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Characterization of serotonin-induced inhibition of excitatory synaptic transmission in the anterior cingulate cortex
© The Author(s). 2017
Received: 11 May 2017
Accepted: 5 June 2017
Published: 12 June 2017
Excitatory synaptic transmission in central synapses is modulated by serotonin (5-HT). The anterior cingulate cortex (ACC) is an important cortical region for pain perception and emotion. ACC neurons receive innervation of projecting serotonergic nerve terminals from raphe nuclei, but the possible effect of 5-HT on excitatory transmission in the ACC has not been investigated. In the present study, we investigated the role of 5-HT on glutamate neurotransmission in the ACC slices of adult mice. Bath application of 5-HT produced dose-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs). Paired pulse ratio (PPR) was significantly increased, indicating possible presynaptic effects of 5-HT. Consistently, bath application of 5-HT significantly decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. After postsynaptic application of G protein inhibitor GDP-β-S, 5-HT produced inhibition of eEPSCs was significantly reduced. Finally, NAN-190, an antagonist of 5-HT1A receptor, significantly reduced postsynaptic inhibition of 5-HT and abolished presynaptic inhibition. Our results strongly suggest that presynaptic as well as postsynaptic 5-HT receptor including 5-HT1A subtype receptor may contribute to inhibitory modulation of glutamate release as well as postsynaptic responses in the ACC.
As one important neurotransmitter in the central nervous system (CNS), serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role in numerous physiological functions. It has been implicated in pain modulation  and emotional disorders . Serotonergic projections to spinal or medullary dorsal horn areas are involved in nociceptive transmission through regulating descending inhibitory pathways [3–5]. Mice lacking serotoninergic neurons in the CNS exhibited enhanced inflammation pain and reduced analgesia in response to opioids and antidepressants . The intrathecal administration of serotonergic agents induced anti-nociceptive actions in animal models . A recent study demonstrates that activation of a specific 5-HT subtype receptor inhibited mechanical allodynia in nerve-injured animals by affecting hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels dendritic function in the anterior cingulate cortex (ACC) .
The ACC is a critical cortical region, which plays a central role in the formation of pain perception and the unpleasantness of pain [1, 9, 10]. Neurons of the ACC receive sensory inputs projecting from other subcortical areas (such as the thalamus) and then project to related sensory regions, including amygdala, midbrain areas, brainstem and spinal cord [11, 12]. Injuries enhanced synaptic transmission in the ACC and inhibition of the ACC potentiation is analgesic in animal models of chronic pain [1, 9, 10]. In the CNS, serotoninergic neurons are mainly located in the raphe nuclei, and they send projecting terminals to different regions of the brain and spinal cord . At the synaptic level, 5-HT has been reported to produce only inhibitory or biphasic modulation of sensory synaptic transmission. For example, 5-HT exerts a biphasic modulation of glutamate transmission in the dorsal horn of the spinal cord [14, 15]. 5-HT produces only inhibitory effects on excitatory or inhibitory transmission in the bed nucleus of the stria terminalis (BNST)  and the periaqueductal grey (PAG) . In forebrain areas including the ACC, neurons are highly innervated by serotoninergic terminals [13, 18]. However, the possible effect of 5-HT on excitatory synaptic transmission in the ACC has not been investigated. In the present study, we perform whole-cell patch-clamp recordings from ACC pyramidal cells and investigate the modulatory effect of 5-HT. We found that bath application of 5-HT produced only inhibitory modulation of excitatory synaptic transmission. Furthermore, we show that 5-HT may produce its inhibitory effects through both presynaptic and postsynaptic mechanisms.
Adult male C57BL/6 mice (8–14 weeks) were used in the experiments and they were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). The animals were housed in plastic boxes with food and water available ad libitum in a colony room with controlled temperature (24 ± 2 °C), humidity (50–60%), and a 12:12 h light-dark cycle. Experiments were performed under protocols approved by the Animal Care and Use Committee at the University of Toronto.
5-HT and guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) were obtained from Sigma–Aldrich (St. Louis, MO, USA). NAN-190, picrotoxin and tetrodotoxin were purchased from Tocris Cookson (Bristol, UK). Drugs were prepared as stock solutions for frozen aliquots at −20 °C. All these drugs were diluted from the stock solution to the final desired concentration in the artificial cerebrospinal fluid (ACSF) before immediate use. All of other chemicals and reagents used were commercially available and of standard biochemical quality.
Brain slice preparation
The general procedures for making the ACC slices were similar to those described previously [19, 20]. Mice were anesthetized with isoflurane in air and then decapitated. Brains were rapidly removed and placed for 2–3 min in an ice-cold and oxygenated ACSF (in mM): containing 124 NaCl, 25 NaHCO3, 2.5 KCl, 1 KH2PO4, 2 CaCl2, 2 MgSO4 and 10 glucose, and continuously gassed with 95% O2/5% CO2. Coronal slices (300 μm) containing the ACC were prepared on a vibratome (Leica VT1200S) in ice-cold ACSF. Slices were then incubated in a room temperature-submerged recovery chamber with oxygenated (95% O2 and 5% CO2) ACSF for at least 1 h.
Whole-cell patch-clamp recording
After recovery, slices were placed in a recording chamber on the stage of an Olympus microscope with infrared digital interference contrast optics for visualization of whole-cell patch-clamp recordings. Recordings were performed at room temperature (21–23 °C) with continuous perfusion of ACSF at a rate of 2 mL/min. For spontaneous excitatory postsynaptic currents (sEPSCs) recording, recording pipettes (3–5 MΩ) were filled with solution containing 145 mM K-gluconate, 5 mM NaCl, 1 mM MgCl2, 0.2 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, and 0.1 mM Na3-GTP, adjusted to pH 7.2 with KOH (280–300 mOsm). sEPSCs were collected in the neurons clamped at −60 mV in the ACSF. For the recording of miniature excitatory postsynaptic currents (mEPSCs), the circulating ACSF was additionally added 1 μM tetrodotoxin. The evoked EPSCs (eEPSCs) were recorded from layer II/III neurons with an Axon 200B amplifier (Molecular Devices) and stimulation was delivered by a bipolar tungsten-stimulating electrode placed in layer V/VI of the ACC. The internal solution containing GDP-β-S was used when the experiment to detect the involvement of postsynaptic G proteins was performed. For the recording of paired-pulse ratio (PPR), a paired pulse paradigm was employed in which two stimuli were delivered at 50 ms inter-stimulus-interval. Picrotoxin (100 μM) was always present to block GABAA receptor-mediated inhibitory synaptic currents in all experiments. Access resistance (15–30 MΩ) was monitored throughout the experiment. Data were discarded if access resistance changed >15% during an experiment.
Whole-cell patch-clamp data were collected and analyzed with Clampex 10.2 and Clampfit 10.2 software (Molecular Devices). For the evoked EPSCs, the amplitudes were normalized and expressed as the percentage of the baseline EPSC amplitude. Miniature and spontaneous EPSCs were detected and analyzed using an event detection program (Mini Analysis Program; Synaptosoft, Inc., Decatur, GA). Analysis of mEPSCs and sEPSCs was performed with cumulative probability plots. For the PPR, the ratio of the slope of the second response to the slope of the first response was calculated and averaged. For comparison between two groups, we used paired or unpaired Student’s t test. For comparison among three groups, we used one-way ANOVA. All data are presented as means ± SEM. In all cases, p < 0.05 was considered statistically significant.
Effect of 5-HT on excitatory transmission in the ACC
Altered paired-pulse ratio by 5-HT
Effect of 5-HT on sEPSC in the ACC
Effect of 5-HT on mEPSC
Postsynaptic inhibition of G proteins attenuated the reduction of EPSC caused by 5-HT
5-HT1A receptors involved in the inhibitory effect of 5-HT
In the present study, we tested the effects of 5-HT on synaptic transmission in the ACC. We found that 5-HT produced reversible inhibitory effects on the excitatory transmission in the ACC. 5-HT significantly decreased the frequency of sEPSC and mEPSCs and increased the PPR of eEPSCs, suggesting that 5-HT inhibited glutamate release from presynaptic terminals. Postsynaptic application of GDP-β-S reduced the inhibitory effects of 5-HT, indicating the involvement of postsynaptic 5-HT receptors in 5-HT produced inhibition. Application of 5-HT1A receptors antagonist inhibited 5-HT produced inhibition of eEPSC amplitude and frequency, implying the involvement of presynaptic 5-HT1A receptors in the inhibition of excitatory transmission.
5-HT innervation in the ACC
In the mammalian brain, 5-HT neurons can be divided into two major groups (rostral group and caudal group) based on cell body localization and their respective projections . The rostral group is located in the mesencephalic and rostral pons, projecting to the forebrain, and the caudal group is located in medulla oblongata, projecting to spinal cord and brain stem . The ACC receives innervation from 5-HT neurons located in the dorsal and median raphe nuclei, which belongs to rostral group . There are at least fourteen different subtype receptors for 5-HT . Most 5-HT receptors belong to the GPCR superfamily with the exception of 5-HT3 receptor, which is a ligand-gated ion channel mediating fast depolarization . In the ACC, various 5-HT receptors have been detected in mouse and rat brain, such as 5-HT1A, 5-HT1B, 5-HT2A and 5-HT7 [8, 30].
Modulation of synaptic transmission by 5-HT
It is well known that 5-HT regulates synaptic transmission by presynaptic and/or postsynaptic mechanisms. For example, in the hippocampus, it has been reported that 5-HT inhibited glutamatergic transmission via both presynaptic and postsynaptic mechanisms . In the (bed nucleus of the stria terminalis, BNST), 5-HT has been shown to suppress glutamatergic neurotransmission through activating presynaptic receptors . However, there is no report of the effects of 5-HT on excitatory transmission in the ACC. In the present study, we found that 5-HT inhibited the excitatory transmission in pyramidal neurons of the ACC in a dose-dependent manner. Moreover, different experiments indicate that 5-HT may produce such inhibition through both presynaptic and postsynaptic mechanisms. In contrast to the ACC, in the spinal cord dorsal horn, a key sensory synapse for pain transmission and modulation, 5-HT produced biphasic modulation of excitatory transmission between primary afferent fibers and spinal cord dorsal horn neurons [14, 32]. These findings suggest that 5-HT modulation is different in various central synapses related to pain and/or emotion.
Neurons in the ACC are involved in pain perception [1, 10, 12, 33] and emotional responses [12, 34]. Long-term plastic changes in excitatory transmission in the ACC modulate not only pain-related behaviors  but also the affective-emotional component of chronic pain [12, 36–38]. Especially, presynaptic potentiation of excitatory transmission in the ACC may be related pain-induced anxiety [12, 36]. Serotonergic projections are known to be important for the regulation of different brain functions, including pain and emotion . Clinical drugs used for the treatment of anxiety such as selective serotonin reuptake inhibitors (SSRIs) increase the level of synaptic 5-HT. In the present study, we show that 5-HT can significantly affect excitatory transmission in the ACC. It is possible that 5-HT induced modulation in the ACC may contribute to the physiological effects of SSRIs. One of 5-HT subtype receptors is 5-HT1A. 5-HT1A subtype receptors are highly expressed in the ACC [25, 40]. It has been reported that 5-HT1A receptors are involved in the development of neuronal circuits regulating anxiety, and 5-HT1A receptor knockout mice shows enhanced anxiety levels . In our study, we found that 5-HT1A mediate 5-HT induced inhibition of excitatory transmission. It is possible that changes in 5-HT1A mediated modulation may contribute to chronic pain and anxiety. Future studies are clearly needed to investigate this possibility, and targeting 5-HT receptors in the ACC may provide new directions for future treatment of chronic pain and emotional disorders.
I would like to thank Melissa Lepp for the help with English editing.
This work is supported by the Canadian Institute for Health Research (CIHR) Michael Smith Chair in Neurosciences and Mental Health, Canada Research Chair, CIHR operating grant (MOP-124807) and project grant (PJT-148648), Azrieli Neurodevelopmental Research Program and Brain Canada.
Availability of data and materials
MZ designed the research; ZT and MY performed the experiment and analyzed the data; MB and MG. Z helped to analyze the data; ZT and MZ wrote the paper. All the authors read and approved the final manuscript.
The author declares that they have no competing interests.
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All experiments were performed under the guidance of National Institutes of Health and with the approval of Animal Care and Use Committee at the University of Toronto, Xi’an Jiaotong University.
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