Synaptic retrograde regulation of the PKA-induced SNAP-25 and Synapsin-1 phosphorylation.

BACKGROUND: Bidirectional communication between presynaptic and postsynaptic components contribute to the homeostasis of the synapse. In the neuromuscular synapse, the arrival of the nerve impulse at the presynaptic terminal triggers the molecular mechanisms associated with ACh release, which can be retrogradely regulated by the resulting muscle contraction. This retrograde regulation, however, has been poorly studied. At the neuromuscular junction (NMJ), protein kinase A (PKA) enhances neurotransmitter release, and the phosphorylation of the molecules of the release machinery including synaptosomal associated protein of 25 kDa (SNAP-25) and Synapsin-1 could be involved. METHODS: Accordingly, to study the effect of synaptic retrograde regulation of the PKA subunits and its activity, we stimulated the rat phrenic nerve (1 Hz, 30 min) resulting or not in contraction (abolished by µ-conotoxin GIIIB). Changes in protein levels and phosphorylation were detected by western blotting and cytosol/membrane translocation by subcellular fractionation. Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry. RESULTS: Here we show that synaptic PKA Cß subunit regulated by RIIß or RIIα subunits controls activity-dependent phosphorylation of SNAP-25 and Synapsin-1, respectively. Muscle contraction retrogradely downregulates presynaptic activity-induced pSynapsin-1 S9 while that enhances pSNAP-25 T138. Both actions could coordinately contribute to decreasing the neurotransmitter release at the NMJ. CONCLUSION: This provides a molecular mechanism of the bidirectional communication between nerve terminals and muscle cells to balance the accurate process of ACh release, which could be important to characterize molecules as a therapy for neuromuscular diseases in which neuromuscular crosstalk is impaired.

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.

Running and swimming prevent the deregulation of the BDNF/TrkB neurotrophic signalling at the neuromuscular junction in mice with amyotrophic lateral sclerosis.

Nerve-induced muscle contraction regulates the BDNF/TrkB neurotrophic signalling to retrogradely modulate neurotransmission and protect the neuromuscular junctions and motoneurons. In muscles with amyotrophic lateral sclerosis, this pathway is strongly misbalanced and neuromuscular junctions are destabilized, which may directly cause the motoneuron degeneration and muscular atrophy observed in this disease. Here, we sought to demonstrate (1) that physical exercise, whose recommendation has been controversial in amyotrophic lateral sclerosis, would be a good option for its therapy, because it normalizes and improves the altered neurotrophin pathway and (2) a plausible molecular mechanism underlying its positive effect. SOD1-G93A mice were trained following either running or swimming-based protocols since the beginning of the symptomatic phase (day 70 of age) until day 115. Next, the full BDNF pathway, including receptors, downstream kinases and proteins related with neurotransmission, was characterized and motoneuron survival was analysed. The results establish that amyotrophic lateral sclerosis-induced damaging molecular changes in the BDNF/TrkB pathway are reduced, prevented or even overcompensated by precisely defined exercise protocols that modulate TrkB isoforms and neurotransmission regulatory proteins and reduce motoneuron death. Altogether, the maintenance of the BDNF/TrkB signalling and the downstream pathway, particularly after the swimming protocol, adds new molecular evidence of the benefits of physical exercise to reduce the impact of amyotrophic lateral sclerosis. These results are encouraging since they reveal an improvement even starting the therapy after the onset of the disease.

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ.

Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes' fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools.

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.

Morphological and attachment changes of Lamellodiscus theroni (Monogenea: Diplectanidae) during its post-larval development on fish.

Species of the genus Lamellodiscus Johnston et Tiegs, 1922 (Monogenea: Diplectanidae) are characterised by a complex haptor bearing many different attachment elements: two pairs of main hooks joined by medial bars, 14 peripheral marginal hooks and one or two lamellodiscs, formed by several overlapping sclerotised plates (lamellae). These haptoral structures appear gradually during parasite development and, therefore, attachment strategies vary with developmental stage. The main aim of this work was to study the developmental changes of Lamellodiscus theroni Amine, Euzet et Kechemir-Issad, 2007 under experimental conditions, with special attention to the gradual variations in attachment strategies and the pathological implications. Throughout the gradual development of the sclerotised structures, six developmental phases were distinguished in L. theroni: phase I, with only 14 peripheral marginal hooks; phase II, with main hooks (ventral and dorsal) formed; phase III, with ventral bar formed; phase IV, with dorsal bars formed; phase V, with dorsal and ventral lamellodiscs formed; and phase VI, adult stage with male copulatory organ formed. During development, parasites attach to different parts of the first and secondary gill lamellae and the mode of attachment changes from unspecific stage, i.e. based on piercing any flat gill tissue in the early stages, through an intermediate stage when ventral and dorsal main hooks are completely functional and parasites become restricted to the interlamellar space, and finally to the definitive adult attachment stage when lamellodiscs are fully developed. The timing of key events in the development of L. theroni was used to establish adequate intervals for anthelmintic drug administration.

[A MODEL OF COMPREHENSIVE CARE FOR COMPLEX CHRONIC PATIENT. EXPERIENCE OF A TERRITORY]. / Un modelo de atención integral al paciente crónico complejo. EXPERIENCIA DE UN TERRITORIO.

INTRODUCTION: The Increase in life expectancy has brought an increase in chronic diseases. The evolution of chronic disease is the cause of several organic and systemic dysfunctions, leading to physical and mental limitations that determine the need for some aid to perform basic vital tasks. Primary health care has a key role in the monitoring of fragility, chronicity, and complexity of population. However, in order to address properly high complexity diseases it is necessary to know and coordinate the different resources existing inside the territory. THE DEVELOPMENT OF THE MODEL FOR ACTION: THE IMPLEMENTATIONS OF A FUNCTIONAL UNIT. The Primary Health Care must ensure equity, accessibility, longitudinally, and continuity of care, bearing in mind that health outcomes must be optimal. There are several health care providers in the Delta del Llobregat SAP, so it was implemented a strategic plan focused on the coordination and/or the reconciliation of all the devices involved in the assistance in order to provide comprehensive attention to the patient. The patients included in this program were to be identified as CCP (Complex chronic Patient), in an evolved and tributary phase of intensive follow-up. CONCLUSIONS. The identification ofpatients listed as CCP and at clinical risk allows a comprehensive monitoring in order to prevent exacerbations and overuse of unscheduled hospital resources.

Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse.

Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity-dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A1 R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M2 action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin-related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally.

Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult.

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.

Adenosine A1 and A2A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 µm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with µ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.

Muscarinic Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs' relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.

Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation.

It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression.

TrkB signaling is correlated with muscular fatigue resistance and less vulnerability to neurodegeneration.

At the neuromuscular junction (NMJ), motor neurons and myocytes maintain a bidirectional communication that guarantees adequate functionality. Thus, motor neurons' firing pattern, which is influenced by retrograde muscle-derived neurotrophic factors, modulates myocyte contractibility. Myocytes can be fast-twitch fibers and become easily fatigued or slow-twitch fibers and resistant to fatigue. Extraocular muscles (EOM) show mixed properties that guarantee fast contraction speed and resistance to fatigue and the degeneration caused by Amyotrophic lateral sclerosis (ALS) disease. The TrkB signaling is an activity-dependent pathway implicated in the NMJ well-functioning. Therefore, it could mediate the differences between fast and slow myocytes' resistance to fatigue. The present study elucidates a specific protein expression profile concerning the TrkB signaling that correlates with higher resistance to fatigue and better neuroprotective capacity through time. The results unveil that Extra-ocular muscles (EOM) express lower levels of NT-4 that extend TrkB signaling, differential PKC expression, and a higher abundance of phosphorylated synaptic proteins that correlate with continuous neurotransmission requirements. Furthermore, common molecular features between EOM and slow soleus muscles including higher neurotrophic consumption and classic and novel PKC isoforms balance correlate with better preservation of these two muscles in ALS. Altogether, higher resistance of Soleus and EOM to fatigue and ALS seems to be associated with specific protein levels concerning the TrkB neurotrophic signaling.

Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development.

During the nervous system development, synapses are initially overproduced. In the neuromuscular junction (NMJ) however, competition between several motor nerve terminals and the synapses they made ends with the maturation of only one axon. The competitive signaling between axons is mediated by the differential activity-dependent release of the neurotransmitter ACh, co-transmitters, and neurotrophic factors. A multiple metabotropic receptor-driven downstream balance between PKA and PKC isoforms modulates the phosphorylation of targets involved in transmitter release and nerve terminal stability. Previously, we observed in the weakest endings on the polyinnervated NMJ that M1 mAChR receptors reduce ACh release through the PKC pathway coupled to an excess of Ca2+ inflow through P/Q- N- and L-type voltage-gated calcium channels (VGCC). This signaling would contribute to the elimination of this nerve terminal. Here, we investigate the involvement of the P/Q-, N-, and L-subtype channels in transgenic B6.Cg-Tg (Thy1-YFP)16-Jrs/J mice during synapse elimination. Then, the axon number and postsynaptic receptor cluster morphologic maturation were evaluated. The results show that both L- and P/Q-type VGCC (but not the N-type) are equally involved in synapse elimination. Their normal function favors supernumerary axonal loss by jointly enhancing intracellular calcium [Ca2+]i. The block of these VGCCs or [Ca2+]i i sequestration results in the same delay of axonal loss as the cPKCßI and nPKCε isoform block or PKA activation. The specific block of the muscle cell's contraction with µ-conotoxin GIIIB also delays synapse maturation, and thus, a retrograde influence from the postsynaptic site regulating the presynaptic CaV1.3 may contribute to the synapse elimination.

The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C27H21N3O5)] or p75(NTR) [Pep5 (C86H111N25O19S2] signaling. The effect of BDNF depends on the M1 and M2 muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M1- and M2-subtype muscarinic pathways.

Silent synapses in neuromuscular junction development.

In the last few years, evidence has been found to suggest that some synaptic contacts become silent but can be functionally recruited before they completely retract during postnatal synapse elimination in muscle. The physiological mechanism of developmental synapse elimination may be better understood by studying this synapse recruitment. This Mini-Review collects previously published data and new results to propose a molecular mechanism for axonal disconnection. The mechanism is based on protein kinase C (PKC)-dependent inhibition of acetylcholine (ACh) release. PKC activity may be stimulated by a methoctramine-sensitive M2-type muscarinic receptor and by calcium inflow though P/Q- and L-type voltage-dependent calcium channels. In addition, tropomyosin-related tyrosine kinase B (trkB) receptor-mediated brain-derived neurotrophic factor (BDNF) activity may oppose the PKC-mediated ACh release depression. Thus, a balance between trkB and muscarinic pathways may contribute to the final functional suppression of some neuromuscular synapses during development.

Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development.

High-resolution immunohistochemistry shows that the receptor protein p75(NTR) is present in the nerve terminal, muscle cell, and glial Schwann cell at the neuromuscular junction (NMJ) of postnatal rats (P4-P6) during the synapse elimination period. Blocking the receptor with the antibody anti-p75-192-IgG (1-5 µg/ml, 1 hr) results in reduced endplate potentials (EPPs) in mono- and polyinnervated synapses ex vivo, but the mean number of functional inputs per NMJ does not change for as long as 3 hr. Incubation with exogenous brain-derived neurotrophic factor (BDNF) for 1 hr (50 nM) resulted in a significant increase in the size of the EPPs in all nerve terminals, and preincubation with anti-p75-192-IgG prevented this potentiation. Long exposure (24 hr) in vivo of the NMJs to the antibody anti-p75-192-IgG (1-2 µg/ml) results in a delay of postnatal synapse elimination and even some regrowth of previously withdrawn axons, but also in some acceleration of the morphologic maturation of the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters. The results indicate that p75(NTR) is involved in both ACh release and axonal retraction during postnatal axonal competition and synapse elimination.

Ultrastructural evidences for chromium(III) immobilization by Escherichia coli K-12 depending on metal concentration and exposure time.

Microorganisms can mediate in heavy metal sequestration through several cellular strategies and pathways. This offers an efficient way to remediate heavy metal polluted environments. This paper describes the ability of Escherichia coli K-12 to capture chromium(III) (Cr(III)) and the ultrastructural effects of this metal on cells, as well as the cellular metal localization and the possible sequestration strategy uses for it. The study was mainly performed by using several electron microscopy techniques and is based on the chromium trivalent concentration and the related exposure time. Transmission electron microscopy (TEM) assay was performed along with field emission scanning electron microscopy (FESEM) for morphological responses. Furthermore, TEM was coupled with an energy dispersive X-ray (TEM-EDX) and TEM with selected area electron diffraction (TEM-SAED) to conduct analytical assays. The exposed cultures to 10 and 12 mM Cr(III) at 12 h and to 5, 7, 10, 12, 13, and 15 mM of Cr(III) at 24 h indicated the presence of multiple electrodense granules that were significantly enriched in chromium and phosphorus content via EDX analysis. Moreover, these granules were observed to be attached to external membrane and/or surrounding cells in the respective ultrathin sections analyzed under TEM. According to these results, E. coli K-12 possesses the ability to immobilize Cr(III) in external polyphosphate granules through a strategy of accumulation, where cell response to Cr(III) toxicity seems to have a dose-dependent and time-dependent relation, thereby offering significant potential for bioremediation in Cr(III)-contaminated areas.

PKA and PKC Balance in Synapse Elimination during Neuromuscular Junction Development.

During the development of the nervous system, synaptogenesis occurs in excess though only the appropriate connections consolidate. At the neuromuscular junction, competition between several motor nerve terminals results in the maturation of a single axon and the elimination of the others. The activity-dependent release of transmitter, cotransmitters, and neurotrophic factors allows the direct mutual influence between motor axon terminals through receptors such as presynaptic muscarinic ACh autoreceptors and the tropomyosin-related kinase B neurotrophin receptor. In previous studies, we investigated the synergistic and antagonistic relations between these receptors and their downstream coupling to PKA and PKC pathways and observed a metabotropic receptor-driven balance between PKA (stabilizes multinnervation) and PKC (promotes developmental axonal loss). However, how much does each kinase contribute in the developmental synapse elimination process? A detailed statistical analysis of the differences between the PKA and PKC effects in the synapse elimination could help to explore this point. The present short communication provides this analysis and results show that a similar level of PKA inhibition and PKC potentiation would be required during development to promote synapse loss.