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The Journal of Neuroscience, July 2, 2003, 23(13):5698-5707
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Properties and Functional Role of Voltage-Dependent Potassium Channels in Dendrites of Rat Cerebellar Purkinje Neurons
Marco Martina, Gui Lan Yao, andBruce P. Bean Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Abstract
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Abstract
Introduction
We characterized the properties and functional roles of voltage-dependent potassium channels in the dendrites of Purkinje neurons studied in rat cerebellar slices. Using outside-out patches formed250 µm away from the soma, we found that depolarization-activated potassium channels were present at high density throughout the dendritic tree. Currents required relatively large depolarizations for activation (midpoint, approximately –10 mV), had rapid activation and deactivation kinetics, and inactivated partially (20–70% over 200 msec) with both fast (time constant, 15–20 msec) and slow (300–400 msec) components. Inactivating and noninactivating components were both blocked potently by external tetraethylammonium (half-block by 150 µM) and 4-aminopyridine (half-block by 110 µM). The voltage dependence, kinetics, and pharmacology suggest a predominant contribution by Kv3 family subunits, and immunocytochemical experiments showed staining for both Kv3.3 and Kv3.4 subunits in the dendritic tree. In the proximal dendrite, potassium channels were activated by passively spread sodium spikes recorded at the same position, and experiments using dual recordings showed that the channels serve to actively dampen back-propagation of somatic sodium spikes. In more distal dendrites, potassium currents were activated by voltage waveforms taken from climbing fiber responses, suggesting that they help shape these responses as well. The requirement for large depolarizations allows dendritic Kv3 channels to shape large depolarizing events while not disrupting spatial and temporal summation of smaller excitatory postsynaptic potentials.
Key words: Kv3; Kv3.3; Kv3.4; tetraethylammonium; 4-aminopyridine; climbing fiber; Purkinje cell
Introduction
A growing body of experimental data has shown the presence of voltage-dependent ion channels in the dendrites of neurons (for review, see Johnston et al., 1996; Spruston et al., 1999
The dendrites of cerebellar Purkinje neurons have active membrane properties (Llinás and Sugimori, 1980
Very little is known about potassium channels in Purkinje neuron dendrites. Current-clamp recordings from dendrites show strong outward rectification, consistent with the presence of large depolarization-activated potassium conductances (Etzion and Grossman, 1998
Materials and Methods
Parasagittal slices of 300 µm thickness were cut from the cerebella of Long–Evans rats using a vibrating tissue slicer (Ted Pella, Redding, CA). Rats (14–21 d of age) were anesthetized by methoxyflurane before decapitation and removal of the cerebellum. All procedures involving animals were approved by the Harvard Medical Area Standing Committee on Animals. After cutting, slices were incubated at 35°C for 20 min and then stored at room temperature.During recording, slices were continuously superfused with physiological extracellular solution containing (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 25 glucose, bubbled with 95% O2 and 5% CO2. Slices were visualized with an Olympus BX50WI (Olympus Optical, Tokyo, Japan) upright microscope using infrared differential interference contrast videomicroscopy (Stuart et al., 1993
Patch pipettes were pulled from borosilicate glass tubing (outer diameter, 1.65 mm; inner diameter, 0.75 mm; Dagan, Minneapolis, MN) and heat polished before use. Pipettes were filled with an internal solution consisting of (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 2 Na2 ATP, 10 HEPES, pH adjusted to 7.3 with KOH. In some experiments, 30 mM KCl was substituted with 30 mM KF, which enhanced seal formation and stabilized outside-out patches. Tip resistances in working solutions were 5–11 M
. The pipettes were brought close to the target while exerting positive pressure (30–60 millibar). This process helped in cleaning off glial cells that often wrap Purkinje cell dendrites. Purkinje cells were easily identified on the basis of their large size and distinctive morphology. Purkinje cells in the vermis were used for these experiments. Distance of the dendrite from which the patch was formed was measured from the center of the soma and off-line from pictures taken with a CCD camera and frame grabber (Scion, Frederick, MD).
We used outside-out patches for characterizing potassium currents because they allow recording of relatively large currents and also facilitate the application of different drugs to study the pharmacological profile of the channels. A potential disadvantage is that properties of channels might change because of changes in the phosphorylation state of the channels (although ATP was included in the internal solution to minimize such changes) or other consequences of dialysis. To evaluate this issue, in early exploratory experiments, we measured potassium currents from dendrites in the cell-attached configuration. It was not possible to use this configuration routinely, because most neurons were spontaneously active and thus it was impossible to control the membrane voltage. In a limited number of cells that did not show spontaneous firing, we determined activation curves in cell-attached patches using absolute voltages calculated after breaking through into the cell and measuring the resting potential. In seven cell-attached patches on dendrites, the voltage of half-maximal activation was –7 ± 3 mV and the slope factor was 20 ± 2 mV, similar to the values of –10 ± 2 mV and 23 ± 1 mV (n = 16) for outside-out patches on dendrites (parameters from fits of the Boltzmann equation raised to the fourth power).
Recordings were performed at 21–24°C using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Signals were low-pass filtered at 5 or 10 KHz (4-pole low-pass Bessel filter on amplifier) and digitized (10–20 kHz) using a Digidata 1321A controlled by pClamp8 software interface (Axon Instruments). Currents were corrected for leak current and capacity transients remaining after electronic capacity compensation using a P/4 leak correction protocol. Currents from outside-out patches were signal-averaged over three trials when determining current–voltage curves and
Dual somatic and dendritic recordings were performed with two Axopatch 200B amplifiers in voltage or current-clamp (I-clamp fast) mode. Capacitive compensation, series resistance compensation, and super-charging (prediction) were used. In voltage-clamp experiments, the compensation ranged from 75 to 85%. Signals from dual current-clamp recordings were sampled at 20 KHz and filtered at 10 KHz. Spontaneous activity was recorded in the presence of 1.2 mM kynurenate and 100 µM picrotoxin to block fast synaptic transmission.
Drugs were applied to patches using quartz microcapillaries in a linear array with control and drug-containing solutions in adjacent capillaries. These experiments were performed using a HEPES-buffered external solution containing (in mM): 140 mM NaCl, 4 mM KCl, 2 mM CaCl2,1mM MgCl2, 25 mM glucose, 10 mM HEPES, pH adjusted to 7.3, with NaOH.
To construct activation curves, chord conductance (G) was calculated from the peak current assuming ohmic behavior and a reversal potential of –95 mV. Activation and inactivation curves were fit with functions on the basis of the Boltzmann function, 1/(1 + exp [±(V – Vh)/k]), where V is the membrane potential, Vh is the potential at which the value of the Boltzmann function is 0.5, and k is the slope factor. Activation curves were fit with a Boltzmann function raised to the fourth power. Inactivation curves were fit with a Boltzmann function plus a constant. The curves shown in figures represent fits to the averaged data obtained from pooling all experiments together. The parameters reported in the text and Table 1 are the values obtained by averaging the results of independent fitting of individual experiments. In principle, this is a better procedure, because when data are averaged before plotting the curves, the apparent slope may be reduced if equally steep curves with slightly different midpoints are averaged. However, the differences between the two sets of parameters were minor.
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Table 1. Comparison of potassium currents in outside-out patches from dendrites and somata of cerebellar Purkinje neurons
Data are reported as mean ± SEM, and error bars in figures also represent SEM. SEMs of fit parameters were obtained by fitting data of individual experiments separately.
Immunocytochemistry. Postnatal 15–16-d-old Long–Evans rats were anesthetized with isoflurane and perfused through the left ventricle with 30 ml of ice-cold saline followed by 200 ml of 4% paraformaldehyde, 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4. The brains were immersed in the same fixative at 4°C for 16 hr and then transferred to 30% sucrose in sodium phosphate buffer for 2–3 d. Twenty-five micrometer thick sagittal sections were cut with a cryostat, mounted on glass slides, and then air-dried and rehydrated in 0.1 M phosphate buffer, pH 7.4, for at least 40 min. The sections were then treated for 10 min in 0.1 M PBS, pH 7.4, containing 0.3% hydrogen peroxide (Sigma, St. Louis, MO) and 0.3% normal goat serum (Sigma) at room temperature and then rinsed with TNT (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) two times for 5 min each. Slices were washed in TNB (TNT plus 1% goat serum; 20–22°C for 1 hr) to minimize unspecific binding and then incubated overnight at 4°C with primary diluted in TNB. Dilutions were 1:1000 for mouse anti-calbindin (Sigma), 1:100 for rabbit anti-Kv3.4 (Alomone Labs, Jeruselum, Israel), and 1:300 for rabbit anti-Kv3.3 (kind gift from Lisa Taylor and Dr. Teresa Perney). Afterward, sections were rinsed in TNT (three times for 10 min each) and then incubated with the following secondary antibodies: Alexa 488 goat anti-mouse (1:100; Molecular Probes, Eugene, OR) and Rhodamine Red-X goat anti-rabbit (1:100; Jackson ImmunoResearch Labs) for 1 hr at room temperature in TNB. Subsequently, sections were washed once in TNT and twice in PBS (10 min each) and then coverslipped in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Sections were photographed using a confocal microscope (Bio-Rad MRC-1024 with 20, 40, or 60x objectives; Bio-Rad, Hercules, CA). Images were merged using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).
In all experiments, control experiments were performed by preincubating sections (taken adjacent to the test sections) with the primary anti-Kv3.4 or anti-Kv3.3 antibodies in the presence of a large molar excess of peptide antigen. For all images shown, adjacent sections treated with an excess of peptide antigen had only faint nonspecific staining when processed identically.
Results
Voltage dependence and kinetics of dendritic potassium current
We characterized the voltage dependence and kinetics of voltage-activated potassium current in outside-out patch recordings taken from Purkinje cell dendrites
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Figure 1. Voltage dependence and kinetics of activation of dendritic potassium currents. A, Potassium currents elicited in an outside-out patch taken from a Purkinje cell dendrite (140 µm from the soma) by 200 msec steps from a holding potential of –90 mV to voltages from –80 to 70 mV (10 mV intervals). B, Peak conductance–voltage relationship for dendritic patches (mean ± SEM; 16 cells). Conductance was calculated assuming a reversal potential of –95 mV and a linear current–voltage curve for open channels. The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [-(V-Vh)/k])] 4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is –46 mV, and the slope factor is 20 mV. This function reaches a midpoint at a value of Vh + 1.67*k, or –13 mV. C, Time course of activation at higher time resolution. D, Time for current to rise from 10 to 90% of its peak value plotted against test pulse voltage for 16 dendritic patches. These parameters are fits to the averaged data. Table 1 shows the values for midpoint, k, and noninactivating fraction obtained by averaging fits to data from individual patches.
Activation was rapid (Fig. 1C), with a 10–90% rise-time of 3.1 ± 0.4 msec at 0 mV (n = 16) (Fig. 1D). Activation kinetics were strongly voltage dependent, and rise-times decreased to near 1 msec for depolarizations positive to 40 mV. Deactivation of the current was also very fast (Fig. 2). Deactivation could generally be fit well by a single exponential, with average time constants of 2.8 ± 0.9 msec at –40 mV and 1.1 ± 0.2 msec at –70 mV (n = 4).
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Figure 2. Rapid deactivation of dendritic potassium currents. A, Potassium currents elicited in a dendritic outside-out patch (taken from dendrite 120 µm from soma) with a 5 msec step to 70 mV to fully activate the channels followed by repolarization to voltages from –110 to –40 mV in 10 mV increments. Tail currents reversed between –80 and –90 mV. B, Collected data (mean ± SEM) from four dendritic patches. Protocols were as in A, except that both shorter (5 msec) and longer (100 msec) activating steps to 70 mV were used. With the longer activating pulses, the reversal potential of tail currents was approximately –50 mV, probably reflecting accumulation of potassium ions on the external surface of the patch. This facilitated the measurement of time constants at –80 and –90 mV. Time constants did not depend on the length of the activation pulse or the direction of current flow.
Dendritic potassium currents showed partial inactivation, typically decaying by 30–50% over 200 msec for voltage steps positive to 0 mV. The time course of decay could generally be fit well by two exponential functions (Fig. 3A). Measured for maximal activation by steps to 70 mV, the faster time constant had an average value of 19 ± 3 msec (n = 26) and the slower time constant was 377 ± 131 msec. On average, the fast inactivating component contributed 24 ± 4% of the total peak current, the slow-inactivating component contributed 31 ± 6%, and 45 ± 5% remained after 200 msec. Figure 3, B and C, shows the steady-state voltage dependence of inactivation, determined by changing the holding potential for 5 sec before a 100 msec test pulse to 70 mV. A notable feature is that inactivation becomes substantial only when depolarizations reach voltages at which significant current is activated (positive to –40 mV). This property differentiates these currents from A-type potassium currents mediated either by Kv4 or Kv1.4 (Pak et al., 1991
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Figure 3. Voltage dependence and kinetics of inactivation of potassium current in dendritic patches. A, Potassium current elicited by a 200 msec pulse from –90 to 70 mV in a dendritic outside-out patch taken 140 µM from the soma. Inactivation could be fit by two exponentials plus a constant, and the experimental trace is overlaid by a fitted curve with 18% of the current decaying with a time constant of 2.5 msec, 43% with a time constant of 68 msec, and 39% nondecaying. B, Voltage dependence of inactivation determined by changing the holding potential for 5 sec before a test pulse to 70 mV (patch taken 70 µM from the soma). C, Peak test pulse current versus prepulse voltage for prepulses of 5 sec (filled circles; mean ± SEM; n = 6) or 100 msec (open circles; mean ± SEM; n = 10). Average values are fit by a Boltzmann function decaying to a non-zero fraction, NI + (1 – NI)/(1 + exp[(V – Vh)/k]), where NI is the fraction of noninactivating current, Vh is the midpoint for the inactivating fraction, and k is the slope factor for the inactivating fraction. For 5 sec prepulses, Vh = –39 mV, k = 11.0 mV, and NI = 0.07. For 100 msec prepulses, Vh = –32 mV, k = 7.3 mV, and NI = 0.63. These parameters are for fits to the averaged data. Table 1 shows the values for Vh, k, and noninactivating fraction obtained by averaging fits to data from individual patches.
When the voltage dependence of the inactivation of dendritic currents by a 5 sec prepulse was fit with a Boltzmann function plus a constant, the average midpoint was –37.6 ± 4.5 mV, and the slope factor was 12 ± 0.6 mV (n = 6). Inactivation was nearly complete with depolarizations beyond approximately –20 mV.
We also determined the voltage dependence of inactivation using 100 msec prepulses, which approximate the time scale of complex spikes and short plateau potentials that dendrites may experience. In this case, inactivation was only partial (
35–40%) even for depolarizations up to 10 mV (Fig. 3C, open circles). The midpoint of inactivation determined using 100 msec prepulses was –33.3 ± 3 mV, and the slope factor was 11.3 ± 3.4 mV, with an average fraction of 58 ± 6% (n = 10) remaining noninactivated. Interestingly, there was considerable heterogeneity among individual dendritic patches in the susceptibility to inactivation by 100 msec prepulses, ranging from 20 to 70% of the total current.
Comparison with somatic currents
Figure 4 illustrates how the current magnitude and degree of inactivation depend on the distance from the soma. Outside-out patches were taken at positions varying from the soma itself to the distal dendrites. Overall, currents in somatic patches (Fig. 4A) were very similar to those in dendritic patches (Table 1). Their voltage dependence was very similar, with half-maximal activation at –12 ± 2 mV (n = 18) and a slope factor of 18 ± 2 mV when fit by a Boltzmann function to the fourth power (Fig. 4B). As for dendritic currents, somatic currents had rapid activation and deactivation kinetics (Table 1). However, there were two trends evident when examining currents as a function of distance from the soma. On average, currents were somewhat smaller with increasing distance from the soma (Fig. 4C). The average current amplitude for a step from –90 to 70 mV was 779 ± 130 pA in somatic patches (25 patches), 440 ± 46 for dendrites
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Figure 4. Dependence of potassium current amplitude and degree of inactivation on distance from soma. A, Potassium currents elicited in a somatic patch by 200 msec steps from a holding potential of –90 mV to voltages from –80 to 70 mV (10 mV intervals). B, Peak conductance–voltage relationship for somatic patches (mean ± SEM; 18 cells). Conductance was calculated assuming a reversal potential of –95 mV and a linear current–voltage curve for open channels. Solid curve is a Boltzmann function raised to the fourth power, with a midpoint potential of –11.5 mV and slope factor 18 mV. C, Magnitude of peak potassium current (step from –90 to 70 mV) in outside-out patches, plotted as a function of distance from the soma from which the patch was formed (closed circles). To facilitate the comparison, only data obtained with 7–11 M
There was also a trend evident in the degree of inactivation with distance from the soma (Fig. 4D), with inactivation being more pronounced with increasing distance from the soma. For depolarizations to 70 mV, the ratio of noninactivating current (measured after 100 msec) to peak current decreased from 0.75 ± 0.02 (n = 28) for somatic patches to 0.67 ± 0.02 (n = 71) for dendrites
Sensitivity to TEA and 4-aminopyridine
The potassium currents in dendritic membranes were highly sensitive to block by external TEA (Fig. 5A). At 0.1 mM, TEA blocked the current by 46 ± 4%, and 1 mM TEA blocked 76 ± 4% of the current. The dose–response curve (Fig. 5B) suggested that
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Figure 5. Pharmacology of dendritic potassium currents. A, Effects of increasing concentrations of external TEA on currents evoked by 200 msec pulses from –90 to 70 mV in a dendritic patch (excised 50 microns from soma). B, Dose–response curve for block of dendritic current by external TEA. The solid curve represents fit by the logistic equation to the data up to 3 mM TEA, 0.8/[1 + (TEA)/Kd] + 0.2, with Kd = 85 µM. C, Effect of 4-AP on currents from a dendritic outside-out patch (excised 75 microns from soma). D, Dose–response curve for block of dendritic current by 4-AP. The solid curve represents fit by the logistic equation with variable slope (Hill coefficient) given by 0.81/(1 + [(4-AP)/Kd]n) + 0.19, with Kd = 86 µM and n = 1.7.
It has been found previously that somatic potassium currents in mouse Purkinje neurons are effectively blocked by external TEA (Raman and Bean, 1999
The potassium currents in the dendrites were also highly sensitive to 4-AP. At 100 µM, 4-AP blocked approximately half the total current (Fig. 5C). The dose–response curve (Fig. 5D) suggests that a component of the total current comprising
The high sensitivity to 4-AP of the potassium current in outside-out patches from rat dendritic membrane is nicely consistent with previous current-clamp recordings from dendrites of guinea-pig Purkinje neurons, in which outward rectification was reduced by low concentrations of 4-AP (Etzion and Grossman, 2001
Immunocytochemistry
These results show that the predominant potassium current in the dendritic membrane of Purkinje cells requires relatively large depolarizations to be activated and has rapid activation and deactivation kinetics as well as high sensitivity to both external TEA and 4-AP. These properties are all characteristic of members of the Kv3 family (Martina et al., 1998
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Figure 6. Immunostaining for Kv3.3 and Kv3.4 subunits in somata and dendrites of Purkinje neurons. A, Confocal image of Purkinje cell layer after staining with antibodies to Kv3.3 (red). B, Same slice stained using primary antibodies to calbindin (green). C, Merged image. D, Higher magnification merged image of staining by Kv3.3 (red) and calbindin (green). E–G, Staining with antibodies to Kv3.4 (E, red), calbindin (F, green), and merged image (G).H, Higher magnification merged image of staining by Kv3.4 (red) and calbindin (green). Note the colocalization of the antibodies in many regions of distal dendrites (including that marked by an arrow). For both Kv3.3 and Kv3.4 staining, controls were run by staining adjacent sections and processing them in parallel except that the antibodies were preincubated with 100-fold excess of antigenic peptide; there was only very faint nonspecific staining of cell body cytoplasm present in the controls.
Activation by action potentials and synaptic potentials
Under what conditions are dendritic potassium channels activated, and what is their functional role? To explore these questions, we made current-clamp recordings of the voltage trajectory of dendritic membrane under various circumstances and then performed voltage-clamp experiments to examine whether those voltage patterns activated dendritic potassium currents in outside-out patches, often formed using the same pipette used to make the current-clamp recordings. Purkinje neurons often fire spontaneous action potentials in a highly regular manner (Häusser and Clark, 1997
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Figure 7. Activation of dendritic potassium current by truncated dendritic spikes. A, Peak amplitude of dendritic action potentials (from spontaneous firing of neurons with no injected current) plotted against distance from soma. The dashed line represents the average voltage at which dendritic K+ currents activate by
However, it is also necessary to establish that activation of the dendritic channels is sufficiently rapid to be activated during the action potential in the dendrite, especially because action potentials in Purkinje neurons are very narrow (0.53 ± 0.02 msec; half-height width; measured in the soma; 23°C; n = 14 cells). The experiment in Figure 7B shows directly that dendritic potassium channels are activated by the action potentials they experience. In this experiment, the recording pipette was first used to make a current-clamp recording from the dendrite (at 35 µm from the soma), and spontaneous spikes with amplitude of
Activation of potassium current by the spikes in the proximal dendrite would be predicted to actively dampen the dendritic spike beyond the truncation from the passive properties of the dendrite. The high sensitivity of the dendritic potassium channels to external TEA provides a way of testing this prediction. Figure 8 shows such an experiment in which a prerecorded waveform taken from current-clamp stimulation of a Purkinje neuron cell body was applied as a voltage-clamp command at the cell body. A second electrode recorded the voltage response at a dendritic location 35 µm away with 300 nM TTX present in the external solution to prevent spontaneous firing of the neuron and avoid eliciting uncontrolled sodium currents in the cell body. As expected, the voltage recorded at the dendrite (height, 35 mV) was much smaller than that at the cell body (94 mV). When 1 mM TEA was added to the external solution, the spikes measured at the dendrite became larger and also broader. This suggests that dendritic potassium channels have the effect of both truncating and narrowing the dendritic spike, at least in the proximal dendrite. In collected data from three cells, the half-duration of dendritic spikes increased from 0.66 ± 0.03 msec in control to 0.87 ± 0.03 msec after block of potassium currents by 1 mM TEA.
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Figure 8. Dendritic potassium channels actively depress and narrow sodium spikes in the dendrite. A, A double recording was performed from soma and dendrite of a Purkinje neuron in the presence of 300 nM TTX. The soma was voltage clamped with a trace of Purkinje neuron spontaneous activity (A). The dendritic pipette recorded in current-clamp passively spread action potential either in control condition or after bath application of 1 mM TEA (B). C, The first action potential in the train (area indicated by arrows) shown on an expanded scale. Note the large increase in spike duration and the incomplete repolarization induced by TEA.
Purkinje neurons receive excitatory synaptic input from the inferior olive by means of climbing fibers, which form powerful 1:1 synaptic connections with Purkinje neurons. Stimulation of a single climbing fiber evokes a large, all-or-none EPSP on which multiple action potentials are superimposed, the "complex spike" (Eccles et al., 1966
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Figure 9. Activation of dendritic potassium current by climbing fiber response. A, Voltage response to climbing fiber stimulation recorded in a dendrite at 75 µm from the soma. B, After forming an outside-out patch using the same pipette at the same location, the recorded voltage response was used as voltage command to record the potassium current elicited in the dendrite by the EPSP.
Discussion
Molecular composition of dendritic potassium channels
The predominant potassium currents in outside-out patches from Purkinje cell dendrites had rapid activation and deactivation kinetics and high sensitivity to external TEA and 4-AP, consistent with Kv3 family potassium channels (Grissmer et al., 1994Our results showing prominent staining for Kv3.4 protein in Purkinje neurons differs from a previous study (Veh et al., 1995
Kv3.1 RNA is present at low levels of expression in Purkinje neurons (Weiser et al., 1994
Different Kv3 family subunits have different kinetic properties. Homomers of Kv3.3 subunits inactivate slowly (hundreds of milliseconds) (Rudy and McBain, 2001
Approximately 20% of the current in dendritic patches was left unblocked by 3 mM TEA or 3 mM 4-AP. Additional analysis will be required to identify the channels underlying this component. Sacco and Tempia (2002
Comparison with somatic potassium channels
Channels in somatic patches had very similar voltage dependence, kinetics, and pharmacology as dendritic channels (Table 1). Our results for somatic potassium channels are very similar to those obtained previously in the somata of mouse Purkinje neurons (Raman and Bean, 1999Purkinje neurons are known to possess both SK-type and BK-type calcium-activated potassium channels (Yool et al., 1988
Functional significance
Unlike many other types of neurons, sodium-dependent action potentials are not effectively propagated in the dendrites of Purkinje neurons because dendritic sodium-channel density is low (Stuart and Häusser, 1994The inactivation properties of Kv3 channels of Purkinje cell dendrites differ considerably from the inactivation of potassium current in dendrites of pyramidal neurons, which is primarily attributable to Kv4 family channels (Maletic-Savatic et al., 1995
Our results with climbing fiber stimulation show that climbing fiber EPSPs are large enough to activate potassium channels, even in fairly distal dendrites, so that the presence of the channels will help determine the magnitude and duration of such EPSPs. Distal dendrites are also known to support slow calcium-dependent spikes and plateau potentials that can reach potentials near 0 mV (Llinás and Sugimori, 1980
In many cases, Kv3 family channels have activation curves with midpoints near 10 mV (Rudy and McBain, 2001
Footnotes
Received Mar. 6, 2003; revised Apr. 16, 2003; accepted Apr. 18, 2003.This work was supported by National Institutes of Health Grants NS36855 and NS38312. We are very grateful to Lisa Taylor and Dr. Teresa Perney (Rutgers, The State University of New Jersey, Newark, Center for Molecular and Behavioral Neuroscience) for the kind gift of antibodies to Kv3.3 and helpful comments on this manuscript.
Correspondence should be addressed to Bruce Bean, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. E-mail: bruce_bean{at}hms.harvard.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235698-10$15.00/0
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