Impulse-dependent extracellular resting dopamine concentration in rat striatum in vivo

Neurochemistry International 62 (2013) 50–57

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Impulse-dependent extracellular resting dopamine concentration in rat striatum in vivo Pan-Li Zuo a, Wei Yao a,b, Liang Sun a, Shu-Ting Kuo a, Qing Li a, Shi-Rong Wang a, Hai-Qiang Dou a, Hua-Dong Xu a, Claire Xi Zhang a, Xin-Jiang Kang a, Zhuan Zhou a,⇑, Bo Zhang a,⇑ a b

State Key Laboratory of Biomembrane and Membrane Biotechnology and the Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing 100871, China Institute of Physiology, Shandong University, School of Medicine, Jinan, Shandong 250012, China

a r t i c l e

i n f o

Article history: Received 26 June 2012 Received in revised form 11 October 2012 Accepted 7 November 2012 Available online 16 November 2012 Keywords: Resting dopamine Undershoot In vivo Amperometry Striatum D2 receptors

a b s t r a c t The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Despite its importance, resting DA level in vivo remains elusive. Here, by high-frequency stimulation of the medial forebrain bundle and immediately following the stimulus-induced DA overflow, we recorded a DA ‘‘undershoot’’ which is a temporal reduction of DA concentration to a level below the baseline. Based on the DA undershoot, we predicted a resting DA concentration of 73 nM in rat striatum in vivo. Simulation studies suggested that removing basal DA by DAT during the post-stimulation inhibition of tonic DA release caused the DA undershoot, and the resting concentration of DA modulated the kinetics of the evoked DA transient. The DA undershoot was eliminated by either blocking D2 receptors with haloperidol or blocking the DA transporter (DAT) with cocaine. Therefore, the impulse-dependent resting DA concentration is in the tens of nanomolar range and is modulated by the presynaptic D2 receptors and the DAT in vivo. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction Midbrain dopaminergic neurons display phasic and tonic firing patterns (Grace, 1991; Schultz, 2002). The fast phasic firing is thought to be involved in encoding reward (Bromberg-Martin et al., 2010; Phillips et al., 2003; Schultz, 2002) and enhancing long-term potentiation (Huang et al., 2004; Reynolds et al., 2001). The slow tonic firing supplies ambient resting DA to regulate cognition and motivation (Bromberg-Martin et al., 2010; Goto and Grace, 2005; Grace, 1991; Schultz, 2002). The resting DA is critical for brain state by determining the activation level of D2 and/or D1 receptors (Dreyer et al., 2010). Low concentration (1.5–30 nM) of DA in striatum in vivo was estimated by several methods, including microdialysis (Jones et al., 1998; Smith et al., 1992), voltammetry (Crespi and Mobius, 1992; Gonon and Buda, 1985; Suaud-Chagny et al., 1992), and simulation with voltammetrically derived data (Venton et al., 2003b). However, microdialysis could underestimate the concentration due to flow-induced

⇑ Corresponding authors. Address: Institute of Molecular Medicine, Peking University, 5 Yiheyuan Road, Beijing 100871, China. Tel./fax: +86 10 6275 3212 (B. Zhang). E-mail addresses: [email protected] (Z. Zhou), [email protected], bert_zhang@ live.com (B. Zhang).

dilution (Jones et al., 1998; Suaud-Chagny et al., 1992) and larger tissue damage (Clapp-Lilly et al., 1999). Voltammetry can measure evoked changes of DA, but not basal DA level, which is an intrinsic burden for voltammetry (Wightman and Zimmerman, 1990). Therefore, the resting concentration of DA and how it is modulated remains to be determined. The extracellular DA level is balanced by DA release and clearance (Cragg and Rice, 2004; Wang et al., 2011). Under resting conditions, the ambient DA concentration is maintained by tonic release vs. clearance (Grace, 1995). Dopaminergic tonic firing is inhibited by the large DA release following intensive electric stimulation at medial forebrain bundle (MFB), a phenomena called ‘‘post-stimulation inhibition’’ (Kuhr et al., 1987; Wightman and Zimmerman, 1990). Theoretically, a temporary blockade of tonic release by the post-stimulation inhibition may reduce the DA level to fall below the pre-stimulation baseline (DA ‘‘undershoot’’) and the extent of this undershoot could reflect the resting concentration of DA (Chen, 2005). In the present work, using amperometric recording (Guo et al., 2012; Wang et al., 2011), for first time we detected a DA undershoot in striatum in vivo after high-frequency stimulation at MFB. This undershoot was modulated by D2 receptors and the DAT. Using this DA undershoot, the resting DA concentration in striatum was estimated to be 73 nM. Simulation studies suggested that different resting concentrations modulated the kinetics of evoked DA overflow.

0197-0186/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.11.006

P.-L. Zuo et al. / Neurochemistry International 62 (2013) 50–57

2. Materials and methods 2.1. Animals and surgical procedures All experiments were performed in accordance with the guidelines of the Animal Research Advisory Committee at Peking University. Male Sprague–Dawley rats (250–400 g) were anesthetized with urethane (1.5 g/kg, i.p.) and fixed in a stereotaxic frame,

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as described previously (Wang et al., 2011). The recording carbon fiber electrode (CFE) was placed in the caudate–putamen (stereotaxic coordinates, in mm from bregma, anterior–posterior (AP), +1.1; medial–lateral (ML), +2.8; dorsal–ventral (DV), +5.5), and the bipolar stimulating electrode (MS303 Plastics One Inc., Roanoke, VA) was positioned in the MFB (AP, 4.2; ML, +1.5; DV, +8.0). A pulse-train (96–768 pulses, 80 Hz) was used to evoke DA release. The trains of stimulus pulses consisted of individual

Fig. 1. DA undershoots following strong electrical pulse stimulations at MFB. (A) Representative traces of DA overflow evoked by 96-pulse stimulations at 80 Hz with current intensities (increasing current intensity from left to right, 0.5, 0.6, 0.7, and 0.8 mA) in striatum in vivo. The arrows point to the undershoot signals expanded in the lower traces. The negative fast transients in Iamp traces at the beginning of the stimulus are stimulus artifacts. (B) Plot-curve of the ‘‘response’’ DA undershoots following different ‘‘stimulating’’ DA overflows in a given rat. The statistic curves are made from 6 plots of 6 rats. The black trace shows a sigmoid fitting with peak undershoot of 60 nM. (C and D) Fast-scan cyclic voltammetry (FSCV) recording illustrating the DA overflow in response to 384 pulses (at 80 Hz) of stimulation. Insets show voltammograms both from the peak of FSCV and 3 s after stimulation of FSCV matched well those produced by in vitro application of 5 lM DA and 5 lM norepinephrine but not by in vitro application of 5 lM 5-HT, and 100 lM ascorbate acid. Similar results were obtained from 4 experiments. (E) Representative trace of DA overflow evoked by 768 pulses at 80 Hz in the striatum. The lower trace shows the expanded undershoot signal. The maximum undershoot (97 nM) occurred about 4 s after stimulation. (F) Summary of overflow-induced the maximum DA undershoot (average 73 ± 7 nM) vs. corresponding DA overflow (average 2.23 ± 0.23 lM). Each spot representing one of 26 rats.

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current spikes of 0.1–1 mA amplitude and 0.2 ms duration. An Ag/ AgCl wire was implanted in the brain as a reference electrode. 2.2. Electrochemistry The DA was recorded with 250-lm-long glass-insulated cylindrical CFEs as described previously (Guo et al., 2012; Wang et al., 2011). DA signals were identified by fast-scan cyclic voltammetry (FSCV), with a voltage scan from 0.4 V to 1.0 V and back at 300 V/s every 100 ms. Constant-potential amperometry (+0.78 V) was used for data analysis, because it has a faster time response than voltammetry, a critical condition for the undershoot detection

during the post-stimulation inhibition of tonic DA release. Amperometric currents were low-pass-filtered at 200 Hz and sampled by the EPC-9/2 amplifier (HEKA Electronic, Lambrecht/Pfalz, Germany) at 4 kHz. The electrochemical data (FSCV and amperometry) were recorded by a Patchmaster-based software (HEKA Electronic), and analyzed by Igor software (WaveMetrics, Lake Oswego, OR). To reduce noise, these data were further digitally filtered at 10 Hz for analysis. To estimate DA overflow and the undershoot signals, the amperometric recordings were calibrated in vitro as 2.0 nM/pA by known concentrations of DA in artificial cerebrospinal fluid (containing 124 mM NaCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 2.5 mM KCl, adjusted to pH 7.4) with 10 Hz low-pass filter

Fig. 2. Simulation of evoked DA overflow and undershoot. (A) Maximal DA undershoot signal in the striatum induced by 96 pulses at 80 Hz. The DA undershoot was 65 nM. Linear regression of the decay phase of the evoked DA overflow showed an apparent Vmax of 2.86 lM/s. The undershoot recovered to baseline with a time constant of 2.2 s in a single exponential regression. (B) Simulated trace (black line) for (A). The parameters were: [DA]p = 50 nM, f = 80 Hz, apparent Vmax = 2.86 lM/s, Km = 0.2 lM and Cr = 65 nM, with a Cr removing time of 3 s. The correlation coefficient r = 0.97. (C) Simulated traces for (A) with (gray line) or without (black line) 3 s post-stimulation inhibition. (D) Poststimulation inhibition increased DA transient recovery time (t1 and t2) while decreasing apparent uptake velocity (V1 and V2) in (C). (E and F) Simulated effect of different Cr on DA transients with (E) or without (F) post-stimulation inhibition. Insets show recovery time as a function of Cr.

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in vitro (Fig. S1) (Garris et al., 1994a). The slow kinetics of resting DA overflows during the undershoot permits low-pass filter and high resolution (nM) of DA overflow detections (Fig. S2). 2.3. Model of evoked DA overflow Following a train of pulse stimulation at MFB, the profiles of DA overflow was similar in amperometry and fast scan cyclic voltammetry (FSCV), except FSCV-overflow had a 150 ms delay (Fig. S2). This latency is negligible for the undershoot of 2–3 s length (see Figs. 1–5). Thus, our simulation did not consider the oxidization effect on DA overflow estimated by amperometric currents (Wang et al., 2011). Amperometric data recorded during 96-pulse stimulations were modeled by assuming that a fixed concentration of DA ([DA]p) at impulse rate f is released with a single pulse and the uptake follows Michaelis–Menten kinetics with a apparent maximal rate of DA uptake Vmax and DAT Michaelis–Menten constant Km (0.2 lM) (Wightman et al., 1988). It can be described as:

dC V max C ¼ ½DAp f  dt Km þ C

ð1Þ

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Following previous simulation approaches by taking the resting DA concentration (Cr) supplied by tonic firing (Grace, 1995) into consideration, the kinetics of DA overflow can be written as (Chen, 2005; Chen and Budygin, 2007; Venton et al., 2003b; Wightman and Zimmerman, 1990):

dðC r þ DCÞ V max ðC r þ DCÞ ¼ ½DAp f  dt K m þ C r þ DC

ð2Þ

DC is the DA concentration difference after stimulation. The inhibition of tonic DA release was performed by removing the Cr within a limited inhibition time during simulation. 2.4. Data analysis and drugs For analysis of the DA undershoot, only signals >3 SD were included. Data analysis was performed using paired or unpaired Student’s t-test in Microsoft Excel and Igor software. Data were presented as mean ± SEM and were considered significant at p < 0.05. Model parameters were evaluated by the correlation coefficients calculated from experimental data and simulated traces (Glantz and Slinker, 1990; Montague et al., 2004). All chemicals

Fig. 3. D2 receptors contributed to DA undershoot. (A) Sample traces of DA overflow before (left) and after haloperidol injection (right) in response to 96 pulses at 80 Hz in striatum (arrows indicate time of measurements for data in (B)). To produce similar DA overflow after haloperidol administration, the pulse-stimulation current were adjusted for recording in right panel. (B) Summary showing haloperidol (Halo) significantly attenuated the undershoot signal with adjusted similar DA overflow. Star symbols in Fig. 3 and Fig. 4 indicate statistical significance (⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001) and a bracket without star symbol indicates no statistical significance (p > 0.05).

Fig. 4. DAT contributed to DA undershoot. (A) Example traces of DA overflow in the striatum induced by 96 pulses at 80 Hz before (left) and after (right) cocaine (Coca) injection (arrows indicate time of measurements for data in (B)). (B) Summary showing cocaine reversed the amplitude of the undershoot signal.

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Fig. 5. Simulations of haloperidol and cocaine effects. (A) Experimental (gray trace) and simulated traces (solid and dashed black traces) of DA overflow induced by 96 pulses at 80 Hz in striatum before and 30 min after haloperidol injection. Parameters of simulation for control trace (left): [DA]p = 43 nM, f = 80 Hz, apparent Vmax = 2.23 lM/s, Km = 0.2 lM and Cr = 50 nM (from undershoot signal); the Cr moving time in the trace was set at 3 s, and the resulting correlation coefficient r = 0.98. After haloperidol injection, no undershoot was observed (right). Two simulated traces with 50 nM (dashed trace, r = 0.91) and 150 nM (solid trace, r = 0.98) of resting DA were applied to fit the data. (B) Experimental (gray) and simulated traces (solid and dashed black lines) before and after cocaine treatment. Parameters of simulation for control trace: [DA]p = 26 nM, f = 80 Hz, apparent Vmax = 1.3 lM/s, Km = 0.2 lM, Cr = 40 nM, and 3 s Cr removing. The correlation coefficient r = 0.99. After cocaine injection, no undershoot was observed (right). Km = 0.9 lM was used for the cocaine trace, other parameters were their pre-cocaine values except that [DA]p was slightly reduced (to 21 nM) to get similar DA overflow after the adjusted stimulation current. The resulting correlation coefficient r = 0.98.

were from Sigma Chemical Co. (St. Louis, MO) except for cocaine (QingHai Pharmaceutical Co., China).

3. Results 3.1. Resting DA concentration estimated by DA undershoot after MFB stimulation To investigate physiological resting DA concentration, the MFB was stimulated with supraphysiological electrical stimuli (96 pulses at 80 Hz). The strong intensity of stimulation triggered a large DA overflow followed by a rapid decline after cessation of stimulation (Fig. 1A), due to neuronal uptake of DA (Cragg and Rice, 2004; Guo et al., 2012). Interestingly, a transient ‘‘undershoot’’ of amperometric current was readily detected after a large DA overflow (Fig. 1A, lower traces), which was probably produced by ‘‘post-stimulation inhibition’’ of tonic DA release (Kuhr et al., 1987; Wightman and Zimmerman, 1990). The undershoot signals were dependent on stimulation strength and recovered to prestimulation levels within only 3–6 s (Fig. 1A, although the poststimulation inhibition of tonic firing at the somatic region can last up to 30 s (Kuhr et al., 1987; Wightman and Zimmerman, 1990). When the values of undershoot were plotted against the peak DA overflows, it decreased with the overflow (Fig. 1B). Sigmoid fitting gave the maximal undershoot of 60 nM (n = 6 rats, 96 pulses at 80 Hz, Fig. 1B). As predicted by a theoretical study (Chen, 2005), the amplitude of the maximal undershoot should represent the

resting DA concentration. The cyclic voltammograms (oxidation– reduction profile) of in vivo recordings confirmed that DA was detected following MFB stimulation (Fig. 1C), because the voltammograms did not match those produced by the in vitro application of 5 lM serotonin or 100 lM ascorbate acid (bottom traces, Fig. 1D). The voltammogram of norepinephrine is close to that of DA (Fig. 1D), but the norepinephrine levels are much lower in striatum (Wightman et al., 1986). Therefore, although in vivo-FSCV is unable to detect DA signal below the baseline, which is used as background current to generate evoked FSCV signals in vivo (Garris et al., 1994a; Wang et al., 2011), the undershoot signals recorded by amperometry were most likely mediated by DA because it is sensitive to the DA specific DAT (see below). We next determined the resting DA concentration via undershoot signal following longer stimuli (768 pulses, 80 Hz) to achieve a maximal and sustained (>10 s) DA undershoot (Fig. 1D). The value of minimal DA undershoot ranged from 20 nM to 138 nM with an average value of 73 ± 7 nM (n = 26, Fig. 1F). Therefore, the averaged resting DA concentration is estimated as 73 ± 7 nM in rat striatum in vivo. The large variation is probably due to different location of the micro probe at a heterogeneous population of DA terminals (Garris et al., 1994b). Slightly different local extracellular volume fraction and tortuosity (Cragg and Rice, 2004; Rice and Nicholson, 1991) could affect the diffusion of DA molecules and might contribute to the variation of DA undershoot. Resting DA were variable at different brain locations, probably resulted in that activation profile of D1 and D2 are brain location-different (Dreyer et al., 2010).

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3.2. Removing resting DA is responsible for DA undershoot Dopaminergic tonic firing at the somatic region is temporally inhibited after a strong stimulus (Kuhr et al., 1987; Wightman and Zimmerman, 1990), which can block up to 93% of the firing via activation of presynaptic D2 receptors (Jones et al., 1999). This ‘‘post-stimulation inhibition’’ predicts to block DA release at the terminal and to cause DA undershoot due to continued DA clearance (Chen, 2005). We examined this theoretic prediction by simulation of evoked DA overflow recorded using amperometry (see Methods for FSCV vs. amperometry). An electrical stimulation of 96 pulses (80 Hz) was used to elicit the maximal DA undershoot (Fig. 2A). The estimated resting concentration (Cr of 65 nM) was obtained by maximal undershoot, and the apparent Vmax (2.86 lM/s) was estimated by fitting the decay phase of DA overflow. We proposed that, immediately after an intensive pulse-stimulation at MFB, resting DA concentration Cr is temporally reduced (removed) jointly by the continuing DA uptake and the post-stimulation inhibition of tonic DA release. Since the recovery underwent 3–6 s, we choose a fixed time (3 s) in this simulation. With the above parameters in Eq. (2) (see Methods), the simulation fits the control recording well with a 3s-undershoot (‘‘Cr removing’’) and resulted in correlation coefficient r = 0.97 (Fig. 2B). The simulation of the rising phase was not as good as the decay phase, probably due to a non-linear Ca2+-dependent vesicular release (Lou et al., 2005). In presence of Cr (i.e. absence of the DA undershoot), the simulation curve could not fit the data because decay time extended (t1 of 0.96 s vs. t2 of 1.61 s) and the apparent uptake velocity decreased (V1 of 2.86 lM/s vs. V2 of 1.75 lM/s) (Fig. 2C and D). Therefore, this simulation supports the hypothesis that removing Cr results in the DA undershoot. We then assessed the effects of different Cr values on DA signals by simulation. With a 3 s removing of Cr, a larger Cr slightly reduced the decay phase of DA overflow (Fig. 2E and inset). Since DA undershoot was triggered only by prolonged high-frequency stimulation (Fig. 1), the post-stimulation inhibition might not occur under physiological conditions (<20 Hz). In the absence of Cr removing, our simulation showed that a larger Cr dramatically slowed down the decay of the evoked DA overflow (Fig. 2F and inset). While resting DA level is 73 nM in average, it ranged in 20–138 nM at different recording sites in striatum (Fig. 1F), implicating that the resting DA concentration could strongly affect the kinetics of evoked DA overflow in response to physiological stimulation. 3.3. D2 receptors and the DAT are both involved in DA undershoot DA probably inhibits its own release through D2 receptors, because the specific D2 receptor agonist quinpirole severely inhibits dopaminergic neuronal firing (93 ± 5%) at the cell body in the ventral tegmental area (Jones et al., 1999). Therefore, we investigated whether D2 receptors regulated tonic release and basal DA level through the post-stimulation inhibition. Haloperidol (i.p., 0.6 mg/ kg), an antagonist of the D2 receptor, increased the DA overflow (control: 1.48 ± 0.13 lM vs. haloperidol: 1.99 ± 0.19 lM, paired t-test, p < 0.05, n = 8) (Wang et al., 2011), with a diminished undershoot (control: 62 ± 17 nM vs. haloperidol: 3 ± 9 nM, paired t-test, p < 0.01, n = 8) (Fig. 3). To minimize the effect of DA overflow amplitude on the undershoot signal, we adjusted the stimuli intensity to evoke a similar amplitude of DA overflow before and 30 min after haloperidol injection. Summarized data showed that haloperidol treatment eliminated the DA undershoot (control: 62 ± 17 nM vs. haloperidol: 5 ± 12 nM, paired t-test, p < 0.001, n = 8) even with a similar DA overflow (control: 1.48 ± 0.13 lM vs. haloperidol: 1.51 ± 0.15 lM, paired t-test, p = 0.31, n = 8) (Fig. 3). Comparing the DA overflow and undershoot signal with

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or without adjustment of stimulation current after haloperidol treatment, we found that stimulation adjustment decreased the DA overflow (1.99 ± 0.19 lM, vs. 1.51 ± 0.15 lM, paired t-test, p < 0.01, n = 8) without significant effect on the DA undershoot (5 ± 12 nM vs. 3 ± 9 nM, paired t-test, p = 0.78, n = 8) (Fig. 3B). Thus, the undershoot signals were dependent on DA/D2 receptor signaling. Since the DA undershoot reflected continued DA clearance beyond the pre-stimulation level, blockade of DA clearance should affect the undershoot. Cocaine, a blocker of the DAT, was used to study the role of DA clearance in the DA undershoot. Thirty minutes after administration of cocaine (i.p., 10 mg/kg), DA overflow dramatically increased (control: 1.82 ± 0.34 lM vs. cocaine: 2.21 ± 0.36 lM, paired t-test, p < 0.05, n = 6), with a diminished undershoot (control: 47 ± 6 nM vs. cocaine: 216 ± 41 nM, paired t-test, p < 0.01, n = 6) (Fig. 4). We adjusted the current intensity of stimuli to obtain a similar DA overflow before and after cocaine treatment. Cocaine eliminated the DA undershoot (control: 47 ± 6 nM vs. cocaine: 205 ± 8 nM, paired t-test, p < 0.001, n = 6, Fig. 4B) even when similar DA overflows were evoked (control: 1.82 ± 0.34 lM vs. cocaine: 1.86 ± 0.31 lM, paired t-test, p = 0.47, n = 6) (Fig. 4). Comparing the DA overflow and the undershoot with or without the adjustment of stimulation current after cocaine challenge, the DA overflow decreased (2.21 ± 0.36 lM vs. 1.86 ± 0.31 lM, paired t-test, p < 0.05, n = 6), while the undershoots were similar (205 ± 8 nM vs. 216 ± 41 nM, paired t-test, p = 0.77, n = 6) (Fig. 4B). These data showed that the DA/DAT was required for the undershoot signal following intensive MFB stimulations. Following injection of either cocaine or haloperidol (i.p.), the basal amperometric currents could slightly (equivalent to 100 nM DA) fluctuate within first 10 min and became stable after then. This slow baseline-shift might partially be due to DA signals, although other oxidizable substances could contribute to the baseline shift as well. 3.4. A simulation of the undershoot We next carried out simulations to study the effects of the antagonists of D2 (haloperidol) and/or DAT (cocaine) on the DA undershoot. In the experiment with haloperidol treatment (Fig. 5A), the following parameters were used for best fit of the decay of DA overflow before haloperidol: Cr = 50 nM, Km = 0.2 lM, apparent Vmax = 2.23 lM/s. Before applying haloperidol, the simulation fits the control recording well with a 3s-undershoot (‘‘Cr removing’’) and resulted in correlation coefficient r = 0.98 (Fig. 5A). After applying haloperidol, the 3s-undershoot was removed, and the simulation could not fit the decay phase of DA overflow at Cr = 50 nM (right panel, Fig. 5A). Since haloperidol increases the resting DA concentration (Reiriz et al., 1994), by increasing Cr to 150 nM the simulation can fits data again (Fig. 5A). These simulations suggested that haloperidol eliminates the undershoot through blocking the post-stimulation inhibition and the enhanced basal DA level. In the experiment with cocaine treatment (Fig. 5B), the following parameters of simulation were used for best fit of the decay of DA overflow before cocaine: [DA]p = 29 nM, f = 80 Hz, apparent Vmax = 1.26 lM/s, Km = 0.2 lM, Cr = 40 nM, and 3 s undershoot. Before applying cocaine, the simulation fits the control recording well with a 3s-undershoot (‘‘Cr removing’’) and resulted in correlation coefficient r = 0.99 (Fig. 5B). After applying cocaine, the 3s-undershoot was removed, the Km was changed from 0.2 lM to 0.9 lM, because the known cocaine effect on DAT (Wu et al., 2001), and [DA]p was adjusted from 29 nM to 21 nM for comparable DA overflow amplitudes (Fig. 5B). These simulations suggested that cocaine eliminates the undershoot mainly through reducing the Km of the DAT.

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4. Discussion In the present study, we showed that the resting DA level was 73 nM in the rat striatum in vivo, as revealed by the maximal undershoot following intensive pulse stimulation at MFB. Furthermore, our experiments and simulations demonstrated that D2 receptor and the DAT were responsible factors for the DA undershoot. 4.1. Undershoot produced by temporal reduction of DA baseline Determination of basal dopamine level is a demand for homeostasis of dopamine signaling and dopamine-related mental health and diseases. Amperometry oxidize nearby molecules at the CFE electrode sensor tip without selectivity among endogenous oxidizable substances (DA, norepinephrine, 5-HT and ascorbic acid). The electrode responds extremely fast and sensitive to changes in analyte concentrations, therefore amperometry offers the best temporal resolution among the available techniques (Robinson et al., 2003; Wang et al., 2011). It would be nice to identify the substance underlining the undershoot signal at real time in vivo, currently there is no available method, because FSCV cannot detect dopamine lower than the baseline. The undershoot signals recorded with amperometry in present study was most likely mediated by DA because (1) undershoot is coupled to the amperometric current of DA, as DA is selectively released in striatum following the MFB stimulation in vivo. Ascorbic acid, 5-HT and norepinephrine contribute unlikely to the undershoot signal (Fig. 1C) (Wightman et al., 1986); (2) although high frequency stimulation could change extracellular pH, amperometry is insensitive to pH changes (Venton et al., 2003b); (3) voltammogram confirmed that, the evoked release in amperometry signals were mediated by DA after cessation of the MFB stimulation (Fig. 1C); (4) the undershoot signals were blocked by the selective DAT antagonist cocaine, which clearly suggested that DA reduction was responsible for the undershoot (Fig. 4); (5) the undershoot signals were blocked by selective D2 receptor antagonist haloperidol, which clearly suggested again that DA reduction was responsible for the undershoot (Fig. 3). Together, these evidences strongly suggest that undershoot signal were produced by temporal reduction of the DA level. Nevertheless, we could not totally rule out the possible small (<5%), if any, contamination on the undershoot signal by other molecules in extracellular space such as dopamine metabolite, DOPAC (Venton et al., 2002, 2003b) and ascorbic acid, although extracellular levels of DOPAC and ascorbic acid were not altered in response to brief electrical stimulation of dopaminergic neurons at a distance from the striatum in anesthetized rats with different electrochemical methods (Ewing et al., 1983; Gonon, 1988; Gonon and Buda, 1985; Gonon et al., 1984; Kuhr et al., 1984). 4.2. The post-stimulation inhibition for DA undershoot Resting DA in vivo is determined by the balance between basal levels of tonic release and clearance. Immediately following intense DA release, there is a transient inhibition of dopaminergic tonic firing, termed ‘‘post-stimulation inhibition’’ (Kuhr et al., 1987; Wightman and Zimmerman, 1990). Since the DA clearance continues during the post-stimulation period, theoretically an undershoot of DA below basal level may occur (Chen, 2005; Chen and Budygin, 2007). To estimate the physiological level of resting DA in vivo, we applied supraphysiological stimulation patterns to the MFB, which readily triggers a transient ‘‘undershoot’’ by clearance of extracellular DA during the post-stimulation inhibition (Figs. 1–5). During MFB stimulation, DA could be released in the

somatodendritic area and subsequently causes somatic autoinhibition of tonic firing in the striatum via D2 (Kuhr et al., 1987). In our DA overflow recordings following the MFB stimulating in striatum in vivo, the undershoot occurs only in amperometry but not voltammetry, probably due to >10 times slower DA overflow in the decay phase (Fig. 1, see also (Venton et al., 2003a)). In amperometric recordings, comparing to the preceding DA overflow, the DA undershoot is at least 20 times smaller. Together, these could explain why the undershoot has not been found previously in either conscious (Garris et al., 1999; Phillips et al., 2003) or anesthetized animals (Garris et al., 1994b; Kulagina et al., 2001), or in tissue recordings (Avshalumov et al., 2003; Kennedy et al., 1992).

4.3. Comparison with previous predicted basal DA in vivo We would like to measure directly the basal DA level in the striatum in vivo. Although this is not currently possible with any methods, the DA overflow undershoot during the post-stimulation inhibition provides a high spatial–temporal-resolution assay concerning the DAT-selected DA signaling. Based on amperometric recordings, our estimation of 73 nM as the resting DA concentration in the striatum in vivo can be compared with previous in vivo-studies using other methods. (1) Our estimation by the undershoot is much higher than that by microdialysis (10 nM in dorsal striatum (Smith et al., 1992), 5 nM in nucleus accumbens (Parsons et al., 1991)). Microdialysis could underestimate resting DA concentration due to flow-induced dilution (Jones et al., 1998; Suaud-Chagny et al., 1992) and larger tissue damage (Clapp-Lilly et al., 1999). (2) Although 73 nM is several times higher than the microdialysis-based estimations, our undershoot-based estimation is impulse-dependent and does not include impulseindependent DA component (Borland and Michael, 2004). (3) Although post-stimulation inhibition of D2 transiently blocked partial dopaminergic activity (Kuhr et al., 1987), there still may be a remaining contribution from impulse-dependent DA (Jones et al., 1999). (4) The capacity of dopaminergic neurons to generate DA transients is slightly impaired in anesthetized animals compared with freely moving animals (Hamilton et al., 1992; Stuber et al., 2005), and dopamine transients could contribute to the average basal extracellular concentration in striatum in living animal with value of 20–30 nM (Owesson-White et al., 2012), therefore, the resting DA might be slightly higher in freely moving animals than our present estimate with anesthetized animal. (5) Finally, although our estimation is much higher than previous estimation based on microdialysis, it is consistent with theoretical predictions (30–220 nM) (Chen, 2005; Chen and Budygin, 2007; Dreyer et al., 2010; Venton et al., 2003b). In conclusion, the present study showed that the resting DA level was 73 nM in the rat striatum in vivo, as revealed by the maximal undershoot. Furthermore, our experiments and simulations demonstrated that D2 receptor and the DAT were key factors for the DA undershoot. A functionally interaction between D2R and DAT has been reported (Lee et al., 2007), thus haloperidol could affect DA undershoot through relieving the inhibition and decreasing DA clearance.

Acknowledgements We thank Dr. Iain Bruce for comments on manuscript. This work was supported by Grants from the National Basic Research Program of China (2012CB518006), the National Natural Science Foundation of China (31221002, 31228010, 31171026, 31100597, 30970660, 30911120491 and 30830043), and a ‘‘985’’ Grant from the Department of Education of China.

P.-L. Zuo et al. / Neurochemistry International 62 (2013) 50–57

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