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.

Electrophysiological properties of identified oxytocin and vasopressin neurones.

To understand the contribution of intrinsic membrane properties to the different in vivo firing patterns of oxytocin (OT) and vasopressin (VP) neurones, in vitro studies are needed, where stable intracellular recordings can be made. Combining immunochemistry for OT and VP and intracellular dye injections allows characterisation of identified OT and VP neurones, and several differences between the two cell types have emerged. These include a greater transient K+ current that delays spiking to stimulus onset, and a higher Na+ current density leading to greater spike amplitude and a more stable spike threshold, in VP neurones. VP neurones also show a greater incidence of both fast and slow Ca2+ -dependent depolarising afterpotentials, the latter of which summate to plateau potentials and contribute to phasic bursting. By contrast, OT neurones exhibit a sustained outwardly rectifying potential (SOR), as well as a consequent depolarising rebound potential, not found in VP neurones. The SOR makes OT neurones more susceptible to spontaneous inhibitory synaptic inputs and correlates with a longer period of spike frequency adaptation in these neurones. Although both types exhibit prominent Ca2+ -dependent afterhyperpolarising potentials (AHPs) that limit firing rate and contribute to bursting patterns, Ca2+ -dependent AHPs in OT neurones selectively show significant increases during pregnancy and lactation. In OT neurones, but not VP neurones, AHPs are highly dependent on the constitutive presence of the second messenger, phosphatidylinositol 4,5-bisphosphate, which permissively gates N-type channels that contribute the Ca2+ during spike trains that activates the AHP. By contrast to the intrinsic properties supporting phasic bursting in VP neurones, the synchronous bursting of OT neurones has only been demonstrated in vitro in cultured hypothalamic explants and is completely dependent on synaptic transmission. Additional differences in Ca2+ channel expression between the two neurosecretory terminal types suggests these channels are also critical players in the differential release of OT and VP during repetitive spiking, in addition to their importance to the potentials controlling firing patterns.

Specificity in the interaction of high-voltage-activated Ca2+ channel types with Ca2+-dependent afterhyperpolarizations in magnocellular supraoptic neurons.

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 µM Cd2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 µM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

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.

Changes in potassium channel modulation may underlie afterhyperpolarization plasticity in oxytocin neurons during late pregnancy.

Oxytocin (OT) neurons exhibit larger afterhyperpolarizations (AHPs) following spike trains during late pregnancy and lactation, times when these neurons fire in bursts and release more OT associated with labor and lactation. Calcium-dependent AHPs mediated by SK channels show this plasticity, and are reduced when the channel complex is phosphorylated by casein kinase 2 (CK2), and increased when dephosphorylated by protein phosphatase (PP)2A, by altering Ca2+ sensitivity. We compared AHP currents in supraoptic OT neurons after CK2 inhibition with 4,5,6,7-tetrabromobenzotriazole (TBB), or PP1-PP2A inhibition with okadaic acid (OA), to determine the roles of these enzymes in AHP plasticity, focusing on the peak current at 100 ms representing the SK-mediated, medium AHP (ImAHP). In slices from virgin and two groups of pregnant rats [embryonic days (E18-19, or E20-21], ImAHPs were evoked with 3-, 10-, and 17-spike trains (20 Hz). With 3-spike trains, TBB increased the ImAHP to the greatest extent in virgin compared with both groups of pregnant animals. A difference between virgins and E20-21 rats was also evident with a 10-spike train but the increases in ImAHPs were similar among groups with 17-spike trains. In contrast, OA, while consistently reducing the ImAHP in all cases, showed no differential effects among groups. In Western blots, CK2α, CK2ß, PP2A-A, PP2A-B, and PP2A-C were found in supraoptic lysates, and expression of CK2α and CK2ß was reduced in E20-21 rats. Coimmunoprecipitation revealed that calmodulin, CK2α, and PP2A-C were associated with SK3 protein. The results suggest that a downregulation of SK3-associated CK2α during late pregnancy may increase the sensitivity of the SK calmodulin (Ca2+) sensor for ImAHP, contributing to the enhanced ImAHP. NEW & NOTEWORTHY The article demonstrates for the first time that enhancement in spike afterhyperpolarizations in oxytocin neurons during pregnancy may be related to a downregulation in the small-conductance Ca2+-activated potassium channels (SK)/calmodulin binding protein casein kinase 2, which phosphorylates the SK channel complex and reduces its Ca2+ sensitivity.

Phosphatidylinositol 4,5-bisphosphate (PIP2 ) modulates afterhyperpolarizations in oxytocin neurons of the supraoptic nucleus.

KEY POINTS: Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2 's control of these AHPs. We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+ ]i , and whole cell Ca2+ currents. Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3 -triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2 . PIP2 depletion reduced spike-evoked Ca2+ entry and voltage-gated Ca2+ currents. PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking. ABSTRACT: Oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium-dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP2 analogue (diC8 -PIP2 ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5-trisphosphate (IP3 )-triggered Ca2+ -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+ ]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+ . Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage-gated Ca2+ channels opened during spike trains.

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.

The calcium-activated slow AHP: cutting through the Gordian knot.

The phenomenon known as the slow afterhyperpolarization (sAHP) was originally described more than 30 years ago in pyramidal cells as a slow, Ca(2+)-dependent afterpotential controlling spike frequency adaptation. Subsequent work showed that similar sAHPs were widely expressed in the brain and were mediated by a Ca(2+)-activated potassium current that was voltage-independent, insensitive to most potassium channel blockers, and strongly modulated by neurotransmitters. However, the molecular basis for this current has remained poorly understood. The sAHP was initially imagined to reflect the activation of a potassium channel directly gated by Ca(2+) but recent studies have begun to question this idea. The sAHP is distinct from the Ca(2+)-dependent fast and medium AHPs in that it appears to sense cytoplasmic [Ca(2+)](i) and recent evidence implicates proteins of the neuronal calcium sensor (NCS) family as diffusible cytoplasmic Ca(2+) sensors for the sAHP. Translocation of Ca(2+)-bound sensor to the plasma membrane would then be an intermediate step between Ca(2+) and the sAHP channels. Parallel studies strongly suggest that the sAHP current is carried by different potassium channel types depending on the cell type. Finally, the sAHP current is dependent on membrane PtdIns(4,5)P(2) and Ca(2+) appears to gate this current by increasing PtdIns(4,5)P(2) levels. Because membrane PtdIns(4,5)P(2) is essential for the activity of many potassium channels, these finding have led us to hypothesize that the sAHP reflects a transient Ca(2+)-induced increase in the local availability of PtdIns(4,5)P(2) which then activates a variety of potassium channels. If this view is correct, the sAHP current would not represent a unitary ionic current but the embodiment of a generalized potassium channel gating mechanism. This model can potentially explain the cardinal features of the sAHP, including its cellular heterogeneity, slow kinetics, dependence on cytoplasmic [Ca(2+)], high temperature-dependence, and modulation.

Essential role for phosphatidylinositol 4,5-bisphosphate in the expression, regulation, and gating of the slow afterhyperpolarization current in the cerebral cortex.

Many neurons of the CNS and peripheral nervous system express a slow afterhyperpolarization that is mediated by a slow calcium-activated potassium current. Previous work has shown that this aftercurrent regulates repetitive firing and is an important target for neuromodulators signaling through receptors coupled to G-proteins of the Gα(q-11) and Gα(s) subtypes. Yet, despite considerable effort, a molecular-level understanding of the potassium current underlying the slow afterhyperpolarization and its modulation has proven elusive. Here, we use a combination of pharmacological and molecular biological approaches in cortical brain slices to show that the functional expression of the slow calcium-activated afterhyperpolarizing current in pyramidal cells is critically dependent on membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and that this dependence accounts for its inhibition by 5-HT(2A) receptors. Furthermore, we show that PtdIns(4,5)P(2) regulates the calcium sensitivity of I(sAHP) in a manner that suggests it acts downstream from the rise in intracellular calcium. These results clarify key functional aspects of the slow afterhyperpolarization current and its modulation by 5-HT(2A) receptors and point to a key role for PtdIns(4,5)P(2) in the gating of this current.

Parallel optical control of spatiotemporal neuronal spike activity using high-speed digital light processing.

Neurons in the mammalian neocortex receive inputs from and communicate back to thousands of other neurons, creating complex spatiotemporal activity patterns. The experimental investigation of these parallel dynamic interactions has been limited due to the technical challenges of monitoring or manipulating neuronal activity at that level of complexity. Here we describe a new massively parallel photostimulation system that can be used to control action potential firing in in vitro brain slices with high spatial and temporal resolution while performing extracellular or intracellular electrophysiological measurements. The system uses digital light processing technology to generate 2-dimensional (2D) stimulus patterns with >780,000 independently controlled photostimulation sites that operate at high spatial (5.4 µm) and temporal (>13 kHz) resolution. Light is projected through the quartz-glass bottom of the perfusion chamber providing access to a large area (2.76 mm × 2.07 mm) of the slice preparation. This system has the unique capability to induce temporally precise action potential firing in large groups of neurons distributed over a wide area covering several cortical columns. Parallel photostimulation opens up new opportunities for the in vitro experimental investigation of spatiotemporal neuronal interactions at a broad range of anatomical scales.

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.

Nonequilibrium calcium dynamics regulate the autonomous firing pattern of rat striatal cholinergic interneurons.

Striatal cholinergic interneurons discharge rhythmically in two patterns associated with different afterhyperpolarization timescales, each dictated by a different calcium-dependent potassium current. Single spiking depends on a medium-duration afterhyperpolarization (mAHP) generated by rapid SK currents that are associated with N-type calcium channels. Periodic bursting is driven by a delayed and slowly decaying afterhyperpolarization (sAHP) current associated with L-type channels. Using calcium imaging we show that the calcium transients underlying these currents exhibit two corresponding timescales throughout the somatodendritic tree. This result is not consistent with spatial compartmentalization of calcium entering through the two calcium channels and acting on the two potassium currents, or with differences in channel gating kinetics of the calcium dependent potassium currents. Instead, we show that nonequilibrium dynamics of calcium redistribution among cytoplasmic binding sites with different calcium binding kinetics can give rise to multiple timescales within the same cytoplasmic volume. The resulting independence of mAHP and sAHP currents allows cytoplasmic calcium to control two different and incompatible firing patterns (single spiking or bursting and pausing), depending on whether calcium influx is pulsatile or sustained. During irregular firing, calcium entry at both timescales can be detected, suggesting that an interaction between the medium and slow calcium-dependent afterhyperpolarizations may underlie this firing pattern.

The mechanism of venom secretion from Duvernoy's gland of the snake Thamnophis sirtalis.

The venom glands of snakes of the families Elapidae and Viperidae are thought to have evolved from Duvernoy's gland of colubrid ancestors. In highly venomous snakes elements of the external adductor musculature of the jaw insert fibers directly onto the capsule of the venom gland. These muscles, upon contraction, cause release of contents by increasing intraglandular pressure. In Thamnophis sirtalis, a colubrid, there is no direct connection between Duvernoy's gland and the adductor musculature. The anatomical arrangement of the gland, skull, adductor muscles, and the integument is such that contraction of the muscles may facilitate emptying of the gland. This hypothesis was tested by electrical stimulation of the muscles, which resulted in significantly greater release of secretion than elicited by controls. The results suggest a possible early step in the evolution of a more intimate association between venom glands and adductor musculature in highly venomous snakes.