Threshold For Action Potential Initiation Research Articles

Consequences of hyperphosphorylated tau on the morphology and excitability of hippocampal neurons in aged tau transgenic mice

The intracellular accumulation of hyperphosphorylated tau characterizes many neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia. A critical role for tau is supported by studies in transgenic mouse models expressing the P301L mutation with accumulation of hyperphosphorylated human tau in hippocampal pyramidal neurons of aged mice. Especially, the somatodendritic mislocalization of hyperphosphorylated tau seems to affect the neuronal network of the hippocampus. To show the consequences of aggregation of hyperphosphorylated tau within hippocampal neurons of aged mice, the CA1 pyramidal cells were analyzed morphologically and electrophysiologically. Here we demonstrate in the P301L pR5 mouse model that hyperphosphorylated tau leads to an increase in stubby spines and filopodia, as well as a decrease in total dendritic length of hippocampal pyramidal neurons due to a decrease in apical dendritic length and nodes. This atrophy is in line with the significant reduction in CA1 long-term potentiation. Furthermore, mutant tau induced a depolarized threshold for action potential initiation and an increased current of inward rectifying potassium channels, which should lead, together with the long-term potentiation decrease, to a decreased excitability of CA1 neurons.

Excavatolide-B Enhances Contextual Memory Retrieval via Repressing the Delayed Rectifier Potassium Current in the Hippocampus.

Memory retrieval dysfunction is a symptom of schizophrenia, autism spectrum disorder (ASD), and absence epilepsy (AE), as well as an early sign of Alzheimer’s disease. To date, few drugs have been reported to enhance memory retrieval. Here, we found that a coral-derived natural product, excavatolide-B (Exc-B), enhances contextual memory retrieval in both wild-type and Cav3.2−/− mice via repressing the delayed rectifier potassium current, thus lowering the threshold for action potential initiation and enhancing induction of long-term potentiation (LTP). The human CACNA1H gene encodes a T-type calcium channel (Cav3.2), and its mutation is associated with schizophrenia, ASD, and AE, which are all characterized by abnormal memory function. Our previous publication demonstrated that Cav3.2−/− mice exhibit impaired contextual-associated memory retrieval, whilst their retrieval of spatial memory and auditory cued memory remain intact. The effect of Exc-B on enhancing the retrieval of context-associated memory provides a hope for novel drug development.

Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment

Hyperphosphorylated tau has a critical role in tauopathies such as Alzheimer’s disease and frontotemporal dementia, impairing neuronal function and eventually leading to neurodegeneration. A critical role for tau is supported by studies in transgenic mouse models that express the P301L tau mutation found in cases of familial frontotemporal dementia, with the accumulation of hyperphosphorylated tau in the hippocampus causing reductions in hippocampal long-term potentiation and impairments in spatial learning and memory. However, what has remained unexplored is the role of hyperphosphorylated tau in reducing neuronal excitability. Here, we show in two complementary P301L tau transgenic mouse models that hyperphosphorylated tau induces a more depolarized threshold for action potential initiation and reduces firing in hippocampal CA1 neurons, which was rescued by the suppression of transgenic tau. Furthermore, using mutagenesis and primary hippocampal neuronal cultures, we reveal that this reduction in neuronal excitability results from the relocation of the axon initial segment (AIS) down the axon in a tau phosphorylation-dependent manner. We also demonstrate that this effect is microtubule-dependent. In addition, pharmacological stabilization was found to prevent both the structural and functional deficits caused by tau hyperphosphorylation. Finally, we demonstrate that the AIS of neurons from tau transgenic mice is further down the axon, which correlates with a reduction in excitability. We therefore propose that a reduction in hippocampal excitability due to a tau-mediated distal relocalization of the AIS contributes to the hippocampal dysfunction observed in tauopathies.

Upregulation of T-type Ca2+ channels in long-term diabetes determines increased excitability of a specific type of capsaicin-insensitive DRG neurons.

BackgroundPrevious studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2–4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca2+ current. In longer–term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to pain. In this study, we examined functional expression of T-type Ca2+ channels in capsaicin-insensitive DRG neurons and excitability of these neurons in longer-term diabetic rats and in thermally hypoalgesic diabetic rats.ResultsHere we have demonstrated that in STZ-diabetes T-type current was upregulated in capsaicin-insensitive low-pH-sensitive small-sized nociceptive DRG neurons of longer-term diabetic rats and thermally hypoalgesic diabetic rats. This upregulation was not accompanied by significant changes in biophysical properties of T-type channels suggesting that a density of functionally active channels was increased. Sensitivity of T-type current to amiloride (1 mM) and low concentration of Ni2+ (50 μM) implicates prevalence of Cav3.2 subtype of T-type channels in the capsaicin-insensitive low-pH-sensitive neurons of both naïve and diabetic rats. The upregulation of T-type channels resulted in the increased neuronal excitability of these nociceptive neurons revealed by a lower threshold for action potential initiation, prominent afterdepolarizing potentials and burst firing. Sodium current was not significantly changed in these neurons during long-term diabetes and could not contribute to the diabetes-induced increase of neuronal excitability.ConclusionsCapsaicin-insensitive low-pH-sensitive type of DRG neurons shows diabetes-induced upregulation of Cav3.2 subtype of T-type channels. This upregulation results in the increased excitability of these neurons and may contribute to nonthermal nociception at a later-stage diabetes.

Action potential threshold of wide dynamic range neurons in rat spinal dorsal horn evoked by manual acupuncture at ST36

This paper investigates the dynamic of action potential initiation in the wide dynamic range (WDR) neurons in spinal dorsal horn of rats evoked by manual acupuncture at ST36 with three different stimulation frequencies. It is observed that the voltage threshold for action potential initiation in WDR neurons is dynamic and varies over a wide range of membrane potential. The broad variations in spike threshold are robust to the acupuncture frequency. It is also shown that the spike threshold is very sensitive to the maximal rate of rise of the action potential upstroke, and there is an obviously inverse relation between them. Our study provides a new perspective on the analysis of acupuncture-evoked signals. The findings could help to interpret how the individual spike evoked by manual acupuncture is generated in WDR neurons, which may contribute to uncover how acupuncture signals are encoded in its afferent neural pathway.

Effects of HIV-1 Tat on enteric neuropathogenesis.

The gastrointestinal (GI) tract presents a major site of immune modulation by HIV, resulting in significant morbidity. Most GI processes affected during HIV infection are regulated by the enteric nervous system. HIV has been identified in GI histologic specimens in up to 40% of patients, and the presence of viral proteins, including the trans-activator of transcription (Tat), has been reported in the gut indicating that HIV itself may be an indirect gut pathogen. Little is known of how Tat affects the enteric nervous system. Here we investigated the effects of the Tat protein on enteric neuronal excitability, proinflammatory cytokine release, and its overall effect on GI motility. Direct application of Tat (100 nm) increased the number of action potentials and reduced the threshold for action potential initiation in isolated myenteric neurons. This effect persisted in neurons pretreated with Tat for 3 d (19 of 20) and in neurons isolated from Tat(+) (Tat-expressing) transgenic mice. Tat increased sodium channel isoforms Nav1.7 and Nav1.8 levels. This increase was accompanied by an increase in sodium current density and a leftward shift in the sodium channel activation voltage. RANTES, IL-6, and IL-1β, but not TNF-α, were enhanced by Tat. Intestinal transit and cecal water content were also significantly higher in Tat(+) transgenic mice than Tat(-) littermates (controls). Together, these findings show that Tat has a direct and persistent effect on enteric neuronal excitability, and together with its effect on proinflammatory cytokines, regulates gut motility, thereby contributing to GI dysmotilities reported in HIV patients.

Action potential initiation in damaged axon initial segment

The effects of physical trauma to nerve cells are of great interest in neuroscience and neuromedicine. The ion channels kinetics are affected by the trauma-induced damage to neural membrane structure. The density of affected channels is at its highest in the nodes and the axon initial segment (AIS). In this study we focus on the consequences of modifying sodium channels kinetics in the AIS to model the effect of damage. It is inspired by a similar study on damaged nodes of Ranvier [1] and by experiments done in vivo on AIS [2,3]. The AIS and the nodes of Ranvier have similar mixtures of channels, but differ in their shapes. Whereas the nodal channels are clustered at high density near a large volume of channel free low capacitance membrane the AIS is long with varying density profiles for each ion channel types. The AIS is the location for action potential (AP) initiation. A neuron was simulated as three sections, each containing multiple compartments: the soma, the initial segment and the myelinated axon. Each compartment was modeled by the classical Hodgkin-Huxley equations using appropriate densities for each type of ion channel. The types of channels and their distributions were based on a previous study looking specifically at cortical pyramidal neurons having Nav1.2 and Nav1.6 at the AIS [4]. The kinetics were modified according to the coupled left-shift (CLS) model [1,5]. This model is based on observations showing that in damaged membranes Na channel kinetic midpoints shift leftward to more hyperpolarized values, effectively rendering the channels hypersensitive. In this study we explore the impact of CLS on the generation of APs in the AIS. The simulation includes NaK-ATPase pumps and inner and outer ion concentrations are monitored. With increased damage, the greater ionic conductance at rest potential (Na-leak conductance) puts greater strain on the pumps. In the case of a single node, hyperexcitability, tonic firing, burst firing, or inexcitability is observed for increasing values of CLS. We explored the effect of damage on the initiation and propagation of APs during repeated stimuli which deplete concentration gradients. Our model allows the investigation of the effect of CLS on the location and duration of the initiation and the threshold duration of Ap-initiatingcurrents. We find that the current threshold for action potential initiation decreases with increased damage or decreased length of the AIS.

Over-Expression of TRESK K+ Channels Reduces the Excitability of Trigeminal Ganglion Nociceptors

TWIK-related spinal cord K+ (TRESK) channel is abundantly expressed in trigeminal ganglion (TG) and dorsal root ganglion neurons and is one of the major background K+ channels in primary afferent neurons. Mutations in TRESK channels are associated with familial and sporadic migraine. In rats, both chronic nerve injury and inflammation alter the expression level of TRESK mRNA. Functional studies indicate that reduction of endogenous TRESK channel activity results in hyper-excitation of primary afferent neurons, suggesting that TRESK is a potential target for the development of new analgesics. However, whether and how enhancing TRESK channel activity would decrease the excitability of primary afferent neurons has not been directly tested. Here, we over-expressed TRESK subunits in cultured mouse TG neurons by lipofectamine-mediated transfection and investigated how this altered the membrane properties and the excitability of the small-diameter TG population. To account for the heterogeneity of neurons, we further divided small TG neurons into two groups, based on their ability to bind to fluorescently-labeled isolectin B (IB4). The transfected TG neurons showed a 2-fold increase in the level of TRESK proteins. This was accompanied by a significant increase in the fraction of lamotrigine-sensitive persistent K+ currents as well as the size of total background K+ currents. Consequently, both IB4-positive and IB4-negative TG neurons over-expressing TRESK subunits exhibited a lower input resistance and a 2-fold increase in the current threshold for action potential initiation. IB4-negative TG neurons over-expressing TRESK subunits also showed a significant reduction of the spike frequency in response to supra-threshold stimuli. Importantly, an increase in TRESK channel activity effectively inhibited capsaicin-evoked spikes in TG neurons. Taken together, our results suggest that potent and specific TRESK channel openers likely would reduce the excitability of primary afferent neurons and therefore are potential therapeutics for the treatment of migraine and other chronic pain symptoms.

Functional Analysis of a Migraine-Associated TRESK K+ Channel Mutation

Recent genetic and functional studies suggest that migraine may result from abnormal activities of ion channels and transporters. A frameshift mutation in the human TWIK-related spinal cord K(+) (TRESK) channel has been identified in migraine with aura patients in a large pedigree. In Xenopus oocytes, mutant TRESK subunits exert a dominant-negative effect on whole-cell TRESK currents. However, questions remain as to whether and how mutant TRESK subunits affect the membrane properties and the excitability of neurons in the migraine circuit. Here, we investigated the functional consequences of the mutant TRESK subunits in HEK293T cells and mouse trigeminal ganglion (TG) neurons. First, we found that mutant TRESK subunits exhibited dominant-negative effects not only on the size of the whole-cell TRESK currents, but also on the level of TRESK channels on the plasma membrane in HEK293T cells. This likely resulted from the heterodimerization of wild-type and mutant TRESK subunits. Next, we expressed mutant TRESK subunits in cultured TG neurons and observed a significant decrease in the lamotrigine-sensitive K(+) current, suggesting that the mutant TRESK subunits have a dominant-negative effect on currents through the endogenous TRESK channels. Current-clamp recordings showed that neurons expressing mutant TRESK subunits had a higher input resistance, a lower current threshold for action potential initiation, and a higher spike frequency in response to suprathreshold stimuli, indicating that the mutation resulted in hyperexcitability of TG neurons. Our results suggest a possible mechanism through which the TRESK mutation increases the susceptibility of migraine headache.

Glutamate modulates the firing rate in oculomotor nucleus motoneurons as a function of the recruitment threshold current

Studies in alert preparations have demonstrated that ocular motoneurons exhibit a phasic–tonic firing rate related to eye velocity and position, respectively. The slopes of these relationships are higher in motoneurons with higher recruitment threshold and have been proposed to depend upon synaptic input. To investigate this hypothesis, motoneurons of the rat oculomotor nucleus were recorded in a brain slice preparation in control conditions and during glutamate (5 μm) application to the bath. Glutamate did not affect membrane potential or input resistance, but produced a decrease in rheobase and depolarization voltage as a function of the current needed for generating a maintained repetitive discharge (recruitment threshold current). In addition, glutamate compressed the range of recruitment threshold current (0.1–0.4 nA) as compared to the control (0.15–0.7 nA). Glutamate exposed motoneurons showed an increase in the tonic frequency gain and the peak frequency. Such increments depended on the recruitment threshold current and the last recruited motoneurons almost doubled the tonic frequency gain (35.2 vs. 57.9 spikes s(−1) nA(−1)) and the peak frequency (52.4 vs. 102.6 spikes s(−1)). Finally, glutamate increased the spike frequency adaptation due to a significant increase in the phasic firing component as compared to the tonic one. In conclusion, glutamate modulates tonic and phasic discharge properties as a function of the recruitment threshold current and, presumably, motoneuron size. These findings contribute to understand the link between cellular functions and motoneuron discharge during oculomotor behaviour.

Differential Effects of Axon Initial Segment and Somatodendritic GABAAReceptors on Excitability Measures in Rat Dentate Granule Neurons

GABA(A) receptors are found on the somatodendritic compartment and on the axon initial segment of many principal neurons. The function of axonal receptors remains obscure, although it is widely assumed that axonal receptors must have a strong effect on excitability. We found that activation of GABA(A) receptors on the dentate granule neuron axon initial segment altered excitability by depolarizing the voltage threshold for action potential initiation under conditions that minimally affected overall cell input resistance. In contrast, activation of somatic GABA(A) receptors strongly depressed the input resistance of granule neurons without affecting the voltage threshold of action potential initiation. Although these effects were observed over a range of intracellular chloride concentrations, average voltage threshold was unaffected when E(Cl) rendered GABA(A) axon initial segment responses explicitly excitatory. A compartment model of a granule neuron confirmed these experimental observations. Low ambient agonist concentrations designed to activate granule neuron tonic currents did not stimulate axonal receptors sufficiently to raise voltage threshold. Using excitatory postsynaptic current (EPSC)-like depolarizations, we show physiological consequences of axonal versus somatic GABA(A) receptor activation. With axonal inhibition, individual excitatory postsynaptic potentials (EPSPs) largely retained their amplitude and time course, but EPSPs that were suprathreshold under basal conditions failed to reach threshold with GABA(A) activation. By contrast, somatic inhibition depressed individual EPSPs because of strong shunting. Our results suggest that axonal GABA(A) receptors have a privileged effect on voltage threshold and that two major measures of neuronal excitability, voltage threshold and rheobase, are differentially affected by axonal and somatic GABA(A) receptor activation.

Axonal sodium channel distribution shapes the depolarized action potential threshold of dentate granule neurons

Intrinsic excitability is a key feature dictating neuronal response to synaptic input. Here we investigate the recent observation that dentate granule neurons exhibit a more depolarized voltage threshold for action potential initiation than CA3 pyramidal neurons. We find no evidence that tonic GABA currents, leak or voltage-gated potassium conductances, or the expression of sodium channel isoform differences can explain this depolarized threshold. Axonal initial segment voltage-gated sodium channels, which are dominated by the Na(V)1.6 isoform in both cell types, distribute more proximally and exhibit lower overall density in granule neurons than in CA3 neurons. To test possible contributions of sodium channel distributions to voltage threshold and to test whether morphological differences participate, we performed simulations of dentate granule neurons and of CA3 pyramidal neurons. These simulations revealed that cell morphology and sodium channel distribution combine to yield the characteristic granule neuron action potential upswing and voltage threshold. Proximal axon sodium channel distribution strongly contributes to the higher voltage threshold of dentate granule neurons for two reasons. First, action potential initiation closer to the somatodendritic current sink causes the threshold of the initiating axon compartment to rise. Second, the proximity of the action potential initiation site to the recording site causes somatic recordings to more faithfully reflect the depolarized threshold of the axon than in cells like CA3 neurons, with distally initiating action potentials. Our results suggest that the proximal location of axon sodium channels in dentate granule neurons contributes to the intrinsic excitability differences between DG and CA3 neurons and may participate in the low-pass filtering function of dentate granule neurons.

Regulation of firing frequency in nociceptive neurons by pro-inflammatory mediators

Nociceptive neurons generate trains of action potentials in response to painful stimuli, and the frequency of firing signals the intensity of the pain. Pro-inflammatory mediators such as prostaglandin E2 (PGE2) enhance the sensation of pain by increasing the frequency of action potential firing in response to a given level of painful stimulus. The mechanism by which the firing frequency is enhanced is discussed in the present review. One hypothesis proposes that the threshold for action potential initiation is lowered because the activation curve of a nociceptor-specific voltage-activated Na current, Na(V)1.8, is shifted to more negative values by PGE2. Recent measurements in our lab show, however, that the action potential threshold in fact changes little when AP firing is accelerated by PGE2. The enhanced firing is, however, abolished by a blocker of an inward current activated by hyperpolarisation, called I(h). The voltage sensitivity of I(h) shifts in the positive direction in small nociceptive neurons when they are exposed to pro-inflammatory mediators, such as PGE2, which activate adenylate cyclase and therefore increase levels of cAMP. By this mechanism the inward current between the resting membrane potential and the threshold for firing of action potentials is enhanced, and the rate of depolarisation in the interval between action potentials is therefore increased. We conclude that the major mechanism responsible for increasing action potential firing following tissue damage or metabolic stress is the hyperpolarisation-activated inward current, I(h), and that other mechanisms play at most a minor role.

Small voltage changes at nerve terminals travel up axons to affect action potential initiation

Nerve terminals are generally considered the destination points for electrical signals, which propagate unidirectionally from the soma to nerve terminals. Here, we demonstrate that small hyperpolarizations or depolarizations (~10 mV), generated under physiological conditions in rat nerve terminals, backpropagate up the axon (~400 – 800 µm), and change the threshold for initiating action potentials and thus firing patterns. These results suggest a novel mechanism for information processing in neurons and neuronal circuits.

Etomidate Reduces Initiation of Backpropagating Dendritic Action Potentials: Implications for Sensory Processing and Synaptic Plasticity During Anesthesia

Anesthetics may induce specific changes that alter the balance of activity within neural networks. Here we describe the effects of the GABA(A) receptor potentiating anesthetic etomidate on sensory processing, studied in a cerebellum-like structure, the electrosensory lateral line lobe (ELL) of mormyrid fish, in vitro. Previous studies have shown that the ELL integrates sensory input and removes predictable features by comparing reafferent sensory signals with a descending electromotor command-driven corollary signal that arrives in part through parallel fiber synapses with the apical dendrites of GABAergic interneurons. These synapses show spike timing-dependent depression when presynaptic activation is associated with postsynaptic backpropagating dendritic action potentials. Under etomidate, almost all neurons become tonically hyperpolarized. The threshold for action potential initiation increased for both synaptic activation and direct intracellular depolarization. Synaptically evoked inhibitory postsynaptic potentials (IPSPs) were also strongly potentiated and prolonged. Current source density analysis showed that backpropagation of action potentials through the apical dendritic arborization in the molecular layer was reduced but could be restored by increasing stimulus strength. These effects of etomidate were blocked by bicuculline or picrotoxin. It is concluded that etomidate affects both tonic and phasic inhibitory conductances at GABA(A) receptors and that increased shunting inhibition at the level of the proximal dendrites also contributes to increasing the threshold for action potential backpropagation. When stimulus strength is sufficient to evoke backpropagation, repetitive association of synaptic excitation with postsynaptic action potential initiation still results in synaptic depression, showing that etomidate does not interfere with the molecular mechanism underlying plastic modulation.

Distinct Membrane Effects of Spinal Nerve Ligation on Injured and Adjacent Dorsal Root Ganglion Neurons in Rats

Painful peripheral nerve injury results in disordered sensory neuron function that contributes to the pathogenesis of neuropathic pain. However, the relative roles of neurons with transected axons versus intact adjacent neurons have not been resolved. An essential first step is identification of electrophysiologic changes in these two neuronal populations after partial nerve damage. Twenty days after spinal nerve ligation (SNL), intracellular recordings were obtained from axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent, intact L4 neurons, as well as from control neurons and others subjected to sham-SNL surgery. Pronounced electrophysiologic changes were seen only in L5 neurons after SNL. Both Aalpha/beta and Adelta neuron types showed increased action potential duration, decreased afterhyperpolarization amplitude and duration, and decreased current threshold for action potential initiation. Aalpha/beta neurons showed resting membrane potential depolarization, and increased repetitive firing during sustained depolarization developed in Adelta neurons. The afterhyperpolarization duration in neurons with C fibers shortened after axotomy. In contrast to the axotomized L5 neurons, neighboring L4 neurons showed no changes in action potential duration, afterhyperpolarization dimensions, or excitability after SNL. Depolarization rate (dV/dt) increased after SNL in L4 Aalpha/beta and Adelta neurons but decreased in L5 neurons. Time-dependent rectification during hyperpolarizing current injection (sag) was greater after SNL in Aalpha/beta L4 neurons compared with L5. Sham-SNL surgery produced only a decreased input resistance in Aalpha/beta neurons and a decreased conduction velocity in medium-sized cells. In the L5 ganglion after axotomy, a novel set of neurons, consisting of 24% of the myelinated population, exhibited long action potential durations despite myelinated neuron conduction velocities, particularly depolarized resting membrane potential, low depolarization rate, and absence of sag. These findings indicate that nerve injury-induced electrical instability is restricted to axotomized neurons and is absent in adjacent intact neurons.

Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons.

Mudpuppy parasympathetic neurons exhibit spontaneous miniature hyperpolarizations (SMHs) that are generated by potassium currents, which are spontaneous miniature outward currents (SMOCs), flowing through clusters of large conductance voltage- and calcium (Ca(2+))-activated potassium (BK) channels. The underlying SMOCs are initiated by a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Perforated-patch whole cell voltage recordings were used to determine whether activation of SMHs contributed to action potential (AP) repolarization or affected the latency to AP generation. Blockade of BK channels by iberiotoxin (IBX, 100 nM) slowed AP repolarization and increased AP duration. Treatment with omega-conotoxin GVIA (3 microM) or nifedipine (10 microM) to inhibit Ca(2+) influx through N- or L-type voltage-dependent calcium channels (VDCCs), respectively, also decreased the rate of AP repolarization and increased AP duration. Elimination of CICR by treatment with either thapsigargin (1 microM) or ryanodine (10 microM) produced no significant change in AP repolarization or duration. Blockade of BK channels with IBX and inhibition of N-type VDCCs with omega-conotoxin GVIA, but not inhibition of L-type VDCCs with nifedipine, decreased the latency of AP generation. A decrease in latency to AP generation occurred with elimination of SMHs by inhibition of CICR following treatment with thapsigargin. Ryanodine treatment decreased AP latency in three of six cells. Apamin (100 nM) had no affect on AP repolarization, duration, or latency to AP generation, but did decrease the hyperpolarizing afterpotential (HAP). Inhibition of L-type VDCCs by nifedipine also decreased HAP amplitude. Inhibition of CICR by either thapsigargin or ryanodine treatment increased the number of APs generated with long depolarizing current pulses, whereas exposure to IBX or omega-conotoxin GVIA depressed excitability. We conclude that CICR, the process responsible for SMH generation, represents a unique mechanism to modulate the response to subthreshold depolarizing currents that drive the membrane potential toward the threshold for AP initiation but does not contribute to AP repolarization. Subthreshold depolarizations would not activate sufficient numbers of VDCCs to allow Ca(2+) influx to elevate [Ca(2+)](i) to the extent needed to directly activate nearby BK channels. However, the elevation in [Ca(2+)](i) is sufficient to trigger CICR from ryanodine-sensitive Ca(2+) stores. Thus CICR acts as an amplification mechanism to trigger a local elevation of [Ca(2+)](i) near a cluster of BK channels to activate these channels at negative levels of membrane potential.

Ion channel properties underlying axonal action potential initiation in pyramidal neurons.

A high density of Na+ channels in the axon hillock, or initial segment, is believed to determine the threshold for action potential initiation in neurons. Here we report evidence for an alternative mechanism that lowers the threshold in the axon. We investigated properties and distributions of ion channels in outside-out patches from axons and somata of layer 5 pyramidal neurons in rat neocortical slices. Na+ channels in axonal patches (<30 microm from the soma) were activated by 7 mV less depolarization than were somatic Na+ channels. A-type K+ channels, which were prominent in somatic and dendritic patches, were rarely seen in axonal patches. We incorporated these findings into numerical simulations which indicate that biophysical properties of axonal channels, rather than a high density of channels in the initial segment, are most likely to determine the lowest threshold for action potential initiation.

Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2+ currents in mouse pancreatic B-cells towards negative potentials.

The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.

Coding and adaptation during mechanical stimulation in the leech nervous system.

The experiments described here were designed to characterise sensory coding and adaptation during mechanical stimulation in the leech (Hirudo medicinalis). A chain of three ganglia and a segment of the body wall connected to the central ganglion were used. Eight extracellular suction pipettes and one or two intracellular electrodes were used to record action potentials from all mechanosensory neurones of the three ganglia. When the skin of the body wall was briefly touched with a filament exerting a force of about 2 mN, touch (T) cells in the central ganglion, but also those in adjacent ganglia (i.e. anterior and posterior), fired one or two action potentials. However, the threshold for action potential initiation was lower for T cells in the central ganglion than for those in adjacent ganglia. The timing of the first evoked action potential in a T cell was very reproducible with a jitter often lower than 100 us. Action potentials in T cells were not significantly correlated. When the force exerted by the filament was increased above 20 mN, pressure (P) cells in the central and neighbouring ganglia fired action potentials. Action potentials in P cells usually followed those evoked in T cells with a delay of about 20 ms and had a larger jitter of 0.5-10 ms. With stronger stimulations exceeding 50 mN, noxious (N) cells also fired action potentials. With such stimulations the majority of mechanosensory neurones in the three ganglia fired action potentials. The spatial properties of the whole receptive field of the mechanosensory neurones were explored by touching different parts of the skin. When the mechanical stimulation was applied for a longer time, i.e. 1 s, only P cells in the central ganglion continued to fire action potentials. P cells in neighbouring ganglia fully adapted after firing two or three action potentials.P cells in adjacent ganglia, having fully adapted to a steady mechanical stimulation of one part of the skin, fired action potentials following stimulation of a different region of the skin. These results indicate that a brief and localised stimulation of the skin can activate more than a dozen different mechanosensory neurones in the three ganglia and after 100 ms of steady stimulation many of these mechanosensory neurones stop firing action potentials and fully adapt. Adaptation occurs primarily at the nerve endings and mechanosensory neurones can quickly respond to mechanical stimulation at a different location on the skin.