Research Article pubs.acs.org/chemneuro
Noradrenergic Modulation of Dopamine Transmission Evoked by Electrical Stimulation of the Locus Coeruleus in the Rat Brain Jin W. Park,† Rohan V. Bhimani,‡ and Jinwoo Park*,†,‡ †
Department of Biotechnical and Clinical Laboratory Sciences and ‡Neuroscience Program, University at Buffalo, State University of New York, Buffalo, New York 14214-3005, United States S Supporting Information *
ABSTRACT: Central norepinephrine (NE) and dopamine (DA) are involved in a variety of physiological functions and behaviors. Accumulating evidence suggests that NE neurons originating from the locus coeruleus (LC) innervate DA neurons of the ventral tegmental area (VTA) and influence VTA-DA neural activity. However, the underlying mechanisms of how LC-NE regulates DA transmission via VTA-DA neurons remain largely unexplored. Herein, we investigated how electrical stimulation of the LC modulates VTA-DA neurotransmission in the nucleus accumbens (NAc). For this study, catecholamine release in the NAc and VTA evoked by electrical stimulation of the LC in urethane-anesthetized rats was simultaneously monitored with carbon-fiber microelectrodes using in vivo multichannel fast-scan cyclic voltammetry for comparison of its extracellular regulation. Pharmacological, anatomical, and electrochemical evidence suggest that electrical stimulation of the LC evokes NE release in the VTA and activates VTA-DA neurons, resulting in DA release in the NAc. The electrically evoked DA in the NAc was regulated by D2 receptors and DA transporters (DAT) as well as α1-adrenergic receptors in the VTA, whereas NE release in the VTA was regulated by α2-adrenergic receptors and NE transporters (NET) not by D2 receptors or DAT. These results suggest that electrical stimulation of LC modulates VTA-DA neurons and DA transmission in the NAc via NE receptors. KEYWORDS: dopamine, norepinephrine, ventral tegmental area, locus coeruleus, fast-scan cyclic voltammetry, nucleus accumbens
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INTRODUCTION Dopamine (DA) and norepinephrine (NE), the major catecholamines in the brain, are involved in a variety of physiological processes and behaviors including learning, attention, and memory.1−4 Accumulating studies have revealed crosstalk between DA and NE neurons that is important for homeostatic control of brain function.5,6 Therefore, determining the mechanisms of these interactions is essential for understanding how disruption of communication between the two systems contributes to many psychiatric disorders and substance abuse.5,6 The primary source of brain NE, the locus coeruleus (LC, A6), projects diffusely throughout the central nervous system, yet dense terminal locations remain highly selective.1,7 LC-NE © XXXX American Chemical Society
neurons are involved in promoting attention and modulating stress and anxiety and are implicated in the processes that underlie addiction to drugs of abuse.2,8 Previous anatomical and physiological studies have shown that the ascending fibers of LC-NE neurons terminate in the vicinity of DA cell bodies in the ventral tegmental area (VTA, A10) and have revealed the presence of NE and its receptors such as β (e.g., β1 and β2)- and α (e.g., α1 and α2)-adrenergic receptors throughout the VTA as well as NE transporters (NET).9−11 VTA-DA neurons, the major source of DA in the mesolimbic DA pathway, innervate Received: February 26, 2017 Accepted: June 8, 2017 Published: June 8, 2017 A
DOI: 10.1021/acschemneuro.7b00078 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 1. Maps of maximal catecholamine (CA) release in the NAc and VTA evoked by electrical stimulation of the LC. A coronal section of the rat brain (AP +1.8 mm from bregma)20 schematically shows the approximate track (left, black dotted line) of the CFM aimed at the anteromedial NAc shell (shaded in green) (A) and the VTA (AP −5.2 mm from bregma, shaded in red)20 (B) with the site of electrolytic lesion (right, red dotted circle). Representative responses evoked by electrical stimulation of the LC in the NAc (C) and VTA (D) at the oxidation peak potential for CA as the CFM was lowered through the brain. The inset shows the voltammogram of CA at the peak concentration value. The red bar under the traces denotes the period of electrical stimulation (60 Hz, 60 pulses, 300 μA). (E) Relative CA concentrations in the NAc evoked by electrical stimulation of the LC (pink line) or VTA (black line). (F) Relative concentration of CA evoked by electrical stimulation of the LC in the VTA. Relative concentration is the concentration at a particular depth (Cdx) divided by the maximum concentration (Cdmax). NAc, nucleus accumbens; DS, dorsal striatum; SN, substantia nigra; VTA, ventral tegmental area.
many limbic structures including the nucleus accumbens (NAc) and play a critical role in reward, motivation, and drug addiction.3,4,12 Although the precise localization of NE receptors and the mechanisms through which they regulate VTA-DA transmission still remains unclear, an increasing number of studies have shown a net excitatory effect of LC-NE on VTA-DA neural activity and DA transmission via α1-adrenergic receptors that are primarily localized on presynaptic neurons in the VTA.2,10,13,14 For example, in vivo studies have shown that electrical stimulation of the LC leads to excitation of VTA-DA neurons15 whereas blockade of α1-adrenergic receptors attenuates psychostimulant-induced DA transmission in the NAc.14 However, little is known about how LC-NE neurons modulate NE transmission at their terminals in the VTA and whether this has downstream effects on DA regulation (release and clearance) in limbic structures. This is due in part to the limitations of conventional in vivo neurochemical techniques for measuring rapid changes of extracellular catecholamines within discrete brain regions, which lack the spatial and temporal resolutions necessary to investigate the underlying mechanisms of these interactions.
In this study, we determined and characterized the regulation of extracellular catecholamines in the NAc and VTA simultaneously evoked by electrical stimulation of the LC in urethane-anesthetized rats using in vivo multichannel fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFMs). This technique allows for real time measurements of the dynamics of electroactive neurochemicals (e.g., monoamines, H2O2, and adenosine) in discrete brain regions with high temporal (milliseconds) and spatial (micrometer) resolution as well as high chemical sensitivity (nanomolar range).16−19 Here, our results suggest that the major catecholamines evoked by phasic electrical stimulation of the LC in the NAc and VTA are DA and NE, respectively, through anatomical, pharmacological, and electrochemical evidence. Furthermore, we demonstrated that electrical stimulation of the LC modulates DA transmission in the NAc via α1-adrenergic receptors in the VTA.
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RESULTS AND DISCUSSION Electrical Stimulation of the LC Evokes Catecholamine Release in the NAc and VTA. LC-NE axons project to and innervate VTA-DA neurons. However, little is known as B
DOI: 10.1021/acschemneuro.7b00078 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
that DA signaling in the NAc is also associated with aversion and salient nonrewarding events.16,27,28 These different roles of DA in the NAc may be due to the topographical organization and regional specificity of VTA-DA subpopulations projecting to the NAc, which are modulated by stimuli of both positive and negative valence in order to generate appropriate behaviors.28,29 Additionally, electrical stimulation of the LC or superfusion of NE in the VTA evokes heterogeneous responses (e.g., excitation or inhibition) of VTA-DA neurons.13,30 Based on these previous studies, our results suggest that a subpopulation of VTA-DA neurons projecting to the medioventral NAc may be excited by phasic LC stimulation. Therefore, DA signaling in the dorsomedial and medioventral NAc may have distinct roles in encoding stimulus outcome relationships however the behavioral consequences of LC-NE and VTA-DA interactions remain a site of future studies. Previous autoradiography, electron microscopy, and immunohistochemical studies have demonstrated that LC-NE neurons terminate in the VTA and the connections occurring between NE varicosities and DA dendrites in the VTA show features typical of extrasynaptic sites, yet the actual number of synapses formed between them are very few ( 5) in anesthetized animals recorded by FSCV primarily depend on electrically evoked release, rather than naturally occurring release, since the firing rate of catecholaminergic neurons is suppressed during anesthesia. A recent in vivo FSCV study revealed that electrical stimulation of the medial forebrain bundle (MFB) in anesthetized rats evokes somatodendritic DA release in the VTA but not NE, and its release was ∼10-fold lower than axon terminal DA release in the NAc.33 This result is in opposition to somatodendritic NE release, where similar concentrations are observed between the LC and its terminals.34 In addition, previous microdialysis and push−pull perfusion studies have revealed appreciable amounts of NE in the VTA of awake, behaving rats, and the basal NE level is similar or even higher than the basal DA level in the VTA.9,35−37 These previous studies suggest that the major catecholamine in the VTA evoked by electrical stimulation of the LC in our study is mainly from LC-NE neurons rather than somatodendritic DA release although they did not demonstrate the origin of NE nor provide a local view of its regulation in the VTA. Dynamics of Catecholamine Release in the NAc and VTA Evoked by LC Stimulation. In the next set of experiments, CFMs were fixed in the NAc or the VTA or both, while the stimulating electrode was lowered from 5.0 to 9.0 mm below the skull surface to determine catecholamine release as a function of stimulating electrode depth in the LC. Catecholamine release was monitored while electrical stimulation (60 Hz, 60 pulses) was delivered to the LC (Figure 2A, shaded in blue) at different depths in increments of 0.2−0.3 mm. Figure 2C shows representative responses in the NAc and VTA evoked by electrical stimulation of the LC. Catecholamine release in both the NAc and VTA was not observed until the stimulating electrode reached a depth of ∼5.0 mm, with maximal release seen at ∼7.0 mm. When averaged from multiple animals, evoked catecholamines in the NAc (black line) and VTA (pink line) were observed at depths of the
to how activation of the LC regulates rapid catecholamine transmission in the VTA and at VTA-DA terminals in the NAc. In this first set of experiments, a bipolar stimulating electrode was positioned near the LC (∼6.5 mm from the skull), while two CFMs were lowered separately through the NAc and VTA in increments of 0.2 to 0.3 mm beginning at ∼6.0 mm to characterize the distribution of catecholamine release evoked by phasic LC stimulation (60 Hz, 60 pulses). Figure 1A,B (left) shows the location of the NAc (green circle) and VTA (red circle) in coronal sections,20 respectively, with the approximate track of the CFM (dotted black line). Figure 1C,D shows representative responses to electrical stimulation of the LC at the oxidation peak potential of catecholamines (∼ +0.65 V) as the CFM was lowered through the brain. Individual background subtracted voltammograms identified the evoked release as catecholamines since they were oxidized on the forward scan to their ortho-quinone forms at ∼ +0.6 to +0.7 V with the reduction peak of the formed quinone at ∼ −0.2 to −0.3 V on the reverse scan.7 The distinct redox potential from the catecholamine voltammogram allows them to be distinguished from interferences such as catecholamine metabolites and ascorbic acid in the extracellular fluid. However, NE and DA cannot be distinguished based on their voltammograms due to their similar oxidation and reduction potentials.21,22 The maximum catecholamine release during LC stimulation was seen near ∼7.5 mm in the NAc and ∼9.0 mm in the VTA below the skull. The normalized average catecholamine concentrations in the NAc evoked by electrical stimulation of the LC (pink line) and VTA (black line) as a function of CFM depth are shown in Figure 1E. Interestingly, the catecholamine evoked by LC stimulation was mainly observed in medioventral NAc (∼7.0−7.8 mm). In contrast, catecholamine release evoked by direct stimulation of the VTA was mostly seen in the dorsomedial subregion (∼6.0−7.0 mm), with less release observed in the medioventral subregion. Figure 1F shows the normalized average catecholamine concentrations in the VTA evoked by LC stimulation as a function of CFM depth in which the maximum catecholamine release was observed between 8.0 and 9.5 mm, consistent with the approximate location of the VTA. At the end of experiments, a lesion was made at the recording site by applying constant current (20 μA for 10 s) to the electrode to verify electrode placement in the NAc and VTA (Figure 1A,B, right, dotted red circles).7 Only data obtained after histological verification of electrode placement and confirmed by electrolytic lesion were included in the normalized average concentrations. The anteromedial NAc coordinate (AP +1.8 mm) in this study was chosen based on previous neurochemical and anatomical studies showing that the predominant catecholamine in the NAc is DA, originating mainly from the VTA.23−25 In detail, very few LC fibers have been observed within the anteromedial NAc where the tissue content of DA and NE are ∼99% and 1%, respectively.23,24 In contrast, the posteromedial NAc receives both DA and NE innervation; however the NE inputs mainly originate in the A1 and A2 noradrenergic nuclei.23−25 Therefore, the major catecholamine in the NAc evoked by LC stimulation is likely DA. Importantly, this evoked release was primarily observed in the medioventral NAc, which is in contrast to direct stimulation of the VTA that shows the greatest response in the dorsomedial NAc.16,24 VTA-DA signaling in the dorsomedial NAc is generally implicated in the processing of rewards and encoding goal directed behaviors.12,26 However, increasing evidence has indicated C
DOI: 10.1021/acschemneuro.7b00078 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
stimulation of the LC as a result of catecholamine release and then decreased back to the prestimulation level once stimulation ceased due to uptake of catecholamines and diffusion from varicosities in the synapse near the microelectrodes.38 Table 1 shows the average maximal catecholamine concentration ([CA]max), the rise time (tr, time to reach the Table 1. Average Maximum Catecholamine Concentration, Rise Time, and Half-Decay Time in the NAc and VTA Evoked by Electrical Stimulation (60 Hz, 60 Pulses) of the LCa rats (n = 5)
CA in the NAc
CA in the VTA
[CA]max (μM) t1/2 (s) tr (s)
0.20 ± 0.05 0.8 ± 0.3 0.6 ± 0.1
0.18 ± 0.02 2.1 ± 0.2b 1.6 ± 0.1b
a
[CA]max is the maximal evoked catecholamine (CA) concentration, rise time (tr) is the time to reach [CA]max and half-decay time (t1/2) is the time for [CA]max to decay to half of the maximum. Values represent the mean ± SEM and were analyzed with the Student’s t test. bSignificant differences between CA in the NAc and the VTA (p < 0.01)
maximum concentration), and the half-decay time (t1/2, the time required to decay to half of the maximum concentration) of the evoked catecholamines in the NAc and VTA. The [CA]max in the NAc (0.20 ± 0.05 μM) evoked by LC stimulation was similar to that in the VTA (0.18 ± 0.02 μM) but approximately 7-fold lower than the [DA]max (1.35 ± 0.33 μM) evoked by direct electrical stimulation of VTA-DA neurons under identical experimental conditions (60 Hz and 60 pulses).24 The rise time in the VTA (tr = 1.6 s) was significantly slower than in the NAc (tr = 0.6 s, t(8) = 8.50, p < 0.01). Furthermore, the half-decay time for the catecholamine in the VTA (t1/2, = 2.1 s) was much slower than that of the catecholamine in the NAc (t1/2 = 0.8 s, t(8) = 3.61, p < 0.01). These values of the catecholamine in the VTA are close to those of NE evoked by LC stimulation in the BNST. Generally, the NE uptake rates in NE rich brain regions receiving dense LC innervation, including the thalamus (t1/2 = 1.3 s) and BNST (t1/2 = 2.5 s), are much slower than for DA uptake in the striatum.7,22,38 Slower uptake in the VTA may allow for the catecholamine to participate in volume transmission, which may affect other brain regions near the VTA greater than the evoked catecholamine in the NAc.10 Simultaneous measurements in the same animal under identical conditions eliminate experimental confounding variables, such as varying levels of anesthesia, and provide direct comparisons of catecholamine regulation between the two brain regions, the NAc and VTA. However, the success of such measurements was low (10 μM) inhibited VTA-DA neurons by activating D2 receptors.57 Therefore, complex interactions exist between LC-NE and VTA-DA neurons due to contributions from both excitatory α1-adrenergic receptors and inhibitory D2 receptors in the VTA. Interestingly, Goertz et al.14 demonstrated that activation of postsynaptic α1adrenergic receptors in VTA-DA neurons also enhanced VTA-DA neural activity. However, it is generally known that activation of presynaptic α1-adrenergic receptors in the VTA enhances VTA-DA neural activity by exciting or inhibiting glutamatergic58 and GABAergic59 neurons, respectively. Therefore, both pre- and postsynaptic α1-adrenergic receptors potentiate VTA-DA neural activity. Taken together, these previous studies support our findings that phasic DA release in the NAc evoked by electrical stimulation of the LC is modulated by both D2 receptors and α1-adrenergic receptors in the VTA. Importantly, microinfusion of lidocaine into the VTA (Figure 4C) nearly eliminated this release, suggesting that DA release in the NAc evoked by LC stimulation is primarily from and modulated through the VTA. Although our neurochemical data cannot distinguish between the contributions of excitation of pre- and postsynaptic α1-adrenergic receptors and inhibition of D2 receptors on VTA-DA neural activity, these results suggest a net excitatory effect of electrical stimulation of LC-NE neurons on DA transmission in the NAc via α1-adrenergic receptors in the VTA. These findings further elucidate the mechanisms of previous studies that have suggested noradrenergic signaling in the VTA may play a critical role in modulating psychostimulant effects on VTA-DA neurons and DA-mediated behaviors via α 1 -adrenergic receptors.14,60,61
receptors may have an excitatory influence on VTA-DA transmission in the NAc. The decrease in [CA]max and t1/2 by the α2-adrenergic receptor antagonist was slightly increased following desipramine administration (Figure 3C,E,G). This might be due to a dampening of the effect of the α2-adrenergic receptor antagonist with time since the NET inhibitor alone did not significantly increase the evoked [CA]max and t1/2 (data not shown). In contrast to DA release in the NAc, which has been characterized in great detail using electroanalytical methods with pharmacological manipulations, relatively few studies have investigated catecholamine regulation in the VTA.40,41,45 Interestingly, an in vivo FSCV study indicated that somatodendritic DA release in the VTA evoked by MFB stimulation was regulated by DAT, not by NET, despite the NET having a higher affinity for DA than for NE.33,44 In contrast, our results demonstrated that the NET and α2-adrenergic receptor inhibitors significantly increased both the LC evoked [CA]max and t1/2 in the VTA whereas the DAT and D2 receptor inhibitors had little effect. Therefore, the major catecholamine in the VTA evoked by LC stimulation is likely NE and is regulated by both presynaptic α2-autoreceptors and NET. However, we cannot ignore the possibility that the terminals of LC-NE neurons may co-release DA and NE in the VTA, similar to the prefrontal cortex and hippocampus, and is an intriguing possibility worth investigating.46,47 Electrical Stimulation of the LC Modulates DA Transmission in the NAc through VTA-DA Neurons. Although electrical stimulation of the LC evoked a time locked release of DA in the NAc and NE in the VTA (Figure 2D,E), systemically administered pharmacological characterization alone does not provide clear evidence whether electrical stimulation of the LC modulates DA transmission in the NAc via VTA-DA neurons or through long-loop indirect anatomical connections as the LC projects diffusely throughout the central nervous system and influences DA release in the NAc.1,8 In order to examine whether LC-NE modulates DA transmission in the NAc directly through the VTA-DA neurons, we microinfused saline followed by the D2 receptor antagonist raclopride, α1-adrenergic receptor antagonist terazosin, or sodium channel blocker lidocaine into the VTA. For these experiments, all drugs were delivered through a microinfusion cannula combined with a bipolar stimulating electrode that was implanted into the VTA, while an additional stimulating electrode was used to deliver electrical stimulation to the LC.7 The success ratio was low (∼40%) for these experiments because of the challenges of unilaterally positioning two separate stimulating electrodes with one CFM and the potential for disrupting the cannula and/or damage of the DA neurons caused by insertion of a microinfusion probe into the VTA, resulting in a sudden decrease or disappearance of the relatively small DA signal in the NAc evoked by LC stimulation. Saline (0.5 μL) followed by raclopride (RA, 0.5 μg), terazosin (TER, 3.0 μg), or lidocaine (LD, 82 μg) were delivered into the VTA over a period of 2 min with a syringe pump and LC evoked release in the NAc was recorded every 4 min. These doses were based on previous studies. 48−51 Figure 4A−C shows representative color plots and DA concentration traces for preinfusion control, saline, raclopride, terazosin, and lidocaine. Saline infusion into the VTA decreased [DA]max to ∼78% (t(9) = 7.99, p < 0.01) from predrug control that gradually recovered with time (Figure 4D, black line). This result was in agreement with a previous study showing saline infusion into the VTA G
DOI: 10.1021/acschemneuro.7b00078 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 5. Effect of electrical stimulation of the LC with different stimulation parameters (frequency and pulse number) on DA and NE in the NAc and VTA, respectively. Effect of frequency (10, 20, 40, and 60 Hz with 60 pulses) (A) in the NAc and (B) in the VTA. Effect of pulse number (10, 20, 40, 80, and 120 with 60 Hz) (C) in the NAc and (D) in the VTA. Maximal catecholamine concentrations in the VTA (red line) and NAc (blue line) with electrical stimulation of the LC in comparison to catecholamine concentrations in the NAc evoked by electrical stimulation of the VTA (black line) with varying (E) stimulation frequencies and (F) number of pulses.
Effects of LC-Electrical Stimulation Conditions on the Catecholamine Regulation in the VTA and NAc. The catecholamine responses in the NAc and VTA to different LC stimulation parameters (pulse number (n) and stimulation frequency (Hz)) were investigated. Figure 5A,B show representative DA and NE responses to increasing the stimulation frequency (10, 20, 40, and 60 Hz with 60 pulses) in the NAc and VTA, respectively. The frequencies from 5 to 20 Hz increased the evoked [DA]max in the NAc, however, [DA]max at higher frequencies (>20 Hz) reached a plateau, independent of the frequencies (Figure 5A,E). In contrast, the evoked [NE]max in the VTA continued to increase, even at higher frequencies (Figure 5B,E). Figure 5C,D shows the responses to LC stimulation with increasing pulse numbers (10, 20, 40, 80, 120 pulses with 60 Hz). [DA]max in the NAc increased at low pulse numbers (≤20) but did not increase over 40 pulses, whereas [NE]max in the VTA increased as a function of pulse number. Measurable NE responses to LC stimulation (S/N ≥ 5) were not often detected in the VTA with fewer than 20 pulses and lower frequencies (
