Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.

INTRODUCTION: Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol triphosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. METHODS: We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine, 2-aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U- 73122). RESULTS: Ryanodine decreases paired-pulse facilitation for intervals <20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals of <20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired-pulse depression for intervals <20 ms. CONCLUSIONS: Ryanodine, IP3 receptors, and phospholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets for improving the safety factor of neuromuscular transmission during repetitive activity of the neuromuscular junction.

Long-term application of adrenergic agonists modulates nociceptive ion channels.

Dorsal root ganglia (DRG) neurons transduce and convey somatosensory information from the periphery to the central nervous system. Adrenergic mediators are known to modulate nociceptive inputs in DRG neurons, acting as up- or down-regulators of neuronal excitability. They are also important in the development of sympathetic neuropathy. ATP-activated P2X channels and capsaicin-activated TRPV1 channels are directly involved in the transduction of nociceptive stimuli. In this work, we show that long-term (up to 3 days) in vitro stimulation of DRG neurons with selective α1-adrenergic agonist increased slow but not fast ATP-activated currents, with no effect on capsaicin currents. Selective agonists for α2, ß1 and ß3-adrenergic receptors decreased capsaicin activated currents and had no effect on ATP currents. Capsaicin currents were associated with increased neuronal excitability, while none of the adrenergic modulators produced change in rheobase. These results demonstrate that chronic adrenergic activation modulates two nociceptive transducer molecules, increasing or decreasing channel current depending on the adrenergic receptor subtype. These observations aid our understanding of nociceptive or antinociceptive effects of adrenergic agonists.

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction.

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-ß-cyclodextrin (MßCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MßCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MßCD were larger than those from control. In addition, treatment with MßCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MßCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

Increased Cholinergic Tone Causes Pre-synaptic Neuromuscular Degeneration and is Associated with Impaired Diaphragm Function.

In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.

Involvement of the PKA pathway and inhibition of voltage gated Ca2+ channels in antihyperalgesic activity of Lippia grata/ß-cyclodextrin.

Neuropathic pain (NP) is a difficult condition to treat because of the modest efficacy of available drugs. New treatments are required. In the study we aimed to investigate the effects of the essential oil from Lippia grata alone or complexed in ß-cyclodextrin (LG or LG-ßCD) on persistent inflammatory and neuropathic pain in a mouse model. We also investigated Ca2+ currents in rat dorsal root ganglion (DRG) neurons. Male Swiss mice were treated with LG or LG/ß-CD (24 mg/kg, i.g.) and their effect was evaluated using an acute inflammatory pleurisy model and nociception triggered by intraplantar injection of an agonist of the TRPs channels. We also tested their effect in chronic pain models: injection of Freund's Complete Adjuvant and partial sciatic nerve ligation (PSNL). In the pleurisy model, LG reduced the number of leukocytes and the levels of TNF-α and IL-1ß. It also inhibited cinnamaldehyde and menthol-induced nociceptive behavior. The pain threshold in mechanical and thermal hyperalgesia was increased and paw edema was decreased in models of inflammatory and neuropathic pain. PSNL increased inflammatory protein contents and LG and LG-ßCD restored the protein contents of TNF-α, NF-κB, and PKA, but not IL-1ß and IL-10. LG inhibited voltage gated Ca2+ channels from DRG neurons. Our results suggested that LG or LG-ßCD produce anti-hyperalgesic effect in chronic pain models through reductions in TNF-α levels and PKA, and inhibited voltage-gated calcium channels and may be innovative therapeutic agents for the management of NP.

Sustained currents through ASIC3 ion channels at the modest pH changes that occur during myocardial ischemia.

Acid-sensing ion channel 3 (ASIC3) is highly expressed on sensory neurons that innervate heart and skeletal muscle and, therefore, is proposed to detect lactic acidosis and to transduce angina and muscle ischemic pain. A difficulty with this idea is that ASIC3 rapidly desensitizes. How can a desensitizing ion channel mediate a persisting sensation such as angina? Here, we show that rat ASIC3 produces a sustained current within the limited range of extracellular pH (7.3 to 6.7) that occurs during cardiac and skeletal muscle ischemia; experiments use patch clamp on transfected cell lines and on fluorescently tagged sensory neurons that innervate rat heart. No such sustained current occurs with ASIC1a (either as homomers or 1a/3 heteromers), whereas ASIC2a/3 heteromers give much larger currents than ASIC3 homomers. The sustained current persists even over tens of minutes because it is caused by a region of pH where there is overlap between inactivation and activation of the channel. Lactate, an anaerobic metabolite, allows the current to activate at slightly more basic pH. Surprisingly, amiloride, which blocks ASICs when they are activated at lower pH, increases ASIC3 current evoked at pH 7.0. Cardiac sensory neurons exhibit a small, perfectly sustained current when pH changes from 7.4 to 7.0. At least some of this current is carried by ASICs because the current is increased by both Zn(2+), an ASIC modulator, and amiloride. We suggest that this sustained mode is the most relevant form of ASIC3 gating for triggering angina and other ischemic pain.

Neuromuscular synapse degeneration without muscle function loss in the diaphragm of a murine model for Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by chorea, incoordination and psychiatric and behavioral symptoms. The leading cause of death in HD patients is aspiration pneumonia, associated with respiratory dysfunction, decreased respiratory muscle strength and dysphagia. Although most of the motor symptoms are derived from alterations in the central nervous system, some might be associated with changes in the components of motor units (MU). To explore this hypothesis, we evaluated morphofunctional aspects of the diaphragm muscle in a mouse model for HD (BACHD). We showed that the axons of the phrenic nerves were not affected in 12-months-old BACHD mice, but the axon terminals that form the neuromuscular junctions (NMJs) were more fragmented in these animals in comparison with the wild-type mice. In BACHD mice, the synaptic vesicles of the diaphragm NMJs presented a decreased exocytosis rate. Quantal content and quantal size were smaller and there was less synaptic depression whereas the estimated size of the readily releasable vesicle pool was not changed. At the ultrastructure level, the diaphragm NMJs of these mice presented fewer synaptic vesicles with flattened and oval shapes, which might be associated with the reduced expression of the vesicular acetylcholine transporter protein. Furthermore, mitochondria of the diaphragm muscle presented signs of degeneration in BACHD mice. Interestingly, despite all these cellular alterations, BACHD diaphragmatic function was not compromised, suggesting a higher resistance threshold of this muscle. A putative resistance mechanism may be protecting this vital muscle. Our data contribute to expanding the current understanding of the effects of mutated huntingtin in the neuromuscular synapse and the diaphragm muscle function.

VAChT overexpression increases acetylcholine at the synaptic cleft and accelerates aging of neuromuscular junctions.

BACKGROUND: Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). METHODS: Chat-ChR2-EYFP (VAChTHyp) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1G93A), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. RESULTS: We show that VAChT is elevated in the spinal cord and at NMJs of VAChTHyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChTHyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChTHyp mice compared to control mice. While the development of NMJs was not affected in VAChTHyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChTHyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChTHyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChTHyp mice. In the SOD1G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice. CONCLUSIONS: The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.

Contrasting phenotypes of putative proprioceptive and nociceptive trigeminal neurons innervating jaw muscle in rat.

BACKGROUND: Despite the clinical significance of muscle pain, and the extensive investigation of the properties of muscle afferent fibers, there has been little study of the ion channels on sensory neurons that innervate muscle. In this study, we have fluorescently tagged sensory neurons that innervate the masseter muscle, which is unique because cell bodies for its muscle spindles are in a brainstem nucleus (mesencephalic nucleus of the 5th cranial nerve, MeV) while all its other sensory afferents are in the trigeminal ganglion (TG). We examine the hypothesis that certain molecules proposed to be used selectively by nociceptors fail to express on muscle spindles afferents but appear on other afferents from the same muscle. RESULTS: MeV muscle afferents perfectly fit expectations of cells with a non-nociceptive sensory modality: Opiates failed to inhibit calcium channel currents (I(Ca)) in 90% of MeV neurons, although ICa were inhibited by GABA(B) receptor activation. All MeV afferents had brief (1 msec) action potentials driven solely by tetrodotoxin (TTX)-sensitive Na channels and no MeV afferent expressed either of three ion channels (TRPV1, P2X3, and ASIC3) thought to be transducers for nociceptive stimuli, although they did express other ATP and acid-sensing channels. Trigeminal masseter afferents were much more diverse. Virtually all of them expressed at least one, and often several, of the three putative nociceptive transducer channels, but the mix varied from cell to cell. Calcium currents in 80% of the neurons were measurably inhibited by mu-opioids, but the extent of inhibition varied greatly. Almost all TG masseter afferents expressed some TTX-insensitive sodium currents, but the amount compared to TTX sensitive sodium current varied, as did the duration of action potentials. CONCLUSION: Most masseter muscle afferents that are not muscle spindle afferents express molecules that are considered characteristic of nociceptors, but these putative muscle nociceptors are molecularly diverse. This heterogeneity may reflect the mixture of metabosensitive afferents which can also signal noxious stimuli and purely nociceptive afferents characteristic of muscle.

Reduced expression of the vesicular acetylcholine transporter and neurotransmitter content affects synaptic vesicle distribution and shape in mouse neuromuscular junction.

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD(HOM)) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD(HOM) mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1-43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD(HOM) neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD(HOM) exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.

Differential effects of Tityus bahiensis scorpion venom on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents.

We examined modification of sodium channel gating by Tityus bahiensis scorpion venom (TbScV), and compared effects on native tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents from rat dorsal root ganglion neurons and cardiac myocytes. In neurons, TbScV dramatically reduced the rate of sodium current inactivation, increased current amplitude, and caused a negative shift in the voltage-dependence of activation and inactivation of tetrodotoxin-sensitive channels. Enhanced activation of modified sodium channels was independent of a depolarizing prepulse. We identified two components of neuronal tetrodotoxin-resistant current with biophysical properties similar to those described for NaV1.8 and NaV1.9. In contrast to its effects on neuronal tetrodotoxin-sensitive current, TbScV caused a small decrease in neuronal tetrodotoxin-resistant sodium current amplitude and the gating modifications described above were absent. A third tetrodotoxin-resistant current, NaV1.5 recorded in rat cardiac ventricular myocytes, was inhibited approximately 50% by TbScV, and the remaining current exhibited markedly slowed activation and inactivation. In conclusion, TbScV has very different effects on different sodium channel isoforms. Among the neuronal types, currents resistant to tetrodotoxin are also resistant to gating modification by TbScV. The cardiac tetrodotoxin-resistant current has complex sensitivity that includes both inhibition of current amplitude and slowing of activation and inactivation.

Sensing muscle ischemia: coincident detection of acid and ATP via interplay of two ion channels.

Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.