γ-Aminobutyric acid alleviates litchi thaumatin-like protein-induced inflammation and reduces gut microbial translocation

Previous research reported litchi thaumatin-like protein(LcTLP)could lead to inflammation,which is a factor causing the adverse reactions after excessive intake of litchi.As a main amino acid in litchi pulp,γ-aminobutyric acid(GABA)was found with anti-inflammatory effect.Therefore,this study aimed to investigate the effects of GABA on LcTLP-induced inflammation through RAW264.7 macrophages and C57BL mice models.In vitro study showed GABA could effectively regulate the level of inflammatory cytokines(interleukin(IL)-1β,IL-6,IL-10,and prostaglandin E2)and Ca2+in cells,and inhibit the phosphorylation of p65,IκB,p38,c-Jun N-terminal kinase(JNK)and extracellular signal-regulated kinase(ERK).These results indicate GABA alleviated inflammation through nuclear factor-κB and mitogen-activated protein kinase pathway signaling pathways.In vivo experiment was performed to verify the anti-inflammatory effect of GABA,and the results demonstrated that GABA reduced the inflammation and oxidative stress in the liver of LcTLP-treated mice,as it down-regulated the pro-inflammatory cytokines,malondialdehyde,aspartate transferase,and alanine transaminase.The relative expression of phosphorylated p38,JNK and ERK in mice liver with GABA treatment were reduced to 65%,39%and 80%of the control group,respectively.Furthermore,GABA treatment enriched probiotic bacteria and decreased pathogenic bacteria in mice gut,which reveals GABA could effectively reduce the translocation of gut microbiota.

Effects ofγ-aminobutyric Acid on Nitrogen Metabolism in Roots and Leaves of Cold-stressed Rice(Oryza sativa L.)During Early Vegetative Growth

Cold stress adversely affects rice growth,particularly at the early vegetative growth stage.In higher plants,nitrogen metabolism plays a central role in amino acid metabolism,plant defense mechanisms and productivity.This report investigated the effects of cold stress and supplementalγ-aminobutyric acid(GABA)under cold stress on nitrogen metabolism in rice seedlings.Cold stress resulted in a greater increase in the transformation to NH_(4)^(+)by nitrate reductase(NR)in roots,it further resulted in lower levels of NO_(3)^(-)content in roots,weakened glutamine glutamate(GOGAT/GS)pathway and elevated glutamate dehydrogenase(GDH)pathway of rice seedlings.Whereas,compared with cold stress,supplementation of GABA(2.5 mmol·L^(-1))could increase relative water content(79.43%)and biomass(34.15%)of rice seedlings.GABA could act as an amplifier of stress signal conduction/transduction to increase NR activity and promote NO_(3)^(-)assimilation in leaves.In addition,GABA elicited the Ca^(2+)signaling pathway which could promote the GDH pathway and GABA shunt,increase the activities of GS and GDH,and the expression of OsGAD2 and OsGDH family.The GABA might increase the ratio of the Glu family and avoid NH4+toxicity in order to raise the concentration of organic compounds and alleviate the harmful consequences of cold stress.Based on these observations,this study proposed that GABA mediated cold tolerance in rice seedlings by activating Ca^(2+)burst and subsequent crosstalk among Ca^(2+)signaling,GDH pathway and GABA shunt.

Environmental cues associated with morphine modulate release of glutamate and γ-aminobutyric acid in ventral subiculum

Objective To investigate whether environmental cues associated with different properties of morphine could regulate the extracellular levels of glutamate and y-aminobutyric acid （GABA） in the hippocampal ventral subiculum, which play a critical role in the reinstatement of drug-seeking behavior induced by environmental cues. Methods Conditioning place preference （CPP） and conditioning place aversion （CPA） models were used to establish environment associated with rewarding and aversive properties of morphine respectively. Microdialysis and high performance liquid chromatography were used to measure the extracelluar level of glutamate and GABA in the ventral subiculum under these environmental cues. Results Exposure to the environmental cues associated with rewarding properties of morphine resulted in a decrease （approximately 11%） of extracellular level of GABA in ventral subiculum, and exposure to the environmental cues associated with aversive properties of morphine resulted in an increase （approximately 230%） of extracellular level of glutamate in ventral subiculum. Conclusion Environmental cues associated with different properties of morphine modulate the release of distinct neurotransmitters in the hippocampal ventral subiculum possibly through different neural circuit.

Modulation of γ-aminobutyric acid on painful sense in central nervous system of morphine-dependent rats

Objective To observe the effects of y-aminobutyric acid （GABA） on the electric activities of pain-excited neurons （PEN） in nucleus accumbens （NAc） in central nervous system （CNS） of morphine-dependent rats. Methods After GABA or the GABAA-receptor antagonist, bicuculline （Bic）, was injected into cerebral ventricles or NAc, right sciatic nerve was stimulated by electrical pulses, which was considered as traumatic pain stimulation. Extracellular recordings methods were used to record the electric activities of PEN in NAc. Results When GABA was injected into intracerebroventricle （ICV） as well as NAc, it could decrease the pain-evoked discharge frequency and prolong the latency of PEN. Bic could interdict the above effects of GABA on the electric activities of PEN. Conclusion Exogenous GABA might have an inhibitory effect on the central pain adjustment. Furthermore, GABA and GABAA receptor participate and mediate the traumatic information transmission process in CNS.

Relationship of nocturnal concentrations of melatonin, gamma-aminobutyric acid and total antioxidants in peripheral blood with insomnia after stroke： study protocol for a prospective non-randomized controlled trial

Melatonin and gamma-aminobutyric acid（GABA） have been shown to regulate sleep. The nocturnal concentrations of melatonin, GABA and total antioxidants may relate to insomnia in stroke patients. In this prospective single-center non-randomized controlled clinical trial performed in the China Rehabilitation Research Center, we analyzed the relationship of nocturnal concentrations of melatonin, GABA and total antioxidants with insomnia after stroke. Patients during rehabilitation of stroke were recruited and assigned to the insomnia group or non-insomnia group. Simultaneously, persons without stroke or insomnia served as normal controls. Each group contained 25 cases. The primary outcome was nocturnal concentrations of melatonin, GABA and total antioxidants in peripheral blood. The secondary outcomes were Pittsburgh Sleep Quality Index, Insomnia Severity Index, Epworth Sleepiness Scale, Fatigue Severity Scale, Morningness-Eveningness Questionnaire（Chinese version）, and National Institute of Health Stroke Scale. The relationship of nocturnal concentrations of melatonin, GABA and total antioxidants with insomnia after stroke was analyzed and showed that they were lower in the insomnia group than in the non-insomnia group. The severity of stroke was higher in the insomnia group than in the non-insomnia group. Correlation analysis demonstrated that the nocturnal concentrations of melatonin and GABA were associated with insomnia after stroke. This trial was registered at Clinical Trials.gov, identifier： NCT03202121.

Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

γ-Aminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology

γ-Aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function. γ-Aminobutyric acid transporter was implicated to be involved in this process. However, the potential role of γ-aminobutyric transporter in testis has not been explored. In this study, we investigated the existence of mouse γ-aminobutyric acid transporter subtype I (mGAT1) in testis. Wild-type and transgenic mice, which overexpressing mGAT1 in a variety of tissues, especially in testis, were primarily studied to approach the profile of mGAT1 in testis. Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type. Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen. Reduced number of spermatids was also found in some seminiferous tubules. Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function.

Effect of the pineal gland on 5-hydroxytryptamine and γ-aminobutyric acid secretion in the hippocampus of male rats during the summer and winter

Objective:The present study aimed to investigate the effect of seasonal variation on neurotransmitter release in the hippocampus of normal rats and rats with pineal excision.Methods:Two time points,the summer and winter solstice,which are the longest and shortest days of the year,respectively,were selected.Male Spraguee Dawley rats that underwent a sham operation without pineal excision were included as a control group.The concentrations of 5-hydroxytryptamine(5-HT)andγ-aminobutyric acid(GABA)were determined by radioimmunoassays and enzyme-linked immunosorbent assays,respectively.Results:In the winter,the 5-HT and GABA levels in normal rats exhibited a significant difference compared with those in the operation group(P<.01).A difference was also noted in GABA levels between the normal group and the sham operation group(P<.05).The concentrations of 5-HT and GABA in the hippocampal tissues of the normal group exhibited a seasonal rhythm consisting of elevation during the summer and reduction during the winter(P<.01),while the GABA levels in the sham operation group exhibited a significant difference,with elevation during the summer and reduction during the winter(P<.01).In the operation group,GABA showed the same trend(P<.01).Conclusion:The seasonal rhythm of neurotransmitter secretion by the hippocampus(5-HT and GABA)consisted of elevation during the summer and reduction during the winter.During the winter,the pineal gland exhibited a reverse regulatory effect on the secretion of 5-HT and GABA in the hippocampus,and it exhibited seasonal selectivity with regard to the regulation of 5-HT.

On-line near-infrared spectroscopy optimizing and monitoring biotransformation process of γ-aminobutyric acid

Near-infrared spectroscopy （NIRS） with its fast and nondestructive advantages can be qualified for the real-time quantitative analysis. This paper demonstrates that NIRS combined with partial least squares （PLS） regression can be used as a rapid analytical method to simultaneously quantify L-glutamic acid （L- GIu） and γ-aminobutyric acid （GABA） in a biotransformation process and to guide the optimization of production conditions when the merits of NIRS are combined with response surface methodology. The high performance liquid chromatography （HPLC） reference analysis was performed by the o-phthaldialdehyde pre-column derivatization. NIRS measurements of two batches of 141 samples were firstly analyzed by PLS with several spectral pre-processing methods. Compared with those of the HPLC reference analysis, the resulting determination coefficients （R2）, root mean square error of prediction （RMSEP） and residual predictive deviation （RPD） of the external validation for the L-GIu concentration were 99.5%, 1.62 g/L, and 11.3, respectively. For the GABA concentration, R2, RMSEP, and RPD were 99.8%, 4.00 g/L, and 16.4, respectively. This NIRS model was then used to optimize the biotransformation process through a Box- Behnken experimental design. Under the optimal conditions without pH adjustment, 200 gjL L-GIu could be catalyzed by 7148 U/L glutamate decarboxylase （GAD） to GABA, reaching 99% conversion at the fifth hour. NIRS analysis provided timely information on the conversion from L-GIu to GABA. The results suggest that the NIRS model can not only be used for the routine profiling of enzymatic conversion, providing a simple and effective method of monitoring the biotransformation process of GABA, but also be considered to be an optimal tool to guide the optimization of production conditions.

Effect of Propofol on Glutamate and γ-aminobutyric Acid Release from Rat Hippocampal Synaptosomes

To investigate the effect of propofol on the release of glutamate and γ-aminobutyric acid （GABA） from rat hippocampal synatosomes, synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid （aCSF）. With the experiment of Ca^2＋-dependent release of glutamate and GABA, dihydrokainic acid （DHK） and nipectic acid were added into aCSF. For the observation of Ca^2＋-independent release of glutamate and GABA, no DHK, nipectic acid and Ca^2＋ were added from aCSF. The release of glutamate and GABA were evoked by 20 μmol/L veratridine or 30 mmol/L KCh The concentration of glutamate and GABA in aCSF was measured by using high-performance liquid chromatography （HPLC）. 30, 100 arid 300 μmol/L propofol significantly inhibited veratridine-evoked Ca^2＋-dependent release of glutamate and GABA （P〈0. 01 or P〈0. 05）, However, propofol showed no effect on elevated KCl-evoked Ca^2＋-dependent release of glutamate and GABA （P〉0, 05）, Veratridine or elevated KCI evoked Ca^2＋-independent release of glutamate and GABA was not affected significantly by propofol （P〉0.05）. Propofol could inhibit Ca^2＋- dependent release of glutamate and GABA, However, it has no effect on the Ca^2＋-independent release of glutamate and GABA,

Expression of gamma-aminobutyric acid type A receptor α_2 subunit in the dorsal root ganglion of rats with sciatic nerve injury

The γ-aminobutyric acid neurotransmitter in the spinal cord dorsal horn plays an important role in pain modulation through primary afferent-mediated presynaptic inhibition. The weakening of γ-aminobutyric acid-mediated presynaptic inhibition may be an important cause of neuropathic pain. γ-aminobutyric acid-mediated presynaptic inhibition is related to the current strength of γ-aminobutyric acid A receptor activation. In view of this, the whole-cell patch-clamp technique was used here to record the change in muscimol activated current of dorsal root ganglion neurons in a chronic constriction injury model. Results found that damage in rat dorsal root ganglion neurons following application of muscimol caused concentration-dependent activation of current, and compared with the sham group, its current strength and γ-aminobutyric acid A receptor protein expression decreased. Immunofluorescence revealed that γ-aminobutyric acid type A receptor α2 subunit protein expression decreased and was most obvious at 12 and 15 days after modeling. Our experimental findings confirmed that the y-aminobutyric acid type A receptor α2 subunit in the chronic constriction injury model rat dorsal root ganglion was downregulated, which may be one of the reasons for the reduction of injury in dorsal root ganglion neurons following muscimol-activated currents.

Incorporation ofγ-aminobutyric acid and cesium cations to formamidinium lead halide perovskites for highly efficient solar cells

The stability issue has become one of the main challenges for the commercialization of perovskite solar cells(PSCs).Formamidinium(FA)-based perovskites have shown great promise owing to their improved thermal and moisture stability.However,these perovskites are suffering from phase transition and separation.Here,a method of incorporating of γ-aminobutyric acid(GABA) and cesium cations into FAPbl_(3) is developed to improve the phase stability.It is demonstrated that the crystallinity of α-FAPbl_(3) phase is greatly improved and the phase transition temperature is significantly dropped.The resultant solar cell therefore obtains a champion power conversion efficiency(PCE) of 23.71%,which is one of the highest efficiencies for methylammonium-free PSCs.Furthermore,it shows an impressively enhanced stability under illumination,exhibiting the great potential of FA-based perovskites for efficient and stable solar cells.

Transgenic mice overexpressingγ-aminobutyric acid transporter subtypeⅠ develop obesity

Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

Electroacupuncture (EA) Speeds Up the Regulation of Hypothalamic Pituitary Adrenal Axis Dysfunction in Acute Surgical Trauma Rats: Mediated by Hypothalamic Gamma-Aminobutyric Acid (GABA)_AReceptors

Hypothalamic Corticotropin-releasing factor (CRF) directly activates the hypothalamic pituitary adrenal axis (HPA axis) during the surgical trauma induced stress response. Electroacupuncture (EA) has been demonstrated to have stress relieving effects in breast surgery, colorectal surgery, prostatectomy and craniotomy. This study was aimed to investigate the hypothesis that EA could regulate hypothalamic CRF in surgical trauma rats. In experiment one, Sprague-Dawley (SD) male rats were divided into intact, model (10% partial hepatectomy), sham EA and EA group. Rats from the Sham EA and EA group were stimulated at ST36-Zusanli and SP6-Sanyiniiao acupoints twice, 24 hours before the surgery and immediately after the surgery. Expressions of hypothalamic CRF and CRFR, GABA receptors, glutamate decarboxylase (GAD), serum adrenocorticotropic hormone (ACTH) and Corticosterone (CORT) were observed at 2, 4, 8 and 24 h after the surgery by radioimmunoassay (RIA), western blot, real-time PCR and immunohistochemistry. In the experiment two, SD male rats were divided into the intact, model, model + vehicle, model + L-838,417 EA and EA + L838,417 group. It was found that hypothalamus CRF, serum ACTH and CORT levels were increased in model group compared with the intact group, and those in the EA group decreased in comparison with the model group. Compared with the model group, hypothalamus-aminobutyric acid (GABA) receptor Aα3 mRNA and protein expressions of the EA group raised strikingly. In conclusion, EA alleviated surgical stress response by improving the GABA synthesis in hypothalamus, thus enhancing GABA receptors’ inhibitory regulation of the HPA axis dysfunction in rats with acute surgical trauma.

Γ-Aminobutyric acid promotes methionine-choline deficient diet-induced nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis（NASH） is one of the most common liver diseases and a major cause of liver fibrosis worldwide.r-Aminobutyric acid（GABA） is one of the most abundant inhibitory neurotransmitters in the central nervous system.Recently,it has been reported that GABAergic signaling pathways are found in various non-neuronal tissues including the immune system and play a functional role.In the present study,we investigated whether administration of GABA has effects on NASH through its immunomodulatory effects.To test this hypothesis,C57BL/6 mice were fed a methionine-choline-deficient（MCD） diet for 8 weeks.After four weeks into MCD feeding,mice were provided with plain water（control） or water containing 2 mg/mL of GABA for the subsequent 4 weeks.Using this MCD diet-induced NASH model,we found that mice receiving GABA showed more severe steatohepatitis and liver fibrosis than control mice.This increased liver damage was confirmed by higher levels of serum alanine transaminase（ALT） and aspartate aminotransferase（AST） compared to the control group.In accordance with increased liver steatohepatitis,NASH-related and inflammatory gene expression（collagen al,tissue inhibitor of metalloproteinase-1,TNF-α） in the liver was markedly increased in GABA-treated mice.Furthermore,GABA directly enhanced production of inflammatory cytokines including IL-6 and TNF-α in LPS activated RAW macrophage cells and increased TIB-73 hepatocyte death.Such effects were abolished when GABA was treated with bicuculline,a competitive antagonist of GABA receptors.These results suggest that oral administration of GABA may be involved in changes of the liver immune milieu and conferred detrimental effects on NASH progression.

Decoding the nexus:branched-chain amino acids and their connection with sleep,circadian rhythms,and cardiometabolic health

The sleep-wake cycle stands as an integrative process essential for sustaining optimal brain function and,either directly or indirectly,overall body health,encompassing metabolic and cardiovascular well-being.Given the heightened metabolic activity of the brain,there exists a considerable demand for nutrients in comparison to other organs.Among these,the branched-chain amino acids,comprising leucine,isoleucine,and valine,display distinctive significance,from their contribution to protein structure to their involvement in overall metabolism,especially in cerebral processes.Among the first amino acids that are released into circulation post-food intake,branched-chain amino acids assume a pivotal role in the regulation of protein synthesis,modulating insulin secretion and the amino acid sensing pathway of target of rapamycin.Branched-chain amino acids are key players in influencing the brain's uptake of monoamine precursors,competing for a shared transporter.Beyond their involvement in protein synthesis,these amino acids contribute to the metabolic cycles ofγ-aminobutyric acid and glutamate,as well as energy metabolism.Notably,they impact GABAergic neurons and the excitation/inhibition balance.The rhythmicity of branchedchain amino acids in plasma concentrations,observed over a 24-hour cycle and conserved in rodent models,is under circadian clock control.The mechanisms underlying those rhythms and the physiological consequences of their disruption are not fully understood.Disturbed sleep,obesity,diabetes,and cardiovascular diseases can elevate branched-chain amino acid concentrations or modify their oscillatory dynamics.The mechanisms driving these effects are currently the focal point of ongoing research efforts,since normalizing branched-chain amino acid levels has the ability to alleviate the severity of these pathologies.In this context,the Drosophila model,though underutilized,holds promise in shedding new light on these mechanisms.Initial findings indicate its potential to introduce novel concepts,particularly in elucidating the intricate connections between the circadian clock,sleep/wake,and metabolism.Consequently,the use and transport of branched-chain amino acids emerge as critical components and orchestrators in the web of interactions across multiple organs throughout the sleep/wake cycle.They could represent one of the so far elusive mechanisms connecting sleep patterns to metabolic and cardiovascular health,paving the way for potential therapeutic interventions.

Effects of gamma-aminobutyric acid receptors on muscarinic receptor-mediated free calcium ion levels in the facial nucleus following facial nerve injury

Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A （GABAA） receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B （GABAB） and C （GABAc） receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.

Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

We used RNA interference （RNAi） to disrupt synthesis of the cortical neuronal y-aminobutyric acid A receptor （GABAAR） al in rats during development, and measured outward K＋ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA （siRNA） for GABAAR al subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR al mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K^currents were decreased, indicating that GABAAR al subunits in developing neurons participate in neuronal function by regulating outward K＋ current.

Synthesis and Anticonvulsant Activity of γ-Aminobutyric Acid Derivatives

Under physiological conditions, γ-aminobutyric acid poorly crosses the blood-brain barrier. It is likely that a non-toxic derivative of γ-aminobutyric acid which enters the brain easily will have useful anticonvulsant activity. 16 derivatives of γ-aminobutyric acid with an imine link to a lipophilic carrier were prepared and tested for anticonvulsant activity; six compounds show anticonvulsant activity.

Connections between 5-HT-containing terminals and 5-HT_(2A) recep tor and γ-aminobutyric acid or glycine co-existed neurons in the rat medullary dorsal horn

Objective: To investigatetheconnectionsbetweenserotonin(5-HT)-containingterminalsand5-HT 2A receptor(5-HT 2A R)/γ-aminobutyricacid(GABA)or5-HT 2A R/glycineco-existedneuronsin theratmedullarydorsalhorn(MDH).Meth ods:Immunofluorescencehistochemicaltriple-stainingfor5-HT,5-HT 2A R,GABAor glycine.Results:5-HT-im-munoreactivefibersandterminalswerechieflylocatedinthesuperficiallaminae(laminaeⅠandⅡ)of theMDH.Neurons exhibiting5-HT 2A R-,GABA-or glycine-immunoreactivitiesweremainlyobservedin the superficiallaminae.Some5-HT 2A R-immunopositiveneuronsalsoexhibitedGABA-or glycine-immunoreactivities.5-HT-containingterminalsmade closecontactswith5-HT 2A R/GABAor5-HT 2A R/glycineco-existedneurons.Conclusion:5-HT 2A R/GABAor5-HT 2A R/glycineco-existinsomeof theneuronsinthesuperficiallaminaeof theMDH.5-HT-immunoreactiveterminalsformclose connectionswith5-HT 2A R/GABAor5-HT 2A R/glycineco-existedneurons.