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The impact of synaptic conductance on action potential waveform: Evoking realistic action potentials with a simulated synaptic conductance

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Abstract

Most current clamp studies trigger action potentials (APs) by step current injection through the recording electrode and assume that the resulting APs are essentially identical to those triggered by orthodromic synaptic inputs. However this assumption is not always valid, particularly when the synaptic conductance is of large magnitude and of close proximity to the axon initial segment. We addressed this question of similarity using the Calyx of Held/MNTB synapse; we compared APs evoked by long duration step current injections, short step current injections and orthodromic synaptic stimuli. Neither injected current protocol evoked APs that matched the evoked orthodromic AP waveform, showing differences in AP height, half-width and after-hyperpolarization. We postulated that this ‘error’ could arise from changes in the instantaneous conductance during the combined synaptic and AP waveforms, since the driving forces for the respective ionic currents are integrating and continually evolving over this time-course. We demonstrate that a simple Ohm's law manipulation of the EPSC waveform, which accounts for the evolving driving force on the synaptic conductance during the AP, produces waveforms that closely mimic those generated by physiological synaptic stimulation. This stimulation paradigm allows supra-threshold physiological stimulation (single stimuli or trains) without the variability caused by quantal fluctuation in transmitter release, and can be implemented without a specialised dynamic clamp system. Combined with pharmacological tools this method provides a reliable means to assess the physiological roles of postsynaptic ion channels without confounding affects from the presynaptic input.

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... Each MNTB neuron receives a large excitatory synapse called the calyx of Held (Held, 1893;Morest, 1968), which originates from the globular bushy cells of the contralateral cochlear nucleus (Spirou et al. 1990;Kuwabara et al. 1991;Smith et al. 1991). In response to a single presynaptic stimulus, this giant synapse generates an excitatory postsynaptic conductance (EPSG) of between 100 and 300 nS (Johnston et al. 2009), which effectively supercharges the MNTB membrane, rapidly bringing it to firing threshold. Although the exuberant size of the presynaptic input confers minimal time delays between the presynaptic and postsynaptic APs, the initial current magnitude and short term depression during repetitive stimulation generate other more subtle problems for information transmission. ...
... However, all active Kv conductances (Fig. 1B) will contribute to the AP amplitude and time course, including Kv1 (see above) and Kv2. Indeed the large synaptic conductance will influence AP waveform (Johnston et al. 2009) and during the peak of an overshooting AP (while V m is positive to E EPSC ) could assist AP repolarization. ...
... Although the large AMPA receptor (AMPAR)-mediated EPSC decays extremely rapidly, a slow component remains (Barnes-Davies & Forsythe, 1995;Taschenberger & von Gersdorff, 2000;Johnston et al. 2009) and summates with repetitive stimulation (>50 Hz), resulting in APs being triggered during sustained depolarization (Taschenberger & von Gersdorff, 2000). Depolarization during the inter-spike period reduces the pool of available sodium channels and can lead to AP failure (Jung et al. 1997;Johnston et al. 2008a). ...
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In this review we take a physiological perspective on the role of voltage-gated potassium channels in an identified neuron in the auditory brainstem. The large number of KCN genes for potassium channel subunits and the heterogeneity of the subunit combination into K(+) channels make identification of native conductances especially difficult. We provide a general pharmacological and biophysical profile to help identify the common voltage-gated K(+) channel families in a neuron. Then we consider the physiological role of each of these conductances from the perspective of the principal neuron in the medial nucleus of the trapezoid body (MNTB). The MNTB is an inverting relay, converting excitation generated by sound from one cochlea into inhibition of brainstem nuclei on the opposite side of the brain; this information is crucial for binaural comparisons and sound localization. The important features of MNTB action potential (AP) firing are inferred from its inhibitory projections to four key target nuclei involved in sound localization (which is the foundation of auditory scene analysis in higher brain centres). These are: the medial superior olive (MSO), the lateral superior olive (LSO), the superior paraolivary nucleus (SPN) and the nuclei of the lateral lemniscus (NLL). The Kv families represented in the MNTB each have a distinct role: Kv1 raises AP firing threshold; Kv2 influences AP repolarization and hyperpolarizes the inter-AP membrane potential during high frequency firing; and Kv3 accelerates AP repolarization. These actions are considered in terms of fidelity of transmission, AP duration, firing rates and temporal jitter. An emerging theme is activity-dependent phosphorylation of Kv channel activity and suggests that intracellular signalling has a dynamic role in refining neuronal excitability and homeostasis.
... Recordings of presynaptic action potentials from large boutons in the brainstem (Forsythe, 1994;Sierksma and Borst, 2017), hippocampus (Alle et al., 2009;Geiger and Jonas, 2000), pituitary gland (Jackson et al., 1991), and cerebellum (Ritzau-Jost et al., 2014) revealed large and stable action potentials, although deviations from an ''all-or-none'' behavior occur due to changes in the steady-state sodium and potassium channel availability (Alle and Geiger, 2006;Johnston et al., 2009;reviewed in Zbili and Debanne, 2019). However, recordings from small conventional boutons, forming >99% of all central synapses, were often restricted to the cell-attached configuration, preventing the measurement of the amplitude of the presynaptic action potential (Rowan et al., 2014;Sasaki et al., 2011;Smith et al., 2004). ...
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Presynaptic action potential spikes control neurotransmitter release and thus interneuronal communication. However, the properties and the dynamics of presynaptic spikes in the neocortex remain enigmatic because boutons in the neocortex are small and direct patch-clamp recordings have not been performed. Here, we report direct recordings from boutons of neocortical pyramidal neurons and interneurons. Our data reveal rapid and large presynaptic action potentials in layer 5 neurons and fast-spiking interneurons reliably propagating into axon collaterals. For in-depth analyses, we establish boutons of mature cultured neurons as models for excitatory neocortical boutons, demonstrating that the presynaptic spike amplitude is unaffected by potassium channels, homeostatic long-term plasticity, and high-frequency firing. In contrast to the stable amplitude, presynaptic spikes profoundly broaden during high-frequency firing in layer 5 pyramidal neurons, but not in fast-spiking interneurons. Thus, our data demonstrate large presynaptic spikes and fundamental differences between excitatory and inhibitory boutons in the neocortex.
... We found that IEM-1460 inhibited the peak amplitude of the postsynaptic MNTB principal cell AP at both age ranges, albeit more strongly in the P8 -P11 group (Fig. 9E). In addition, IEM-1460 reduced the depolarizing afterpotential significantly for the P8 -P11 group (Fig. 9D), which is probably due to the slow decay of EPSCs ( Fig. 1C; Johnston et al., 2009) and the slow discharge of the unmyelinated axon capacitance (Borst et al., 1995;Kim et al., 2010). The AP half-width remained unaffected in both age groups, whereas the AP peak delay was increased in A B C D1 D2 D3 Figure 6. ...
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GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca2+ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca2+ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ∼80% of the AMPAR population are permeable to Ca2+ at immature P4-P5 synapses. However, after the onset of hearing, Ca2+ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18-P22 and P30-P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30-P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.SIGNIFICANCE STATEMENT The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca2+ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca2+ permeable. However, we find that Ca2+-permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca2+-permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.
... Whether PNNs attract these Na ϩ and K ϩ ions is unclear. However, ChABC increases the diffusion of calcium ions in cortical and hippocampal brain slices, but not the monovalent cation tetramethylammonium (Hrabetová et al., 2009). Increased diffusion of calcium ions could have an effect similar to the application of extracellular high divalent solutions. ...
Article
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Perineuronal nets (PNNs) are specialized complexes of extracellular matrix molecules that surround the somata of fast-spiking neurons throughout the vertebrate brain. PNNs are particularly prevalent throughout the auditory brainstem, which transmits signals with high speed and precision. It is unknown whether PNNs contribute to the fast-spiking ability of the neurons they surround. Whole-cell recordings were made from medial nucleus of the trapezoid body (MNTB) principal neurons in acute brain slices from postnatal day 21 (P21) to P27 mice. PNNs were degraded by incubating slices in chondroitinase ABC (ChABC) and were compared to slices that were treated with a control enzyme (penicillinase). ChABC treatment did not affect the ability of MNTB neurons to fire at up to 1000 Hz when driven by current pulses. However, f–I (frequency–intensity) curves constructed by injecting Gaussian white noise currents superimposed on DC current steps showed that ChABC treatment reduced the gain of spike output. An increase in spike threshold may have contributed to this effect, which is consistent with the observation that spikes in ChABC-treated cells were delayed relative to control-treated cells. In addition, parvalbumin-expressing fast-spiking cortical neurons in >P70 slices that were treated with ChABC also had reduced excitability and gain. The development of PNNs around somata of fast-spiking neurons may be essential for fast and precise sensory transmission and synaptic inhibition in the brain.
... Information transmission along the auditory nerve is compromised and centrally compensated following sound-induced trauma (Kujawa & Liberman, 2009) but we postulate that other activity-dependent modulatory changes in the CNS can further influence auditory processing; for example, by changes in the subunit composition of the synaptic receptors. Such changes would change channel open time, and so be reflected in the decay kinetics of synaptic currents (Magleby & Stevens, 1972;Raman et al. 1994;Koike-Tani et al. 2005) thereby modifying synaptic integration and neuronal output (Johnston et al. 2009). Thus, by examining the kinetics of synaptic currents and comparing this to subunit expression, we can gain insights into activity-dependent mechanisms affecting auditory processing. ...
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Key points: LSO principal neurons receive AMPAR & NMDAR-mediated EPSCs and glycinergic IPSCs. Both EPSCs and IPSCs have slow kinetics in prehearing animals, which on maturation accelerate to sub-millisecond decay time-constant. This correlates with glutamate and glycine receptor subunit mRNA levels. The NMDAR-EPSCs accelerate over development to achieve decay time-constants of 2.5 ms. This is the fastest NMDAR-mediated EPSC reported. Loud sounds slow AMPAR-EPSC decay, increasing GluA1 and decreasing GluA4 mRNA. Modelling of Interaural Intensity Difference suggests that the increased EPSC duration after AT shifts IID to the right and compensates for hearing loss. Two months after AT the EPSC decay times had recovered to control values. Synaptic transmission in the LSO matures by P20, with EPSCs and IPSCs having fast kinetics. AT changes the AMPAR subunits expressed and slows the EPSC time-course at synapses in the central auditory system. Abstract: Damaging levels of sound (acoustic trauma, AT) diminish peripheral synapses, but what is the impact on the central auditory pathway? Developmental maturation of synaptic function and hearing were characterized in the mouse lateral superior olive (LSO) from P7 to P96 using voltage-clamp and auditory brainstem responses (ABR). IPSCs and EPSCs show rapid acceleration during development, so that decay kinetics converge to similar sub-millisecond time-constants (τ, 0.87 ± 0.11 ms, 0.77 ± 0.08 ms, respectively) in adult mice. This correlated with LSO mRNA levels for glycinergic and glutamatergic ionotropic receptor subunits; confirming a switch from Glyα2 to Glyα1 for IPSCs and increased expression of GluA3 and GluA4 subunits for EPSCs. The NMDAR-EPSC decay τ accelerated from > 40 ms in prehearing animals, to 2.6 ± 0.4 ms in adults, as GluN2C expression increased. In vivo induction of AT at around P20, disrupted IPSC and EPSC integration in the LSO, so that one week later the AMPAR-EPSC decay was slowed and mRNA for GluA1 increased while GluA4 decreased. In contrast, GlyR IPSC and NMDAR-EPSC decay times were unchanged. Computational modelling confirmed that matched IPSC and EPSC kinetics are required to generate mature interaural level difference (IID) functions, and that longer-lasting EPSCs compensates to maintain binaural function with raised auditory thresholds after AT. We conclude that LSO excitatory and inhibitory synaptic drive matures to identical time-courses; that AT changes synaptic AMPARs by expression of subunits with slow kinetics (which recover over two months) and that loud sounds reversibly modify excitatory synapses in the brain, changing synaptic function for several weeks after exposure. This article is protected by copyright. All rights reserved.
... As described here and previously for SGNs and other auditory neurons (Brew and Forsythe, 1995;Smith, 1995;Schwarz and Puil, 1997;Bal and Oertel, 2001), LVA current inhibition results in slowed adaptation. Within the auditory pathway, EPSCs are often severalfold larger than the threshold required to elicit postsynaptic APs (Johnston et al., 2009;Grant et al., 2010;Rutherford et al., 2012), minimizing timing delays and ensuring fast AP transmission. Rapid adaptation ensures that auditory neurons fire phasically in response to these large depolarizing stimuli, thus maintaining temporal fidelity and phase synchronization (Johnston et al., 2010). ...
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Unlabelled: Spiral ganglion neurons (SGNs) relay acoustic code from cochlear hair cells to the brainstem, and their stimulation enables electrical hearing via cochlear implants. Rapid adaptation, a mechanism that preserves temporal precision, and a prominent feature of auditory neurons, is regulated via dendrotoxin-sensitive low-threshold voltage-activated (LVA) K(+) channels. Here, we investigated the molecular physiology of LVA currents in SGNs cultured from mice following the onset of hearing (postnatal days 12-21). Kv1.1- and Kv1.2-specific toxins blocked the LVA currents in a comparable manner, suggesting that both subunits contribute to functional heteromeric channels. Confocal immunofluorescence in fixed cochlear sections localized both Kv1.1 and Kv1.2 subunits to specific neuronal microdomains, including the somatic membrane, juxtaparanodes, and the first heminode, which forms the spike initiation site of the auditory nerve. The spatial distribution of Kv1 immunofluorescence appeared mutually exclusive to that of Kv3.1b subunits, which mediate high-threshold voltage-activated currents. As Kv1.2-containing channels are positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5-bisphosphate (PIP2) availability on SGN electrophysiology. Reducing PIP2 production using wortmannin, or sequestration of PIP2 using a palmitoylated peptide (PIP2-PP), slowed adaptation rate in SGN populations. PIP2-PP specifically inhibited the LVA current in SGNs, an effect reduced by intracellular dialysis of a nonhydrolysable analog of PIP2. PIP2-PP also inhibited heterologously expressed Kv1.1/Kv1.2 channels, recapitulating its effect in SGNs. Collectively, the data identify Kv1.1/Kv1.2 heteromeric channels as key regulators of action potential initiation and propagation in the auditory nerve, and suggest that modulation of these channels by endogenous phosphoinositides provides local control of membrane excitability. Significance statement: Rapid spike adaptation is an important feature of auditory neurons that preserves temporal precision. In spiral ganglion neurons, the primary afferents in the cochlea, adaptation is regulated by heteromeric ion channels composed of Kv1.1 and Kv1.2 subunits. These subunits colocalize to common functional microdomains, such as juxtaparanodes and the somatic membrane. Activity of the heteromeric channels is controlled by cellular availability of PIP2, a membrane phospholipid. This mechanism provides an intrinsic regulation of output from the auditory nerve, which could be targeted for therapeutic adjustment of hearing sensitivity.
... We found that SNL induced a significant increase in amplitude (SNL 16.44 ± 1.17 mV versus naïve 11.46 ± 1.11 mV and sham 11.58 ± 1.10 mV, p < 0.01) but not frequency (numbers per 1 min) of the ADP ( Figure 4B). Unexpectedly, although rest membrane potential (RMP) is closely related to ADP amplitude [34], we did not observe any significant difference on RMP among naïve, sham and SNL groups (p > 0.05). Similar as the ADP amplitude, the duration of ADP was also increased significantly in SNL rats (4652 ± 1372 ms) as compared with that in naïve (1001 ± 488 ms) and sham (1381 ± 364 ms) rats (p < 0.05, Figure 4C). ...
Article
Background Despite high prevalence of anxiety accompanying with chronic pain, the mechanisms underlying pain-related anxiety are largely unknown. With its well-documented role in pain and emotion processing, the amygdala may act as a key player in pathogenesis of neuropathic pain-related anxiety. Pain-related plasticity and sensitization of CeA (central nucleus of the amygdala) neurons have been shown in several models of chronic pain. In addition, firing pattern of neurons with spike output can powerfully affect functional output of the brain nucleus, and GABAergic neurons are crucial in the modulation of neuronal excitability. In this study, we first investigated whether pain-related plasticity (e.g. alteration of neuronal firing patterns) and sensitization of CeA neurons contribute to nerve injury-evoked anxiety in neuropathic rats. Furthermore, we explored whether GABAergic disinhibition is responsible for regulating firing patterns and intrinsic excitabilities of CeA neurons as well as for pain-related anxiety in neuropathic rats.ResultsWe discovered that spinal nerve ligation (SNL) produced neuropathic pain-related anxiety-like behaviors in rats, which could be specifically inhibited by intra-CeA administration of anti-anxiety drug diazepam. Moreover, we found potentiated plasticity and sensitization of CeA neurons in SNL-induced anxiety rats, of which including: 1) increased burst firing pattern and early-adapting firing pattern; 2) increased spike frequency and intrinsic excitability; 3) increased amplitude of both after-depolarized-potential (ADP) and sub-threshold membrane potential oscillation. In addition, we observed a remarkable reduction of GABAergic inhibition in CeA neurons in SNL-induced anxiety rats, which was proved to be important for altered firing patterns and hyperexcitability of CeA neurons, thereby greatly contributing to the development of neuropathic pain-related anxiety. Accordantly, activation of GABAergic inhibition by intra-CeA administration of muscimol, a selective GABAA receptors agonist, could inhibit SNL-induced anxiety-like behaviors in neuropathic rats. By contrast, suppression of GABAergic inhibition by intra-CeA administration of bicuculline, a selective GABAA receptors antagonist, produced anxiety-like behavior in normal rats.Conclusions This study suggests that reduction of GABAergic inhibition may be responsible for potentiated plasticity and sensitization of CeA neurons, which likely underlie the enhanced output of amygdala and neuropathic pain-related anxiety in SNL rats.
... Consistent with this finding, EPSCs from LES rats exhibited a slow component of decay not observed in controls. Although the amplitude of the slow component is only about 5% of the total EPSC, due to its long time constant it makes a significant contribution to the total EPSC charge integral and may generate the prominent ADP observed in the AP from LES rats (Johnston et al. 2009), unlikely the presynaptic ADP in the calyx of Held terminal, which is generated by a Na ϩ conductance (Kim et al. 2010). During high-frequency stimulation, the broader AP and ADP may interfere with the removal of Na ϩ channel inactivation and may activate low-threshold K ϩ channels, both of which would extend the refractory period of the MNTB neuron thus leading to postsynaptic failures during high-frequency stimulation. ...
Article
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Auditory brainstem circuits rely on fast, precise, and reliable neurotransmission to process auditory information. To determine the fundamental role of myelination in auditory brainstem function, we examined the evoked auditory brainstem response (ABR) from the Long Evans shaker (LES) rat, which lacks myelin due to a genetic deletion of myelin basic protein. In control rats, the ABR evoked by a click consisted of five well-defined waves (denoted waves I-V). In LES rats, wave I, IV and V were present, but waves II and III were undetectable indicating disrupted function in the earliest stages of CNS auditory processing. In addition, the developmental shortening of the interval between waves I and IV, that normally occurs in control rats, was arrested and resulted in a significant increase in the central conduction time in LES rats. In brainstem slices, action potential transmission between the calyx of Held terminals and the Medial Nucleus of the Trapezoid Body (MNTB) neurons was delayed and less reliable in LES rats, although the resting potential, threshold, input resistance, and length of the axon initial segment of the postsynaptic MNTB neurons were normal. The amplitude of glutamatergic EPSCs and the degree of synaptic depression during high frequency stimulation were not different between LES rats and controls, but LES rats exhibited a marked slow component to the EPSC decay and a much higher rate of presynaptic failures. Together, these results indicate that loss of myelin disrupts brainstem auditory processing, increasing central conduction time and reducing the reliability of neurotransmission.
... Information transmission along the auditory nerve is compromised and centrally compensated following sound-induced trauma (Kujawa & Liberman, 2009) but we postulate that other activity-dependent modulatory changes in the CNS can further influence auditory processing; for example, by changes in the subunit composition of the synaptic receptors. Such changes would change channel open time, and so be reflected in the decay kinetics of synaptic currents (Magleby & Stevens, 1972;Raman et al. 1994;Koike-Tani et al. 2005) thereby modifying synaptic integration and neuronal output (Johnston et al. 2009). Thus, by examining the kinetics of synaptic currents and comparing this to subunit expression, we can gain insights into activity-dependent mechanisms affecting auditory processing. ...
... There is some ambiguity in most extracellular in vivo studies as to whether the recorded prespike-EPSP waveform originates in the same neuronal element as the compound APs. This problem has been addressed by statistical analysis off-line (Mc Laughlin et al., 2008;Englitz et al., 2009), although this approach might also miss failures if the statistic criteria for variation in the waveform are too stringent, especially considering that the postsynaptic AP waveform is influenced by changes in the synaptic conductance (Johnston et al., 2009). Recently, with the development of in vivo whole-cell patch recording, the original description of failures occurring at the calyx of Held/MNTB synapse (Kopp-Scheinpflug et al., 2003) could be corroborated ( Fig. 2d; Lorteije et al., 2009). ...
Article
The aim of this review is to consider the various forms and functions of transmission across the calyx of Held/MNTB synapse and how its modulation might contribute to auditory processing. The calyx of Held synapse is the largest synapse in the mammalian brain which uses the conventional excitatory synaptic transmitter, glutamate. It is sometimes portrayed as the 'ultimate' in synaptic signalling: it is a synaptic relay in which a single axon forms one synaptic terminal onto one specific target neuron. Questions that are often raised are: "Why does such a large and secure synapse need any form of modulation? Surely it is built simply to guarantee firing an action potential in the target neuron? If this synapse is so secure, why is a synapse needed at all?" Investigating these questions explains some general limitations of transmission at synapses and provides insight into the ionic basis of neuronal function by bringing together in vivo and in vitro approaches. We will start by defining the firing behaviour of MNTB neurons in vitro (in response to synaptic stimulation or current injection) and in vivo (in response to sound) and examining the reasons for different types of firing under the two conditions. Then we will consider some of the mechanisms by which transmission can be regulated. We will finish by discussing the following hypothesis: modulation and adaptation of presynaptic and postsynaptic conductances at the calyx of Held relay synapse are aimed at maximising the security of sound onset encoding while providing secondary information on frequency spectrum, harmonic envelope and duration of sound throughout the later part of the response.
... Moreover, I NaR also produces a fast rising DAP that accelerates the firing of a subsequent AP (Fig. 1c), and thus the precise timing of APs during a spike train (Fig. 8c,d). Postsynaptic MNTB neurons also exhibit a DAP (Johnston et al., 2009), and a developmental increase in I NaR , which improves their ability to fire at higher frequencies, and I NaR expression is altered by deafness (Leão et al., 2006). Of course, speed, precision, and reliability of spiking are also augmented through the myelination of axons, which increases during early postnatal development (Vabnick and Shrager, 1998). ...
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Axonal and nerve terminal action potentials often display a depolarizing after potential (DAP). However, the underlying mechanism that generates the DAP, and its impact on firing patterns, are poorly understood at axon terminals. Here, we find that at calyx of Held nerve terminals in the rat auditory brainstem the DAP is blocked by low doses of externally applied TTX or by the internal dialysis of low doses of lidocaine analog QX-314. The DAP is thus generated by a voltage-dependent Na(+) conductance present after the action potential spike. Voltage-clamp recordings from the calyx terminal revealed the expression of a resurgent Na(+) current (I(NaR)), the amplitude of which increased during early postnatal development. The calyx of Held also expresses a persistent Na(+) current (I(NaP)), but measurements of calyx I(NaP) together with computer modeling indicate that the fast deactivation time constant of I(NaP) minimizes its contribution to the DAP. I(NaP) is thus neither sufficient nor necessary to generate the calyx DAP, whereas I(NaR) by itself can generate a prominent DAP. Dialysis of a small peptide fragment of the auxiliary β4 Na(+) channel subunit into immature calyces (postnatal day 5-6) induced an increase in I(NaR) and a larger DAP amplitude, and enhanced the spike-firing precision and reliability of the calyx terminal. Our results thus suggest that an increase of I(NaR) during postnatal synaptic maturation is a critical feature that promotes precise and resilient high-frequency firing.
... The average peak voltages of APs followed a similar trend to that observed for the measures of AP amplitudes with average values becoming more depolarized until they overshot 0 mV at P5 (RMP: 1.7 ± 7.0 mV; SMP: 3.3 ± 9.3 mV) and then moving toward more hyperpolarized values through P14 (RMP: −15.4 ± 8.0 mV; SMP: −12.1 ± 8.0 mV; Supplemental Fig. 4B). The peak values at older ages (P8 and P14) were similar to those reported from MNTB neurons of P10-19 mice and rats at near-physiological temperatures (Johnston et al. 2009). AP half-widths measured at the RMP were significantly longer than those measured at the SMP from E17 to P0 (RMP 2.4 ± 0.9 ms, 1.5 ± 0.3 ms J Physiol 588.22 and 1.3 ± 0.3 ms, SMP 1.9 ± 0.3 ms, 1.4 ± 0.2 ms and 1.2 ± 0.2 ms for E17, E18 and P0, respectively; Fig. 8C; P < 0.02) indicating that depolarized RMP at younger ages played a role in broadening APs. ...
Article
Maturation of principal neurons of the medial nucleus of the trapezoid body (MNTB) was assessed in the context of the developmental organization and activity of their presynaptic afferents, which grow rapidly to form calyces of Held and to establish mono-innervation between postnatal days (P)2 and 4. MNTB neurons and their inputs were studied from embryonic day (E)17, when the nucleus was first discernable, until P14 after the onset of hearing. Using a novel slice preparation containing portions of the cochlea, cochlear nucleus and MNTB, we determined that synaptic inputs form onto MNTB neurons at E17 and stimulation of the cochlear nucleus can evoke action potentials (APs) and Ca(2+) signals. We analysed converging inputs onto individual MNTB neurons and found that competition among inputs was resolved quickly, as a single large input, typically larger than 4 nA, emerged from P3-P4. During calyx growth but before hearing onset, MNTB cells acquired their mature, phasic firing property and quantitative real-time PCR confirmed a coincident increase in low threshold K(+) channel mRNA. These events occurred in concert with an increase in somatic surface area and a 7-fold increase in the current threshold (30 to >200 pA) required to evoke action potentials, as input resistance (R(in)) settled from embryonic values greater than 1 GΩ to approximately 200 MΩ. We postulate that the postsynaptic transition from hyperexcitability to decreased excitability during calyx growth could provide a mechanism to establish the mature 1:1 innervation by selecting the winning calyceal input based on synaptic strength. By comparing biophysical maturation of the postsynaptic cell to alterations in presynaptic organization, we propose that maturation of synaptic partners is coordinated by synaptic activity in a process that is likely to generalize to other neural systems.
... APs were evoked by passing long depolarizing pulses (100 ms) of increasing magnitude (-20 to 130 pA in 50 pA steps) through the patch electrode (Xi and Xu 1996;Heflin and Cook, 2007;Yu et al., 2008). AP amplitudes were measured from the resting potential, and AP duration were defined as the width at half of the AP amplitude (Geiger and Jonas, 2000;Bean, 2007;Johnston et al., 2009). It has been reported that the photoactive hypericin exhibits enhanced in vitro cytotoxicity on light activation (Theodossiou et al., 2008). ...
Article
Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John's Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels. We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na(+), I(A) and I(K) currents. Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I(A) and delayed rectifier I(K) potassium currents. In contrast, hypericin exerted no significant effect on both Na(+) peak current and its decay kinetics. CONCLUSIONS AND IMPLICATions: Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I(A) and I(K) currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.
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The medial nucleus of the trapezoid body (MNTB) relays auditory information important for sound source localization. MNTB neurons faithfully preserve the temporal patterning of action potentials (APs) occurring in their single giant input synapse, even at high frequencies. The aim of this work was to examine the postsynaptic potassium conductances that shape the transfer of auditory information across this glutamatergic synapse. We used whole cell patch techniques to record from MNTB neurons in thin slices of rat brainstem. Two types of potassium conductance were found which had a strong influence on an MNTB neuron's postsynaptic response. A small low voltage threshold current, Id, limited the response during each EPSP to a single brief AP. Id was specifically blocked by dendrotoxin (DTX), resulting in additional APs during the tail end of the EPSP. Thus DTX degraded the temporal fidelity of synaptic transmission, since one presynaptic AP then led to several postsynaptic APs. A second conductance was a fast delayed rectifier with a high voltage activation threshold, that rapidly repolarised APs and thus facilitated high frequency AP responses. Together, these two conductances allow high frequency auditory information to be passed accurately across the MNTB relay synapse and separately, such conductances may perform analogous functions elsewhere in the nervous system.
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Neocortical neurons display a wide range of dendritic morphologies, ranging from compact arborizations to highly elaborate branching patterns. In vitro electrical recordings from these neurons have revealed a correspondingly diverse range of intrinsic firing patterns, including non-adapting, adapting and bursting types. This heterogeneity of electrical responsivity has generally been attributed to variability in the types and densities of ionic channels. We show here, using compartmental models of reconstructed cortical neurons, that an entire spectrum of firing patterns can be reproduced in a set of neurons that share a common distribution of ion channels and differ only in their dendritic geometry. The essential behaviour of the model depends on partial electrical coupling of fast active conductances localized to the soma and axon and slow active currents located throughout the dendrites, and can be reproduced in a two-compartment model. The results suggest a causal relationship for the observed correlations between dendritic structure and firing properties and emphasize the importance of active dendritic conductances in neuronal function.
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1. Whole-cell patch recordings were used to examine the EPSC generated by the calyx of Held in neurones of the medial nucleus of the trapezoid body (MNTB). Each neurone receives a somatic input from a single calyx (giant synapse). 2. A slow NMDA receptor-mediated EPSC peaked in 10 ms and decayed as a double exponential with time constants of 44 and 147 ms. A fast EPSC had a mean rise time of 356 microseconds (at 25 degrees C), while the decay was described by a double exponential with time constants of 0.70 and 3.43 ms. 3. Cyclothiazide slowed the decay of the fast EPSC, indicating that it is mediated by AMPA receptors. The slower time constant was slowed to a greater extent than the faster time constant. Cyclothiazide potentiated EPSC amplitude, partly by a presynaptic mechanism. 4. The metabotropic glutamate receptor (mGluR) agonists, 1S,3S-ACPD, 1S,3R-ACPD and L-2-amino-4-phosphonobutyrate (L-AP4) reversibly depressed EPSC amplitude. A dose-response curve for 1S,3S-ACPD gave an EC50 of 7 microM and a Hill coefficient of 1.2. 5. Analysis of the coefficient of variation ratio showed that the above mGluR agonists acted presynaptically to reduce the probability of transmitter release. Adenosine and baclofen also depressed transmission by a presynaptic mechanism. 6. alpha-Methyl-4-carboxyphenylglycine (MCPG; 0.5-1 mM) did not antagonize the effects of 1S,3S-ACPD, while high concentrations of L-2-amino-3-phosphonopropionic acid (L-AP3; 1 mM) and 4-carboxy-3-hydroxyphenyglycine (4C3HPG; 500 microM) depressed transmission. 7. There was a power relationship between [Ca2+]o and EPSC amplitude with co-operativity values ranging from 1.5 to 3.4. 8. The mechanism by which mGluRs modulate transmitter release appeared to be independent of presynaptic Ca2+ or K+ currents, since ACPD caused no change in the level of paired-pulse facilitation or the duration of the presynaptic action potential (observed by direct recording from the terminal), indicating that the presynaptic mGluR transduction mechanism may be coupled to part of the exocytotic machinery. 9. Our data are not consistent with the presence at the calyx of Held of any one known mGluR subtype. Comparison of the time course and pharmacology of the fast EPSC with data from cloned AMPA receptors is consistent with the idea that GluR-Do subunits dominate the postsynaptic channels.
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1. Using a combination of patch-clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. 2. Voltage-clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low-threshold dendrotoxin (DTX)-sensitive current (ILT) and a high-threshold DTX-insensitive current (IHT). 3. IHT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mM). 4. The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. 5. An mRNA probe corresponding to the C-terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. 6. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. 7. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1- like current is essential for cells to respond to a sustained train of high-frequency stimuli. 8. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high-frequency inputs.
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Kv3 channels are thought to be essential for the fast-spiking (FS) phenotype in GABAergic interneurons, but how these channels confer the ability to generate action potentials (APs) at high frequency is unknown. To address this question, we developed a fast dynamic-clamp system (approximately 50 kHz) that allowed us to add a Kv3 model conductance to CA1 oriens alveus (OA) interneurons in hippocampal slices. Selective pharmacological block of Kv3 channels by 0.3 mm 4-aminopyridine or 1 mm tetraethylammonium ions led to a marked broadening of APs during trains of short stimuli and a reduction in AP frequency during 1 sec stimuli. The addition of artificial Kv3 conductance restored the original AP pattern. Subtraction of Kv3 conductance by dynamic clamp mimicked the effects of the blockers. Application of artificial Kv3 conductance also led to FS in OA interneurons after complete K+ channel block and even induced FS in hippocampal pyramidal neurons in the absence of blockers. Adding artificial Kv3 conductance with altered deactivation kinetics revealed a nonmonotonic relationship between mean AP frequency and deactivation rate, with a maximum slightly above the original value. Insertion of artificial Kv3 conductance with either lowered activation threshold or inactivation also led to a reduction in the mean AP frequency. However, the mechanisms were distinct. Shifting the activation threshold induced adaptation, whereas adding inactivation caused frequency-dependent AP broadening. In conclusion, Kv3 channels are necessary for the FS phenotype of OA interneurons, and several of their gating properties appear to be optimized for high-frequency repetitive activity.
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The time course of synaptic conductance is important in temporal precision of information processing in the neuronal network. The AMPA receptor (AMPAR)-mediated EPSCs at the calyx of Held become faster in decay time as animals mature. To clarify how desensitization and deactivation of AMPARs contribute to developmental speeding of EPSCs, we compared the decay time of quantal EPSCs (qEPSCs) with the deactivation and desensitization times of AMPAR currents induced in excised patches by fast glutamate application (AMPA patch currents). Both the deactivation and desensitization times of AMPA patch currents became markedly faster from postnatal day 7 (P7) to P14 and changed little thereafter. In individual neurons, throughout development (P7-P21), the time constants of deactivation and fast desensitization in AMPA patch currents were similar to each other and close to the qEPSC decay time constant. Cyclothiazide (CTZ) abolished the fast desensitization, prolonged deactivation of AMPA patch currents, and slowed the decay time of EPSCs. The effects of CTZ on AMPA patch currents were unchanged throughout development, whereas its effect on EPSCs became weaker as animals matured. In single-cell reverse transcription-PCR analysis, glutamate receptor subunit 4 (GluR4) flop increased from P7 to P14 and changed little thereafter. At P7, the GluR4 flop abundance had an inverse correlation with the qEPSC decay time. These results together suggest that both desensitization and deactivation of AMPARs are involved in the EPSC decay time, but the contribution of desensitization decreases during postnatal development at the calyx of Held.
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Despite identification of >100 potassium channel subunits, relatively little is known about their roles in synaptic transmission. To address this issue we recorded presynaptic potassium currents (IPK) directly from the calyx of Held terminal in brainstem slices of rats. IPK was composed of a 4-aminopyridine (4-AP)-sensitive component and a smaller 4-AP-insensitive component composed of an iberiotoxin-sensitive current and an unidentified slowly activating potassium current. IPK could also be separated into a tetraethylammonium (TEA; 1 mm)-sensitive high-voltage-activated component and a margatoxin (10 nm)-sensitive low-voltage-activated component, which was also blocked by dendrotoxin-I (200 nm) and tityustoxin-Kalpha (100 nm). In outside-out patches excised from calyceal terminals, TEA (1 mm) consistently and to a large extent attenuated IPK, whereas margatoxin attenuated IPK only in a subset of patches (three of seven). Immunocytochemical examination using Kv subtype-specific antibodies indicated that multiple Kv1 and Kv3 subtypes were present at the calyceal terminal. In paired presynaptic and postsynaptic whole-cell recordings, TEA (1 mm) increased both the duration and peak amplitude of presynaptic action potentials and simultaneously potentiated EPSCs. Margatoxin alone had no such effect but reduced the amount of depolarization required for action potential generation, thereby inducing a burst of spikes when the nerve terminal was depolarized for a prolonged period. Thus, at the calyx of Held terminal, Kv3 channels directly regulate evoked transmitter release, whereas Kv1 channels reduce nerve terminal excitability, thereby preventing aberrant transmitter release. We conclude that both Kv3 and Kv1 channels contribute differentially to maintaining the fidelity of synaptic transmission at the calyx of Held.
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Principal neurones of the mouse medial nucleus of the trapezoid body (MNTB) possess multiple voltage-gated potassium currents, including a transient outward current (or A-current), which is characterized here. The A-current exhibited rapid voltage-dependent inactivation and was half inactivated at resting membrane potentials. Following a hyperpolarizing pre-pulse to remove inactivation, the peak transient current was 1.07 nA at -17 mV. The pharmacological characteristics of this A-current were consistent with Kv4 subunits in expression studies; the A-current was resistant to block by tetraethylammonium and dendrotoxin-I but sensitive to millimolar concentrations of 4-aminopyridine and 5 microM hanatoxin. Immunohistochemistry confirmed that Kv4.3 sub-units are present in the MNTB. In a single-compartment model of an MNTB neurone, the A-current served to accelerate the decay of the initial action potentials in a stimulus train and suggested that removal of A-current steady-state inactivation could raise firing threshold for non-calyceal synaptic inputs. This A-type current was not observed in the rat.