Theta-Frequency Bursting and Resonance in Cerebellar Granule Cells: Experimental Evidence and Modeling of a Slow K+-Dependent Mechanism

The Journal of Neuroscience, February 1, 2001, 21(3):759–770 Theta-Frequency Bursting and Resonance in Cerebellar Granule Cells: Experimental Evidence and Modeling of a Slow K1Dependent Mechanism Egidio D’Angelo,1,2 Thierry Nieus,1 Arianna Maffei,1 Simona Armano1 Paola Rossi,1 Vanni Taglietti,1 Andrea Fontana,3 and Giovanni Naldi4 Department of Molecular/Cellular Physiology and Instituto Nazionale per la Fisica della Materia, University of Pavia, I-27100 Pavia, Italy, 2Department of Evolutionary and Functional Biology, University of Parma, Parma, Italy, 3Department of Nuclear and Theoretical Physics, University of Pavia, Pavia, Italy, and 4Department of Mathematics and Applications, University of Milano Bicocca, Milan, Italy 1 Neurons process information in a highly nonlinear manner, generating oscillations, bursting, and resonance, enhancing responsiveness at preferential frequencies. It has been proposed that slow repolarizing currents could be responsible for both oscillation/burst termination and for high-pass filtering that causes resonance (Hutcheon and Yarom, 2000). However, different mechanisms, including electrotonic effects (Mainen and Sejinowski, 1996), the expression of resurgent currents (Raman and Bean, 1997), and network feedback, may also be important. In this study we report theta-frequency (3–12 Hz) bursting and resonance in rat cerebellar granule cells and show that these neurons express a previously unidentified slow re- polarizing K 1 current (IK-slow ). Our experimental and modeling results indicate that IK-slow was necessary for both bursting and resonance. A persistent (and potentially a resurgent) Na 1 current exerted complex amplifying actions on bursting and resonance, whereas electrotonic effects were excluded by the compact structure of the granule cell. Theta-frequency bursting and resonance in granule cells may play an important role in determining synchronization, rhythmicity, and learning in the cerebellum. Neurons process information by generating action potentials organized either in regular discharges (fast repetitive firing) or in bursts (Connors and Gutnik, 1990), which can occur repetitively when they are sustained by slow membrane potential oscillations (Wang and Rinzel, 1999). Moreover, some neurons respond better to a preferential input frequency, a property called resonance (Hutcheon and Yarom, 2000). Oscillations, bursting, and resonance have been related to synchronization of neuronal activity and to the emergence of brain rhythms (Llinas, 1988). Oscillations and bursting can arise from various mechanisms that involve slow depolarizing and repolarizing currents. Noteworthy examples are provided by the interaction of a persistent Na 1 current (INa-p) with a slow K 1 current (IM) in hippocampal pyramidal neurons (Gutfreund et al., 1995; Pape and Driesang, 1998) or with an inward rectifier current (Ih) in entorhinal neurons (Alonso and Llinas, 1989; Dickson et al., 2000). In thalamic neurons, depolarization and delayed repolarization are determined by low-threshold Ca 21 current (IT) activation and inactivation and are regulated by other currents, including Ih (McCormick and Huguenard, 1992). In invertebrate cells, Ca 21dependent bursts are terminated by a Ca 21-dependent K 1 current (IAHP) (Wang and Rinzel, 1999). In addition, bursting emerges when a membrane potential difference is established between dendrites and soma, causing rebound depolarization after a spike (Mainen and Sejinowski, 1996). Rebound depolarization may also be caused by currents activated by spike repolarization, e.g., by a resurgent Na 1 current (Raman and Bean, 1997). Despite the multitude of mechanisms that potentially generate oscillation and bursting, resonance essentially requires a slow repolarizing current that reduces neuronal excitability at low input frequency (Hutcheon et al., 1996a,b; Hutcheon and Yarom, 2000). The most apparent discharge mode of cerebellar granule cells is fast repetitive firing (Gabbiani et al., 1994; D’Angelo et al., 1995). However, a more complex electrical behavior has been suggested by the observation of spike bursting unveiled by pharmacological manipulation (D’Angelo et al., 1998). In this study, we report that cerebellar granule cells also show thetafrequency resonance, and we suggest that both bursting and resonance are based on a slow K 1 current. This conclusion is supported by a mathematical model that provides a realistic reconstruction of granule cell electroresponsiveness. Theta-frequency bursting and resonance in granule cells may play an important role in determining synchronization (Maex and DeShutter, 1998), rhythmicity (Pellerin and Lamarre, 1997; Hartmann and Bower, 1998), and learning (D’Angelo et al., 1999; Armano et al., 2000) at the major input stage of the cerebellum. Received Aug. 10, 2000; revised Oct. 30, 2000; accepted Nov. 3, 2000. This work was supported by European Community Grants PL97 0182 and PL97 6060, and by Instituto Nazionale per la Fisica della Materia. We acknowledge Marja-Leena Linne and Massimiliano Zaniboni for contributing to preliminary simulations, and Lia Forti and Elisabetta Sola for their helpful comments on this manuscript. Correspondence should be addressed to Egidio D’Angelo, Department of Cellular/Molecular Physiology and Pharmacology, Via Forlanini 6, I-27100 Pavia, Italy. E-mail: [email protected]. Copyright © 2001 Society for Neuroscience 0270-6474/01/210759-12$15.00/0 Key words: bursting; resonance; M-current; cerebellum; granule cell; modeling MATERIALS AND METHODS W hole-cell patch-clamp recordings. C erebellar granule cells were recorded in acute cerebellar slices obtained from 20 62-d-old rats. Slice preparation and patch-clamp recordings were performed as reported previously (Rossi et al., 1994, 1998; D’Angelo et al., 1995, 1997, 1998, 1999; Armano et al., 2000). D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells 760 J. Neurosci., February 1, 2001, 21(3):759–770 Current-clamp recordings were performed at 30°C. The extracellular solution contained (in mM): NaC l 120, KC l 2, MgSO4 1.2, NaHC O3 26, K H2PO4 1.2, C aC l2 2, glucose 11, and bicuculline 0.01, and was equilibrated with 95% O2 and 5% C O2, pH 7.4. The pipette solution contained (in mM): K-gluconate 126 (or C s2SO4 78), KC l 4 (or C sC l 4), NaC l 4, MgSO4 1, C aC l2 0.02, BAP TA 0.1, glucose 15, ATP 3, GTP 0.1, H EPES 5; pH was adjusted to 7.2 with KOH (or C sOH). Voltage-clamp recordings were performed at room temperature (25.5°C). The extracellular solution contained (in mM): NaC l 100, KC l 2, K H2PO4 1.2, MgSO4 1.2, NaHC O3 26, glucose 11, Tetraethyl-ammonium C l 2 (TEA) 20, 4-amino-piridine (4-AP) 4, Ni 21 2, tetrodotoxin (TTX) 0.001, and bicuculline 0.01, and was equilibrated with 95% O2 and 5% C O2, pH 7.4. The pipette solution contained (in mM): K-gluconate 126, NaC l 4, MgSO4 1, C aC l2 0.02, BAP TA 0.1, glucose 15, ATP 3, GTP 0.1, H EPES 5; pH was adjusted to 7.2 with KOH. TEA, 4-AP, TTX, and bicuculline were obtained from Sigma (St. L ouis, MO). The glutamate receptor antagonists D-2-amino-5phosphonovaleric acid (APV), 7-chlorokinurenic acid (7-C l-Kyn), and 6-cyano-7-nitroquinoxaline-2,3-dione (C NQX) were obtained from Tocris Cookson (Bristol, UK). Data were recorded with an Axopatch 200B amplifier, digitized with a Digidata 1200 interface (500 msec /point), and analyzed with PC lamp software (Axon Instruments, Foster C ity, CA). In voltage-clamp recordings, leak subtraction was performed by using a P/4 protocol. All data are reported as mean 6 SD. Mathematical modeling. A mathematical model of rat cerebellar granule cell electroresponsiveness (D’Angelo et al., 1995, 1998; Brickley et al., 1996) was constructed using the N EURON simulator (Hines and C arnevale, 1997). Because granule cells have a compact electrotonic structure (Silver et al., 1992; D’Angelo et al., 1993, 1995), a singlecompartment model was used. The experimental value of membrane capacitance (3 pF; see refences above) was used to calculate the granule cell surface, assuming a spherical shape and a specific membrane capacitance of 1 mF/cm 2. Mathematical methods. The mathematical problem in neuronal simulation is to solve the set of differential equations representing membrane voltage, intracellular C a 21 concentration, and channel gating dynamics [see for example Yamada et al. (1998)]. Voltage was obtained as the time integral of the equation: dV/dt 5 21/Cm * $(@ gi*~V 2 Vi!# 1 iinj%, (1) where V is membrane potential, Cm is membrane capacitance, gi is ionic conductance, Vi is reversal potential (the subscript i indicates different channels), and iinj is the injected current. Membrane conductances were represented using Hodgkin –Huxley-like models (Hodgkin and Huxley, 1952) of the type: g i 5 Gmaxi * xizi * yi, (2) where Gmaxi is the maximum ionic conductance, xi and yi are state variables (probabilities ranging from 0 to 1) for a gating particle, and zi is the number of such gating particles in ionic channel i. x and y (with the suffix i omitted) were related to the first-order rate constants a and b by the equations: x ` 5 a x/~ax 1 bx! t x 5 1/~ax 1 bx! y` 5 ay/~ay 1 by!, (3) ty 5 1/~ay 1 by!, (4) where a and b are f unctions of voltage. The equations used to parameterize a and b and the state variables x`, tx, y`, and ty for different ionic channels are shown in Table 1. The state variable kinetics were: dx/dt 5 ~ x ` 2 x ! / t x, (5) d y/dt 5 ~ y ` 2 y ! / t y. (6) The model included a leakage current and voltage-dependent Na 1, C a 21, and K 1 conductances (see Table 1). Nernst equilibrium potentials were calculated from ionic concentrations used in current-clamp recordings. The C a 21 equilibrium potential was updated after changes in the intracellular C a 21 concentration. All ionic currents used in the model have been identified in cerebellar granule cells in situ, when the excitable response has assumed its mature pattern (.P20) (D’Angelo et al., 1997). Gating kinetics were corrected using a Q10 5 3 according to the relation Q10 (Tsim –Texp)/10 (Gutfreund Figure 1. Granule cell electroresponsiveness during step current injection. A, The injection of current steps (from 28 to 6 pA, resting potential 5 262 mV) causes inward rectification in the hyperpolarizing direction. Spikes are activated around 240 mV. The tracing at 4 pA shows a single spike, the tracing at 6 pA shows spikes clustered in two bursts, and the tracing at 8 pA shows regular repetitive firing. B, Just-threshold response illustrating spike fast afterhyperpolarization ( fAHP), slow afterhyperpolarization (sAHP), and afterdepolarization (ADP). The neuron in B is different from that in A (spikes are truncated). Recordings in this and the following figures were performed in the presence of 10 mM bicuculline. et al., 1995) to account for differences between simulation temperature (Tsim 5 30°C) and experimental temperature (Texp). Maximum ionic conductances were corrected for ionic concentration differences between voltage- and current-clamp recordings. A f urther adjustment (usually ,30%) of current densities allowed us to fine tune the excitable response [for f urther explanations see Traub and L linas (1979); Traub et al. (1991); Vanier and Bower (1999)]. Leak age current. Mature rat cerebellar granule cells in situ have an aspecific and a GABA-A receptor-dependent leakage (Brickley et al., 1996). In the model, leakage consisted of a 5.68 3 10 2 5 S/cm 2 conductance with reversal potential at 259 mV ( gL), and of a 2.17 3 10 2 5 S/cm 2 C l 2 conductance with reversal potential at 265 mV (accounting for 28% of the total input conductance) (Armano et al., 2000). No qualitative difference was observed in model responses by setting GABA-A receptor leakage to zero (data not shown). Na 1 currents (INa-f, INa-p, INa-r). Mature rat cerebellar granule cells in situ express a fast and persistent Na 1 current (INa-f and INa-p) (D’Angelo et al., 1998) and probably also a resurgent Na 1 current (INa-) (E. D’Angelo and J. Magistretti, unpublished observations). The INa-f model was based on Gutfreund et al. (1995), and inactivation was slowed down around threshold to reproduce spike adaptation during bursts (Mainen et al., 1995). INa-f density was set to reproduce repetitive firing. INa-p was reproduced from Gutfreund et al. (1995) and shifted by 22 mV to match spike threshold. INa-p density (1/65 INa-f) was set to reproduce Na 1- D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells J. Neurosci., February 1, 2001, 21(3):759–770 761 Figure 2. Resonance in a cerebellar granule cell (same cell as in Fig. 1 A). A, Injection of sinusoidal currents at various frequencies (0.5– 40 Hz) reveals resonance in burst spike frequency, which was measured by dividing the time period between the first and last spike in a burst by the number of interspike intervals. The plot shows that the resonance frequency was 6 Hz, with sinusoidal currents of 66 pA (F) and 8 Hz with 68 pA (D). At frequencies higher than those shown in the plot, just one or no spikes were generated, and spike frequency fell to zero. B, After 1 mM TTX perfusion, injection of sinusoidal currents at various frequencies reveals resonance in the maximum depolarization reached during the positive phase of the sinusoidal voltage response. The plot shows that the resonance frequency was 6 Hz, with sinusoidal currents of 6 6 pA (F) and 8 Hz with 68 pA (D). Beyond the resonance peak, the sinusoidal voltage response decreased monotonically until 40 Hz (data not shown). dependent plateaus (D’Angelo et al., 1998). INa-r was reconstructed from Raman and Bean (1997). INa-r density (initially 1/26 INa-f) was regulated in different simulations. Ca 21 current (ICa). The IC a model was derived from high-threshold C a 21 currents (mostly N-type) measured in mature rat cerebellar granule cells in situ (Rossi et al., 1994). IC a had fast second-order activation kinetics and slow voltage-dependent inactivation. IC a density was halved to account for different extracellular C a 21 concentrations. K 1 currents (IK-V, IK-Ca, IK-A, IK-slow, IK-IR). Mature rat cerebellar granule cells in situ express IK-V, IK-C a, and IK-A (Cull-C andy et al., 1989; Bardoni and Belluzzi, 1993), IK-IR (Rossi et al., 1998), and IK-slow (this study). IK-V is a voltage-dependent K 1 current resembling other neuronal delayed rectifiers, and its model has been adapted from Gutfreund et al. (1995). IK-V was shifted by 25 mV to match INa-f. IK-C a is a voltageand C a 21-dependent K 1 current corresponding to “big-K” channel recordings from granule cells in culture (Fagni et al., 1991), the kinetics of which are largely determined by those of the associated C a 21 channels and intracellular C a 21 fluctuations. The IK-C a model is the same as in Gabbiani et al. (1994), and IK-C a density was set close to that of IK-V. IA is a fast-activating, fast-inactivating voltage-dependent K 1 current, which was reproduced from data reported by Bardoni and Belluzzi (1993). IK-slow is a slow C a 21-independent TEA-insensitive K 1 current, which was reconstructed from data shown in Figure 5. IK-slow voltage dependence was shifted by 210 mV (28 mV accounting for liquidjunction potential and 22 mV required to maintain a proper matching with INa-p). IK-IR is a fast inward rectifier current that was reconstructed from data reported by Rossi et al. (1998). Ca 21 dynamics. The intracellular C a 21 concentration, [C a 21], was calculated through the equation: d @ Ca21#/dt 5 2 IC a/~2F*Ad! 2 ~bC a~@Ca21# 2 @Ca21#0!! (7) where d is the depth of a shell adjacent to the cell surface of area A, bC a determines the loss of C a 21 ions from the shell approximating the effect of fluxes, ionic pumps, diff usion, and buffers (Traub and L linas, 1979; McCormick and Huguenard, 1992; DeSchutter and Smolen, 1998), and C a0 is resting [C a 21]. Once IC a and IKC a had been set, C a 21 dynamics were adapted to yield C a 21 transients of ;1 mM, similar to those reported by Gabbiani et al. (1994), and to reproduce the excitable response (Traub and L linas, 1979; Traub et al., 1991). Parameters used in Equation 7 were d 5 200 nm, bC a51.5, and C a0 5 100 nM. C a0 was measured in rat cerebellar granule cells in culture (Irving et al., 1992; Marchetti et al., 1995) and experimentally maintained by appropriate BAP TA-C a 21 buffers (see above). Resting membrane potential. Resting membrane potential in the model settled at 280 mV, reflecting a prominent contribution of IK-IR. Although resting membrane potential measured in rat cerebellar granule cells ranges from 260 to 285 mV (D’Angelo et al., 1995, 1998; Brickley et al., 1996; Watkins and Mathie, 1996; Rossi et al., 1998; Armano et al., 2000), at 280 mV the model was rather insensitive to manipulation of ionic conductances, which provided a usef ul reference potential for subsequent simulations. RESULTS Bursting and resonance in granule cell Intrinsic granule cell electroresponsiveness was investigated in current-clamp recordings. During step current injection (Fig. 1 A), granule cells showed inward rectification in the hyperpolarizing direction. Just-threshold depolarizing currents generated spikes, which could be clustered in doublets–triplets or longer bursts occurring at a frequency of 3–10/sec (Fig. 1 A, B) [also see D’Angelo et al. (1998), their Figs. 1, 2]. Spikes were followed by a fast afterhyperpolarization (AHP), an afterdepolarization, and a slow afterhyperpolarization (Fig. 1 B). When stronger depolarizing currents were injected, firing became regular, and afterdepolarization and slow afterhyperpolarization were no longer observed (Fig. 1 A, top trace). It should be noted that recordings were performed in the presence of 10 mM bicuculline preventing granule cell rhythmic inhibition by Golgi cells (Brickley et al., 1996) and that spontaneous EPSPs were too rare (;0.1/sec) to significantly affect spike generation (no difference was noted after application of the glutamate receptor blockers 10 mM C NQX, 100 mM APV, 762 J. Neurosci., February 1, 2001, 21(3):759–770 D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells and 50 mM 7-C l-kyn; n 5 3; data not shown). Thus, intrinsic membrane mechanisms should generate spike bursts, slow afterhyperpolarization (Alonso and L linas, 1989; Gutfreund et al., 1995; Pape and Driesang, 1998), and spike afterdepolarization (Azouz et al., 1996; Raman and Bean, 1997). Injection of sinusoidal currents of appropriate amplitude generated spike bursts in correspondence with the positive phase of the stimulus (Fig. 2 A). Spike frequency within bursts increased and then decreased with injected current frequency, therefore showing resonance. The resonance frequency was 8.9 6 3.2 Hz (n 5 8; average data are shown in Fig. 10 A). Resonance was also observed in the absence of spikes (1 mM TTX in the bath) (Fig. 2 B). In this case, the maximum depolarization reached during the positive phase of the sinusoidal voltage response showed a resonance frequency of 8.1 6 2.9 Hz (n 5 8; average data are shown in Fig. 10 B). Regarding both spike frequency and membrane potential measurements, the resonance frequency tended to increase slightly with the intensity of the injected current (Fig. 2 A, B; see Fig. 10 A, B). Evidence for a K 1-dependent mechanism in oscillation and resonance The results shown in Figures 1 and 2 suggest that granule cells combine membrane mechanisms that generate fast repetitive firing, with others responsible for slow oscillations and resonance in the theta-frequency range. Accordingly, previous observations unveiled oscillations that sustained spike bursting by reducing K 1 conductances with TEA and showed that they depended on a persistent TTX-sensitive Na 1 current (D’Angelo et al., 1998). Here we investigated the ionic dependence of the slow oscillatory mechanism and of resonance during bath application of 1 mM Ni 21, which fully blocks granule cell Ca 21 currents (Rossi et al., 1994; Tottene et al., 1996; D’Angelo et al., 1997, 1998), and of 4 mM 4-AP and 20 mM TEA, which block the granule cell K 1 currents IK-V, IK-C a, and IK-A (Cull-Candy et al., 1989; Bardoni and Belluzzi, 1993). When these ionic channel blockers were used, depolarizing current steps sustained large-size oscillations surmounted by a solitary spike when the patch pipette solution contained K 1 (n 5 5) but not when it contained Cs 1 (n 5 6) as the main intracellular cation (Fig. 3A). In these recordings, a marked adaptation prevented repetitive spike activation and bursting. Oscillations and solitary spikes were blocked by 1 mM TTX, unveiling their Na 1 dependence. Likewise, resonance was observed when the patch pipette contained K 1 (n 5 3) but not when it contained Cs 1 (n 5 3) (Fig. 3B). Because Cs 1 prevents K 1 permeation through K 1 channels, and because Ca 21 channels in these recordings are blocked, oscillation and resonance are likely to involve a TEAinsensitive Ca 21-independent K 1 current. Isolation of a slow K 1 current in granule cells A TEA-insensitive Ca 21-independent slow outward current (IK-slow) was isolated by performing voltage-clamp recordings in the presence of 20 mM TEA, 4 mM 4-AP, 1 mM Ni 21, and 1 mM TTX (Fig. 4). IK-slow could not be measured in cells internally perfused with Cs 1 rather than K 1 (data not shown), which revealed its K 1 dependence, and was reversibly reduced by 48.7 6 8.4% (n 5 4) by extracellular perfusion of 1 mM Ba 21 (Fig. 4 A). During application of depolarizing voltage steps from the holding potential of 280 mV (Fig. 4 B), IK-slow activated around 240 mV, and its amplitude increased by increasing the test potential. Figure 3. K 1 dependence of slow oscillation and resonance. Currentclamp recordings were performed in the presence of 20 mM TEA, 4 mM 4-AP, and 1 mM Ni 21. A, A sustained slow oscillation is observed in granule cells recorded with K 1-containing patch pipette during step current injection (10 pA from 280 mV). A solitary action potential is generated in a different cell recorded with a Cs 1-containing patch pipette. In both cases, excitable responses were abolished by 1 mM TTX. B, Resonance curves in a cell recorded with K 1 (F) and in another cell recorded with Cs 1 (E) inside the patch pipette. Comparable voltage responses in neurons shown in A and B were obtained by properly adjusting the intensity of injected current (lower with Cs 1- than with K 1-containing pipettes). IK-slow rising phase was well fitted by a single exponential function (Fig. 4 B, inset), indicating first-order activation kinetics. Exponential time constants ranged from 10 to .100 msec for test potentials between 240 and 0 mV, reaching values two orders of magnitude higher than those of other granule cell outward currents (Cull-Candy et al., 1989; Bardoni and Belluzzi, 1993). IK-slow persisted for .1 sec, but in six of nine cells it showed a slight tendency to inactivate at positive test potentials (,15% after 1 sec at 130 mV). Voltage jumps to different potentials after a 300 msec conditioning pulse at 130 mV (when IK-slow was almost fully activated) generated tail currents that relaxed with an exponential time course (Fig. 4C). Exponential fitting (Fig. 4 B, inset) yielded the activation time constant, the instantaneous tail current amplitude (time 5 0), and the steady-state current. According to the Nernstian equilibrium potential of K 1 ions, the instantaneous tail current was zero at 274.8 6 5.5 mV (n 5 4) and shifted to 237.7 6 6.1 mV (n 5 4) when extracellular K 1 was raised to 25 mM by equimolar Na 1 substitution. Voltage dependence of the steady-state current and the activation time constant obtained from data shown in Figure 4, A and D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells J. Neurosci., February 1, 2001, 21(3):759–770 763 Figure 5. Gating properties of IK-slow (average data from 9 granule cells, mean 6 SEM). A, Average I–V relationship (E) fitted with Equation 8 (solid line). The brok en line is the normalized steady-state activation curve (x` (K-slow)) obtained with Equation 9. B, Average activation time constant (t(K-slow)) versus membrane potential (E). The fitting line was obtained from Equation 4 and the kinetic f unctions shown in C. C, Voltage dependence of the kinetic constants a and b (see Eq. 3 and Table 1). 4 Figure 4. Isolation of a slow K 1 current, IK-slow , in the presence of 20 mM TEA, 4 mM 4-AP, 1 mM Ni 21, and 1 mM TTX. A, IK-slow was generated by a voltage pulse from 280 to 130 mV. IK-slow was reversibly inhibited by application of 1 mM Ba 21. B, IK-slow activation was investigated by applying 1 sec, 10 mV depolarizing voltage steps from the holding potential of 280 mV (a short pre-step was applied to inactivate IK-A ) (Bardoni and Belluzzi, 1993). The inset shows exponential fitting to the rising phase of a current recorded at 0 mV with the function I( t) 5 Iss * (1 - exp(2t/tact )), where Iss 5 53 pA is the steady-state current and t act 5 33.5 msec is the activation time constant. C, IK-slow deactivation was investigated by using voltage jumps to different potentials after a 300 msec conditioning pulse at 130 mV (holding potential 5 280 mV). The inset shows exponential fitting to a tail current recorded at 210 mV with the function I( t) 5 Iss 1 I0 * exp(2t/ tdeact ), where (Io 1 Iss ) 5 48.9 pA is the instantaneous current, Iss 5 39.2 pA is the steady-state current, and tdeact 5 46.9 msec is the deactivation time constant. D, Voltage dependence of steady-state amplitude of deactivation curves (Iss , Œ), and of time constants obtained by exponential fitting to activation (tact , E) and deactivation (tdeact , F) curves. The inset shows intersection of the linear regression curve to instantaneous tail current amplitude with the voltage axis at 271.4 mV. Data in B–D were obtained from the same granule cell. D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells 764 J. Neurosci., February 1, 2001, 21(3):759–770 Table 1. Voltage-dependent conductance parameters Conductance state variables gNa-f Activation Inactivation gNa-r Activation Inactivation gNa-p Activation gK-V Activation gK-A Activation Inactivation gK-IR Activation gK-C a Activation gC a Activation Inactivation gK-slow Activation n Gmax (S/cm2) Vrev (mV) a (sec21) b (sec21) 3 1 0.013 87.39 0.9(V 1 19)/(1 2 exp(2(V 1 19)/10)) 0.315 exp(20.3(V 1 44)) 36 exp(20.055(V 1 44)) 4.5/(1 1 exp(2(V 1 11)/5)) 1 1 5e24 87.39 0.00024 2 0.015(V 2 4.5)/((exp(2(V 2 4.5)/6.8) 2 1)) 0.96*exp(2(V 1 80)/62.5) 0.14 1 0.047(V 1 44)/(exp((V 1 44)/0.11) 2 1)) 0.03*exp((V 1 83.3)/16.1) 1 2e24 87.39 0.091(V 1 42)/(1 2 exp(2(V 1 42)/5)) x` 5 1/(1 1 exp(2(V 1 42)/5)) t` 5 5/(a 1 b) 20.062(V 1 42)/(1 2 exp((V 1 42)/5)) 4 0.003 284.69 0.13(V 1 25)/(1 2 exp(2(V 1 25)/10)) 1.69 exp(20.0125(V 1 35)) 3 1 0.004 284.69 14.67/(1 1 exp(2(V 1 9.17)/23.32)) 0.33/(1 1 exp((V 1 111.33)/12.84)) x` 5 1/(1 1 exp(2(V 1 46.7)/19.8)) y` 5 1/(1 1 exp((V 1 78.8)/8.4)) 2.98(exp(2(V 1 18.28)/19.47)) 0.31/(1 1 exp(2(V 1 49.95)/8.9)) 1 9e24 284.69 0.4 exp(20.041(V 1 83.94)) 0.51 exp(0.028(V 1 83.94)) 1 0.004 284.69 2.5/(1 1 1.5e 2 3/[Ca] exp(20.085V)) 1.5/(1 1 [Ca]/(0.15e 2 3 exp(20.085V))) 2 1 4.6e24 129.33 * 0.15 exp(0.063 (V 1 29.06)) 0.0039 exp(20.055(V 1 48)) 0.089 exp(20.039(V 1 18.66)) 0.0039 exp(0.012(V 1 48)) 1 3.5e24 284.69 0.008 exp(0.025(V 1 30)) x` 5 1/(1 1 exp(2(V 1 30)/6)) 0.008 exp(20.05(V 1 30)) This Table reports the equations used to calculate ax, bx, ay, and by (see Eqs. 3 and 4) for the membrane conductances used in the model (in a few cases x` and y` are reported; see Eq. 9). The number of gating particles (n), maximum conductance (Gmax), and ionic reversal potential (* resting value for Ca21) used to calculate ionic currents are also shown. B, are shown in Figure 4 D. The I–V curve showed outward rectification, and the activation time constant showed a bellshaped voltage dependence. It should be noted that time constants measured from activation and deactivation currents coincided, consistent with a first-order gating mechanism. However, deactivation currents were preferred to activation currents for gating reconstruction, because they covered a more extended membrane potential range and were free of potential contamination by IK-A, which is not completely blocked even by 4 mM 4-AP in cerebellar granule cells (Bardoni and Belluzzi, 1993). Figure 5 shows average data obtained from nine granule cell recordings. The I–V curve (Fig. 5A) was fitted with a Boltzman equation of the form [see Rossi et al. (1998)]: I ~ V ! 5 @ G max~V 2 Vrev!#/@1 1 exp~~V 2 V1/2!/k!# (8) where Gmax 5 0.8 nS is the maximum conductance, Vrev 5 270.2 mV is the reversal potential, V1/2 5 220 mV is the half-activation potential, and k 5 6 mV 2 1 is the activation voltage dependence. These parameters were used to reconstruct steady-state IK-slow activation (Fig. 5A) by using the equation: G ~ V ! /G max 5 1/@1 1 exp~~V 2 V1/2!/k!#. (9) The relationship between activation time constant and voltage (Fig. 5B) was fitted by Equation 4 using the kinetic constants shown in Figure 5C. As in other ionic current models (Gutfreund et al., 1995; Mainen et al., 1995), independent expressions for the activation time constant and steady-state activation improved data representation. These results allowed IK-slow reconstruction according to a first-order Hodgkin–Huxley kinetic scheme (Yamada et al., 1998) (see Materials and Methods; Table 1). Mathematical reconstruction of intrinsic excitability To investigate the role of IK-slow in oscillations, bursting, and resonance, we developed a mathematical model of granule cell excitability (see Materials and Methods; Table 1). With INa-f, IC a, IK-V, IK-C a, IK-A, and IK-IR, the model generated inward rectification and fast repetitive firing (Gabbiani et al., 1994). The model was then endowed with a persistent Na 1 current (INa-p) (D’Angelo et al., 1998) and IK-slow, and was extended to test the potential contribution of a resurgent Na 1 current (INa-r) (Raman and Bean, 1997). With INa-p, IK-slow, and INa-r, just-threshold step current injection generated membrane potential oscillations and spike bursts, with the spikes showing fast- and slow-afterhyperpolarization and afterdepolarization (Fig. 6 A, B). With stronger current injection, the model generated regular repetitive firing (Fig. 6 A; see Fig. 8). Moreover, the model reproduced TEAinduced bursting (see Fig. 9) and intrinsic resonance (see Fig. 10). The ionic mechanism of slow oscillations Model simulations showed that INa-p and IK-slow were sufficient to generate regular theta-frequency oscillations, which were elimi- D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells J. Neurosci., February 1, 2001, 21(3):759–770 765 and GC a were set to zero, reproducing the pharmacological application of 20 mM TEA and 1 mM Ni 21 (Fig. 7C; compare Fig. 2). Moreover, the model generated a solitary Ca 21 action potential when GK-V, GK-C a, and GNa were set to zero, reproducing the pharmacological application of 20 mM TEA and 1 mM TTX (Fig. 7D) [compare Fig. 3 in D’Angelo et al. (1998) and Fig. 10 in D’Angelo et al. (1997)]. Modeling results therefore indicated that slow oscillations in granule cells depend on the interaction of INa-p and IK-slow but are independent from Ca 21 currents. Repetitive firing Figure 8 A shows repetitive firing in the model. As in experimentally recorded granule cell responses (D’Angelo et al., 1995, 1998; Brickley et al., 1996), spikes showed negligible adaptation, and first-spike latency decreased whereas spike frequency increased when the injected current intensity was raised. The frequency– intensity ( f–I ) plot was almost linear between 0 and 100 Hz, with a slope of 7.3 spikes per pA 2 1 z sec 2 1. Spikes were generated by a sudden INa-f raise followed by activation of IK-V and the IC a/IK-C a system (Fig. 8 B). The intracellular Ca 21 wave peaked after the spike and decayed to zero in ;3 msec. INa-f, IK-V, IC a, and IK-C a accounted for most of the active current during the spike and the fast AHP, whereas other currents were two orders of magnitude smaller. INa-p increased during the spike prepotential and persisted for several milliseconds. INa-r increased just after the spike. IK-A, IK-IR, and IK-slow were mostly active during the interspike trajectory. Thus, whereas INa-p and INa-r enhanced spike activation, IK-A, IK-IR, and IK-slow delayed it. The regulatory action of INa-r and IK-A on spike frequency is shown in the plots of Figure 8 A. TEA-induced bursting Figure 9A illustrates theta-frequency bursting in the model after partial IK-C a blockage, simulating experimental TEA application (D’Angelo et al., 1998, their Figs. 6 and 8). A relatively modest IK-C a blockage caused spike doublets–triplets, whereas stronger blockage caused marked membrane potential oscillations surmounted by adapting spike bursts. Thus, IK-C a prevented activation of the INa-p/IK-slow oscillatory mechanism. Bursting was enhanced by INa-r (Fig. 9A, compare left with right column), but no bursting was generated by INa-r alone when the INa-p/IK-slow mechanism was turned off (Fig. 9A, bottom panel ). TEA-induced bursting was characterized by a remarkable rise in INa-p associated with a progressive IK-slow activation (Fig. 9B, compare Fig. 7B). During the burst, INa-f inactivation caused spike amplitude adaptation. Figure 6. Mathematical modeling of granule cell excitability. A, Model responses to 2 pA step current injection from 280 mV. The model generates inward rectification in subthreshold responses, followed by regular repetitive firing with almost no adaptation. B, Slow oscillations, slow afterhyperpolarization, occasional uncoupling of spike prepotential from upstroke, and spike bursts can be generated by the model by using just-threshold stimulation (10.5 pA in the top and middle tracings, 12 pA in the bottom tracing). The shape of oscillations and bursting could be modified by changing the GNa-r or GK-slow intensities. nated when either of these currents was turned off (Fig. 7A). During oscillations, INa-p and IK-slow showed activation/deactivation cycles describing stable orbits in the phase plane (Fig. 7B). The histerisis observed in IK-slow (and to a lesser extent in INa-p) trajectory reflected its delayed gating during membrane potential changes. The model generated repetitive oscillations when GK-V, GK-C a, Resonance Injecting the granule cell model with sinusoidal currents of different frequency generated resonant responses. Resonance in burst spike frequency is shown in Figure 10 A, and resonance in maximum membrane depolarization (with spikes blocked by setting INa-f, INa-p, and INa-r to zero mimicking TTX block) is shown in Figure 10 B. In both cases a family of resonant curves is shown, one of which matches average experimental measurements from a set of five granule cells. The model was used to investigate the ionic mechanisms of resonance. Resonance was eliminated by blocking IK-slow, which determined the ascending branch of resonance curves (Fig. 11 A, B). The descending branch of resonance curves was determined by passive membrane filtering, as demonstrated by its persistence when the ionic channels involved in resonance were blocked. In addition, IK-A markedly accelerated the descending 766 J. Neurosci., February 1, 2001, 21(3):759–770 D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells Figure 7. The oscillatory mechanism in granule cells. A, Simulation of stable oscillations sustained by GK-slow and GNa-p during injection of an 11 pA current step. Oscillations are eliminated by turning off either GK-slow or GNa-p. All other active conductances were set to zero except GK-IR , which was used to keep input conductance close to its normal value. B, Time course and phase-plane trajectory of IK-slow and INa-p during membrane potential oscillations (dotted line). C, Voltage responses to an 11 pA current step simulating TEA (GK-V 5 0, GK-C a 5 0), and Ni 21 (GC a 5 0) application. The brok en line simulates subsequent application of TTX (GNa 5 0). D, Voltage responses to an 11 pA current step simulating TEA (GK-V 5 0, GK-C a 5 0) and TTX (GNa 5 0) application. Same calibration in A, C, and D. Figure 8. Ionic mechanisms of repetitive firing. A, Model tracings show regular firing with negligible adaptation at .100 Hz. The top plot ( f–I plot) reports firing frequency in control conditions (F) and after having turned off IK-A (brok en line) or INa-r (dotted line). The bottom plot shows first-spike latency in control conditions (F) and after having turned off IK-A (brok en line). B, Ionic currents and [C a 21] changes during repetitive firing. The right set of tracings is an enlargement of currents associated with an action potential. Note that both INa-p and INa-r are activated after the spike. D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells J. Neurosci., February 1, 2001, 21(3):759–770 767 Figure 9. Ionic mechanisms of bursting. A, Tracings show intensification of bursting and spike adaptation by progressively increasing GK-C a inhibition during injection of 11 pA current steps. Stronger GK-C a inhibition is needed to generate bursting when GNa-r is set to zero. No bursting is generated when GNa-p and GK-slow are turned off, but GNa-r is left active. B, Ionic currents and [Ca 21] changes during bursting elicited with IK-C a reduction to 37% of its normal value. Note that IK-slow and INa-p are greatly enhanced during bursting compared with repetitive firing. Figure 10. Ionic mechanisms of resonance. A, Injection of sinusoidal currents causes oscillatory bursting in the model. Tracings are generated by a 66 pA sinusoidal current superimposed on a 12 pA current step. Insets show higher spike frequency in bursts generated at 10 Hz than at 2 Hz. The plot shows model resonance with three different sinusoidal current intensities (64, 66, or 6 8 pA superimposed on a constant 12 pA current step). The curve generated with 66 pA is a good match with the average experimental response (F; mean 6 SD; n 5 5). B, Same as in A, except that maximum membrane depolarization during the positive phase of sinusoidal voltage responses is measured with INa-f , INa-p , and INa-r set to zero. This result is compared with experimental recordings in the presence of 1 mM TTX (F; mean 6 SD; n 5 5, same cells as in A). As with real granule cells, the model shows resonance ;10 Hz. 768 J. Neurosci., February 1, 2001, 21(3):759–770 branch. Enhanced activation of IK-slow at low frequency and IK-A at high frequency is shown in Figure 11C. It should be noted that resonance was not eliminated by blocking INa-p (Fig. 11 A, B), which showed minor frequency-dependent changes (Fig. 11C). Finally, INa-r (in the case of burst spike frequency) decelerated the descending branch of resonance curves. DISCUSSION This study shows that in addition to generating fast repetitive firing, cerebellar granule cells generate oscillations, bursting, and resonance in the theta-frequency range. Experimental and modeling results indicated that these aspects of intrinsic excitability require a slow K 1 current (IK-slow) to be generated. IK-slow is a Ca 21-independent TEA-insensitive K 1 current activating in the spike threshold region with slow kinetics (10 –100 msec). A current like IK-slow has not been reported previously in cerebellar granule cells, although a persistent TEA-resistant current component was noted in cell culture (Cull-Candy et al., 1989) [also see Bardoni and Belluzzi (1993), their Fig. 10 B]. IK-slow biophysical and pharmacological properties are similar to those of IM of vertebrate neurons (Brown and Adams, 1980; Adams et al., 1982a,b) and are suitable to generate the delayed repolarizing feedback and high-pass filtering required for bursting and resonance. Indeed, IM has been reported to sustain oscillations and resonance in amygdaloid neurons (Pape and Driesang, 1998) and in cortical pyramidal neurons (Gutfreund et al., 1995). Alternative mechanisms that might be invoked to explain bursting and resonance are unlikely to occur in cerebellar granule cells. (1) A slow inward rectifier current (Ih) (Dickson et al., 2000) and a slow Ca 21-dependent K 1 current (IAHP) (Wang Figure 11. Resonance regulation. This Figure shows resonance being regulated by injection of a 66 pA sinusoidal current superimposed on a 12 pA current step. A, Resonance in burst spike frequency in different conditions: F, control; M, IK-slow 5 0; L, GK-A 5 0; D, GNa-p 5 0; ƒ, GNa-r 5 0. No resonance could be observed in the model when INa-p , IK-slow , and INa-r were turned off (thin dotted line). B, Resonance in maximum membrane depolarization during the positive phase of sinusoidal voltage responses in different conditions: control (D, INa-p , INa-f , INa-r 5 0; TTX condition); M, IK-slow 5 0, L, IK-A 5 0; F, INa-p active. No resonance could be observed in the model when INa-p , IK-slow , and INa-r were turned off (thin dotted line). C, INa-p , IK-slow , and IK-A at three different frequencies (thick line, 8 Hz; thin line, 2 Hz; broken line, 14 Hz). Note the different frequency-dependent activation of these currents. D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells and Rinzel, 1999) are apparently not expressed in granule cells. Granule cell inward rectification is fully explained by a fast K 1-dependent inward rectifier (Fig. 6 A) (Rossi et al., 1998), and granule cell slow afterhyperpolarization is fully explained by IK-slow (Fig. 6 B). It should also be noted that the IAHP blocker apamin did not affect the excitable response, and oscillations and bursting persisted in the presence of C a 21 channel blockers (Fig. 3) (D’Angelo et al., 1998). Moreover, contrary to what would be expected from IAHP, granule cell slow AHP occurred with just-threshold stimulation disappearing rather than being enhanced with higher spike frequency and was not associated with any spike frequency adaptation. (2) A resurgent current, INa-r, facilitated but proved not sufficient to induce bursting and resonance (Fig. 8 A). (3) Finally, return currents from the dendrites, which might generate somatic rebound depolarization and spike bursting, are unlikely to be effective because of the granule cell compact electrotonic structure. Actually, with a 10 MV somatodendritic resistance (calculated assuming an axial resistance of 150 V/cm in a 20 mm dendrite) and a somatodendritic ratio between 1 and 2 (Silver et al., 1992; D’Angelo et al., 1993; Gabbiani et al., 1994), no bursting is expected even if active conductances are expressed in the dendrites (Mainen and Sejinowsky, 1996). Modeling reliability was based on the extensive characterization of membrane currents and the compact electrotonic structure of cerebellar granule cells (for details, see Materials and Methods). Indeed, patch-clamp recordings from mature cerebellar granule cells in situ have been used (1) to reconstruct IC a (Rossi et al., 1994), IK-IR (Rossi et al., 1998), IK-A D’Angelo et al. • Bursting and Resonance in Cerebellar Granule Cells (Bardoni and Belluzzi, 1993), and IK-slow (this study), (2) to identif y different Na 1 current components (including INa-f, INa-p, and INa-r) (D’Angelo and Magistretti, unpublished results), and (3) to characterize the pharmacological properties of excitability (D’Angelo et al., 1998). Patch-clamp recordings from granule cells in culture were used to reconstruct IK-C a (Fagni et al., 1991; Gabbiani et al., 1994). Although f urther investigation is required to clarif y C a 21 dynamics and the biophysical properties of the Na 1 current, mathematical modeling allowed us to investigate the role of IK slow in relationship to other excitable properties of the granule cell. In addition to modeling, IK slow involvement in oscillations, bursting, and resonance may be investigated by IK slow selective pharmacological blockage or by IK slow electronic antagonism /expression through a dynamic-clamp circuit (Hutcheon et al., 1996a,b). Experimental and modeling observations allow the following reconstruction of the ionic mechanisms of rat cerebellar granule cell electroresponsiveness. (1) INa-f and IK-V were the core of a fast oscillatory mechanism sustaining fast repetitive firing, as in the classical Hodgkin– Huxley model (Hodgkin and Huxley, 1952). IK-A increased spike latency (Connor and Stevens, 1971), and the IC a–IK-C a system stabilized repetitive firing by enhancing fast AHP and Na 1 channel deinactivation [see also Gabbiani et al. (1994)]. (2) INa-p and IK-slow were the core of a slow oscillatory mechanism sustaining theta-frequency oscillation and bursting. INa-p has been reported to sustain oscillations in association with IM in neocortical pyramidal neurons (Gutfreund et al., 1995) and in association with Ih in enthorinal pyramidal neurons (Alonso and Llinas, 1989; Dickson et al., 2000). IK-slow caused delayed repolarization, terminating the positive phase of the oscillation promoted by INa-p. (3) Emergence of bursting was regulated by the IC a–IK-C a system (D’Angelo et al., 1998) through the fast AHP, which reduced the depolarizing action of INa-p (Azouz et al., 1996) and INa-r (Raman and Bean, 1997) after the spike. (4) Resonance depended on high-pass filtering caused by IK-slow (generating the ascending branch of the resonance curve) in association with low-pass filtering caused by passive membrane properties (generating the descending branch of the resonance curve) and was amplified by INa-p. Thus, resonance involved subthreshold changes in membrane excitability [corresponding to subthreshold impedance resonance reported by Hutcheon et al. (1996a,b)], consistent with the identification of IK-slow as a “resonator” current and INa-p as an “amplifier” current“ (Hutcheon and Yarom, 2000). In addition, the model suggested that IK-A, which is inactivated at low but not high frequency, accelerated the descending branch of the resonance curve. Finally, the model predicted that resonance in burst spike frequency could be intensified by spike clustering promoted by INa-r. Thus, resonance and bursting may have in common their dependence on IK-slow as well as that on INa-r. Granule cells are excitatory interneurons relaying information conveyed by mossy fibers into the cerebellar cortex (Marr, 1969). 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