Activation of Parabrachial Nucleus Glutamatergic Neurons Accelerates Reanimation from Sevoflurane Anesthesia in Mice.

BACKGROUND: The parabrachial nucleus (PBN), which is a brainstem region containing glutamatergic neurons, is a key arousal nucleus. Injuries to the area often prevent patient reanimation. Some studies suggest that brain regions that control arousal and reanimation are a key part of the anesthesia recovery. Therefore, we hypothesize that the PBN may be involved in regulating emergence from anesthesia. METHODS: We investigated the effects of specific activation or inhibition of PBN glutamatergic neurons on sevoflurane general anesthesia using the chemogenetic "designer receptors exclusively activated by designer drugs" approach. Optogenetic methods combined with polysomnographic recordings were used to explore the effects of transient activation of PBN glutamatergic neuron on sevoflurane anesthesia. Immunohistochemical techniques are employed to reveal the mechanism by which PBN regulated sevoflurane anesthesia. RESULTS: Chemogenetic activation of PBN glutamatergic neurons by intraperitoneal injections of clozapine-N-oxide decreased emergence time (mean ± SD, control vs. clozapine-N-oxide, 55 ± 24 vs. 15 ± 9 s, P = 0.0002) caused by sevoflurane inhalation and prolonged induction time (70 ± 15 vs. 109 ± 38 s, n = 9, P = 0.012) as well as the ED50 of sevoflurane (1.48 vs. 1.60%, P = 0.0002), which was characterized by a rightward shift of the loss of righting reflex cumulative curve. In contrast, chemogenetic inhibition of PBN glutamatergic neurons slightly increased emergence time (56 ± 26 vs. 87 ± 26 s, n = 8, P = 0.034). Moreover, instantaneous activation of PBN glutamatergic neurons expressing channelrhodopsin-2 during steady-state general anesthesia with sevoflurane produced electroencephalogram evidence of cortical arousal. Immunohistochemical experiments showed that activation of PBN induced excitation of cortical and subcortical arousal nuclei during sevoflurane anesthesia. CONCLUSIONS: Activation of PBN glutamatergic neurons is helpful to accelerate the transition from general anesthesia to an arousal state, which may provide a new strategy in shortening the recovery time after sevoflurane anesthesia.

Ethanol inhibits histaminergic neurons in mouse tuberomammillary nucleus slices via potentiating GABAergic transmission onto the neurons at both pre- and postsynaptic sites.

AIM: Ethanol, one of the most frequently used and abused substances in our society, has a profound impact on sedation. However, the neuronal mechanisms underlying its sedative effect remain unclear. In this study, we investigated the effects of ethanol on histaminergic neurons in the tuberomammillary nucleus (TMN), a brain region thought to be critical for wakefulness. METHODS: Coronal brain slices (250 µm thick) containing the TMN were prepared from GAD67-GFP knock-in mice. GAD67-GFP was used to identify histaminergic neurons in the TMN. The spontaneous firing and membrane potential of histaminergic neurons, and GABAergic transmission onto these neurons were recorded using whole-cell patch-clamp recordings. Drugs were applied through superfusion. RESULTS: Histaminergic and GAD67-expressing neurons in the TMN of GAD67-GFP mice were highly co-localized. TMN GFP-positive neurons exhibited a regular spontaneous discharge at a rate of 2-4 Hz without burst firing. Brief superfusion of ethanol (64, 190, and 560 mmol/L) dose-dependently and reversibly suppressed the spontaneous firing of the neurons in the TMN; when synaptic transmission was blocked by tetrodotoxin (1 µmol/L), ethanol caused hyperpolarization of the membrane potential. Furthermore, superfusion of ethanol markedly increased the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs), which were abolished in the presence of the GABAA receptor antagonist bicuculline (20 µmol/L). Finally, ethanol-mediated enhancement of sIPSCs and mIPSCs was significantly attenuated when the slices were pretreated with the GABAB agonist baclofen (30 µmol/L). CONCLUSION: Ethanol inhibits the excitability of histaminergic neurons in mouse TMN slices, possibly via potentiating GABAergic transmission onto the neurons at both pre- and postsynaptic sites.

[Advances in the study of the effect of basal forebrain on sleep-wake regulation].

The basal forebrain(BF) is known to participate in the control of motion, attention, learning and memory, and it also plays a key role in sleep-wake regulation. Although there is a strong heterogeneity among neurons in the BF, the main types are cholinergic, gamma-aminobutyric acid (GABAergic) and glutamatergic neurons. This review provided the research progress in the regulation of sleep-wakefulness behavior by the 3 neurons in the BF. The cholinergic neurons play roles in activation of cortex and promote phase transition between sleep and wakefulness. The cortical projecting GABAergic neurons, which accept the projections from the adjacent cholinergic and glutamatergic neurons, contribute to awakening and the maintenance of normal wakefulness. The GABAergic interneurons may promote sleepiness by inhibiting the wake-active neurons which excite the cortical neurons. The glutamatergic neurons regulate sleep and wakefulness by interacting with neighbor cholinergic and cortical projecting GABAergic neurons or through the direct projection to the cortex as well.

[Peoniflorin activates Nrf2/ARE pathway to alleviate the Abeta(1-42)-induced hippocampal neuron injury in rats].

This study was to investigate the effect of peoniflorin on the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream signal molecules in the hippocampus of Alzheimer's disease (AD) rats for exploring the mechanism of peoniflorin protecting hippocampal neurons. AD model rats were established by bilateral intrahippocampal injection of beta-amyloid(1-42) (Abeta(1-42)) and divided randomly into 3 groups: AD model group, peoniflorin low-dose (15 mg x kg(-1)) group and peoniflorin high-dose (30 mg x kg(-1)) group. The vehicle control rats were given bilateral intrahippocampal injection of solvent with the same volume. After peoniflorin or saline was administered (ip) once daily for 14 days, the hippocampuses of all animals were taken out for measuring the expressions of Nrf2, heme oxygenase-1 (HO-1) and gamma-glutamylcysteine synthethase (gamma-GCS) mRNA by reverse transcription PCR, determining the contents of glutathione (GSH), malondialdehyde (MDA) and carbonyl protein (CP) using colorimetric method, and for assaying the expressions of neuronal apoptosis inhibitory protein (NAIP) and Caspase-3 by immunohistochemical staining method. The results showed that peoniflorin markedly increased the expressions of Nrf2, HO-1 and gamma-GCS mRNA, enhanced the level of GSH and decreased the contents of MDA and CP in the hippocampus, as compared with the model group. Peoniflorin also improved the NAIP expression and reduced the Caspase-3 expression in the hippocampus neurons. In conclusion, peoniflorin protects against the Abeta(1-42)-mediated oxidative stress and hippocampal neuron injury in AD rats by activating the Nrf2/ARE pathway.

[Pharmacokinetics of tramadol hydrochloride in the extracellular fluid of mouse frontal cortex studied by in vivo microdialysis].

The paper aims to explore the studying method for the pharmacokinetics of drugs in target organs, the pharmacokinetic process of tramadol hydrochloride in the extracellular fluid of frontal cortex (FrCx) of mice was investigated. Six male mice (Kunming strain) were anaesthetized (urethane, 1.8 g x kg(-1), ip) and secured on a stereotaxic frame. A microdialysis probe was implanted into the FrCx and perfused with artificial cerebrospinal fluid at a flow rate of 2 microL x min(-1). One hour later, mice were administrated (ip) with tramadol hydrochloride (50 mg x kg(-1)) and dialysates were collected continuously at 12-min intervals (24 microL each) for 6 h. The tramadol concentration in dialysates was determined by HPLC-Ultraviolet detection method, and the concentration-time curve and pharmacokinetic parameters of tramadol were calculated with DAS software. The results showed that the pharmacokinetic process of tramadol in the FrCx extracellular fluid of mice was fitted to a two-compartment open model, and the main pharmacokinetic parameters t1/2alpha, t1/2beta, t(max), C(max) and AUC(0-infinity) were (0.27 +/- 0.05) h, (2.72 +/- 0.24) h, (0.50 +/- 0.10) h, (2 110.37 +/- 291.22) microg x L(-1) and (4 474.51 +/- 441.79) microg x L(-1) x h, respectively. In conclusion, a studying method for pharmacokinetics of drugs in the target organ is established, which is simple and feasible. Tramadol hydrochloride shows a two-compartment model in the extracellular fluid of the mouse FrCx, and the distribution- and elimination half-life are 0.5 h and 2.7 h, respectively.

[Pharmacokinetics--pharmacodynamics of modafinil in mice].

To guide the reasonable clinical application of modafinil (MOD), pharmacokinetics and pharmacodynamics of MOD in mice and the correlation between them were investigated. Male mice (Kunming strain) were given a single oral dose of MOD (120 mg x kg(-1)). The plasma concentration of MOD was measured by HPLC and the pharmacokinetic parameters were calculated with DAS 3.0 software. For another batch of male Kunming strain mice, their locomotor activities were recorded by an infrared ray passive sensor after a same oral dose of MOD, and the synchronization and correlation between the changes of MOD plasma concentration and the locomotor activity induced by MOD were compared and analyzed. The results showed that the plasma concentration-time curve of MOD was fitted to two-compartment open model with a first order absorption. The main pharmacokinetic parameters t1/2alpha, t1/2beta, t(max), C(max) and AUC(0-inifinity) were 0.42 h, 3.10 h, 1.00 h, 41.34 mg x L(-1) and 142.22 mg x L(-1) x h, respectively. MOD significantly increased locomotor activity and the effect lasted for about 4 h. The changes of MOD plasma concentration and the locomotor activity induced by MOD were synchronous. In conclusion, there is a significant correlation between the effect of MOD and its plasma concentration after administration of 120 mg x kg(-1) in mice.

[Protective effect of paeonol on neurotoxicity induced by Abeta1-42 and underlying mechanisms].

OBJECTIVE: To investigate the protective effect of paeonol on amyloid beta1-42 (Abeta1-42)-induced neurotoxicity and its mechanism. METHOD: Hippocampal neurons of well-grown newborn SD rats and differentiated SH-SY5Y cell lines were cultured with various concentrations of paeonol (1, 5, 10 micromol x L(-1), respectively) for 6 hours and then incubated with Abeta1-42 oligomer (30 micromol x L(-1)) for 24 hours and 48 hours, respectively. The neuron apoptosis was observed by Heochst33258. Annexin V/PI double stain flow cytometry assay was adopted for determining SH-SY5Y cell apoptosis rate. And the expression of BDNF and Bcl-2 mRNA was detected by RT-PCR. RESULT: Compared with the model group, various concentrations of paeonol (1, 5, 10 micromol x L(-1)) significantly reduced the hippocampal neurons karyopycnosis, decreased the rate of SH-SY5Y cell apoptosis to 22.4%, 18.1% and 16.4%, respectively, and improved the expressions of BDNF and Bcl-2 mRNA. CONCLUSION: Paeonol relieves Abeta1-42 oligomer-induced neuron injury by increasing BDNF and Bcl-2 expressions.

[Advances in the study of histaminergic systems and sleep-wake regulation].

Histaminergic neurons solely originate from the tuberomammillary nucleus (TMN) in the posterior hypothalamus and send widespread projections to the whole brain. Experiments in rats show that histamine release in the central nervous system is positively correlated with wakefulness and the histamine released is 4 times higher during wake episodes than during sleep episodes. Endogeneous prostaglandin E2 and orexin activate histaminergic neurons in the TMN to release histamine and promote wakefulness. Conversely, prostaglandin D2 and adenosine inhibit histamine release by increasing GABA release in the TMN to induce sleep. This paper reviews the effects and mechanisms of action of the histaminergic system on sleep-wake regulation, and briefly discusses the possibility of developing novel sedative-hypnotics and wakefulness-promoting drugs related to the histaminergic system.

Sevoflurane depresses neurons in the medial parabrachial nucleus by potentiating postsynaptic GABAA receptors and background potassium channels.

Despite persistent clinical use for over 170 years, the neuronal mechanisms by which general anesthetics produce hypnosis remain unclear. Previous studies suggest that anesthetics exert hypnotic effects by acting on endogenous arousal circuits. Recently, it has been shown that the medial parabrachial nucleus (MPB) is a novel wake-promoting component in the dorsolateral pons. However, it is not known whether and how the MPB contributes to anesthetic-induced hypnosis. Here, we investigated the action of sevoflurane, a widely used volatile anesthetic agent that best represents the drug class of halogenated ethers, on MPB neurons in mice. Using in vivo fiber photometry, we found that the population activities of MPB neurons were inhibited during sevoflurane-induced loss of consciousness. Using in vitro whole-cell patch-clamp recordings, we revealed that sevoflurane suppressed the firing rate of MPB neurons in concentration-dependent and reversible manners. At a concentration equal to MAC of hypnosis, sevoflurane potentiated synaptic GABAA receptors (GABAA-Rs), and the inhibitory effect of sevoflurane on the firing rate of MPB neurons was completely abolished by picrotoxin, which is a selective GABAA-R antagonist. At a concentration equivalent to MAC of immobility, sevoflurane directly hyperpolarized MPB neurons and induced a significant decrease in membrane input resistance by increasing a basal potassium conductance. Moreover, pharmacological blockade of GABAA-Rs in the MPB prolongs induction and shortens emergence under sevoflurane inhalation at MAC of hypnosis. These results indicate that sevoflurane inhibits MPB neurons through postsynaptic GABAA-Rs and background potassium channels, which contributes to sevoflurane-induced hypnosis.

High cortical delta power correlates with aggravated allodynia by activating anterior cingulate cortex GABAergic neurons in neuropathic pain mice.

Patients with chronic pain often report being sensitive to pain at night before falling asleep, a time when the synchronization of cortical activity is initiated. However, how cortical activity relates to pain sensitivity is still unclear. Because sleep is characterized by enhanced cortical delta power, we hypothesized that enhanced cortical delta power may be an indicator of intensified pain. To test this hypothesis, we used pain thresholds tests, EEG/electromyogram recordings, c-Fos staining, and chemogenetic and pharmacological techniques in mice. We found that sleep deprivation or pharmacologic enhancement of EEG delta power by reserpine and scopolamine dramatically decreased mechanical pain thresholds, but not thermal withdrawal latency, in a partial sciatic nerve ligation model of neuropathic pain mice. On the contrary, suppression of EEG delta power using a wake-promoting agent modafinil significantly attenuated mechanical allodynia. Moreover, when EEG delta power was enhanced, c-Fos expression decreased in most regions of the cortex, except the anterior cingulate cortex (ACC), where c-Fos was increased in the somatostatin- and parvalbumin-positive GABAergic neurons. Chemogenetic activation of GABAergic neurons in ACC enhanced EEG delta power and lowered mechanical pain thresholds simultaneously in naive mice. However, chemogenetic inhibition of ACC GABAergic neurons could not block mechanical allodynia. These results provided compelling evidence that elevated EEG delta power is accompanied with aggravated neuropathic pain, whereas decreased delta power attenuated it, suggesting that enhanced delta power can be a specific marker of rising chronic neuropathic pain and that wake-promoting compounds could be used as analgesics in the clinic.

Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice.

To evaluate the antinociceptive and hypnotic effects of pregabalin, we established a neuropathic pain-like model in mice using partial sciatic nerve ligation (PSNL), and examined thermal hyperalgesia, mechanical allodynia, electroencephalogram, rota-rod testing, and c-Fos expression in the anterior cingulate cortex. Gabapentin was used as a reference drug in the study. Pregabalin administered i.g. at 12.5 and 25mg/kg prolonged the duration of thermal latencies by 1.4- and 1.6-fold and increased the mechanical threshold by 2.2- and 3.1-fold 3h after administration, respectively, but did not affect motor coordination in PSNL mice, compared with vehicle control. Pregabalin (12.5 and 25mg/kg) given at 6:30 increased the amount of non-rapid eye movement sleep in a 4-h period by 1.3- and 1.4-fold, respectively, in PSNL mice. However, pregabalin (25mg/kg) given at 20:30 did not alter the sleep pattern in normal mice. Immunohistochemical study showed that PSNL increased c-Fos expression in the neurons of anterior cingulate cortex by 2.1-fold, which could be reversed by pregabalin. These results indicate that pregabalin is an effective treatment for both neuropathic pain and sleep disturbance in PSNL mice.

[Genetical investigation on a rare genealogy of congenital camptodactyly].

Introduced a rare genealogy of autosomal dominant inheritance disease, which cardinal signs were congenital camptodactyly and functional disturbance of the proximal interphalangeal joints of the II, III, IV, V fingers. Part cases had congenital high myopia.

Dopamine is involved in food-anticipatory activity in mice.

When food is available during a restricted and predictable time of the day, mammals exhibit food-anticipatory activity (FAA), an increase in locomotor activity preceding the presentation of food. Although many studies have attempted to locate the food-entrainable circadian oscillator in the central nervous system, the pathways that mediate food entrainment are a matter of controversy. The present study was designed to determine the role of dopaminergic and histaminergic systems on FAA. Mice were given access to food for 2 h (ZT12-ZT14), and FAA was defined as the locomotor activity that occurred 2 h before the availability of food. Dopamine D(1) receptor (R), D(2)R, and histamine H(1)R-specific antagonists were used to clarify the role of dopamine and histamine receptors in FAA induced by food restriction (FR). FAA was monitored by infrared locomotor activity sensors. Mice were sacrificed at ZT12 on the 14th day of FR, and monoamine concentrations were determined by high-performance liquid chromatography coupled to electrochemical detection (HPLC-ECD). The results showed that pretreatment with the D(1)R antagonist SCH23390 at 1, 3, or 10 µg/kg significantly reduced FAA by 19% (p < 0.05), 26% (p < 0.05), or 19% (p < 0.01), respectively, and the D(2)R antagonist raclopride at 22, 67, or 200 µg/kg significantly reduced FAA by 16% (p < 0.05), 36% (p < 0.01), or 41% (p < 0.01), respectively, as compared with vehicle control. Moreover, coadministration of SCH23390 (10 µg/kg) and raclopride (200 µg/kg) synergistically inhibited FAA by 57% (p < 0.01) as compared with vehicle control. Consistently, the levels of dopamine and its metabolites in the striatum and midbrain were significantly increased during FAA, even with the pretreatment of D(1)R and D(2)R antagonists. However, pretreatment with pyrilamine at 2.5, 5, or 10 mg/kg did not significantly reduce FAA, although it reduced the locomotor activity during the dark period in ad libitum mice. These results strongly indicate that the dopaminergic system plays an essential role in the FAA in mice.

Safranal enhances non-rapid eye movement sleep in pentobarbital-treated mice.

AIMS: Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, C(10) H(14) O) is an active ingredient in the saffron, which is used in traditional medicine. It has been reported to have sedative and anti-epileptic effects, but its hypnotic effects remain uncertain. The aim of this study was to evaluate effects of safranal on sleep-wake cycle. METHODS: We established hypnotic-model mice treated with a low dose of pentobarbital 20 mg/kg, and administered different doses of safranal, vehicle, or diazepam. The change of sleep-wake cycle was assessed by sleep recording and c-Fos expression in the brain was analyzed by immunohistochemistry. RESULTS: Safranal increased the duration of non-rapid eye movement (NREM) sleep, shortened NREM sleep latency, and enhanced the delta power activity of NREM sleep. Immunohistochemical evaluation revealed that safranal increased c-Fos expression in the ventrolateral preoptic nucleus (VLPO), one of the putative sleep centers, and decreased it in the arousal histaminergic tuberomammillary nuclei (TMN). CONCLUSION: These findings indicate that safranal enhances NREM sleep in pentobarbital-treated mice. The hypnotic effects of safranal may be related to the activation of the sleep-promoting neurons in the VLPO and the simultaneous inhibition of the wakefulness-promoting neurons in the TMN, suggesting that safranal may be a hypnotic substance.

Altered sleep-wake characteristics and lack of arousal response to H3 receptor antagonist in histamine H1 receptor knockout mice.

Histaminergic neurons play an important role in the regulation of sleep-wake behavior through histamine H(1) receptors (H(1)R). Blockade of the histamine H(3) receptor (H(3)R) is proposed to induce wakefulness by regulating the release of various wake-related transmitters, not only histamine. In the present study, we characterized sleep-wake cycles of H(1)R knockout (KO) mice and their arousal responses to an H(3)R antagonist. Under baseline conditions, H(1)R KO mice showed sleep-wake cycles essentially identical to those of WT mice but with fewer incidents of brief awakening (<16-sec epoch), prolonged durations of non-rapid eye movement (NREM) sleep episodes, a decreased number of state transitions between NREM sleep and wakefulness, and a shorter latency for initiating NREM sleep after an i.p. injection of saline. The H(1)R antagonist pyrilamine mimicked these effects in WT mice. When an H(3)R antagonist, ciproxifan, was administered i.p., wakefulness increased in WT mice in a dose-dependent manner but did not increase at all in H(1)R KO mice. In vivo microdialysis revealed that the i.p. application of ciproxifan increased histamine release from the frontal cortex in both genotypes of mice. These results indicate that H(1)R is involved in the regulation of behavioral state transitions from NREM sleep to wakefulness and that the arousal effect of the H(3)R antagonist completely depends on the activation of histaminergic systems through H(1)R.

Orexin A promotes histamine, but not norepinephrine or serotonin, release in frontal cortex of mice.

AIM: To investigate the effects of orexin A on release of histamine, norepinephrine, and serotonin in the frontal cortex of mice. METHODS: Samples for measuring histamine, norepinephrine, and serotonin contents were collected by in vivo microdialysis of the frontal cortex of anesthetized mice. The histamine, noradrenaline, and serotonin content in dialysates were measured by HPLC techniques. RESULTS: Intracrebroventricular injection of orexin A at doses of 12.5, 50, and 200 pmol per mouse promoted histamine release from the frontal cortex in a dose-dependent manner. At the highest dose given, 200 pmol, orexin A significantly induced histamine release, with the maximal magnitude being 230% over the mean basal release. The enhanced histamine release was sustained for 140 min, and then gradually returned to the basal level. However, no change in norepinephrine or serotonin release was observed under application of the same dose of orexin A. CONCLUSION: These results suggest that the arousal effect of orexin A is mainly mediated by histamine, not by norepinephrine or serotonin.

An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats.

The adenosine A(2A) receptor (A(2A)R) has been demonstrated to play a crucial role in the regulation of the sleep process. However, the molecular mechanism of the A(2A)R-mediated sleep remains to be elucidated. Here we used electroencephalogram and electromyogram recordings coupled with in vivo microdialysis to investigate the effects of an A(2A)R agonist, CGS21680, on sleep and on the release of histamine and GABA in the brain. In freely moving rats, CGS21680 applied to the subarachnoid space underlying the rostral basal forebrain significantly promoted sleep and inhibited histamine release in the frontal cortex. The histamine release was negatively correlated with the amount of non-rapid eye movement sleep (r = - 0.652). In urethane-anesthetized rats, CGS21680 inhibited histamine release in both the frontal cortex and medial pre-optic area in a dose-dependent manner, and increased GABA release specifically in the histaminergic tuberomammillary nucleus but not in the frontal cortex. Moreover, the CGS21680-induced inhibition of histamine release was antagonized by perfusion of the tuberomammillary nucleus with a GABA(A) antagonist, picrotoxin. These results suggest that the A(2A)R agonist induced sleep by inhibiting the histaminergic system through increasing GABA release in the tuberomammillary nucleus.