Anticonvulsive Effects and Pharmacokinetic Profile of Cannabidiol (CBD) in the Pentylenetetrazol (PTZ) or N-Methyl-D-Aspartate (NMDA) Models of Seizures in Infantile Rats

In spite of use of cannabidiol (CBD), a non-psychoactive cannabinoid, in pediatric patients with epilepsy, preclinical studies on its effects in immature animals are very limited. In the present study we investigated anti-seizure activity of CBD (10 and 60 mg/kg administered intraperitoneally) in two models of chemically induced seizures in infantile (12-days old) rats. Seizures were induced either with pentylenetetrazol (PTZ) or N-methyl-D-aspartate (NMDA). In parallel, brain and plasma levels of CBD and possible motor adverse effects were assessed in the righting reflex and the bar holding tests. CBD was ineffective against NMDA-induced seizures, but in a dose 60 mg/kg abolished the tonic phase of PTZ-induced generalized seizures. Plasma and brain levels of CBD were determined up to 24 h after administration. Peak CBD levels in the brain (996 ± 128 and 5689 ± 150 ng/g after the 10- and 60-mg/kg doses, respectively) were reached 1–2 h after administration and were still detectable 24 h later (120 ± 12 and 904 ± 63 ng/g, respectively). None of the doses negatively affected motor performance within 1 hour after administration, but CBD in both doses blocked improvement in the bar holding test with repeated exposure to this task. Taken together, anti-seizure activity of CBD in infantile animals is dose and model dependent, and at therapeutic doses CBD does not cause motor impairment. The potential risk of CBD for motor learning seen in repeated motor tests has to be further examined.

Nux Vomica 200 CH reduced acute hypnotic effect of alcohol in young toads

Potentized Nux Vomica has been reported to produce antialcoholic effect in mice, rats and toads. The effect relates to consumption of alcohol and alcohol-induced loss of righting reflex (RR). RR’s maintain normal erect posture of an animal and are centrally controlled in the midbrain. In the present study young toads, Duttaphrynus melanostictus were first treated with Nux vomica 200 CH and then partially immersed in 209 mM ethanol solution in such a way that their head remained above the level of ethanol solution. Toadlets were removed from the ethanol solution every 10 min, tested for the loss of RR and returned to the ethanol solution. Toadlets were placed in a supine position on a dry flat surface. Failure to right within 60 sec was considered as the loss of RR. The experiment was repeated 10 times. Control toadlets were pretreated with 90% ethanol instead of Nux Vomica 200 CH. The percentages of toadlets showing loss of RR, both in the control as well as in the Nux-treated groups, were shown in graphs against the duration of exposure to ethanol solution. Differences in the percentage distribution between the control and the treatment groups losing RR were tested by χ2 test. All the experiments were conducted at room temperature. The percentage of toadlets losing RR increased with time of exposure to ethanol solution. The increase was significantly higher with the control than with the Nux-treated group. Nux Vomica 200 CH might have influenced the mid-brain of toadlets thereby countering the hypnotic effect of ethanol in the toadlets.

Biological effects of high-diluted substances and periodic table of elements

Background and Aims. There are several experimental evidences for the effects of high-diluted substances (see e.g. C. Taddei-Ferretti, A. Cotugno 1997, on effects of high-diluted drugs on the prevention and control of mice teratogenicity induced by purine derivatives; N.C. Sukul, C. Taddei-Ferretti, S.P. Sinha Babu, A. De, B. Nandi, A. Sukul, R. Dutta-Nag 2000, on high-diluted Nux vomica countering alcohol-induced loss of righting reflex in toads). Also the physical characterization and mechanism of action of high-diluted drugs have been studied (see e.g. N.C. Sukul, A. Sukul, High dilution effects: Physical and biochemical basis 2004). However, further experimental researches are needed to clarify how physical characteristics of a drug are linked to its global biological effects. Considerations on some high-diluted mineral remedies will be developer here. Methods. In Organon, sect. 119, S. Hahnemann writes: «As certainly each species of plants is different from every other one with regard to external appearance, way of life and growth, taste and smell, and as certainly each mineral, each salt is different from the others with regard to external, internal, physical and chemical qualities [...], so certainly all these vegetal and mineral substances have pathogenetic – and thus also curative – effects different among themselves [...]». This statement may be taken as basis for considering the characteristics of some elements, as ordered in the periodic table, in relation to those of some high-diluted mineral remedies. Conclusions. The elements were previously ordered in the periodic table according to the atomic weight chemically determined, and later more precisely according to the atomic number (number of protons). Then also the electronic configuration was taken into account: properties depending on atomic mass and deep electrons are not periodical, while chemical and several physical properties are linked to external electrons which have periodical configuration. In particular, let us consider the group of elements C, P, S, Cl and the group of elements Ca, Mg, K, Na. One may conclude that the four elements of the first group (respectively receiver-or-donor of 4 electrons, receiver of 3, of 2, of 1 electron), which, according to H. Bernard, are linked to the fixed human constitutions, are close among themselves in the periodic table, while they are very distant from the four elements of the second group (respectively donor of 2, of 2, of 1, of 1 electron), which are close among themselves and are linked to the changing constitutional stages.

Identification of a Novel Secreted Metabolite Cyclo(Phenylalanyl-Prolyl) from Batrachochytrium Dendrobatidis and its effect on Galleria Mellonella.

The fungus, Batrachochytrium dendrobatidis, is the causative agent of chytridiomycosis and a leading cause of global decline in amphibian populations . The first stages of chytridiomycosis include: inflammation, hyperkeratosis, lethargy, loss of righting reflex, and disruption of internal electrolyte levels leading to eventual death of the host. Previous work indicates that B. dendrobatidis can produce immunomodulatory compounds and other secreted molecules that regulate the growth of the fungus. In this study, filtrates of the fungus grown in media and water were subjected to ultra performance liquid chromatography-mass spectrometry and analyzed using Compound Discoverer 3.0. Identification of cyclo(phenylalanyl-prolyl), chitobiose, and S-adenosylmethionine were verified by their retention times and fragmentation patterns from B. dendrobatidis supernatants. Previous studies have analyzed the effects of B. dendrobatidis on amphibian models, in vitro, or in cell culture. We studied the effects of live B. dendrobatidis cells, spent culture filtrates containing secreted metabolites, and cyclo(pheylalanyl-prolyl) on wax moth larvae ( Galleria mellonella) . Concentrated filtrates caused melanization within 24 hours, while live B. dendrobatidis caused melanization within 48 hours. Our results indicate B. dendrobatidis produces secreted metabolites previously unreported. These findings provide another alternative for the use of a non-amphibian model system to study pathogenicity traits in this fungus.

Homeopathic drugs complementary, antidotal and inimical to Nux vomica produce stronger anti-alcoholic effect on toads than Nux.

Background In homeopathy some drugs are known to act as complementary, antidotal or inimical to a particular drug. Practitioners can follow this rule when they apply one drug following another. Potentized Nux vomica can reduce acute hypnotic effect of alcohol on toads. Sulphur and Sepia are reported to be complementary to Nux-vom, while Coffea cruda and Zincum met are antidotal and inimical to Nux, respectively .The four drugs have been tested on the toad model to find out their actual therapeutic relationship with Nux vom. Objective To verify the complementary effect of Sulphur and Sepia, antidotal effect of Coffea and inimical effect of Zincum in relation to Nux vom in the toad model. Methods Five batches of toads, each comprising 20 individuals, were treated by partial immersion in a drug diluted with distilled water 1:500 for 20 min. The control consisted of 90% ethanol diluted with distilled water 1:500. The drugs were Nux vom 200 CH, Sulphur 200 CH, Sepia 200 CH, Coffea 200 CH and Zincum 200 CH. Toads of each batch were separately exposed to 260mM ethanol solution and tested every 10 min to see if they had lost their righting reflex (RR). For this, each toad was laid on its dorsal surface. If it failed to turn on its ventrum in a cut-off time of 60 sec it was considered to have lost it’s RR. Four more batches of toads were pretreated with Nux vom 200 CH and subsequently treated separately by Sulphur 200 CH, Sepia 200 CH, Coffea 200 CH and Zincum 200 CH. All the toads were then exposed to 260 mM ethanol solution to record their tolerance to ethanol anesthesia in terms of time to lose RR. Results Toads treated with the five drugs took significantly longer time (P

Investigation of Potassium Chloride for Euthanasia of Anesthetized Marine Toads (Rhinella marina)

Euthanasia techniques in amphibians are poorly described and sparsely validated. This study investigated potassium chloride (KCl) for euthanasia of anesthetized marine toads ( Rhinella marina ). Twenty three toads were immersed in buffered MS-222 (2 g/L) for five minutes (min) beyond loss of righting reflex, manually removed, and randomly administered KCl (n = 6/group) via one of three routes: intracardiac at 10 mEq/kg (IC), intracoelomic at 100 mEq/kg (ICe), or immersion at 4500 mEq/L (IMS) or no treatment (C) (n = 5/group). Doppler sounds were assessed continuously from prior to treatment until two min post-treatment and every five min thereafter until sound cessation or resumption of spontaneous movement. Plasma potassium concentration (K+) was measured at the time of Doppler sound cessation in ICe and IMS. In IC, ICe, IMS, and C, Doppler sound cessation occurred in 4/6, 6/6, 6/6, and 1/5 toads with median (range) or mean + SD times of 0.23 (0-4.65), 17.5 + 9.0, 40.6 + 10.9, and >420 min, respectively. Nonsuccess in 2/6 toads in IC was suspected due to technique failure. Plasma K+ exceeded the limits of detection (>9 mmol/L) in 12/12 toads in ICe and IMS. Five of six toads in C resumed spontaneous movement at median (range) times of 327 (300-367) min. KCl delivered via an intracardiac, intracoelomic, or immersion routes resulted in Doppler sound cessation in 16 of 18 toads and may be appropriate for euthanasia of anesthetized marine toads.

Return of the Righting Reflex Does Not Portend Recovery of Cognitive Function in Anesthetized Rats

As the number of individuals undergoing general anesthesia rises globally, it becomes increasingly important to understand how consciousness and cognition are restored after anesthesia. In rodents, levels of consciousness are traditionally captured by physiological responses such as the return of righting reflex (RORR). However, tracking the recovery of cognitive function is comparatively difficult. Here we use an operant conditioning task, the 5-choice serial reaction time task (5-CSRTT), to measure sustained attention, working memory, and inhibitory control in male and female rats as they recover from the effects of several different clinical anesthetics. In the 5-CSRTT, rats learn to attend to a five-windowed touchscreen for the presentation of a stimulus. Rats are rewarded with food pellets for selecting the correct window within the time limit. During each session we tracked both the proportion of correct (accuracy) and missed (omissions) responses over time. Cognitive recovery trajectories were assessed after isoflurane (2% for 1 h), sevoflurane (3% for 20 min), propofol (10 mg/kg I.V. bolus), ketamine (50 mg/kg I.V. infusion over 10 min), and dexmedetomidine (20 and 35 μg/kg I.V. infusions over 10 min) for up to 3 h following RORR. Rats were classified as having recovered accuracy performance when four of their last five responses were correct, and as having recovered low omission performance when they missed one or fewer of their last five trials. Following isoflurane, sevoflurane, and propofol anesthesia, the majority (63–88%) of rats recovered both accuracy and low omission performance within an hour of RORR. Following ketamine, accuracy performance recovers within 2 h in most (63%) rats, but low omission performance recovers in only a minority (32%) of rats within 3 h. Finally, following either high or low doses of dexmedetomidine, few rats (25–32%) recover accuracy performance, and even fewer (0–13%) recover low omission performance within 3 h. Regardless of the anesthetic, RORR latency is not correlated with 5-CSRTT performance, which suggests that recovery of neurocognitive function cannot be inferred from changes in levels of consciousness. These results demonstrate how operant conditioning tasks can be used to assess real-time recovery of neurocognitive function following different anesthetic regimens.

Contribution of GlyR α3 Subunits to the Sensitivity and Effect of Ethanol in the Nucleus Accumbens

The glycine receptor (GlyR), a ligand-gated ion channel, is critical for inhibitory neurotransmission in brainstem, spinal cord, and in supraspinal regions. Recent data from several laboratories have shown that GlyRs are expressed in the brain reward circuitry and that α1 and α2 are the principal subunits expressed in the nucleus accumbens (nAc). In the present study, we studied the sensitivity to ethanol of homomeric and heteromeric α3 GlyR subunits in HEK293 cells and dissociated neurons from the nAc. Finally, we explored ethanol-related behaviors in a Glra3 knockout mouse (Glra3–/–). Studies in HEK293 cells showed that while homomeric α3 GlyR subunits were insensitive to ethanol, heteromeric α3β GlyR subunits showed higher sensitivity to ethanol. Additionally, using electrophysiological recordings in dissociated accumbal neurons, we found that the glycine current density increased in Glra3–/– mice and the GlyRs were less affected by ethanol and picrotoxin. We also examined the effect of ethanol on sedation and drinking behavior in Glra3–/– mice and found that the duration in the loss of righting reflex (LORR) was unchanged compared to wild-type (WT) mice. On the other hand, using the drinking in the dark (DID) paradigm, we found that Glra3–/– mice have a larger ethanol consumption compared to WT mice, and that this was already high during the first days of exposure to ethanol. Our results support the conclusion that heteromeric α3β, but not homomeric α3, GlyRs are potentiated by ethanol. Also, the increase in GlyR and GABAAR mediated current densities in accumbal neurons in the KO mice support the presence of compensatory changes to α3 knock out. The increase in ethanol drinking in the Glra3–/– mice might be associated to the reduction in β and compensatory changes in other subunits in the receptor arrangement.

A Single Low Dose of Dexmedetomidine Efficiently Attenuates Esketamine-Induced Overactive Behaviors and Neuronal Hyperactivities in Mice

Introduction: Esketamine (Esk) (S(+)-ketamine) is now used as an alternative to its racemic mixture, i. e., ketamine in anesthesia. Esk demonstrated more powerful potency and rapid recovery in anesthesia and less psychotomimetic side effects comparing with ketamine, but Esk could still induce psychological side effects in patients. This study was to investigate whether dexmedetomidine (Dex) can attenuate the Esk-induced neuronal hyperactivities in Kunming mice.Methods: Dexmedetomidine 0.25, 0.5, and 1 mg/kg accompanied with Esk 50 mg/kg were administrated on Kunming mice to assess the anesthesia quality for 1 h. The indicators, such as time to action, duration of agitation, duration of ataxia, duration of loss pedal withdrawal reaction (PWR), duration of catalepsy, duration of righting reflex (RR) loss, duration of sedation, were recorded for 1 h after intraperitoneal administration. The c-Fos expression in the brain was detected by immunohistochemistry and Western Blot after 1 h of administration. Considering the length of recovery time for more than 1 h in Dex and Dex with Esk groups, other mice were repeatedly used to evaluate recovery time from the administration to emerge from anesthesia.Results: Dexmedetomidine dose-dependently increased recovery time when administrated with Esk or alone. Dex combined with Esk efficiently attenuated the duration of agitation, ataxia, and catalepsy. Dex synergically improved the anesthesia of Esk by increasing the duration of sedation, loss of RR, and loss of PWR. Esk induced the high expression of c-Fos in the cerebral cortex, hippocampus, thalamus, amygdala, hypothalamus, and cerebellum 1 h after administration. Western Blot results indicated that Dex at doses of 0.25, 0.5, and 1 mg/kg could significantly alleviate the Esk-induced c-Fos expression in the mice brain.Conclusion: Dexmedetomidine ranged from 0.25 to 1 mg/kg could improve the anesthesia quality and decreased the neuronal hyperactivities and the overactive behaviors when combined with Esk. However, Dex dose-dependently increased the recovery time from anesthesia. It demonstrated that a small dose of Dex 0.25 mg/kg could be sufficient to attenuate Esk-induced psychotomimetic side effects without extension of recovery time in Kunming mice.