Modulatory effects of two novel agonists for serotonin receptor 7 on emotion, motivation and circadian rhythm profiles in mice

Serotonin receptor 7, i.e. 5-HT(7) protein coded by Htr7 gene, was discovered in supra-chiasmatic nucleus (SCN) of the hypothalamus but is widespread in the forebrain. Studies have shown that this receptor is involved in learning/memory, regulation of mood and circadian rhythms. The modulatory effects of two novel agonists, LP-211 and LP-378, were assessed in male adult CD-1 mice with a battery of behavioral tests. Exp. 1 (Black/White Boxes, BWB: Adriani et al., 2009) and Exp. 2 (Dark/Light, D/L; Novelty-seeking, N-S) show: a) that LP-211 administration (acutely, at a 0.25 mg/kg dose i.p.) increases locomotion and BWB exploration; b) that the time spent away from an aversive, lit chamber (i.e., stress-induced anxiety) and in a new environment (i.e., novelty-induced curiosity) are both reduced. Sub-chronic LP-211 (at a 2.5 mg/kg dose i.p.) reveals a sensitization of locomotor-stimulant properties over 4-5 days. In Exp. 3 (BWB), a three- to four-fold dosage (acutely, at 0.83 mg/kg i.p.) is needed with LP-378 to increase locomotion and BWB exploration. In Exp. 4, mice under constant-light conditions reveal the expected spontaneous lengthening (1.5 h per day) of circadian rhythms. A significant phase advance is induced by LP-211 (at a 0.25 mg/kg dose i.p., administered around activity offset), with onset of activity taking place 6 h earlier than in controls. In summary, LP-211 is able to act consistently onto exploratory motivation, anxiety-related profiles, and spontaneous circadian rhythm. In the next future, agonist modulation of 5-HT(7) receptors might turn out to be beneficial for sleep and/or anxiety disorders. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

5-HT2C Receptor Desensitization Moderates Anxiety in 5-HTT Deficient Mice: From Behavioral to Cellular Evidence

Background:

Desensitization and blockade of 5-HT_2C receptors (5-HT_2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT_2CR desensitization in specific brain areas.

Methods:

Mice lacking the 5-HT reuptake carrier (5-HTT^-/-) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT_2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT_2CR–induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos–induced expression) levels.

Results:

Although 5-HTT^-/- mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT_2CR–mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR–mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT^-/- mutants. These indices of tolerance to 5-HT_2CR stimulation were associated neither with altered levels of 5-HT_2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT_2CR was higher in 5-HTT^-/- mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT_2CR–like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT_2CR agonist RO-60,0175 was absent in 5-HTT^-/- mutants.

Conclusions:

Such blunted responsiveness of the 5-HT_2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT^-/- mice.     

Clozapine Acts as an Agonist at Serotonin 2A Receptors to Counter MK-801-Induced Behaviors through a βArrestin2-Independent Activation of Akt

The G protein-coupled serotonin 2A receptor (5-HT2AR) is a prominent target for atypical antipsychotic drugs, such as clozapine. Although clozapine is known to inhibit 5-HT2AR signaling through G protein-dependent mechanisms, it differs from classic GPCR antagonists, in that it also induces 5-HT2AR internalization and activates Akt signaling via a 5-HT2AR-mediated event. In this regard, clozapine may also be considered a functionally selective agonist. The cognate neurotransmitter at the 5-HT2AR, serotonin, also induces 5-HT2AR internalization and Akt phosphorylation. Serotonin promotes interactions with the scaffolding and regulatory protein, βarrestin2, which results in the recruitment and activation of Akt. These interactions prove to be critical for serotonin-induced, 5-HT2AR-mediated behavioral responses in mice. Herein, we sought to determine whether clozapine also utilizes βarrestin2-mediated mechanisms to induce 5-HT2AR signaling, and whether this interaction contributes to its behavioral effects in mice. We demonstrate that unlike serotonin, clozapine-mediated 5-HT2AR internalization and Akt phosphorylation is independent of receptor interactions with βarrestin2. Moreover, clozapine-mediated suppression of MK-801 and phencyclidine (PCP)-induced hyperlocomotion is βarrestin2 independent, although it is dependent upon Akt. These results demonstrate that pharmacologically oppositional ligands, serotonin and clozapine, utilize differential mechanisms to achieve the same 5-HT2AR-meadiated downstream events: Akt phosphorylation and receptor internalization. Although βarrestin2 has no effect on clozapine''s actions in vivo, Akt phosphorylation is required for clozapine''s efficacy in blocking MK-801- and PCP-induced models of schizophrenic behaviors in mice.     

5-HT(1A) and 5-HT(7) receptor crosstalk in the regulation of emotional memory: Implications for effects of selective serotonin reuptake inhibitors

This study utilized pharmacological manipulations to analyze the role of direct and indirect activation of 5-HT₇ receptors (5-HT₇Rs) in passive avoidance learning by assessing emotional memory in male C57BL/6J mice. Additionally, 5-HT₇R binding affinity and 5-HT₇R-mediated protein phosphorylation of downstream signaling targets were determined. Elevation of 5-HT by the selective serotonin reuptake inhibitor (SSRI) fluoxetine had no effect by itself, but facilitated emotional memory performance when combined with the 5-HT_1AR antagonist NAD-299. This facilitation was blocked by the selective 5-HT₇R antagonist SB269970, revealing excitatory effects of the SSRI via 5-HT₇Rs. The enhanced memory retention by NAD-299 was blocked by SB269970, indicating that reduced activation of 5-HT_1ARs results in enhanced 5-HT stimulation of 5-HT₇Rs. The putative 5-HT₇R agonists LP-44 when administered systemically and AS19 when administered both systemically and into the dorsal hippocampus failed to facilitate memory. This finding is consistent with the low efficacy of LP-44 and AS19 to stimulate protein phosphorylation of 5-HT₇R-activated signaling cascades. In contrast, increasing doses of the dual 5-HT_1AR/5-HT₇R agonist 8-OH-DPAT impaired memory, while co-administration with NAD-299 facilitated of emotional memory in a dose-dependent manner. This facilitation was blocked by SB269970 indicating 5-HT₇R activation by 8-OH-DPAT. Dorsohippocampal infusion of 8-OH-DPAT impaired passive avoidance retention through hippocampal 5-HT_1AR activation, while 5-HT₇Rs appear to facilitate memory processes in a broader cortico-limbic network and not the hippocampus alone.     

Severe Serotonin Depletion after Conditional Deletion of the Vesicular Monoamine Transporter 2 Gene in Serotonin Neurons: Neural and Behavioral Consequences

The vesicular monoamine transporter type 2 gene (VMAT2) has a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of VMAT2 under control of the serotonin transporter (slc6a4) promoter. VMAT2^sert−cre mice showed a major (−95%) depletion of 5-HT levels in the brain with no major alterations in other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2^sert−cre mice but developed normal innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT_1A autoreceptor coupling to G protein, as assessed with agonist-stimulated [³⁵S]GTP-γ-S binding, was observed in the raphe area, indicating an adaptive change to reduced 5-HT transmission. Behavioral evaluation in adult VMAT2^sert−cre mice showed an increase in escape-like reactions in response to tail suspension and anxiolytic-like response in the novelty-suppressed feeding test. In an aversive ultrasound-induced defense paradigm, VMAT2^sert−cre mice displayed a major increase in escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, although in reduced amounts, in synaptosomes from VMAT2^sert−cre mouse brain. These findings are coherent with the notion that 5-HT has an important role in anxiety, and provide new insights into the role of endogenous 5-HT in defense behaviors.     

Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity

Both the glutamatergic and serotonergic (5-HT) systems are implicated in the modulation of mood and anxiety. Descending cortical glutamatergic neurons regulate 5-HT neuronal activity in the midbrain raphe nuclei through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. To analyze the functional role of GLUA1-containing AMPA receptors in serotonergic neurons, we used the Cre-ERT2/loxP-system for the conditional inactivation of the GLUA1-encoding Gria1 gene selectively in 5-HT neurons of adult mice. These Gria1^5-HT−/− mice exhibited a distinct anxiety phenotype but showed no alterations in locomotion, depression-like behavior, or learning and memory. Increased anxiety-related behavior was associated with significant decreases in tryptophan hydroxylase 2 (TPH2) expression and activity, and subsequent reductions in tissue levels of 5-HT, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine in the raphe nuclei. However, TPH2 expression and activity as well as monoamine levels were unchanged in the projection areas of 5-HT neurons. Extracellular electrophysiological recordings of 5-HT neurons revealed that, while α1-adrenoceptor-mediated excitation was unchanged, excitatory responses to AMPA were enhanced and the 5-HT_1A autoreceptor-mediated inhibitory response to 5-HT was attenuated in Gria1^5-HT−/− mice. Our data show that a loss of GLUA1 protein in 5-HT neurons enhances AMPA receptor function and leads to multiple local molecular and neurochemical changes in the raphe nuclei that dysregulate 5-HT neuronal activity and induce anxiety-like behavior.     

Ranunculus ternatus Thunb extract attenuates renal fibrosis of diabetic nephropathy via inhibiting SMYD2

ContextRanunculus ternatus Thunb (Ranunculaceae), (RTT) is used clinically for the treatment of tuberculosis or as tumour adjuvant therapy, but its potential effect on diabetic nephropathy (DN) has not been studied.ObjectiveTo investigate the effect of RTT extract in renal fibrosis of DN.Materials and methodsC57BL/6 mice were randomly divided into four groups (n = 12). Diabetes mellitus (DM) mice were induced by streptozotocin (STZ, 55 mg/kg/day) for five consecutive days and treated by RTT extract (2 g/kg). Afterward, blood glucose, HE and Masson staining were assayed. The expression levels of Vimentin, ɑ-SMA, TNF-ɑ, NF-κB p-p65, NF-κB p65, SMYD2, H3K36me3, H3K4me3 were determined by western blots. Firbronectin was respectively assayed by western blot and immunofluorescent staining.ResultsRTT extract significantly ameliorated renal injury and renal fibrosis in the renal tissue of STZ-induced diabetic mice as demonstrated by the decreased expression level of Fibronectin (65%), Vimentin and α-SMA (75% & 53%). In addition, the levels of TNF-α (57%), NF-κB p-p65 and NF-κB p65 (35% & 25%) were elevated in the DN mice. Importantly, these were alleviated after RTT extract treatment. Moreover, we observed that the protein levels of SMYD2 (30%), H3K36me3 and H3K4me3 (53% & 75%) were reduced in DN mice after treatment with RTT extract.Discussion and conclusionsRTT extract mediates antifibrotic effects and anti-inflammatory responses in STZ-induced DN mainly through suppressing SMYD2 activation and H3K36me3 and H3K4me3 protein expression. RTT extract might have therapeutic potential against high glucose-induced nephropathy.     

A comprehensive study on the relieving effect of Lilium brownii on the intestinal flora and metabolic disorder in p-chlorphenylalanine induced insomnia rats

ContextThe bulb of Lilium brownii F. E. Brown (Liliaceae) (LB) is a common Chinese medicine to relieve insomnia.ObjectiveTo investigate the molecular mechanism of LB relieving insomnia.Materials and methodsInsomnia model was induced by intraperitoneally injection p-chlorophenylalanine (PCPA) in Wistar rats. Rats were divided into three groups: Control, PCPA (400 mg/kg, i.p. 2 days), LB (598.64 mg/kg, oral 7 days). The levels of 5-hydroxytryptamine (5-HT), norepinephrine (NE), melatonin (MT), and the expression of GABA_A, 5-HT1A and MT receptors, as well as pathological changes in hypothalamus, were evaluated. 16S rDNA sequencing and UPLC-MS/MS were used to reveal the change of the intestinal flora and metabolic profile.ResultsThe adverse changes in the abundance and diversity of intestinal flora and faecal metabolic phenotype altered by PCPA in rats were reversed after LB treatment, accompanied by the up-regulated levels of 5-HT as 8.14 ng/mL, MT as 16.16 pg/mL, 5-HT1A R and GABA_A R, down-regulated level of NE as 0.47 ng/mL, and the improvement of pathological phenomena of cells in the hypothalamus. And the arachidonic acid metabolism and tryptophan metabolism pathway most significantly altered by PCPA were markedly regulated by LB. Besides, it was also found that LB reduced the levels of kynurenic acid related to psychiatric disorders and trimethylamine-N-oxide associated with cardiovascular disease.ConclusionThe mechanism of LB relieving insomnia involves regulating flora and metabolites to resemble the control group. As a medicinal and edible herb, LB could be considered for development as a health-care food to relieve increasing insomniacs in the future.     

Serotonin Transporter and Tryptophan Hydroxylase Gene Variations Mediate Working Memory Deficits of Cocaine Users

Cocaine users consistently develop working memory (WM) impairments but the mediating molecular mechanisms are unknown so far. Recent evidence suggests that the serotonin (5-HT) system is altered by chronic cocaine use, while also being involved in WM processing. Thus, we investigated the effects of genetic variations impacting 5-HT activity and of peripheral 5-HT transporter (5-HTT) mRNA expression on WM performance in cocaine users and stimulant naive controls. Two hundred twenty participants (126 cocaine users, 94 controls) were assessed with visuospatial, spatial, and verbal WM tasks, genotyped for the length polymorphism in the promoter region of the 5-HTT (5-HTTLPR), the variable number of tandem repeats in the second intron of the 5-HTT (VNTR In2), two single-nucleotide polymorphisms (rs4570625 and rs1386497) in the tryptophan hydroxylase-2 (TPH2) gene and quantified for peripheral 5-HTT mRNA expression in whole-blood samples. Several significant gene × environment interactions between 5-HT genotypes and cocaine use on WM emerged: in cocaine users, the long/long (5-HTTLPR), 9+10/9+10 (VNTR In2) and C/C (TPH2 rs1386497) genotypes were risk alleles for WM impairments, whereas in healthy controls these polymorphisms were associated with improved WM performance. Analogously, high 5-HTT mRNA levels were associated with worse executive WM performance in cocaine users but with increased performance in controls. These gene × environment interactions suggest that the 5-HT system has an important role in the development of cognitive deficits in chronic cocaine users. Hence, pharmacological compounds targeting 5-HT neurotransmission might be promising for the treatment of cognitive deficits in cocaine dependence.     

Liver 5-HT7 receptors: A novel regulator target of fibrosis and inflammation-induced chronic liver injury in vivo and in vitro

Background and aimHepatocellular cancer (HCC) is the sixth most common cancer and liver fibrosis is strongly associated with HCC. Treatment options are limited, and preventive strategies should be developed. An important step in the beginning of liver fibrosis is a strong inflammatory response. 5-HT7 is the last recognized member of the serotonin receptor family and is expressed in both central nerve system and peripheral system and have a lot of functions like learning, memory, smooth muscular relaxation, in the control of circadian rhythms and thermoregulation, pain and migraine, schizophrenia, anxiety, cognitive disturbances, and even inflammation.MethodsWe therefore examined the biochemical, histopathological and molecular effects of the 5-HT7 receptor agonist and antagonist on inflammatory liver fibrogenesis in animal models of progressive cirrhosis: a mouse model induced by carbon tetrachloride (CCl4) and in Hep3b cells.Results5-HT7 expression was observed in the liver in vivo and in vitro in CCl4-induced damage. 5-HT7 receptor agonist but not the antagonist reduced liver markers in mice and in Hep3b cells in carbon tetrachloride (CCl4) induced damage. 5-HT7 agonist, but not antagonist, protected liver tissue from oxidative stress in fibrosis. 5-HT7 agonist but not antagonist induces anti-inflammatory, anti-fibrinotic and anti-cytokine features in liver fibrosis in vivo and in vitro.Conclusions5-HT7 receptors have modulatory function and are an up-and-coming pharmacological target in the inflammatory fibrotic process. 5-HT7 receptor agonist LP-44 showed significant hepatoprotective effects against liver fibrosis, and LP-44 might become a useful therapeutic target for chronic liver inflammation and fibrosis.     

Cloning,expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7(b))

1. The rat 5-hydroxytryptamine (5-HT)₇ receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT₇ receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.
2. A truncated splice variation in the human 5-HT₇ receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT_7(b) receptor and the long form of the receptor as h5-HT_7(a).
3. The h5-HT_7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [³H]-5-carboxyamidotryptamine (5-CT; K_d=0.28±0.06 nM, B_max=7.3±1.7 pmol mg⁻¹ protein). The rank order of affinities (pK_i) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).
4. The h5-HT_7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC₅₀): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.
5. In a similar fashion to that described for the 5-HT_7(a) receptor, PCR studies suggested that the 5-HT_7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.
6. It is concluded that the h5-HT₇ receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT₇ receptor gene.

     

Characterization of 5-HT receptors mediating contraction and relaxation of the longitudinal muscle of guinea-pig distal colon in vitro

A range of agonists and antagonists were used to characterize the receptors through which 5-hydroxytryptamine (5-HT) contracts and relaxes the longitudinal muscle of segments of guinea-pig distal colon, in vitro. 5-HT contracted the longitudinal muscle over the concentration range 10^–9 to 10^–4 mol/l. The 5-HT3 receptor agonist, 2-methyl-5-HT, produced concentration dependent contractions over the range 10^–6 to 10^–4 mol/l. 5-methoxytryptamine, an agonist at 5-HT4 receptors, caused contractions over a concentration range of 10^–8 to 10^–4 mol/l. The 5-HT4 antagonist, SDZ 205-557 (5 × 10^–7 mol/l) substantially suppressed the responses to low concentrations of 5-HT and to 5-methoxytryptamine, but had no effect on the responses to higher concentrations of 5-HT. In contrast, the 5-HT3 antagonist, granisetron (10^–6 mol/l), blocked the effect of 2-methyl-5-HT and substantially depressed responses to high concentrations of 5-HT, but had no effect on lower concentrations of 5-HT. Granisetron produced a small reduction in the response to 5-methoxytryptamine. Tetrodotoxin (TTX) (3 × 10^–7 mol/l) almost abolished the response to 5-methoxytryptamine and markedly suppressed the response to 2-methyl-5-HT, but the responses to 5-HT were only partially reduced. The 5-HT, antagonist, methiothepin 10^–6 mol/l. depressed the response to 5-HT 10^–7 to 10^–4 mol/l. and blocked its TTX insensitive component. The 5-HT₂ antagonist, ketanserin, in concentrations up to 10^–5 mol/l, had no effect on the contractions evoked by 5-HT.The response to 5-HT was substantially depressed by hyoscine (3 × 10^–6 mol/l. The tachykinin antagonist, spantide 10^–5 mol/l. depressed the response to 5-HT but to a lesser extent than hyoscine. Spantide and hyoscine combined completely blocked the contractile responses to 5-HT Responses to 2-methyl-5-HT were partially suppressed by hyoscine (3 x 10^–6 mol/l. and spantide (10^–5 mol/l) and completely blocked when both byoscine and spantide were present. Contractions evoked by 5-methoxytryptamine were partially blocked by hyoscine (3 × 10^–6 mol/l) and were unaffected by spantide (10^–5 mol/l), but a combination of hyoscine and spantide completely blocked such responses.When the excitatory transmission was blocked with hyoscine (3 × 10^–6 mol/l) and spantide 10^–5 mol/l) and the tone of the muscle raised, an inhibitory response to 5-HT was revealed that had a threshold concentration between 10^–7 mol/l) and 3 × 10^–7 mol/l, and a maximum effect at 10^–4 mol/l. It was blocked by TTX (3 × 10^–7 mol/l) and granisetron 10^–6 mol/l. while N-nitro-l-arginine (NOLA) (10^–4 mol/l) and SDZ 205-557 (5 × 10^–7 mol/l) had no effect. Apamin A 10^–6 mol/l. partially suppressed this response.It is concluded that 5-HT3, 5-HT4 and 5-HT₁-like receptors mediate contraction of the longitudinal muscle of the distal colon. The 5-HT3 and 5-HT4 receptors are located on the excitatory motor neurons innervating the longitudinal muscle and the 5-HT₁-like receptor is located on the muscle. 5-HT3 receptors are also found on inhibitory neurons to the muscle. Correspondence to: D. J. Woollard at the above address     

Electrophysiological Effects of Repeated Administration of Agomelatine on the Dopamine,Norepinephrine, and Serotonin Systems in the Rat Brain

Agomelatine is a melatonergic MT1/MT2 agonist and a serotonin (5-HT) 5-HT_2C antagonist. The effects of 2-day and 14-day administration of agomelatine were investigated on the activity of ventral tegmental area (VTA) dopamine (DA), locus coeruleus (LC) norepinephrine (NE), and dorsal raphe nucleus (DRN) 5-HT neurons using in vivo electrophysiology in rats. The 5-HT_1A transmission was assessed at hippocampus CA3 pyramidal neurons. After a 2-day regimen of agomelatine (40 mg/kg/day, i.p.), an increase in the number of spontaneously active VTA-DA neurons (p<0.001) and in the firing rate of LC-NE neurons (p<0.001) was observed. After 14 days, the administration of agomelatine induced an increase in: (1) the number of spontaneously active DA neurons (p<0.05), (2) the bursting activity of DA neurons (bursts/min, p<0.01 and percentage of spikes occurring in bursts, p<0.05), (3) the firing rate of DRN-5-HT neurons (p<0.05), and (4) the tonic activation of postsynaptic 5-HT_1A receptors located in the hippocampus. The increase in 5-HT firing rate was D2 dependent, as it was antagonized by the D2 receptor antagonist paliperidone. The enhancement of NE firing was restored by the 5-HT_2A receptor antagonist MDL-100,907 after the 14-day regimen. All the effects of agomelatine were antagonized by a single administration of the melatonergic antagonist {"type":"entrez-protein","attrs":{"text":"S22153","term_id":"100005","term_text":"pir||S22153"}}S22153 (except for the increase in the percentage of spikes occurring in burst for DA neurons). The present results suggest that (1) agomelatine exerts direct (2 days) and indirect (14 days) modulations of monoaminergic neuronal activity and (2) the melatonergic agonistic activity of agomelatine contributes to the enhancement of DA and 5-HT neurotransmission.     

Anticolitis activity of chinese herbal formula Yupingfeng powder via regulating colonic enterochromaffin cells and serotonin

Objective:

To investigate whether traditional Chinese herbal formula Yupingfeng (YPF) powder has an anti-inflammatory effect on colonic inflammation, and to explore the mechanism involved.

Materials and Methods:

YPF powder was orally administrated to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice at the dose of 3, 6, and 12 g/kg/d for 7 consecutive days. Body weight, stool consistency, histopathological score, and myeloperoxidase (MPO) activity were tested to evaluate the effect of YPF powder on colonic inflammation while colonic enterochromaffin (EC) cell density and serotonin 5-hydroxytryptamine (5-HT) content were investigated to identify the effect of YPF powder on colonic 5-HT availability.

Results:

The results showed that the body weight of colitis mice was markedly decreased by 10, 12, 14, and 17% at 1, 3, 5, and 7 days (P < 0.05), whereas stool consistency score (3.6 vs. 0.4, P < 0.05), histopathological score (3.6 vs. 0.3, P < 0.05), and MPO activity (2.7 vs. 0.1, P < 0.05) in colitis mice were significantly increased compared to that of the normal mice; YPF powder treatment dose-dependently increased the body weight (7–13% increase) and decreased the stool consistency score (0.4–1.4 decrease), histopathological score (0.2–0.7 decrease), and MPO activity (0.1–0.9 decrease) in colitis mice. Colonic EC cell density (70% increase) and 5-HT content (40% increase) were markedly increased in colitis mice (P < 0.05), YPF powder treatment dose-dependently reduced EC cell density (20–50% decrease), and 5-HT content (5–27% decrease) in colitis mice.

Conclusion:

The findings demonstrate that the anti-inflammatory effect of YPF powder on TNBS - induced colitis may be mediated via reducing EC cell hyperplasia and 5-HT content. The important role of YPF powder in regulating colonic EC cell number and 5-HT content may provide an alternative therapy for colonic inflammation.KEY WORDS: Colonic inflammation, enterochromaffin cell, serotonin, ulcerative colitis     

Prenatal Poly I:C Challenge Affects Behaviors and Neurotransmission via Elevated Neuroinflammation Responses in Female Juvenile Rats

BackgroundExposure to polyriboinosinic-polyribocytidylic acid (Poly I:C) in pregnant rats has been reported to cause schizophrenia-like behaviors and abnormal neurotransmissions in adult, particularly male, offspring. However, what is less well understood are the effects of maternal Poly I:C exposure on adolescent behaviors and neurotransmission in female juvenile rats.MethodsFemale adolescent Poly I:C offspring were constructed by treating with 5 mg/kg Poly I:C on timed pregnant rats (gestation day 15). A battery of behavioral tests was conducted during postnatal day 35–60. Neurotransmitter receptors and inflammation markers in brain regions were evaluated by RT-qPCR on postnatal day 60.ResultsOpen field, elevated plus maze, and forced swimming tests revealed that prenatal Poly I:C exposure led to elevated anxiety-like and depression-like behaviors in female adolescent offspring. Deficits in pre-pulse inhibition and social interaction were also observed. However, the Poly I:C rats had better performance than the controls in the novel object recognition memory test, which demonstrated a behavioral phenotype with improved cognitive function. Prenatal Poly I:C exposure caused brain region–specific elevation of the P2X7 receptor- and NF-κB-NLRP3-IL-1β inflammatory signaling in female juvenile rats. Prenatal Poly I:C exposure decreased expression of GABA_A receptor subunits Gabrb3 in the prefrontal cortex and Gabrb1 and dopamine D2 receptor in the hippocampus, but increased NMDA receptor subunit Grin2a in the prefrontal cortex, 5-HT2A in the hippocampus, and Gabrb3 and D2 receptor in the nucleus accumben.ConclusionsPrenatal Poly I:C challenge causes behavioral deficits and brain-specific neurotransmission changes via elevated neuroinflammation responses in female adolescent offspring rats.     

Para-halogenated phenethylamines: Similar serotonergic effects in rats by different mechanisms

The serotonin (5-HT) behavioral syndrome in rats served as a model to test for possible acute serotonergic effects of parahalogenated phenethylamines. p-Chloro-, p-chloro-β-methyl-, p-fluoro-, p-bromo-, and p-iodophenethylamine produced the same 5-HT behavioral syndrome as did p-chloroamphetamine, but unlike the latter did not deplete brain 5-HT 3 days after injection. Pretreatment of rats with the 5-HT depletor p-chlorophenylalanine (pCPA) prevented the seronergic effects of both chloro-derivatives, and partially prevented the effects of bromo- and iodophenethylamine. 5-Hydroxytryptophan restored the behavioral responses to these compounds in pCPA-pretreated rats. pCPA treatment did not prevent the behavioral effects of p-fluorophenethylamine. Similarlt, zimelidine, a 5-HT uptake inhibitor, prevented the serotonergic behavioral effects of all compounds tested except p-fluorophenethylamine. Taken as a group, parahalogenated phenethylamines are short-acting serotonergic compounds which, unlike p-chloroamphetamine, do not produce long-lasting depletion of brain 5-HT p-Chlorophenethylamine and its β-methyl analog apparently activate central 5-HT receptors indirectly, i.e., by 5-HT release; p-fluorophenethylamine is a direct 5-HT agonist. The p-bromo- and p-iodo-derivatives apparently possess both properties.     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

5-HT potentiation of the GABAA response in the rat sacral dorsal commissural neurones

1. The modulatory effect of 5-hydroxytryptamine (5-HT) on the γ-aminobutyric acid_A (GABA_A) response was investigated in the neurones freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions.
2. 5-HT potentiated GABA-induced Cl⁻ current (I_GABA) without affecting the reversal potential of I_GABA and the apparent affinity of GABA to its receptor.
3. α-Methyl-5-HT mimicked the potentiation effect of 5-HT on I_GABA while ketanserine blocked it. 1-Oleoyl-2-acetyl-glycerol (OAG) potentiated I_GABA, and the effect of 5-HT on I_GABA was occluded by OAG pretreatment. In the presence of chelerythrine, 5-HT failed to potentiate I_GABA, suggesting that protein kinase C (PKC) is involved in the pathway through which the activation of the 5-HT₂ receptor potentiates the I_GABA.
4. The facilitatory effect of 5-HT on I_GABA remained in the presence of BAPTA-AM. LiCl also had no effect on 5-HT-induced potentiation of I_GABA.
5. H-89, genistein, okadaic acid and pervanadate all had no effects on 5-HT potentiation of I_GABA. Pertussis toxin treatment for 6–8 h did not block the facilitatory effect of 5-HT on I_GABA.
6. The present results show that GABA_A receptor in the rat SDCN could be modulated in situ by 5-HT, one of the major transmitters involved in the supraspinal control of nociception, and that the phosphorylation of GABA_A receptor by PKC may be sufficient to support such modulation. The results also strongly support the hypothesis that the cotransmission by 5-HT and GABA has an important role in the spinal cord.

     

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC₅₀ value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC₅₀, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.KEY WORDS: 2-Hydroxytyrosol, Metarhizium sp., Tyrosinase inhibitor, Melanine formation, B16 melanoma cells