Climatic modulation of neurotransmitter release in amphibian neuromuscular junctions: role of dynorphin-A.

Amphibian neuromuscular junctions (NMJs) become relatively more silent during the dry winter season in Australia. During the dry, calcium sensitivity is reduced, whereas calcium dependence remains unchanged. Endogenous opioid peptides play an important role in the regulation of the physiological functions of active and dormant vertebrates. Previous findings suggest that dynorphin-A is more potent than other opiates in decreasing evoked neurotransmission in amphibian NMJs. Dynorphin-A has been shown not to alter the amplitude or the frequency of miniature quantal neurotransmitter release. In the present study, we report that dynorphin-A exerted a more pronounced inhibitory effect on evoked neurotransmitter release during the dry (hibernating period) when compared with the wet (active period) season. Dynorphin-A increased the frequency and decreased the amplitude of miniature neurotransmitter release only at relatively high concentration during the dry season. In the present study, we propose that dynorphin-A suppresses evoked neurotransmitter release and thus contraction of skeletal muscles, while allowing subthreshold activation of the NMJ by miniature neurotransmission, thus preventing any significant neuromuscular remodeling. The inhibitory effect of dynorphin-A on evoked transmitter release is reduced by increasing the extracellular calcium concentration.

Seasonal factors influence quantal transmitter release and calcium dependence at amphibian neuromuscular junctions.

Amphibian neuromuscular junctions (NMJs) are composed of hundreds of neurotransmitter release sites that exhibit nonuniform transmitter release probabilities and demonstrated seasonal modulation. We examined whether recruitment of release sites is variable when the extracellular calcium concentration ([Ca2+]o) is increased in the wet and dry seasons. The amount of transmitter released from the entire nerve terminal increases by approximately the fourth power as [Ca2+]o is increased. Toad (Bufo marinus) NMJs were visualized using 3,3'-diethyloxardicarbocyanine iodide [DiOC2(5)] fluorescence, and focal loose patch extracellular recordings were used to record the end-plate currents (EPCs) from small groups of release sites. Quantal content (m̄e ), average probability of quantal release (pe ), and the number of active release sites (ne ) were determined for different [Ca2+]o Our results indicated that the recruitment of quantal release sites with increasing [Ca2+]o differs spatially (between different groups of release sites) and also temporally (in different seasons). These differences were reflected by the nonuniform alterations in pe and ne Most release site groups demonstrated an increase in both pe and ne when [Ca2+]o increased. In ~30% of release site groups examined, pe decreased while ne increased only during the active period (wet season). Although the dry season induced parallel right shift in the quantal release versus extracellular calcium concentration when compared with the wet season, the dependence of quantal content on [Ca2+]o was not changed. These results demonstrate the flexibility, reserve, and adaptive capacity of neuromuscular junctions in maintaining appropriate levels of neurotransmission.

Nicotine enhances both excitatory and inhibitory synaptic inputs to inspiratory-activated airway vagal preganglionic neurons.

The airway vagal preganglionic neurons (AVPNs) supply the essential excitatory drive to the postganglionic neurons and dominate the neural control of the airway both physiologically and pathophysiologically. The AVPNs express multiple subunits of nicotinic acetylcholine receptors (nAChRs), but the influences of exogenous nicotine and endogenous acetylcholine are unknown. This study examined the effects of nicotine and endogenous acetylcholine on retrogradely labelled, functionally identified inspiratory-activated AVPNs (IA-AVPNs) using the patch-clamp technique. Nicotine (10 µmol l(-1)) significantly increased the frequency and amplitude of the spontaneous EPSCs of IA-AVPNs, and these effects were insensitive to methyllycaconitine (MLA, 100 nmol l(-1)), an antagonist of the α7 type of nAChR, but was prevented by dihydro-ß-erythroidine (DHßE, 3 µmol l(-1)), an antagonist of the α4ß2 type of nAChR. Nicotine caused a tonic inward current in IA-AVPNs, which was reduced by MLA or DHßE alone, but was not abolished by co-application of MLA and DHßE. Nicotine caused a significant increase in the frequency of GABAergic and glycinergic spontaneous IPSCs and significantly increased the amplitude of glycinergic spontaneous IPSCs, all of which were prevented by DHßE. Nicotine had no effects on the miniature EPSCs or miniature IPSCs following pretreatment with TTX. Under current clamp, nicotine caused depolarization and increased the firing rate of IA-AVPNs during inspiratory intervals. Neostigmine (10 µmol l(-1)), an acetylcholinesterase inhibitor, mimicked the effects of nicotine. These results demonstrate that nicotine and endogenous ACh enhance the excitatory and inhibitory synaptic inputs of IA-AVPNs and cause a postsynaptic excitatory current and that the nicotinic effects are mediated presynaptically by activation of the α4ß2 type of nAChR and postsynaptically by activation of multiple nAChRs, including α7 and α4ß2 types.

Influence of cannabinoids upon nerve-evoked skeletal muscle contraction.

Endocannabinoids play important roles in regulating CNS synaptic function and peripheral metabolism, but cannabinoids can also act acutely to modulate contraction strength in skeletal muscle. Nerve terminals and the skeletal muscle sarcolemma express components of the cannabinoid signaling system. Endocannabinoids, N-arachidonylethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2-AG), are produced by skeletal muscle. They may be involved in the acute regulation of neuromuscular transmission, by adjusting the parameters for quantal acetylcholine release from the motor nerve terminal. Downstream of neuromuscular transmission, cannabinoids may also act to limit the efficiency of excitation-contraction coupling. Improved understanding of the distinct signaling actions of particular cannabinoid compounds and their receptor/transduction systems will help advance our understanding of the role of endocannabinoids in skeletal muscle physiology. Cannabinoids might also offer the potential to develop new pharmacotherapeutics to treat neuromuscular disorders that affect muscle strength.

What are Neurotransmitter Release Sites and Do They Interact?

It has long been known that each neuron in both the central and peripheral nervous system has a large number of active zones. Nonetheless, how active zones are regulated to maintain a homeostatic release state and response to the constantly changing environment remains poorly understood. Due to its relatively simple structure and easy accessibility, the neuromuscular synapse (NM-synapse) continues to be used as a model synapse to examine the basic nature of synaptic neurotransmission. In the NM-synapse, quantal neurotransmitter release can occur spontaneously or triggered by invading nerve impulses. Past research has indicated that some active zones tend to be involved more with spontaneous quantal release than evoked quantal release. Furthermore, evoked quantal release has been shown to be highly non-uniform between active zones along nerve terminal branches. How these large numbers of active zones along the same nerve terminal are functionally correlated remains unclear. This review starts with the basic features of quantal neurotransmitter release, then progresses to the current knowledge on how the active zones interact with each other along the same nerve terminal.

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus.

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February-March) when these toads are active, to toads obtained from dry season (October-November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.

Activation of α1-adrenoceptors facilitates excitatory inputs to medullary airway vagal preganglionic neurons.

In mammals, the neural control of airway smooth muscle is dominated by a subset of airway vagal preganglionic neurons in the ventrolateral medulla. These neurons are physiologically modulated by adrenergic/noradrenergic projections, and weakened α2-adrenergic inhibition of them is indicated to participate in the pathogenesis and exacerbation of asthma. This study tests whether these neurons are modulated by α1-adrenoceptors, and if so, how. In anesthetized adult rats, microinjection of the α1A-adrenoceptor agonist A61603 (1 pmol) unilaterally into the medullary region containing these neurons caused a significant increase in airway resistance, which was prevented by intraperitoneal atropine (0.5 mg/kg). In rhythmically firing medullary slices of newborn rats, A61603 (10 nM) caused depolarization in both the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons that were retrogradely labeled, and a significant increase in the spontaneous firing rate. Under voltage clamp, A61603 significantly enhanced the spontaneous excitatory inputs to both types of neurons and caused a tonic inward current in the inspiratory-activated neurons along with significantly increased peak amplitude of the inspiratory inward currents. The responses in vitro were prevented by α1A-adrenoceptor antagonist RS100329 (1 µM), which alone significantly inhibited the spontaneous excitatory inputs to both types of the neurons. After pretreatment with tetrodotoxin (1 µM), A61603 (10 or 100 nM) had no effect on either type of neuron. We conclude that in rats, activation of α1-adrenoceptors in the medullary region containing airway vagal preganglionic neurons increases airway vagal tone, and that this effect is primarily mediated by facilitation of the excitatory inputs to the preganglionic neurons.