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Presynaptic α2-adrenoceptor modulates glutamatergic synaptic transmission in rat nucleus accumbens in vitro
Neuroscience Letters 665 (2018) 117–122
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Research article
Presynaptic α2-adrenoceptor modulates glutamatergic synaptic transmission in rat nucleus accumbens in vitro ⁎
Shi-Yu Peng1, Bin Li1, Kang Xi, Jian-Jun Wang , Jing-Ning Zhu
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State Key Laboratory of Pharmaceutical Biotechnology and Department of Biological Science and Technology, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China
A R T I C L E I N F O
A B S T R A C T
Keywords: α2-Adrenoceptor Nucleus accumbens shell Glutamatergic transmission Excitatory postsynaptic current Presynaptic modulation
The nucleus accumbens (NAc), integrating information from the prefrontal cortex and limbic structures, plays a critical role in reward and emotion regulation. Previous studies have reported that the NAc shell receives direct noradrenergic projections, and activation of α2-adrenoceptor (α2-AR) in the NAc shell decreases the fear or anxiety level of rats. However, the underlying mechanism is still little known. Intriguingly, glutamatergic neurotransmission in the NAc shell is closely related to reward and emotion. Here, using brain slice preparations and whole-cell patch clamp recordings, we examined the effect of activation of α2-AR on glutamatergic neurotransmission in the NAc shell. Perfusing slice with α2-AR selective agonist clonidine (CLON) reduced the evoked excitatory postsynaptic currents (EPSCs) on the NAc shell neurons. This inhibitory effect on AMPAmediated glutamatergic EPSCs was blocked by the α2-AR selective antagonist yohimbine (YOH). Notably, CLON reduced the frequency but not the amplitude of miniature EPSCs. Furthermore, CLON decreased the first EPSC amplitude but increased the paired-pulse facilitation on the NAc shell neurons, and it did not affect postsynaptic AMPA/NMDA ratio, revealing a presynaptic mechanism of α2-AR-mediated inhibition on glutamatergic transmission. In addition, the modulation on glutamatergic transmission by α2-AR was independent of presynaptic NMDA receptor. These results suggest that noradrenergic afferent inputs may suppress glutamatergic synaptic transmission via presynaptic α2-AR in the NAc shell, and actively participate in rewarding and emotional processes via the NAc.
1. Introduction The nucleus accumbens (NAc), a key structure of the mesolimbic circuitry, receives excitatory inputs from the medial prefrontal cortex, basolateral amygdala, thalamus and hippocampus and, in turn, projects to other basal ganglia nuclei, which send feedback projections into the prefrontal cortex [21]. This anatomical organization places the NAc as an integrator responsible for several important higher brain functions, including rewarding and emotional processing [23,36]. Based on cytomorphologic and immunohistochemical characteristics, the NAc is anatomically divided into two distinct structures, the core and the shell [21]. The latter has been implicated in the cognitive processing of reward, such as pleasurable stimuli, motivational salience and positive reinforcement [3,55]. Moreover, the NAc shell also mediates the anxiety behaviors in response to threatening stimuli [2,34]. Notably, most of the inputs into NAc are glutamatergic, which not only encode the cues and descriptive features [17,28], but also promote synaptic plasticity to strengthen the pertinent environmental cues after addictive
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drug use [47,48]. In addition, it has been reported that a delayed glutamate release occurs in the NAc following a conditioned emotional response [46]. Interestingly, neuroanatomical and tracing studies have revealed a moderate dense of noradrenergic projections from the locus coeruleus [45], ventrolateral medulla [30], and nucleus of the solitary tract [15] to the NAc, indicating that the central noradrenergic system and norepinephrine (NE) may regulate rewarding and emotional processing through the NAc. Central noradrenergic system is one of the most important neuromodulatory systems in the central nervous system [45]. It is generally implicated in regulation of many emotional functions, including anxiety [1], and reward [18,54]. The concentration of NE increases rapidly in the locus coeruleus, amygdala and hypothalamus in fear/anxiety rats [42,52]. Importantly, microinjection of α2-adrenoceptor (α2-AR) selective agonist clonidine (CLON) in the NAc shell decreased the level of fear/anxiety in rats in the elevated plus maze test [31]. However, the underlying mechanism remains unclear. Therefore, considering the great contribution of glutamatergic
Corresponding authors. E-mail addresses: [email protected] (J.-J. Wang), [email protected] (J.-N. Zhu). These authors contributed equally.
https://doi.org/10.1016/j.neulet.2017.11.060 Received 13 October 2017; Received in revised form 25 November 2017; Accepted 27 November 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
Neuroscience Letters 665 (2018) 117–122
S.-Y. Peng et al.
Fig. 1. Activation of α2-AR inhibits the glutamatergic transmission in the NAc shell neurons. A The image shows the placement of stimulating electrode and the recorded NAc shell neuron on a coronal brain slice. B Raw traces show the APMA receptormediated eEPSC on NAc shell neuron in the presence of TTX and SR95531. Note that the fast evoked EPSC was totally blocked by AMPA/KA receptor antagonist NBQX. C Bath application of CLON, a selective α2-AR agonist, decrease the amplitude of eEPSCs, and this effect could be washed out. D Bar graphs show the effect of CLON on the AMPA-mediated eEPSC. E Application of YOH, a selective α2-AR antagonist, blocked the CLON-induced inhibitory effect on AMPA-mediated eEPSCs in NAc shell neurons. F Bar graphs show the blocking effect of YOH on the CLON-induced inhibition of eEPSCs. G Application of YOH blocked the NA-induced inhibitory effect on AMPA-mediated eEPSCs in NAc shell neurons. H Bar graphs show the blocking effect of YOH on the NAinduced inhibition of eEPSCs. Data are shown as mean ± SEM; **P < 0.01, ***P < 0.001 and n.s., no statistical difference.
transmission in NAc to reward and emotion regulation, in the present study, using whole-cell patch clamp recordings, we investigated the role of α2-AR in the modulation of glutamatergic transmission in the NAc shell. The results show that activation of α2-AR inhibits the glutamatergic transmission in the NAc shell presynaptically.
The slices were stored for at least 1 h in a holding chamber filled with 95% O2 and 5% CO2 oxygenated ACSF at 35 ± 0.2 °C before transferring to a recording chamber.
2. Materials and methods
Visualized whole-cell recordings were performed as our previous reports [20,56]. All the excitatory postsynaptic current (EPSC) recordings were made with electrodes filled with a solution containing the following (in mM): 120 cesium methanesulfonate, 20 CsCl, 10 HEPES, 0.2 EGTA, 10 sodium phosphocreatine, 5 QX-314, 4 Na2-ATP, 0.4 NaGTP, pH 7.2 (290–330 mOsm). Electrode resistance in the bath solution was 5–7 MΩ and series resistance (< 25 MΩ) was monitored continuously and stable to within 20%. Whole-cell patch clamp recordings were performed from the medium spiny neurons, the principle/projection neurons in the NAc shell, using an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA) and data were filtered at 2 kHz and digitized at 5 kHz using pClamp10 software. For recording evoked EPSCs (eEPSCs), a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME) was placed about 100 μm rostral to the recording electrode and at the same depth of the recorded neuron. eEPSCs were evoked by an ISO-flex stimulus isolation unit (A.M.P.I., Jerusalem, Israel). The amplitude of each EPSC was measured
2.2. Whole-cell patch clamp recordings
2.1. Animals and brain slice preparations Sprague-Dawley rats were individually housed on a 12-h light/dark cycle with water available ad libitum. Coronal brain slice containing the NAc shell was prepared from rats of either sex, in compliance with US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23, revised 1996). All efforts were made to minimize the number of animals used and discomfort. After the animal was decapitated under sodium pentobarbital (40 mg/kg) anesthesia, the brain was quickly removed and cooled in ice-cold standard artificial cerebrospinal fluid (ACSF, composition in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 26 NaHCO3, 2 CaCl2 and 10 D-glucose) saturated with 95% O2/5% CO2. Coronal slices containing NAc shell were cut with a vibroslicer (VT 1200S, Leica Microsystem, Wetzlar, Germany) according to the rat brain atlas [40]. 118
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Fig. 2. Activation of α2-AR reduces frequency but not amplitude of mEPSCs in the NAc shell neurons. A, B Raw traces showing mEPSCs (indicated by the inverted triangles in panel a) recorded in NAc shell neurons before and during the application of CLON. The superimposed traces in panel b show the presences of mEPSCs within 30 min with and without CLON application. C, D Cumulative distribution plots of mEPSC inter-event interval and amplitude in the NAc shell neuron showing CLON decreased the frequency rather than the amplitude of mEPSCs. Bar graphs showing the averaged mEPSC frequency and amplitude in the absence and presence of CLON. Data are shown as mean ± SEM; *P < 0.05 and n.s., no statistical difference.
containing the NAc shell and used concentric bipolar electrode to stimulate anterior commissure fibers to the NAc (Fig. 1A), which contain glutamatergic afferents from the prefrontal cortex, ventral hippocampus, and basolateral amygdala to the NAc shell [8]. Glutamatergic EPSCs were evoked by local electrical stimulation in the presence of GABAA receptor antagonist SR95531 (20 μM). As illustrated in Fig. 1B, the tested 5 NAc shell neurons were clamped at −70 mV, and the recorded eEPSCs were totally blocked by the potent and selective AMPA receptor antagonist NBQX (20 μM), indicating that the recorded eEPSCs are mediated by AMPA receptor. Then we examined the effect of the selective α2-AR agonist CLON on eEPSCs in NAc shell to address whether activation of α2-AR could modulate glutamate transmission. CLON (10 μM) induced a significant decrease in the amplitude of eEPSCs to 54.91 ± 4.81% of the control (n = 8, P < 0.001, paired t-test; Fig. 1C and D). This inhibitory effect of CLON on glutamatergic eEPSCs could be washed out (n = 8, P = 0.417, paired t-test; Fig. 1C and D). Furthermore, bath application of yohimbine (YOH, 10 μM), a selective α2AR antagonist, blocked the CLON-induced inhibition on eEPSCs. As shown in Fig. 1E and F, YOH blocked the CLON-induced decrease in the amplitude of AMPA-eEPSCs from 51.04 ± 5.13% (n = 5, P < 0.001, paired t-test) to 96.62 ± 4.13% (n = 5, P = 0.459, paired t-test). To evaluate the contribution of α2-AR to the net influence of ARs in NAc, we further examined the effect of YOH on the NE-induced modulation on glutamate transmission in NAc shell. The results showed that application of NA (10 μM) decreased the amplitude of AMPA-eEPSCs to 66.19 ± 4.73% of the control (n = 5, P < 0.01, paired t-test; Fig. 1G and H), and this inhibitory effect was attenuated by 10 μM YOH (91.60 ± 3.79%; n = 5, P < 0.001, paired t-test; Fig. 1G and H). These results indicate that α2-AR activation inhibits the glutamatergic synaptic transmission and contributes greatly to the modulation of NE on glutamatergic neurotransmission in the NAc shell neurons.
from a 5 ms baseline current. The AMPA receptor-mediated eEPSCs were recorded when the holding potential is at −70 mV. The miniature EPSCs (mEPSCs) were recorded in the presence of the tetrodetoxin (TTX, 0.3 μM; Alomone Laboratory, Jerusalem, Israel) and SR95531 (20 μM; Tocris Bioscience), which is used to block GABAA receptormediated inhibitory postsynaptic currents. Paired stimuli (50 ms interval) were delivered every 20 s, and the paired pulse ratio was calculated as EPSC2/EPSC1 to evaluate whether the effect on EPSCs is presynaptic or postsynaptic. In addition, the AMPA/NMDA ratio was measured by the AMPA-eEPSCs recorded in the presence of NMDA antagonist D-APV (50 μM; Tocris Bioscience, Bristol, UK) and the NMDA-eEPSCs recorded in the presence of AMPA receptor antagonist NBQX (20 μM; Tocris Bioscience) at a holding potential of +40 mV. In these conditions, both AMPA and NMDA receptors are activated by synaptically released glutamate. NMDA antagonist (+)-5-methyl10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK 801; 1 mM; Sigma, St. Louis, MO) was loaded in the recoding pipette to block the postsynaptic NMDA receptor [7]. 2.3. Statistical analysis All numeric data are performed as mean ± S.E.M. Statistical analyses were performed with Prism software (version 6.02, GraphPad, La Jolla, CA). The Student’s t-test was used to further determine the differences between group means. Paired student’s t-test and KolmogorovSmirrov (KS) test were used when appropriate. P-values of < 0.05 were considered to be significant. 3. Results 3.1. Activation of α2-AR inhibits eEPSCs mediated by AMPA in NAc shell neurons We performed whole-cell patch clamp recording in brain slice 119
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3.2. Activation of α2-AR suppresses frequency rather than amplitude of mEPSCs in NAc shell neurons
Anatomical and electrophysiological studies have revealed a convergence of prefrontal cortex and limbic system afferents onto single NAc neurons, suggesting that the integration of cortical and limbic information may be processed at the level of single NAc neuron [19]. NAc lesions cause disruptions of array of cognitive and affective processes, including operant and emotional learning, response inhibition and behavioral flexibility [41]. Deep brain stimulation at 100–150 Hz in the NAc decreases the rating of anxiety and depression in treatment-resistant depression patients [5]. As an important subregion, the NAc shell receives extensive glutamatergic projections from emotional centers, such as prefrontal cortex, hippocampus, basolateral amygdala, ventral tegmental area and dorsal raphe nucleus [33,43]. These glutamatergic neurotransmissions are critical for rewarding and emotional processing, and even have been recognized as a primary target for drugs of abuse to induce addiction-related pathophysiological motivational states [50]. Furthermore, AMPA receptor in the NAc shell is necessary for morphine withdrawal-induced negative affective states in rats [44]. Therefore, the present result that activation of α2-AR inhibits glutamatergic transmission suggests that the noradrenergic afferent inputs and α2-AR in the NAc shell may actively participate in reward and emotion regulations. Accumulating evidence indicates that α2-AR may play a functional role in NAc. Systemic administration of CLON reduces extracellular dopamine concentration in the brain in the NAc in vivo using microdialysis [37]. Moreover, microinjection of dexmedetomidine (selective α2-AR agonist) decreases NE level in NAc, suggesting that the α2-AR, as a presynaptic autoreceptor, may directly modulate the NA release [25]. In the present study, the results clearly suggest that α2-AR may also act as a presynaptic heteroreceptor to inhibit glutamatergic transmission. The presynaptic action of α2-AR on glutamatergic synaptic transmission was confirmed by the findings that CLON reduced the frequency rather than amplitude of mEPSCs, increased paired-pulse facilitations and did not change the AMPA/NMDA ratio on the NAc shell neurons. These results strongly indicate a reduction in the probability of glutamate release by the activation of α2-AR, and are consistent with the previous reports on the role of heterosynaptic activation of α2-ARs in the modulation of glutamate transmission in other brain regions, such as ventral tegmental area, hippocampus, brainstem and spinal cord [4,6,26,39]. Therefore, α2-AR may play a multiple role in NAc, including not only regulating NE release as an autoreceptor but also modulating glutamatergic transmission as a heteroreceptor. The central noradrenergic system, originating mainly from the locus coeruleus in brainstem, is closely related to arousal and attention, learning and memory, nociception, as well as emotion regulation in mammals [1,11,12]. It has been well known that the noradrenergic projections innervate various brain regions in the limbic system, in which adrenoceptors are also extensively expressed. NA release in the NAc shell enhances representations in memory for emotionally arousing events via α-AR [29]. Suppression of noradrenergic activity within the NAc shell with the β-AR antagonist propranolol blocks the facilitating effect of concurrently administration of glucocorticoid receptor agonist on memory consolidation in both the appetitive and aversive learning tasks [57]. β-AR also disrupts long-term reward-related memory reconsolidation [16]. Notably, presynaptic α2-AR also holds a key position in mediating the emotion regulation of the central noradrenergic system. Chronic activation of α2-AR protects rodent prefrontal cortexrelated cognition from detrimental effects of stress [22]. Intraperitoneal administration of CLON reverses the anxiety behaviors induced by sleep-deprivation in mice [49]. Similarly, pre-treatment of CLON potentiates ethanol induced anxiolysis [51]. Besides, it has been shown that activation of α2-AR increases the punished response in the vogel conflict test, which is often used to monitor the potential anxiolytic properties of drugs [32,35]. Moreover, stimulation of α2-AR in amygdala inhibits cellular firing rate [9] and suppresses the long-term potentiation and depression [14], whereas activation of α2-AR in prefrontal cortex increases the excitability of pyramidal neurons [10].
Next, we studied the effect of α2-AR activation on mEPSCs in NAc shell neurons. In the presence of TTX and SR95531, bath application of CLON (10 μM) significantly decreased the frequency of mEPSCs (P < 0.001, K-S test; Fig. 2A, B and C left panel), with a decrease from 2.20 ± 0.28 Hz to 1.08 ± 0.26 Hz (n = 6, P = 0.018, paired t-test; Fig. 2C right panel). In contrast, the amplitude of the mEPSCs was not affected by CLON (P = 0.056, K-S test; Fig. 2A, 2 B and 2D left panel). The averaged mEPSC amplitude in the presence and absence of CLON was 15.51 ± 1.84 pA and 15.86 ± 1.85 pA (n = 6, P = 0.780, paired t-test; Fig. 2D right panel), respectively. These results indicate that α2AR may also modulate the spontaneous glutamatergic synaptic transmission. Since inhibition of mEPSC amplitude reflects a reduction in the sensitivity and/or the number of postsynaptic ionotropic glutamate receptors, the results further suggest that the suppression of α2-AR activation on glutamatergic transmission may be through a presynaptic mechanism. 3.3. α2-AR activation increases the paired-pulse facilitation of EPSCs and does not affect AMPA/NMDA ratio on the NAc shell neurons To confirm whether the activation of α2-AR inhibits glutamatergic transmission via a presynaptic mechanism, we further examined the effect of CLON on the paired pulse ratio (PPR), which is generally used to determine the changes in neurotransmitter release. As shown in Fig. 3A left panel and 3B, stimuli elicited a pair of eEPSCs with the second eEPSC amplitude larger than the first one. Application of CLON (10 μM) reduced the peak amplitude of both EPSCs. However, CLON significantly enhanced the paired pulse facilitation of eEPSCs from 1.41 ± 0.17 to 1.91 ± 0.19 (n = 6, P < 0.001, paired t-test; Fig. 3A–C), strongly suggesting that the inhibition of eEPSCs induced by CLON in NAc shell is mediated by a presynaptic mechanism. A change in the ratio of AMPA- to NMDA-dependent currents is a measure of postsynaptic changes in strength at central synapses [13,27,53]. Therefore, we further measured the effect of CLON on the AMPA/NMDA ratio in the NAc shell neurons and found that application of CLON (10 μM) did not affect the AMPA/NMDA ratio (1.01 ± 0.15 vs 0.99 ± 0.13; n = 5, P = 0.938, paired t-test; Fig. 3D and E), confirming the presynaptic mechanism. Moreover, since presynaptic NMDA receptor may be involved in glutamate release in the NAc shell [24], we examined the CLON-induced inhibition of eEPSCs with internal solution containing MK 801 to block NMDA receptor in the postsynaptic recorded neuron [7,38], in the presence and absence of DAPV. As shown in Fig. 3F and G, CLON caused a decrease in the amplitude of eEPSCs to 60.20 ± 6.54% (n = 5, P < 0.01, paired t-test; Fig. 3F) in the absence of D-APV and to 61.20 ± 4.71% (n = 5, P < 0.01, paired t-test; Fig. 3F) in the presence of D-APV, and D-APV did not affect the CLON-induced inhibition of eEPSCs (n = 5, P = 0.772, paired t-test; Fig. 3G). All these results suggest that activation the presynaptic inhibition of α2-AR on the glutamatergic transmission in NAc shell is independent of presynaptic NMDA receptor. 4. Discussion The present study demonstrates that presynaptic α2-AR modulates glutamatergic synaptic transmission in NAc shell. Selective α2-AR agonist CLON inhibits the AMPA-mediated eEPSCs in NAc shell neurons. The CLON-induced suppression of glutamatergic eEPSCs is blocked by YOH, a selective α2-AR antagonist. Furthermore, activation of α2-AR decreases the frequency of mEPSCs but increases the pairedpulse facilitation, indicating that the inhibitory effect on glutamatergic transmission in NAc shell was mediated by the activation of heterosynaptic presynaptic α2-AR. NAc is well known to be involved in reward and emotion regulation. 120
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Fig. 3. Activation of α2-AR increases the paired-pulse ratio in the NAc shell neurons. A Raw traces in the left panel showing the eEPSCs evoked by paired stimuli (50 ms interval) in the absence and presence of CLON. The right panel showing the first eEPSC during CLON application is scaled to the amplitude of the first eEPSC collected in control condition. B Scatter plots presenting the amplitude of the first and second eEPSCs obtained from the same neuron illustrated in A. C Group data of the mean paired-pulse ratio obtained in the absence and presence of CLON. The plots show the paired-pulse ratio for each experiment in the absence and presence of CLON. D The AMPA- and NMDA-mediated eEPSCs recorded at +40 mV in the absence and presence of CLON in the NAc shell neurons. E Group data show the averaged AMPA/NMDA ratio in the absence and presence of CLON. F Bath application of CLON, with MK 801 in internal solution, decreased the amplitude of eEPSCs, which was not affected by application of D-APV. G Bar graphs show the effect of CLON, with MK 801 in internal solution, on eEPSCs in the absence and presence of D-APV. Data are shown as mean ± SEM; **P < 0.01, ***P < 0.001 and n.s., no statistical difference.
Therefore, our findings that presynaptic α2-AR activation suppresses glutamatergic synaptic transmission in NAc shell provide a new insight into the mechanism underlying the reward and emotional functions of the central noradrenergic system.
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Acknowledgements This work was supported by the grants 31330033, 31471112, 31500848, 81671107, and the NSFC/RGC Joint Research Scheme 31461163001 from the National Natural Science Foundation of China; Fundamental Research Funds for the Central Universities 020814380071 from the Ministry of Education, China; and the grant BK20140599 from the Natural Science Foundation of Jiangsu Province, China. References [1] G. Aston-Jones, M. Ennis, V.A. Pieribone, W.T. Nickell, M.T. Shipley, The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network, Science 234 (1986) 734–737. [2] C.H. Beck, H.C. Fibiger, Conditioned fear-induced changes in behavior and in the expression of the immediate early gene c-fos: with and without diazepam pretreatment, J. Neurosci. 15 (1995) 709–720.
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Research article
Presynaptic α2-adrenoceptor modulates glutamatergic synaptic transmission in rat nucleus accumbens in vitro ⁎
Shi-Yu Peng1, Bin Li1, Kang Xi, Jian-Jun Wang , Jing-Ning Zhu
T
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State Key Laboratory of Pharmaceutical Biotechnology and Department of Biological Science and Technology, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China
A R T I C L E I N F O
A B S T R A C T
Keywords: α2-Adrenoceptor Nucleus accumbens shell Glutamatergic transmission Excitatory postsynaptic current Presynaptic modulation
The nucleus accumbens (NAc), integrating information from the prefrontal cortex and limbic structures, plays a critical role in reward and emotion regulation. Previous studies have reported that the NAc shell receives direct noradrenergic projections, and activation of α2-adrenoceptor (α2-AR) in the NAc shell decreases the fear or anxiety level of rats. However, the underlying mechanism is still little known. Intriguingly, glutamatergic neurotransmission in the NAc shell is closely related to reward and emotion. Here, using brain slice preparations and whole-cell patch clamp recordings, we examined the effect of activation of α2-AR on glutamatergic neurotransmission in the NAc shell. Perfusing slice with α2-AR selective agonist clonidine (CLON) reduced the evoked excitatory postsynaptic currents (EPSCs) on the NAc shell neurons. This inhibitory effect on AMPAmediated glutamatergic EPSCs was blocked by the α2-AR selective antagonist yohimbine (YOH). Notably, CLON reduced the frequency but not the amplitude of miniature EPSCs. Furthermore, CLON decreased the first EPSC amplitude but increased the paired-pulse facilitation on the NAc shell neurons, and it did not affect postsynaptic AMPA/NMDA ratio, revealing a presynaptic mechanism of α2-AR-mediated inhibition on glutamatergic transmission. In addition, the modulation on glutamatergic transmission by α2-AR was independent of presynaptic NMDA receptor. These results suggest that noradrenergic afferent inputs may suppress glutamatergic synaptic transmission via presynaptic α2-AR in the NAc shell, and actively participate in rewarding and emotional processes via the NAc.
1. Introduction The nucleus accumbens (NAc), a key structure of the mesolimbic circuitry, receives excitatory inputs from the medial prefrontal cortex, basolateral amygdala, thalamus and hippocampus and, in turn, projects to other basal ganglia nuclei, which send feedback projections into the prefrontal cortex [21]. This anatomical organization places the NAc as an integrator responsible for several important higher brain functions, including rewarding and emotional processing [23,36]. Based on cytomorphologic and immunohistochemical characteristics, the NAc is anatomically divided into two distinct structures, the core and the shell [21]. The latter has been implicated in the cognitive processing of reward, such as pleasurable stimuli, motivational salience and positive reinforcement [3,55]. Moreover, the NAc shell also mediates the anxiety behaviors in response to threatening stimuli [2,34]. Notably, most of the inputs into NAc are glutamatergic, which not only encode the cues and descriptive features [17,28], but also promote synaptic plasticity to strengthen the pertinent environmental cues after addictive
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drug use [47,48]. In addition, it has been reported that a delayed glutamate release occurs in the NAc following a conditioned emotional response [46]. Interestingly, neuroanatomical and tracing studies have revealed a moderate dense of noradrenergic projections from the locus coeruleus [45], ventrolateral medulla [30], and nucleus of the solitary tract [15] to the NAc, indicating that the central noradrenergic system and norepinephrine (NE) may regulate rewarding and emotional processing through the NAc. Central noradrenergic system is one of the most important neuromodulatory systems in the central nervous system [45]. It is generally implicated in regulation of many emotional functions, including anxiety [1], and reward [18,54]. The concentration of NE increases rapidly in the locus coeruleus, amygdala and hypothalamus in fear/anxiety rats [42,52]. Importantly, microinjection of α2-adrenoceptor (α2-AR) selective agonist clonidine (CLON) in the NAc shell decreased the level of fear/anxiety in rats in the elevated plus maze test [31]. However, the underlying mechanism remains unclear. Therefore, considering the great contribution of glutamatergic
Corresponding authors. E-mail addresses: [email protected] (J.-J. Wang), [email protected] (J.-N. Zhu). These authors contributed equally.
https://doi.org/10.1016/j.neulet.2017.11.060 Received 13 October 2017; Received in revised form 25 November 2017; Accepted 27 November 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Activation of α2-AR inhibits the glutamatergic transmission in the NAc shell neurons. A The image shows the placement of stimulating electrode and the recorded NAc shell neuron on a coronal brain slice. B Raw traces show the APMA receptormediated eEPSC on NAc shell neuron in the presence of TTX and SR95531. Note that the fast evoked EPSC was totally blocked by AMPA/KA receptor antagonist NBQX. C Bath application of CLON, a selective α2-AR agonist, decrease the amplitude of eEPSCs, and this effect could be washed out. D Bar graphs show the effect of CLON on the AMPA-mediated eEPSC. E Application of YOH, a selective α2-AR antagonist, blocked the CLON-induced inhibitory effect on AMPA-mediated eEPSCs in NAc shell neurons. F Bar graphs show the blocking effect of YOH on the CLON-induced inhibition of eEPSCs. G Application of YOH blocked the NA-induced inhibitory effect on AMPA-mediated eEPSCs in NAc shell neurons. H Bar graphs show the blocking effect of YOH on the NAinduced inhibition of eEPSCs. Data are shown as mean ± SEM; **P < 0.01, ***P < 0.001 and n.s., no statistical difference.
transmission in NAc to reward and emotion regulation, in the present study, using whole-cell patch clamp recordings, we investigated the role of α2-AR in the modulation of glutamatergic transmission in the NAc shell. The results show that activation of α2-AR inhibits the glutamatergic transmission in the NAc shell presynaptically.
The slices were stored for at least 1 h in a holding chamber filled with 95% O2 and 5% CO2 oxygenated ACSF at 35 ± 0.2 °C before transferring to a recording chamber.
2. Materials and methods
Visualized whole-cell recordings were performed as our previous reports [20,56]. All the excitatory postsynaptic current (EPSC) recordings were made with electrodes filled with a solution containing the following (in mM): 120 cesium methanesulfonate, 20 CsCl, 10 HEPES, 0.2 EGTA, 10 sodium phosphocreatine, 5 QX-314, 4 Na2-ATP, 0.4 NaGTP, pH 7.2 (290–330 mOsm). Electrode resistance in the bath solution was 5–7 MΩ and series resistance (< 25 MΩ) was monitored continuously and stable to within 20%. Whole-cell patch clamp recordings were performed from the medium spiny neurons, the principle/projection neurons in the NAc shell, using an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA) and data were filtered at 2 kHz and digitized at 5 kHz using pClamp10 software. For recording evoked EPSCs (eEPSCs), a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME) was placed about 100 μm rostral to the recording electrode and at the same depth of the recorded neuron. eEPSCs were evoked by an ISO-flex stimulus isolation unit (A.M.P.I., Jerusalem, Israel). The amplitude of each EPSC was measured
2.2. Whole-cell patch clamp recordings
2.1. Animals and brain slice preparations Sprague-Dawley rats were individually housed on a 12-h light/dark cycle with water available ad libitum. Coronal brain slice containing the NAc shell was prepared from rats of either sex, in compliance with US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23, revised 1996). All efforts were made to minimize the number of animals used and discomfort. After the animal was decapitated under sodium pentobarbital (40 mg/kg) anesthesia, the brain was quickly removed and cooled in ice-cold standard artificial cerebrospinal fluid (ACSF, composition in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 26 NaHCO3, 2 CaCl2 and 10 D-glucose) saturated with 95% O2/5% CO2. Coronal slices containing NAc shell were cut with a vibroslicer (VT 1200S, Leica Microsystem, Wetzlar, Germany) according to the rat brain atlas [40]. 118
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Fig. 2. Activation of α2-AR reduces frequency but not amplitude of mEPSCs in the NAc shell neurons. A, B Raw traces showing mEPSCs (indicated by the inverted triangles in panel a) recorded in NAc shell neurons before and during the application of CLON. The superimposed traces in panel b show the presences of mEPSCs within 30 min with and without CLON application. C, D Cumulative distribution plots of mEPSC inter-event interval and amplitude in the NAc shell neuron showing CLON decreased the frequency rather than the amplitude of mEPSCs. Bar graphs showing the averaged mEPSC frequency and amplitude in the absence and presence of CLON. Data are shown as mean ± SEM; *P < 0.05 and n.s., no statistical difference.
containing the NAc shell and used concentric bipolar electrode to stimulate anterior commissure fibers to the NAc (Fig. 1A), which contain glutamatergic afferents from the prefrontal cortex, ventral hippocampus, and basolateral amygdala to the NAc shell [8]. Glutamatergic EPSCs were evoked by local electrical stimulation in the presence of GABAA receptor antagonist SR95531 (20 μM). As illustrated in Fig. 1B, the tested 5 NAc shell neurons were clamped at −70 mV, and the recorded eEPSCs were totally blocked by the potent and selective AMPA receptor antagonist NBQX (20 μM), indicating that the recorded eEPSCs are mediated by AMPA receptor. Then we examined the effect of the selective α2-AR agonist CLON on eEPSCs in NAc shell to address whether activation of α2-AR could modulate glutamate transmission. CLON (10 μM) induced a significant decrease in the amplitude of eEPSCs to 54.91 ± 4.81% of the control (n = 8, P < 0.001, paired t-test; Fig. 1C and D). This inhibitory effect of CLON on glutamatergic eEPSCs could be washed out (n = 8, P = 0.417, paired t-test; Fig. 1C and D). Furthermore, bath application of yohimbine (YOH, 10 μM), a selective α2AR antagonist, blocked the CLON-induced inhibition on eEPSCs. As shown in Fig. 1E and F, YOH blocked the CLON-induced decrease in the amplitude of AMPA-eEPSCs from 51.04 ± 5.13% (n = 5, P < 0.001, paired t-test) to 96.62 ± 4.13% (n = 5, P = 0.459, paired t-test). To evaluate the contribution of α2-AR to the net influence of ARs in NAc, we further examined the effect of YOH on the NE-induced modulation on glutamate transmission in NAc shell. The results showed that application of NA (10 μM) decreased the amplitude of AMPA-eEPSCs to 66.19 ± 4.73% of the control (n = 5, P < 0.01, paired t-test; Fig. 1G and H), and this inhibitory effect was attenuated by 10 μM YOH (91.60 ± 3.79%; n = 5, P < 0.001, paired t-test; Fig. 1G and H). These results indicate that α2-AR activation inhibits the glutamatergic synaptic transmission and contributes greatly to the modulation of NE on glutamatergic neurotransmission in the NAc shell neurons.
from a 5 ms baseline current. The AMPA receptor-mediated eEPSCs were recorded when the holding potential is at −70 mV. The miniature EPSCs (mEPSCs) were recorded in the presence of the tetrodetoxin (TTX, 0.3 μM; Alomone Laboratory, Jerusalem, Israel) and SR95531 (20 μM; Tocris Bioscience), which is used to block GABAA receptormediated inhibitory postsynaptic currents. Paired stimuli (50 ms interval) were delivered every 20 s, and the paired pulse ratio was calculated as EPSC2/EPSC1 to evaluate whether the effect on EPSCs is presynaptic or postsynaptic. In addition, the AMPA/NMDA ratio was measured by the AMPA-eEPSCs recorded in the presence of NMDA antagonist D-APV (50 μM; Tocris Bioscience, Bristol, UK) and the NMDA-eEPSCs recorded in the presence of AMPA receptor antagonist NBQX (20 μM; Tocris Bioscience) at a holding potential of +40 mV. In these conditions, both AMPA and NMDA receptors are activated by synaptically released glutamate. NMDA antagonist (+)-5-methyl10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK 801; 1 mM; Sigma, St. Louis, MO) was loaded in the recoding pipette to block the postsynaptic NMDA receptor [7]. 2.3. Statistical analysis All numeric data are performed as mean ± S.E.M. Statistical analyses were performed with Prism software (version 6.02, GraphPad, La Jolla, CA). The Student’s t-test was used to further determine the differences between group means. Paired student’s t-test and KolmogorovSmirrov (KS) test were used when appropriate. P-values of < 0.05 were considered to be significant. 3. Results 3.1. Activation of α2-AR inhibits eEPSCs mediated by AMPA in NAc shell neurons We performed whole-cell patch clamp recording in brain slice 119
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3.2. Activation of α2-AR suppresses frequency rather than amplitude of mEPSCs in NAc shell neurons
Anatomical and electrophysiological studies have revealed a convergence of prefrontal cortex and limbic system afferents onto single NAc neurons, suggesting that the integration of cortical and limbic information may be processed at the level of single NAc neuron [19]. NAc lesions cause disruptions of array of cognitive and affective processes, including operant and emotional learning, response inhibition and behavioral flexibility [41]. Deep brain stimulation at 100–150 Hz in the NAc decreases the rating of anxiety and depression in treatment-resistant depression patients [5]. As an important subregion, the NAc shell receives extensive glutamatergic projections from emotional centers, such as prefrontal cortex, hippocampus, basolateral amygdala, ventral tegmental area and dorsal raphe nucleus [33,43]. These glutamatergic neurotransmissions are critical for rewarding and emotional processing, and even have been recognized as a primary target for drugs of abuse to induce addiction-related pathophysiological motivational states [50]. Furthermore, AMPA receptor in the NAc shell is necessary for morphine withdrawal-induced negative affective states in rats [44]. Therefore, the present result that activation of α2-AR inhibits glutamatergic transmission suggests that the noradrenergic afferent inputs and α2-AR in the NAc shell may actively participate in reward and emotion regulations. Accumulating evidence indicates that α2-AR may play a functional role in NAc. Systemic administration of CLON reduces extracellular dopamine concentration in the brain in the NAc in vivo using microdialysis [37]. Moreover, microinjection of dexmedetomidine (selective α2-AR agonist) decreases NE level in NAc, suggesting that the α2-AR, as a presynaptic autoreceptor, may directly modulate the NA release [25]. In the present study, the results clearly suggest that α2-AR may also act as a presynaptic heteroreceptor to inhibit glutamatergic transmission. The presynaptic action of α2-AR on glutamatergic synaptic transmission was confirmed by the findings that CLON reduced the frequency rather than amplitude of mEPSCs, increased paired-pulse facilitations and did not change the AMPA/NMDA ratio on the NAc shell neurons. These results strongly indicate a reduction in the probability of glutamate release by the activation of α2-AR, and are consistent with the previous reports on the role of heterosynaptic activation of α2-ARs in the modulation of glutamate transmission in other brain regions, such as ventral tegmental area, hippocampus, brainstem and spinal cord [4,6,26,39]. Therefore, α2-AR may play a multiple role in NAc, including not only regulating NE release as an autoreceptor but also modulating glutamatergic transmission as a heteroreceptor. The central noradrenergic system, originating mainly from the locus coeruleus in brainstem, is closely related to arousal and attention, learning and memory, nociception, as well as emotion regulation in mammals [1,11,12]. It has been well known that the noradrenergic projections innervate various brain regions in the limbic system, in which adrenoceptors are also extensively expressed. NA release in the NAc shell enhances representations in memory for emotionally arousing events via α-AR [29]. Suppression of noradrenergic activity within the NAc shell with the β-AR antagonist propranolol blocks the facilitating effect of concurrently administration of glucocorticoid receptor agonist on memory consolidation in both the appetitive and aversive learning tasks [57]. β-AR also disrupts long-term reward-related memory reconsolidation [16]. Notably, presynaptic α2-AR also holds a key position in mediating the emotion regulation of the central noradrenergic system. Chronic activation of α2-AR protects rodent prefrontal cortexrelated cognition from detrimental effects of stress [22]. Intraperitoneal administration of CLON reverses the anxiety behaviors induced by sleep-deprivation in mice [49]. Similarly, pre-treatment of CLON potentiates ethanol induced anxiolysis [51]. Besides, it has been shown that activation of α2-AR increases the punished response in the vogel conflict test, which is often used to monitor the potential anxiolytic properties of drugs [32,35]. Moreover, stimulation of α2-AR in amygdala inhibits cellular firing rate [9] and suppresses the long-term potentiation and depression [14], whereas activation of α2-AR in prefrontal cortex increases the excitability of pyramidal neurons [10].
Next, we studied the effect of α2-AR activation on mEPSCs in NAc shell neurons. In the presence of TTX and SR95531, bath application of CLON (10 μM) significantly decreased the frequency of mEPSCs (P < 0.001, K-S test; Fig. 2A, B and C left panel), with a decrease from 2.20 ± 0.28 Hz to 1.08 ± 0.26 Hz (n = 6, P = 0.018, paired t-test; Fig. 2C right panel). In contrast, the amplitude of the mEPSCs was not affected by CLON (P = 0.056, K-S test; Fig. 2A, 2 B and 2D left panel). The averaged mEPSC amplitude in the presence and absence of CLON was 15.51 ± 1.84 pA and 15.86 ± 1.85 pA (n = 6, P = 0.780, paired t-test; Fig. 2D right panel), respectively. These results indicate that α2AR may also modulate the spontaneous glutamatergic synaptic transmission. Since inhibition of mEPSC amplitude reflects a reduction in the sensitivity and/or the number of postsynaptic ionotropic glutamate receptors, the results further suggest that the suppression of α2-AR activation on glutamatergic transmission may be through a presynaptic mechanism. 3.3. α2-AR activation increases the paired-pulse facilitation of EPSCs and does not affect AMPA/NMDA ratio on the NAc shell neurons To confirm whether the activation of α2-AR inhibits glutamatergic transmission via a presynaptic mechanism, we further examined the effect of CLON on the paired pulse ratio (PPR), which is generally used to determine the changes in neurotransmitter release. As shown in Fig. 3A left panel and 3B, stimuli elicited a pair of eEPSCs with the second eEPSC amplitude larger than the first one. Application of CLON (10 μM) reduced the peak amplitude of both EPSCs. However, CLON significantly enhanced the paired pulse facilitation of eEPSCs from 1.41 ± 0.17 to 1.91 ± 0.19 (n = 6, P < 0.001, paired t-test; Fig. 3A–C), strongly suggesting that the inhibition of eEPSCs induced by CLON in NAc shell is mediated by a presynaptic mechanism. A change in the ratio of AMPA- to NMDA-dependent currents is a measure of postsynaptic changes in strength at central synapses [13,27,53]. Therefore, we further measured the effect of CLON on the AMPA/NMDA ratio in the NAc shell neurons and found that application of CLON (10 μM) did not affect the AMPA/NMDA ratio (1.01 ± 0.15 vs 0.99 ± 0.13; n = 5, P = 0.938, paired t-test; Fig. 3D and E), confirming the presynaptic mechanism. Moreover, since presynaptic NMDA receptor may be involved in glutamate release in the NAc shell [24], we examined the CLON-induced inhibition of eEPSCs with internal solution containing MK 801 to block NMDA receptor in the postsynaptic recorded neuron [7,38], in the presence and absence of DAPV. As shown in Fig. 3F and G, CLON caused a decrease in the amplitude of eEPSCs to 60.20 ± 6.54% (n = 5, P < 0.01, paired t-test; Fig. 3F) in the absence of D-APV and to 61.20 ± 4.71% (n = 5, P < 0.01, paired t-test; Fig. 3F) in the presence of D-APV, and D-APV did not affect the CLON-induced inhibition of eEPSCs (n = 5, P = 0.772, paired t-test; Fig. 3G). All these results suggest that activation the presynaptic inhibition of α2-AR on the glutamatergic transmission in NAc shell is independent of presynaptic NMDA receptor. 4. Discussion The present study demonstrates that presynaptic α2-AR modulates glutamatergic synaptic transmission in NAc shell. Selective α2-AR agonist CLON inhibits the AMPA-mediated eEPSCs in NAc shell neurons. The CLON-induced suppression of glutamatergic eEPSCs is blocked by YOH, a selective α2-AR antagonist. Furthermore, activation of α2-AR decreases the frequency of mEPSCs but increases the pairedpulse facilitation, indicating that the inhibitory effect on glutamatergic transmission in NAc shell was mediated by the activation of heterosynaptic presynaptic α2-AR. NAc is well known to be involved in reward and emotion regulation. 120
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Fig. 3. Activation of α2-AR increases the paired-pulse ratio in the NAc shell neurons. A Raw traces in the left panel showing the eEPSCs evoked by paired stimuli (50 ms interval) in the absence and presence of CLON. The right panel showing the first eEPSC during CLON application is scaled to the amplitude of the first eEPSC collected in control condition. B Scatter plots presenting the amplitude of the first and second eEPSCs obtained from the same neuron illustrated in A. C Group data of the mean paired-pulse ratio obtained in the absence and presence of CLON. The plots show the paired-pulse ratio for each experiment in the absence and presence of CLON. D The AMPA- and NMDA-mediated eEPSCs recorded at +40 mV in the absence and presence of CLON in the NAc shell neurons. E Group data show the averaged AMPA/NMDA ratio in the absence and presence of CLON. F Bath application of CLON, with MK 801 in internal solution, decreased the amplitude of eEPSCs, which was not affected by application of D-APV. G Bar graphs show the effect of CLON, with MK 801 in internal solution, on eEPSCs in the absence and presence of D-APV. Data are shown as mean ± SEM; **P < 0.01, ***P < 0.001 and n.s., no statistical difference.
Therefore, our findings that presynaptic α2-AR activation suppresses glutamatergic synaptic transmission in NAc shell provide a new insight into the mechanism underlying the reward and emotional functions of the central noradrenergic system.
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