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1Neuroscience Graduate Program, Oregon Health and Science University; and 2Oregon Hearing Research Center/ Vollum Institute, Portland, Oregon
Submitted 18 May 2006; accepted in final form 15 November 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Young and Davis 2002). Auditory nerve fibers terminate on dendrites of giant cells and fusiform cells in the DCN deep layer, and their input is modified by various interneurons within the cochlear nucleus (Nelken and Young 1994; Zhang and Oertel 1993b,c, 1994). The fusiform cells of the DCN also receive excitation through a system of granule cells and their associated parallel fibers in the DCN molecular layer. Some of the fibers that drive the granule cells originate in medullary somatosensory nuclei and may convey ear and head position. Indeed, one proposed function of the DCN has been that it contributes to orienting toward specific sounds by integrating auditory, somatosensory, and vestibular input (Kanold and Young 2001; May 2000; Oertel and Young 2004; Young and Davis 2002). Cartwheel cells (CWCs) are inhibitory interneurons that receive parallel fiber input and form synapses on fusiform cells and among themselves in the molecular and fusiform cell layer of the DCN (Berrebi and Mugnaini 1991; Mugnaini et al. 1987). The output of DCN principal neurons in vivo or in vitro has been characterized by inhibition of background spontaneous activity, and it is believed that CWCs play an important role in this process, being spontaneously active and capable of providing powerful feed-forward inhibition of parallel fiber input (Young and Davis 2002; Zhang and Oertel 1994). However, in vivo recordings have been made in immobilized animals where many of the somatosensory and vestibular inputs to granule cells are not activated, making the physiological activity of the molecular layer poorly understood.
CWCs are unique in the cochlear nucleus for their ability to generate complex spikes (Manis et al. 1994; Zhang and Oertel 1993a). Complex spikes, also called "bursts", consist of brief (<100 ms), clusters of high-frequency (>100 Hz) action potentials superimposed on an underlying slow depolarization and are seen in several neuronal cell types (Athanassiadis et al. 2005; Brumberg et al. 2000; Chagnac-Amitai et al. 1990; Deschenes et al. 1982; Jung et al. 2001; Kandel and Spencer 1961; Niespodziany and Poulain 1995; Schmolesky et al. 2002). The slow underlying depolarization of CWC complex spikes is believed to be Ca2+ dependent, whereas the fast action potentials riding on the slow wave are Na+ dependent as are single action potentials (the "simple spike") in these same cells (Golding and Oertel 1997). The terms "complex spike" and "simple spike" imply electrophysiological similarity of the CWCs to the cerebellar Purkinje cells and are in keeping with other studies highlighting morphological, genetic, and molecular parallels between these two cell types (Berrebi and Mugnaini 1991; Berrebi et al. 1990; Mugnaini and Morgan 1987). However, several features that differentiate these cell types suggest that a more careful investigation of CWC firing properties is needed. For example, Purkinje cells fire complex spikes only in response to climbing fiber activity; this activity is believed to play a key role in induction of synaptic plasticity at climbing fiber and parallel fiber synapses (Hansel and Linden 2000; Ito 2001; Konnerth et al. 1992). By contrast, CWCs may fire complex spikes spontaneously or in response to glutamatergic parallel fibers or glycinergic inputs from other CWCs (Golding and Oertel 1996; Tzounopoulos et al. 2004; Zhang and Oertel 1993a). Thus the computational meaning of complex spikes may be different in the two cell types. We therefore have investigated the channel types underlying firing of complex and simple spikes and what conditions promote each mode of firing, in slices of mouse DCN. Our results identify the role of multiple Ca2+ channels and Ca2+-dependent K+ channels as well as Na+ currents in promoting and shaping the complex spike.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Brain stem slices containing the DCN were prepared from ICR mice aged 16–23 days (Harlan, Indianapolis, IN). Mice were anesthetized with isoflurane and then decapitated in accord with the regulations of the Institutional Animal Care and Use Committee of Oregon Health and Science University. Subsequently, a block of brain stem was isolated and horizontal slices of 210-µm thickness were cut with a vibrating slicer (VT1000S, Leica, Deerfield, IL). Dissection and slicing were done in a warm (
30°C) solution, which was composed of (in mM) 130 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, and 10 glucose and was saturated with 95% O2-5% CO2. The chamber containing DCN slices were incubated at 34.5°C for the first hour and left at room temperature thereafter. For recording, a slice was transferred to the recording chamber on the stage of Olympus BX51WI microscope, and DCN cells were visualized with infrared differential interference contrast videomicroscopy. The bathing solution for recording was the same as that used for dissection and was perfused at 2–3 ml/min to the recording chamber by a peristaltic pump (Minipulse 3, Gilson, Middleton, WI). The temperature of the solution at the recording chamber was maintained at 33°C by a heating water jacket around perfusion tubing or by an in-line heating device.
Medium-sized cells in the molecular and fusiform cell layers of DCN were identified as CWCs if they showed complex spikes spontaneously or on injection of depolarizing current. The majority of data presented in this study were obtained with gramicidin perforated-patch recording (Kyrozis and Reichling 1995; Rhee et al. 1994) unless otherwise specified. The pipette solution for perforated patch recording consisted of (in mM) 140 KCl, 10 NaCl, and 10 HEPES (pH adjusted to 7.25 with KOH), and gramicidin was added just before use at a final concentration of 10–40 µg/ml from a stock solution of 10–30 mg/ml DMSO. The tip of the recording pipette was filled with the same solution but without gramicidin. For examination of the effect of increased intracellular Ca2+ buffering, conventional whole cell recording was employed: a standard internal solution containing (in mM) 113 K-gluconate, 4.5 MgCl2, 14 trisphosphocreatine, 9 HEPES, 0.1 EGTA, 4 Na-ATP, and 0.3 Tris-GTP (pH adjusted to 7.3 with KOH), was modified to have higher EGTA at 5 mM or to include 20 mM BAPTA (tetrapotassium salt) in place of equimolar K-gluconate. Patch pipettes for recording electrodes of 2–6 M
resistances were prepared by pulling thick-walled filamented borosilicate glass capillaries (1B120F-4, World Precision Instruments, Sarasota, FL) and wrapped with Parafilm along one-third of length from the tip to reduce capacitance. The liquid junction potentials measured (according to Neher 1992) and then corrected (for the reference junction, with JPCalc) (Barry 1994) were 2.8, 16, and 13.7 mV for the 140 mM KCl-based, 5 mM EGTA-containing, and 20 mM BAPTA-containing pipette solutions, respectively; the values for latter two solutions were subtracted from the voltage data obtained with each solution off-line.
Recordings were made with a BVC-700A (Dagan, Minneapolis, MN) or MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in conjunction with pClamp 9.2 software (Molecular Devices). Data were digitized with Digidata 1322A (Molecular Devices) at 20 kHz (current-clamp) or 10 kHz (voltage-clamp) and low-pass filtered at 10 kHz (current-clamp) or 3 kHz (voltage-clamp). Pipette capacitance was compensated in all current-clamp recordings. For perforated-patch recording, after the electrode had formed a seal (>1G) on the cell membrane in voltage clamp, the progression of perforation (reduction in series resistance, Rs) was monitored in current clamp by periodic bridge balancing and by observing the growth in amplitudes of spontaneous fast spikes (see Terminology). Within 40 min of forming a seal, the Rs dropped to 20–40 M; cells in which the Rs did not go <40 M were excluded from analysis. Occasionally, the perforated patch spontaneously ruptured, thus establishing whole cell configuration. There were several signs of patch rupture when the recording pipette contained the 140 mM KCl-based solution in the preceding text, listed in the order of occurrence: an abrupt positive shift in voltage trace by 7–10 mV accompanied with an increase in amplitude of fast spikes (due to Rs reduction; supplemental Fig. 1 Bi),1 very large depolarizing glycinergic/GABAergic postsynaptic potentials when the corresponding synaptic blockers were not present, and changes in pattern and waveforms of spikes characteristic of whole cell recording with a KCl-based internal solution (supplemental Fig. 1Bii). In some cases, the Vm shift occurred slowly, obscuring detection of patch rupture, but the striking effects of dialysis with KCl eventually confirmed the rupture. Extracellular recordings were done in voltage-clamp mode (VH = 0 mV) with a bath solution-filled recording pipette loosely attached to a cell.
Data were analyzed with Clampfit (pClamp 9.2) and Microsoft Excel. The membrane potential (Vm) between spikes of spontaneously active CWCs often fluctuated over a broad (20 mV) range (see RESULTS). When there were not such fluctuations in basal Vm, an approximate level of interspike trough potentials, Vtrough, (Fig. 1Ei) was used as a representative value of Vm. The thresholds of fast spikes were defined by the potential at the inflection point of the rising phase of the spike waveform (Fig. 1Ab, 
). For the repolarizing phase of a fast spike, the most negative Vm before the afterdepolarization, which may be a consequence of the fast afterhyperpolarization (fAHP), was measured and termed pFR (potential of fast spike repolarization; Fig. 1Ab, 
). The half-width of a fast spike was measured at the mid-point between the threshold and the peak of the spike. For comparison of spike waveform parameters between different conditions in one cell, the initial spikes in response to step current injections of a given amount were selected to obtain measurements unless otherwise specified. The threshold and pFR of spontaneous simple spikes were measured for some cells for inter-cellular comparison. For cells firing simple spikes with stable Vtrough, the threshold and pFR were measured from randomly selected spikes. However, for some cells in which the basal Vm during trains of simple spikes fluctuated (e.g., control trace in Fig. 9Aii), spikes sitting on the most negative deflections of basal Vm were chosen for measurement of the two parameters.
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). The waveform of our extracellular spikes generally resembled that of the first derivative of the intracellular fast spike; the extracellular interspikelet interval is similar to the interval between the points of maximum rise slope of two adjacent intracellular spikelets. If all the spikelets are uniform in waveform, the extracellular interspikelet interval should be the same as the intracellular ones. For a typical complex spike in which the successive spikelets are smaller in amplitude and slower rising, the extracellular interspikelet interval is likely to be shorter than the intracellularly recorded interspikelet interval at least for later appearing, small spikelets. Thus the sum of interspikelet intervals of an extracellular complex spike may be a slight underestimate of that of the underlying intracellular complex spike. To document changes in the shape of Ca2+ spikes, rough indices of amplitude and decay time, the (maximum, if multi-peaked) peak-to-trough amplitude and the (last, if multi-peaked) peak-to-trough duration, respectively, were used. These were measured and averaged, for each control and drug condition, from all HTSs or LTSs (see RESULTS for definition) evoked during 12 runs of an incremental (100–700 pA) current step protocol given from a Vm more negative than –75 mV. Voltage-clamp mode was used to measure steady-state currents (Fig. 3B). The pipette and whole cell capacitance were partially compensated, but the Rs was not. Statistical presentation of data were given as means ± SD, and the difference between two groups of data were tested using two-tailed t-test (paired or unpaired) at the 0.05 level of significance. Drug application
All the pharmacological agents were applied by bath perfusion. Blockers of fast glutamatergic, GABAergic and glycinergic transmission [20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX), 100 µM 2-amino-5-phosphonovaleric acid (APV), 10 µM SR95531, and 0.5 µM strychnine] were added to the bath solution after an initial examination of spiking properties unless otherwise specified. Drugs were obtained from Sigma-Aldrich (St Louis, MO) with the exception of SR95531 (Tocris Cookson, Ellisville, MO), tetrodotoxin (TTX), 
-conotoxin-GVIA, iberiotoxin, apamin (Alomone Labs, Jerusalem, Israel), -agatoxin-TK, -agatoxin-IVA and -conotoxin-MVIIC (Peptide International, Louisville, KY). When applying peptide toxins, 0.5 mg/ml cytochrome C (for agatoxins, conotoxins), or 0.1 mg/ml bovine serum albumin (BSA, for iberiotoxin) was included in the drug perfusate to reduce nonspecific binding, and control traces were obtained in the presence of cytochrome C or BSA alone before drug application began. Agatoxins, conotoxins, and iberiotoxin were perfused for 
15 min using recirculation. When CdCl2 or NiCl2 was used, KH2PO4 in the bathing solution was replaced with KCl to prevent precipitation.
Terminology
Fast action potentials riding on the slow depolarization of a complex-spike waveform became slower and smaller as they arose from more depolarized membrane potentials (Manis et al. 1994). However, the initial fast spikes could have similar thresholds and amplitude to simple spikes. We used the term "spikelets" to refer specifically to the spikes comprising a complex spike and "fast spikes" to indicate both simple spikes and initial spikelets of complex spikes. Figure 12 diagrams several types of spike discussed here.
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Whole cell recording with the standard K-gluconate solution was generally avoided because of the following changes, which developed within 15 min from the moment of patch break-through in CWCs: Vm became hyperpolarized resulting in loss of spontaneous activity, later, complex spikes would occur more frequently and at a lower stimulus level in response to step depolarizations, and fast spikes repolarized less, the pFR becoming depolarized by 3.2 ± 3.4 mV (n = 24). These changes progressed more slowly when using recording pipettes of smaller bore and therefore must be related to the dialysis of cytoplasmic constituents. Changing the major anion of the internal solution to methylsulfate or methanesulfonate did not prevent time-dependent changes. On the other hand, with gramicidin perforated-patch recording, spontaneous spike activity was maintained, although slight depolarization of Vm, as apparent from increase in spiking frequency, was often seen during early periods of recording. Most importantly, the increase with time in complex spiking on routine step current protocols did not occur, as it did with whole cell recording. Supplemental Fig. 1A illustrates changes in spike properties of a cell that had been recorded initially in perforated-patch mode and then in whole cell mode with the K-gluconate based solution after rupture of patch.
A sudden 8-mV jump in Vm and corresponding shift in spike parameters (threshold, pFR) were observed when the perforated-patch ruptured during recordings with the 140 mM KCl-based internal solution. This shift in potential might suggest that a junction potential existed across the gramicidin perforated patch between the pipette solution and the cytoplasm, such that the potential at the recording electrode had been more negative than the true Vm; alternatively, it may be that a Donnan potential developed on patch rupture (Horn and Marty 1988). Similar positive Vm shifts at patch rupture during gramicidin recording have been reported and considerations for correction of the recorded Vm by adding the magnitude of shift have been addressed (Atherton and Bevan 2005; Brockhaus and Ballanyi 1998; Hallworth et al. 2003). In our recordings, however, the initial 8-mV upward shift in Vm later appeared to sag back toward the original potential (supplemental Fig. 1Bi); during the 20-s period after rupture, peaks of fast spikes gradually declined in spiking cells, and in silent cells the Vm settled to a final level 3–5 mV more depolarized than the potential before the sudden jump. This led us to wonder what correction should be applied to the recorded Vm, if any. We have found that when gramicidin perforated-patch recordings were done with a pipette solution composed of (in mM) 140 K-gluconate, 1 MgCl2, and 10 HEPES, no shift of Vm was noticeable at patch rupture; this indicates that there may be no junction potential between this solution and the cytoplasm. To estimate the potential difference between the 140 KCl solution and the cytoplasm, we employed dual perforated-patch recording on single cells with the 140 K-gluconate solution in one recording pipette and the 140 KCl solution in the other. The difference in Vm read-out between the two recording electrodes was 14.7 ± 1.3 mV (n = 10), and when the patch under the K-gluconate pipette was ruptured to form a whole cell configuration, the potential difference did not change. The mean value, 14.7 mV, was corrected for liquid junction potentials of each solution (13.3 and 2.8 mV, respectively) to become 4.2 mV, which is the estimated potential difference across the perforated-patch between the 140 KCl solution and the cytoplasm. Because the offsets to be considered for the Vm recorded with 140 KCl solution were similar in magnitude and of opposite direction (–2.8 for liquid junction potential, +4.2 mV for the patch potential) leaving 1.4 mV, we opted not to apply any correction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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CWCs were identified as medium-sized neurons located in the molecular and fusiform cell layers of the DCN that fired complex spikes in response to depolarizing current injection. Of the 335 CWCs recorded with gramicidin perforated-patch method and not previously exposed to pharmacological agents, 33.7% (n = 113) were silent and the rest, 76.3% (n = 222), were spontaneously spiking. Silent cells had a mean resting potential of –82.3 ± 2.7 mV (range –76 –88 mV; n = 113). Among spontaneously active cells, a range of spiking patterns was observed with respect to the tendency to fire complex spikes: 72.1% (n = 160) showed only simple spikes ("all-simple-spiking"), whereas the others (27.9%, n = 62) showed different frequencies of complex spikes along with simple spikes ("complex spiking"). Another characteristic feature of CWCs was a slow fluctuation or oscillation in the basal profile of Vm. The Vm excluding spikes (base Vm) coursed between levels close to and far from the spike thresholds, generating periods of spiking interposed with deep hyperpolarized periods of silence (Fig. 1A). Such fluctuation in base Vm was seen in most complex-spiking cells and in 24.5% of all-simple-spiking cells (together, 44.8% of spontaneously spiking cells). The remainder of all-simple-spiking cells maintained relatively stable base Vm while firing quasi-regularly at 10–30 Hz (Fig. 1Ei) or irregularly at a lower frequency. Silent CWCs were heterogeneous with respect to their tendency to fire complex spikes or exhibit fluctuating base Vm behavior when made to spike with small depolarizing holding currents. Elimination of spontaneous synaptic potentials by applying a cocktail of blockers of ionotropic glutamate, GABAA, and glycine receptors did not eliminate spontaneous spike activity of CWCs, suggesting that CWCs’ spiking characteristics—complex spikes and slow Vm fluctuation—are intrinsically determined.
Variety of complex spikes
In this section, we will describe at some length the appearance and variety of complex spikes as these detailed characteristics helped make clear what factors promote complex versus simple spiking. We noticed that spontaneous complex spikes of individual CWCs were often not uniform: some were distinct, whereas others appeared to blend with neighboring simple spikes, especially on the rising phase of their underlying depolarization, which made the beginning of such complex spikes unclear. However, these are all recognized as complex spikes based on a core motif in their waveform, including the terminal part in the rising phase where the slope was steepest and the presence of two to three spikelets having intervals of <4 ms. We used the term "prompt" spike to refer to a distinct form of the complex spike, which is separated from a previous spike by 30 ms and has an initially fast rise consisting of three to four spikelets at intervals <6 ms (Figs. 1Aa, and 12). The less distinct complex spikes, which appeared to follow directly from one or more simple spikes having 7- to 25-ms interspike intervals, were termed "delayed" spikes (Figs. 1A, b–d, and 12). Spontaneous complex spikes were often preceded by a hyperpolarization. Typically, in a given cell, the prompt complex spike occurred after a long (>100 ms) hyperpolarized phase of slow Vm oscillation, and the delayed spike after a short hyperpolarization (Fig. 1Ai). However, some cells showed only delayed complex spikes even after prolonged hyperpolarizations (Fig. 1Aii). Spontaneous complex spikes occurring in the middle or end of a prolonged spiking phase without a clearly preceding hyperpolarization were also observed in some cells. It was also possible to recognize prompt and delayed complex spikes in extracellular recordings (Fig. 1B; n = 65 cells) (Tzounopoulos et al. 2004). To summarize, we distinguished two main classes of spontaneous complex spike: prompt spikes arising quickly from a preceding hyperpolarization and delayed spikes arising soon after simple spike activity.
The repolarizing phase of a complex spike was steeper when there were more spikelets, as shown from comparison of prompt complex spikes with random variation in spikelet number observed in same cells (Fig. 1C). Sometimes the extra spikelet(s) was much slower than the preceding ones ("slow spikelet") and appeared to occur from the repolarizing phase of the underlying slow depolarization (Fig. 1C, 3rd pair). These data suggest that current activated during spikelets drive repolarization of the complex spike; in a later section, we show that part of this current is due to SK K+ channels.
The ability to fire complex spikes on depolarizing current injection distinguished all-simple-spiking or silent CWCs from other neurons in the DCN (Fig. 1E). Most CWCs, when given incremental depolarizing current steps (
20–25 pA, 200–300 ms) from a quiescent state near –80 mV (with hyperpolarizing bias current for spontaneously active cells), fired only simple spikes as their first active response (Fig. 2 Ai). With increasing step depolarizations, they generated complex spikes of which we distinguished two types based on their thresholds. Typically, one complex spike appeared first at onset of the depolarizing step followed by simple spikes throughout (the "onset" spike). Then with larger depolarizations, additional complex spikes arose after the onset spike, interspersed with simple spikes, and are termed "late" spikes. Late complex spikes became more frequent as depolarization increased (Fig. 2Ai). Occasional deviations from this pattern included, for example, cases in which the cells first gave rise to late complex spikes (as in Fig. 6Ai, top) and then with more current generated the onset spike. In some cells, the initial spike response to depolarizing steps was one or more complex spikes with or without following simple spikes (Fig. 2Aii). Stronger depolarizations made the next responses follow the more typical pattern. The onset spike for these cells occurred immediately on the stimulus regardless of the magnitude of stimuli, but for other cells, the onset spike appeared delayed when first seen at the threshold stimulus, and the delay decreased on increasing stimuli (Fig. 2Ai). To examine the dependence of complex spikes on the preceding potential, onset spikes were triggered with a two-step current protocol (Fig. 2Bi). As the conditioning Vm was gradually made more positive than –80 mV, the onset spike became delayed and eventually could not be triggered. The level of Vm above which an onset spike (limited to those arising within 40 ms) could not be evoked with this protocol varied between –70 and –65 mV across different cells (Fig. 2Bii). A transient depolarization at the onset of a current step was often observed at stimulus levels subthreshold to the appearance of a delayed onset complex spike (Fig. 2Ai, 80 pA trace) or at failure of an onset spike (Fig. 2Bi, right bottom).
Afterdepolarization as a trigger for complex spikes
The waveform of CWC simple spikes had a characteristic bump-like afterdepolarization (ADP) following the end of repolarization (Fig. 1Ab) (Manis et al. 1994; Zhang and Oertel 1993a). When the first spikelet of a prompt complex spike and an isolated simple spike having the same threshold belonging to the same cell were superimposed, the waveforms appeared indistinguishable, except that the pFR of the former tended to be slightly (1.5 mV) less negative than that of the latter. Considering this similarity and that the simple spike’s ADP peaks within 3–6 ms of the peak of the spike, it seemed possible that the prompt complex spike starts from a simple spike having a larger ADP such that the ADP triggers the second spikelet and the rest of a complex-spike waveform, the core motif. In the same manner, the delayed complex spike might arise where a simple spike’s ADP is not depolarized enough to give rise to a fast spike at its peak but enough that another simple spike arises on its decay phase, and so on, until the threshold for the core burst is reached on a spike’s ADP (Fig. 1A, b–d). Examples in which successive ADPs accumulate but decay without reaching such threshold are seen in the beginning of the fourth spike cluster in Fig. 1Aii and the 80-pA response in Fig. 2Ai. Is there a systematic relationship between the threshold for a fast spike, the peak of the ADP, and the probability of firing complex spikes? Spontaneous simple spikes’ thresholds and pFRs were measured for subsets of complex-spiking and all-simple-spiking CWCs (Table 1). The pFR was measured as an indirect index of the basal ADP level of simple spikes because the ADP itself appeared to vary in amplitude in some cells (supplemental Fig. 2). The mean pFR and the mean difference between pFR and threshold of spontaneous simple spikes were found to be significantly different between all-simple-spiking cells and complex-spiking cells (P < 0.001 for both), such that spontaneous complex spikes were not seen in cells in which the fAHP brought the membrane potential farther from threshold.
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2/3 (20 of 29) of the spontaneously complex-spiking cells tested, but they could also elicit complex spikes (Figs. 1Di and 11E, left) from some prompt complex-spiking cells and silent cells. These features will be used in the following sections to explore the effect of selective channel blockers. Effect of Na+ channel block
Application of the voltage-dependent Na+ channel blocker TTX (0.5 µM) eliminated fast action potentials. TTX did not affect the resting potential of silent cells, but in spiking cells the Vm in TTX ranged between –75 and –50 mV. For all-simple-spiking cells that fired quasi-regularly without base Vm oscillation, the Vm in TTX was less negative than the interspike trough potential (Vtrough) before TTX by 10 ± 5.6 mV (n = 20). Depolarizing current evoked slow spikes in a pattern reminiscent of complex spikes (109 of 109 cells given TTX; Fig. 3A) . Typically, a lone slow spike could be observed at the onset of a step depolarization, and then, with larger depolarizing steps, more slow spikes appeared (Fig. 4 Ai). The slow spike at onset of depolarization appeared graded in amplitude with the amount of current injected, whereas the later-occurring ones were all-or-none. The threshold current for the late slow spikes was 293 ± 83 pA (n = 21, Vm held at –80 mV), and this was 112 ± 91 pA more than that required for the late complex spikes before TTX application in the same cell. A more detailed study of slow spikes is presented in later sections of RESULTS.
CWCs have a persistent Na+ current. When stepping to potentials less negative to –70 mV from a holding potential of –80 mV, a persistent inward current was observed that was blocked by TTX (Fig. 3Bi). Accordingly, a negative slope conductance region existed in the steady-state current-voltage (I-V) plot of CWCs, which started between –70 and –75 mV extending to about –55 mV where the plot terminated due to limitation in voltage control (Fig. 3Bii). The I-V plots varied in their vertical position with respect to the 0-current axis. For many silent cells, the plot crossed 0-current axis once at their resting potential with the negative slope region located above the 0-current axis. On the other hand, the entire plot lay below the 0-current axis for many spontaneously spiking cells. The current at the crest of I-V plot was –14 ± 21 pA (n = 52) for spiking cells and 78 ± 51 pA (n = 32) for silent cells (t-test, P < 0.001). TTX eliminated, or in some silent cells reduced the negative slope region (Fig. 3B). The difference currents obtained by subtracting the currents in the presence of TTX from those in its absence show that the persistent Na+ current activated near –75 mV and reached –139 ± 31 pA (n = 15) at –55 mV. In current clamp, the Vm was unstable above –75 mV and drifted in the depolarizing direction until cycles of fast spikes were generated. It is likely that this drift arose from activation of persistent Na+ current. Similar observations were made with sharp electrode recordings (Hirsch and Oertel 1988).
Effect of general Ca2+ channel block and properties of the slow spikes
Ca2+ currents are believed to contribute to the underlying depolarization of complex spikes (Golding and Oertel 1997). We thus examined how the removal of regenerative Ca2+ currents with Cd2+ or by replacing Ca2+ with Mg2+ affected firing. As Cd2+ (200 µM, n = 3; 100 µM, n = 2) entered the recording chamber, there was a transient period of enhanced complex spiking in all CWCs (Fig. 5 A, middle). After a longer exposure to Cd2+, cells fired broadened simple spikes at high rates and in long clusters. Eventually only a two-spikelet pair remained at the onset of a step depolarization (Fig. 5A, bottom) or at the beginning of spontaneous, simple spike clusters. The simple spikes in Cd2+ appeared broadened more toward their base, and comparison of the spike width at 40% of amplitude yielded a more significant result than that of the half-width (40%-width, 0.45 ± 0.07 vs. 0.64 ± 0.17 ms, n = 5, paired t-test, P = 0.021; half-width, P = 0.047). Cd2+ also caused a hyperpolarization of Vm by <10 mV, but this was not studied further. Removal of external Ca2+ (replaced with Mg2+, n = 3) also abolished complex spikes and broadened the base of simple spikes (40%-width 0.52 ± 0.06 vs. 0.63 ± 0.08 ms, paired t-test, P = 0.01). However, in the absence of Ca2+, CWCs eventually became depolarized and were unable to fire more than a few spikes even with a hyperpolarizing holding current.
In the presence of TTX, the slow spike triggered at the onset of a depolarizing step only occurred when the preceding potential was more negative than approximately –65 mV (Fig. 4B), suggesting that the onset spike had a lower threshold than later slow spikes (see also Fig. 4Ai). The slow spike at the onset will be referred to as the low-threshold spike (LTS), and the later ones, the high-threshold spike (HTS). Expecting that these TTX-insensitive slow spikes were mediated by Ca2+, Cd2+ was added (50 µM, n = 2; 200 µM, n = 4; 500 µM, n = 3). HTSs, but not LTS, were abolished by Cd2+ at all the concentrations tried (n = 9; Fig. 5Bi). The low-threshold nature and the relative resistance to Cd2+ suggest that T-type Ca2+ channels mediate LTS (Ertel 2004). Ni2+, a more efficient blocker of T-type Ca2+ channels, was therefore applied at 500 µM plus 100 µM Cd2+ (n = 1), or alone at 500 µM (n = 1; Fig. 5Bii), or at 1 mM (n = 1). Under these conditions, HTSs were eliminated and the maximum peak-to-trough amplitude of the LTS evoked with a series of hyperpolarizing presteps (as in Fig. 4B) was reduced by 73, 63, and 87%, respectively. Removal of external Ca2+ also eliminated all slow spikes in TTX (n = 3). The differences in threshold and in sensitivity to Cd2+ and Ni2 indicate that the LTS and HTS were Ca2+ spikes that were mediated by different subtypes of Ca2+ channels. The forms of Ca2+ spikes were not as stereotypical as those of Na+ spikes; the amplitude and width of HTS or LTS varied within a CWC depending on the Vm from which spikes were evoked and on the amount of current injected. HTSs were more frequent and larger when evoked from a more depolarized Vm, and multi-peaked broader forms could be seen in some CWCs with threshold current injection (Fig. 4A). The magnitude of depolarizing current steps just sufficient to elicit HTS or LTS and the maximum amplitude of these spikes differed among CWCs, as did the onset and late complex spikes in control solutions. Moreover, the amplitude of the LTS varied between cells, and could be quite small (Fig. 10Bi).
To determine whether Ca2+ channels shape the ADP and fAHP, spikes evoked with short pulses of current were recorded in the presence of 100 µM Cd2+ plus 500 µM Ni2+ (Fig. 5C; n = 8). The short pulse-evoked fast spikes broadened toward the base of the spike (width at –40 mV, roughly just below half-amplitude level, 0.52 ± 0.06 vs. 1.16 ± 0.38 ms, n = 8, paired t-test, P = 0.001) and repolarized to less negative potentials. The changes in repolarization suggested involvement of Ca2+-mediated outward currents. To identify the subtype channels underlying these effects, further experiments were conducted with specific blockers of Ca2+ channel and Ca2+-activated K+ channel subtypes.
Effect of intracellular Ca2+ buffering
To test the possibility that Ca2+-activated K+ conductances served as repolarizing currents, intracellular Ca2+ was buffered using whole cell recording with an EGTA- or BAPTA-containing pipette solutions. As mentioned in METHODS, whole cell recording, with 0.1 mM EGTA, led to hyperpolarization, increased complex spiking, and a small depolarizing shift in pFR. Raising the EGTA to 5 mM or including 20 mM BAPTA caused progressive changes in spike waveforms far larger than those seen with 0.1 mM EGTA.
In recordings using 5 mM EGTA in the patch pipette, the pFR became less negative by 8.1 ± 1.4 mV after 15 min of dialysis (n = 6). Moreover, complex spikes occurred more readily with depolarizing current injection and had a broader appearance and several slow spikelets (Fig. 6Ai). The half-width of fast spikes after 15 min was 0.45 ± 0.03 ms (n = 6), which was not significantly longer than that measured from 6 whole cell recordings with an internal solution containing 0.1 mM EGTA (0.42 ± 0.04 ms, t-test, P = 0.16). In the presence of TTX, 5 mM EGTA, caused the HTSs to become prolonged and to acquire multiple peaks (n = 3; Fig. 6Aii). The LTS did not noticeably change except that an HTS often appeared to occur on top of the LTS (Fig. 6Aii). The effect of a faster Ca2+ buffer, BAPTA, at 20 mM, was also examined. Complex spikes broadened in <1 min after patch break-through (Fig. 6Bi, top), and then a marked loss of fAHP ensued. Simple spikes disappeared leaving only complex spikes consisting of a half-repolarizing fast spike and many slow spikelets (Fig. 6Bi, middle; n = 7). Eventually the pFR became very depolarized (by 41.3 ± 4.3 mV after >15 min; n = 7), and the complex spike waveform became narrower, appearing more like a broadened simple spike with inflections in its repolarizing phase (Fig. 6Bi, bottom, initial 2 spikes; half-width 1.22 ± 0.53 ms, n = 7). In the presence of TTX, a similar broadening-narrowing sequence in waveforms of HTSs was observed as BAPTA diffused intracellularly (Fig. 6Bii; n = 3).
Effect of BK and SK channel block
Because blocking Ca2+ channels or buffering intracellular Ca2+ slowed spike repolarization, we turned to selective blockers of Ca2+-activated K+ channels, using iberiotoxin for the large-conductance Ca2+-activated K+ (BK) channel and apamin for the small-conductance Ca2+-activated K+ (SK) channel. Incubation in iberiotoxin (100 nM) led to spontaneous firing of complex spikes in all-simple-spiking cells (n = 6). In two cells that were silent and gave only simple spikes at rheobase (i.e., the smallest suprathreshold depolarizations), iberiotoxin also led to firing of complex spikes. Iberiotoxin generally increased firing of complex spikes (Fig. 7 A), but the degree of increase was quite variable; among five spontaneously complex-spiking cells monitored with intra- or extracellular recording, the increase in spiking was 0, 116, 154, 390, and 536% (paired t-test, n = 5, P = 0.10). Along with this enhancement of complex spiking was a reduction in the fAHP (pFR depolarized by 13.9 ± 4.8 mV, n = 9; Figs. 7Ai, inset, and 7B) and increase in the half-width of fast spikes (0.42 ± 0.08 vs. 0.49 ± 0.11 ms, n = 9, paired t-test, P = 0.007). The less negative pFR in iberiotoxin was accompanied by a shortening of the first interspikelet interval for the one cell that had spontaneous, prompt, complex spikes (Fig. 7Ai, inset) and conversion of delayed onset complex spikes to prompt ones for other cells (Fig. 7Aiii); prompt complex spikes appeared in spontaneous or evoked activity of cells that did not display such complex spikes in control conditions. The decrease in the first interspikelet interval for spontaneous prompt complex spikes, when jointly assessed with measurements from four extracellularly recorded complex-spiking cells, all of which displayed prompt complex spikes in control conditions (e.g., Fig. 7Aii), was significant (4.0 ± 0.5 vs. 1.8 ± 0.2 ms, paired t-test, n = 5, P < 0.001). The reduced fAHP and boosted complex spiking in iberiotoxin was also observed for spikes evoked by short pulses (n = 3). The three tested cells generated only simple spikes in control conditions; two of these became able to fire complex spikes robustly (Fig. 7B). In the presence of TTX, iberiotoxin increased the amplitudes of Ca2+ spikes (Fig. 7C, n = 7). The peak-to-trough amplitude of HTSs increased by 51 ± 17% (from 17.8 ± 4.4 mV, paired t-test, n = 7, P < 0.001), whereas the peak-to-trough time did not change (9.9 ± 2.0 vs. 10.0 ± 1.7 ms, paired t-test, n = 7, P = 0.8). For the LTS, four of the seven cells showed an increase in the peak-to-trough amplitude (by 70 ± 6%, paired t-test, P = 0.014). Among the other three cells that had no or barely noticeable LTS, two gained a clear LTS in iberiotoxin. Thus BK channels played a key role in determining the shape of fast spikes and the likelihood of generating complex spikes.
Blockade of SK channels using apamin (100 nM, n = 7, or 50 nM, n = 9), also increased the tendency to fire complex spikes. Four of the five all-simple-spiking cells as well as complex-spiking cells (n = 5) started to fire broad, prompt or short-delay (preceded by just one simple spike with the interval <10 ms) complex spikes, isolated or clustered with more complex spikes and few interposing simple spikes (Fig. 8 A). For the five complex-spiking cells and one extracellularly recorded complex-spiking cell, the frequency of spontaneous complex spikes increased in apamin (0.9 ± 0.8 vs. 2.7 ± 1.4 Hz, 15 –1440% increase, n = 6, paired t-test, P = 0.015). Silent cells’ firing at rheobase also became predominantly complex spiking in apamin (n = 7 of 8 cells). The usual initial response to step current injections, either all simple spiking or an onset complex spike followed by simple spikes, became replaced in apamin with all complex spiking, whereas responses to larger current injections were also dominated by complex spikes (Fig. 8B). For the cell shown in Fig. 8B, a transitional effect of apamin as the drug washed in was also recorded (supplemental Fig. 3A): the train of simple spikes following the onset spike depolarized more steeply, giving rise to late complex spikes at a higher frequency than in control. Thus apamin appeared to impair CWCs’ ability to fire simple spikes with a stable Vtrough.
We measured interspikelet intervals of spontaneous complex spikes and plotted these for successive spikes in the waveform. To monitor spikes of uniform shape, we only included those spikes preceded by >150 ms of silence; this was done for six cells including one extracellular recording. Figure 8C shows two sets of such plots for two cells, the second one (ii) extracellularly recorded. Both cells in control conditions had delayed complex spikes, starting with an interspikelet/spike interval >5 ms, and the first interval varied rather widely [7.6 ± 4.2 (n = 18) and 15.0 ± 3.6 ms (n = 73), for Fig. 8C, i and ii]. In apamin, both cells showed marked decrease in the duration and variability of the first interspikelet interval [3.8 ± 0.2 (n = 25) and 5.2 ± 0.9 (n = 92) ms, for i and ii], so that the complex spikes in apamin were prompt or of short delay. These changes in the first interspikelet interval in apamin occurred in all of the five complex-spiking cells and one extracellularly recorded cell (n = 6; duration, –4.5 ± 3.5 ms, paired t-test; P = 0.025; F-test, P < 0.001 for every cell). When short pulse-evoked complex spikes were examined (n = 4, 2 of complex-spiking cells and 2 silent cells), the first interspikelet interval was also found significantly decreased by apamin (Fig. 8Dii; –0.9 ± 0.5 ms, paired t-test, P = 0.041). Compared with the effects of BK channel block, which also include the shortening of first interspikelet interval, the pFR and the half-width of fast spikes were not affected by apamin (Fig. 8D, i–iv; pFR of short pulse evoked spikes, –0.1 ± 0.5 mV, n = 16, paired t-test, P = 0.23). However, the slope of the first interspikelet interval appeared steeper in apamin, and this may have led to a shortening of the interval (Fig. 8D, i and ii). The complex spikes in apamin appeared prolonged with more spikelets but repolarized to a lesser extent (Fig. 8D, i and ii). To measure complex spikes’ duration, we summed the interspikelet intervals of each complex spike preceded by >150 ms of silent period, beginning from the first interval 
5 ms to exclude the long intervals before the abrupt rise of underlying slow depolarization in delayed complex spikes. The sum of interspikelet intervals thus obtained from spontaneous complex spikes was compared between control and apamin-treated conditions. For the five complex-spiking cells and one extracellularly recorded cell, the complex spike duration increased from 6.6 ± 1.1 ms in control to 17.6 ± 8.8 ms in apamin (n = 6, 50 –470% increase, paired t-test, P = 0.034), along with the change in the number of spikelets (number of included intervals +1) from 3.3 ± 0.5 in control to 6.7 ± 2.8 in apamin (n = 6, 40 –360% increase, paired t-test, P = 0.045).
In 9 of 16 cells treated with apamin, short pulses that initially evoked simple spikes continued to do so in apamin although their spontaneous activity or response to longer step depolarizations became predominantly complex spiking. However, the simple spikes triggered by short current pulses in the presence of apamin revealed a larger, or more slowly decaying, ADP compared with that in control conditions. The corresponding depolarizing shifts of Vm at 10 ms from the peak of the averaged evoked spikes were significant (Fig. 8Dv; 4.7 ± 3.5 mV, n = 9, paired t-test, P = 0.004). For Ca2+ spikes in TTX, apamin caused a general enhancement of HTSs, including broadened appearance with more peaks, larger amplitude and higher frequency compared with those evoked by same current injection in control conditions. However, the HTS in apamin had a slowed repolarization and afterhyperpolarization (Fig. 8E; peak-to-trough time, 12.3 ± 1.8 vs. 17.5 ± 3.5 ms, n = 6, paired t-test, P = 0.004; peak-to-peak amplitude, 17.6 ± 3.8 vs. 20.7 ± 3.9 mV, n = 6, paired t-test, P = 0.026). The LTS did not appear changed in apamin. Thus SK channels determine the duration of complex spikes and likelihood of complex spikes, possibly by regulating the duration and size of the ADP.
Given the effects of blocking SK and BK channels on spike waveforms, the results of buffering intracellular Ca2+ with EGTA and BAPTA were revisited. Although complications of whole cell recording were necessarily superimposed, 5 mM EGTA appeared to inhibit partially BK channels because it reduced the fAHP only moderately, whereas the signs of SK channel block, broadening of complex spikes and the HTS, were more obvious. This suggests that BK channels may lie in close proximity to Ca2+ channels and the slow buffering by EGTA affects the Ca2+ concentration near the BK channels only little. BAPTA (20 mM), on the other hand, caused a far greater loss in fast spike repolarization than iberiotoxin. It is unclear whether this was due to incomplete block of BK channels by iberiotoxin, as the sensitivity of BK channels to iberiotoxin may vary with subunit composition (Meera et al. 2000), or due to consequences of whole cell dialysis or extremely low intracellular Ca2+. It is also possible that the eventual narrowing of complex spikes with 20 mM BAPTA may not be related to the block of BK and SK channels because multi-spikelet waveforms persisted during the combined application of apamin and iberiotoxin (extracellular recording, n = 3; not shown).
Specific block of subtype Ca2+ channels
N-TYPE.
Application of -conotoxin-GVIA (1–3 µM), which specifically blocks N-type channels had no effect on either the firing pattern or on the shape of spikes (n = 2 without TTX, n = 3 with TTX).
P/Q-TYPE.
The presence of P/Q type Ca2+ channels was probed with -agatoxin-TK, -agatoxin-IVA, or -conotoxin-MVIIC. -agatoxin-TK (100–200 nM; n = 16), -agatoxin-IVA (200 nM; n = 1) and -conotoxin-MVIIC (2–3 µM; n = 5) each caused increased complex spiking. Thirteen all-simple-spiking cells as well as 2 complex-spiking cells started to fire clusters of complex spikes and higher frequency simple spikes in the presence of these toxins (Fig. 9A). The frequency of spontaneous complex spikes for the two complex-spiking cells and three extracellularly recorded complex-spiking cells was increased in P/Q channel blockers (0.8 ± 0.6 vs. 1.9 ± 0.8 Hz, n = 5, paired t-test, P = 0.043). The spontaneous complex spikes in the presence of P/Q channel blockers were often broader with more spikelets than those in control conditions (Fig. 9Aii). Plots of interspikelet intervals versus their duration were generated and analyzed as with apamin-treated cells (Fig. 9B). The duration of spontaneous complex spikes for one of the two complex-spiking cells and three extracellularly recorded cells, increased from 4.4 ± 0.9 to 10.0 ± 1.2 ms in P/Q channel blockers (n = 4; paired t-test, P = 0.011). The number of spikelets included was 2.7 ± 0.5 in control and 3.8 ± 1.2 in the drug (n = 4, paired t-test, P = 0.083). The duration of the first interspikelet interval, unlike with apamin, did not decrease ( 3.9 ± 5.0 ms, n = 4, paired t-test, P = 0.22). The one complex-spiking cell not included in the preceding text fired only two-spikelet complex spikes after >150 ms of hyperpolarized periods in control condition and continued to do so in P/Q channel blocker but with increased occurrence of delayed complex spikes following the two-spikelet one. For this cell, the interspikelet interval of the two-spikelet complex spikes increased from 4.7 ± 0.6 (n = 23) to 5.5 ± 0.9 ms (n = 20; P = 0.002). In the response to depolarizing current pulses, P/Q channel blockers caused faster trains of simple spikes, such that the maximum frequency of simple spike train not giving rise to a late complex spike during a 325-ms current step was increased (78 ± 30 vs. 115 ± 55 Hz, n = 15, paired t-test, P = 0.001). Also, the late complex spikes appeared at a lower current levels in the presence of P/Q channel blockers (140 ± 78 vs. 37 ± 33 pA, n = 12, paired t-test, P < 0.001), suggesting that the excitability of CWCs had increased (Figs. 9C and supplemental Fig. 3C). The late complex spikes were broadened, while the onset spike did not appear so (Figs. 9C and supplemental Fig. 3C). The lowering of the threshold for complex spikes by P/Q channel blockers was reminiscent of the effects of apamin (supplemental Fig. 3, A and B vs. C), suggesting that the Ca2+ influx through P/Q-type Ca2+ channels serves to activate SK channels. However, differences between the effect of P/Q blockers and that of apamin were noted in short pulse-evoked spikes, in induced complex spikes, and in Ca2+ spikes. P/Q blockers caused the pFR of short pulse-evoked spikes to become less negative by 1.8 ± 1.4 mV (n = 11, paired t-test, P = 0.002), whereas apamin did not affect the pFR. The less negative pFR led to a more depolarized ADP (Fig. 9D), and 1 of the 10 cells that under control conditions responded to short pulses with only simple spikes became able to fire complex spikes. In one cell, the averaged pFR of spikes evoked by short pulses did not change in the presence of P/Q channel blockers; in this cell, the amplitude and decay of the ADP also were unaffected by P/Q channel blockers. All-simple-spiking cells were induced to fire spontaneous complex spikes along with slow Vm oscillations by both the SK and P/Q-type Ca2+ channel blockers. A difference in these cases between the block of these two channels was that the complex spike occurring after a hyperpolarized phase was prompt or just briefly delayed when SK channels were blocked, but only delayed when P/Q channels were blocked (Figs. 8B, left traces vs. 9Ai or supplemental Fig. 3, B vs. C).
The shapes of Ca2+</
) are indicated for 2 fast spikes in b. B: an example of extracellularly recorded complex-spiking activity with expanded views below. 
, prompt complex spikes; 
) and shorter interspikelet intervals of complex spikes in ibtx (red) were shown by superimposing indicated spikes on the left. Aii: extracellular recording of a complex-spiking cell. Two prompt complex spikes along with simple spikes are shown in the control trace, whereas in ibtx all spikes are complex spikes. Inset: indicated complex spikes shown in expanded scales to reveal a decrease in the first interspikelet interval by ibtx. Aiii: less negative pFR and facilitation of complex spiking by ibtx shown in current step responses. Note change from a delayed complex spike to 2 prompt spikes at onset. B: using short stimulus pulses, complex spikes were triggered in ibtx in a cell for which only simple spikes were seen in control solution. Three traces are superimposed for each condition; 50-pA bias current in both conditions. C: ibtx enlarged the amplitudes of both the LTS and HTSs in TTX, 0.5 µM. Traces in A–C are from 5 different cells.
