Absence Of Action Potentials Research Articles

Frequency of Spontaneous Neurotransmission at Individual Boutons Corresponds to the Size of the Readily Releasable Pool of Vesicles.

Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential (AP) across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle (SV) fusing (Pv) and in the number of vesicles available for immediate release, known as the readily releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of APs. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses. Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release.

Microgeometrical dendritic factors predict electrical decoupling between somatic and dendritic compartments in magnocellular neurosecretory neurons.

It is generally assumed that dendritic release of neuropeptides from magnocellular neurosecretory neurons (MNNs), a critical process involved in homeostatic functions, is an activity-dependent process that requires backpropagating action potentials (APs). Still, growing evidence indicates that dendritic release can occur in the absence of APs, and axonal APs have been shown to fail to evoke dendritic release. These inconsistencies strongly suggest that APs in MNNs may fail to backpropagating into dendrites. Here we tested whether simple factors of electrical signal attenuation could lead to effective decoupling between cell's body and dendritic release site within typical geometrical characteristics of MNN. We developed a family of linear mathematical models of MNNs and evaluated whether the somato-dendritic transfer of electrical signals is influenced by the geometrical characteristics. We determined the prerequisites for critically strong dendritic attenuation of the somatic input which are sufficient to explain the failure of APs initiated in the soma to backpropagating into dendritic compartments. Being measured in 100 μm from soma voltage attenuations down to 0.1 and 0.01 of the input value were chosen as the markers of electrical decoupling of dendritic sites from the soma, considering 0.1 insufficient for triggering dendritic spikes and 0.01 indistinguishable from background noise. The tested micro-geometrical factors were the dendritic stem diameter, varicosities, and size of peri-dendritic space limited by glial sheath wrapping. Varicosities increased the attenuation along homogeneous proximal dendrites by providing an increased current leak at the junction with the proximal dendritic section. The glial sheath wrapping a dendrite section promoted greater attenuation by increasing longitudinal resistance of the interstitial peri-dendritic space thus playing the insulating role. These decoupling effects were strengthened in the case of the dendritic stems with thinner diameters of and/or increased conductivity of the membrane. These micro-geometrical factors are biophysically realistic and predict electrical decoupling between somatic and dendritic compartments in MNNs.

Plateau potentials contribute to myotonia in mouse models of myotonia congenita.

It has long been accepted that myotonia (muscle stiffness) in patients with muscle channelopathies is due to myotonic discharges (involuntary firing of action potentials). In a previous study, we identified a novel phenomenon in myotonic muscle: development of plateau potentials, transient depolarizations to near -35mV lasting for seconds to minutes. In the current study we examined whether plateau potentials contribute to myotonia. A recessive genetic model (ClCadr mice) with complete loss of muscle chloride channel (ClC-1) function was used to model severe myotonia congenita with complete loss of ClC-1 function and a pharmacologic model using anthracene-9-carboxylic acid (9AC) was used to model milder myotonia congenita with incomplete loss of ClC-1 function. Simultaneous measurements of action potentials and myoplasmic Ca2+ from individual muscle fibers were compared to recordings of whole muscle force generation. In ClCadr muscle both myotonia and plateau potentials lasted 10s of seconds to minutes. During plateau potentials lasting 1-2min, there was a gradual transition from high to low intracellular Ca2+, suggesting a transition in individual fibers from myotonia to flaccid paralysis in severe myotonia congenita. In 9AC-treated muscles, both myotonia and plateau potentials lasted only a few seconds and Ca2+ remained elevated during the plateau potentials, suggesting plateau potentials contribute to myotonia without causing weakness. We propose, that in myotonic muscle, there is a novel state in which there is contraction in the absence of action potentials. This discovery provides a mechanism to explain reports of patients with myotonia who suffer from electrically silent muscle contraction lasting minutes.

Insulin differentially modulates GABA signalling in hippocampal neurons and, in an age-dependent manner, normalizes GABA-activated currents in the tg-APPSwe mouse model of Alzheimer's disease.

We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. In young mice, insulin (1nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.

Closing the Gap Between Mammalian and Invertebrate Peripheral Nerve Injury: Protocol for a Novel Nerve Repair.

BackgroundOutcomes after peripheral nerve injuries are poor despite current nerve repair techniques. Currently, there is no conclusive evidence that mammalian axons are capable of spontaneous fusion after transection. Notably, certain invertebrate species are able to auto-fuse after transection. Although mammalian axonal auto-fusion has not been observed experimentally, no mammalian study to date has demonstrated regenerating axolemmal membranes contacting intact distal segment axolemmal membranes to determine whether mammalian peripheral nerve axons have the intrinsic mechanisms necessary to auto-fuse after transection.ObjectiveThis study aims to assess fusion competence between regenerating axons and intact distal segment axons by enhancing axon regeneration, delaying Wallerian degeneration, limiting the immune response, and preventing myelin obstruction.MethodsThis study will use a rat sciatic nerve model to evaluate the effects of a novel peripheral nerve repair protocol on behavioral, electrophysiologic, and morphologic parameters. This protocol consists of a variety of preoperative, intraoperative, and postoperative interventions. Fusion will be assessed with electrophysiological conduction of action potentials across the repaired transection site. Axon-axon contact will be assessed with transmission electron microscopy. Behavioral recovery will be analyzed with the sciatic functional index. A total of 36 rats will be used for this study. The experimental group will use 24 rats and the negative control group will use 12 rats. For both the experimental and negative control groups, there will be both a behavior group and another group that will undergo electrophysiological and morphological analysis. The primary end point will be the presence or absence of action potentials across the lesion site. Secondary end points will include behavioral recovery with the sciatic functional index and morphological analysis of axon-axon contact between regenerating axons and intact distal segment axons.ResultsThe author is in the process of grant funding and institutional review board approval as of March 2020. The final follow-up will be completed by December 2021.ConclusionsIn this study, the efficacy of the proposed novel peripheral nerve repair protocol will be evaluated using behavioral and electrophysiologic parameters. The author believes this study will provide information regarding whether spontaneous axon fusion is possible in mammals under the proper conditions. This information could potentially be translated to clinical trials if successful to improve outcomes after peripheral nerve injury.International Registered Report Identifier (IRRID)PRR1-10.2196/18706

Neuronal Ca2+ signalling at rest and during spontaneous neurotransmission

Action potential driven neuronal signalling drives several electrical and biochemical processes in the nervous system. However, neurons can maintain synaptic communication and signalling under resting conditions independently of activity. Importantly, these processes are regulated by Ca2+ signals that occur at rest. Several studies have suggested that opening of voltage-gated Ca2+ channels near resting membrane potentials, activation of NMDA receptors in the absence of depolarization or Ca2+ release from intracellular stores can drive neurotransmitter release as well as subsequent signalling in the absence of action potentials. Interestingly, recent studies have demonstrated that manipulation of resting neuronal Ca2+ signalling yielded pronounced homeostatic synaptic plasticity, suggesting a critical role for this resting form of signalling in regulation of synaptic efficacy and neuronal homeostasis. Given their robust impact on synaptic efficacy and neuronal signalling, neuronal resting Ca2+ signals warrant further mechanistic analysis that includes the potential role of store-operated Ca2+ entry in these processes.

Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (I MI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Excitatory and Inhibitory Neurons Utilize Different Ca2+ Sensors and Sources to Regulate Spontaneous Release

Spontaneous neurotransmitter release (mini) is an important form of Ca2+-dependent synaptic transmission that occurs in the absence of action potentials. A molecular understanding of this process requires an identification of the underlying Ca2+ sensors. Here, we address the roles of the relatively low-and high-affinity Ca2+ sensors, synapotagmin-1 (syt1) and Doc2α/β, respectively. We found that both syt1 and Doc2 regulate minis, but, surprisingly, their relative contributions depend on whether release was from excitatory or inhibitory neurons. Doc2α promoted glutamatergic minis, while Doc2β and syt1 both regulated GABAergic minis. We identified Ca2+ ligand mutations in Doc2 that either disrupted or constitutively activated the regulation of minis. Finally, Ca2+ entry via voltage-gated Ca2+ channels triggered miniature GABA release by activating syt1, but had no effect on Doc2-driven minis. This work reveals an unexpected divergence in the regulation of spontaneous excitatory and inhibitory transmission in terms of both Ca2+ sensors and sources.

Reduced Heart Rate in Mice Harboring an SR Luminal Ca2+ Sensor Mutation (E4872Q) is Linked to Abnormal Ca2+ Release and Pacemaker Function in Isolated Cardiocytes Derived from the Mutant RyR Clone

Rationale: The coupled-clock theory of cardiac pacemaker normal automaticity integrates numerous facets of pacemaker cell Ca2+ cycling and electrophysiology. Details of intrinsic RyR molecular mechanisms that regulate spontaneous RyR activation to generate local Ca2+ releases (LCRs) of the “Ca2+ clock”, that drive normal automaticity, however, have not been elucidated. Objective: We hypothesized that spontaneous RyR activation to generate LCRs of the “Ca2+ clock” will be attenuated in cells harboring mutant SR luminal Ca2+ sensor (E4872Q). Methods and Results: We measured the spontaneous beating rate, action potential (AP) triggered RyR Ca2+ releases, characteristics of spontaneous LCRs in single ES derived cardiocytes from wild type (WT) and the RyR mutant (E4872Q) mouse embryonic stem cells (ES) with intact sarcolemma and in permeabilized cells in the absence of AP's, and the expression of SR Ca2+ proteins in SAN lysates. We also measured the heart rate in vivo, which was 15% lower in mutant mice vs WT mice. Compared to WT cardiocytes, cells harboring the RyR mutation had a reduced spontaneous AP firing rate and reduced spontaneous RyR Ca2+ release. Expression of RyR protein was reduced, and calsequestrin and calreticulin were increased in mutant vs that of WT cells. Conclusions: Numerous luminal SR Ca2+ sensing mechanisms linked to regulation of spontaneous RyR activation, regulator of the spontaneous AP firing rate of pacemaker cells. Alterations in spontaneous RyR activation in RyR mutant cells were a mechanism for a reduction of their AP firing rate and for reduced heart rate in mutant mice in vivo.

Molecular Mechanisms for Synchronous, Asynchronous, and Spontaneous Neurotransmitter Release

Most neuronal communication relies upon the synchronous release of neurotransmitters, which occurs through synaptic vesicle exocytosis triggered by action potential invasion of a presynaptic bouton. However, neurotransmitters are also released asynchronously with a longer, variable delay following an action potential or spontaneously in the absence of action potentials. A compelling body of research has identified roles and mechanisms for synchronous release, but asynchronous release and spontaneous release are less well understood. In this review, we analyze how the mechanisms of the three release modes overlap and what molecular pathways underlie asynchronous and spontaneous release. We conclude that the modes of release have key fusion processes in common but may differ in the source of and necessity for Ca(2+) to trigger release and in the identity of the Ca(2+) sensor for release.

Presynaptic NMDA Receptor Mechanisms for Enhancing Spontaneous Neurotransmitter Release

NMDA receptors (NMDARs) are required for experience-driven plasticity during formative periods of brain development and are critical for neurotransmission throughout postnatal life. Most NMDAR functions have been ascribed to postsynaptic sites of action, but there is now an appreciation that presynaptic NMDARs (preNMDARs) can modulate neurotransmitter release in many brain regions, including the neocortex. Despite these advances, the cellular mechanisms by which preNMDARs can affect neurotransmitter release are largely unknown. Here we interrogated preNMDAR functions pharmacologically to determine how these receptors promote spontaneous neurotransmitter release in mouse primary visual cortex. Our results provide three new insights into the mechanisms by which preNMDARs can function. First, preNMDARs can enhance spontaneous neurotransmitter release tonically with minimal extracellular Ca(2+) or with major sources of intracellular Ca(2+) blocked. Second, lowering extracellular Na(+) levels reduces the contribution of preNMDARs to spontaneous transmitter release significantly. Third, preNMDAR enhance transmitter release in part through protein kinase C signaling. These data demonstrate that preNMDARs can act through novel pathways to promote neurotransmitter release in the absence of action potentials.

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest(1). The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness(2-4). This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated. One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram(5,6). These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state(7). During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell(8); absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake. Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS. We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated. One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake. Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS. We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

Intracellular Ca2+ and TRPM5‐mediated membrane depolarization produce ATP secretion from taste receptor cells

ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca(2+) and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca(2+) release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca(2+) has a dual role: (1) Ca(2+) opens TRPM5 channels to depolarize receptor cells and (2) Ca(2+) plus membrane depolarization opens ATP-permeable gap junction hemichannels.

003 Gap junctional uncoupling with carbenoxolone slows conduction and increases vulnerability to ventricular arrhythmias in structurally normal hearts: an optical mapping study

<h3>Background</h3> Gap junctional uncoupling is thought to contribute to arrhythmogenesis through its effects on conduction slowing and creation of heterogeneities, often in the context of action potential or structural abnormalities. However, it is uncertain as to whether moderate conduction slowing through gap junctional uncoupling on its own, in the absence of action potential or structural changes, is sufficient to be pro-arrhythmic. We used optical mapping: (1) to study the effects of carbenoxolone, a gap junction uncoupler, on conduction velocity and action potential duration, and (2) to assess the effects of moderate conduction slowing and gap junctional uncoupling on arrhythmia vulnerability in structurally normal hearts. <h3>Methods</h3> Normal rat hearts (n=12) were isolated and perfused with Krebs-Henseleit solution, then loaded with a voltage-sensitive dye (RH237) and perfused with an excitation-contraction uncoupler (10 μM blebbistatin) to eliminate motion artefact. Transmembrane voltage (V_m) transients were recorded using a 256-photodiode array and epicardial activation was mapped across the anterolateral left ventricle during ventricular pacing (stimulus 1 mA, cycle length 180 ms). Hearts were then perfused with 30 μM carbenoxolone (CBX; n=6) or control (n=6) for 30 min and epicardial activation mapped every 2 min. Subsequently, hearts were subjected to programmed ventricular stimulation with up to three extrastimuli and assigned an Inducibility Quotient depending on ease of arrhythmia inducibility. <h3>Results</h3> Carbenoxolone significantly slowed conduction across the left ventricular epicardial surface (Conduction velocity index: Baseline 100%, CBX 70% ± 2%, p&lt;0.01) whereas there was no change in conduction velocity in control hearts after 30 min (Baseline 100%, Controls 98%±3%, p=NS). Carbenoxolone did not affect action potential duration (Baseline: APD50: 52±2 ms, APD75: 70±3 ms, APD90: 90±3 ms; CBX: APD50: 54±4 ms; APD75: 74±6 ms; APD90: 94±5 ms; all p=NS). Inducibility of ventricular arrhythmias was significantly increased in CBX hearts compared with control hearts during programmed stimulation (Inducibility quotient: CBX: 5.0±1.0; Control: 2.5±1.0; p&lt;0.05). <h3>Conclusions</h3> Gap junctional uncoupling with carbenoxolone produced a 30% reduction in conduction velocity without changes in action potential duration, and resulted in increased arrhythmia vulnerability in structurally normal hearts. These results suggest that moderate conduction slowing produced by gap junctional uncoupling in isolation, that is, in the absence of changes in action potential duration or structural abnormalities, is sufficient to increase susceptibility to ventricular arrhythmias, and therefore highlight the importance of gap junctional uncoupling in ventricular arrhythmogenesis.

From biological gastroenterology to fundamental neurosciences: How studies in gastric emptying have led to the discovery of a new mechanism of neuronal functioning

Gastric emptying undergoes complex regulation by the nervous system, which organizes in particular the inhibition of duodenum motility after a rise in intra-gastric pressure: the gastro-duodenal inhibitory reflex. It was first shown in mammals that this reflex could be organized by a sympathetic ganglion, the coeliac plexus. The excitation of gastric mechanosensitive fibres leads in this ganglion to the release of a neurotransmitter, which in turn activates ganglionic neurones leading to inhibition of the duodenum contractions. It rapidly became apparent that this reflex presented striking properties since it was organized in the absence of action potentials along the nerve fibres. Then it was shown that the neurotransmitter released in the coeliac plexus was gaseous: nitric oxide (NO). The nature of the mechanism conducting, without action potentials, the excitation along the nerve fibres was recently determined. This mechanism necessitates the integrity of particular areas of the neuronal membrane (the lipid rafts) and the activation in cascade of the following second messenger sequence: ceramide, calcium, NO and guanosine cyclic monophosphate (c-GMP). These results show how studies in biological gastroenterology have led to the rethinking of one of the central dogmas in neuroscience according to which excitation is only conducted along the nerves by the action potential.

Presynaptic Miniature Gabaergic Currents in Developing Interneurons

Miniature synaptic currents have long been known to represent random transmitter release under resting conditions, but much remains to be learned about their nature and function in central synapses. In this work, we describe a new class of miniature currents ("preminis") that arise by the autocrine activation of axonal receptors following random vesicular release. Preminis are prominent in gabaergic synapses made by cerebellar interneurons during the development of the molecular layer. Unlike ordinary miniature postsynaptic currents in the same cells, premini frequencies are strongly enhanced by subthreshold depolarization, suggesting that the membrane depolarization they produce belongs to a feedback loop regulating neurotransmitter release. Thus, preminis could guide the formation of the interneuron network by enhancing neurotransmitter release at recently formed synaptic contacts.

Dendritic NMDA Receptors Activate Axonal Calcium Channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.

Somatic Ca2+ transients do not contribute to inspiratory drive in preBötzinger Complex neurons

PreBötzinger Complex (preBötC) neurons are postulated to underlie respiratory rhythm generation. The inspiratory phase of the respiratory cycle in vitro results from preBötC neurons firing synchronous bursts of action potentials (APs) on top of 10-20 mV, 0.3-0.8 s inspiratory drive potentials. Is the inspiratory drive in individual neurons simply the result of the passive integration of inspiratory-modulated synaptic currents or do active processes modulate these currents? As somatic Ca(2+) is known to increase during inspiration, we hypothesized that it affects inspiratory drive. We combined whole cell recording in an in vitro slice preparation with Ca(2+) microfluorometry to detect single inspiratory neuron somatic Ca(2+) transients with high temporal resolution ( approximately mus). In neurons loaded with either Fluo-4 or Oregon Green BAPTA 5 N, we observed Ca(2+) transients associated with each AP. During inspiration, significant somatic Ca(2+) influx was a direct consequence of activation of voltage-gated Ca(2+) channels by APs. However, when we isolated the inspiratory drive potential in active preBötC neurons (by blocking APs with intracellular QX-314 or by hyperpolarization), we did not detect somatic Ca(2+) transients; yet, the parameters of inspiratory drive were the same with or without APs. We conclude that, in the absence of APs, somatic Ca(2+) transients do not shape the somatic inspiratory drive potential. This suggests that in preBötC neurons, substantial and widespread somatic Ca(2+) influx is a consequence of APs during the inspiratory phase and does not contribute substantively to the inspiratory drive potential. Given evidence that the Ca(2+) buffer BAPTA can significantly reduce inspiratory drive, we hypothesize that dendritic Ca(2+) transients amplify inspiratory-modulated synaptic currents.

Why Do Hair Cells Have Ribbons? Focus on “Synaptic Ribbon Enables Temporal Precision of Hair Cell Afferent Synapse by Increasing the Number of Readily Releasable Vesicles: A Modeling Study”

Hair cells, like retinal photoreceptors and bipolar cells, encode stimuli with graded changes in membrane potential and transmit that signal through ribbon synapses to drive action potentials in associated afferent neurons ([Sterling and Matthews 2005][1]). Thus one would expect transmitter release