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Background: Nesfatin-1 is a newly identified satiety peptide that has regulatory effects on food intake and glucose metabolism, and is located in the hypothalamic nuclei, including the supraoptic nucleus (SON). In this study, we have investigated the hypothesis that nesfatin-1 neurons are activated by refeeding and intraperitoneal (ip) glucose injection and that the glutamatergic system has regulatory influences on nesfatin-1 neurons in the SON. Materials and methods: The first set of experiments analyzed activation of nesfatin-1 neurons after refeeding as a physiological stimulus and the effectiveness of the glutamatergic system on this physiological stimulation. The subjects were randomly divided into three groups: fasting group, refeeding group and antagonist (CNQX+refeeding) group. The second set of experiments analyzed activation of nesfatin-1 neurons by glucose injection as a metabolic stimulus and the effectiveness of the glutamatergic system on this metabolic stimulation. The subjects were randomly divided into three groups: saline group, glucose group and antagonist (CNQX+glucose) group. Results: Refeeding significantly increased the number of activated nesfatin-1 neurons by approximately 66%, and intraperitoneal glucose injection activated these neurons by about 55%, compared to the fasting and saline controls. The injections of glutamate antagonist (CNQX) greatly decreased the number of activated nesfatin-1 neurons. Conclusions: This study suggested that nesfatin-1 neurons were activated by peripheral and/or metabolic signals and that this effect was mediated through the glutamatergic system.

Background GLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark. Results Wild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development. Conclusions Our work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

Thalamic stroke leads to ataxia if the cerebellum-receiving ventrolateral thalamus (VL) is affected. The compensation mechanisms for this deficit are not well understood, particularly the roles that single neurons and specific neuronal subpopulations outside the thalamus play in recovery. The goal of this study was to clarify neuronal mechanisms of the motor cortex involved in mitigation of ataxia during locomotion when part of the VL is inactivated or lesioned. In freely ambulating cats, we recorded the activity of neurons in layer V of the motor cortex as the cats walked on a flat surface and horizontally placed ladder. We first reversibly inactivated ∼10% of the VL unilaterally using glutamatergic transmission antagonist CNQX and analyzed how the activity of motor cortex reorganized to support successful locomotion. We next lesioned 50%–75% of the VL bilaterally using kainic acid and analyzed how the activity of motor cortex reorganized when locomotion recovered. When a small part of the VL was inactivated, the discharge rates of motor cortex neurons decreased, but otherwise the activity was near normal, and the cats walked fairly well. Individual neurons retained their ability to respond to the demand for accuracy during ladder locomotion; however, most changed their response. When the VL was lesioned, the cat walked normally on the flat surface but was ataxic on the ladder for several days after lesion. When ladder locomotion normalized, neuronal discharge rates on the ladder were normal, and the shoulder-related group was preferentially active during the stride’s swing phase. NEW & NOTEWORTHY This is the first analysis of reorganization of the activity of single neurons and subpopulations of neurons related to the shoulder, elbow, or wrist, as well as fast- and slow-conducting pyramidal tract neurons in the motor cortex of animals walking before and after inactivation or lesion in the thalamus. The results offer unique insights into the mechanisms of spontaneous recovery after thalamic stroke, potentially providing guidance for new strategies to alleviate locomotor deficits after stroke.

Our previous study has found that aureusidin can inhibit inflammation by targeting myeloid differentiation 2 (MD2) protein. Structural optimization of aureusidin gave rise to a derivative named CNQX. LPS was used to induce inflammation in intestinal macrophages; flow cytometry, PI staining and Hoechst 33342 staining were used to detect the apoptotic level of macrophages; enzyme‐linked immunosorbent assay (ELISA) was utilized to detect the expression level of inflammatory factors (including IL‐1β, IL‐18 and TNF‐α); immunofluorescence staining was used to investigate the expression of MD2; Western blot was employed to measure the protein level of TLR4, MD2, MyD88 and p‐P65. As a result, CNQX with IC50 of 2.5 μM can significantly inhibit the inflammatory damage of macrophages, decrease apoptotic level, reduce the expression level of inflammatory factors and simultaneously decrease the expression level of TLR4, MD2, MyD88 as well as p‐P65. Caco‐2 cell line was used to simulate the intestinal mucosal barrier in vitro, LPS was employed to induce cell injury in Caco‐2 (to up‐regulate barrier permeability), and CNQX with IC50 of 2.5 μl was used for intervention. Flow cytometry was used to detect the apoptotic level of Caco‐2 cells, trans‐epithelial electric resistance (TEER) was measured, FITC‐D was used to detect the permeability of the intestinal mucosa, and Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2). As a result, CNQX decreased the apoptotic level of Caco‐2 cells, increased TEER value, decreased the expression levels of MyD88, TLR4 and MD2, and increased the protein levels of tight junction proteins (including occludin and claudin‐1). C57BL/6 wild‐type mice were treated with drinking water containing Dextran sulphate sodium (DSS) to establish murine chronic colitis model. After CQNX intervention, we detected the bodyweight, DAI score and H&E tissue staining to evaluate the life status and pathological changes. Immunohistochemistry (IHC) staining was used to detect the expression of MD2 protein, tight junction protein (including occludin and claudin‐1). Transmission electron microscopy and FITC‐D were used to detect intestinal mucosal permeability. Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2) in the intestinal mucosa tissue. Consequently, CNQX can inhibit the intestinal inflammatory response in mice with colitis, inhibit the mucosal barrier injury, increase the expression of tight junction proteins (including occludin and claudin‐1) and decrease the expression levels of MyD88, TLR4 and MD2. Mechanistically, pull‐down and immunoprecipitation assays showed that CNQX can inhibit the activation of TLR4/MD2‐NF‐κB by binding to MD2 protein. Collectively, in this study, we found that CNQX can suppress the activation of TLR4 signals by targeting MD2 protein, thereby inhibiting inflammation and mucosal barrier damage of chronic colitis.

Dopamine signaling in nucleus accumbens (NAc) is modulated by γ-aminobutyric acid (GABA), acting through GABA-A and GABA-B receptors: dysregulation of GABAergic control of dopamine function may be important in behavioral deficits in schizophrenia. We investigated the effect of GABA-A (muscimol) and GABA-B (baclofen) receptor agonists on electrically stimulated dopamine release. Furthermore, we explored whether drug-induced changes were disrupted by pretreatment with phencyclidine, which provides a well-validated model of schizophrenia. Using brain slices from female rats, fast-scan cyclic voltammetry was used to measure electrically stimulated dopamine release in NAc shell. Both muscimol and baclofen caused concentration-dependent attenuation of evoked dopamine release: neither effect was changed by dihydro-β-erythroidine, a nicotinic acetylcholine receptor antagonist, or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), precluding indirect mechanisms using these transmitter systems in the GABAergic actions. In slices taken from rats pretreated with phencyclidine, the attenuation of evoked dopamine release by baclofen was abolished, but the attenuation by muscimol was unaffected. Since phencyclidine pretreatment was followed by drug-free washout period of at least a week, the drug was not present during recording. Therefore, disruption of GABA-B modulation of dopamine is due to long-term functional changes resulting from the treatment, rather than transient changes due to the drug's presence at test. This enduring dysregulation of GABA-B modulation of accumbal dopamine release provides a plausible mechanism through which GABA dysfunction influences accumbal dopamine leading to behavioral changes seen in schizophrenia and may provide a route for novel therapeutic strategies to treat the condition.

Linalool, a major odorous constituent in essential oils extracted from lavender, is known to have a wide range of physiological effects on humans including pain management. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is involved in transmission of orofacial nociceptive responses through thin myelinated A[Formula: see text] and unmyelinated C primary afferent fibers. Up to date, the orofacial antinociceptive mechanism of linalool concerning SG neurons of the Vc has not been completely clarified yet. To fill this knowledge gap, whole-cell patch-clamp technique was used in this study to examine how linalool acted on SG neurons of the Vc in mice. Under a high chloride pipette solution, non-desensitizing and repeatable linalool-induced inward currents were preserved in the presence of tetrodotoxin (a voltage-gated Na[Formula: see text]channel blocker), CNQX (a non-NMDA glutamate receptor antagonist), and DL-AP5 (an NMDA receptor antagonist). However, linalool-induced inward currents were partially suppressed by picrotoxin (a GABA[Formula: see text] receptor antagonist) or strychnine (a glycine receptor antagonist). These responses were almost blocked in the presence of picrotoxin and strychnine. It was also found that linalool exhibited potentiation with GABA- and glycine-induced responses. Taken together, these data show that linalool has GABA- and glycine-mimetic effects, suggesting that it can be a promising target molecule for orofacial pain management by activating inhibitory neurotransmission in the SG area of the Vc.

BackgroundIncreasing number of studies provide evidence that the vagus nerve stimulation (VNS) dampens inflammation in peripheral visceral organs. However, the effects of afferent fibers of the vagus nerve (AFVN) on anti-inflammation have not been clearly defined. Here, we investigate whether AFVN are involved in VNS-mediated regulation of hepatic production of proinflammatory cytokines.MethodsAn animal model of hepatitis was generated by intraperitoneal (i.p.) injection of concanavalin A (ConA) into rats, and electrical stimulation was given to the hepatic branch of the vagus nerve. AFVN activity was regulated by administration of capsaicin (CAP) or AP-5/CNQX and the vagotomy at the hepatic branch of the vagus nerve (hVNX). mRNA and protein expression in target tissues was analyzed by RT-PCR, real-time PCR, western blotting and immunofluorescence staining. Hepatic immune cells were analyzed by flow cytometry.ResultsTNF-α, IL-1β, and IL-6 mRNAs and proteins that were induced by ConA in the liver macrophages were significantly reduced by the electrical stimulation of the hepatic branch of the vagus nerve (hVNS). Alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum and the number of hepatic CD4+and CD8+T cells were increased by ConA injection and downregulated by hVNS. CAP treatment deteriorated transient receptor potential vanilloid 1 (TRPV1)-positive neurons and increased caspase-3 signals in nodose ganglion (NG) neurons. Concomitantly, CAP suppressed choline acetyltransferase (ChAT) expression that was induced by hVNS in DMV neurons of ConA-injected animals. Furthermore, hVNS-mediated downregulation of TNF-α, IL-1β, and IL-6 expression was hampered by CAP treatment and similarly regulated by hVNX and AP-5/CNQX inhibition of vagal feedback loop pathway in the brainstem. hVNS elevated the levels of α7 nicotinic acetylcholine receptors (α7 nAChR) and phospho-STAT3 (Tyr705; pY-STAT3) in the liver, and inhibition of AFVN activity by CAP, AP-5/CNQX and hVNX or the pharmacological blockade of hepatic α7 nAChR decreased STAT3 phosphorylation.ConclusionsOur data indicate that the activity of AFVN contributes to hepatic anti-inflammatory responses mediated by hVNS in ConA model of hepatitis in rats.

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca2+ [Ca2+]i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca2+]i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 μM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca2+]i, and cytotoxicity, respectively. Contribution of Ca2+-permeable and impermeable AMPARs was compared using either CNQX, or the Ca2+-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca2+]i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg's effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca2+]i, and cytotoxicity, and that both Ca2+-permeable and impermeable AMPARs contribute the elevations in [Ca2+]i.

RATIONALE: The development of addiction is accompanied by a shift in the mechanisms motivating cocaine use from nucleus accumbens (NAc) dopamine D(1) receptor (D(1)R) signaling to glutamate AMPA-kainate receptor (AMPA-R) signaling. OBJECTIVE: Here we determined whether similar shifts occur for NAc-D(2)R signaling and following systemic manipulation of D(1)R, D(2)R, and AMPA-R signaling. METHODS: Male rats were given short-access (20 infusions/day) or extended-access to cocaine (24-hr/day, 96 infusions/day, 10 days). Motivation for cocaine was assessed following 14-days of abstinence using a progressive-ratio schedule. Once responding stabilized, the effects of NAc-D(2)R antagonism (eticlopride; 0-10.0 μg/side) and systemic D(1)R (SCH-23390; 0-1.0 mg/kg), D(2)R (eticlopride; 0-0.1 mg/kg), and AMPA-R (CNQX; 0-1.5 mg/kg) antagonism, and NAc-dopamine-R gene expression (Drd1/2/3) were examined. RESULTS: Motivation for cocaine was markedly higher in the extended- versus short-access group confirming the development of an addiction-like phenotype in the extended-access group. NAc-infused eticlopride decreased motivation for cocaine in both the short- and extended-access groups although low doses (0.1-0.3-μg) were more effective in the short-access group and high doses (3-10-μg/side) tended to be more effective in the extended-access group. Systemic administration of eticlopride (0.1 mg/kg) was more effective in the extended-access group, and systemic administration of CNQX was effective in the extended-, but not short-access group. NAc-Drd2 expression was decreased in both the short- and extended-access groups. CONCLUSION: These findings indicate that in contrast to NAc-D(1)R, D(2)R remain critical for motivating cocaine use with the development of an addiction-like phenotype. These findings also indicate that shifts in the mechanisms motivating cocaine use impact the response to both site-specific and systemic pharmacological treatment.

Previous studies showed that mecamylamine a noncompetitive and nonspecific blocker of nicotinic acetylcholine receptors (nAChRs), stimulates the activity of the dorsal raphe nucleus (DRN) serotonergic neurons and DRN serotonin (5-HT) release. In the present study, the mechanisms involved in these mecamylamine-induced effects were examined using electrophysiology and calcium-imaging studies, both performed in Wistar rat midbrain slices. Mecamylamine (0.5-9 μM), bath administered, increased the firing frequency of identified 5-HT DRN neurons by a maximum of 5% at 3 μM. This effect was accompanied by a 112 % increase in the frequency of spontaneous excitatory postsynaptic currents of 5-HT DRN neurons. It was blocked by the AMPA/kainate receptor blocker CNQX (10 μM) and by the specific α4β2 nAChRs blocker dihydro-β-erythroidine (100 nM) but was not affected by tetrodotoxin (TTX, 500 nM). Simultaneously, mecamylamine produced a 58 % decrease in the frequency of GABAergic spontaneous inhibitory postsynaptic currents, an effect that was not influenced by TTX. Calcium-imaging studies support the results obtained with the electrophysiological studies by showing that mecamylamine (3 μM) increases the activity of a cell population located in the midline of the DRN, which was sensitive to the inhibitory effects of 8-OH-DPAT, an agonist at 5-HT1A receptors. It is assumed that mecamylamine, in low concentrations, acts as an agonist of α4β2 nAChRs present on the glutamatergic DRN terminals, thus increasing intra-raphe glutamate release. This stimulatory effect is reinforced by the decrease in DRN GABA release, which is dependent on the mecamylamine-induced blockade of α7 nAChRs located on DRN GABAergic terminals. We hypothesize that at least a part of mecamylamine antidepressant effects described in animal models of depression are mediated by an increase in DRN 5-HT release.

In experiments on mice spinal cord slices, it was found that intense apoptosis of motoneurons develops in these slices rather rapidly (6 hours). This process can be significantly suppressed by treatment with the NMDA glutamate receptor antagonist MK-801 (50 μM) and AMPA/kainite non-NMDA glutamate receptor antagonist CNQX (100 μM). Such treatment considerably increased the percentage of viable motoneurons in the ventral horns of the spinal cord. The results obtained confirm the statement that glutamate excitotoxicity could be a significant possible factor responsible for apoptosis of motoneurons under conditions of spinal cord impairment.

Background and purpose Perampanel is a newly approved anticonvulsant uniquely targeting AMPA receptors, which mediate the most abundant form of excitatory synaptic transmission in the brain. However, the network mechanism underlying the anti-epileptic effect of the AMPAergic inhibition remains to be explored. Experimental approach The mechanism of perampanel action was studied with the basolateral amygdala network containing pyramidal-inhibitory neuronal resonators in seizure models of 4-aminopyridine (4-AP) and electrical kindling. Key results Application of either 4-AP or electrical kindling to the basolateral amygdala readily induces AMPAergic transmission-dependent reverberating activities between pyramidal-inhibitory neuronal resonators, which are chiefly characterized by burst discharges in inhibitory neurons and corresponding recurrent inhibitory postsynaptic potentials in pyramidal neurons. Perampanel reduces post-kindling "paroxysmal depolarizing shift" especially in pyramidal neurons and, counterintuitively, eliminates burst activities in inhibitory neurons and inhibitory synaptic inputs onto excitatory pyramidal neurons to result in prevention of epileptiform discharges and seizure behaviours. Intriguingly, similar effects can be obtained with not only the AMPA receptor antagonist CNQX but also the GABAA receptor antagonist bicuculline, which is usually considered as a proconvulsant. Conclusion and implications Ictogenesis depends on the AMPA receptor-dependent recruitment of pyramidal-inhibitory neuronal network oscillations tuned by dynamic glutamatergic and GABAergic transmission. The anticonvulsant effect of perampanel then stems from disruption of the coordinated network activities rather than simply decreased neuronal excitability or excitatory transmission. Positive or negative modulation of epileptic network reverberations may be pro-ictogenic or anti-ictogenic, respectively, constituting a more applicable rationale for the therapy against seizures.

The Kolliker-Fuse (KF) nucleus is a part of the parabrachial complex, located in the dorsolateral pons. It is involved in the chemoreflex-evoked cardiovascular and respiratory changes, but the role of GABA and glutamate in cardiovascular chemoreflex has not been shown yet. This study was performed to determine the role of GABA, glutamate, and their interaction in the KF, in cardiovascular chemoreflex in anesthetized rat. The antagonists were microinjected into the KF, and arterial pressure, heart rate, and single-unit responses were recorded simultaneously. The chemoreflex was evoked by i.v. injection of KCN, consisted of a short pressor followed by long bradycardia responses. Both responses were significantly attenuated by injection of a synaptic blocker (CoCl2) into the KF, confirming involvement of the KF in generating the reflex. Microinjection of AP5, an NMDA receptor antagonist, into the KF significantly attenuated the pressor and bradycardia responses, while blocking the AMPA receptors by CNQX had no significant effect. Blockade of GABAA receptors by bicuculline methiodide (BMI) potentiated both responses. Co-injection of BMI and CNQX potentiated the responses too. Co-injection of BMI and AP5 had no significant effect on the pressor response but significantly attenuated the bradycardia response. In conclusion, the KF plays a role in generating cardiovascular chemoreflex via its glutamate NMDA but not AMPA receptors. GABA inhibits both components of this reflex through GABAA receptors. There is an interaction between GABAA and NMDA receptors in regulating the bradycardia response of the reflex. Single-unit results were also presented which were correlated with and supported the homodynamic findings.

Experimental and clinical data strongly support vagus nerve stimulation (VNS) as a novel treatment in migraine. Vagus nerve stimulation acutely suppresses cortical spreading depression (CSD) susceptibility, an experimental model that has been used to screen for migraine therapies. However, mechanisms underlying VNS efficacy on CSD are unknown. Here, we interrogated the central and peripheral mechanisms using VNS delivered either invasively (iVNS) or noninvasively (nVNS) in male Sprague-Dawley rats. Cortical spreading depression susceptibility was evaluated 40 minutes after the stimulation. iVNS elevated the electrical CSD threshold more than 2-fold and decreased KCl-induced CSD frequency by 22% when delivered to intact vagus nerve. Distal vagotomy did not alter iVNS efficacy (2-fold higher threshold and 19% lower frequency in iVNS vs sham). By contrast, proximal vagotomy completely abolished iVNS effect on CSD. Pharmacological blockade of nucleus tractus solitarius, the main relay for vagal afferents, by lidocaine or glutamate receptor antagonist CNQX also prevented CSD suppression by nVNS. Supporting a role for both norepinephrine and serotonin, CSD suppression by nVNS was inhibited by more than 50% after abrogating norepinephrinergic or serotonergic neurotransmission alone using specific neurotoxins; abrogating both completely blocked the nVNS effect. Our results suggest that VNS inhibits CSD through central afferents relaying in nucleus tractus solitarius and projecting to subcortical neuromodulatory centers providing serotonergic and norepinephrinergic innervation to the cortex.

Experiments were done to investigate the role of glutamatergic systems in the median preoptic nucleus (MnPO) in the water ingestion induced by administration of angiotensin II (ANG II) in the subfornical organ (SFO) in the awake rat. Microdialysis methods were utilized to quantify the extracellular content of glutamate (Glu) in the region of MnPO. Microinjection of ANG II (10−10 M) into the SFO significantly increased the release of Glu in the MnPO in the rats under the condition that water is available for drinking and the rats under the condition that water is not available for drinking. The amount of initial maximal increases in the Glu levels elicited by the ANG II injection was quite similar in drinking and non-drinking rats, whereas the duration of the response was much longer in non-drinking than in drinking rats. The amount of water ingestion in 20 min immediately after the ANG II injection was significantly enhanced by previous injections of N-methyl-d-aspartate (NMDA, 10 μM) into the MnPO, while the ANG II-induced water ingestion was attenuated by pretreatment with the NMDA antagonist dizocilpine (MK-801, 10 μM). The amount of water intake elicited by the ANG II injection into the SFO was enhanced by previous injections of either the non-NMDA agonist kainic acid (KA, 50 μM) or quisqualic acid (QA, 50 μM) into the MnPO. On the contrary, the ANG II-induced drinking response was diminished by pretreatment with the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM) in the MnPO. Each injection of NMDA, KA, and QA into the MnPO produced drinking behavior. These results imply that the glutamatergic neural pathways to the MnPO may transmit the information for eliciting drinking in response to ANG II acting at the SFO. Our data further provide evidence that the ANG II-induced dipsogenic response may be mediated through both NMDA and non-NMDA glutamatergic receptor mechanisms in the MnPO.

BACKGROUND Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP). We hypothesized that the nucleus tractus solitarius (NTS), a primary central site that integrates pancreatic afferents apart from the thoracic spinal dorsal horn, plays a key role in the pathogenesis of visceral hypersensitivity in a rat model of CP. AIM To investigate the role of the NTS in the visceral hypersensitivity induced by chronic pancreatitis. METHODS CP was induced by the intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. Pancreatic hyperalgesia was assessed by referred somatic pain via von Frey filament assay. Neural activation of the NTS was indicated by immunohistochemical staining for Fos. Basic synaptic transmission within the NTS was assessed by electrophysiological recordings. Expression of vesicular glutamate transporters (VGluTs), N-methyl-D-aspartate receptor subtype 2B (NR2B), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subtype 1 (GluR1) was analyzed by immunoblotting. Membrane insertion of NR2B and GluR1 was evaluated by electron microscopy. The regulatory role of the NTS in visceral hypersensitivity was detected via pharmacological approach and chemogenetics in CP rats. RESULTS TNBS treatment significantly increased the number of Fos-expressing neurons within the caudal NTS. The excitatory synaptic transmission was substantially potentiated within the caudal NTS in CP rats (frequency: 5.87 ± 1.12 Hz in CP rats vs 2.55 ± 0.44 Hz in sham rats, P < 0.01; amplitude: 19.60 ± 1.39 pA in CP rats vs 14.71 ± 1.07 pA in sham rats; P < 0.01). CP rats showed upregulated expression of VGluT2, and increased phosphorylation and postsynaptic trafficking of NR2B and GluR1 within the caudal NTS. Blocking excitatory synaptic transmission via the AMPAR antagonist CNQX and the NMDAR antagonist AP-5 microinjection reversed visceral hypersensitivity in CP rats (abdominal withdraw threshold: 7.00 ± 1.02 g in CNQX group, 8.00 ± 0.81 g in AP-5 group and 1.10 ± 0.27 g in saline group, P < 0.001). Inhibiting the excitability of NTS neurons via chemogenetics also significantly attenuated pancreatic hyperalgesia (abdominal withdraw threshold: 13.67 ± 2.55 g in Gi group, 2.00 ± 1.37 g in Gq group, and 2.36 ± 0.67 g in mCherry group, P < 0.01). CONCLUSION Our findings suggest that enhanced excitatory transmission within the caudal NTS contributes to pancreatic pain and emphasize the NTS as a pivotal hub for the processing of pancreatic afferents, which provide novel insights into the central sensitization of painful CP.

Rats express a positive emotional state by emitting 50-kHz ultrasonic vocalization (USV) calls in response to drug exposure. This study demonstrated the linking of glutamate receptors in the nucleus accumbens (NAc) to vocal expression of 50-kHz USV calls after repeated cocaine administration in freely moving rats. Repeated systemic injections of cocaine (20 mg/kg/day, i.p.) for seven consecutive days increased the number of 50-kHz USV calls. Intra-NAc core infusion of the broad-glutamate receptor antagonist, γDGG (50 nmol/side), decreased the repeated cocaine-induced increase in the number of 50-kHz USV calls. Intra-NAc core infusion of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK801 (2 nmol/side), but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainic acid receptor antagonist, CNQX disodium salt (2 nmol/side), decreased the number of 50-kHz USV calls that had been elevated by repeated exposure to cocaine. Intra-NAc core infusion of the group I metabotropic glutamate receptor subtype 5 (mGluR5), MPEP (0.5 nmol/side), MTEP (15 nmol/side) and inositol-1,4,5-trisphosphate receptor blocker, xestospongin C (0.004 nmol/side) decreased the cocaine-induced increase in the number of USV calls. These data suggest that the NMDA receptor- and mGluR5-dependent increase in intracellular Ca2+ concentrations in the NAc core is linked to a positive emotional state after repeated exposure to cocaine in rats.

Background Oligomeric amyloid-β peptide (Aβ) is associated with dysfunctional neuronal networks and neuronal loss in the development of Alzheimer's disease (AD). Ischemic postconditioning protects against post-ischemic excitotoxicity, oxidative stress, and inflammatory process that have also been implicated in the pathogenesis of AD. Evaluating the roles of ischemic postconditioning in oligomeric Aβ-induced neurotoxicity and underlying signal events may provide potential strategy for medical therapy in AD. Objectives The aim of the present study was to explore whether and how a brief ischemic postconditioning protects against Aβ neurotoxicity in rat hippocampus. Methods Oligomeric Aβ25-35 (20 nmol/rat) or Aβ1-42 (5 nmol/rat) was infused by intracerebroventricular injection in adult male Sprague-Dawley rats. Ischemic postconditioning, a brief episode of global brain ischemia (3 min), was conducted at 1, 3, or 7 days after Aβ treatment, respectively. Results A brief ischemic postconditioning reduced neuronal loss and inhibited the activation of MLK3, MKK3/6, and P38MAPKs in rat hippocampal CA1 and CA3 subfields after Aβ oligomer infusion. An N-methyl-D-aspartate (NMDA) receptor antagonist amantadine, but not non-NMDA receptor antagonist CNQX, reversed the MLK3-MKK3/6-P38MAPK signal events and beneficial effect of ischemic postconditioning on neuronal survival. Such reversion was also realized by NVP-AAM077, a GluN2A-subunit-selective NMDA receptor antagonist. Moreover, posttreatment with low doses of NMDA (5 nmol-40 nmol/rat) suppressed the Aβ-induced P38MAPK signaling and imitated the neuroprotection of ischemic postconditioning against Aβ neurotoxicity. Conclusions Ischemic postconditioning provides neuroprotection against Aβ neurotoxicity by moderate upregulation of NMDA receptor signaling, especially GluN2A-containing NMDA receptor pathway, and thereafter downregulation of MLK3-MKK3/6-P38MAPK signal events.

Whole-cell patch clamp recordings demonstrated that in the dentate gyrus (DG) as well as in the CA3 area of mouse hippocampal slices the prototypic P2X7 receptor (R) agonist dibenzoyl-ATP (Bz-ATP) induced inward current responses both in neurons and astrocytes. Whereas the selective P2X7R antagonist A438079 strongly inhibited both neuronal and astrocytic currents, a combination of ionotropic glutamate receptor (CNQX, AP-5) and GABAA-R (gabazine) antagonists depressed the Bz-ATP-induced current responses in the DG (granule cells) and CA3 neurons only. It was concluded that Bz-ATP activated astrocytic P2X7Rs and thereby released glutamate and GABA to stimulate nearby neurons. The residual A438079-resistant current response of astrocytes was suggested to be due to the stimulation of P2XRs of the non-P2X7-type. Further, we searched for presynaptic P2X7Rs at the axon terminals of DG and CA3 pyramidal neurons innervating CA3 and CA1 cells, respectively. Bz-ATP potentiated the frequency of spontaneous postsynaptic currents (sPSCs) in CA1 but not CA3 pyramidal cells. However, the Bz-ATP effect in CA1 cells was inhibited by gabazine or the astrocytic toxin fluorocitrate suggesting stimulation of P2X7Rs at stratum radiatum astrocytes located near to interneurons and synapsing onto CA1 neurons. Our data suggest that functional P2X7Rs are missing at neurons in the tri-synaptic network of the rodent hippocampus, but are present at nearby astrocytes indirectly regulating network activity.

Morphine addiction is known as a serious social problem. Medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) are two important sites of the brain that contribute to this type of addiction, and a complicated relation exists in between. In addition, neurotransmitters like glutamate and γ--Amino Butyric Acid (GABA) play an important role in the formation of these relations. Thus, the present study was undertaken to investigate these relations by evaluating the level of associated changes in the indicated neurotransmitters in the VTA, using HPLC method. This was performed after electrical stimulation and inducing lesion of mPFC and through microinjections of N-Methyl-D-Aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, respectively AP5 and CNQX, into the VTA of addicted rats. Our results showed that intra-peritoneal (i.p.) administration of morphine in 9 days in the morphine group, and also electrical stimulation (100 μA) of mPFC, receiving (i.p.) morphine, caused an increase in the glutamate release in the VTA, compared to the control group, but the increase of glutamate levels in the VTA in the morphine-stimulation group was not significant, compared to the morphine group. Moreover, GABA release into this area was decreasing in morphine and morphine- stimulation groups, compared to the control group. Our findings also showed that electrical lesion (0.4 mA) of mPFC, and also microinjection of glutamate antagonists into the VTA, receiving (i.p.) morphine in rats, caused a decrease of glutamate in the VTA. Therefore, it could be concluded that the relation between mPFC and VTA is highly effective in the formation of reward system.

Neuronal amyloid β1–42 (Aβ1–42) accumulation is considered an upstream event in Alzheimer’s disease pathogenesis. Here we report the mechanism on synaptic activity-independent Aβ1–42 uptake in vivo. When Aβ1–42 uptake was compared in hippocampal slices after incubating with Aβ1–42, In vitro Aβ1–42 uptake was preferentially high in the dentate granule cell layer in the hippocampus. Because the rapid uptake of Aβ1–42 with extracellular Zn2+ is essential for Aβ1–42-induced cognitive decline in vivo, the uptake mechanism was tested in dentate granule cells in association with synaptic activity. In vivo rapid uptake of Aβ1–42 was not modified in the dentate granule cell layer after co-injection of Aβ1–42 and tetrodotoxin, a Na+ channel blocker, into the dentate gyrus. Both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not modified after co-injection of CNQX, an AMPA receptor antagonist, which blocks extracellular Zn2+ influx, Both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not also modified after either co-injection of chlorpromazine or genistein, an endocytic repressor. The present study suggests that Aβ1–42 and Zn2+ are synaptic activity-independently co-taken up into dentate granule cells in the normal brain and the co-uptake is preferential in dentate granule cells in the hippocampus. We propose a hypothesis that Zn-Aβ1–42 oligomers formed in the extracellular compartment are directly incorporated into neuronal plasma membranes and form Zn2+-permeable ion channels.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca2+ (Cai) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 μM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2–5 mV depolarization and a modest Cai rise whereas Cai was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 μM), CNQX caused a 5–8 mV depolarization and also the Cai rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 μM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Cai rise was blocked by the L-type voltage-activated Ca2+ channel inhibitor nifedipine (100 μM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca2+ channel-mediated neuronal Cai rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K+ channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate‐induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia‐induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D‐AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA‐produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate‐induced calcium influx. Together, our results suggest that VEGF reduces glutamate‐induced calcium influx in astrocytes via enhancing PKCα‐mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium‐permeable to calcium‐impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate‐mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.

Acute occlusal interference following improper occlusal alteration in dental practice can induce chronic masticatory muscle pain. The underlying mechanism has not been clarified. Synaptic plasticity in the anterior cingulate cortex (ACC) plays a key role in the chronic pain state. This study investigated the role of synaptic plasticity in the ACC in acute occlusal interference–induced chronic masticatory muscle pain. A rat model of experimental occlusal interference (EOI) was established. In vivo local field potential (LFP) recording was conducted to evaluate the change of synaptic strength and plasticity from the medial thalamus (MT) to the ACC after EOI application. The effects of microdialysis of antagonists of glutamate receptors into the ACC on synaptic transmission from the MT to the ACC were examined. Furthermore, the influence of inhibiting glutamate receptors in the ACC on EOI-induced mechanical hyperalgesia in the masseter muscles of rats was investigated. The amplitude of LFP in the ACC evoked by MT stimulation was significantly potentiated since 14 d of EOI application. Long-term potentiation of LFP in the ACC was reliably induced by theta burst stimulation to the MT in control rats but was occluded in 14-d EOI rats. Microdialysis of AMPA/kainate receptor antagonist CNQX into the ACC attenuated LFP in the ACC evoked by stimulating the MT in control and EOI rats. Administration of NMDA receptor subunit NR2B antagonist Ro 25-6981 into the ACC significantly alleviated the potentiation of MT stimulation-evoked LFP in the ACC of EOI rats without affecting that in control rats. EOI-induced hyperalgesia in the bilateral masseter muscles of rats was dose-dependently relieved after microdialysis of Ro 25-6981 into ACC. These findings provide direct evidence that prolonged acute occlusal interference potentiates synaptic transmission in the ACC, which in turn mediates chronic masticatory muscle pain.