Single Action Potentials Research Articles

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.

Voltage-gated potassium channels involved in regulation of physiological function in MrgprA3-specific itch neurons

Itch is described as an unpleasant or irritating skin sensation that elicits the desire or reflex to scratch. MrgprA3, one of members of the Mrgprs family, is specifically expressed in a subpopulation of dorsal root ganglion (DRG) in the peripheral nervous system (PNS). These MrgprA3-expressing DRG neurons have been identified as itch-specific neurons. They can be activated by the compound, chloroquine, which is used as a drug to treat malaria. In the present study, we labeled these itch-specific neurons using the method of molecular genetic markers, and then studied their electrophysiological properties. We also recorded the cutaneous MrgprA3− neurons retrogradely labeled by Dil dye (MrgprA3−-Dil). We first found that MrgprA3+ neurons have a lower excitability than MrgprA3− neurons (MrgprA3−-non-Dil and MrgprA3−-Dil). The number of action potential (AP) was reduced more obviously in MrgprA3+ neurons than that of in MrgprA3− neurons. In most cases, MrgprA3+ neurons only generated single AP; however, in MrgprA3− neurons, the same stimulation could induce multiple AP firing due to the greater voltage-gated potassium (Kv) current existence in MrgprA3+ than in MrgprA3− neurons. Thus, Kv current plays an important role in the regulation of excitability in itch-specific neurons.

Roles of specific Kv channel types in repolarization of the action potential in genetically identified subclasses of pyramidal neurons in mouse neocortex.

The action potential (AP) is a fundamental feature of excitable cells that serves as the basis for long-distance signaling in the nervous system. There is considerable diversity in the appearance of APs and the underlying repolarization mechanisms in different neuronal types (reviewed in Bean BP. Nat Rev Neurosci 8: 451-465, 2007), including among pyramidal cell subtypes. In the present work, we used specific pharmacological blockers to test for contributions of Kv1, Kv2, or Kv4 channels to repolarization of single APs in two genetically defined subpopulations of pyramidal cells in layer 5 of mouse somatosensory cortex (etv1 and glt) as well as pyramidal cells from layer 2/3. These three subtypes differ in AP properties (Groh A, Meyer HS, Schmidt EF, Heintz N, Sakmann B, Krieger P. Cereb Cortex 20: 826-836, 2010; Guan D, Armstrong WE, Foehring RC. J Neurophysiol 113: 2014-2032, 2015) as well as laminar position, morphology, and projection targets. We asked what the roles of Kv1, Kv2, and Kv4 channels are in AP repolarization and whether the underlying mechanisms are pyramidal cell subtype dependent. We found that Kv4 channels are critically involved in repolarizing neocortical pyramidal cells. There are also pyramidal cell subtype-specific differences in the role for Kv1 channels. Only Kv4 channels were involved in repolarizing the narrow APs of glt cells. In contrast, in etv1 cells and layer 2/3 cells, the broader APs are partially repolarized by Kv1 channels in addition to Kv4 channels. Consistent with their activation in the subthreshold range, Kv1 channels also regulate AP voltage threshold in all pyramidal cell subtypes.

Single CA3 pyramidal cells trigger sharp waves in vitro by exciting interneurones.

The CA3 hippocampal region generates sharp waves (SPW), a population activity associated with neuronal representations. The synaptic mechanisms responsible for the generation of these events still require clarification. Using slices maintained in an interface chamber, we found that the firing of single CA3 pyramidal cells triggers SPW like events at short latencies, similar to those for the induction of firing in interneurons. Multi-electrode records from the CA3 stratum pyramidale showed that pyramidal cells triggered events consisting of putative interneuron spikes followed by field IPSPs. SPW fields consisted of a repetition of these events at intervals of 4-8ms. Although many properties of induced and spontaneous SPWs were similar, the triggered events tended to be initiated close to the stimulated cell. These data show that the initiation of SPWs in vitro is mediated via pyramidal cell synapses that excite interneurons. They do not indicate why interneuron firing is repeated during a SPW. Sharp waves (SPWs) are a hippocampal population activity that has been linked to neuronal representations. We show that SPWs in the CA3 region of rat hippocampal slices can be triggered by the firing of single pyramidal cells. Single action potentials in almost one-third of pyramidal cells initiated SPWs at latencies of 2-5ms with probabilities of 0.07-0.76. Initiating pyramidal cells evoked field IPSPs (fIPSPs) at similar latencies when SPWs were not initiated. Similar spatial profiles for fIPSPs and middle components of SPWs suggested that SPW fields reflect repeated fIPSPs. Multiple extracellular records showed that the initiated SPWs tended to start near the stimulated pyramidal cell, whereas spontaneous SPWs could emerge at multiple sites. Single pyramidal cells could initiate two to six field IPSPs with distinct amplitude distributions, typically preceeded by a short-duration extracellular action potential. Comparison of these initiated fields with spontaneously occurring inhibitory field motifs allowed us to identify firing in different interneurones during the spread of SPWs. Propagation away from an initiating pyramidal cell was typically associated with the recruitment of interneurones and field IPSPs that were not activated by the stimulated pyramidal cell. SPW fields initiated by single cells were less variable than spontaneous events, suggesting that more stereotyped neuronal ensembles were activated, although neither the spatial profiles of fields, nor the identities of interneurone firing were identical for initiated events. The effects of single pyramidal cell on network events are thus mediated by different sequences of interneurone firing.

Single Vesicle Recording in Hippocampal ‘Xenapses’ Reveals Diffusional Dispersion of SV Proteins After Fusion

In order to maintain neuronal transmission after exocytosis of synaptic vesicles (SVs), the vesicular proteins have to be cleared away from the active zone. Until now it remained controversial whether SV components remain clustered during translocation from sites of exocytosis or disperse by free diffusion. To adress this question, we developed a novel purely presynaptic neuronal preparation which enables single vesicle recording by TIRFM.Using click-chemistry we functionalized micropatterned coverglasses with protein domains of synaptic cell adhesion molecules, serving as artificial postsynapses. On these host substrates purely presynaptic boutons form ‘en face’ directly onto the coverslip, termed ‘xenapses’. Serial section TEM as well as focused-ion-beam SEM showed that xenapses contain a few hundred SVs, many of them docked in several clusters at the bottom membrane. 4Pi and TIRF-STORM confirmed the existence of several active zones. Thus, xenapses offer the unique opportunity to record exocytosis of single vesicles by TIRFM. Using fusion constructs of the pH-sensitive pHluorin, single fusion events were visible as diffraction-limited spots on stimulation with single action potentials. We could localize fusion events synchronous to action potentials with ∼20 nm precision and follow the fate of released SV proteins. We observed diffusional dispersion of vesicular proteins post fusion with diffusion constants in the range of 0.1 - 1 µm²/s. Thus, our results point to free diffusion as mechanism for fast clearance.

Mechanism of Slow Inactivation in Prokaryotic Voltage-Gated Sodium Channel

Voltage-gated sodium channels (Nav) play a crucial role in electrically excitable cells by mediating the rapid influx of Na+ and thereby initiating the action potential in response to a depolarizing stimulus. In excitable cells, Nav activates and then undergoes fast and slow inactivation. Fast inactivation occurs within the time frame of a single action potential, whereas slow inactivation is produced by prolonged depolarization, mediating the availability of sodium channels over extended period of time. Mutations that alter slow inactivation are associated with several diseases such as hyperkalemic periodic analysis and long-QT syndrome. However, the molecular mechanism of slow inactivation and modulation by drug binding are still not completely known. In order to establish a structural framework of slow inactivation in molecular level, we used the prokaryotic homologue NavSP1 from Silicibacter pomeroyi as a model system. NavSP1 shares very similar overall architecture, activation, inactivation properties, and similar drug binding sites with the eukaryotic homologues and thereby proves to be an excellent model to be studied in native environment. We investigated how channel opening or the activation of voltage sensor initiates slow inactivation by evaluating the changes in conformational dynamics of the channel pore in the presence and absence of the voltage sensor. Our approach combined structural dynamics by EPR spectroscopy (CW and DEER methods) and functional studies by electrophysiological methods. Evaluation of the changes in the conformational dynamics and solvent accessibility in the turret and selectivity filter region show structural rearrangement of the outer pore region during slow inactivation.

Development of Fast-Response GCaMP6 Calcium Sensors for Monitoring Neuronal Action Potential

Green Fluorescent-Calmodulin Proteins (GCaMPs) have been the reporters of choice for visualizing neuronal network activity in vivo. With the GCaMP6 generation sufficient brightness was achieved for single action potential (AP) detection1. However, the Ca2+-response kinetics of GCaMP6 probes remained limiting to separating individual APs. The aim was to accelerate the Ca2+ response kinetics of GCaMP6 probes by point mutations in the Ca2+.CaM.RS20 complex2. A series of probes retaining high fluorescence dynamic ranges was generated with in vitro dissociation constants (Kd) for Ca2+ in the μM range (0.1-3.3 μM) and Hill coefficients of 1.7 to 4.2. Two highlighted probes termed GCaMP6fast and GCaMP6bright had in vitro half times (t1/2) at 37°C for Ca2+ rise of 1.3 ms and 3.3 ± 0.2 ms and decay t1/2-s of 2.8 ± 0.1 and 3.5 ± 0.1 ms, respectively, compared to the 10 ± 1 ms rise and 63 ± 6 ms decay times for GCaMP6f. Fluorescence changes on Ca2+ association were highly cooperative and characterized by a rate limiting conformational change. In vivo Ca2+ responses associated with AP firing patterns were tested in cultured hippocampal slices by two-photon imaging at 28°C. The kinetic performance of GCaMP6fast and GCaMP6bright was compared with that of GCaMP6f. Ca2+ decay kinetics were determined by monitoring fluorescence changes evoked by 5 APs fired at 100 Hz. t1/2 values for GCaMP6fast and GCaMP6bright were 58 ms and 126 ms, respectively. With an up to 7-fold faster in vivo decay kinetics than GCaMP6f, GCaMP6fast and GCaMP6bright are promising tools for monitoring brain activity.1Podor et al., Neurophotonics, Epub 2015 Apr 30.2Helassa et al., Scientific Reports, accepted.This work was funded by Wellcome Trust (094385/Z/10/Z to KT), BBSRC (BB/M02556X/1 to KT), CIHR (MOP-123514 to AF) and NSERC (RGPIN-170421 to AF).

Nicotinic acetylcholine receptor-mediated GABAergic inputs to cholinergic interneurons in the striosomes and the matrix compartments of the mouse striatum

The striatum consists of two neurochemically distinct compartments: the striosomes (or patches) and the extrastriosomal matrix. Although striatal neurons are strongly innervated by intrinsic cholinergic interneurons, acetylcholinesterase is expressed more abundantly in the matrix than in the striosomes. At present, little is known about the different cholinergic functions of the striatal compartments. In this study, we examined gamma-aminobutyric acidergic (GABAergic) inputs to cholinergic interneurons in both compartments. We found that nicotinic receptor-mediated GABAergic responses were evoked more frequently in the matrix than in the striosomes. Furthermore, a single action potential of cholinergic neurons induced nicotinic receptor-mediated GABAergic inputs to the cholinergic neurons themselves, suggesting mutual connections that shape the temporal firing pattern of cholinergic neurons. The nicotinic receptor-mediated GABAergic responses were attenuated by continuous application of acetylcholine or the acetylcholinesterase inhibitor eserine and were enhanced by desformylflustrabromine, a positive allosteric modulator of the α4β2 subunit containing a nicotinic receptor. These results suggest that the nicotinic impact on the GABAergic responses are not uniform despite the massive and continuous cholinergic innervation. It has been reported that differential activation of neurons in the striosomes and the matrix produce a repetitive behavior called stereotypy. Drugs acting on α4β2 nicotinic receptors might provide potential tools for moderating the imbalanced activities between the compartments.

Asynchronous presynaptic glutamate release enhances neuronal excitability during the post-spike refractory period.

Many excitatory synapses in the brain release glutamate with both synchronous and asynchronous components. Immediately following an action potential, neurons display a reduced excitability due to the post-spike afterhyperpolarization (AHP). This gives rise to a relative refractory period. When an action potential is evoked by glutamate synaptic input possessing asynchronous release, the delayed glutamate release events act to depolarize the neuron during the AHP and overcome the relative refractory period. These results demonstrate a new role for asynchronous release in regulating post-spike excitability and the relative refractory period in central neurons. Post-spike afterhyperpolarizations (AHPs) functionally inhibit neuronal excitability for tens to hundreds of milliseconds following each action potential. This imposes a relative refractory period during which synaptic excitation is less effective at evoking spikes. Here we asked whether some synapses have mechanisms in place that allow them to overcome the AHP and drive spiking in target cells during this period of reduced excitability. We examined glutamate synapses onto oxytocin and vasopressin neurons in the paraventricular nucleus of the hypothalamus. These synapses can display pronounced asynchronous glutamate release following a single presynaptic spike, with the time course of release being similar to that of the post-spike AHP. To test whether asynchronous release is more effective at overcoming the relative refractory period, we evoked a single action potential with either a brief synchronous depolarization or an asynchronous potential and then assessed excitability at multiple time points following the spike. Neurons receiving asynchronous depolarizing synaptic inputs had a shorter relative refractory period than those receiving synchronous depolarizations. Our data demonstrate that synapses releasing glutamate in an asynchronous and delayed manner are ideally adapted to counter the AHP. By effectively overcoming the relative refractory period, the kinetics of excitatory synaptic input can play an important role in controlling post-spike excitability.

Development and physiology of GABAergic feedback excitation in parvalbumin expressing interneurons of the mouse basolateral amygdala.

We have previously shown that in the basolateral amygdala (BLA), action potentials in one type of parvalbumin (PV)-expressing GABAergic interneuron can evoke a disynaptic feedback excitatory postsynaptic potential (fbEPSP) onto the same presynaptic interneuron. Here, using whole-cell recordings from PV-expressing interneurons in acute brain slices we expand on this finding to show that this response is first detectable at 2-week postnatal, and is most prevalent in animals beyond 3weeks of age (>P21). This circuit has a very high fidelity, and single action potential evoked fbEPSPs display few failures. Reconstruction of filled neurons, and electron microscopy show that interneurons that receive feedback excitation make symmetrical synapses on both the axon initial segments (AIS), as well as the soma and proximal dendrites of local pyramidal neurons, suggesting fbEPSP interneurons are morphologically distinct from the highly specialized chandelier neurons that selectively target the axon initial segment of pyramidal neurons. Single PV interneurons could trigger very large (~ 1nA) feedback excitatory postsynaptic currents (fbEPSCs) suggesting that these neurons are heavily reciprocally connected to local glutamatergic principal cells. We conclude that in the BLA, a subpopulation of PV interneurons forms a distinct neural circuit in which a single action potential can recruit multiple pyramidal neurons to discharge near simultaneously and feed back onto the presynaptic interneuron.

Simulation Of The Electrical Field Generated By Bipolar And Ganglion Cells In The Electrically Stimulated Retina

Event Abstract Back to Event Simulation Of The Electrical Field Generated By Bipolar And Ganglion Cells In The Electrically Stimulated Retina Paul Werginz1* and Frank Rattay1 1 Vienna University of Technology, Institute for Analysis and Scientific Computing, Austria Motivation Recording of the (summed) response of bipolar (BCs) and ganglion cells (GCs) is essential for examination of cell activation in in-vitro experiments in order to gain a better understanding of the electrically stimulated retina. Here, we describe a proof of concept to simulate these electrical fields during extracellular stimulation. Material and Methods The response of BCs and GCs to extracellular electrical stimulation is computed with multi-compartment models. Our modeling discretizes a simplified 2-D cell geometry in space, using cylindrical and spherical compartments. Current flow between compartments and across the cell membrane leads to a system of ordinary differential equations (Eq. 1 [1]). Ionic currents are computed with a Hodgkin-Huxley-like model approach for 35 degree Celsius [2]. Here the extracellular field generated by a stimulating electrode is computed by a simple analytical solution for point sources in homogeneous media (Eq. 2) Extracellular recordings were computed with the same formula; see [3] for details. The used model of the ribbon synapse at BC terminals is presented in [4]. Eq. 1 Eq. 2 Results A single extracellular action potential is bi- or triphasic, depending on the position of the recording electrode and the site of spike initiation. For direct GC stimulation recordings at or close to the soma resulted in biphasic waveforms whereas recordings at the distal axon resulted in triphasic waveforms (Fig. 1). Additionally, the amplitude of the recorded waveforms varies upon the location of the recording electrode, the geometric properties of the model neuron (e.g. fiber diameter in Fig. 1B) and ion channel equipment along the neural membrane. When BC input was fed to the model GC bursts of action potentials were generated and in the extracellular recording the synaptic input could be displayed (Fig. 2). Calcium currents that drive the synaptic signaling cascade in BC terminal compartments, however, could not be revealed because of the stimulus artifact. Discussion Direct stimulation of GCs from the epiretinal space generated responses that are qualitatively similar to data obtained in in-vitro experiments [5]. Periodic synaptic input during repetitive stimulation led to bursting spiking activity in GCs. Applied stimulus amplitudes resulted in synaptic (indirect) activation whereas no direct GC spikes were generated. The spiking rate is decreased for later pulses because of vesicle depletion in the ribbon synapses. Recorded intra- and extracellular responses revealed the synaptic inputs, however, the signal was rather small in the extracellular recording. Conclusion The presented initial modeling study shows that it is generally feasible to simulate the extracellular field generated by retinal neurons during electrical stimulation.

Electrophysiological property and chemical sensitivity of primary afferent neurons that innervate rat whisker hair follicles.

Whisker hair follicles are sensory organs that sense touch and perform tactile discrimination in animals, and they are sites where sensory impulses are initiated when whisker hairs touch an object. The sensory signals are then conveyed by whisker afferent fibers to the brain for sensory perception. Electrophysiological property and chemical sensitivity of whisker afferent fibers, important factors affecting whisker sensory processing, are largely not known. In the present study, we performed patch-clamp recordings from pre-identified whisker afferent neurons in whole-mount trigeminal ganglion preparations and characterized their electrophysiological property and sensitivity to ATP, serotonin and glutamate. Of 97 whisker afferent neurons examined, 67% of them are found to be large-sized (diameter ≥45 µm) cells and 33% of them are medium- to small-sized (diameter <45 µm) cells. Almost every large-sized whisker afferent neuron fires a single action potential but many (40%) small/medium-sized whisker afferent neurons fire multiple action potentials in response to prolonged stepwise depolarization. Other electrophysiological properties including resting membrane potential, action potential threshold, and membrane input resistance are also significantly different between large-sized and small/medium-sized whisker afferent neurons. Most large-sized and many small/medium-sized whisker afferent neurons are sensitive to ATP and/or serotonin, and ATP and/or serotonin could evoke strong inward currents in these cells. In contrast, few whisker afferent neurons are sensitive to glutamate. Our results raise a possibility that ATP and/or serotonin may be chemical messengers involving sensory signaling for different types of rat whisker afferent fibers.

AMPA receptor inhibition by synaptically released zinc

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

The increase of intrinsic excitability of layer V pyramidal cells in the prelimbic medial prefrontal cortex of adult mice after peripheral inflammation

Symptoms including depression and hypofunction of cognitive and decision-making are commonly associated with chronic pain. Recent studies have shown that the state of the medial prefrontal cortex (mPFC) neurons is important in diverse high-order cognitive and emotional activity in animals and humans. The mPFC layer V neurons mainly integrate information from other brain areas. The abnormal activity and function of pyramidal neurons influence the signal processing in the mPFC. Here we observed the changes of the excitability of the prelimbic mPFC neurons by whole-cell current-clamp recordings in adult mouse with inflammatory pain induced by complete Freund's adjuvant (CFA). Results showed that resting membrane potential and membrane input resistance did not differ between CFA-treated and control animals. Single action potential (AP) did not differ in terms of amplitude, but rheobase, half AP duration, and decay time were significantly decreased, and voltage threshold was more hyperpolarized in CFA group. Although the firing adaptation did not differ in two groups, the repetitive AP firing number and initial firing rate were significantly increased in CFA group. These data suggest that the increase in the intrinsic excitability of prelimbic mPFC layer V pyramidal neurons may be involved, at least in part in peripheral inflammatory pain.

Functional Deficiencies in Fragile X Neurons Derived from Human Embryonic Stem Cells.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by silencing of the FMR1 gene. In this study, we describe for the first time the properties of neurons developed from human embryonic stem cells (hESCs) that carry the FMR1 mutation and are grown in culture for extended periods. These neurons are retarded compared with controls in several morphological and functional properties. In vitro neural differentiation of FX hESCs can thus serve as a most relevant system for the analysis of molecular mechanisms underlying the impaired neuronal functions in FXS.

Motor unit number estimation as a complementary test to routine electromyography in the diagnosis of amyotrophic lateral sclerosis

Electromyographic (EMG) abnormalities that reveal denervation and reinnervation caused by lower motor neuron degeneration do not reflect the number of motor units that determines muscle strength. Consequently, motor unit activity potential (MUAP) parameters do not reflect muscle dysfunction.The aim of the study was to compare the value of motor unit number estimation (MUNE) and MUAP parameters as indicators of clinical muscle dysfunction in patients with amyotrophic lateral sclerosis (ALS), and to analyze the role of MUNE as a supplement to the EMG criteria for the diagnosis of ALS.In 25 patients with ALS, MUNE by the multipoint incremental method in the abductor digiti minimi (ADM) and quantitative EMG in the first dorsal interosseous (FDI) were obtained. The Medical Research Council (MRC) scale was used to evaluate clinical muscle dysfunction. A strong correlation between the number of motor units evaluated by MUNE and ADM clinical function by the MRC scale was found (P<0.001). An increased value of surface-detected single motor action potential was associated with a decreased MRC score for ADM (P<0.1). No relation was found between MUAP parameters in FDI and MRC scores. Our data support the value of the MUNE method for the detection of motor unit loss in ALS, and it could be postulated that MUNE studies may be considered complementary tests for ALS in a future revision of ALS criteria.

Ionotropic glutamate receptor GluA4 and T-type calcium channel Cav 3.1 subunits control key aspects of synaptic transmission at the mouse L5B-POm giant synapse.

The properties and molecular determinants of synaptic transmission at giant synapses connecting layer 5B (L5B) neurons of the somatosensory cortex (S1) with relay neurons of the posteriomedial nucleus (POm) of the thalamus have not been investigated in mice. We addressed this by using direct electrical stimulation of fluorescently labelled single corticothalamic terminals combined with molecular perturbations and whole‐cell recordings from POm relay neurons. Consistent with their function as drivers, we found large‐amplitude excitatory postsynaptic currents (EPSCs) and multiple postsynaptic action potentials triggered by a single presynaptic action potential. To study the molecular basis of these two features, ionotropic glutamate receptors and low voltage‐gated T‐type calcium channels were probed by virus‐mediated genetic perturbation. Loss of GluA4 almost abolished the EPSC amplitude, strongly delaying the onset of action potential generation, but maintaining the number of action potentials generated per presynaptic action potential. In contrast, knockdown of the Cav3.1 subunit abrogated the driver function of the synapse at a typical resting membrane potential of −70 mV. However, when depolarizing the membrane potential to −60 mV, the synapse relayed single action potentials. Hence, GluA4 subunits are required to produce an EPSC sufficiently large to trigger postsynaptic action potentials within a defined time window after the presynaptic action potential, while Cav3.1 expression is essential to establish the driver function of L5B‐POm synapses at hyperpolarized membrane potentials.

Estimation of the Electrode-Fiber Bioelectrical Coupling From Extracellularly Recorded Single Fiber Action Potentials.

Selective peripheral neural interfaces are currently capable of detecting minute electrical signals from nearby nerve fibers as single fiber action potential (SFAP) waveforms. Each detected single unit has a distinct shape originating from the unique bioelectrical coupling that exists between the neuroprosthetic electrode, the nerve fiber and the extracellular milieu. The bioelectrical coupling manifests itself as a series of low-pass Bessel filters acting on the action currents along the nerve fiber. Here, we present a method to estimate the electrode-fiber bioelectrical coupling through a quantitative analysis of the spectral distribution of the single units extracellularly recorded with the thin-film longitudinal intrafascicular electrode (tfLIFE) in an in vivo mammalian peripheral nerve animal model. The bioelectrical coupling estimate is an estimate of the electrode sensitivity function traversed by the nerve fiber, suggesting that it is as a means to directly measure the spatial relationship between the nerve fiber and electrode. It not only reflects a shape change to the SFAP, but has implications for in situ nerve fiber location tracking, in situ diagnostics of nerves and neuroproshetic electrodes, and assessment of the biocompatibility of neural interfaces and the health of the reporting nerve fibers.

Nicotine-Mediated ADP to Spike Transition: Double Spiking in Septal Neurons.

The majority of neurons in lateral septum (LS) are electrically silent at resting membrane potential. Nicotine transiently excites a subset of neurons and occasionally leads to long lasting bursting activity upon longer applications. We have observed simultaneous changes in frequencies and amplitudes of spontaneous action potentials (AP) in the presence of nicotine. During the prolonged exposure, nicotine increased numbers of spikes within a burst. One of the hallmarks of nicotine effects was the occurrences of double spikes (known also as bursting). Alignment of 51 spontaneous spikes, triggered upon continuous application of nicotine, revealed that the slope of after-depolarizing potential gradually increased (1.4 vs. 3mV/ms) and neuron fired the second AP, termed as double spiking. A transition from a single AP to double spikes increased the amplitude of after-hyperpolarizing potential. The amplitude of the second (premature) AP was smaller compared to the first one, and this correlation persisted in regard to their duration (half-width). A similar bursting activity in the presence of nicotine, to our knowledge, has not been reported previously in the septal structure in general and in LS in particular.

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal-like neurons of entorhinal-cortex layer II.

Two types of principal neurons, stellate cells and pyramidal-like cells, are found in medial entorhinal-cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex-hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (Vh ) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near-threshold Vh levels, and was accompanied by an enhancement of excitability and spike-timing precision. This DAP was sensitive to voltage-gated Na(+)-channel block with TTx, but not to partial removal of extracellular Ca(2+). Application of 5-μM anandamide, which inhibited the resurgent and persistent Na(+) -current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal-like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular-Ca(2+) removal, and was sensitive to the low-voltage-activated Ca(2+)-channel blocker, NNC55-0396. This Ca(2+) dependence was not observed in the residual DAP evoked in pyramidal-like cells from likewise negative Vh levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer-II neurons, one Na(+)-dependent and active at near-threshold Vh levels in both stellate and-pyramidal-like cells, the other Ca(2+)-dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization.