Mechanisms of pacemaking in mammalian neurons.

Many neurons in the mammalian brain show pacemaking activity: rhythmic generation of action potentials in the absence of sensory or synaptic input. Slow pacemaking of neurons releasing modulatory transmitters is easy to rationalize. More surprisingly, many neurons in the motor system also show pacemaking activity, often rapid, including cerebellar Purkinje neurons that fire spontaneously at 20-100 Hz, as well as key neurons in the basal ganglia, including subthalamic nucleus neurons and globus pallidus neurons. Although the spontaneous rhythmic firing of pacemaking neurons is phenomenologically similar to cardiac pacemaking, the underlying ionic mechanism in most neurons is quite different than for cardiac pacemaking. Few spontaneously active neurons rely on HCN 'pacemaker' channels for their activity. Most commonly, a central element is 'persistent' sodium current, steady-state subthreshold current carried by the same voltage-dependent sodium channels that underlie fast action potentials. Persistent sodium current is a steeply voltage-dependent current with a midpoint near -60 mV, which results in regenerative spontaneous depolarization once it produces a net inward current when summed with all other background currents, often at voltages as negative as -70 mV. This 'engine' of pacemaking is present in almost all neurons and must be held in check in non-pacemaking neurons by sufficiently large competing outward currents from background potassium channels. The intrinsic propensity of neurons to fire spontaneously underlies key normal functions such as respiration and generates the complex background oscillatory circuits revealed in EEGs, but can also produce out-of-control oscillations of overall brain function in epilepsy, ataxia and tremor.

Estimation of persistent sodium-current density in rat hippocampal mossy fibre boutons: Correction of space-clamp errors.

We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (iNaP) in rat hippocampal mossy fibre boutons. Cox's method for correcting space-clamp errors was extended to the case of an isopotential compartment with attached neurites. The method was applied to voltage-ramp experiments, in which iNaP is assumed to gate instantaneously. The raw estimates of iNaP led to predicted clamp currents that were at variance with observation, hence an algorithm was devised to improve these estimates. Optionally, the method also allows an estimate of the membrane specific capacitance, although values of the axial resistivity and seal resistance must be provided. Assuming that membrane specific capacitance and axial resistivity were constant, we conclude that seal resistance continued to fall after adding TTX to the bath. This might have been attributable to a further deterioration of the seal after baseline rather than an unlikely effect of TTX. There was an increase in the membrane specific resistance in TTX. The reason for this is unknown, but it meant that iNaP could not be determined by simple subtraction. Attempts to account for iNaP with a Hodgkin-Huxley model of the transient sodium conductance met with mixed results. One thing to emerge was the importance of voltage shifts. Also, a large variability in previously reported values of transient sodium conductance in mossy fibre boutons made comparisons with our results difficult. Various other possible sources of error are discussed. Simulations suggest a role for iNaP in modulating the axonal attenuation of EPSPs. KEY POINTS: We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (iNaP) in rat hippocampal mossy fibre boutons, using a KCl-based internal (pipette) solution and correcting for the liquid junction potential (2 mV). Space-clamp errors and deterioration of the patch-clamp seal during the experiment were corrected for by compartmental modelling. Attempts to account for iNaP in terms of the transient sodium conductance met with mixed results. One possibility is that the transient sodium conductance is higher in mossy fibre boutons than in the axon shaft. The analysis illustrates the need to account for various voltage shifts (Donnan potentials, liquid junction potentials and, possibly, other voltage shifts). Simulations suggest a role for iNaP in modulating the axonal attenuation of excitatory postsynaptic potentials, hence analog signalling by dentate granule cells.

Persistent sodium currents in neurons: potential mechanisms and pharmacological blockers.

Persistent sodium current (INaP) is an important activity-dependent regulator of neuronal excitability. It is involved in a variety of physiological and pathological processes, including pacemaking, prolongation of sensory potentials, neuronal injury, chronic pain and diseases such as epilepsy and amyotrophic lateral sclerosis. Despite its importance, neither the molecular basis nor the regulation of INaP are sufficiently understood. Of particular significance is a solid knowledge and widely accepted consensus about pharmacological tools for analysing the function of INaP and for developing new therapeutic strategies. However, the literature on INaP is heterogeneous, with varying definitions and methodologies used across studies. To address these issues, we provide a systematic review of the current state of knowledge on INaP, with focus on mechanisms and effects of this current in the central nervous system. We provide an overview of the specificity and efficacy of the most widely used INaP blockers: amiodarone, cannabidiol, carbamazepine, cenobamate, eslicarbazepine, ethosuximide, gabapentin, GS967, lacosamide, lamotrigine, lidocaine, NBI-921352, oxcarbazepine, phenytoine, PRAX-562, propofol, ranolazine, riluzole, rufinamide, topiramate, valproaic acid and zonisamide. We conclude that there is strong variance in the pharmacological effects of these drugs, and in the available information. At present, GS967 and riluzole can be regarded bona fide INaP blockers, while phenytoin and lacosamide are blockers that only act on the slowly inactivating component of sodium currents.

Development of electrophysiological properties of fusiform neurons from the dorsal cochlear nucleus of mice before and after hearing onset.

The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature DCN fusiform neurons fall into two qualitatively distinct types: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the prehearing phase (P4-P13), we found that most fusiform neurons are quiet, with active neurons emerging after hearing onset at P14. Subthreshold properties underwent significant changes before hearing onset, whereas changes to the action potential waveform occurred mainly after P14, with the depolarization and repolarization phases becoming markedly faster and half-width significantly decreased. The activity threshold in posthearing neurons was more negative than in prehearing cells. Persistent sodium current (INaP) was increased after P14, coinciding with the emergence of spontaneous firing. Thus, we suggest that posthearing expression of INaP leads to hyperpolarization of the activity threshold and the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.NEW & NOTEWORTHY Auditory brainstem neurons express unique electrophysiological properties adapted for their complex physiological functions that develop before hearing onset. Fusiform neurons of the DCN present two firing states, quiet and active, but the origin of these states is not known. Here, we showed that the quiet and active states develop after hearing onset at P14, along with changes in action potentials, suggesting an influence of auditory input on the refining of fusiform neuron's excitability.

Dynamics of ramping bursts in a respiratory neuron model.

Intensive computational and theoretical work has led to the development of multiple mathematical models for bursting in respiratory neurons in the pre-Bötzinger Complex (pre-BötC) of the mammalian brainstem. Nonetheless, these previous models have not captured the pre-inspiratory ramping aspects of these neurons' activity patterns, in which relatively slow tonic spiking gradually progresses to faster spiking and a full-blown burst, with a corresponding gradual development of an underlying plateau potential. In this work, we show that the incorporation of the dynamics of the extracellular potassium ion concentration into an existing model for pre-BötC neuron bursting, along with some parameter adjustments, suffices to induce this ramping behavior. Using fast-slow decomposition, we show that this activity can be considered as a form of parabolic bursting, but with burst termination at a homoclinic bifurcation rather than as a SNIC bifurcation. We also investigate the parameter-dependence of these solutions and show that the proposed model yields a greater dynamic range of burst frequencies, durations, and duty cycles than those produced by other models in the literature.

The novel persistent sodium current inhibitor PRAX-562 has potent anticonvulsant activity with improved protective index relative to standard of care sodium channel blockers.

OBJECTIVE: This study investigates the effects of PRAX-562 on sodium current (INa ), intrinsic neuronal excitability, and protection from evoked seizures to determine whether a preferential persistent INa inhibitor would exhibit improved preclinical efficacy and tolerability compared to two standard voltage-gated sodium channel (NaV ) blockers. METHODS: Inhibition of INa  was characterized using patch clamp analysis. The effect on intrinsic excitability was measured using evoked action potentials recorded from hippocampal CA1 pyramidal neurons in mouse brain slices. Anticonvulsant activity was evaluated using the maximal electroshock seizure (MES) model, and tolerability was assessed by measuring spontaneous locomotor activity (sLMA). RESULTS: PRAX-562 potently and preferentially inhibited persistent INa induced by ATX-II or the SCN8A mutation N1768D (half-maximal inhibitory concentration [IC50 ] = 141 and 75 nmol·L-1 , respectively) relative to peak INa tonic/resting block (60× preference). PRAX-562 also exhibited potent use-dependent block (31× preference to tonic block). This profile is considerably different from standard NaV blockers, including carbamazepine (CBZ; persistent INa IC50 = 77 500 nmol·L-1 , preference ratios of 30× [tonic block], less use-dependent block observed at various frequencies). In contrast to CBZ, PRAX-562 reduced neuronal intrinsic excitability with only a minor reduction in action potential amplitude. PRAX-562 (10 mg/kg po) completely prevented evoked seizures without affecting sLMA (MES unbound brain half-maximal efficacious concentration = 4.3 nmol·L-1 , sLMA half-maximal tolerated concentration = 69.7 nmol·L-1 , protective index [PI] = 16×). In contrast, CBZ and lamotrigine (LTG) had PIs of approximately 5.5×, with significant overlap between doses that were anticonvulsant and that reduced locomotor activity. SIGNIFICANCE: PRAX-562 demonstrated robust preclinical anticonvulsant activity similar to CBZ but improved compared to LTG. PRAX-562 exhibited significantly improved preclinical tolerability compared with standard NaV blockers (CBZ and LTG), potentially due to the preference for persistent INa . Preferential targeting of persistent INa may represent a differentiated therapeutic option for diseases of hyperexcitability, where standard NaV blockers have demonstrated efficacy but poor tolerability.

Post-stroke epileptogenesis is associated with altered intrinsic properties of hippocampal pyramidal neurons leading to increased theta resonance.

Brain insults like stroke, trauma or infections often lead to blood-brain barrier-dysfunction (BBBd) frequently resulting into epileptogenesis. Affected patients suffer from seizures and cognitive comorbidities that are potentially linked to altered network oscillations. It has been shown that a hippocampal BBBd in rats leads to in vivo seizures and increased power at theta (3-8 Hz), an important type of network oscillations. However, the underlying cellular mechanisms remain poorly understood. At membrane potentials close to the threshold for action potentials (APs) a subpopulation of CA1 pyramidal cells (PCs) displays intrinsic resonant properties due to an interplay of the muscarine-sensitive K+-current (IM) and the persistent Na+-current (INaP). Such resonant neurons are more excitable and generate more APs when stimulated at theta frequencies, being strong candidates for contributing to hippocampal theta oscillations during epileptogenesis. We tested this hypothesis by characterizing changes in intrinsic properties of hippocampal PCs one week after post-stroke epileptogenesis, a model associated with BBBd, using slice electrophysiology and computer modeling. We find a higher proportion of resonant neurons in BBBd compared to sham animals (47 vs. 29%), accompanied by an increase in their excitability. In contrast, BBBd non-resonant neurons showed a reduced excitability, presented with lower impedance and more positive AP threshold. We identify an increase in IM combined with either a reduction in INaP or an increase in ILeak as possible mechanisms underlying the observed changes. Our results support the hypothesis that a higher proportion of more excitable resonant neurons in the hippocampus contributes to increased theta oscillations and an increased likelihood of seizures in a model of post-stroke epileptogenesis.

The effect of lamotrigine and other antiepileptic drugs on respiratory rhythm generation in the pre-Bötzinger complex.

OBJECTIVE: Lamotrigine and other sodium-channel blocking agents are among the most commonly used antiepileptic drugs (AEDs). Because other sodium channel blockers, such as riluzole, can severely alter respiratory rhythm generation during hypoxia, we wanted to investigate if AEDs can have similar effects. This is especially important in the context of sudden unexpected death in epilepsy (SUDEP), the major cause of death in patients suffering from therapy-resistant epilepsy. Although the mechanism of action is not entirely understood, respiratory dysfunction after generalized tonic-clonic seizures seems to play a major role. METHODS: We used transverse brainstem slice preparations from neonatal and juvenile mice containing the pre-Bötzinger complex (PreBötC) and measured population as well as intracellular activity of the rhythm-generating network under normoxia and hypoxia in the presence or absence of AEDs. RESULTS: We found a substantial inhibition of the gasping response induced by the application of sodium channel blockers (lamotrigine and carbamazepine). In contrast, levetiracetam, an AED-modulating synaptic function, had a much smaller effect. The inhibition of gasping by lamotrigine was accompanied by a significant reduction of the persistent sodium current (INap) in PreBötC neurons. Surprisingly, the suppression of persistent sodium currents by lamotrigine did not affect the voltage-dependent bursting activity in PreBötC pacemaker neurons, but led to a hypoxia-dependent shift of the action potential rheobase in all measured PreBötC neurons. SIGNIFICANCE: Our results contribute to the understanding of the effects of AEDs on the vital respiratory functions of the central nervous system. Moreover, our study adds further insight into sodium-dependent changes occurring during hypoxia and the contribution of cellular properties to the respiratory rhythm generation in the pre-Bötzinger complex. It raises the question of whether sodium channel blocking AEDs could, in conditions of extreme hypoxia, contribute to SUDEP, an important issue that warrants further studies.

Cenobamate (XCOPRI): Can preclinical and clinical evidence provide insight into its mechanism of action?

Approximately one-third of people living with epilepsy are unable to obtain seizure control with the currently marketed antiseizure medications (ASMs), creating a need for novel therapeutics with new mechanisms of action. Cenobamate (CBM) is a tetrazole alkyl carbamate derivative that received US Food and Drug Administration approval in 2019 for the treatment of adult partial onset (focal) seizures. Although CBM displayed impressive seizure reduction in clinical trials across all seizure types, including focal aware motor, focal impaired awareness, and focal to bilateral tonic-clonic seizures, the precise mechanism(s) through which CBM exerts its broad-spectrum antiseizure effects is not known. Experimental evidence suggests that CBM differentiates itself from other ASMs in that it appears to possess dual modes of action (MOAs); that is, it predominately blocks persistent sodium currents and increases both phasic and tonic γ-aminobutyric acid (GABA) inhibition. In this review, we analyze the preclinical efficacy of CBM alongside ASMs with similar MOAs to better understand the mechanism(s) through which CBM achieves such broad-spectrum seizure protection. CBM's preclinical performance in tests, including the mouse 6-Hz model of treatment-resistant seizures, the chemoconvulsant seizure models of generalized epilepsy, and the rat hippocampal kindling model of focal epilepsy, was distinct from other voltage-gated sodium channel blockers and GABAA modulators. This distinction, in light of its proposed mechanism(s) of action, provides insight into the impressive clinical efficacy of CBM in the adult patient with focal onset epilepsy. The results of this comparative reverse translational analysis suggest that CBM is a mechanistically distinct ASM that offers an important advancement in drug development for treatment of therapy-resistant epilepsy.

Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks.

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 µm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.

Sodium-activated potassium channels moderate excitability in vascular smooth muscle.

KEY POINTS: We report that a sodium-activated potassium current, IKNa , has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells. IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice. The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild-type with raised blood pressure when challenged with vasoconstrictive agents. IKNa is negatively regulated by angiotensin II acting through Gαq protein-coupled receptors. In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+ -dependent action potentials. ABSTRACT: Although several potassium currents have been reported to play a role in arterial smooth muscle (ASM), we find that one of the largest contributors to membrane conductance in both conduit and resistance ASMs has been inadvertently overlooked. In the present study, we show that IKNa , a sodium-activated potassium current, contributes a major portion of macroscopic outward current in a critical physiological voltage range that determines intrinsic cell excitability; IKNa is the largest contributor to ASM cell resting conductance. A genetic knockout (KO) mouse strain lacking KNa channels (KCNT1 and KCNT2) shows only a modest hypertensive phenotype. However, acute administration of vasoconstrictive agents such as angiotensin II (Ang II) and phenylephrine results in an abnormally large increase in blood pressure in the KO animals. In wild-type animals Ang II acting through Gαq protein-coupled receptors down-regulates IKNa , which increases the excitability of the ASMs. The complete genetic removal of IKNa in KO mice makes the mutant animal more vulnerable to vasoconstrictive agents, thus producing a paroxysmal-hypertensive phenotype. This may result from the lowering of cell resting K+ conductance allowing the cells to depolarize more readily to a variety of excitable stimuli. Thus, the sodium-activated potassium current may serve to moderate blood pressure in instances of heightened stress. IKNa may represent a new therapeutic target for hypertension and stroke.

mGluR5-dependent modulation of dendritic excitability in CA1 pyramidal neurons mediated by enhancement of persistent Na+ currents.

KEY POINTS: High-frequency stimulation (HFS) of the Schaffer collateral pathway activates metabotropic glutamate receptor 5 (mGluR5) signalling in the proximal apical dendrites of CA1 pyramidal neurons. The synaptic activation of mGluR5-mediated calcium signalling causes a significant increase in persistent sodium current (INa,P ) in the dendrites. Increased INa,P by HFS underlies potentiation of synaptic inputs at both the proximal and distal dendrite, leading to an enhanced probability of action potential firing associated with decreased action potential thresholds. Therefore, HFS-induced activation of intracellular mGluR5 serves an important role as an instructive signal for potentiation of upcoming inputs by increasing dendritic excitability. ABSTRACT: Dendritic Na+ channels in pyramidal neurons are known to amplify synaptic signals, thereby facilitating action potential (AP) generation. However, the mechanisms that modulate dendritic Na+ channels have remained largely uncharacterized. Here, we report a new form of short-term plasticity in which proximal excitatory synaptic inputs to hippocampal CA1 pyramidal neurons transiently elevate dendritic excitability. High-frequency stimulations (HFS) to the Schaffer collateral (SC) pathway activate mGluR5-dependent Ca2+ signalling in the apical dendrites, which, with calmodulin, upregulates specifically Nav1.6 channel-mediated persistent Na+ currents (INa,P ) in the dendrites. This HFS-induced increase in dendritic INa,P results in transient increases in the amplitude of excitatory postsynaptic potentials induced by both proximal SC and distal perforant path stimulation, leading to the enhanced probability of AP firing associated with decreased AP thresholds. Taken together, our study identifies dendritic INa,P as a novel target for mediating activity-dependent modulation of dendritic integration and neuronal output.

Persistent sodium current modulates axonal excitability in CA1 pyramidal neurons.

Axonal excitability is an important determinant for the accuracy, direction, and velocity of neuronal signaling. The mechanisms underlying spike generation in the axonal initial segment and transmitter release from presynaptic terminals have been intensely studied and revealed a role for several specific ionic conductances, including the persistent sodium current (INaP ). Recent evidence indicates that action potentials can also be generated at remote locations along the axonal fiber, giving rise to ectopic action potentials during physiological states (e.g., fast network oscillations) or in pathological situations (e.g., following demyelination). Here, we investigated how ectopic axonal excitability of mouse hippocampal CA1 pyramidal neurons is regulated by INaP . Recordings of field potentials and intracellular voltage in brain slices revealed that electrically evoked antidromic spikes were readily suppressed by two different blockers of INaP , riluzole and phenytoin. The effect was mediated by a reduction of the probability of ectopic spike generation while latency was unaffected. Interestingly, the contribution of INaP to excitability was much more pronounced in axonal branches heading toward the entorhinal cortex compared with the opposite fiber direction toward fimbria. Thus, excitability of distal CA1 pyramidal cell axons is affected by persistent sodium currents in a direction-selective manner. This mechanism may be of importance for ectopic spike generation in oscillating network states as well as in pathological situations.

Baicalein reverts L-valine-induced persistent sodium current up-modulation in primary cortical neurons.

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

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 (IH), a transient, A-type, potassium current (IA), a background, 'persistent' (INaP) sodium current and a transient, low voltage activated (LVA) calcium current (ICaLVA). Brain slice pharmacology, in good agreement with computer simulations, showed that spontaneous firing occurred independently of IH, IA 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.

Downregulation of Spermine Augments Dendritic Persistent Sodium Currents and Synaptic Integration after Status Epilepticus.

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.

Activation of ß-noradrenergic receptors enhances rhythmic bursting in mouse olfactory bulb external tufted cells.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the nucleus locus coeruleus. Despite the well-documented role of norepinephrine and ß-adrenergic receptors in neonatal odor preference learning, identified cellular physiological actions of ß-receptors in the MOB have remained elusive. ß-Receptors are expressed at relatively high levels in the MOB glomeruli, the location of external tufted (ET) cells that exert an excitatory drive on mitral and other cell types. The present study investigated the effects of ß-receptor activation on the excitability of ET cells with patch-clamp electrophysiology in mature mouse MOB slices. Isoproterenol and selective ß2-, but not ß1-, receptor agonists were found to enhance two key intrinsic currents involved in ET burst initiation: persistent sodium (INaP) and hyperpolarization-activated inward (Ih) currents. Together, the positive modulation of these currents increased the frequency and strength of ET cell rhythmic bursting. Rodent sniff frequency and locus coeruleus neuronal firing increase in response to novel stimuli or environments. The increase in ET excitability by ß-receptor activation may better enable ET cell rhythmic bursting, and hence glomerular network activity, to pace faster sniff rates during heightened norepinephrine release associated with arousal.

Multiple timescale mixed bursting dynamics in a respiratory neuron model.

Experimental results in rodent medullary slices containing the pre-Bötzinger complex (pre-BötC) have identified multiple bursting mechanisms based on persistent sodium current (I NaP) and intracellular Ca2+. The classic two-timescale approach to the analysis of pre-BötC bursting treats the inactivation of I NaP, the calcium concentration, as well as the Ca2+-dependent inactivation of IP 3 as slow variables and considers other evolving quantities as fast variables. Based on its time course, however, it appears that a novel mixed bursting (MB) solution, observed both in recordings and in model pre-BötC neurons, involves at least three timescales. In this work, we consider a single-compartment model of a pre-BötC inspiratory neuron that can exhibit both I NaP and Ca2+ oscillations and has the ability to produce MB solutions. We use methods of dynamical systems theory, such as phase plane analysis, fast-slow decomposition, and bifurcation analysis, to better understand the mechanisms underlying the MB solution pattern. Rather surprisingly, we discover that a third timescale is not actually required to generate mixed bursting solutions. Through our analysis of timescales, we also elucidate how the pre-BötC neuron model can be tuned to improve the robustness of the MB solution.

Generation of resonance-dependent oscillation by mGluR-I activation switches single spiking to bursting in mesencephalic trigeminal sensory neurons.

The primary sensory neurons supplying muscle spindles of jaw-closing muscles are unique in that they have their somata in the mesencephalic trigeminal nucleus (MTN) in the brainstem, thereby receiving various synaptic inputs. MTN neurons display bursting upon activation of glutamatergic synaptic inputs while they faithfully relay respective impulses arising from peripheral sensory organs. The persistent sodium current (IN aP ) is reported to be responsible for both the generation of bursts and the relay of impulses. We addressed how IN aP is controlled either to trigger bursts or to relay respective impulses as single spikes in MTN neurons. Protein kinase C (PKC) activation enhanced IN aP only at low voltages. Spike generation was facilitated by PKC activation at membrane potentials more depolarized than the resting potential. By injection of a ramp current pulse, a burst of spikes was triggered from a depolarized membrane potential whereas its instantaneous spike frequency remained almost constant despite the ramp increases in the current intensity beyond the threshold. A puff application of glutamate preceding the ramp pulse lowered the threshold for evoking bursts by ramp pulses while chelerythrine abolished such effects of glutamate. Dihydroxyphenylglycine, an agonist of mGluR1/5, also caused similar effects, and increased both the frequency and impedance of membrane resonance. Immunohistochemistry revealed that glutamatergic synapses are made onto the stem axons, and that mGluR1/5 and Nav1.6 are co-localized in the stem axon. Taken together, glutamatergic synaptic inputs onto the stem axon may be able to switch the relaying to the bursting mode.

Suppression of epileptiform activity by a single short-duration electric field in rat hippocampus in vitro.

The mechanisms behind the therapeutic effects of electrical stimulation of the brain in epilepsy and other disorders are poorly understood. Previous studies in vitro have shown that uniform electric fields can suppress epileptiform activity through a direct polarizing effect on neuronal membranes. Such an effect depends on continuous DC stimulation with unbalanced charge. Here we describe a suppressive effect of a brief (10 ms) DC field on stimulus-evoked epileptiform activity in rat hippocampal brain slices exposed to Cs(+) (3.5 mM). This effect was independent of field polarity, was uncorrelated to changes in synchronized population activity, and persisted during blockade of synaptic transmission with Cd(2+) (500 µM). Antagonists of A(1), P(2X), or P(2Y) receptors were without effect. The suppressive effect depended on the alignment of the external field with the somato-dendritic axis of CA1 pyramidal cells; however, temporal coincidence with the epileptiform activity was not essential, as suppression was detectable for up to 1 s after the field. Pyramidal cells, recorded during epileptiform activity, showed decreased discharge duration and truncation of depolarizing plateau potentials in response to field application. In the absence of hyperactivity, the applied field was followed by slow membrane potential changes, accompanied by decreased input resistance and attenuation of the depolarizing afterpotential following action potentials. These effects recovered over a 1-s period. The study suggests that a brief electric field induces a prolonged suppression of epileptiform activity, which can be related to changes in neuronal membrane properties, including attenuation of signals depending on the persisting Na(+) current.