Contributions of T-type calcium channel isoforms to neuronal firing

Low voltage-activated (LVA) T-type calcium channels play critical roles in the excitability of many cell types and are a focus of research aimed both at understanding the physiological basis of calcium channel-dependent signaling and the underlying pathophysiology associated with hyperexcitability disorders such as epilepsy. These channels play a critical role towards neuronal firing in both conducting calcium ions during action potentials and also in switching neurons between distinct modes of firing. In this review the properties of the CaV3.1, CaV3.2 and CaV3.3 T-type channel isoforms is discussed in relation to their individual contributions to action potentials during burst and tonic firing states as well their roles in switching between firing states.     

A novel disease connection for TRPM2 channels

Low voltage-activated (LVA) T-type calcium channels play critical roles in the excitability of many cell types and are a focus of research aimed both at understanding the physiological basis of calcium channel-dependent signaling and the underlying pathophysiology associated with hyperexcitability disorders such as epilepsy. These channels play a critical role towards neuronal firing in both conducting calcium ions during action potentials and also in switching neurons between distinct modes of firing. In this review the properties of the Ca_V3.1, Ca_V3.2 and Ca_V3.3 T-type channel isoforms is discussed in relation to their individual contributions to action potentials during burst and tonic firing states as well their roles in switching between firing states.     

Action Potential Modulates Ca-Dependent and Ca-Independent Secretion in a Sensory Neuron

Neurotransmitter release normally requires calcium triggering. However, the somata of dorsal root ganglion (DRG) neurons possess a calcium-independent but voltage-dependent secretion (CIVDS) in addition to the classic calcium-dependent secretion (CDS). Here, we investigated the physiological role of CIVDS and the contributions of CIVDS and CDS induced by action potentials (APs) in DRG soma. Using membrane capacitance measurements, caged calcium photolysis, and membrane capacitance kinetics analysis, we demonstrated that AP-induced secretion had both CIVDS and CDS components. Following physiological stimuli, the dominant component of AP-induced secretion was either CIVDS for spontaneous firing or CDS for high-intensity stimuli. AP frequency modulates CDS-coupled exocytosis and CIVDS-coupled endocytosis but not CIVDS-coupled exocytosis and CDS-coupled endocytosis. Finally, CIVDS did not contribute to excitatory postsynaptic currents induced by APs in DRG presynaptic terminals in the spinal cord. Thus, CIVDS is probably an essential physiological component of AP-induced secretion in the soma. These findings bring novel insights into primary sensory processes in DRG neurons.     

Ionic currents influencing spontaneous firing and pacemaker frequency in dopamine neurons of the ventrolateral periaqueductal gray and dorsal raphe nucleus (vlPAG/DRN): A voltage-clamp and computational modelling study

Dopamine (DA) neurons of the ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) fire spontaneous action potentials (APs) at slow, regular patterns in vitro but a detailed account of their intrinsic membrane properties responsible for spontaneous firing is currently lacking. To resolve this, we performed a voltage-clamp electrophysiological study in brain slices to describe their major ionic currents and then constructed a computer model and used simulations to understand the mechanisms behind autorhythmicity in silico. We found that vlPAG/DRN DA neurons exhibit a number of voltage-dependent currents activating in the subthreshold range including, a hyperpolarization-activated cation current (I_H), a transient, A-type, potassium current (I_A), a background, ‘persistent’ (I_NaP) sodium current and a transient, low voltage activated (LVA) calcium current (I_CaLVA). Brain slice pharmacology, in good agreement with computer simulations, showed that spontaneous firing occurred independently of I_H, I_A or calcium currents. In contrast, when blocking sodium currents, spontaneous firing ceased and a stable, non-oscillating membrane potential below AP threshold was attained. Using the DA neuron model we further show that calcium currents exhibit little activation (compared to sodium) during the interspike interval (ISI) repolarization while, any individual potassium current alone, whose blockade positively modulated AP firing frequency, is not required for spontaneous firing. Instead, blockade of a number of potassium currents simultaneously is necessary to eliminate autorhythmicity. Repolarization during ISI is mediated initially via the deactivation of the delayed rectifier potassium current, while a sodium background ‘persistent’ current is essentially indispensable for autorhythmicity by driving repolarization towards AP threshold.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

A model of the electrophysiological properties of nucleus reticularis thalami neurons.

A model of the electrophysiological properties of rodent nucleus reticularis thalami (NRT) neurons of the dorsal lateral thalamus was developed using Hodgkin-Huxley style equations. The model incorporated voltage-dependent rate constants and kinetics obtained from recent voltage-clamp experiments in vitro. The intrinsic electroresponsivity of the model cell was found to be similar to several empirical observations. Three distinct modes of oscillatory activity were identified: 1) a pattern of slow rhythmic burst firing (0.5-7 Hz) usually associated with membrane potentials negative to approximately -70 mV which resulted from the interplay of ITs and IK(Ca); 2) at membrane potentials from approximately -69 to -62 mV, rhythmic burst firing in the spindle frequency range (7-12 Hz) developed and was immediately followed by a tonic tail of single spike firing after several bursts. The initial bursting rhythm resulted from the interaction of ITs and IK(Ca), with a slow after-depolarization due to ICAN which mediated the later tonic firing; 3) with further depolarization of the membrane potential positive to approximately -61 mV, sustained tonic firing appeared in the 10-200-Hz frequency range depending on the amplitude of the injected current. The frequency of this firing was also dependent on the maximum conductance of the leak current, IK(leak), and an interaction between the fast currents involved in generating action potentials, INa(fast) and IK(DR), and the persistent Na+ current, INa(P). Transitions between different firing modes were identified and studied parametrically.     

Determinants of voltage-gated potassium channel surface expression and localization in Mammalian neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K+ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca2+ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K⁺ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca²⁺ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Amphetamine elicited potential changes in vertebrate and invertebrate central neurons

The effects of amphetamine on potential changes in both vertebrate and invertebrate central neurons and factors affecting the potential changes were tested. The animals studied included mice, newborn rat and African snail. Seizure was elicited after lethal doses of d-amphetamine (75 mg/kg, i.p.) administration in mice. Repetitive firing of the action potentials were elicited after d-amphetamine (1-30 microM) administration in thin thalamic brain slices of newborn rat. Bursting firing of action potentials in the giant African central RP4 neuron were also elicited after d-amphetamine or l-amphetamine (0.27 mM) administration. The amphetamine elicited bursting firing of action potentials was not blocked even after high concentrations of d-tubocurarine, atropine, haloperidol, hexamethonium administration. Therefore, the amphetamine elicited potential changes may not be directly related to the activation of the receptors of the neuron. The bursting firing of action potentials elicited by amphetamine occurred 20-30 min after amphetamine administration extracellularly, even after high concentrations of d-amphetamine administration (0.27, 1 mM). However, the bursting firing of potentials occurred immediately if amphetamine was administrated intracellularly at lower concentration. Extracellular application of ruthenium red, the calcium antagonist, abolished the amphetamine elicited bursting firing of action potentials. If intracellular injection of EGTA, a calcium ion chelator, or injection with high concentrations of magnesium, the bursting firing of potentials were immediately abolished. These results suggested that the active site of amphetamine may be inside of the neuron and the calcium ion in the neuron played an important role on the bursting of potentials. In two-electrode voltage clamped RP4 neuron, amphetamine, at 0.27 mM, decreased the total inward and steady outward currents of the RP4 neuron. d-Amphetamine also decreased the calcium, Ia and the steady-state outward currents of the RP4 neuron. Besides, amphetamine elicited a negative slope resistance (NSR) if membrane potential was in the range of -50 to -10 mV. The NSR was decreased in cobalt substituted calcium free and sodium free solution. The effects of secondary messengers on the amphetamine elicited potential changes were tested. The bursting firing of action potentials elicited by amphetamine in central snail neurons decreased following extracellular application of H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after extracellular application of H7 (1,(5-isoquinolinesulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neuron may be associated with the cyclic AMP second messenger system; on the other hand, it may not be associated with the G protein and protein kinase C activity. It is concluded that amphetamine elicited potential changes in both vertebrate and invertebrate central neurons. The changes are closely related to the ionic currents and second messengers of the neurons.     

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

A unified model for two modes of bursting in GnRH neurons

Gonadotropin-releasing hormone (GnRH) neurons exhibit at least two intrinsic modes of action potential burst firing, referred to as parabolic and irregular bursting. Parabolic bursting is characterized by a slow wave in membrane potential that can underlie periodic clusters of action potentials with increased interspike interval at the beginning and at the end of each cluster. Irregular bursting is characterized by clusters of action potentials that are separated by varying durations of interburst intervals and a relatively stable baseline potential. Based on recent studies of isolated ionic currents, a stochastic Hodgkin-Huxley (HH)-like model for the GnRH neuron is developed to reproduce each mode of burst firing with an appropriate set of conductances. Model outcomes for bursting are in agreement with the experimental recordings in terms of interburst interval, interspike interval, active phase duration, and other quantitative properties specific to each mode of bursting. The model also shows similar outcomes in membrane potential to those seen experimentally when tetrodotoxin (TTX) is used to block action potentials during bursting, and when estradiol transitions cells exhibiting slow oscillations to irregular bursting mode in vitro. Based on the parameter values used to reproduce each mode of bursting, the model suggests that GnRH neurons can switch between the two through changes in the maximum conductance of certain ionic currents, notably the slow inward Ca²⁺ current I _s, and the Ca²⁺ -activated K⁺ current I _KCa. Bifurcation analysis of the model shows that both modes of bursting are similar from a dynamical systems perspective despite differences in burst characteristics.     

Modulation of membrane potential and ionic currents by the AT1 and AT2 receptors of angiotensin II

Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca²⁺-activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.     

Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells]

We have investigated the electrical response of patched GH3 cells to Growth-Hormone Releasing-Hormone (GH-RH). GH-RH (100 nM) enhanced firing frequency of action potentials. This is accompanied by membrane depolarization (5-10 mV) and conductance increase. Voltage clamp studies reveal that GH-RH potentiates calcium inward currents and a calcium-dependent chloride current; transient outward current is diminished. These changes in membrane conductance account for the cytosolic free calcium rise shown by Indo-1 fluorescence measurements.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.     

Burst firing of action potentials in central snail neurons elicited by d-amphetamine: effect of anticonvulsants

The effect of anticonvulsants on the burst firing of action potentials in snail central neuron elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac. Oscillation of membrane potential and burst firing of action potentials were elicited by d-amphetamine in a concentration-dependent manner. Voltage clamped studies revealed that d-amphetamine elicited a negative slope resistance (NSR) in steady-state I-V curve between - 40 and - 10 mV. The burst firing of action potentials was alleviated following extracellular application of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. The NSR elicited by d-amphetamine was blocked by phenytoin. However, the NSR was not altered if carbamazepine was added. These results suggest that of the four anticonvulsants tested, only phenytoin could alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron.