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A Reconfiguration of Ca_V2 Ca²⁺ Channel Current and Its Dopaminergic D₂ Modulation in Developing Neostriatal Neurons

Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

Submitted 3 May 2005; accepted in final form 17 August 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The modulatory effect of D₂ dopamine receptor activation on calcium currents was studied in neostriatal projection neurons at two stages of rat development: postnatal day (PD)14 and PD40. D₂-class receptor agonists reduced whole cell calcium currents by about 35% at both stages, and this effect was blocked by the D₂ receptor antagonist sulpiride. Nitrendipine partially occluded this modulation at both stages, indicating that modulation of Ca_V1 channels was present throughout this developmental interval. Nevertheless, modulation of Ca_V1 channels was significantly larger in PD40 neurons. -Conotoxin GVIA occluded most of the Ca²⁺ current modulation in PD14 neurons. However, this occlusion was greatly decreased in PD40 neurons. -Agatoxin TK occluded a great part of the modulation in PD40 neurons but had a negligible effect in PD14 neurons. The data indicate that dopaminergic D₂-mediated modulation undergoes a change in target during development: from Ca_V2.2 to Ca_V2.1 Ca²⁺ channels. This change occurred while Ca_V2.2 channels were being down-regulated and Ca_V2.1 channels were being up-regulated. Presynaptic modulation mediated by D₂ receptors reflected these changes; Ca_V2.2 type channels were used for release in young animals but very little in mature animals, suggesting that changes took place simultaneously at the somatodendritic and the synaptic membranes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Neostriatal projection neurons express a diverse array of voltage-gated Ca²⁺ channels (Bargas et al. 1994). The different types of Ca²⁺ channels exhibit striking dissimilar roles during repetitive firing and transmitter release (Perez-Garci et al. 2003; Tecuapetla et al. 2005; Vilchis et al. 2000). For example, L-type Ca²⁺ (Ca_V1) channels activate near spike threshold and contribute to set the range of frequencies for evoked discharge—the dynamic range (Hernandez-Lopez et al. 1997, 2000; Perez-Garci et al. 2003). By this token, dopaminergic D₁ receptor activation facilitates neuronal excitability by enhancing Ca²⁺ current through L-type Ca²⁺ channels (Hernandez-Lopez et al. 1997; Surmeier et al. 1995). Activation of dopaminergic D₂ receptors reduces L-type Ca²⁺ currents, leading to a decrease in firing (Hernandez-Lopez et al. 2000; Olson et al. 2005). These actions partially explain the so-called facilitatory and repressing actions of dopamine on striatal output at the molecular level (Prescott et al. 2003).

However, not only Ca_V1 Ca²⁺ channels but also Ca_V2 Ca²⁺ channels may be controlled by dopamine. Consequently, this study asked if D₂ receptor activation also regulates Ca_V2 Ca²⁺ channels in striatal neurons. The dopaminergic modulation of Ca_V2 Ca²⁺ channels may be important given the roles of these channels in spiny cells (Perez-Garci et al. 2003; Tecuapetla et al. 2005; Vilchis et al. 2000). Moreover, because the contribution of diverse Ca_V2 Ca²⁺ channels may change during development (Chameau et al. 1999), and because medium spiny neurons have a protracted development (Tepper et al. 1998) with respect to other neurons, it is important to see what happens with dopaminergic modulation during possible channel reconfigurations.

The relevance of this question becomes evident when recalling that Ca_V2 channels supply the Ca²⁺ necessary to activate Ca²⁺-dependent K⁺ channels (Vilchis et al. 2000) in mature neurons. In turn, these K⁺ channels generate the postspike afterhyperpolarization (AHP) that makes up the interspike interval. The AHP controls the firing of spiny cells by setting the gain for a given stimulus (Bargas et al. 1999; Perez-Garci et al. 2003; Pineda et al. 1992; Vilchis et al. 2000). In addition, Ca_V2 channels are in charge of GABA release at the synaptic terminals of spiny neurons (Tecuapetla et al. 2005). In this place too, there is evidence that dopamine exerts a presynaptic modulation of GABA release (Guzman et al. 2003). Thus theoretically, dopamine could control neostriatal output at two different levels through the regulation of Ca_V2 Ca²⁺ channels: first, at the somatodendritic membrane through the regulation of the firing mechanism, and second, at the synaptic terminals (Cooper and Stanford 2001; Guzman et al. 2003) through the regulation of transmitter release. Accordingly, the main objective of this study was to find evidence of this dual regulation. In relation to this, it was recently shown that activation of muscarinic M₁ receptors controls both the interspike interval and the transmitter release at the synaptic terminals of spiny neurons (Perez-Rosello et al. 2005). Because muscarinic M₁ receptors and dopaminergic D₂ receptors seem to be linked to the same signaling cascade (Hernandez-Lopez et al. 2000; Rakhilin et al. 2004), it seems interesting to ask if these receptors share some physiological actions in neostriatal cells, and if not, why. This work has been published in abstract form (Salgado et al. 2004).

	METHODS

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Preparation of slices

The protocols followed the National University of Mexico and National Institutes of Health guidelines for the use of animals in biomedical experiments. Briefly, and as described elsewhere (Bargas et al. 1999), male Wistar rats of postnatal days 14 (PD14) and 40 (PD40), from our animal house, were deeply anesthetized, and their brains were quickly removed into ice-cold saline (4°C) containing (in mM) 126 NaCl, 3 KCl, 1 MgCl₂, 2 CaCl₂, 25 NaHCO₃, and 11 glucose (pH 7.4 with NaOH, 298 mOsm/l with glucose; aerated with 95% O₂-5% CO₂). Parasagittal neostriatal slices (300 µm thick) were cut in 4°C saline using a vibratome (Ted Pella, Reading, CA). Slices were transferred to room temperature saline (23–25°C) and left to recover for 1 h.

Voltage-clamp recordings of calcium currents

Neostriatal neurons from PD14 or PD40 rats were acutely dissociated using procedures similar to those previously described (Bargas et al. 1999). Briefly, after a 1- to 6-h incubation, slices were removed into HEPES-buffered saline, and the dorsal striatum was dissected. Neostriatal slices were placed in the same HEPES solution, now containing 1 mg/ml of pronase E type XIV (Sigma, St. Louis, MO) at 32°C. After about 20 min, the slices were removed into a low-Ca²⁺ HEPES-buffered saline. Slices were rinsed and dissociated mechanically with a graded series of fire-polished Pasteur pipettes. The cell suspension (2 ml) was plated into a 35-mm Lux petri dish mounted on the stage of an inverted microscope containing 1 ml of the following recording saline (in mM): 0.001 TTX, 140 NaCl, 3 KCl, 5 BaCl₂, 2 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4 with NaOH; 298 mOsm/l with glucose). After allowing the cells to settle, superfusion began at about 1 ml/min with saline of the same composition. Recordings were made only from medium-sized neurons (10–12 µm soma diam). Whole cell currents recordings used standard techniques. Electrodes were pulled from borosilicate glass (WPI, Sarasota, FL) in a Flaming-Brown puller (Sutter Instrument Corp., Novato, CA) and fire polished before use. The internal saline contained (in mM) 180 N-methyl-D-glucamine (NMDG), 40 HEPES, 4 MgCl₂, 10 EGTA, 2 Na₂ATP, 0.2 Na₃GTP, and 0.1 leupeptin (pH = 7.2 with H₂SO₄, 265–270 mOsm/l). Electrode DC resistances were 4–8 M in the bath. Recordings were obtained with an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA), and controlled and monitored with a PC clone running pClamp (version 5) with a 125-kHz DMA interface (Axon Instruments). After seal rupture, the series resistance (<15 M) was compensated (70–80%) and monitored before and after drug application. Current-voltage relationships (I-V plots) before and during drug applications were built from currents evoked with both 20-ms step voltage commands, from –80 to 50 mV in 10-mV steps, and with responses to voltage ramp commands (0.7 mV/ms) from –80 to 50 mV. Because the results from both methods coincided (see Fig. 1), most figures only show representative I-V plots built from the responses to voltage ramps.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Ca2+ current density does not change between PD14 and PD40 developmental stages. A: representative family of currents evoked by 20-ms step voltage commands from –80 to 50 mV, in 10-mV steps, in a PD14 cell. B: family of currents obtained with the same protocol in a PD40 cell. C: current evoked with a depolarizing ramp command from –80 to 50 mV and 180-ms duration (0.7-mV/ms depolarizing rate) in a PD14 cell (same cell as in A). D: similar current response evoked with the same ramp protocol in a PD40 cell (same cell as in B). E: current-voltage relationship of the PD14 cell (I-V plot) built with measurements from both steps (filled circles) and ramp (continuous line) evoked responses. There is close agreement between both measurements. For clarity, next figures only show I-V plots built after ramp commands. F: I-V plot of a PD40 cell built as in E. G: histogram comparing averaged current amplitudes (means ± SE) of PD14 and PD40 neurons. H: current was divided by cell capacitance to report a quantity proportional to current density. Histogram shows that current density does not change between these stages (PD14 and PD40). Currents were carried by 5 mM Ba2+. All records were from medium-sized neostriatal neurons.

View larger version (26K): [in this window] [in a new window]   FIG. 8. Action of dopaminergic D2-class receptors on Ca2+ currents are mediated by a voltage-independent, Ca2+-dependent, diffusible pathway. A: D2-class receptor agonists reduce Ca2+ current in the presence of 1 mM EGTA. B: D2-class receptor agonists reduce Ca2+ current in the presence of 10 mM EGTA. Amount of modulated current did not change when A and B were compared. C: action of D2-class receptor agonists on Ca2+ current were virtually abolished in the presence of 15 mM BAPTA; suggesting that these actions are mediated by a Ca2+-dependent diffusible pathway. D: histogram summarizing these results. Differences were significant only when comparing EGTA and BAPTA samples (P < 0.001; ANOVA with post hoc Tukey tests). E: double pulse protocol to 0 mV. Note that the 2nd pulse is preceded by a depolarizing command to 80 mV. F: current evoked after the depolarizing commands to 0 mV before and after the depolarizing prepulse to 80 mV. Note that both control (black traces) and modulated current (gray trace; quinelorane) were enhanced in the same proportion, indicating that there is a constitutive voltage-dependent modulation but that quinelorane does not change it significantly in PD14 neurons. G: similar results were obtained in PD40 neurons. H and I: histograms summarizing samples of experiments (n = 6) as in F and G, respectively.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Activation of dopaminergic D2 receptors reversibly modulates Ca2+ currents in both young and adult neurons. A: time-course showing that 2 successive applications of the dopaminergic D2 receptor agonist quinelorane (10 µM) reduced whole cell Ca2+ current (gray symbols). It is also shown that these current reductions were reversible. Bars indicate time of drug application and wash off. Stability of current amplitude when no drug was applied is also shown (empty symbols; measurements taken from another neuron). B: representative records of experiment in A. Note that both steady-state amplitude and tail current amplitude were decreased by the D2 agonist. Numbers correspond to labels in A. C: box plot showing distribution of percentage of run down found in Ca2+ current when no drug was applied (n = 15). PD40 neuron. Similar results were obtained in PD14 neurons.

Neostriatal slices from juvenile (PD14) or mature young rats (PD40) were transferred to a custom plexiglass recording chamber and superfused with oxygenated saline (3–6 ml/min) as above. Individual neurons were visualized (x40 water immersion objective) under differential interference contrast (DIC) enhanced visual guidance using infrared videomicroscopy in an upright microscope (Diaphot, Nikon, Melville, NY) adapted with a CCD camera (CCD-100, Dage-MTI, Michigan City, IN). Synaptic events were evoked with a bipolar concentric tungsten electrode (12 µm at the "pencil shaped" tip; FHC, Bowdoinham, ME) located at the globus pallidus to stimulate antidromically the axons of spiny cells (Guzman et al. 2003; Tecuapetla et al. 2005). Paired shock stimulation (45–50 ms of interstimulus interval; 0.2- to 0.4-ms duration; 1- to 4-V strength; frequency of 0.1 Hz) was delivered with a computer interface. Isolation units (Digitimer, Hertfordshire, UK) between the computer and the stimulating electrodes were used to adjust stimulus parameters during the experiment. Distance between recording and stimulating electrode was about 1 mm. Synaptic responses in these conditions were of moderate amplitude, but some experiments were performed with either strong or weak stimulation strength to check if D₂-class receptor actions were present. Thus evoked inhibitory postsynaptic currents (eIPSCs) had amplitude variation but without exhibiting failures except at the weaker stimulation strengths. Traces shown are the average of 4-min recordings (24 traces) taken once the amplitude had been stabilized in a given condition. A hyperpolarizing voltage command (10 mV) continuously monitored input conductance. Internal solution was (in mM) 72 KH₂PO₄, 36 KCl, 2 Mg Cl₂, 10 HEPES, 1.1 EGTA, 2 Na₂ATP, 0.2 Na₃GTP, 5 QX 314 (to prevent neuronal firing and enhance input resistance), and 0.5% biocytin (pH = 7.2, 275 mOsM/l). Bath solution contained 10 µM 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline disodium salt (CNQX) and 50 µM D-(–)-2-amino-5 phosphonovaleric acid (AP5) to block glutamatergic currents so that synaptic responses were eIPSC sensitive to bicuculline. Cells with resting potential more negative than –70 mV (at 0 current), input resistance about 100–200 M, and holding current (in voltage clamp mode) 0.02 nA to maintain a holding potential near the resting potential of the cell were chosen. Whole cell recordings were made using an Axoclamp 2B amplifier (Axon Instruments). Whole cell access resistances were in the range of 5–20 M. Access resistance was continuously monitored, and experiments were abandoned if changes >20% were encountered. No cell capacitance, series resistance, or liquid junction potential (2 mV) compensations were made. All recordings were filtered at 1–3 KHz and digitized with an AT-MIO-6040E interface and a DAQ (NI-DAQ) board (National Instruments, Austin, TX) in a PC clone. On-line data acquisition used custom programs made by L. Carrillo in the LabVIEW environment (National Instruments). The NI-DAQ board was used to save the data on binary files in the computer hard disk for further off-line analysis.

eIPSCs obtained with different simulation strengths (weak to strong; see intensity-amplitude plots in Tecuapetla et al. 2005) and different ages were chosen to have an array of different amplitudes for eIPSCs. These were used to perform a mean-variance analysis as described in Clements and Silver (2000).

Intracellular recordings

Slices obtained as above (see Preparation of slices), from PD40 rats, were also recorded in a submerged chamber and superfused with the same saline at 1 ml/min (34–36°C). Intracellular recordings were performed with sharp microelectrodes filled with 3 M K-acetate (DC resistances: 80–120 M) and the help of an active bridge electrometer (Neuro Data, Cygnus Tech, DWG) (Pineda et al. 1992). Records were digitized and saved on VHS tapes (40 KHz) and analyzed off-line in a PC clone. Stimulation consisted of intracellular injections of constant current steps to evoke the AHP after a single action potential (Pineda et al. 1992). Stimulus were of suprathreshold intensity and given at a holding potential of –55 to –60 mV by adjusting constant current. Bridge balance as well as recovery periods (without DC current) were monitored between sample records. After recording, some neurons were injected with biocytin as previously described (Galarraga et al. 1999). All neurons identified in this study were medium-sized spiny projection neurons.

Drugs used

Drugs were dissolved in the bath saline from daily made stock solutions using a gravity-driven superfusion system. Equilibrated concentrations of the drugs were achieved in 4–5 min. L--amino-3-hydroxy-5-methyl-isoxazolepropionate and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine/kainate (AMPA/KA) antagonist, CNQX (10 µM), N-methyl-D-aspartate (NMDA) antagonist, AP5 (50 µM), nitrendipine, bicuculline, QX-314, TTX, sulpiride, quinelorane, and quinpirole were all purchased from Sigma-RBI (St. Louis, MO). Calcium channels antagonists -conotoxin GVIA (-CgTx) and -agatoxin TK (-AgaTK) were obtained from both Peptides international (Louisville, KY) and Alomone Labs (Jerusalem, Israel). Almost all drugs were dissolved in water to get stock solutions and added to the superfusate to reach the final concentration. Nitrendipine was disolved in dimethylsulfoxide (DMSO; 0.01%).

Data analysis

Digitized data were imported for analysis and graphing into commercial software (origin v.6. Microcal, Northampton, MA). Mean ± SE of all I_Ca²⁺ and IPSCs are reported. Free-distribution statistical tests were used to assess statistical significance; Mann-Whitney U test or Wilcoxon's t-test (depending on nonpaired or paired samples) and one-way ANOVA with post hoc Tukey's test were used to assess significance between multiple samples in which quinelorane modulation was tested in the presence or the absence of Ca²⁺ channel blockers (for details see Vilchis et al. 2000).

To approximate the contribution for each type of Ca²⁺ channel, we took the amount of Ca²⁺ current blocked by a given antagonist (nitrendipine for L-type; -AgaTK for P/Q-type; and -CgTx for N-type) as the contribution of a given channel type to the whole cell calcium current. Current through R-type Ca²⁺ channels was calculated as the amount of current left after all three blockers were added together. The sum was normalized to 100% so that L + N + P/Q + R = 100%. To determine the amount of quinelorane modulation for each type of Ca²⁺ channel, we compared quinelorane modulation on Ca²⁺ currents in the absence and presence of the different Ca²⁺ channels blockers and introduced the data into a system of linear equations (for details, see Vilchis et al. 2002).

	RESULTS

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Ca²⁺ currents expressed in dissociated neostriatal neurons at two different developmental stages

Figure 1A shows a family of Ca²⁺currents obtained in response to depolarizing step voltage commands (see METHODS) from a PD14 neuron. Ba²⁺ was the charge carrier. Figure 1B shows a similar experiment done in a PD40 neuron. Figure 1C shows the Ca²⁺ current evoked in the same PD14 neuron (Fig. 1A) after a depolarizing ramp command from –80 to 50 mV. A similar experiment is shown in Fig. 1D for the PD40 neuron. Figure 1, E and F, shows the current-voltage relationships (I-V plots) obtained from the experiments depicted in Fig. 1, A–D. Filled circles are measurements from the currents depicted in Fig. 1, A and B, measured at the end of the voltage step. Continuous traces are taken from currents depicted in Fig. 1, C and D, plotted as a function of ramp voltage. Note that ramp-evoked I-V plots look as the "fits" of the I-V plots obtained with step commands; indicating a virtual agreement between both recording methods. For the sake of clarity, most figures will show I-V plots obtained with ramp commands only. However, most experiments were done with both protocols. Histogram in Fig. 1G compares mean current amplitudes at both developmental stages. In PD14 neurons, peak current amplitude was 234 ± 14 (SE) pA (n = 49), whereas it was 254 ± 12 pA in PD40 neurons [n = 54; not significant (NS); Mann-Whitney U test]. Because current density could change during development, this parameter was compared in Fig. 1H by dividing ionic current over whole cell capacitance (C_N). C_N in PD14 cells was 7.3 ± 0.2 pF, whereas it was 7.5 ± 0.3 pF in PD40 cells (NS; Mann-Whitney U test), indicating that somatic surface does not change between these developmental stages. Accordingly, normalized currents were 32 ± 2.2 pA/pF in PD14 and 34 ± 1.4 pA/pF in PD40 cells (Fig. 1H; NS; Mann-Whitney U test). These measurements made us confident that the total number of Ca²⁺ channels does not increase between these two developmental stages. Experiments to document possible changes in kinetics or voltage dependence were not done in this work (cf., Fig. 1, A and B). Instead we focused on the channel types that contribute to the whole cell current, because, even if channel numbers do not increase, the channel types that compose the current may change. To test this expectation, a pharmacological dissection of Ca²⁺ currents at both stages, PD14 and PD40, was done with the help of known Ca²⁺ channel antagonists.

Figure 2A shows that, in PD14 neurons, 10 µM nitrendipine blocked 29 ± 3% (n = 15) of the Ca²⁺ current, whereas in PD40 neurons, the percentage of Ca²⁺ current blocked by dihydropyridine was 23 ± 2% (n = 24; this difference was not statistically significant; Mann Whitney U test; Fig. 2C). Therefore it was concluded that L-type Ca²⁺ channels do not change their numbers between these developmental stages. However, further study is needed to see if the subtypes of L-type channels (_1D and _1C) do change (Olson et al. 2005).

View larger version (35K): [in this window] [in a new window]   FIG. 2. CaV2.1 (P/Q-type) Ca2+ channels increase, whereas CaV2.2 (N-type) Ca2+ channels decrease in neostriatal neurons between the PD14 and the PD40 developmental stages. A and B: sensitivity of Ca2+ current to dihydropyridine (10 µM nitrendipine) was not significantly different when comparing PD14 and PD40 cells. C: histogram summarizing results in 2 neuronal samples. D and E: sensitivity of Ca2+ current to -agatoxin TK (-AgaTK; 400 nM) is significantly different when comparing PD14 and PD40 cells. More current is blocked by -AgaTK in PD40 neurons. F: histogram summarizing results in 2 neuronal samples (P < 0.0001). G and H: sensitivity of Ca2+ current to -conotoxin GVIA (-CgTx; 1 µM) is significantly different when comparing PD14 and PD40 cells. More current is blocked by -CgTx in PD14 neurons. I: histogram summarizing results in 2 neuronal samples (P < 0.002). J and K: amount of Ca2+ current left: CaV2.3 or R-type current, after all 3 blockers (nitrendipine, -AgaTK, and -CgTx) were administered together to PD14 (J) and PD40 (K) neurons, was not significantly different. L: histogram summarizing results in 2 neuronal samples.

Equivalent experiments were done using the Ca_V2.2 (N-type) channel blocker, -CgTx (1 µM). This peptide reduced Ca²⁺ current in PD14 cells by 43 ± 2% (n = 11; Fig. 2G), whereas it reduced Ca²⁺ current in PD40 cells by 28 ± 2% (n = 8; Fig. 2H). This difference was statistically significant (Fig. 2I; P < 0.002; Mann-Whitney U test), indicating that N-type channels decrease their numbers and their relative contribution to the whole cell Ca²⁺ current during the PD14 to PD40 developmental window. Comparable findings have been reported in cultured pyramidal neurons (Chameau et al. 1999).

Finally, to isolate the Ca_V2.3 (R-type) current, we added the three blockers together (nitrendipine, -AgaTK, and -CgTx) at the above concentrations and measured the current that was left. Less than 15% of the initial current was left in both PD14 and PD40 cells (Fig. 2, J–L), indicating that, as L-type channels, R-type channels do not change their numbers during this developmental period (Bargas et al. 1994; Foehring et al. 2000).

In PD14 cells, the percentage that each current type contributes to the whole cell current was (see METHODS and Vilchis et al. 2002) as follows: L-type, 27%; P/Q-type, 22%; N-type, 39%; R-type, 12% (Fig. 3A). In PD40 cells, the percentages were as follows: L-type, 22%; P/Q-type, 38%; N-type, 26%; R-type, 14% (Fig. 3B). Therefore the results show that expression of Ca_V2.2 (N-type) channels diminishes, whereas that of Ca_V2.1 (P/Q-type) channels augments during this developmental interval. Because one of the main functional roles of N- and P/Q-type Ca²⁺ channels in spiny neurons is the triggering of transmitter release (Tecuapetla et al. 2005), we chose to start searching into the functional significance of these changes by hypothesizing that GABA released from the terminals of mature spiny neurons should be mainly triggered by P/Q-type Ca²⁺ channels.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Contribution of different Ca2+ channel types to the whole cell Ca2+ current. CaV2.2 channels decline during development, whereas CaV2.1 channels increase with age. Other channel types remain constant during development.

To test the above hypothesis, we observed eIPSCs elicited by the activation of axon collaterals that interconnect spiny neurons. These were obtained by electrically stimulating the globus pallidus to antidromically activate striofugal axons and, therefore axon collaterals that stay inside the neostriatum and interconnect spiny neurons (Guzman et al. 2003). At the PD14 stage, both N- and P/Q-types Ca²⁺ channels trigger GABA release from these terminals (Tecuapetla et al. 2005) (Fig. 4, A and D): -CgTx and -AgaTK reduced the amplitude of antidromically eIPSCs by 65 ± 7 (n = 10) and 90 ± 5% (n = 8), respectively (Tecuapetla et al. 2005)—both peptides were used at saturating concentrations (1 µM and 400 nM, respectively).

View larger version (34K): [in this window] [in a new window]   FIG. 4. -CgTx sensitivity of synaptic terminals declines with development. A: 1 µM -CgTx greatly reduced (65%) inhibitory postsynaptic potentials (IPSCs) in PD14 neurons. Note transition from paired pulse depression to paired pulse facilitation during -CgTx action (inset). B: IPSC sensitivity to -CgTx was greatly reduced in PD40 neurons (18%). In the case shown, -CgTx had no effect. Note paired pulse depression in the control (inset). C: histogram summarizes effect of -CgTx in 2 neuronal samples (P < 0.009). D and E: sensitivity of IPSCs to -AgaTK (400 nM) was strong at both developmental stages. Blockage of P/Q-type channels enhanced paired pulse ratio at both stages. Also note that the same concentration of -AgaTK had a faster action in PD40 neurons. F: histogram summarizes effect of -AgaTK in 2 neuronal samples (NS). IPSCs were evoked by electrically stimulating the globus pallidus to antidromically activate striofugal axons and therefore axon collaterals that interconnect spiny neurons (Guzman et al. 2003); 10 µM cyano-2,3-dihydroxy-7-nitro-quinoxaline disodium salt (CNQX) and 50 µM D-(–)-2-amino-5 phosphonovaleric acid (AP5) were present in the bath saline during the experiment. Antidromically evoked IPSCs were completely blocked by 10 µM bicuculline (data not shown, but see Tecuapetla et al. 2005).

Dopaminergic D₂ receptor modulation of Ca²⁺ currents in young and mature neurons

An important characteristic of medium spiny neurons is their having D₂ dopaminergic receptors whose activation modulates Ca²⁺ currents. However, only the modulation of Ca_V1 Ca²⁺ channels has been extensively studied (Hernandez-Lopez et al. 2000; Olson et al. 2005; Rakhilin et al. 2004). Here we report that modulation of Ca_V2 Ca²⁺ channels is also very important. Figure 5 confirms the D₂ modulation of Ca²⁺ current in neostriatal cells, showing that this modulation is reversible during the time course of a typical experiment. This modulation reduces the slower tail currents recorded after a step command (Fig. 5B, inset), finding that has been taken as evidence of L-type current modulation (Olson et al. 2005; Rakhilin et al. 2004). D₂ modulation of Ca²⁺ current was observed in 80% of the recorded dissociated neurons at each developmental stage.

Figure 6, A–C, shows that the D₂ modulation of Ca²⁺ current (10 µM quinelorane) is present in both juvenile (PD14) and mature (PD40) neurons. This modulation comprises about the same amount of current reduction at both developmental stages (Fig. 6C; NS). Quinelorane acted in a concentration-dependent fashion (data not shown) at both stages. Thus 10 µM quinelorane reduced the current in PD14 by 35 ± 2% (n = 12) of neurons and by 36 ± 3% (n = 12) in PD40 neurons (NS; Mann-Whitney U and ANOVA tests). All the modulation produced by quinelorane and other D₂ receptor agonists (e.g., quinpirole) was virtually blocked by 1 µM of the unselective D₂ receptor class antagonist, sulpiride (1 µM), at both stages (n = 12; data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 6. Amount of D2 receptor modulation of Ca2+ current was the same in young and mature neurons. A and B: amount of Ca2+ current reduction by D2 receptor agonists (10 µM quinelorane in this case) is about the same in juvenile (PD14: A) and mature (PD40: B) neurons. C: histogram summarizes results from juvenile and mature neuronal samples (NS). D and E: amount of IPSC reduction by D2 receptor agonists (1 µM quinelorane in this case) is not significantly different in juvenile (PD14) and mature (PD40) synaptic terminals. Note that the paired pulse ratio (Guzman et al. 2003) was enhanced by quinelorane in both cases. F: histogram summarizes results from juvenile and mature samples (NS). IPSCs were evoked as in Fig. 4.

In juvenile animals (PD14), the mean paired pulse ratio (PPR) of eIPSCs after a pair of stimulus (Fig. 6D, inset) in the control condition was 1.3 ± 0.16 and in the presence of D₂ receptor agonists was 1.9 ± 0.26 (n = 12; P < 0.002; Wilcoxon's t-test). In mature animals (PD40; Fig. 6E, inset), control PPR was 0.82 ± 0.1 and in the presence of D₂ receptor agonists it was 0.97 ± 0.08 (n = 6; P < 0.05; Wilcoxon's t-test). Because the PPR value is linearly proportional to the probability of release, the paired pulse stimulation has been an accepted protocol to search for presynaptic actions (e.g., Baldelli et al. 2005). Thus the modulation is suggested to be presynaptic in both juvenile and mature animals (Guzman et al. 2003). However, to further support this inference, a mean-variance analysis was performed (Clements and Silver 2000) and accompanied with an independent analysis of amplitude histograms.

Figure 7A shows an amplitude histogram of spontaneous IPSCs (sIPSCs) recorded in a neostriatal neuron (Fig. 7B). These events may come from interneurons or spiny cell terminals and fitted to Gaussian functions with a first modal amplitude of 8 pA (range, 5–12 pA), similar to that reported for other GABAergic synapses (e.g., Ling and Benardo 1999). The second mode was 16 pA (range, 12–20 pA) (cf., Paulsen and Heggelund 1994). Thereafter, some eIPSCs were recorded after applying quasi-minimal stimulation strength. The amplitudes histogram of these eIPSCs was multimodal (Fig. 7, C and D), obtaining values of 9 and 17 pA for the first modes; the interval between peaks was 8–9 pA (Ling and Benardo 1999). Similarity between sIPSCs and eIPSCs histograms supports the notion that terminals that interconnect spiny neurons are fairly homogeneous (Tecuapetla et al. 2005), and their modal values can now be used to independently ascertain the results of the mean-variance analysis (Clements and Silver 2000). eIPSCs were obtained from a range of eIPSCs amplitudes from different experiments (see intensity-amplitude plots in Tecuapetla et al. 2005). Mean eIPSCs amplitudes were graphed against their peak variance (Fig. 7E, black circles) and the resultant plot could be fitted wit a parabola (see Clements and Silver 2000) that yielded a weighted quantal amplitude, Qw = 8 ± 2 pA, notably similar to the first modal amplitude in the histograms. In the presence of the D₂-class receptor agonist, quinelorane (1 µM), the cases appeared intermingled, with control cases of lower mean amplitudes at the initial portion of the parabola (Fig. 7E, gray circles), and linear fits of the two samples were not significantly different. Qw was 9 ± 2 pA for the D₂ cases (NS; Mann-Whitney U test; also see amplitude histograms). Therefore mean-variance analysis supported the view that D₂ receptor actions are presynaptic (i.e., initial parabola slopes do not diverge; Clements and Silver 2000).

View larger version (34K): [in this window] [in a new window]   FIG. 7. Mean-variance analysis indicates that action of dopaminergic D2-class receptor agonists on synaptic transmission is presynaptic. A: amplitude histogram of spontaneous IPSCs (sIPSCs) recorded in a neostriatal neuron. Two modes are evident: 8 and 16 pA. B: sample records from which the histogram for sIPSCs was built. C: amplitude histogram of an evoked IPSC (eIPSC) recorded in the same neostriatal neuron. Histogram is multimodal, 1st 2 modes are 9 and 17 pA. Interval between modes is 8–9 pA. eIPSCs were evoked by electrically stimulating the globus pallidus to antidromically activate striofugal axons and therefore axon collaterals that interconnect spiny neurons. D: sample records of eIPSCs for each mode, including failures. E: mean peak amplitudes of eIPSCs taken from different experiments in which different stimulation strengths were used were graphed against their corresponding peak variances (black circles). A parabola (continuous line) of the form (Clements and Silver 2000) was fitted to these data. From this fit, an approximation of quantal amplitude (Qw) could be obtained: 8 pA. Note the similarity of values with values obtained with amplitude histograms (A–D). Mean peak amplitudes of eIPSCs recorded in the presence of quinelorane (1 µM) were also graphed against their peak variances (gray circles), Qw = 9 pA (NS). Because data from both control and test samples share initial slope of parabola, analysis supports that the action of D2-class receptors is presynaptic (Clements and Silver 2000). F: family of eIPSCs obtained with quasi-minimal stimulation. Observe few failures in the control condition (1st event), and many more failures during quinelorane. Experiments as this one allow a direct assessment of P. Bottom: average recording of individual trials: note that PPR increases when P decreases.

D₂-class receptor–mediated actions on other ion channels have been linked to intracellular Ca²⁺ mobilization and calcineurin (CaN) (Hernandez-Lopez et al. 2000; Hu et al. 2005; Rakhilin et al. 2004). However, it was shown (Fig. 8, A–D) that the percentage of D₂-mediated modulation on Ca²⁺ currents was not significantly different when the neurons were internally perfused with either 1 or 10 mM EGTA: 34 ± 2 and 36 ± 3%, respectively (NS; n = 6; Mann-Whitney U test). Nevertheless, D₂-class receptor–mediated actions were virtually abolished when 15 mM BAPTA were perfused instead (Fig. 8, C and D), suggesting that D₂ receptor actions were mediated by a Ca²⁺-dependent diffusible pathway (Dargan and Parker 2003; Stefani et al. 2002). Figure 8F shows Ca²⁺ current evoked with the double pulse protocol (depicted in Fig. 8E; see METHODS) in a PD14 neuron. It can be seen that the amount of current modulated by the D₂ receptor agonists did not change significantly (Fig. 8H; Wilcoxon's t-test). The same result was found on PD40 neurons (Fig. 8, G and I) (Hernandez-Lopez et al. 2000). The results suggest that the signaling pathway used by D₂-class receptors to modulate Ca_V2 Ca²⁺ channels is a diffusible Ca²⁺-sensitive cascade, as is the case of Ca_V1 Ca²⁺ channels (Hernandez-Lopez et al. 2000; Hu et al. 2005; Olson et al. 2005; Rakhilin et al. 2004). In view of these results, the next question was whether this modulation was being mediated by the same Ca²⁺ channels at both developmental stages.

Ca²⁺ channels mediating dopaminergic D₂ receptor modulation change during development

Because the amount of D₂ modulation on the Ca²⁺ current was the same in juvenile (PD14) and mature (PD40) neurons, it was decided to use different Ca²⁺ channel blockers in conjunction to the D₂-class receptor agonist, quinelorane (10 µM), to see if the channels being modulated were the same in juvenile and mature neurons. The calcium channel blockers, nitrendipine (10 µM), -AgaTK (400 nM), and -CgTx (1 µM) were used, the rationale being that, if one channel type mediates the D₂-induced modulation, its blockage would occlude a part of this modulation (Perez-Rosello et al. 2005; Vilchis et al. 2002). However, if a channel type does not mediate most of the D₂-induced modulation, its blockage would not occlude the modulation significantly because percentage of modulation is calculated from the current left after the action of a given blocker. Nonetheless, the percentages of current components vary somewhat from cell to cell, and multiple samples were compared with the same control samples (quinelorane's effect without channel blockers). Therefore both parametric and nonparametric (Kruskal-Wallis) ANOVA tests were used to compare all samples altogether taken into account intrasampling variance. Both tests yielded significant differences when the D₂-class agonist was administered (F = 15.2; K-W: 53.8; P < 0.0001 in both cases). Therefore post hoc (Tukey's test) pair to pair comparisons were used to contrast different groups. At the PD14 stage, pretreatment with nitrendipine did not completely block quinelorane action but reduced it from 35 ± 2 to 26 ± 2%; (n = 11; Fig. 9A; 9% occlusion; P < 0.05; ANOVA with post hoc Tukey's test), as expected after a block of a modulated channel. This shows that D₂ receptor activation modulates Ca_V1 Ca²⁺ channels at this stage, but also shows that there is a remaining modulation on Ca_V2 Ca²⁺ channels. Still, a more important occlusion of the D₂ agonist effect by the L-type channel blocker was observed in PD40 neurons: quinelorane action was reduced from 36 ± 3 to 17 ± 1% (n = 11; Fig. 9B; 19% occlusion; P < 0.0001; ANOVA with post hoc Tukey's test). When the degree of occlusion by the L-type channel blocker was compared between both developmental stages, the difference was statistical significant (P < 0.04; ANOVA and post hoc Tukey's test). These experiments show that the dopaminergic D₂ modulation of L-type Ca²⁺ channels is not only preserved but enhanced significantly with age. This suggests that the modulation of the dynamic firing range (Perez-Garci et al. 2003) enhances its importance with network development.

View larger version (31K): [in this window] [in a new window]   FIG. 9. Ca2+ currents modulated by D2 receptor activation change during development. A: previous blockage of CaV1 Ca2+ channels (L-type) by nitrendipine (10 µM) in PD14 neurons does not impede the action of the D2-receptor agonist, quinelorane (10 µM), on Ca2+ current, indicating an important D2-modulation on CaV2 Ca2+ channels. However, a partial occlusion of D2 modulation was present and statistically significant. B: similarly, blockage of L-type channels partially occludes quinelorane action on the Ca2+ current of PD40 neurons. However, partial occlusion was significantly larger in PD40 neurons (P < 0.04). C: quinelorane can act on Ca2+ current of a PD14 neuron after block of CaV2.1 Ca2+ channels (P/Q-type) by -AgaTK (400 nM). In fact, the action of the D2 receptor agonist was not significantly different with and without -AgaTK. D: -AgaTK significantly occluded the action of quinelorane on the Ca2+ current of a PD40 neuron. Occlusion of the D2 receptor agonist action by -AgaTK was significantly different when comparing both developmental stages (P < 0.05). E: quinelorane had a much reduced action on the Ca2+ current of a PD14 neuron after blocking CaV2.2 Ca2+ channels (N-type) with -CgTx (1 µM). F: -CgTx does not occlude quinelorane action on the Ca2+ current of PD40 neurons. Occlusion of the D2 receptor agonist action by -CgTx was significantly different when comparing both developmental stages (P < 0.001).

A completely different outcome was obtained with the N-type channel blocker -CgTx. In the presence of this blocker, the D₂ agonist action on Ca²⁺ current of PD14 neurons was reduced from 35 ± 2 to 12 ± 2% (n = 8; Fig. 9E; 66% occlusion; P < 0.0001; ANOVA with post hoc Tukey's test). Therefore -CgTx resulted in the most potent agent capable to occlude the dopaminergic modulation in juvenile neurons, suggesting that modulation of Ca_V2 Ca²⁺ channels by dopamine is more important than modulation of Ca_V1 Ca²⁺ channels at this stage. In contrast, -CgTx was unable to significantly decrease the action of D₂ agonists on the Ca²⁺ current of PD40 neurons. Ca²⁺ current reduction by quinelorane in the presence of -CgTx in PD40 neurons was from 36 ± 3 to 31 ± 3% (n = 8; Fig. 9F; P < 0.9; ANOVA and post hoc Tukey's test). When the degree of occlusion by the N-type channel blocker was compared between both developmental stages, the difference was statistical significant (P < 0.0001; ANOVA and post hoc Tukey's test). This means that modulation of N-type channels by D₂ receptor activation is large or very important in juvenile (PD14) neurons, but it is much smaller in mature (PD40) neurons.

A summary of all samples compared can be seen in Fig. 10. It was observed that the most potent blocker of the D₂ agonist action in young (PD14) neurons was -CgTx, followed by nitrendipine. However, in mature neurons (PD40), both nitrendipine and -AgaTK were potent blockers of the D₂-agonist action on Ca²⁺ currents, but -CgTx did not longer occlude dopaminergic actions. In summary, a considerable switch in target for D₂ receptor signaling occurs in medium spiny neurons during development.

View larger version (19K): [in this window] [in a new window]   FIG. 10. Action of D2 receptor activation on different components of the Ca2+ current. A: most potent blocker of D2 receptor agonist action on the Ca2+ current of PD14 neurons was -CgTx (***P < 0.0001), indicating that dopaminergic modulation of N-type channels is very important at juvenile stages of development. Regulation of L-type channels is also present at juvenile stages (*P < 0.05). B: modulation of both L- and P/Q-types Ca2+ channels becomes very important with neuronal maturation (***P < 0.0001). However, modulation of N-type channels, important at juvenile stages, becomes virtually nonexisting at a mature age (PD40).

If the above data are correct, the following predictions can be made to further corroborate these findings: first, the conjoined administration of nitrendipine (10 µM) and -AgaTK (400 nM) will virtually abolish quinelorane action on the Ca²⁺ current of PD40 neurons, but not in PD14 neurons (cf., Fig. 11, A and B). Conversely, the conjoined administration of nitrendipine (10 µM) and -CgTx (1 µM) will almost totally block the action of the D₂ receptor agonist on the Ca²⁺ current of PD14 neurons, but not in the case of PD40 neurons (cf., Fig. 11, D and E). Thus after L-type and P/Q-type Ca²⁺ channels were blocked, quinelorane was still capable to reduce the remaining current by 19 ± 1% (n = 6) in PD14 neurons (Fig. 11C). In contrast, when L-type and P/Q-type Ca²⁺ channels were blocked in PD40 neurons, quinelorane action was virtually abolished to 3 ± 1% (n = 6: Fig. 11C; P < 0.0001; ANOVA with post hoc Tukey's test). Conversely, when L-type and N-type channels were blocked in PD14 neurons, almost no quinelorane action was left: 6 ± 0.7% (n = 6; Fig. 11, D and F). The opposite findings were observed at the PD40 stage: quinelorane was still capable to modulate the remaining Ca²⁺ current by 18 ± 2% in the same conditions (n = 6; Fig. 11F; P < 0.0001; ANOVA with post hoc Tukey's test). Globally, both parametric and nonparametric (Kruskal-Wallis) ANOVA tests were significant (F = 50.7; K-W: 18.5; P < 0.0001 for both tests). In summary, besides modulating Ca_V1 Ca²⁺ channels, D₂ receptor activation also modulates Ca_V2 Ca²⁺ channels. However, the modulation is mainly on Ca_V2.2 Ca²⁺ channels (N-type) in juvenile striatal neurons (PD14), whereas it is mainly on Ca_V2.1 Ca²⁺ channels (P/Q-type) in mature striatal neurons (PD40).

View larger version (31K): [in this window] [in a new window]   FIG. 11. Different pairs of Ca2+ channels explain most dopaminergic D2 modulation of Ca2+ current in young and mature neurons. A: D2 receptor agonist, quinelorane (10 µM), still had a substantial action on the Ca2+ currents of young (PD14) neurons after L-type and P/Q-type Ca2+channel types were blocked (with 10 µM nitrendipine plus 400 nM -AgaTK). B: action of the D2 receptor agonist on the Ca2+ currents of mature (PD40) neurons was virtually abolished after L-type and P/Q-type Ca2+channel types were blocked (with 10 µM nitrendipine plus 400 nM -AgaTK). C: histogram summarizes quinelorane action on the Ca2+ current of young and mature neurons in the presence of L- and P/Q-type Ca2+ channels blockage (*P < 0.0001). D: action of the D2 receptor agonist on the Ca2+ currents of young (PD14) neurons was almost abolished after L-type and N-type Ca2+channel types were blocked (with 10 µM nitrendipine plus 1 µM -CgTx). E: D2 receptor agonist still had a substantial action on the Ca2+ currents of mature (PD40) neurons after L-type and N-type Ca2+channel types were blocked. F: histogram summarizes quinelorane action on the Ca2+ current of young and mature neurons in the presence of L- and N-type Ca2+ channels blockage (*P < 0.0001).

To study whether the change of target affects presynaptic inhibition (Guzman et al. 2003), the effect of the D₂ receptor agonist, quinelorane (1 µM), in the presence of -CgTx (1 µM) was examined on the eIPSCs generated by antidromic activation of the axon collaterals. Figure 12A shows that -CgTx (1 µM) almost abolished the D₂ agonist mediated reduction of IPSCs in PD14 terminals: from a 50 ± 8% reduction in the control (Guzman et al. 2003) to only 10 ± 2% reduction in the presence of -CgTx (n = 3; Fig. 12A; P < 0.02; Mann-Whitney U test). A posterior application of -AgaTK (400 nM) blocked the remaining eIPSC (Fig. 12A), confirming that P/Q-type channels were also present at the terminals and that quinelorane only had its full action if the main target, N-type channels, were present in juvenile terminals.

View larger version (19K): [in this window] [in a new window]   FIG. 12. The D2-mediated modulation of IPSCs is occluded by CaV2.2 channel blockage in young but not in mature neurons. A: time-course of IPSC amplitude recorded in the presence of 1 µM -CgTx. -CgTx virtually abolished the action of quinelorane (1 µM) on the IPSCs recorded from PD14 neurons (cf., Fig. 6D). Insets 1 and 2 show representative records in the presence of -CgTx. A subsequent addition of -AgaTK shows that P/Q-type channels are present in terminals because the peptide blocked all the IPSCs; thus the dopaminergic agonist does not act on P/Q-type channels at this developmental stage. B: time-course of IPSC amplitude recorded in the presence of 1 µM -CgTx. -CgTx did not affect the action of quinelorane (1 µM) on the IPSCs recorded from PD40 neurons (cf., Fig. 6E). Insets 1 and 2 show representative records in the presence of -CgTx. Addition of quinelorane reduced the paired pulse depression. It is concluded that the dopaminergic agonist does not act on N-type channels at this developmental stage. C: histogram shows different degree that -CgTx has on dopaminergic D2 effects at these 2 different developmental stages (P < 0.009).

AHP in medium spiny neurons is negligibly affected by D₂ receptor agonists

Because P/Q-type Ca²⁺ channels are a main source of Ca²⁺ to activate the Ca²⁺ dependent K⁺ channels that generate the AHP in medium spiny neurons (Vilchis et al. 2000), D₂ receptor agonists were tested on the AHP of mature spiny neurons after a single action potential. Two D₂ receptor selective agonists were used: 5 µM quinelorane and 5 µM quinpirole. Figure 13 shows that, in mature spiny neurons, no change in AHP was observed in n = 6/18 neurons (33%; Fig. 13, A–C), and a mild change was observed in n = 12/18 neurons (66%; Fig. 13, D–F) for a mean overall reduction of 1.3 ± 0.26 mV (20%). Note that even when D₂-mediated modulation of Ca²⁺ currents was seen in 80% of dissociated cells, only about 60% of intracellularly recorded (PD40) neurons exhibited AHP modulation. This is a number more in agreement with the expected number of cells presenting D₂-type responses, probably suggesting that not all members of the D₂ receptor family (D₂, D₃, D₄) participate in the same specific cellular functions.

View larger version (18K): [in this window] [in a new window]   FIG. 13. Activation of D2 receptors negligibly affect the afterhyperpolarization (AHP) in medium spiny neurons. A: action potential was evoked by a brief depolarization (top) from a holding potential of –55 mV (bottom). B: addition of a D2-selective agonist, in this case quinpirole (5 µM), does not reduce or modify AHP. C: superimposition of records in A and B. D: similar experiment in another spiny neuron held at –60 mV. E: In this case, addition of D2 receptor agonist produced a small reduction of the AHP. F: superimposition of C and D. Inset: behavior of a sample of mature spiny neurons: one-half of the neurons had no AHP modification after D2 agonists and one-half had a minor change.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Dopaminergic modulation of Ca²⁺ currents undergoes a shift in target during development

Previous studies have shown that medium spiny neurons express functional D₂-class receptors (e.g., Aizman et al. 2000; Surmeier et al. 1993, 1996). These receptors increase their number between the third and fourth postnatal weeks (Broaddus and Bennett 1990), when an important escalation in the numbers of dopaminergic synaptic terminals occurs (Antonopoulos et al. 2002).

This work reports that N and P/Q-type channels are modulated in neostriatal projection neurons by D₂-class receptor activation. If only the whole cell Ca²⁺ current is observed, no difference could be seen in the amount of current modulated when comparing young and mature neurons. Moreover, inhibitory transmission between spiny neurons was similarly modulated in young (Guzman et al. 2003</