The M2 muscarinic receptor, in association to M1 , regulates the neuromuscular PKA molecular dynamics.

Muscarinic acetylcholine receptor 1 subtype (M1 ) and muscarinic acetylcholine receptor 2 subtype (M2 ) presynaptic muscarinic receptor subtypes increase and decrease, respectively, neurotransmitter release at neuromuscular junctions. M2 involves protein kinase A (PKA), although the muscarinic regulation to form and inactivate the PKA holoenzyme is unknown. Here, we show that M2 signaling inhibits PKA by downregulating Cß subunit, upregulating RIIα/ß and liberating RIß and RIIα to the cytosol. This promotes PKA holoenzyme formation and reduces the phosphorylation of the transmitter release target synaptosome-associated protein 25 and the gene regulator cAMP response element binding. Instead, M1 signaling, which is downregulated by M2 , opposes to M2 by recruiting R subunits to the membrane. The M1 and M2 reciprocal actions are performed through the anchoring protein A kinase anchor protein 150 as a common node. Interestingly, M2 modulation on protein expression needs M1 signaling. Altogether, these results describe the dynamics of PKA subunits upon M2 muscarinic signaling in basal and under presynaptic nerve activity, uncover a specific involvement of the M1 receptor and reveal the M1 /M2 balance to activate PKA to regulate neurotransmission. This provides a molecular mechanism to the PKA holoenzyme formation and inactivation which could be general to other synapses and cellular models.

PRE-CLINICAL SKILLS: A competency-based assessment integrated course implemented early in the curriculum to prepare second-year medical students prior to entering clinical settings.

This article was migrated. The article was marked as recommended. Objective: This Case Study describes the experience of Rovira i Virgili University School of Medicine (URV) with the early introduction of pre-clinical skills learning in the undergraduate medical curricula to monitor and assessing these competencies as a prerequisite for medical students accessing their training in clinical settings. Course Development: A PRE-CLINICAL SKILLS course has been developed to guarantee medical student's performance in managing clinically relevant basic medical sciences to interpret clinical scenarios, to develop technical communication skills and to value professional behavior throughout the first two years of medical education. The set of pre-clinical competencies evaluated in this course as well as the corresponding assessment methods have been established according to an international reference standards review work in collaboration with the regional quality assurance agency. An integrated formative assessment is being used. Course Advantages, Added Values and Outcome Measures: Since the academic year 2009-2010 about 130 students from second-year of Medicine follows this integrated trunk-course while being enrolled in parallel in other core courses as Physiology, Anatomy, and Histology. The program doesn't include lectures but only learning activities to train and monitor the successful achievement of the pre-clinical skills by medical students. A good majority of the participants achieve readiness for start training in clinical settings. As a whole, this course is useful ensuring patient's safety by identifying weaknesses acquisition of pre-clinical skills and predicting medical students who will have difficulty during their clinical training. Conclusions: Reflecting on our experience, we believe that the named course "PRE-CLINICAL SKILLS" overcomes the disadvantages of the traditional teaching methods. Helping students to conceptualize rather than memorize and encouraging them to integrate clinically relevant basic medical sciences concepts and principles by training pre-clinical skills in this competence-based assessment course prior entering into clinical settings.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release.

Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.