Shine bright: considerations on the use of fluorescent substrates in living monoaminergic neurons in vitro

The biogenic monoamines dopamine （DA）, norepinephrine （NE） and serotonin （5-hydroxytryptamine, 5-HT） are major neuromodulators in the mammalian central nervous system （CNS）. DA containing neurons are found in i） the mesolimbic system in which cell bodies in the ventral tegmental area （VTA） project axons into the amygdala, cortex, hippocampus and the nucleus accumbens; and ii） the nigrostriatal system in which cell bodies located in the substantia nigra pars compacta send their axons into the dorsolateral parts of the striatum （Bjorklund and Dunnett, 2007）. The central noradrenergic neurons are concentrated in distinct brainstem nuclei with the locus coeruleus （LC） being the most prominent nucleus which projects a diffusely arborizing axonal network to most areas of the CNS （Szabadi, 2013）.

Antidepressant-Like Effect of Tetramethylpyrazine in Mice and Rats

The aim of this study was to investigate the potential antidepressive-like effect of tetramethylpyrazine (TMP), one of available blood-activating and stasis-eliminating components from traditional Chinese medicines, and its mechanism of the antidepressant-like action. Forced-swimming, tail-suspension, reserpine-induced hypothermia, akinesia and ptosis, 5-hydroxytryptophan (5-HTP)-induced head-twitch, and potentiation of noradrenaline (NE) toxicity tests, were per-formed to assess the potential antidepressant-like activity of TMP and to study the mechanism by which TMP exerts the antidepressant-like action. Intragastric (ig) administration of TMP markedly reduced the duration of immobility during forced-swimming tests and tail-supension test in rats and mice. TMP partialy reversed reserpine-induced hypothermia, ptosis and akinesia, and potentiated NE toxicity in mice, and these are similar to those of clomipramine;however, TMP did not potentiate 5-HTP-induced head-twitch response (HTR) in mice, and this is different from that of fluoxetine (FLU). The present data provide evidences that TMP possesses potent antidepressant-like activity, and it might be an adrenergic component of pharmacological activity, and its mechanism of antidepressant-like action is similar to that of clomipramine, and different from that of FLU.

Involvement of A5/A7 noradrenergic neurons and B2 serotonergic neurons in nociceptive processing:a fiber photometry study

In the central nervous system,the A6 noradrenaline(NA)and the B3 serotonin(5-HT)cell groups are well-recognized players in the descending antinociceptive system,while other NA/5-HT cell groups are not well characterized.A5/A7 NA and B25-HT cells project to the spinal horn and form descending pathways.We recorded G-Ca MP6 green fluorescence signal intensities in the A5/A7 NA and the B25-HT cell groups of awake mice in response to acute tail pinch stimuli,acute heat stimuli,and in the context of a non-noxious control test,using fiber photometry with a calcium imaging system.We first introduced G-Ca MP6 in the A5/A7 NA or B25-HT neuronal soma,using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamineβ-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus(AAV-Tet O(3 G)-G-Ca MP6).After confirming the specific expression patterns of G-Ca MP6,we recorded G-Ca MP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli.G-Ca MP6 fluorescence intensity in the A5,A7,and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after,it returned to baseline fluorescence intensity.This was not observed in the non-noxious control test.The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B25-HT neurons but the non-noxious stimuli do not.The present study suggests that A5/A7 NA or B25-HT neurons play important roles in nociceptive processing in the central nervous system.We suggest that A5/A7/B2 neurons may be new therapeutic targets.All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University(MD17105)on February 22,2018.

Jiaotai Pill (交泰丸) Alleviates Insomnia through Regulating Monoamine and Organic Cation Transporters in Rats

Objective To reveal the effect and mechanism of Jiaotai Pill(交泰丸,JTP)on insomniac rats.Methods The insomniac model was established by intraperitoneal injection of p-chlorophenylalanine(PCPA).In behavioral experiments,rats were divided into control,insomniac model,JTP[3.3 g/(kg•d)],and diazepam[4 mg/(kg•d)]groups.The treatment effect of JTP was evaluated by weight measurement(increasement of body weight),open field test(number of crossings)and forced swimming test(immobility time).A high performance liquid chromatography-electrochemical detection(HPLC-ECD)method was built to determine the concentration of monoamine transmitters in hypothalamus and peripheral organs from normal,model,JTP,citalopram[30 mg/(kg•d)],maprotiline[40 mg/(kg•d)]and bupropion[40 mg/(kg•d)]groups.Expressions of serotonin transporter(SERT),dopamine transporter(DAT),and norepinephrine transporter(NET)were analyzed by quantitative polymerase chain reaction(qPCR)and Western blot in normal,model and JTP groups.A high performance liquid chromatography-electrospray ionization mass spectrometry(HPLC-ESI-MS/MS)method was established to determine the pharmacokinetics,urine cumulative excretion of metformin in vivo,and tissue slice uptake in vitro,which were applied to assess the activity of organic cation transporters(OCTs)in hypothalamus and peripheral organs.Results Compared with the insomniac model group,the body weight and spontaneous locomotor were increased,and the immobility time was decreased after treatment with JTP(P<0.01).Both serotonin and dopamine contents in hypothalamus and peripheral organs were increased(P<0.01).The norepinephrine content was increased in peripheral organs and decreased in hypothalamus(P<0.05 or P<0.01).At the same time,SERT,DAT,OCT1,OCT2,and OCT3 were down-regulated in hypothalamus and peripheral organs(P<0.05).NET was down-regulated in peripheral organs and up-regulated in hypothalamus(P<0.05 or P<0.01).Moreover,the activity of OCTs in hypothalamus and peripheral organs was inhibited(P<0.05).Conclusion JTP alleviates insomnia through regulation of monoaminergic system and OCTs in hypothalamus and peripheral organs.