Co-created in vivo pharmacology practical classes using the novel organism Lumbriculus variegatus.

Co-creation within higher education emphasizes learner empowerment to promote collaboration between the students and staff, enabling students to become active participants in their learning process and the construction of resources with academic staff. Concurrently, a diminishing number of higher education institutions offer in vivo practical classes, resulting in an in vivo skills shortage. To address this, and to actively engage students in their own learning, we describe the co-creation of a student-led drug trial using Lumbriculus variegatus. Under blinded conditions, final-year undergraduate biomedical science students, under the tutelage of academic staff and fellow students, were involved in the co-creation of an in vivo practical class to determine the effects of histamine and histamine receptor inverse agonists mepyramine and loratadine. Throughout this process, undergraduate- and masters-level students played key roles in every aspect of practical delivery and data analysis. Herein, students demonstrated the test compounds, both in isolation and in combination, resulted in reduced stereotypical movements of L. variegatus (p < .05, n ≥ 6). 15% of students in the class responded to a feedback survey (n = 8) after the class. Students reported the class provided "real life" insights into in vivo research and enabled the development of hands-on skills which would be useful in applying in their future careers. All students reported that they enjoyed the class with 25% (n = 2) reporting concerns about animal use in research, enabling useful discussions about animals in research. Moreover, these student-led in vivo trials add to the pharmacological knowledge of L. variegatus promoting education-led research.

Developing a new undergraduate pharmacology core curriculum: The British Pharmacological Society Delphi Method.

The British Pharmacological Society (BPS) developed a new core curriculum for undergraduate pharmacology degrees. To do this, a modification of the Delphi Process was used. Initially, a pharmacology educator workshop was hosted to explore the core attributes expected of pharmacology graduates. We then developed these discussions into knowledge, skills, and attitudes statements and sent them, in the form of a questionnaire, to our Expert Group, which included pharmacology professionals from across academia and industry. In an iterative process, the Expert Group were asked to rank each statement according to how much they agreed it was a core graduate attribute. Where there was disagreement, statements were modified according to feedback. After three rounds of questionnaires, we had a draft core curriculum which was then finalized through a discussion workshop with the education community. In this workshop, practical aspects of curriculum implementation were discussed and the potential for the Society to develop resources to support it considered. The revised core curriculum is freely available on the Society website: https://www.bps.ac.uk/media-library-assets/library/undergraduate-pharmacology-core-curriculum. Several examples exist of the curriculum making an impact within and beyond the United Kingdom, where it has been utilized in a quality assurance context, as a tool for curriculum review and also to guide building new programs. Through a series of further expert workshops, the BPS Education and Training committee is currently developing more granular learning outcomes to accompany the core curriculum alongside recommended resources to enable delivery. In addition, this expanded curriculum is also being reviewed and updated to ensure it is fully inclusive and represents the diversity of pharmacology educators and learners worldwide.

Lumbriculus variegatus: A novel organism for in vivo pharmacology education.

Pharmacology graduates require an understanding of both in vitro and in vivo drug responses but there has been a decline in animal use in pharmacology education over the last 30 years. To address this, we present the novel invertebrate model, Lumbriculus variegatus, for in vivo testing of drugs in a teaching environment. We have developed two novel behavioral assays: the stereotypical movement assay, which measures the effect of drugs on the ability of L. variegatus to perform stereotypical movements following tactile stimulation, and the free locomotion assay, which measures drug effects on unstimulated movement. We report the effects of compounds with diverse pharmacodynamic properties on L. variegatus using these assays. The ryanodine receptor antagonist, dantrolene, altered the unstimulated movement of L. variegatus at 5 µM, whereas stimulated movement was inhibited at ≥25 µM. Lidocaine, a voltage-gated sodium channel blocker, and quinine, a nonselective sodium and potassium channel blocker, reduced both stimulated and unstimulated L. variegatus movement at ≥0.5 mM. Inhibitory effects of quinine persisted for up to 24 h after drug removal, whereas lidocaine effects were reduced 10 min after drug removal. Herein, we provide proof-of-concept utilization of L. variegatus as an organism for use in in vivo pharmacology education but without regulatory constraints or the need for specialized equipment and training.

Improved quality and quantity of written feedback is associated with a structured feedback proforma.

Facilitating the provision of detailed, deep and useful feedback is an important design feature of any educational programme. Here we evaluate feedback provided to medical students completing short transferable skills projects. Feedback quantity and depth were evaluated before and after a simple intervention to change the structure of the feedback-provision form from a blank free-text feedback form to a structured proforma that asked a pair of short questions for each of the six domains being assessed. Each pair of questions consisted of asking the marker 'what was done well?' and 'what changes would improve the assignment?' Changing the form was associated with a significant increase in the quantity of the feedback and in the amount and quality of feedback provided to students. We also observed that, for these double-marked projects, the marker designated as 'marker 1' consistently wrote more feedback than the marker designated 'marker 2'.

PKCepsilon regulates behavioral sensitivity, binding and tolerance to the CB1 receptor agonist WIN55,212-2.

The cannabinoid CB1 receptor (CB1) is one of the most abundant G protein-coupled receptors in the brain, but little is known about the mechanisms that modulate CB1 receptor signaling. Here, we show that inhibition or null mutation of the epsilon isozyme of protein kinase C (PKCepsilon) selectively enhances behavioral responses to the CB1 agonist WIN55,212-2 in mice, but not to the structurally unrelated CB1 agonist CP55,940. Binding affinity for [(3)H] WIN55,212-2 was increased in brain membranes from PKCepsilon(-/-) mice compared with PKCepsilon(+/+) mice. There was no difference in binding of the inverse agonist [(3)H] SR141716A. In addition, repeated administration of WIN55,212-2 produced greater analgesic and thermal tolerance in PKCvarepsilon(-/-) mice compared with PKCepsilon(+/+)mice. These results indicate that PKCvarepsilon selectively regulates behavioral sensitivity, CB1 receptor binding and tolerance to WIN55,212-2.

A blocker of N- and T-type voltage-gated calcium channels attenuates ethanol-induced intoxication, place preference, self-administration, and reinstatement.

There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 also attenuated the reinforcing and rewarding properties of ethanol, measured by operant self-administration and the expression of an ethanol conditioned place preference, and abolished stress-induced reinstatement of ethanol seeking. NP078585 did not affect alcohol responses in mice lacking N-type calcium channels. These results suggest that selective calcium channel inhibitors may be useful in reducing acute ethanol intoxication and alcohol consumption by human alcoholics.

Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits.

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Acute functional tolerance to ethanol mediated by protein kinase Cepsilon.

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCepsilon) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCepsilon (-/-) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCepsilon and resistance of GABA(A) receptors to ethanol. In contrast, in PKCepsilon (-/-) mice, GABA(A) receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCepsilon (-/-) and PKCepsilon (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCepsilon signaling pathway that contributes to GABA(A) receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCepsilon and does not alter PKCepsilon modulation of ethanol preference.

Deletion of N-type calcium channels alters ethanol reward and reduces ethanol consumption in mice.

N-type calcium channels are modulated by acute and chronic ethanol exposure in vitro at concentrations known to affect humans, but it is not known whether N-type channels are important for behavioral responses to ethanol in vivo. Here, we show that in mice lacking functional N-type calcium channels, voluntary ethanol consumption is reduced and place preference is developed only at a low dose of ethanol. The hypnotic effects of ethanol are also substantially diminished, whereas ethanol-induced ataxia is mildly increased. These results demonstrate that N-type calcium channels modulate acute responses to ethanol and are important mediators of ethanol reward and preference.

The endogenous cannabinoid system regulates seizure frequency and duration in a model of temporal lobe epilepsy.

Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract Delta9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1 ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.

Evidence for a physiological role of endocannabinoids in the modulation of seizure threshold and severity.

The anticonvulsant effect of cannabinoids has been shown to be mediated through activation of the cannabinoid CB(1) receptor. This study was initiated to evaluate the effects of endogenously occurring cannabinoids (endocannabinoids) on seizure severity and threshold. The anticonvulsant effect of the endocannabinoid, arachidonylethanolamine (anandamide), was evaluated in the maximal electroshock seizure model using male CF-1 mice and was found to be a fully efficacious anticonvulsant (ED(50)=50 mg/kg i.p.). The metabolically stable analog of anandamide, (R)-(20-cyano-16,16-dimetyldocosa-cis-5,8,11,14-tetraenoyl)-1'-hydroxy-2'-propylamine (O-1812), was also determined to be a potent anticonvulsant in the maximal electroshock model (ED(50)=1.5 mg/kg i.p.). Furthermore, pretreatment with the cannabinoid CB(1) receptor specific antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) completely abolished the anticonvulsant effect of anandamide as well as O-1812 (P< or =0.01, Fisher exact test), indicating a cannabinoid CB(1) receptor-mediated anticonvulsant mechanism for both endocannabinoid compounds. Additionally, the influence of cannabinoid CB(1) receptor endogenous tone on maximal seizure threshold was assessed using SR141716A alone. Our data show that SR141716A (10 mg/kg i.p.) significantly reduced maximal seizure threshold (CC(50)=14.27 mA) compared to vehicle-treated animals (CC(50)=17.57 mA) (potency ratio=1.23, lower confidence limit=1.06, upper confidence limit=1.43), indicating the presence of an endogenous cannabinoid tone that modulates seizure activity. These data demonstrate that anandamide and its analog, O-1812, are anticonvulsant in a whole animal model and further implicate the cannabinoid CB(1) receptor as a major endogenous site of seizure modulation.