Neuroscience Letters 559 (2014) 61–66
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Dopamine inhibits neurons from the rat dorsal subcoeruleus nucleus through the activation of ␣2 -adrenergic receptors Nian Yang a , Kai-Yuan Zhang b , Fu-Fan Wang b , Zhi-An Hu a,∗∗ , Jun Zhang a,∗ a b
Department of Physiology, Third Military Medical University, Chongqing 400038, PR China Student Brigade, Third Military Medical University, Chongqing 400038, PR China
h i g h l i g h t s • Dopamine induces a hyperpolarization on SubCD neurons and inhibited their firing. • Dopamine dose dependently elicits a TTX-resistant outward current on SubCD neurons. • Dopamine inhibits SubCD neurons via ␣2 -adrenergic but not its endogenous receptors.
a r t i c l e
i n f o
Article history: Received 7 September 2013 Received in revised form 20 November 2013 Accepted 21 November 2013 Keywords: Dopamine Dorsal subcoeruleus nucleus ␣2 -adrenergic receptors
a b s t r a c t Previous studies have revealed that the central dopaminergic system may participate in regulating sleep/wakefulness. In particular, rapid eye movement (REM) sleep behavior disorder (RBD) occurs in patients with Parkinson’s disease (PD), highlighting the possible connection between dopamine and REM sleep-related neural structures. The dorsal subcoeruleus nucleus (SubCD) is a critical structure for the generation and maintenance of REM sleep. Thus, the present study investigated the modulatory effects of dopamine on SubCD neurons. Using whole-cell patch clamp recordings, we first observed that dopamine induced a hyperpolarization of the membrane potentials in SubCD neurons and thus inhibited their firing. We determined that a dose-dependent and tetrodotoxin-resistant postsynaptic outward current underpinned this inhibitory effect on SubCD neurons induced by dopamine. Finally, using pharmacological agents, we revealed that the dopamine-elicited outward current in SubCD neurons was mediated by ␣2 -adrenergic receptors, but not by the dopamine receptors, including D1-like and D2-like receptors. These results suggest that the central dopaminergic system may play a role in the regulation of REM sleep through the effect of dopamine on SubCD neurons. The relationship between the loss of this effect and the RBD in PD is discussed. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Central dopaminergic system plays a crucial role in several basic nervous functions, such as motor control, reward processing, and mood [1,2]. Intriguingly, several lines of evidence, including lesion studies of the central dopaminergic system [3,4], genetic manipulations of the activity of dopaminergic system [5,6], and neuropharmacological studies employing the injection of dopamine and dopaminergic agents systemically or locally in several brain structures [7,8], raise a strong possibility that dopamine may also modulate sleep/wakefulness. Recently, interest in the modulatory mechanisms of dopamine on sleep/wakefulness has
∗ Corresponding author. Tel.: +86 23 68775266. ∗∗ Corresponding author. Tel.: +86 23 68752254. E-mail addresses: [email protected] (Z.-A. Hu), [email protected] (J. Zhang). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.037
increased [9]. In particular, clinical observations in patients with Parkinson’s disease (PD), which is caused by the degeneration of dopaminergic neurons, have revealed sleep disturbances, especially the rapid eye movement (REM) sleep behavior disorder (RBD) [10]. Taken together, these results highlight a possible connection between dopamine and REM sleep-related neural structures. REM sleep is a complex neural process, and several important brain structures are involved [11]. Among these structures, dorsal subcoeruleus nucleus (SubCD) is thought critical for the generation and maintenance of REM sleep [12], because lesions of the SubCD dramatically affect REM sleep [13] and may even completely suppress it [14]. Importantly, c-Fos immunostaining has revealed that neuronal activity within the SubCD is highest during REM sleep [15]. Furthermore, increasing neuronal activity in the SubCD through microinjections of either GABAA receptor antagonist bicuculline [15,16] or glutamatergic receptor agonist kainic acid [15,17] can produce a REM-like state, while inactivating the SubCD through microinjections of tetrodotoxin (TTX) decreases the amount of REM
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N. Yang et al. / Neuroscience Letters 559 (2014) 61–66
sleep [18]. These results strongly suggest that the neuronal activity of the SubCD is associated with REM sleep and modulation of the neuronal activity may affect REM sleep. Intriguingly, the infusion of dopamine through a microdialysis probe into the SubCD also affects REM sleep [19]. However, whether dopamine influences neuronal activity within the SubCD and the mechanisms that may underlie a putative dopaminergic influence are unclear. In the present study, we investigated the electrophysiological effects of dopamine on SubCD neurons and the receptor mechanisms underlying these effects. The relationship between a loss of the dopaminergic effects and the RBD in PD is discussed. 2. Materials and methods 2.1. Brain slice preparation Coronal brainstem slices (300–400 m) containing the SubCD were prepared from Sprague-Dawley rats aged 8–13 days. Under sodium pentobarbital (40 mg/kg) anesthesia, rats were decapitated and the brainstem was rapidly removed into 95% O2 and 5% CO2 oxygenated ice-cold cutting solution (composition in mM: 220 sucrose, 2.5 KCl, 1.25 NaH2 PO4 , 6 MgCL2 , 26 NaHCO3 , 1 CaCl2 , and 10 d-glucose). The coronal slices were cut with a VT1200 vibroslicer (Leica, Germany) and then incubated in oxygenated artificial cerebrospinal fluid (ACSF, composition in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2 PO4 , 1.3 MgSO4 , 26 NaHCO3 , 2 CaCl2 , and 20 d-glucose) at room temperature for at least 1 h. During the recording session, the slices were continuously superfused with oxygenated ACSF at a rate of approximately 2 ml/min at room temperature in a submerged chamber. 2.2. Recorded SubCD region verification and whole-cell patch clamp recording Whole-cell recordings were performed on SubCD neurons with borosilicate glass pipettes (3–5 M) filled with an internal solution (composition in mM: 130 K-gluconate, 5 KCl, 2 MgCl2 , 10 HEPES, 0.1 EGTA, 2 Na2 ATP, 2 Na2 -GTP, 4 Na2 -phosphocreatine, adjusted to pH 7.25 with 1 M KOH). First, the location of the SubCD was identified and SubCD neurons in the slices were verified by using a BX51WI microscope (Olympus, Japan) with infrared differential interference contrast (IR-DIC) illumination. According to the rat brain atlas [20], SubCD is located just ventral to the mesencephalic trigeminal nucleus and the locus coeruleus, which both contain a high density of large neurons and can easily be identified. Moreover, cells are more scattered in the SubCD as compared with the surrounding regions [21]. Therefore, it is not difficult to locate the SubCD precisely by using IR-DIC. The recording sites were restricted to a SubCD region that was in the coronal brain slice obtained immediately rostral to the facial nerve (7n) and located dorsomedial to the motor trigeminal nucleus (Mo5) in this slice. This region of the SubCD mainly contains SubCD reticular neurons that are closely associated with REM sleep [21,22]. The patch clamp recordings were acquired with a MultiClamp-700B amplifier (Molecular Device, USA). For whole-cell recording, neurons were first held at a membrane potential of −70 mV. Notably, throughout the entire experiment, the series resistance was continually monitored, and a recorded neuron was excluded from analysis if the series resistance changed by more than 15% or exceeded 20 M. In addition, only neurons with a membrane resistance higher than 150 M were included in the final analysis. After recording a stable baseline for at least 15 min, the responses of SubCD neurons to dopamine, noradrenaline, and dopaminergic and noradrenergic agents were observed. Using the Digidata-1440A interface (Molecular Device,
USA), recordings of membrane potentials or whole-cell currents were low pass filtered at 5 kHz and 2 kHz, respectively, and then digitized at 10 kHz. 2.3. Drugs All drugs except tetrodotoxin (TTX) were purchased from Sigma, USA. TTX was obtained from Refine, China. Drugs were bath applied. TTX, dopaminergic and noradrenergic antagonists, and fusaric acid were continually superfused for at least 15 min before dopamine was applied. 2.4. Data capture and analysis Data capture and analyses were conducted with pClamp 10 (Molecular Device, USA) and Origin (OriginLab, USA) software. Student’s t-test was employed for statistical analysis and P-values of
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Dopamine inhibits neurons from the rat dorsal subcoeruleus nucleus through the activation of ␣2 -adrenergic receptors Nian Yang a , Kai-Yuan Zhang b , Fu-Fan Wang b , Zhi-An Hu a,∗∗ , Jun Zhang a,∗ a b
Department of Physiology, Third Military Medical University, Chongqing 400038, PR China Student Brigade, Third Military Medical University, Chongqing 400038, PR China
h i g h l i g h t s • Dopamine induces a hyperpolarization on SubCD neurons and inhibited their firing. • Dopamine dose dependently elicits a TTX-resistant outward current on SubCD neurons. • Dopamine inhibits SubCD neurons via ␣2 -adrenergic but not its endogenous receptors.
a r t i c l e
i n f o
Article history: Received 7 September 2013 Received in revised form 20 November 2013 Accepted 21 November 2013 Keywords: Dopamine Dorsal subcoeruleus nucleus ␣2 -adrenergic receptors
a b s t r a c t Previous studies have revealed that the central dopaminergic system may participate in regulating sleep/wakefulness. In particular, rapid eye movement (REM) sleep behavior disorder (RBD) occurs in patients with Parkinson’s disease (PD), highlighting the possible connection between dopamine and REM sleep-related neural structures. The dorsal subcoeruleus nucleus (SubCD) is a critical structure for the generation and maintenance of REM sleep. Thus, the present study investigated the modulatory effects of dopamine on SubCD neurons. Using whole-cell patch clamp recordings, we first observed that dopamine induced a hyperpolarization of the membrane potentials in SubCD neurons and thus inhibited their firing. We determined that a dose-dependent and tetrodotoxin-resistant postsynaptic outward current underpinned this inhibitory effect on SubCD neurons induced by dopamine. Finally, using pharmacological agents, we revealed that the dopamine-elicited outward current in SubCD neurons was mediated by ␣2 -adrenergic receptors, but not by the dopamine receptors, including D1-like and D2-like receptors. These results suggest that the central dopaminergic system may play a role in the regulation of REM sleep through the effect of dopamine on SubCD neurons. The relationship between the loss of this effect and the RBD in PD is discussed. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Central dopaminergic system plays a crucial role in several basic nervous functions, such as motor control, reward processing, and mood [1,2]. Intriguingly, several lines of evidence, including lesion studies of the central dopaminergic system [3,4], genetic manipulations of the activity of dopaminergic system [5,6], and neuropharmacological studies employing the injection of dopamine and dopaminergic agents systemically or locally in several brain structures [7,8], raise a strong possibility that dopamine may also modulate sleep/wakefulness. Recently, interest in the modulatory mechanisms of dopamine on sleep/wakefulness has
∗ Corresponding author. Tel.: +86 23 68775266. ∗∗ Corresponding author. Tel.: +86 23 68752254. E-mail addresses: [email protected] (Z.-A. Hu), [email protected] (J. Zhang). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.037
increased [9]. In particular, clinical observations in patients with Parkinson’s disease (PD), which is caused by the degeneration of dopaminergic neurons, have revealed sleep disturbances, especially the rapid eye movement (REM) sleep behavior disorder (RBD) [10]. Taken together, these results highlight a possible connection between dopamine and REM sleep-related neural structures. REM sleep is a complex neural process, and several important brain structures are involved [11]. Among these structures, dorsal subcoeruleus nucleus (SubCD) is thought critical for the generation and maintenance of REM sleep [12], because lesions of the SubCD dramatically affect REM sleep [13] and may even completely suppress it [14]. Importantly, c-Fos immunostaining has revealed that neuronal activity within the SubCD is highest during REM sleep [15]. Furthermore, increasing neuronal activity in the SubCD through microinjections of either GABAA receptor antagonist bicuculline [15,16] or glutamatergic receptor agonist kainic acid [15,17] can produce a REM-like state, while inactivating the SubCD through microinjections of tetrodotoxin (TTX) decreases the amount of REM
62
N. Yang et al. / Neuroscience Letters 559 (2014) 61–66
sleep [18]. These results strongly suggest that the neuronal activity of the SubCD is associated with REM sleep and modulation of the neuronal activity may affect REM sleep. Intriguingly, the infusion of dopamine through a microdialysis probe into the SubCD also affects REM sleep [19]. However, whether dopamine influences neuronal activity within the SubCD and the mechanisms that may underlie a putative dopaminergic influence are unclear. In the present study, we investigated the electrophysiological effects of dopamine on SubCD neurons and the receptor mechanisms underlying these effects. The relationship between a loss of the dopaminergic effects and the RBD in PD is discussed. 2. Materials and methods 2.1. Brain slice preparation Coronal brainstem slices (300–400 m) containing the SubCD were prepared from Sprague-Dawley rats aged 8–13 days. Under sodium pentobarbital (40 mg/kg) anesthesia, rats were decapitated and the brainstem was rapidly removed into 95% O2 and 5% CO2 oxygenated ice-cold cutting solution (composition in mM: 220 sucrose, 2.5 KCl, 1.25 NaH2 PO4 , 6 MgCL2 , 26 NaHCO3 , 1 CaCl2 , and 10 d-glucose). The coronal slices were cut with a VT1200 vibroslicer (Leica, Germany) and then incubated in oxygenated artificial cerebrospinal fluid (ACSF, composition in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2 PO4 , 1.3 MgSO4 , 26 NaHCO3 , 2 CaCl2 , and 20 d-glucose) at room temperature for at least 1 h. During the recording session, the slices were continuously superfused with oxygenated ACSF at a rate of approximately 2 ml/min at room temperature in a submerged chamber. 2.2. Recorded SubCD region verification and whole-cell patch clamp recording Whole-cell recordings were performed on SubCD neurons with borosilicate glass pipettes (3–5 M) filled with an internal solution (composition in mM: 130 K-gluconate, 5 KCl, 2 MgCl2 , 10 HEPES, 0.1 EGTA, 2 Na2 ATP, 2 Na2 -GTP, 4 Na2 -phosphocreatine, adjusted to pH 7.25 with 1 M KOH). First, the location of the SubCD was identified and SubCD neurons in the slices were verified by using a BX51WI microscope (Olympus, Japan) with infrared differential interference contrast (IR-DIC) illumination. According to the rat brain atlas [20], SubCD is located just ventral to the mesencephalic trigeminal nucleus and the locus coeruleus, which both contain a high density of large neurons and can easily be identified. Moreover, cells are more scattered in the SubCD as compared with the surrounding regions [21]. Therefore, it is not difficult to locate the SubCD precisely by using IR-DIC. The recording sites were restricted to a SubCD region that was in the coronal brain slice obtained immediately rostral to the facial nerve (7n) and located dorsomedial to the motor trigeminal nucleus (Mo5) in this slice. This region of the SubCD mainly contains SubCD reticular neurons that are closely associated with REM sleep [21,22]. The patch clamp recordings were acquired with a MultiClamp-700B amplifier (Molecular Device, USA). For whole-cell recording, neurons were first held at a membrane potential of −70 mV. Notably, throughout the entire experiment, the series resistance was continually monitored, and a recorded neuron was excluded from analysis if the series resistance changed by more than 15% or exceeded 20 M. In addition, only neurons with a membrane resistance higher than 150 M were included in the final analysis. After recording a stable baseline for at least 15 min, the responses of SubCD neurons to dopamine, noradrenaline, and dopaminergic and noradrenergic agents were observed. Using the Digidata-1440A interface (Molecular Device,
USA), recordings of membrane potentials or whole-cell currents were low pass filtered at 5 kHz and 2 kHz, respectively, and then digitized at 10 kHz. 2.3. Drugs All drugs except tetrodotoxin (TTX) were purchased from Sigma, USA. TTX was obtained from Refine, China. Drugs were bath applied. TTX, dopaminergic and noradrenergic antagonists, and fusaric acid were continually superfused for at least 15 min before dopamine was applied. 2.4. Data capture and analysis Data capture and analyses were conducted with pClamp 10 (Molecular Device, USA) and Origin (OriginLab, USA) software. Student’s t-test was employed for statistical analysis and P-values of
