Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system. Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons. However, the role of ASIC1b is still elusive in terms of its functions and expression profile. In this study, we probed the role of ASIC1b in acid-induced muscle pain in Asic1b-knockout (Asic1b–/–) and Asic1b-Cre transgenic (Asic1bCre) mice. We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type (Asic1b+/+) but not Asic1b–/– mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in Asic1b+/+ mice, but the duration of hyperalgesia was significantly shortened in Asic1b–/– mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in Asic1b+/+ mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from Asic1bCre-TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current (IASIC) in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the IASIC in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on IASIC in about half of the ASIC1b-expressing DRG neurons. In situ hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.

The amount of neurotransmitter released from a presynaptic terminal is the product of the quantal content (number of vesicles) and the presynaptic quantal size (QSpre, amount of transmitter per vesicle). QSpre varies with synaptic use, but its regulation is poorly understood. The motor nerve terminals at the neuromuscular junction (NMJ) contain TGF-β receptors. We present evidence that TGF-β2 regulates QSpre at the NMJ. Application of TGF-β2 to the rat diaphragm NMJ increased the postsynaptic response to both spontaneous and evoked release of acetylcholine, whereas antibodies to TGF-β2 or its receptor had the converse effect. L-vesamicol and bafilomycin blocked the actions of TGF-β2, indicating that TGF-β2 acts by altering the extent of vesicular filling. Recordings of the postsynaptic currents from the diaphragm were consistent with TGF-β2 having this presynaptic action and a lesser postsynaptic effect on input resistance. TGF-β2 also decreased quantal content by an atropine-sensitive pathway, indicating that this change is secondary to cholinergic feedback on vesicular release. Consequently, the net actions of TGF-β2 at the NMJ were to amplify the postsynaptic effects of spontaneous transmission and to diminish the number of vesicles used per evoked stimulus, without diminishing the amount of acetylcholine released.motoneuron | quantal size | synaptic plasticity | synaptic vesicle | motor nerve terminal S ynapses need to be efficient and malleable to account for the dynamic features of neuronal networks. The number of synaptic vesicles released with each stimulus (quantal content) and the postsynaptic response to each vesicle (quantal size) are actively regulated to achieve this malleability and are major determinants of whether synaptic transmission occurs (1). The amount of neurotransmitter released from each vesicle (presynaptic quantal size, QSpre) appears to vary inversely with synaptic activity and is a component of quantal size (2-4). However, it is unclear whether QSpre is actively regulated as an important component of the characteristics of a synapse.QSpre cannot be measured directly at most synapses; variation in QSpre is inferred from the analysis of either evoked (EPP) or spontaneous postsynaptic potentials. This inference tacitly assumes that the function of QSpre is to influence postsynaptic quantal size. Although this influence may occur in many circumstances, the existence of negative feedback on vesicle release (5) challenges the universality of this assumption. Because any QSpre-dependent change in the amount of neurotransmitter released should be attenuated rapidly by an opposite change in quantal content, upregulation of QSpre would restrict the number of vesicles released per evoked stimulus but would not acutely affect evoked postsynaptic currents unless the negative feedback loop was ineffectual. In that case, the regulation of QSpre would not be readily apparent from studies of EPPs.The major features of synapses are profoundly shaped by extracellular signals from the pre-and postsynap...

Neurotrophin-3 (NT-3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT-3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency-induced reduction in NT-3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1-43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve-mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1-43 were impaired in mature NT-3-deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype-dependent variation in the number of synaptic vesicles near the active zone. NT-3(+/-) mice had normal soleus muscle fiber numbers but their fibers had smaller cross-sectional areas and were more densely-packed than wild-type littermates. Moreover, the muscles of adult NT-3-deficient animals were weaker than those of wild-type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT-3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.

Cytoplasmic polyadenylation element binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein that downregulates translation of multiple plasticity-related proteins (PRPs) at the glutamatergic synapses. Activity-induced synthesis of PRPs maintains long-lasting synaptic changes that are critical for memory consolidation and chronic pain manifestation. CPEB3-knockout (KO) mice show aberrant hippocampus-related plasticity and memory, so we investigated whether CPEB3 might have a role in nociception-associated plasticity. CPEB3 is widely expressed in the brain and peripheral afferent sensory neurons. CPEB3-KO mice with normal mechanosensation showed hypersensitivity to noxious heat. In the complete Freund's adjuvant (CFA)-induced inflammatory pain model, CPEB3-KO animals showed normal thermal hyperalgesia and transiently enhanced mechanical hyperalgesia. Translation of transient receptor potential vanilloid 1 (TRPV1) RNA was suppressed by CPEB3 in dorsal root ganglia (DRG), whereas CFA-induced inflammation reversed this inhibition. Moreover, CPEB3/TRPV1 double-KO mice behaved like TRPV1-KO mice, with severely impaired thermosensation and thermal hyperalgesia. An enhanced thermal response was recapitulated in non-inflamed but not inflamed conditional-KO mice, with cpeb3 gene ablated mostly but not completely, in small-diameter nociceptive DRG neurons. CPEB3-regulated translation of TRPV1 RNA may play a role in fine-tuning thermal sensitivity of nociceptors.