Kv2.1 Potassium Channels Regulate Repetitive Burst Firing in Extratelencephalic Neocortical Pyramidal Neurons.

Coincidence detection and cortical rhythmicity are both greatly influenced by neurons' propensity to fire bursts of action potentials. In the neocortex, repetitive burst firing can also initiate abnormal neocortical rhythmicity (including epilepsy). Bursts are generated by inward currents that underlie a fast afterdepolarization (fADP) but less is known about outward currents that regulate bursting. We tested whether Kv2 channels regulate the fADP and burst firing in labeled layer 5 PNs from motor cortex of the Thy1-h mouse. Kv2 block with guangxitoxin-1E (GTx) converted single spike responses evoked by dendritic stimulation into multispike bursts riding on an enhanced fADP. Immunohistochemistry revealed that Thy1-h PNs expressed Kv2.1 (not Kv2.2) channels perisomatically (not in the dendrites). In somatic macropatches, GTx-sensitive current was the largest component of outward current with biophysical properties well-suited for regulating bursting. GTx drove ~40% of Thy1 PNs stimulated with noisy somatic current steps to repetitive burst firing and shifted the maximal frequency-dependent gain. A network model showed that reduction of Kv2-like conductance in a small subset of neurons resulted in repetitive bursting and entrainment of the circuit to seizure-like rhythmic activity. Kv2 channels play a dominant role in regulating onset bursts and preventing repetitive bursting in Thy1 PNs.

PIP2 alters of Ca2+ currents in acutely dissociated supraoptic oxytocin neurons.

Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca2+ channels that provide Ca2+ for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP2 ) is a phospholipid membrane component that has been previously shown to modulate Ca2+ channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP2 modulates these channels, and for the first time show how PIP2 modulates CaV channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP2 depletion via wortmannin (0.5 µmol/L) inhibits Ca2+ channel currents in OT but not VP neurons. Additionally, it hyperpolarizes voltage-dependent activation of the channels by ~5 mV while leaving the slope of activation unchanged, properties unaffected in VP neurons. We also identified key differences in baseline currents between the cell types, wherein VP whole cell Ca2+ currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca2+ channels in OT neurons, which we show to be mostly an effect on N-type Ca2+ channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca2+ channel currents. We conclude that PIP2 is a modulator that enhances current through N-type channels. This has implications for the afterhyperpolarization (AHP) of OT neurons, as previous work from our laboratory demonstrated the AHP is inhibited by wortmannin, and that its primary activation is from intracellular Ca2+ contributed by N-type channels.

Functional roles of Kv1-mediated currents in genetically identified subtypes of pyramidal neurons in layer 5 of mouse somatosensory cortex.

We used voltage-clamp recordings from somatic outside-out macropatches to determine the amplitude and biophysical properties of putative Kv1-mediated currents in layer 5 pyramidal neurons (PNs) from mice expressing EGFP under the control of promoters for etv1 or glt. We then used whole cell current-clamp recordings and Kv1-specific peptide blockers to test the hypothesis that Kv1 channels differentially regulate action potential (AP) voltage threshold, repolarization rate, and width as well as rheobase and repetitive firing in these two PN types. We found that Kv1-mediated currents make up a similar percentage of whole cell K+ current in both cell types, and only minor biophysical differences were observed between PN types or between currents sensitive to different Kv1 blockers. Putative Kv1 currents contributed to AP voltage threshold in both PN types, but AP width and rate of repolarization were only affected in etv1 PNs. Kv1 currents regulate rheobase, delay to the first AP, and firing rate similarly in both cell types, but the frequency-current slope was much more sensitive to Kv1 block in etv1 PNs. In both cell types, Kv1 block shifted the current required to elicit an onset doublet of action potentials to lower currents. Spike frequency adaptation was also affected differently by Kv1 block in the two PN types. Thus, despite similar expression levels and minimal differences in biophysical properties, Kv1 channels differentially regulate APs and repetitive firing in etv1 and glt PNs. This may reflect differences in subcellular localization of channel subtypes or differences in the other K+ channels expressed. NEW & NOTEWORTHY In two types of genetically identified layer 5 pyramidal neurons, α-dendrotoxin blocked approximately all of the putative Kv1 current (on average). We used outside-out macropatches and whole cell recordings at 33°C to show that despite similar expression levels and minimal differences in biophysical properties, Kv1 channels differentially regulate action potentials and repetitive firing in etv1 and glt pyramidal neurons. This may reflect differences in subcellular localization of channel subtypes or differences in the other K+ channels expressed.

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

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

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

Electrophysiological properties of genetically identified subtypes of layer 5 neocortical pyramidal neurons: Ca²âº dependence and differential modulation by norepinephrine.

We studied neocortical pyramidal neurons from two lines of bacterial artificial chromosome mice (etv1 and glt; Gene Expression Nervous System Atlas: GENSAT project), each of which expresses enhanced green fluorescent protein (EGFP) in a different subpopulation of layer 5 pyramidal neurons. In barrel cortex, etv1 and glt pyramidal cells were previously reported to differ in terms of their laminar distribution, morphology, thalamic inputs, cellular targets, and receptive field size. In this study, we measured the laminar distribution of etv1 and glt cells. On average, glt cells were located more deeply; however, the distributions of etv1 and glt cells extensively overlap in layer 5. To test whether these two cell types differed in electrophysiological properties that influence firing behavior, we prepared acute brain slices from 2-4-wk-old mice, where EGFP-positive cells in somatosensory cortex were identified under epifluorescence and then studied using whole cell current- or voltage-clamp recordings. We studied the details of action potential parameters and repetitive firing, characterized by the larger slow afterhyperpolarizations (AHPs) in etv1 neurons and larger medium AHPs (mAHPS) in glt cells, and compared currents underlying the mAHP and slow AHP (sAHP) in etv1 and glt neurons. Etv1 cells exhibited lower dV/dt for spike polarization and repolarization and reduced direct current (DC) gain (lower f-I slope) for repetitive firing than glt cells. Most importantly, we found that 1) differences in the expression of Ca(2+)-dependent K(+) conductances (small-conductance calcium-activated potassium channels and sAHP channels) determine major functional differences between etv1 and glt cells, and 2) there is differential modulation of etv1 and glt neurons by norepinephrine.

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex.

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Whole cell recording from an organotypic slice preparation of neocortex.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons.

Potassium channels regulate numerous aspects of neuronal excitability, and several voltage-gated K(+) channel subunits have been identified in pyramidal neurons of rat neocortex. Previous studies have either considered the development of outward current as a whole or divided currents into transient, A-type and persistent, delayed rectifier components but did not differentiate between current components defined by α-subunit type. To facilitate comparisons of studies reporting K(+) currents from animals of different ages and to understand the functional roles of specific current components, we characterized the postnatal development of identified Kv channel-mediated currents in pyramidal neurons from layers II/III from rat somatosensory cortex. Both the persistent/slowly inactivating and transient components of the total K(+) current increased in density with postnatal age. We used specific pharmacological agents to test the relative contributions of putative Kv1- and Kv2-mediated currents (100 nM α-dendrotoxin and 600 nM stromatoxin, respectively). A combination of voltage protocol, pharmacology, and curve fitting was used to isolate the rapidly inactivating A-type current. We found that the density of all identified current components increased with postnatal age, approaching a plateau at 3-5 wk. We found no significant changes in the relative proportions or kinetics of any component between postnatal weeks 1 and 5, except that the activation time constant for A-type current was longer at 1 wk. The putative Kv2-mediated component was the largest at all ages. Immunocytochemistry indicated that protein expression for Kv4.2, Kv4.3, Kv1.4, and Kv2.1 increased between 1 wk and 4-5 wk of age.

Experimental evidence of improved transthoracic defibrillation with electroporation-enhancing pulses.

There is considerable work on defibrillation wave form optimization. This paper determines the impedance changes during defibrillation, then uses that information to derive the optimum defibrillation wave form. METHODS PART I: Twelve guinea pigs and six swine were used to measure the current wave form for square voltage pulses of a strength which would defibrillate about 50% of the time. In guinea pigs, electrodes were placed thoracically, abdominally and subcutaneously using two electrode materials (zinc and steel) and two electrode pastes (Core-gel and metallic paste). RESULTS PART I: The measured current wave form indicated an exponentially increasing conductance over the first 3 ms, consistent with enhanced electroporation or another mechanism of time-dependent conductance. We fit this current with a parallel conductance composed of a time-independent component (g0 = 1.22 +/- 0.28 mS) and a time-dependent component described by g delta (1-e(-t/tau)), where g delta = 0.95 +/- 0.20 mS and tau = 0.82 +/- 0.17 ms in guinea pigs using zinc and Cor-gel. Different electrode placements and materials had no significant effect on this fit. From our fit, we determined the stimulating wave form that would theoretically charge the myocardial membrane to a given threshold using the least energy from the defibrillator. The solution was a very short, high voltage pulse followed immediately by a truncated ascending exponential tail. METHODS PART II: The optimized wave forms and similar nonoptimized wave forms were tested for efficacy in 25 additional guinea pigs and six additional swine using methods similar to Part I. RESULTS PART II: Optimized wave forms were significantly more efficacious than similar nonoptimized wave forms. In swine, a wave form with the short pulse was 41% effective while the same wave form without the short pulse was 8.3% effective (p < 0.03) despite there being only a small difference in energy (111 J versus 116 CONCLUSIONS: We conclude that a short pulse preceding a defibrillation pulse significantly improves efficacy, perhaps by enhancing electroporation.

Experimental verification of theoretical predictions concerning the optimum defibrillation waveform.

The efficacy of electrical therapy at terminating ventricular fibrillation is highly dependent on the waveform used. We present experimental results which test one theory for defibrillation waveform dependence. Forty-four defibrillation waveforms (22 monophasic, 22 biphasic) were designed according to the theoretical construct of Fishier (2000). The waveforms were then tested on 67 male guinea pigs (46 for monophasic, 21 for biphasic waveforms) using a custom designed defibrillator and 12-mm subcutaneous disc electrodes. There was considerable agreement between the theoretical and experimental results. For example, as predicted, the ascending exponential waveform of 1 ms proved to be the most effective (86.4%) monophasic waveform, where efficacy is the number of successful shocks divided by the total number delivered. In addition, the efficacy decrease with duration increase was accurately predicted by the model for monophasic waveforms. For biphasic waveforms, as predicted by the model, when the first phase was optimized, an increase in second phase duration caused an increase in defibrillation efficacy (10 of 11 tested duration pairs). We conclude that the theoretical framework adequately explains the mechanism by which the defibrillation waveform affects efficacy for monophasic waveforms and, in at least one aspect, biphasic waveforms.

Analysis of the defibrillation efficacy for 5-ms waveforms.

INTRODUCTION: Empirical studies have shown that biphasic defibrillation waveforms are more efficacious than monophasic waveforms. However, a more systematic approach to waveform development might be more productive. This study tested 147 multiphasic waveforms uniformly sampled from all possible 5-ms waveforms. METHODS AND RESULTS: One hundred ninety-eight guinea pigs (850-1,050 g) received 30 episodes of ventricular fibrillation followed by transthoracic defibrillation. The first 10 shocks were used to determine the ED(50) for a biphasic control. Then, 20 waveforms including 2 controls were tested once at the ED(50). Of the 147 waveforms tested here, 21 waveforms showed equivalent or greater efficacies than the biphasic control, with one being statistically more efficacious (P < 0.05). Two fundamental assumptions were addressed: (1) similarly efficacious waveforms are analytically similar, and (2) a single optimal waveform can be described. The mean percentage of similarly efficacious waveforms with similar shapes was greater than zero in the most efficacious 21 waveforms (P = 0.023), but less efficacious waveforms showed randomly distributed shapes. Cluster analysis revealed that the best waveforms share a major phase containing most of the defibrillation energy. The optimal waveform shape extrapolated from the sample waveforms was a 2.5/1-ms biphasic-type waveform (highest correlation r = 0.701, P < 0.001). CONCLUSION: This work supports the assumption that efficacious waveforms are similarly shaped and the notion that one single optimum exists.

The defibrillation efficacy of high frequency alternating current sinusoidal waveforms in guinea pigs.

There have been few basic studies of alternating current (AC) defibrillation, despite growing interest in the ability of AC to terminate or alter ongoing fibrillation. Based on fibrillation threshold testing, it has been suggested that cardiac tissue is most sensitive to long duration, low strength AC stimulation at around 50 Hz. This has not been directly tested for defibrillation. Two subcutaneous electrodes were placed 40 mm apart on opposing aspects of the guinea pig thorax. Seven seconds were allowed to elapse between fibrillation initiation and defibrillation. The tested waveforms were at 50, 100, 200, 500, and 1000 Hz with 2, 4, 8, 16, and 32-cycles. The efficacy of every waveform was measured using a single stimulus in a large population of animals. Forty-one guinea pigs were used in the fixed energy group. Thirty-three guinea pigs were used in the fixed amplitude group with additional 1-cycle waveforms tested. The 200-Hz and the 2-cycle waveforms were significantly more efficacious than those at other frequencies (P < 0.02) and other durations (P < 0.001). The 50-Hz waveforms were the least successful. Amplitude, not duration or energy, was the determinate of efficacy for 2-cycle (the most efficacious) waveforms. Unlike low strength stimulation, defibrillation strength stimuli are most effective with high frequency (200 Hz) pulses (2 cycles).