Transmitter Release Research Articles

Neuromuscular Transmission in a Biological Context.

Neuromuscular transmission is the process by which motor neurons activate muscle contraction and thus plays an essential role in generating the purposeful body movements that aid survival. While many features of this process are common throughout the Animal Kingdom, such as the release of transmitter in multimolecular "quanta," and the response to it by opening ligand-gated postsynaptic ion channels, there is also much diversity between and within species. Much of this diversity is associated with specialization for either slow, sustained movements such as maintain posture or fast but brief movements used during escape or prey capture. In invertebrates, with hydrostatic and exoskeletons, most motor neurons evoke graded depolarizations of the muscle which cause graded muscle contractions. By contrast, vertebrate motor neurons trigger action potentials in the muscle fibers which give rise to all-or-none contractions. The properties of neuromuscular transmission, in particular the intensity and persistence of transmitter release, reflect these differences. Neuromuscular transmission varies both between and within individual animals, which often have distinct tonic and phasic subsystems. Adaptive plasticity of neuromuscular transmission, on a range of time scales, occurs in many species. This article describes the main steps in neuromuscular transmission and how they vary in a number of "model" species, including C. elegans , Drosophila , zebrafish, mice, and humans. © 2024 American Physiological Society. Compr Physiol 14:5641-5702, 2024.

Intersectin-1 enhances calcium-dependent replenishment of the readily releasable pool of synaptic vesicles during development.

Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca2+-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca2+-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca2+-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca2+ channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.

Tetrabenazine, a vesicular monoamine transporter 2 inhibitor, inhibits vesicular storage capacity and release of monoamine transmitters in mouse brain tissue

AbstractBackground and PurposeTetrabenazine (TBZ), used for treating hyperkinetic disorders, inhibits vesicular monoamine transporter‐2 (VMAT‐2), which sequesters monoamines into vesicles for exocytosis. However, our knowledge of the effect of TBZ on monoaminergic transmission is limited. Herein, we provide neurochemical evidence regarding the effect of VMAT‐2 inhibition on vesicular neurotransmitter release from the prefrontal cortex (PFC) and striatum (STR) (brain regions involved in characteristic TBZ treatment side effects). The interaction between TBZ and MDMA was also assessed regarding motor behaviour in mice.Experimental ApproachVesicular storage capacity and release of [3H]‐noradrenaline ([3H]‐NA), [3H]‐dopamine ([3H]‐DA), [3H]‐serotonin ([3H]‐5‐HT), and [3H]‐acetylcholine ([3H]‐ACh) was studied in mouse PFC and STR ex vivo slice preparations using electrical field stimulation. Additionally, locomotor activity was assessed in vehicle‐treated mice and compared with that of MDMA, TBZ, and co‐administered animals (n = 6) using the LABORAS system.Key ResultsTBZ lowered the storage capacity and inhibited the vesicular release of [3H]‐NA and [3H]‐DA from the PFC, and [3H]‐DA and [3H]‐5‐HT from the STR in a concentration‐dependent manner. Unlike vesamicol (vesicular ACh uptake inhibitor), TBZ failed to inhibit the vesicular release of [3H]‐ACh from the PFC. When the vesicular storage of the investigated monoamines was inhibited by TBZ in the PFC and STR, MDMA induced the release of transmitters through transporter reversal; MDMA dose dependently increased locomotor activity in vivo.Conclusion and ImplicationsOur observations provide neurochemical evidence explaining the mechanism of VMAT‐2 inhibitors in the brain and support the involvement of dopaminergic and noradrenergic transmission in hyperkinetic movement disorders.

Modulation of Neurotransmission by Acid-Sensing Ion Channels.

Interstitial pH fluctuations occur normally in the brain and significantly modulate neuronal functions. Acid-sensing ion channels (ASICs), which serve as neuronal acid chemosensors, play important roles in synaptic plasticity, learning, and memory. However, the specific mechanisms by which ASICs influence neurotransmission remain elusive. Here, we report that ASICs modulate transmitter release and axonal excitability at a glutamatergic synapse in the rat and mouse hippocampus. Blocking ASIC1a channels with the tarantula peptide psalmotoxin 1 down-regulates basal transmission and alters short-term plasticity. Notably, the effect of psalmotoxin 1 on ASIC-mediated modulation is age-dependent, occurring only during a limited postnatal period (postnatal weeks 2-6). This finding suggests that protons, through the activation of ASICs, may act as modulators in synapse formation and maturation during early development.

Synapse-to-synapse plasticity variability balanced to generate input-wide constancy of transmitter release.

Basal synaptic strength can vary greatly between synapses formed by an individual neuron because of diverse probabilities of action potential (AP) evoked transmitter release ( Pr ). Optical quantal analysis on large numbers of identified Drosophila larval glutamatergic synapses shows that short-term plasticity (STP) also varies greatly between synapses made by an individual type I motor neuron (MN) onto a single body wall muscle. Synapses with high and low P r and different forms and level of STP have a random spatial distribution in the MN nerve terminal, and ones with very different properties can be located within 200 nm of one other. While synapses start off with widely diverse basal P r at low MN AP firing frequency and change P r differentially when MN firing frequency increases, the overall distribution of P r remains remarkably constant due to a balance between the numbers of synapses that facilitate and depress as well as their degree of change and basal synaptic weights. This constancy in transmitter release can ensure robustness across changing behavioral conditions.

Endocytosis of Synaptic Vesicle in Motor Nerve Endings of FUS Transgenic Mice with a Model of Amyotrophic Lateral Sclerosis.

In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.

Deficiency in transmitter release triggers homeostatic transcriptional changes that increase presynaptic excitability.

Weakening of synaptic transmission at the Drosophila larval neuromuscular junction triggers two forms of homeostatic compensation, one that increases the probability of glutamate release per action potential (Pr) and another that increases motoneuron (MN) activity. We investigated the molecular changes in MNs that underlie the increase in MN activity. RNA-seq analysis on MNs whose glutamate release is weakened by knockdown of components of the MN transmitter release machinery reveals a reduction in expression of a group of genes that encode potassium channels and their positive modulators. These results identify a mechanism of compensation for weakened synaptic transmission by MNs, which engages a transcriptional program in those cells to increase firing and, thereby, ensure sufficient locomotory drive.

Distinct Neuromodulatory Effects of Endogenous Orexin and Dynorphin Corelease on Projection-Defined Ventral Tegmental Dopamine Neurons.

Dopamine (DA) neurons in the ventral tegmental area (VTA) respond to motivationally relevant cues, and circuit-specific signaling drives different aspects of motivated behavior. Orexin (ox; also known as hypocretin) and dynorphin (dyn) are coexpressed lateral hypothalamic (LH) neuropeptides that project to the VTA. These peptides have opposing effects on the firing activity of VTADA neurons via orexin 1 (Ox1R) or kappa opioid (KOR) receptors. Given that Ox1R activation increases VTADA firing, and KOR decreases firing, it is unclear how the coreleased peptides contribute to the net activity of DA neurons. We tested if optical stimulation of LHox/dyn neuromodulates VTADA neuronal activity via peptide release and if the effects of optically driven LHox/dyn release segregate based on VTADA projection targets including the basolateral amygdala (BLA) or the lateral or medial shell of the nucleus accumbens (lAcbSh, mAchSh). Using a combination of circuit tracing, optogenetics, and patch-clamp electrophysiology in male and female orexincre mice, we showed a diverse response of LHox/dyn optical stimulation on VTADA neuronal firing, which is not mediated by fast transmitter release and is blocked by antagonists to KOR and Ox1R signaling. Additionally, where optical stimulation of LHox/dyn inputs in the VTA inhibited firing of the majority of BLA-projecting VTADA neurons, optical stimulation of LHox/dyn inputs in the VTA bidirectionally affects firing of either lAcbSh- or mAchSh-projecting VTADA neurons. These findings indicate that LHox/dyn corelease may influence the output of the VTA by balancing ensembles of neurons within each population which contribute to different aspects of reward seeking.

Graded control of Purkinje cell outputs by cAMP through opposing actions on axonal action potential and transmitter release.

All-or-none signalling by action potentials (APs) in neuronal axons is pivotal for the precisely timed and identical size of outputs to multiple distant targets. However, technical limitations with respect to measuring the signalling in small intact axons have hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in slice and culture, we demonstrate that the timing and amplitude of axonal outputs are gradually modulated by cAMP depending on the length of axon. During the propagation in long axon, APs were attenuated and slowed in conduction by cAMP via specifically decreasing axonal Na+ currents. Consequently, the Ca2+ influx and transmitter release at distal boutons are reduced by cAMP, counteracting its direct facilitating effect on release machinery as observed at various CNS synapses. Together, our tour de force functional dissection has unveiled the axonal distance-dependent graded control of output timing and strength by intracellular signalling. KEY POINTS: The information processing in the nervous system has been classically thought to rely on the axonal faithful and high-speed conduction of action potentials (APs). We demonstrate that the strength and timing of axonal outputs are weakened and delayed, respectively, by cytoplasmic cAMP depending on the axonal length in cerebellar Purkinje cells (PCs). Direct axonal patch clamp recordings uncovered axon-specific attenuation of APs by cAMP through reduction of axonal Na+ currents. cAMP directly augments transmitter release at PC terminals without changing presynaptic Ca2+ influx or readily releasable pool of vesicles, although the extent is weaker compared to other CNS synapses. Two opposite actions of cAMP on PC axons, AP attenuation and release augmentation, together give rise to graded control of synaptic outputs in a manner dependent on the axonal length.

Stress-induced impairment of parasympathetic NO-mediated inhibition of sympathetic vasoconstriction in submucosal arteriole of rat rectum.

In the gastrointestinal tract, nitrergic inhibition of the arteriolar contractility has not been demonstrated. Here, we explored whether neurally-released nitric oxide (NO) inhibits sympathetic vasoconstrictions in the rat rectal arterioles. Changes in sympathetic vasoconstrictions and their nitrergic modulation in rats exposed to water avoidance stress (WAS, 10days, 1h per day) were also examined. In rectal submucosal preparations, changes in arteriolar diameter were monitored using video microscopy. In control or sham-treated rats, electrical field stimulation (EFS)-induced sympathetic vasoconstrictions were increased by the neuronal nitric oxide synthase (nNOS) inhibitor L-NPA (1μM) and diminished by the cyclic guanosine monophosphate-specific phosphodiesterase 5 (PDE5) inhibitor tadalafil (10nM). In phenylephrine-constricted, guanethidine-treated arterioles, EFS-induced vasodilatations were inhibited by the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN-4096 (1μM) but not L-NPA. Perivascular nNOS-immunoreactive nitrergic fibres co-expressing the parasympathetic marker vesicular acetylcholine transporter (VAChT) were intermingled with tyrosine hydroxylase (TH)-immunoreactive sympathetic fibres expressing soluble guanylate cyclase (sGC), a receptor for NO. In WAS rats in which augmented sympathetic vasoconstrictions were developed, L-NPA failed to further increase the vasoconstrictions, while tadalafil-induced inhibition of the vasoconstrictions was attenuated. Phenylephrine- or α,β-methylene ATP-induced vasoconstrictions and acetylcholine-induced vasodilatations were unaltered by WAS. Thus, in arterioles of the rat rectal submucosa, NO released from parasympathetic nerves appears to inhibit sympathetic vasoconstrictions presumably by reducing sympathetic transmitter release. In WAS rats, sympathetic vasoconstrictions are augmented at least partly due to the diminished pre-junctional nitrergic inhibition of transmitter release without changing α-adrenoceptor or P2X-purinoctor mediated vasoconstriction and endothelium-dependent vasodilatation.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 β-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

Cholinergic modulation in the vertebrate auditory pathway.

Acetylcholine (ACh) is a prevalent neurotransmitter throughout the nervous system. In the brain, ACh is widely regarded as a potent neuromodulator. In neurons, ACh signals are conferred through a variety of receptors that influence a broad range of neurophysiological phenomena such as transmitter release or membrane excitability. In sensory circuitry, ACh modifies neural responses to stimuli and coordinates the activity of neurons across multiple levels of processing. These factors enable individual neurons or entire circuits to rapidly adapt to the dynamics of complex sensory stimuli, underscoring an essential role for ACh in sensory processing. In the auditory system, histological evidence shows that acetylcholine receptors (AChRs) are expressed at virtually every level of the ascending auditory pathway. Despite its apparent ubiquity in auditory circuitry, investigation of the roles of this cholinergic network has been mainly focused on the inner ear or forebrain structures, while less attention has been directed at regions between the cochlear nuclei and midbrain. In this review, we highlight what is known about cholinergic function throughout the auditory system from the ear to the cortex, but with a particular emphasis on brainstem and midbrain auditory centers. We will focus on receptor expression, mechanisms of modulation, and the functional implications of ACh for sound processing, with the broad goal of providing an overview of a newly emerging view of impactful cholinergic modulation throughout the auditory pathway.

The Impact of Chronic Magnesium Deficiency on Excitable Tissues-Translational Aspects.

Neuromuscular excitability is a vital body function, and Mg2+ is an essential regulatory cation for the function of excitable membranes. Loss of Mg2+ homeostasis disturbs fluxes of other cations across cell membranes, leading to pathophysiological electrogenesis, which can eventually cause vital threat to the patient. Chronic subclinical Mg2+ deficiency is an increasingly prevalent condition in the general population. It is associated with an elevated risk of cardiovascular, respiratory and neurological conditions and an increased mortality. Magnesium favours bronchodilation (by antagonizing Ca2+ channels on airway smooth muscle and inhibiting the release of endogenous bronchoconstrictors). Magnesium exerts antihypertensive effects by reducing peripheral vascular resistance (increasing endothelial NO and PgI2 release and inhibiting Ca2+ influx into vascular smooth muscle). Magnesium deficiency disturbs heart impulse generation and propagation by prolonging cell depolarization (due to Na+/K+ pump and Kir channel dysfunction) and dysregulating cardiac gap junctions, causing arrhythmias, while prolonged diastolic Ca2+ release (through leaky RyRs) disturbs cardiac excitation-contraction coupling, compromising diastolic relaxation and systolic contraction. In the brain, Mg2+ regulates the function of ion channels and neurotransmitters (blocks voltage-gated Ca2+ channel-mediated transmitter release, antagonizes NMDARs, activates GABAARs, suppresses nAChR ion current and modulates gap junction channels) and blocks ACh release at neuromuscular junctions. Magnesium exerts multiple therapeutic neuroactive effects (antiepileptic, antimigraine, analgesic, neuroprotective, antidepressant, anxiolytic, etc.). This review focuses on the effects of Mg2+ on excitable tissues in health and disease. As a natural membrane stabilizer, Mg2+ opposes the development of many conditions of hyperexcitability. Its beneficial recompensation and supplementation help treat hyperexcitability and should therefore be considered wherever needed.

Astrocytic HILPDA promotes lipid droplets generation to drive cognitive dysfunction in mice with sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is manifested as a spectrum of disturbed cerebral function ranging from mild delirium to coma. However, the pathogenesis of SAE has not been clearly elucidated. Astrocytes play important roles in maintaining the function and metabolism of the brain. Most recently, it has been demonstrated that disorders of lipid metabolism, especially lipid droplets (LDs) dyshomeostasis, are involved in a variety of neurodegenerative diseases. The aim of this study was to investigate whether LDs are involved in the underlying mechanism of SAE. The open field test, Y-maze test, and contextual fear conditioning test (CFCT) were used to test cognitive function in SAE mice. Lipidomics was utilized to investigate alterations in hippocampal lipid metabolism in SAE mice. Western blotting and immunofluorescence labeling were applied for the observation of related proteins. In the current study, we found that SAE mice showed severe cognitive dysfunction, including spatial working and contextual memory. Meanwhile, we demonstrated that lipid metabolism was widely dysregulated in the hippocampus by using lipidomic analysis. Furthermore, western blotting and immunofluorescence confirmed that LDs accumulation in hippocampal astrocytes was involved in the pathological process of cognitive dysfunction in SAE mice. We verified that LDs can be inhibited by specifically suppress hypoxia-inducible lipid droplet-associated protein (HILPDA) in astrocytes. Meanwhile, cognitive dysfunction in SAE was ameliorated by reducing A1 astrocyte activation and inhibiting presynaptic membrane transmitter release. The accumulation of astrocytic lipid droplets plays a crucial role in the pathological process of SAE. HILPDA is an attractive therapeutic target for lipid metabolism regulation and cognitive improvement in septic patients.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Sympathetic Pathophysiology in Hypertension Origins: The Path to Renal Denervation.

The importance of the sympathetic nervous system in essential hypertension has been recognized in 2 eras. The first was in early decades of the 20th century, through to the 1960s. Here, the sympathetic nervous system was identified as a target for the treatment of hypertension, and an extensive range of antiadrenergic therapies were developed. Then, after a period of lapsed interest, in a second era from 1985 on, the development of precise measures of human sympathetic nerve firing and transmitter release allowed demonstration of the importance of neural mechanisms in the initiation and maintenance of the arterial blood pressure elevation in hypertension. This led to the development of a device treatment of hypertension, catheter-based renal denervation, which we will discuss.

RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

Detecting unitary synaptic events with machine learning

Spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs) are fundamental electrophysiological events produced by quantal vesicular transmitter release at synapses. Their analysis can provide important information regarding pre- and postsynaptic function. However, the small signal relative to recording noise requires expertise and considerable time for their identification. Furthermore, many mEPSCs smaller than ~8 pA are not well resolved (e.g., those produced at distant synapses or synapses with few receptor channels). Here, we describe an automated approach to detect mEPSCs using a machine learning-based tool. This method, which can be easily generalized to other one-dimensional signals, eliminates inter-observer bias, provides an estimate of its sensitivity and specificity and permits reliable detection of small (e.g., 5 pA) spontaneous unitary synaptic events.

Blood oxygen regulation via P2Y12R expressed in the carotid body

BackgroundPeripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown.MethodsThe presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals.ResultsInitially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia.ConclusionsPeripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.