Role Of Action Potentials Research Articles

Effects of Cooling Temperatures via Thermal K2P Channels on Regeneration of High-Frequency Action Potentials at Nodes of Ranvier of Rat Aβ-Afferent Nerves.

Temperature-sensitive two-pore domain potassium channels (thermal K2P) are recently shown to cluster at nodes of Ranvier (NRs) and play a key role in action potential (AP) regeneration and conduction on Aβ-afferent nerves. Cooling temperatures affect AP regeneration and conduction on Aβ-afferent nerves but the underlying mechanisms are not completely understood. Here, we have performed patch-clamp recordings directly at the NR in an ex vivo trigeminal nerve preparation. We have characterized the effects of cooling temperatures on intrinsic electrophysiological properties and AP regeneration at the NR on rat Aβ-afferent nerves, and determined whether and how thermal K2P channels may be involved in the effects of cooling temperatures. We show that cooling temperatures from 35°C to 15°C decrease outward leak currents, increase input resistance, depolarize resting membrane potential (RMP), broaden AP width and increase latency of AP threshold at the NR. We further demonstrate that cooling temperatures impair regeneration of high-frequency AP trains at the NR. The effects of cooling temperatures on the intrinsic electrophysiological properties and regeneration of high-frequency AP trains at the NR can be partially reversed by BL-1249 (BL), arachidonic acid (AA), and intra-axonal protons, three thermal K2P activators, indicating the involvement of thermal K2P channels. Moreover, we show that at cooling temperatures there are interplays among thermal K2P channels, RMPs, and voltage-gated Na+ channels, which together limit regeneration of high-frequency AP trains at the NR. Our findings demonstrate a new role of thermal K2P channels in temperature-dependent conduction of high-frequency sensory signals.

Neuronal activity and AMPA-type glutamate receptor activation regulates the morphological development of oligodendrocyte precursor cells.

Myelination is initiated when oligodendrocyte precursor cells (OPC) contact target axons. Neuronal activity promotes myelination through actions that may involve OPC AMPA and NMDA glutamate receptors (AMPAR, NMDAR). Therefore, activity and AMPAR/NMDAR activation are predicted to promote the morphological development of OPC. AMPAR can regulate OPC development, but this analysis was not performed in situ and the role of action potentials was not examined. Hence, the influence of activity and AMPAR on OPC morphology and development remain untested in the CNS where axon-glial interactions are preserved. Data on NMDAR are mixed with conflicting results from in vitro and in vivo work. To gain a fuller understanding of activity-dependent OPC development in situ, we explored the role of AMPAR and NMDAR in cerebellar slice cultures that permit the study of endogenous OPC development and myelination. The structure of individual OPC was resolved from cells labeled with membrane targeted GFP. Morphological data were then validated against assays of OPC development. Blocking either activity or AMPAR impaired the morphological development of OPC and promoted proliferation and differentiation. Increasing the pool of oligodendrocytes by blocking activity or AMPAR failed to promote myelination. Instead both myelination and the expression of myelin basic protein were reduced by these treatments suggesting that full differentiation to a myelinating phenotype did not occur. Blocking NMDAR left OPC proliferation, differentiation and morphology unchanged. These data indicate an important role for AMPAR but not NMDAR in mediating the activity-dependent signals that regulate OPC morphology, development and myelination.

The role of action potentials in determining neuron-type-specific responses to nitric oxide.

The electrical activity in developing and mature neurons determines the intracellular calcium concentration ([Ca(2+)]i), which in turn is translated into biochemical activities through various signaling cascades. Electrical activity is under control of neuromodulators, which can alter neuronal responses to incoming signals and increase the fidelity of neuronal communication. Conversely, the effects of neuromodulators can depend on the ongoing electrical activity within target neurons; however, these activity-dependent effects of neuromodulators are less well understood. Here, we present evidence that the neuronal firing frequency and intrinsic properties of the action potential (AP) waveform set the [Ca(2+)]i in growth cones and determine how neurons respond to the neuromodulator nitric oxide (NO). We used two well-characterized neurons from the freshwater snail Helisoma trivolvis that show different growth cone morphological responses to NO: B5 neurons elongate filopodia, while those of B19 neurons do not. Combining whole-cell patch clamp recordings with simultaneous calcium imaging, we show that the duration of an AP contributes to neuron-specific differences in [Ca(2+)]i, with shorter APs in B19 neurons yielding lower growth cone [Ca(2+)]i. Through the partial inhibition of voltage-gated K(+) channels, we increased the B19 AP duration resulting in a significant increase in [Ca(2+)]i that was then sufficient to cause filopodial elongation following NO treatment. Our results demonstrate a neuron-type specific correlation between AP shape, [Ca(2+)]i, and growth cone motility, providing an explanation to how growth cone responses to guidance cues depend on intrinsic electrical properties and helping explain the diverse effects of NO across neuronal populations.

Role of sodium channels in the spontaneous excitability of early embryonic cardiomyocytes.

Sodium channels play an important role in action potentials. Moreover, some evidences recently suggested that sodium channels were responsible for murine sinoatrial node pacemaking. The aim of this study was to investigate the role of sodium channels in pacemaking in embryonic cardiomyocytes in early development stage (EDS). Whole-cell patch-clamp technique was employed to record sodium current of murine early embryonic cardiomyocytes. Current clamp technique was used to record the effect of 0.1, 1 and 10 μM tetrodotoxin (TTX) on embryonic cardiomyocytes pacemaking. Electro- physiology properties of sodium channels in embryonic cardiomyocytes corresponded to Nav1.5, and the IC₅₀ of TTX was 5.24 μM. TTX at 0.1 μM concentration had no effects on the pacemaking. TTX at 1 μM concentration, however, dramatically slowed the spontaneous beating rate from 73.975 ± 10.478 to 50.268 ± 10.476 cycle/min (P < 0.05), and the maximum upstroke velocity (dV/dtmax) of phase 4 from 0.074 ± 0.006 to 0.046 ± 0.007 V/s (P < 0.01). Furthermore, 1 μM TTX reduced the dV/dtmax of phase 0 from 16.405 ± 0.056 to 12.801 ± 0.084 V/s (P < 0.01), and increased the period of phase 4 from 710.342 ± 110.983 to 1320.618 ± 250.483 ms (P < 0.05). TTX at 1 μM also had some effects on the peak of phase 0 decreasing it from 40.621 ± 3.012 to 37.407 ± 2.749 mV (P < 0.05). But TTX at 1 μM had no effects on the period of phase 0. In some cells (9/13), TTX at 10 μM caused complete cessation of spontaneous action potentials. Our results suggested that the main expression subtype of sodium channels was Nav1.5 of early embryonic cardiomyocytes. And TTX-resistant sodium channels contributed to the initiation of action potentials of early embryonic cardiomyocytes, while TTX-sensitive sodium channels were not involved in initiation of action potentials.

Simultaneous Patch-Clamping and Calcium Imaging in Developing Dendrites

Calcium imaging has been used extensively to explore the role of action potential (AP) firing in the development of neuronal structure and synaptic function because increases in intracellular calcium ([Ca(2+)]i) reliably and, within a certain range, linearly reflect neuronal spiking activity. Patterns of APs in individual cells can be deduced from calcium recordings, which have typically been performed at the level of cell bodies. However, neurons are particularly susceptible to phototoxicity when they are illuminated at the soma. Furthermore, for some imaging experiments (e.g., those that address the interactions between dendrites and axons during synapse formation), the cell body of a given neuron may simply not be in the field of view. In these situations, it would be helpful to determine the spiking patterns of a neuron from the calcium activity in its subcellular compartments such as stretches of dendrites or axons. Here, we describe an approach for determining the relationship between AP firing and dendritic calcium transients by simultaneously imaging calcium transients in small dendritic stretches of hippocampal pyramidal neurons in slice cultures from neonatal rats and recording spiking activity with whole-cell patch-clamp recordings in these neurons. These experiments allow us to correlate the electrophysiological spiking pattern with the accompanying changes in the calcium concentration in individual dendritic segments.

Simulating Voltage-Gated Na and K Ion Channel Kinetics Using Hodgkin- Huxley Mode

Voltage-gated sodium channels play an important role in action potentials. If enough channels open during a change in the cell's membrane potential, a small but significant number of sodium (Na+) ions will move into the cell reducing its electrochemical gradient and further depolarizing the cell. Voltage-gated Na+channels play a fundamental role in the excitability of nerve and muscle cells. Na+channels both open and close more quickly than potassium (K+) channels, producing an influx of positive charge (Na+) toward the beginning of the action potential and an efflux (K+) toward the end. The study of K+ channels is essential as they appear to be more diverse in structure and function than any other types of ion channel. K+ channels shape the action potential, set the membrane potential, and determine firing rates. There already are some drugs in clinical use that target K+ channels which improve our ability to regulate excitability.In this research, we study the influence of voltage dependence on channel activation and inactivation by simulating different channel subtypes as well as the effect of different kinetic parameters on membrane excitability.

Modeling and simulation of ion channels and action potentials in taste receptor cells

Based on patch clamp data on the ionic currents of rat taste receptor cells, a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components, including voltage-gated Na(+) currents and outward delayed rectifier K(+) currents. Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants. The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed. Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.

Endothelium as target for large-conductance calcium-activated potassium channel openers.

The endothelium is a highly active organ responsible for vasculatory tone and structure, angiogenesis, as well as hemodynamic, humoral, and inflammatory responses. The endothelium is constantly exposed to blood flow, sheer stress and tension. Endothelial cells are present as a vasculature in every tissue of the body and react to and control its microenvironment. A variety of ion channels are present in the plasma membranes of endothelial cells. These include potassium channels such as inwardly rectifying potassium (K(ir)) channels, voltage-dependent (K(v)) channels, ATP-regulated potassium (K(ATP)) channels and three types of calcium-activated potassium channels (K(Ca)), the large (BK(Ca)), intermediate (IK(Ca)), and small (SK(Ca)) -conductance potassium channels. Potassium current plays a critical role in action potentials in excitable cells, in setting the resting membrane potential, and in regulating neurotransmitter release. Mitochondrial isoforms of potassium channel contribute to the cytoprotection of endothelial cells. Prominent among potassium channels are families of calcium-activated potassium channels, and especially large-conductance calcium-activated potassium channels. The modulation of BK(Ca) channels, which are voltage- and calcium-dependent, has been intensively studied. The BK(Ca) channels show large expression dynamics in endothelial cells and tissue-specific expression of large numbers of alternatively spliced isoforms. In this review, a few examples of the modulatory mechanisms and physiological consequences of the expression of BK(Ca) channels are discussed in relation to potential targets for pharmacological intervention.

Effect of thermal acclimation on action potentials and sarcolemmal K+channels from Pacific bluefin tuna cardiomyocytes

To sustain cardiac muscle contractility relatively independent of temperature, some fish species are capable of temporarily altering excitation-contraction coupling processes to meet the demands of their environment. The Pacific bluefin tuna, Thunnus orientalis, is a partially endothermic fish that inhabits a wide range of thermal niches. The present study examined the effects of temperature and thermal acclimation on sarcolemmal K(+) currents and their role in action potential (AP) generation in bluefin tuna cardiomyocytes. Atrial and ventricular myocytes were enzymatically isolated from cold (14 degrees C)- and warm (24 degrees C)-acclimated bluefin tuna. APs and current-voltage relations of K(+) channels were measured using the whole cell current and voltage clamp techniques, respectively. Data were collected either at the cardiomyocytes' respective acclimation temperature of 14 or 24 degrees C or at a common test temperature of 19 degrees C (to reveal the effects of acclimation). AP duration (APD) was prolonged in cold-acclimated (CA) cardiomyocytes tested at 14 degrees C compared with 19 degrees C and in warm-acclimated (WA) cardiomyocytes tested at 19 degrees C compared with 24 degrees C. This effect was mirrored by a decrease in the density of the delayed-rectifier current (I(Kr)), whereas the density of the background inward-rectifier current (I(K1)) was unchanged. When CA and WA cardiomyocytes were tested at a common temperature of 19 degrees C, no significant effects of temperature acclimation on AP shape or duration were observed, whereas I(Kr) density was markedly increased in CA cardiomyocytes. I(K1) density was unaffected in CA ventricular myocytes but was significantly reduced in CA atrial myocytes, resulting in a depolarization of atrial resting membrane potential. Our results indicate the bluefin AP is relatively short compared with other teleosts, which may allow the bluefin heart to function at cold temperatures without the necessity for thermal compensation of APD.

Kinetic Properties of the Cardiac L-Type Ca 2+ Channel and Its Role in Myocyte Electrophysiology: A Theoretical Investigation

The L-type Ca 2+ channel (Ca V1.2) plays an important role in action potential (AP) generation, morphology, and duration (APD) and is the primary source of triggering Ca 2+ for the initiation of Ca 2+-induced Ca 2+-release in cardiac myocytes. In this article we present: 1), a detailed kinetic model of Ca V1.2, which is incorporated into a model of the ventricular mycoyte where it interacts with a kinetic model of the ryanodine receptor in a restricted subcellular space; 2), evaluation of the contribution of voltage-dependent inactivation (VDI) and Ca 2+-dependent inactivation (CDI) to total inactivation of Ca V1.2; and 3), description of dynamic Ca V1.2 and ryanodine receptor channel-state occupancy during the AP. Results are: 1), the Ca V1.2 model reproduces experimental single-channel and macroscopic-current data; 2), the model reproduces rate dependence of APD, [Na +] i, and the Ca 2+-transient (CaT), and restitution of APD and CaT during premature stimuli; 3), CDI of Ca V1.2 is sensitive to Ca 2+ that enters the subspace through the channel and from SR release. The relative contributions of these Ca 2+ sources to total CDI during the AP vary with time after depolarization, switching from early SR dominance to late Ca V1.2 dominance. 4), The relative contribution of CDI to total inactivation of Ca V1.2 is greater at negative potentials, when VDI is weak; and 5), loss of VDI due to the Ca V1.2 mutation G406R (linked to the Timothy syndrome) results in APD prolongation and increased CaT.

Voltage-gated potassium conductances in Gymnotus electrocytes AB

Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na +-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1–11] there were four K +-dependent conductances. Membrane depolarization activated a delayed rectifier (I K) and an A-type (I A) current. I A displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The I K activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex “hyperpolarizing responses” (HRs) described under current-clamp. IR1 shows “instantaneous” activation, is blocked by Ba 2+ and Cs + and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba 2+ and Cs +. This current fits the depolarizing phase of the HR. The EOD waveform of Gymnotus carapo is more complex than that of other gymnotiform fish species, the complexity originates in the voltage responses generated through the interactions of three Na + and four K + voltage- and time-dependent conductances although the innervation pattern also contributes [Trujillo-Cenóz O, Echagüe JA (1989) Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J Comp Physiol A 165:343–351].

Measurement of Action Potential-Induced Presynaptic Calcium Domains at a Cultured Neuromuscular Junction

Spatially localized Ca(2+) domains are thought to play a key role in action potential (AP)-evoked neurotransmitter release at fast synapses. We used a stage-scan confocal spot-detection method and the low-affinity Ca(2+) indicator Oregon Green 488 BAPTA-5N to study the spatiotemporal profile of presynaptic AP-induced Ca(2+) domains. Families of scanned AP-induced fluorescence transients were detected from spot locations separated by 200-300 nm, within the vicinity of Ca(2+) entry sites. Typically, the largest transient in a particular scan peaked within approximately 1 msec and decayed with rapid (tau(1) of 1.7 msec) and slow components (tau(2) of 16 msec, tau(3) of 78 msec). As the spot was incrementally displaced, transients progressively exhibited a slowing in their time-to-peak and a loss of the fast decay component. Three-dimensional graphs of fluorescence versus time and spot displacement revealed the presence of AP-induced fluorescence domains that dissipated within approximately 7 msec. The size of fluorescence domains were estimated from the full-width at half-maximum of gaussian fits to isochronal DeltaF/F plots and ranged from 0.6 to 3.0 micrometer, with a mean +/- SD of 1.6 +/- 0.6 micrometer. Model simulations of a localized Ca(2+) entry site predicted the major features of the fluorescence transients and suggested that, within approximately 1 msec of the initiation of the Ca(2+) current, both the fluorescence domain and the underlying Ca(2+) domain do not extend significantly beyond the site of entry. Consistent with this prediction, the intracellular addition of EGTA (up to 2 mM) accelerated the decay of the measured transients but did not affect the domain size.

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones.

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by Lambert-Eaton syndrome IgG: Blandino JKW, Viglione MP, Bradley WA, Oie HK, Kim YI. Department Biomedical Engineering, University Virginia School Medicine, Charlottesville, VA. 22908. J Membr Biol 1995;143:153–163

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by Lambert-Eaton syndrome IgG: Blandino JKW, Viglione MP, Bradley WA, Oie HK, Kim YI. Department Biomedical Engineering, University Virginia School Medicine, Charlottesville, VA. 22908. J Membr Biol 1995;143:153–163

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by lambert-eaton syndrome IgG

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na+ channels are only weakly tetrodotoxin (TTX)-sensitive (Kd = 215 nM). With the membrane potential maintained at -60 to -80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (> 20 pA). Similar all-or-nothing spikes were also observed with anodal break excitation. Removal of external Ca2+ did not affect the action potential production, whereas 5 microM TTX or substitution of Na+ with choline abolished it. Action potentials elicited in the Ca(2+)-free condition were reversibly blocked by 4 mM MnCl2 due to the Mn(2+)-induced inhibition of voltage-dependent sodium currents (INa). Therefore, Na+ channels, not Ca2+ channels, underlie the excitability of SCLC cells. Whole-cell INa was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na(+)-selective channel. INa evoked by depolarizing test potentials (-60 to +40 mV) exhibited a transient time course and activation/inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m infinity and h infinity, also revealed biophysical similarity between SCLC and neuronal Na+ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at -20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit ICa and INa in bovine adrenal chromaffin cells, also significantly inhibited INa in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na+ channel conductance; (ii) fundamental characteristics of SCLC Na+ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na+ channels are an additional target of the pathological IgG present in the patients' sera.

Role of action potentials in the postnatal development of retinogeniculate connections

Role of action potentials in the postnatal development of retinogeniculate connections

A role for action-potential activity in the development of neuronal connections in the kitten retinogeniculate pathway

The role of action potentials in the development of proper synaptic connections in the mammalian CNS was studied in the kitten retinogeniculate pathway. Our basic finding is that there is improper segregation of retinal inputs onto LGN cells after prolonged retinal action-potential blockade. Retinal ganglion cell firing was silenced from birth by repeated monocular injections of TTX. The resulting ganglion cell connections in the LGN were studied electrophysiologically after the action-potential blockade was ended. Most cells in the deprived LGN layers received excitatory input from both ON-center and OFF-center type ganglion cells, whereas LGN cells normally receive inputs only from ON-center or OFF-center ganglion cells, but not from both types. Improper segregation of ON and OFF inputs has never been reported after other types of visual deprivation that do not block ganglion cell activity. Control experiments showed that receptive fields in the nondeprived LGN layers were normal, that ganglion cell responses remained normal, and that there was no obvious ganglion cell loss. We also showed that individual LGN cells with ON and OFF excitatory inputs were not present in normal neonatal kittens. Two other types of improper input segregation in response to action-potential blockade were also found in the deprived LGN layers. (1) A greater than normal number of LGN cells received both X- and Y-type ganglion cell input. (2) Almost half of the cells at LGN layer borders were excited binocularly. Recovery of LGN normality was rapid and complete after blockade that lasted for only 3 weeks from birth, but little recovery was seen after about 11 weeks of blockade. The susceptibility to action-potential blockade decreased during the first 3 postnatal weeks. These findings may result from axon-terminal sprouting or from the failure of axon terminals to retract. The results are consistent with the idea that normally synchronous activity of neighboring ganglion cells of like center-type may be used in the refinement of retinogeniculate synaptic connections.

Chromaffin cell action potentials and their possible role in adrenaline secretion from rat adrenal medulla.

1. The role of action potentials in adrenaline secretion was investigated in the rat adrenal medulla. The effects of various treatments on adrenaline secretion from the perfused adrenal medulla were compared with the effects of similar treatments on spike frequency in dissociated adrenal chromaffin cells. 2. KCl concentrations between 10 and 20 mM increased the extracellularly recorded spike frequency of dissociated adrenal chromaffin cells. Upon perfusion by a KCl concentration of 30 mM there was an initial brief burst of spikes followed by a period of inactivity in the continued presence of 30 mM-Kcl. Tetrodotoxin (TTX, 6 microM) decreased the amplitude and frequency of the KCl evoked spikes. 3. The rate of adrenaline secretion from the isolated perfused rat adrenal gland increased as the KCl concentration was raised to 10 and up to 120 mM. Secretion which was evoked by KCl concentrations between 10 and 20 mM was partially inhibited by TTX. At KCl concentrations of 30 mM or greater evoked secretion was no longer affected by TTX. 4. CoCl2 (5 mM) blocked KCl increase of spike frequency and also blocked stimulation of adrenaline secretion by all concentrations of KCl tested. 5. Tetraethylammonium chloride (10 mM), which decreased spike frequency but greatly prolonged the spike duration, enhanced secretion induced by 15 mM-Kcl. 6. The results are consistent with the following interpretation. The TTX insensitive portion of the KCl stimulated adrenaline secretion is due to Ca influx through voltage dependent Ca channels which are open as a consequence of the steady-state level of KCl depolarization. The TTX sensitive portion of secretion is indicative of an extra increment of Ca influx during spike activity enhanced by KCl. This increment of Ca influx may occur through voltage dependent Ca channels whose activation is facilitated by the voltage changes caused during the TTX sensitive Na component of the spike and possibly through the Na channel itself. 7. Stimulation of secretion by acetylcholine (ACh) in the perfused adrenal medulla was half maximal at 15 microM and began to saturate around 50 microM. Release was partially inhibited by TTX only when the concentration of ACh was 10 microM or less. The possible role of action potentials in ACh stimulated adrenaline release is discussed.

The action potential of chick dorsal root ganglion neurones maintained in cell culture.

1. The directly evoked action potential of dissociated, embryonic, chick, dorsal root ganglion (DRG) neurones maintained in cell culture is prolonged compared to spinal cord cell spikes and the re-polarization phase is marked by a plateau. 2. Evidence was obtained that both Ca2+ and Na+ carry inward current across the active soma membrane. Ca2+ because: overshooting spikes persist in tetrodotoxin (TTX) or Na+-free media; in the presence of TTX (or absence of Na+) spike size varies directly with extracellular Ca2+ and spikes are eliminated by Co2+. Na+ because: spikes persist in the presence of Co2+ or Ca2+-free media; in the presence of Co2+ (or absence of Ca2+) spike varies directly with extracellular Na+ and spikes are blocked by TTX. 3. On the other hand, Ca2+ plays less if any role in action potentials conducted along sensory nerve cell processes. Conducted spikes could not be evoked in TTX containing or Na+-free media. 4. A long-lasting depolarization follows the action potential in some neurones. This depolarization is associated with an increase in membrane conductance and appears to drive the membrane potential to ca. -30mV. It persists when conducted impulses are blocked so it is probably not a recurrent synaptic potential. 5. It is suggested that combined Ca2+-Na+ spikes observed in isolated sensory neurones in vitro reflect the action potential of adult sensory cells but the possibility that they represent an early stage in development is also discussed.

Electrophysiology of the Neurosecretory Cell

This chapter discusses the electrophysiology of the neurosecretory (NS) cell. The NS cell possesses the unique characteristic that at least one of its axon collaterals terminates in the neurohemal structure. The chapter discusses the electrical properties of the NS cell membrane, the role of action potentials in the endocrine activity of NS cells, and synaptic control of NS cells. In electrophysiological studies of NS neurons it is extremely important to identify definitively a cell under study as a NS cell either morphologically and/or electrophysiologically. All the identified NS neurons so far examined in invertebrate and vertebrate species have been proved to generate and conduct action potentials. Ionic mechanisms for resting and action membrane potentials have been studied mostly in invertebrate NS neurons. A number of studies have depicted the mechanism of excitation-secretion coupling as follows. Depolarization of NS axon terminals, which is produced physiologically by action potentials conducted along the NS axons, induces an increase in calcium permeability of the terminal membrane, and as a result causes a calcium influx along the electrochemical potential gradient. Intracellular calcium ions in turn evoke the exocytotic release of neurohormones and binding proteins. Studies employing both techniques of antidromic identification and iontophoretic drug application have provided suggestive evidence for a particular substance to be a neurotransmitter which directly controls the activity of NS neurons in the mammalian hypothalamus.