2+</sup> transients, an intermediate step of ECC, using an acute in cellulo model of uncontrolled hyperglycemia (25?mM, 48?h.). Control and elevated glucose-exposed muscle fibers cultured for five days displayed four distinct patterns of AP-induced Ca<sup>2+</sup> transients (phasic, biphasic, phasic-delayed, and phasic-slow decay); most control muscle fibers show phasic AP-induced Ca<sup>2+</sup> transients, while most fibers exposed to elevated D-glucose displayed biphasic Ca<sup>2+</sup> transients upon single field stimulation. We hypothesize that these changes in the temporal profile of the AP-induced Ca<sup>2+</sup> transients are due to changes in the intrinsic excitable properties of the muscle fibers. We propose that these changes accompany early stages of diabetic myopathy.
Project description:Cardiac small conductance Ca<sup>2+</sup>-activated K<sup>+</sup> (SK) channels are activated solely by Ca<sup>2+</sup>, but the SK current (I<sub>SK</sub>) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca<sup>2+</sup>/Mg<sup>2+</sup> block. Thus, Ca<sup>2+</sup> has a biphasic effect on I<sub>SK</sub>, activating at low [Ca<sup>2+</sup>] yet inhibiting I<sub>SK</sub> at high [Ca<sup>2+</sup>]. We examined the effect of I<sub>SK</sub> rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca<sup>2+</sup> transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of I<sub>SK</sub> during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca<sup>2+</sup>] reached ∼10 μM. During the rest of the AP stimulus, I<sub>SK</sub> either plateaued or gradually increased as the cell repolarized and submembrane [Ca<sup>2+</sup>] decreased further. We used a six-state gating model combined with intrinsic and Ca<sup>2+</sup>/Mg<sup>2+</sup>-dependent rectification to simulate I<sub>SK</sub> and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of I<sub>SK</sub> recording during AP clamp showing that intrinsic rectification limits I<sub>SK</sub> at high V<sub>m</sub> during the early and plateau phase of APs. Furthermore, the initial rise of Ca<sup>2+</sup> transients activates, but higher [Ca<sup>2+</sup>] blocks SK channels, yielding a transient outward-like I<sub>SK</sub> trajectory. During the decay phase of Ca<sup>2+</sup>, the Ca<sup>2+</sup>-dependent block is released, causing I<sub>SK</sub> to rise again and contribute to repolarization. Therefore, I<sub>SK</sub> is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.
Project description:Astrocytes exhibit spatially-restricted near-membrane microdomain Ca<sup>2+</sup>transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. Here we show that <i>Drosophila</i> astrocytes exhibit spontaneous, activity-independent microdomain Ca<sup>2+</sup> transients in their fine processes. Astrocyte microdomain Ca<sup>2+</sup> transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca<sup>2+</sup> transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O<sub>2</sub> and regulate gas exchange. Many astrocyte microdomain Ca<sup>2+</sup> transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca<sup>2+</sup> transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.
Project description:Information processing by brain circuits depends on Ca<sup>2+</sup>-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca<sup>2+</sup> in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca<sup>2+</sup> indicator Cal-590 is Ca<sup>2+</sup>-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca<sup>2+</sup> kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca<sup>2+</sup> or spike-evoked Ca<sup>2+</sup> entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca<sup>2+</sup> entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.
Project description:Little is known about the properties and function of ion channels that affect synaptic terminal-resting properties. One particular subthreshold-active ion channel, the Kv7 potassium channel, is highly localized to axons, but its role in regulating synaptic terminal intrinsic excitability and release is largely unexplored. Using electrophysiological recordings together with computational modeling, we found that the K<sub>V</sub>7 current was active at rest in adult hippocampal mossy fiber synaptic terminals and enhanced their membrane conductance. The current also restrained action potential-induced Ca<sup>2+</sup> influx via N- and P/Q-type Ca<sup>2+</sup> channels in boutons. This was associated with a substantial reduction in the spike half-width and afterdepolarization following presynaptic spikes. Further, by constraining spike-induced Ca<sup>2+</sup> influx, the presynaptic K<sub>V</sub>7 current decreased neurotransmission onto CA3 pyramidal neurons and short-term synaptic plasticity at the mossy fiber-CA3 synapse. This is a distinctive mechanism by which K<sub>V</sub>7 channels influence hippocampal neuronal excitability and synaptic plasticity.