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Dopaminergic neurons of the substantia nigra pars compacta are autonomous pacemakers. This activity is responsible for the sustained release of dopamine necessary for the proper functioning of target structures like the striatum. Somatodendritic L-type Ca2+ channels have long been viewed as important, if not necessary, for this activity. The studies reported here challenge this viewpoint. Using a combination of optical and electrophysiological approaches in brain slices, it was found that antagonism of L-type Ca2+ channel effectively stopped dendritic Ca2+ oscillations, but left autonomous pacemaking unchanged. Moreover, damping intracellular Ca2+ oscillations with exogenous buffer had little effect on pacemaking rate. Although not necessary for pacemaking, L-type channels helped support pacemaking when challenged with cationic channel blockers. Simulations suggested that the insensitivity to antagonism of L-type channels reflected the multichannel nature of the pacemaking process. The robustness of pacemaking underscores its biological importance and provides a framework for understanding how therapeutics targeting L-type Ca2+ channels might protect dopaminergic neurons in Parkinson's disease without compromising their function.
Dopamine (DA) releasing neurons of the substantia nigra pars compacta (SNc) are critical to a broad array of psychomotor functions, including action selection and reward based learning (Schultz, 2007; Yin et al., 2008; Cohen and Frank, 2009). A key feature of these neurons is their steady, autonomous pacemaking. This self-generated activity maintains extracellular DA levels and the DA receptor signaling necessary for normal network operation in basal ganglia structures like the striatum (Albin et al., 1989; Gonon and Bloch, 1998). Although synaptic signals are capable of altering ongoing spike activity in vivo (Grace and Bunney, 1984; Tepper et al., 1998), the rate and regularity of the autonomous activity is remarkably robust in the face of a variety of pharmacological and molecular perturbations (e.g.,Paladini et al., 2003; Chan et al., 2007).
The mechanisms underlying autonomous pacemaking have been the subject of investigation for over a decade. At present, the dominant view is that in adult SNc dopaminergic neurons, pacemaking is dependent upon Ca2+ channels that open at relatively hyperpolarized membrane potentials (Nedergaard et al., 1993; Mercuri et al., 1994; Amini et al., 1999; Wilson and Callaway, 2000; Puopolo et al., 2007). Based upon the ability of dihydropyridines (DHPs) to slow or stop pacemaking, this channel has been assumed to be of the Cav1 or L-type (Nedergaard et al., 1993; Mercuri et al., 1994; Chan et al., 2007; Puopolo et al., 2007). However, the DHP concentrations necessary to achieve significant slowing or silencing in these acute physiological experiments are typically several orders of magnitude greater than those predicted by binding studies to be required for near complete channel antagonism (Koschak et al., 2001; Sinnegger-Brauns et al., 2009). In principle, there are several factors that might contribute to this mismatch. For example, the affinity of DHPs for L-type channels is state-dependent being roughly a thousand-fold lower at negative membrane potentials (Bean, 1984). Another major limitation is the difficulty in achieving binding equilibrium in brain slices. Lipophilic DHPs not only must diffuse tens of microns into the slice, they must partition into the membrane and diffuse to a binding site buried in the lipid bilayer (Lipkind and Fozzard, 2003). This makes equilibrium slow to achieve, taking as much as an hour (Herbette et al., 1989). Because electrophysiological recordings are typically stable for a much shorter period of time, high DHP concentrations have been used to achieve measurable levels of channel antagonism rapidly. This approach has a number of caveats, not the least of which is that because DHPs have very high membrane partition coefficients (Herbette et al., 1989), their concentration can reach levels that begin to have non-specific effects on other ion channels, complicating the interpretation of effects on complex phenomena, like pacemaking.
Determining the role of L-type Ca2+ channels in pacemaking is not only of theoretical importance, but of clinical importance as well. Ca2+ ions entering SNc DA neurons through L-type Ca2+ channels during pacemaking elevates cellular vulnerability to toxins used to create animal models of Parkinson's disease (PD) (Chan et al., 2007). The potential linkage of these channels to PD has been strengthened by epidemiological studies showing a decreased disease risk in humans treated for hypertension with DHPs (Becker et al., 2008). But if L-type channels are critical to pacemaking, this therapeutic benefit must come at a functional cost, like a drop in pacemaking rate and DA in target structures. Using optical and electrophysiological approaches to assay acute L-type channel antagonism, we show that although these channels contribute to the robustness of pacemaking, they are not necessary.
Acute midbrain coronal slices (220 μm-thick) were obtained from wild-type Male C57BL6 (Charles River) between postnatal ages 21 and 32 days, unless specified otherwise. The handling of mice and all procedures performed on them were approved by the institutional Animal Care and Use Committee and were in accordance with the ‘National Institutes of Health Guide to the Care and Use of Laboratory Animals’ and Society for Neuroscience guidelines. Mice were anesthetized with a ketamine/xylazine mixture followed by a transcardial perfusion with ice-cold, oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 25 dextrose, pH 7.3, osmolality 315-320 mOsm/L. After perfusion, mice were decapitated and brains were removed rapidly, followed by sectioning in ice-cold oxygenated ACSF using a vibratome (VT1000S Leica Microsystems, Nusslock, Germany). Midbrain slices recovered in ACSF at 34°C for thirty minutes prior to electrophysiological recordings. Slices were transferred to a small volume (<0.5 ml) recording chamber that was mounted on a fixed-stage, upright microscope (BX51, Olympus America) equipped with IR-DIC (0.9 numerical aperture, NA) with de Sénarmont compensation (Olympus). Experiments were performed at 33-34°C, unless specified otherwise. The recording chamber was superfused with carbogen-saturated ACSF with a flow rate of 2–3 ml min-1 running through an in-line heater (SH-27B with TC-324B controller; Warner Instruments). Neuronal somata and proximal dendrites were visualized by video microscopy at high magnification (60×, 0.9 NA water immersion objective, Olympus). Cell-attached and whole-cell patch clamp recordings were performed in DA neurons from the mid-to-ventral tier of the SNc. Recording patch electrodes (resistance of 3-4 MΩ) were prepared with a Sutter Instruments horizontal puller (Novato, CA) using borosilicate glass with filament (Novato, CA). For all voltage and current clamp recordings, patch electrodes were filled with internal solution containing (in mM): 135 K-MeSO4, 5 KCl, 5 HEPES, 0.05 EGTA, 10 phosphocreatine-di(tris), 2 ATP-Mg, 0.5 GTP-Na; the pH and osmolality were adjusted to 7.3 and 290-300 mOsm/L, respectively. The liquid junction potential in our recording ACSF was 7 mV and not corrected for. Conventional tight-seal (>3 GΩ) whole-cell patch-clamp and cell-attached recordings were made on visually identified neurons based on 1) size and somatodendritic morphology; 2) regular spiking between 1-4 Hz, 3) presence of a voltage sag during delivery of a hyperpolarizing pulse (-250 pA current injection); and presence of the HCN current recorded in voltage clamp (data not shown). For Na+ current recordings, voltage control and space clamp was improved by lowering the external concentration of Na+ to 20 mM and adding 50 μM Cd2+ to the perfusate; patch electrodes were filled with a cesium-based internal containing in mM: 120 Cs-MeSO3, 15 CsCl, 8 NaCl, 10 HEPES, 0.2 EGTA, 3 ATP-Mg, 0.3 GTP-Na, 10 TEA-Cl, pH 7.3, and osmolality 280 mOsm/L. TTX-sensitive sodium currents were recorded in voltage-clamp with ramps from -70 mV to -40 mV for 500 ms (ramp speed of 0.06 mV/ms) and peak currents were measured. Signals were filtered at 1–4 kHz and digitized at 5–20 kHz with a Digidata 1322A (Molecular Devices). For current-clamp recordings, the amplifier bridge circuit was adjusted to compensate for electrode resistance and monitored. Electrode capacitance was also compensated. If series resistance increased >20% during recording, the data were discarded.
For dendritic Ca2+ measurements, SNc DA neurons in tissue slices (as described above) were loaded with Alexa Fluor 594 (50 μM) and Fluo-4 (200 μM) through the patch pipette. All experiments were performed at 32-34° C. Images were acquired with a 60×/0.9NA water-immersion lens. Dyes were allowed to equilibrate for at least 15 minutes prior to imaging. The two-photon excitation source was a Chameleon-ultra2 tunable laser system (680 nm to 1080 nm) utilizing Ti:sapphire gain medium with all-solid-state active components and a computer-optimized algorithm to ensure reproducible excitation wavelength, average power, and peak power (Coherent Laser Group, Santa Clara, CA). Optical signals were acquired using 810 nm excitation beam (80MHz pulse repetition frequency and ~250fs pulse duration) to simultaneously excite Alexa and Fluor-4 dyes. Laser power attenuation was achieved with two Pockel cell electro-optic modulators (models 350-80, Con Optics, Danbury, CT). The two cells are aligned in series to provide enhanced modulation range for fine control of the excitation dose (0.1% steps over four decades). The laser-scanned images were acquired with a Bio-Rad Radiance MPD system (Hemel Hempstead, England, UK). The fluorescence emission was collected by external or non-de-scanned photomultiplier tubes (PMTs). The green fluorescence (500-550 nm) was detected by a bialkali-cathode PMT and the red fluorescence (580-640 nm) was collected by a multi-alkali-cathode (S-20) PMT. The system digitizes the current from detected photons to 12 bits. The laser light transmitted through the sample was collected by the condenser lens and sent to another PMT to provide a bright-field transmission image in registration with the fluorescent images. Measurements were taken in a sample plane along dendritic segments (100-150 mm from the soma). Line scan signals were acquired (as described above) at 6 ms per line and 512 pixels per line with 0.18 μm pixels and 10 μs pixel dwell time. The time between the control and isradipine treatment measurements was 5-7 minutes.
Electrophysiological data collected was analyzed using ClampFit 10.1 (Molecular Devices), Igor Pro 6.0 (Wavemetrics, CA) or Matlab (Mathworks, Inc., Natick, MA). The stimulation, display, and analysis software for the two-photon imaging data was analyzed using a custom-written shareware package, WinFluor, PicViewer and PowerCAL kindly provided by John Dempster (Strathclyde University, Glasgow, Scotland; UK). Data was summarized either using box-plots for small sample sizes or with parametric statistics (mean ± standard error) for larger samples. Statistical analysis was done with SigmaStat 3.5 (Systat Software, Chicago, IL) using non-parametric testing Mann-Whitney Rank Sum Test for comparing between two groups or Kruskal Wallis ANOVA with Dunnet's post hoc for multiple group comparison. Before-after statistical analyses were performed using the Wilcoxon Signed Rank Test. Probability (p) threshold for statistical significance was p<0.05.
Reagents were purchased from Sigma (St.Louis, MO) except K-MeSO4 (MP Biomedicals), GTP-Na (Roche Diagnostics), isradipine, ZD7288 (Tocris-Cookson), tetrodotoxin and α-dendrotoxin (Alomone Labs), and BAPTA tetrasodium salt, Alexa594, Fura-2, Fluo-4 (Invitrogen/Molecular Probes). Drugs stocks solutions were prepared in deionized water, DMSO or methanol; on the day of experiment, stocks were diluted to final concentrations in ACSF to achieve a final solvent concentration of less than 0.01% v/v.
Simulations were performed with NEURON, version 5.9 (Hines and Carnevale, 2001). The model neuron was constructed of a cylindrical soma (L=15 μm, D=15 μm) with axial resistivity of 70 Ω/cm, membrane capacitance 1 μF/cm2; the temperature was 35° C and all conductances had Q10 values of 3-4. The density of the simulated channels (S/cm2) inserted into the membrane were: Nav1 (3.5e-2), Kv1 (3e-5), Kv2 (2.5e-3), Kv4fast (8.5e-5), Kv4slow (2e-5), Kv7 (7e-7), BK (1e-3), SK (1e-5), HCN (1.25e-4), Cav1.3 (5e-5), Cav2 (1e-5), Leak (3.5e-6); Ena=50 mV, Ek=-90 mV, Eleak/HCN=-20 mV. Kinetics and voltage dependence of channel gating were adjusted to fit experimentally derived values in either SNc DA neurons or other basal ganglia neurons (Mercer et al., 2007); densities were adjusted to fit experimental results and to achieve pacemaking in a normal range. Mod files for Ca2+ diffusion, buffering and pumping were adapted to fit the kinetics observed experimentally using Fluo-4. All of the NEURON mod files used in simulation will be posted on the NeuronDB web site (http://senselab.med.yale.edu/neurondb) or are available on request.
Simulations of the modulated DHP receptor were performed with Matlab using the framework proposed by Bean (1984). The channel was assumed to have two affinity states. A high affinity state at depolarized (inactivated) membrane potentials and a low affinity state at hyperpolarized (resting) membrane potentials. The dissociation constant for high affinity state was assumed to be equal to that found in equilibrium binding studies, whereas the dissociation constant for low affinity state was assumed to be a thousand fold higher (Bean, 1984). The macroscopic balance between these states was assumed to be governed by voltage-dependent inactivation of the channel (Bean, 1984); the relationship between steady-state inactivation and voltage for Cav1.3 channels was taken from the work of Koschak et al. (2001).
To speed drug delivery to cells buried (30-75 μm below the surface) in a slice, 5 μM isradipine was applied – a concentrations 25-50 times that calculated to achieve near complete blockade of Cav1.3 channels at the depolarized membrane potentials traversed by SNc DA neurons (Bean, 1984). In cell-attached patch recordings (to minimize rundown), application of 5 μM isradipine for 10 minutes failed to slow pacemaking (Fig. 1a,b). This was not because isradipine had not antagonized L-type Ca2+ channels. The antagonism could be clearly seen in whole cell recordings of SNc DA neurons where oscillations in dendritic Ca2+ concentration were monitored using the Ca2+ dye fluo-4 (200 μM). In proximal dendrites (<~70 μm from the soma), isradipine clearly reduced the oscillations in Ca2+ concentration (Fig. 1b). In the more distal (>80 μm from the soma) dendrites, 5 μM isradipine at this time point eliminated any detectable oscillation in Ca2+ concentration and lowered modal fluorescence (Fig. 1c,d). Again, at the time point where these measurements were made, there was no change in spike rate (Fig. 1e). Spectral analysis of the fluorescence signal in these distal dendrites consistently failed to detect power at the pacemaking frequency (Fig. 1f).
Another observation that has been taken as evidence of the centrality of L-type channels in pacemaking is that following block of voltage-dependent Na+ channels with tetrodotoxin (TTX), the membrane potential continues to oscillate at near the pacemaking frequency (Yung et al., 1991; Nedergaard et al., 1993). These slow oscillatory potentials (SOPs) are widely held to drive pacemaking and trigger Na+ spikes. But, like the oscillations in dendritic Ca2+ concentration, the SOPs were eliminated by application of low micromolar concentrations of isradipine (Fig. 2a) – concentrations that did not change pacemaking rate when Na+ channels were left unblocked (e.g., Fig.1a). To better tests the inferences drawn above, slices were incubated in 200 nM isradipine for 60 minutes and then patched. If the rate limiting step in DHP antagonism is diffusion within the lipid bilayer, then at this time point the number of bound channels should be within 10% of the equilibrium value (Herbette et al., 1989); assuming a modal membrane potential of -60 mV (a conservative estimate), over 90% of the Cav1.3 channels should be antagonized at this point. As predicted from the work with exposure to higher isradipine concentrations, there was no detectable change in pacemaking rate or regularity of SNc DA neurons sampled with cell-attached recordings from slices treated in this way (Fig. 2b,d). However, invariably these cells lacked TTX-insensitive SOPs (Fig. 2c), arguing that Cav1.3 Ca2+ channels were effectively antagonized. Spectral analysis of the membrane potential in these cells occasionally detected some residual power near the pacemaking rate with exposure to 200 nM isradipine, but never with higher concentrations (Fig. 2e).
The SOPs seen in the presence of TTX reflect the interplay between dendritic L-type Ca2+ channels and small conductance, Ca2+-activated K+ (SK) channels (Ping and Shepard, 1996; Wilson and Callaway, 2000). If this interplay was necessary for pacemaking, then disrupting the efficient activation of SK channels by chelating intracellular Ca2+ should suppress pacemaking. However, whole cell dialysis with BAPTA (5 mM) did not disrupt pacemaking, but rather led to a slow acceleration of pacemaking (Fig. 3a), in spite of its elimination of the SOP seen in the presence of TTX (Fig. 2b) and its predicted effect on the trajectory of the spike after-hyperpolarization (Fig. 3c). Finally, the temporal properties of the SOPs – which clearly depend upon L-type channels – were not consistent with a causal role in pacemaking. First, the frequency of spiking during pacemaking was not strongly correlated with the SOP frequency in the same cell after TTX application (Fig. 3d). Second, the coefficient of variation in interval duration during spiking was an order of magnitude smaller than that of the SOP (median CV spiking=1.9e-4; median CV oscillation=3.2e-2, n=9, p<0.05); and examination of successive intervals consistently showed random scatter in the SOP interval, even when the interspike interval in the same cell was very regular (Fig. 3e).
The insensitivity of pacemaking to DHPs seen in our work differs from that seen in younger, acutely isolated SNc DA neurons from mice (Puopolo et al., 2007). One possible explanation is the work with acutely isolated neurons was performed at room temperature, some 10° C cooler than the experiments described here. To test this hypothesis, SNc DA neurons in slices were recorded at 23-25° C. At this temperature, pacemaking rate in cell-attached recordings was roughly half that seen near body temperature, in agreement with previous work (Guatteo et al., 2005). However, application of isradipine (5 μM) for 10-15 minutes failed to significantly slow the discharge rate at 25° C (control median = 0.67 Hz; +isradipine = 0.78 Hz, n=4, p>0.05; Fig. S1). Again, this wasn't because isradipine had failed to antagonize L-type channels, as the SOPs were absent when the membrane patch was disrupted to monitor transmembrane voltage (Fig. S1). One interpretation of these results is that the dendrites or axon initial segment (AIS), which are sheared off during acute isolation, contribute to pacemaking, rendering it sensitive to antagonism of L-type Ca2+ channels. Underscoring the importance of these regions to the pacemaking process, SNc DA neurons in tissue slices also were insensitive to TRP channel antagonists 2-APB (20 μM, n=4, p>0.05) and SKF 96365 (20 μM, n=4, p>0.05), in contrast to the situation reported in acutely isolated DA neurons (Kim et al., 2007).
The prevailing Ca2+ channel dependent model of pacemaking hypothesizes that dendritic and somatic Ca2+ oscillations are coupled, synchronizing the somatodendritic membrane potential and driving spike generation (Wilson and Callaway, 2000). A key feature of the coupled oscillator model is strong electrical coupling between somatic and dendritic compartments, leading to synchronization of the membrane potential. This means that oscillations in Ca2+ concentration should be phase-locked in dendritic and somatic compartments. To test these two aspects of the model, line scans assessing changes in Ca2+ concentration were performed at both proximal and distal dendritic regions of SNc DA neurons during pacemaking; averages of the Ca2+-induced fluorescence just before and after the spike were then calculated. In agreement with this aspect of the model, the proximal and distal dendritic Ca2+ signals were tightly phase locked in all neurons examined (n=4), rising with the depolarization that preceded the spike and peaking just after the spike (Fig. 4a). Computer simulations with a single compartment model of an SNc DA neuron containing low threshold Cav1.3 Ca2+ channels yielded a very similar relationship between intracellular Ca2+ concentration and membrane potential (Fig. 4b), arguing that the slow depolarization preceding the spike was driving Ca2+ channel opening throughout the dendritic tree.
Although the dendritic tree might be electrotonically compact for slowly changing signals, like those in the interspike period, there should be greater attenuation of more rapidly changing voltages unless there are active processes involved (Holmes and Rall, 1992). Previous work has shown that spikes originate in the axon initial segment (AIS) of SNc DA neurons and that, in the absence of pacemaking, these action potentials are actively propagated into distal dendritic regions (Hausser et al., 1995). However, action potential propagation is strongly influenced by even small changes in membrane potential (Gentet and Williams, 2007), making it uncertain whether there is back-propagation during pacemaking. If conducted into the dendrites, these spikes should reset dendritic oscillations that rely upon voltage dependent Ca2+ channels. To test this possibility, ectopic spikes were generated in the somatic region by a brief current pulse and the effects on somatic and dendritic oscillations monitored (Fig. 4c). In all of the neurons studied (n=4), ectopic spikes occurring in the first half of the pacemaking cycle phase delayed the next spike, suggesting that the pacemaking mechanism had been reset (Fig. 4d). In some cells (like the one illustrated in Fig. 4), the spike only propagated into the proximal dendritic regions as judged by the change in Ca2+-dependent fluorescence (Fig. 4e), suggesting that resetting the somatic component of the pacemaker was sufficient to reset the entire pacemaking process. These results are consistent with strong somatodendritic coupling posited in previous models, but argue that the Na+ channel-dependent spike itself is a key part of the pacemaking mechanism, not simply an epiphenomenon riding on top of the SOP, and that this proximally generated event determines pacemaking rate. This conclusion is consistent with the work presented above, showing that pacemaking continues unaltered in frequency or regularity in the absence of dendritic Ca2+ oscillations and the SOP.
In an attempt to gain a better conceptual foothold on the mechanisms controlling pacemaking, a computational model was created using NEURON. The model consisted of a single ‘somatic’ compartment; biophysically accurate models of channels thought to govern pacemaking were incorporated at densities resembling those seen experimentally (see Materials and Methods). The model generated regular, autonomous spiking at 1-2 Hz (Fig. 5a). As observed experimentally (Puopolo et al., 2007), the Cav1.3 Ca2+ current was significantly larger than the Nav1 Na+ current in the interspike interval (Fig. S2); however, both currents were significantly smaller than those generated by ‘leak’ channels, as in previous simulations (Amini et al., 1999). Reducing the Nav1 channel density to zero prevented spiking but did not stop sub-threshold oscillations, but subsequent block of Cav1.3 channels did, stabilizing the membrane potential near -40 mV.
Having verified that the basic behavior of the model resembled that seen experimentally, the interaction between Cav1.3 channels and other cationic channels in regulating pacemaking was explored. Eliminating Cav1.3 channels had little effect on pacemaking rate, in spite of clear effects on the voltage trajectory (Fig. 5b). Similarly, completely eliminating HCN channels had little effect on pacemaking rate. In both cases, the loss of inward current was compensated for by alterations in other conductances (Fig. S2). The combined removal of HCN and Cav1.3 channels, however, stopped spiking (Fig. 5b).
These results suggest that pacemaking is a robust, cooperative process, capable of withstanding perturbations in the availability of any one channel. To test this idea, recordings were made from SNc DA neurons and combinations of channels were antagonized pharmacologically. Selective block of HCN channels with saturating concentrations of ZD 7288 (10 μM), had little or no effect on pacemaking rate (Fig. 5c,d). However, as predicted by the simulations, blocking HCN channels rendered pacemaking sensitive to antagonism of L-type Ca2+ channels, as application of isradipine (5 μM) in the presence of ZD 7288 (10 μM) silenced every SNc DA neuron tested (n=5, Fig. 5c,d). It is worth noting that higher concentrations of ZD 7288 (50-100 μM) antagonized non-HCN channels (Zolles et al., 2006) and stopped pacemaking on their own (Fig. 5d).
Why do high concentrations of DHPs, like isradipine, reliably slow or stop pacemaking in SNc DA neurons (Nedergaard et al., 1993; Mercuri et al., 1994; Ping and Shepard, 1996; Chan et al., 2007)? The work presented thus far argues that significant slowing or silencing is not attributable to antagonism of Cav1.3 Ca2+ channels and must be due to reductions in other inward currents that help support pacemaking. There are three potential targets: Nav1 Na+, HCN and cation ‘leak’ channels. Point clamp experiments using slow, depolarizing voltage ramps revealed that 20 μM isradipine modestly, but consistently reduced peak Na+ currents in SNc DA neurons (median reduction = 27%, n=4, P<0.05). In contrast, isradipine did not reduce the amplitude of the voltage sag produced by hyperpolarizing current steps (median reduction = 0.04%, n=6, P>0.05). However, input conductance measured by small hyperpolarizing steps from -60 mV was significantly reduced by isradipine, suggesting that cationic leak channels were being partially blocked (median reduction = 31%, n=9, P<0.05). Reducing Na+ and leak channel density together by 20% consistently silenced pacemaking in the model, making this explanation plausible (Fig. 6a).
With sustained application of silencing concentrations of isradipine, pacemaking restarts after several hours (Chan et al., 2007). It could be restarted immediately by application of Kv1 channel antagonists (Fig. 6b,c), suggesting the delayed restarting seen previously depends upon functional down-regulation of these channels. The model captured this recovery, as the silencing produced by partial block of Na+ and leak channels was reversed by block of Kv1 channels (Fig. 6a). More importantly, this result reinforces the proposition that pacemaking reflects the interaction of a network of ion channels that is capable of withstanding perturbations in individual components.
Our principal conclusion is that although L-type Ca2+ channels contribute to the oscillatory behavior of SNc DA neurons, they are not necessary for autonomous pacemaking (Nedergaard et al., 1993; Mercuri et al., 1994; Wilson and Callaway, 2000; Puopolo et al., 2007). Our work shows that there is an interactive network of ion channels in SNc DA neurons that is capable of sustaining pacemaking even when parts of it, like L-type Ca2+ channels, are antagonized. This finding adds another dimension to an array of other observations showing that the rate of autonomous spiking in SNc DA neurons is insensitive to a variety of extrinsic and intrinsic perturbations (Paladini et al., 2003; Chan et al., 2007) – a testament to the importance of pacemaking in the maintenance of DA in target structures involved in movement, learning and cognition.
The view that somatodendritic L-type Ca2+ channels contribute to pacemaking in SNc DA neurons has a long history. Initially, it was found that in the presence of the Na+ channel blocker TTX the membrane potential of SNc DA neurons continued to oscillate at near the pacemaking frequency (Yung et al., 1991; Nedergaard et al., 1993). This SOP (slow oscillatory potential) was sensitive to blockade of Ca2+ channels and, more importantly, to DHPs that antagonize L-type Ca2+ channels (Nedergaard et al., 1993; Mercuri et al., 1994). The hyperpolarizing phase of this oscillation is brought about by activation of Ca2+-activated SK K+ channels (Ping and Shepard, 1996). The ability of DHPs to suppress the SOPs has been repeated by several groups (e.g.,Nedergaard et al., 1993; Chan et al., 2007). It was natural to infer from the temporal similarity of the oscillatory potential and pacemaking that the two processes were causally linked. This inference was supported by the ability of DHPs to slow or stop the regular spiking of SNc DA neurons (Nedergaard et al., 1993; Mercuri et al., 1994). However, in previous experiments silencing typically required DHP concentrations in the low micromolar range; several orders of magnitude above the KD of DHPs for the Cav1.3 L-type Ca2+ channels that drive the SOP.
Our work argues that the ability of DHPs at these high concentrations to slow or stop pacemaking has little to do with L-type Ca2+ channels. There are five key pieces of experimental evidence on this point. First, Ca2+ imaging studies showed that intracellular fluctuations in Ca2+ concentration attributable to opening of L-type Ca2+ channels were significantly attenuated or eliminated by application of the DHP isradipine at concentrations that did not alter pacemaking rate. Second, concentrations of isradipine that blocked SOPs, did not alter pacemaking rate. Third, SOPs were less regular and often slower than pacemaking. Fourth, chelation of intracellular Ca2+ did not stop or slow pacemaking, but rather accelerated it. Lastly, the ability of an ectopic somatic spike to reset the Ca2+ oscillation throughout the dendritic tree, even when there was no evidence that the spike invaded the distal dendrites, argues that Na+-dependent spikes originating in the AIS (Hausser et al., 1995) pace the somatodendritic oscillations, determining their rate and regularity, not the other way around.
Simulations that incorporated realistic descriptions of the ion channels known to participate in pacemaking provided an explanation for much of the experimental data. Not only did the model generate autonomous spiking of the right rate and regularity, it also produced SOPs when Na+ channels were blocked that strongly resembled those seen experimentally. The model also sustained pacemaking in the absence of L-type Ca2+ channels, showing how the compensatory changes in other conductances, served to maintain the discharge rate. Thus, the channels expressed by SNc DA neurons form an interactive network that is capable of maintaining pacemaking in the face of perturbations in the channel availability. This level of robustness should provide a means of withstanding fluctuations in channel availability as a consequence of neuromodulatory challenge or synaptically driven activity.
That said, the robustness of the model and real SNc DA neurons was limited. Combined antagonism of Cav1.3 and HCN channels stopped pacemaking, although antagonism of either alone had little or no effect – just as observed experimentally. The sensitivity of SNc DA neurons to the combined disruption of a mixed population of ion channels helps explain the effects of high DHP concentrations on pacemaking. At DHP concentrations that consistently induced silencing, isradipine reduced not only L-type Ca2+ channel currents, but Na+ channel currents and ‘leak’ channel currents as well. Simulations showed how this combined antagonism could account for cessation of pacemaking. This also means that experimental interventions that compromise the pacemaking mechanism could lead to a spurious elevation in the sensitivity to DHPs. Our assessment of DHP sensitivity relied primarily on cell-attached patch (cap) recordings that did not alter the intracellular environment the way whole cell or sharp electrode recordings can. In cap recordings, there was not a graded slowing of pacemaking with increasing DHP concentration; rather, at a well-defined time point after bath application, cells either continued at a normal rate, became irregular, or they stopped entirely (as also described by Nedergaard et al., 1993). In our hands, this transition occurred at DHP concentrations where other channels besides L-type Ca2+ channels were being blocked. This was also true in our simulations. As a consequence, the slowing of pacemaking at lower DHP concentrations described by Mercuri et al. (1994) or anecdotally by Nedergaard et al., (1993) in brain slices can only be explained (within our framework) by positing that either 1) their sharp electrode recordings disrupted the capacity of these collateral ionic mechanisms to compensate for the loss of L-type channels or 2) because of lipid phase partitioning (DHP partition coefficients >1000 (Herbette et al., 1989)) and extended drug exposure, DHP concentration in the membrane had risen to levels where non-L-type channels were being affected, in spite of the fact bath concentrations were below 10 μM. The difficulty of this pharmacokinetic problem was one of the primary motivations for our use of Ca2+ imaging to monitor fluctuations in dendritic Ca2+ concentration during drug application, giving us a much more direct way of monitoring effective membrane concentrations of bath applied DHPs. Although potentially an issue in brain slices, pharmacokinetics isn't a major concern with acutely isolated neurons. But, there are other issues. In their biophysical study, Puopolo et al. (2007) reported that low micromolar, selective concentrations of nimodipine had variable effects on pacemaking of acutely isolated DA neurons recorded with whole cell methods – sometimes slowing or stopping pacemaking, sometimes having no effect. This variability is most readily attributed to the one variable that is poorly controlled with this approach – the degree to which dendritic and axonal arbors are truncated. Although not necessary, these are regions that very likely contribute in a material way to the robustness of pacemaking (Tepper et al., 1987; Grace and Onn, 1989; Hausser et al., 1995; Amini et al., 1999; Wilson and Callaway, 2000).
The robustness of pacemaking in SNc DA neurons has implications for neuroprotective therapies. Ca2+ entry through L-type Ca2+ channels renders SNc DA neurons vulnerable to a range of toxins used to create animal models of PD (Chan et al., 2007). Moreover, DHP use in humans is associated with a decreased incidence of PD (Becker et al., 2008). Isradipine is now being tested in clinical trials for its ability to modify PD progression. At the doses to be used in this study, isradipine achieves stable serum concentrations of ~2 ng/ml, which is close to the serum concentration that is neuroprotective in animal models of PD (unpublished observations). If we assume that brain and serum concentrations are similar (Kupsch et al., 1996), isradipine should be roughly 5 nM in the extracellular space bathing SNc DA neurons, which should antagonize between 70-80% of their Cav1.3 Ca2+ channels, assuming a modal membrane potential of -50 mV (Bean, 1984; Sinnegger-Brauns et al., 2009). Our results show that the neuroprotection afforded by this antagonism should not come at the cost of a reduction in basal pacemaking rate in SNc DA neurons.
grants from the Hartman Foundation, Falk Trust, USAMRMC (W81 XWH-071-0170) and NIH (NS047085) supported this work. We would like to thank Karen Saporito, Sasha Ulrich, David Wokosin, and Ema Ilijic for their technical support and scientific contributions.