Effects Of Pentobarbital Research Articles

Effect of Lower Body Negative Pressure (LBNP) on Blood Pressure in Awake Rats and Effects of Anesthesia

Lower body negative pressure (LBNP) has been used for decades in a variety of settings and species to model orthostatic challenge (OC) and baroreceptor unloading. However, due to physical constraints, measurements of blood pressure (BP) during LBNP have always been made in anesthetized animals, where cardiovascular reflexes are blunted or even blocked. In terminal procedures, for example, urethane is often used in an attempt to preserve some measure of autonomic reflex function during OC. More recently, investigators have used isoflurane in animals fitted with BP telemeters in order to perform longitudinal studies, but the extent to which various cardiovascular reflexes may be preserved is unknown. Here we have developed a novel method to apply lower body negative pressure in awake rats instrumented for BP measurement. Rats were placed into a tubular restraining device (5 cm diameter X 21 cm length), that is divided into an upper and lower chamber by two adjacent latex septa with small holes (~ 2 cm). Rats were positioned so that the septa form a seal at the lower margin of the thorax, thereby creating independent upper and lower chambers. Negative pressure was then applied to the lower chamber using a high-volume vacuum and the pressure controlled using an adjustable damper. BP measurements in response to progressive levels of LBNP (-3 to -15 mmHg) were first made in awake rats, followed by induction of anesthesia and repetition of the LBNP protocol. The effects of pentobarbital (50mg/kg), ketamine/xylazine (70/6 mg/kg) and isoflurane (3%) were determined on separate days. BP and heart rate (HR) responses are shown in the Table. We found that BP was remarkably preserved in awake rats up to at least -9 mmHg LBNP, and thereafter (at -12 and -15 mmHg) decreased modestly. There was a robust baroreceptor-mediated increase in HR beginning at -6 mmHg. By contrast, under each anesthetic, blood pressure decreased dramatically and progressively with increasing LBNP, and the reflex increases in HR were entirely blocked (and actually inverted). These data indicate that the awake rat has robust mechanisms to preserve cardiovascular function during OC, and that these mechanisms are severely compromised during anesthesia. University of Cincinnati College of Medicine Innovation Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unexpected epileptogenic effect of lethal doses of pentobarbital: a case report

Background: Barbiturate poisoning is rare but potentially fatal. Case Report: We reported a case of barbiturate poisoning in a 28-year-old woman who recovered from lethal pentobarbital deliberate self-poisoning. The initial blood pentobarbital concentration was 61 mg/L, corresponding to a potentially lethal dose. Despite the ingestion of a high dose of pentobarbital, the electroencephalogram revealed an unattended pattern compatible with possible nonconvulsive status epilepticus. Following resuscitation maneuvers, appropriate care, and antiseizure medication, the patient awakened after 7 days. The evolution was excellent without neurological deficits at 2 months.Conclusion: Despite the expected and known effects of high-dose pentobarbital in reducing and suppressing cortical activity in the brain, the present case demonstrates that lethal dose of pentobarbital may have an epileptogenic effect. Our hypothesis was that the mechanism of the origin of such a picture is a relatively abrupt decrease in toxic doses of pentobarbital, resulting in a withdrawal phenomenon.

Pharmacological Analysis of GABAA Receptor and Sigma1R Chaperone Interaction: Research Report I-Investigation of the Anxiolytic, Anticonvulsant and Hypnotic Effects of Allosteric GABAA Receptors' Ligands.

Two groups of facts have been established in previous drug development studies of the non-benzodiazepine anxiolytic fabomotizole. First, fabomotizole prevents stress-induced decrease in binding ability of the GABAA receptor's benzodiazepine site. Second, fabomotizole is a Sigma1R chaperone agonist, and exposure to Sigma1R antagonists blocks its anxiolytic effect. To prove our main hypothesis of Sigma1R involvement in GABAA receptor-dependent pharmacological effects, we performed a series of experiments on BALB/c and ICR mice using Sigma1R ligands to study anxiolytic effects of benzodiazepine tranquilizers diazepam (1 mg/kg i.p.) and phenazepam (0.1 mg/kg i.p.) in the elevated plus maze test, the anticonvulsant effects of diazepam (1 mg/kg i.p.) in the pentylenetetrazole-induced seizure model, and the hypnotic effects of pentobarbital (50 mg/kg i.p.). Sigma1R antagonists BD-1047 (1, 10, and 20 mg/kg i.p.), NE-100 (1 and 3 mg/kg i.p.), and Sigma1R agonist PRE-084 (1, 5, and 20 mg/kg i.p.) were used in the experiments. Sigma1R antagonists have been found to attenuate while Sigma1R agonists can enhance GABAARs-dependent pharmacological effects.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32–76%; median, min-max) in comparison to H60 and H164 (24%,11–33% and 13%, 8–44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

N-methyl-d-aspartate receptors and glycinergic transmission, respectively, mediate muscle relaxation and immobility of pentobarbital in mice

Pentobarbital-induced anesthesia is believed to be mediated by enhancement of the inhibitory action of γ-aminobutyric acid (GABA)ergic neurons in the central nervous system. However, it is unclear whether all components of anesthesia induced by pentobarbital, such as muscle relaxation, unconsciousness, and immobility in response to noxious stimuli, are mediated only through GABAergic neurons. Thus, we examined whether the indirect GABA and glycine receptor agonists gabaculine and sarcosine, respectively, the neuronal nicotinic acetylcholine receptor antagonist mecamylamine, or the N-methyl-d-aspartate receptor channel blocker MK-801 could enhance pentobarbital-induced components of anesthesia. Muscle relaxation, unconsciousness, and immobility were evaluated by grip strength, the righting reflex, and loss of movement in response to nociceptive tail clamping, respectively, in mice. Pentobarbital reduced grip strength, impaired the righting reflex, and induced immobility in a dose-dependent manner. The change in each behavior induced by pentobarbital was roughly consistent with that in electroencephalographic power. A low dose of gabaculine, which significantly increased endogenous GABA levels in the central nervous system but had no effect on behaviors alone, potentiated muscle relaxation, unconsciousness, and immobility induced by low pentobarbital doses. A low dose of MK-801 augmented only the masked muscle-relaxing effects of pentobarbital among these components. Sarcosine enhanced only pentobarbital-induced immobility. Conversely, mecamylamine had no effect on any behavior. These findings suggest that each component of anesthesia induced by pentobarbital is mediated through GABAergic neurons and that pentobarbital-induced muscle relaxation and immobility may partially be associated with N-methyl-d-aspartate receptor antagonism and glycinergic neuron activation, respectively.

Sodium Pentobarbital Suppresses Breast Cancer Cell Growth Partly via Normalizing Microcirculatory Hemodynamics and Oxygenation in Tumors

Breast cancer remains the leading cause of cancer-related death among women worldwide. Sodium pentobarbital was found to play an inhibitory role in glioma growth in rats. In this study, we aimed to evaluate the effects of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and its impacts on the microcirculatory changes on both skin and tumor surface in mice bearing subcutaneous xenograft. Cell counting assay was used to assess the antiproliferative effect of sodium pentobarbital on MDA-MB-231 breast cancer cells. Subcutaneous xenograft model was established to study the role of sodium pentobarbital on in vivo tumor growth. Speed-resolved blood perfusion, hemoglobin oxygen saturation (SO2, %), total hemoglobin tissue concentration (ctTHb, μM), and red blood cell (RBC) tissue fraction (%) were examined simultaneously by using enhanced perfusion and oxygen saturation system to investigate the effects of sodium pentobarbital on microcirculatory hemodynamics and oxygenation. Sodium pentobarbital suppressed breast tumor growth both in vitro and in vivo. Cutaneous blood flux in nutritive capillaries with low-speed flow was significantly increased in tumor-bearing mice, and high-dose sodium pentobarbital treatment cause a reduction in this low-speed blood flux, whereas sodium pentobarbital therapy caused an elevated blood flux in larger microvessels with mid and high speed in a dose-dependent manner. Different doses of sodium pentobarbital exerted different actions on SO2, ctTHb, and RBC tissue fraction. Collectively, the inhibitory effect of sodium pentobarbital on breast tumor growth was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors. SIGNIFICANCE STATEMENT: This study is the first to demonstrate the inhibiting effect of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and such an inhibition was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors.

Pentobarbital may protect against neurogenic inflammation after surgery via inhibition of substance P release from peripheral nerves of rats

The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100–1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

Essential Oil from the Leaves of Chromolaena odorata, and Sesquiterpene Caryophyllene Oxide Induce Sedative Activity in Mice.

Chromolaena odorata (L.) R.M.King & H.Rob. essential oil (COEO) was investigated for its sedative activity in mice. The results showed that COEO significantly reduced mice locomotor activity and the most efficient concentrations were 0.04 and 0.00004 mg/cage (volume of the cage 61.2L). Analysis of chemical composition of the oil indicated that caryophyllene oxide (43.75%) was the major compound and bioactivity-guided fractionation of the oil was performed to isolate the compound responsible for activity. The data clearly identified sesquiterpene caryophyllene oxide as the compound inducing COEO sedative activity and it was effective in decreasing mice locomotor activity by 56% and 57% at 0.0004 and 0.04 mg/cage, respectively. In order to understand the action mechanisms, caryophyllene oxide was tested for its effects on the central nervous system (CNS) by using a caffeine pre-excited mice test and a pentobarbital sleeping-induced test in mice. The results showed that caryophyllene oxide is a potent CNS depressant. Nevertheless, it fails to potentiate the effects of pentobarbital on the GABAergic system, nor did flumazenil, a GABAA receptor antagonist, reversed its effects. It was especially interesting to note that β-caryophyllene, the precursor of caryophyllene oxide, demonstrated a similar pattern of sedative activity, and the present work further extends actual knowledge on these naturally occurring sesquiterpenes. The findings in this study reveal the new activity of caryophyllene oxide as an innovative way to manage sleep and CNS-related disorders, and demonstrates a satisfactory effect of two interesting sesquiterpene compounds on the CNS.

Sedative-Hypnotic Effects of Different Extracts and Fractions of Capparis Spinosa L. in Mice

Introduction: Capparis spinosa L. (Caper) is an aromatic plant growing in dry regions around the Mediterranean area. C. spinosa is shown to have several compounds such as tannins, sterols, alkaloids, polyphenols and flavonoids. Previous studies showed that flavonoids have sedative and hypnotic effects. The aim of this study was evaluating the sedative-hypnotic effects of various extracts and fractions of C. spinosa in different doses. Methods and Results: Maceration method was used for extraction of the aerial part of the plant. All experiments conducted on male NMRI mice (18-25g weight). For evaluation the hypnotic and sedative effects of plant, open field and pentobarbital induced sleep test were used. In this study, animals were treated with different doses of aqueous extract, methanolic extract, methanolic fraction and dichloromethane fraction (i.p.). In pentobarbital induced sleep test, the duration of lack of righting reflex was reported. In open field test, the locomotor activities of mice were calculated by measuring total distance moved in the open field arena. According to the results, the dichloromethane fraction decreased the total distance moved in the open field test (ED50 = 66.6 mg/kg) and also increased the sleep duration in pentobarbital induced sleep test (ED50 =18.7 mg/kg) compared with other extract and fractions groups. Conclusion: In conclusion, the dichloromethane fraction of C. spinosa has hypnotic and sedative effects compared to the other extract and fraction groups. Further studies are necessary to find the active components responsible for the several effects of the dichloromethane fraction and the exact mechanism of action of these effects.

Verification and Defined Dosage of Sodium Pentobarbital for a Urodynamic Study in the Possibility of Survival Experiments in Female Rat.

Objectives To evaluate the effects of pentobarbital dosages on lower urinary tract function and to define an appropriate dosage of sodium pentobarbital that would be suitable for urodynamic studies in which recovery from anesthesia and long term survive were needed for subsequent experiment. Methods Twenty-four 8-week-old, female, virgin, Sprague-Dawley rats (200-250 g) were used in this study. Rats in study groups received gradient doses of pentobarbital intraperitoneally, and those in the control group received urethane intraperitoneally. External urethral sphincter electromyography (EUS-EMG) was recorded simultaneously during cystometry and leak point pressure tests. The toe-pinch reflex was used to determine the level of anesthesia. Results Micturition was normally induced in both the urethane group and 32 mg/kg pentobarbital group. However, in groups of 40 mg/kg or 36 mg/kg pentobarbital, micturition failed to be induced; instead, nonvoiding contractions accompanied by EUS-EMG tonic activity were observed. There were no significant differences in leak point pressure or EUS-EMG amplitude or frequency between the urethane and 32 mg/kg pentobarbital groups. Conclusions This study confirmed significant dose-dependent effects of pentobarbital on lower urinary tract function and 32 mg/kg pentobarbital as an appropriate dosage for recovery urodynamic testing, which enable the achievement of expected essential micturition under satisfactory anesthesia in female rats.

Nrf2 Antioxidative System is Involved in Cytochrome P450 Gene Expression and Activity: A Delay in Pentobarbital Metabolism in Nrf2-Deficient Mice.

NF-E2-related factor 2 (Nrf2) is a transcriptional regulator of biologic defense proteins, such as antioxidant proteins and phase II detoxification enzymes. Cytochrome P450 (P450) enzymes have been shown to regulate phase I metabolism of various drugs and are partially regulated by Nrf2; however, the influence of Nrf2 on drug pharmacokinetics is not known. Here, we showed that Nrf2 depletion prolonged the effect of pentobarbital, a sleep-promoting drug. Pretreatment with phenobarbital, a P450 inducer, shortens the sleeping time associated with pentobarbital-induced sedation in wild-type (WT) mice; however, this effect was not observed in Nrf2-/- mice. Furthermore, the blood pentobarbital concentration was higher in Nrf2-/- mice than in WT mice at 30-60 minutes, and the phenobarbital-induced enhancement of its clearance was attenuated in Nrf2-/- mice compared with WT mice. Total P450 content was decreased in Nrf2-/- mouse livers, and the phenobarbital-induced increase in P450 content was lower in Nrf2-/- mice than WT mice. Cyp1a2, Cyp2a5, Cyp2c29, and Cyp2e1 gene expression levels under physiologic conditions and Cyp1a2, Cyp2a5, and Cyp2b10 gene expression levels under phenobarbital-treated conditions were lower in Nrf2-/- mice compared with WT mice. Additionally, pentobarbital metabolism in liver microsomes was attenuated by Nrf2 depletion. Taken together, these findings suggested that Nrf2 influenced pentobarbital pharmacokinetics through the regulation of drug metabolism and P450 gene expression. Thus, Nrf2-mediated regulation of P450 may contribute to the biologic defense against increased reactive oxygen species production. SIGNIFICANCE STATEMENT: NF-E2-related factor 2 (Nrf2) plays a critical role in the cellular defense against oxidative stress. Nrf2-/- mice with reduced ability to eliminate reactive oxygen species (ROS) showed a significant delay in emergence from pentobarbital-induced sleep, which was associated with decreased P450 activities and gene expression. Our findings provide that Nrf2 dysfunction or ROS that exceed a threshold level of the eliminating ability of the Nrf2 system may reduce P450 activity.

The Effect of a Novel Theanine Complex (JDS‐MT‐003) on Sleep in a Pentobarbital‐Induced Sleep Model in Mice

ObjectiveJDS‐MT‐003 is a novel theanine complex that is hypothesized to have superior effects compared to L‐theanine, an amino acid found naturally in tea. L‐theanine has been clinically shown to positively affect sleep quality, mood, and cognitive performance. Structurally similar to the excitatory transmitter glutamate, L‐theanine induces its calming effects by decreasing the binding of glutamate and increasing production of the inhibitory neurotransmitter GABA. The purpose of the following preclinical study was to examine the effects of JDS‐MT‐003 compared to L‐theanine on sleep quality in a pentobarbital‐induced sleep model in mice.MethodsThirty‐five male BALB/c mice were randomized to receive one of the following treatments: Pentobarbital (42 mg/kg), Pentobarbital + Theanine (20 mg/kg) or Pentobarbital + JDS‐MT‐003 (20 mg/kg). L‐theanine and JDS‐MT‐003 samples were resuspended in physiological saline for oral administration. Forty‐five minutes following oral treatment with L‐theanine or JDS‐MT‐003 mice were injected with a hypotonic dose of pentobarbital (42 mg/kg) into the left side of the abdomen. Pentobarbital is a hypnotic drug prescribed for insomnia that can induce sleep but also impair sleep architecture. Mice were then placed in individual cages and subjected to measurements of sleep latency and duration. Sleep latency was measured as the period between pentobarbital injection and sleep onset. Sleep duration was measured as the time elapsed between reflex loss and recovery. Mice that failed to fall asleep within 10 minutes after pentobarbital injection were excluded from the experiments.ResultsAnimals in the JDS‐MT‐003 group showed the greatest sleep duration and the lowest sleep latency times compared to all other groups (p &lt; 0.05). Mean sleep duration times (mean ± SD) in minutes were as follows: 51.01 ± 2.32 (Pentobarbital), 61.64 ± 2.18 (Pentobarbital + Theanine), and 86.41 ± 4.45 (Pentobarbital + JDS‐MT‐003). Mean sleep latency times (mean ± SD) in minutes were as follows: 3.86 ± 0.07 (Pentobarbital), 3.64 ± 0.09 (Pentobarbital + Theanine), and 3.11 ± 0.17 (Pentobarbital + JDS‐MT‐003). Overall, a greater percentage of animals fell asleep in the Pentobarbital + JDS‐MT‐003 group compared to the Pentobarbital + Theanine group (62.5% vs. 37.5%, respectively). No significant adverse events were observed.ConclusionsThese results demonstrate that JDS‐MT‐003, a novel theanine complex, prolongs sleep duration and decreases sleep latency significantly more than L‐theanine when combined with pentobarbital. These data suggest that JDS‐MT‐003 can counteract the negative effects of pentobarbital and other hypnotic sleep drugs on sleep quality. Moreover, these data support the use of JDS‐MT‐003 as a potential treatment option to improve overall sleep quality in individuals having trouble falling and staying asleep.Support or Funding InformationThis study was conducted at Firat University and funded by JDS Therapeutics LLC

Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor β3 Subunits.

Transgenic mouse studies suggest that γ-aminobutyric acid type A (GABAA) receptors containing β3 subunits mediate important effects of etomidate, propofol, and pentobarbital. Zebrafish, recently introduced for rapid discovery and characterization of sedative-hypnotics, could also accelerate pharmacogenetic studies if their transgenic phenotypes reflect those of mammals. The authors hypothesized that, relative to wild-type, GABAA-β3 functional knock-out (β3) zebrafish would show anesthetic sensitivity changes similar to those of β3 mice. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mutagenesis was used to create a β3 zebrafish line. Wild-type and β3 zebrafish were compared for fertility, growth, and craniofacial development. Sedative and hypnotic effects of etomidate, propofol, pentobarbital, alphaxalone, ketamine, tricaine, dexmedetomidine, butanol, and ethanol, along with overall activity and thigmotaxis were quantified in 7-day postfertilization larvae using video motion analysis of up to 96 animals simultaneously. Xenopus oocyte electrophysiology showed that the wild-type zebrafish β3 gene encodes ion channels activated by propofol and etomidate, while the β3 zebrafish transgene does not. Compared to wild-type, β3 zebrafish showed similar morphology and growth, but more rapid swimming. Hypnotic EC50s (mean [95% CI]) were significantly higher for β3 versus wild-type larvae with etomidate (1.3 [1.0 to 1.6] vs. 0.6 [0.5 to 0.7] µM; P < 0.0001), propofol (1.1 [1.0 to 1.4] vs. 0.7 [0.6 to 0.8] µM; P = 0.0005), and pentobarbital (220 [190 to 240] vs. 130 [94 to 179] μM; P = 0.0009), but lower with ethanol (150 [106 to 213] vs. 380 [340 to 420] mM; P < 0.0001) and equivalent with other tested drugs. Comparing β3 versus wild-type sedative EC50s revealed a pattern similar to hypnosis. Global β3 zebrafish are selectively insensitive to the same few sedative-hypnotics previously reported in β3 transgenic mice, indicating phylogenetic conservation of β3-containing GABAA receptors as anesthetic targets. Transgenic zebrafish are potentially valuable models for sedative-hypnotic mechanisms research.

Taurine potentiates the anticonvulsive effect of the GABAA agonist muscimol and pentobarbital in the immature mouse hippocampus.

The high incidence of epileptic seizures in neonates and their frequent refractoriness to pharmacologic therapies require identification of new therapeutical options. Therefore, we investigated whether the modulatory effect of taurine on γ-aminobutyric acid (GABA)A receptors can enhance the anticonvulsive potential of the GABAA receptor agonist muscimol and of the barbiturate pentobarbital. We performed field potential recordings in in toto hippocampus preparations of immature (postnatal days 4-7) C57Bl/6 mouse pups. Spontaneous epileptiform activity was induced by the continuous presence of the potassium channel blocker 4-aminopyridine and the glycinergic antagonist strychnine in Mg2+ -free solutions. Bath application of 0.1μmol/L muscimol increases the occurrence of recurrent epileptiform discharges, whereas they are significantly attenuated in a dose-dependent manner by muscimol in concentrations between 0.5 and 5μmol/L. Taurine at concentrations between 0.1 and 0.5mmol/L induces a proconvulsive effect, but upon coapplication, it significantly augments the anticonvulsive effect of moderate muscimol doses (0.5-1μmol/L). In addition, the anticonvulsive effect of 100 and 200μmol/L pentobarbital is increased significantly in the presence of 0.5μmol/L taurine. These observations demonstrate that taurine can indeed enhance the anticonvulsive effects of muscimol and pentobarbital, suggesting that taurine may act as a positive modulator on GABAA receptors. Thus, interfering with the modulatory taurine binding site of GABAA receptors or the interstitial taurine concentration may provide new therapeutical options for anticonvulsive therapies in neonates.

Vigilance demand and the effects of stimulant drugs in a five-choice reaction-time procedure in mice.

Stimulant drugs used for treating attention-deficit hyperactivity disorder (ADHD) increase signal-detection accuracy in five-choice serial reaction-time procedures. These increases may result from drug-induced increases in control exerted by the stimuli that prompt responses, which was assessed in the present study. Mice were trained with food reinforcement to nose poke into one of five holes after its illumination (signal), and effects of methylphenidate, d-amphetamine, and pentobarbital were assessed. Subsequently, the time from trial onset to signal was changed from fixed to variable for one group of subjects. A 'warning' stimulus (change in ambient lighting) preceding the signal was added for a second group. Effects of the drugs were reassessed. Dose-related increases in accuracy of signal detection (nose pokes in hole where a signal was displayed) were obtained with methylphenidate and d-amphetamine, but not with pentobarbital. When the presignal time was variable, increases in signal detection were not obtained with either stimulant. When a warning stimulus preceded the signal, the increases in accuracy were similar to those obtained without the warning stimulus. Hence, a procedure that increased vigilance demand (using a variable prestimulus period) eliminated the effects of drugs useful in treating ADHD, whereas a procedure that decreased vigilance demand (adding the warning light) had no appreciable effects on the response to stimulant drugs. Taken together, the present results suggest that the five-choice serial reaction-time has predictive validity for selecting drugs effective for treating ADHD, although effects can depend critically on the stimulus conditions used and the vigilance required by the procedure.

\u22069-Tetrahydrocannabinol, a major marijuana component, enhances the anesthetic effect of pentobarbital through the CB1 receptor

Purpose∆9-Tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), major psychoactive constituents of marijuana, induce potentiation of pentobarbital-induced sleep in mice. We have elucidated the mechanism of enhancement of the anesthetic effect of pentobarbital by cannabinoids.MethodsWe carried out pharmacological experiment and cannabinoid1 (CB1) receptor binding assay using CB1 antagonists to clarify whether the CB1 receptor is involved in the synergism or not. The affinities of cannabinoids for the CB1 receptor in the mouse brain synaptic membrane were evaluated using a specific CB1 ligand, [3H]CP55940.ResultsAlthough the potentiating effect of ∆9-THC on pentobarbital-induced sleep was attenuated by co-administration of CB1 receptor antagonists, such as SR141716A and AM251, at a dose of 2 mg/kg, intravenously (i.v.) to mice, the CBD-enhanced pentobarbital-induced sleep was not inhibited by SR141716A. The inhibitory constant (Ki) values of ∆9-THC and CBD were 6.62 and 2010 nM, respectively, showing a high affinity of ∆9-THC and a low affinity of CBD for the CB1 receptor, respectively. A high concentration of pentobarbital (1 mM) did not affect specific [3H]CP55940 binding on the mouse brain synaptic membrane.ConclusionsThese results suggest that binding of ∆9-THC to the CB1 receptor is involved in the synergism with pentobarbital, and that potentiating effect of CBD with pentobarbital may differ from that of ∆9-THC. We successfully demonstrated that ∆9-THC enhanced the anesthetic effect of pentobarbital through the CB1 receptor.

Expression and Purification of Delta Sleep-Inducing Peptide Fused with Protein Transduction Domain and Human Serum Albumin in Pichia pastoris.

Sleep is a natural part of every individual's life. Delta sleep-inducing peptide (DSIP) is a nonapeptide that could promote sleep through the induction of slow wave sleep. However, little is known about the pharmacological effect of DSIP on insomnia. The main objective of this study was to analyze the pharmacological effect of DSIP on insomnia. We designed a fusion protein containing N-terminal TAT-based transduction domain followed by human serum albumin and DSIP and designated this protein as PHD fusion protein. The PHD fusion protein were expressed in Pichia pastoris and purified. Mice were administered single subcutaneous injections three concentrations of PHD fusion protein (0.5, 1, 2 mg/kg), and the pharmacological activity of PHD fusion protein was studied using classic pentobarbitalinduced sleep test. We expressed the PHD fusion protein in P. pastoris; furthermore, the PHD fused protein was purified to near homogeneity by DEAE Sepharose FF, Phenyl Sepharose HP and Blue Sepharose 6 FF. Our result showed that the increase of pentobarbital-induced hypnotic effect characterized by reducing sleep latency and prolonged sleep duration was observed for increasing concentrations of PHD fusion protein (P<0.05); moreover, different dose of PHD fusion protein could induce the mice to re-sleep in a dose-dependent manner, whereas higher doses of PHD fusion protein (1.0, 2.0 mg/kg) significantly increased the rate of sleep re-onset compared with the vehicle group of mice (P<0.05). PHD fusion protein increased the hypnotic effects of pentobarbital by reducing sleep latency and prolonged sleep duration. The present study suggested PHD fusion protein could be a new drug candidate for insomnia.

Propofol Anesthesia Is Reduced in Phospholipase C-Related Inactive Protein Type-1 Knockout Mice.

The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pretreatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phosphorylation of cortical the GABAA-R β3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R β3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.

Sedative and Motor Incoordination Effects of Ethanol in Mice Lacking CD14, TLR2, TLR4, or MyD88.

In our companion article, we examined the role of MyD88-dependent signaling in ethanol (EtOH) consumption in mice lacking key components of this inflammatory pathway and observed differential effects on drinking. Here, we studied the role of these same signaling components in the acute sedative, intoxicating, and physiological effects of EtOH. Toll-like receptor 4 (TLR4) has been reported to strongly reduce the duration of EtOH-induced sedation, although most studies do not support its direct involvement in EtOH consumption. We examined TLR4 and other MyD88 pathway molecules to determine signaling specificity in acute EtOH-related behaviors. We also studied other GABAergic sedatives to gauge the EtOH specificity and potential role for GABA in EtOH's sedative and intoxicating effects in the mutant mice. Loss of righting reflex (LORR) and recovery from motor incoordination were studied following acute injection of EtOH or other sedative drugs in male and female control C57BL/6J mice versus mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We also examined EtOH-induced hypothermia and blood EtOH clearance in these mice. Male and female mice lacking TLR4 or MyD88 showed reduced duration of EtOH-induced LORR and faster recovery from EtOH-induced motor incoordination in the rotarod test. MyD88 knockout mice had slightly faster recovery from EtOH-induced hypothermia compared to control mice. None of the mutants differed from control mice in the rate of blood EtOH clearance. All of the mutants showed similar decreases in the duration of gaboxadol-induced LORR, but only mice lacking TLR4 were less sensitive to the sedative effects of pentobarbital. Faster recovery from diazepam-induced motor impairment was observed in CD14, TLR4, and MyD88 null mice of both sexes. TLR4 and MyD88 were key mediators of the sedative and intoxicating effects of EtOH and GABAergic sedatives, indicating a strong influence of TLR4-MyD88 signaling on GABAergic function. Despite the involvement of TLR4 in EtOH's acute behaviors, it did not regulate EtOH consumption in any drinking model as shown in our companion article. Collectively, our studies demonstrate differential effects of TLR-MyD88 components in the acute versus chronic actions of EtOH.

Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

BackgroundPentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.MethodsUsing oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition.ResultsCombining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.ConclusionsOur novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.