Excitation of rat hippocampal interneurons via modulation of endogenous agonist activity at the α7 nicotinic ACh receptor

The α7 subtype of the nicotinic acetylcholine receptor (α7 nAChR) is prominently expressed in the hippocampus where it is thought to play a role in the regulation of cognitive function. In this study, we have investigated the effects of 5-hydroxyindole (5-HI), a positive modulator of the α7 nAChR, on GABAergic activity in hippocampal CA1 stratum radiatum interneurons in acute rat brain slices. Superfusion of 5-HI (100 μ m ) increased the mean frequency and amplitude of spontaneous IPSCs (sIPSCs). The potentiation was occluded by pretreatment of slices with: (1) a high concentration of the broad-spectrum agonist nicotine to desensitize the α7 receptor, (2) an α7 nAChR antagonist, and (3) tetrodotoxin to block action potential firing. These results indicate that facilitation by 5-HI was mediated by the α7 nAChR and required neuronal excitation. In contrast, 5-HI had no effect on sIPSCs recorded in hippocampal slices from younger animals, even though the expression of functional α7 nAChRs was confirmed by agonist application experiments. In these slices, 5-HI only enhanced sIPSCs after pretreatment with the acetylcholinesterase inhibitor Bw284c51. Taken together, our results suggest that 5-HI facilitates GABAergic transmission via excitation of the α7 nAChR, and that this effect requires the presence of the endogenous agonist ACh in the extracellular environment of the receptor.     

Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca2+ signal by local application or perfusion of ACh at the mouse neuromuscular junction

Local application of acetylcholine (ACh; 0.3 mM, 20 microl) elicited bi-phasic elevation of intracellular Ca2+ concentrations (contractile fast and non-contractile slow Ca2- signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 microM), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 microM), nitrendipine (1 microM), verapamil (1 microM) and diltiazem (1 microM), or a Na+ channel blocker tetrodotoxin (TTX; 0.1 microM) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 microM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 microM) also elicited a slow Ca2+ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 microM) antagonized against the ACh perfusion-elicited slow Ca2+ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.     

Nitric oxide, cAMP and the biphasic muscarinic modulation of ACh release at the lizard neuromuscular junction

In this study, we characterized the pharmacology and physiology of the automodulation of ACh release at the lizard neuromuscular junction (NMJ). The activation of muscarinic ACh receptors generated a biphasic modulation of synaptic transmission. Muscarine-induced activation of M₃ receptors (0–12 min) decreased release, whereas M₁ activation (> 12 min) enhanced release. Both phases of the biphasic effect are dependent on nitric oxide. However, cAMP acting via protein kinase A is also necessary for the M₁ effect. In summary, we present a novel biphasic role for muscarine and implicate M₃ receptors in the inhibition and M₁ receptors in the enhancement of transmitter releaseat the cholinergic lizard NMJ.     

Increased ratio of rapsyn to ACh receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse

The metabolic turnover of nicotinic ACh receptors (AChR) at the neuromuscular synapse is regulated over a tenfold range by innervation status, muscle electrical activity and neural agrin, but the downstream effector of such changes has not been defined. The AChR-associated protein rapsyn is essential for forming AChR clusters during development. Here, rapsyn was tagged with enhanced green fluorescent protein (EGFP) to begin to probe its influence at the adult synapse. In C2 myotubes, rapsyn–EGFP participated with AChR in agrin-induced AChR cluster formation. When electroporated into the tibialis anterior muscle of young adult mice, rapsyn–EGFP accumulated in discrete subcellular structures, many of which colocalized with Golgi markers, consistent with the idea that rapsyn assembles with AChR in the exocytic pathway. Rapsyn–EGFP also targeted directly to the postsynaptic membrane where it occupied previously vacant rapsyn binding sites, thereby increasing the rapsyn to AChR ratio. At endplates displaying rapsyn–EGFP, the metabolic turnover of AChR (labelled with rhodamine-α-bungarotoxin) was slowed. Thus, the metabolic half-life of receptors at the synapse may be modulated by local changes in the subsynaptic ratio of rapsyn to AChR.     

Chronic corticosterone treatment-induced ultrastructural changes at rat neuromuscular junction

Background: Chronic exposure to glucocorticoids affects both the structure and function of vertebrate skeletal muscles. As little is known about the effects of such steroids on the neuromuscular junctions (NMJs) of different muscle fiber types, the influence of chronic corticosterone (CORT) administration on the ultrastructure of NMJs of soleus (SOL) and extensor digitorum longus (EDL) was studied. Methods: Ten Fischer 344 male rats, the same animals used previously, were either injected daily with 5–10 mg CORT or received vehicle as control animals for 3 months and were sacrificed at 5 months of age. Muscles were bathed in situ in 4% phosphate buffered glutaraldehyde for ten minutes, then removed and conventional electron microscopic procedures were followed. Qualitative and quantitative observations of nerve terminal ultrastructures were statistically treated with multivariate analysis of variance to determine differences between control and CORT-treated animals. Results: Fast-twitch EDL muscles were more affected by CORT-treatment than slow-twitch SOL muscles. Morphometric analysis of NMJ's in CORT-treated rats revealed significant decrease in fiber diameter, nerve terminal area and synaptic vesicle density, but a significant increase in synaptic cleft (P<0.05). The NMJ's underwent partial denervation and reinnervation processes as demonstrated by large areas of presynaptic nerve terminal occupied by microtubules and electron dense granular material. Conclusions: Chronic CORT-treatments induced degenerative changes which were more pronouced in fast-twitch EDL muscles than slow-twitch SOL muscles, suggesting that pattern or amount of activity affect the CORT-treatment outcome. These steriod-induced stress changes are similar to those observed in aging and disuse studies of NMJ. Thus, glucocorticoid hormones may play an etiological role in the homeostasis of the NMJ in response to various stimuli. © 1995 Wiley-Liss, Inc.     

Independent release of supranormal acetylcholine quanta at the rat neuromuscular junction

This electrophysiological study deals with the occurrence and with the mode of release of unusually large miniature end-plate potentials at the rat neuromuscular junction during physiological conditions. A specific limit for the normal miniature end-plate potential amplitude at each cell studied was determined after fitting the observed frequency-amplitude histogram to a Gaussian distribution. The relative abundance of giant miniature end-plate potentials was 4.15% at room temperature. The occurrence of giant miniature end-plate potentials was temperature dependent. The percentage of giant miniature end-plate potentials was 5.8% and 0.61% at 35 degrees C and at 16 degrees C, respectively. The amplitude-independence of the intervals between miniature end-plate potentials was demonstrated at room temperature as well as at 35 degrees C and at 16 degrees C. The results of this study show that giant miniature end-plate potentials are produced by acetylcholine packets which are released independently and that they are not a consequence of the synchronous release of several normal-sized quanta. Moreover, the results indicate that during physiological conditions a minor but regular proportion of the spontaneous release of acetylcholine is made up of larger packets, which produce miniature end-plate potentials of supranormal amplitude.     

Substance P preferentially inhibits large conductance nicotinic ACh receptor channels in rat intracardiac ganglion neurons

The effects of substance P (SP) on nicotinic acetylcholine (ACh)-evoked currents were investigated in parasympathetic neurons dissociated from neonatal rat intracardiac ganglia using standard whole cell, perforated patch, and outside-out recording configurations of the patch-clamp technique. Focal application of SP onto the soma reversibly decreased the peak amplitude of the ACh-evoked current with half-maximal inhibition occurring at 45 microM and complete block at 300 microM SP. Whole cell current-voltage (I-V) relationships obtained in the absence and presence of SP indicate that the block of ACh-evoked currents by SP is voltage independent. The rate of decay of ACh-evoked currents was increased sixfold in the presence of SP (100 microM), suggesting that SP may increase the rate of receptor desensitization. SP-induced inhibition of ACh-evoked currents was observed following cell dialysis and in the presence of either 1 mM 8-Br-cAMP, a membrane-permeant cAMP analogue, 5 microM H-7, a protein kinase C inhibitor, or 2 mM intracellular AMP-PNP, a nonhydrolyzable ATP analogue. These data suggest that a diffusible cytosolic second messenger is unlikely to mediate SP inhibition of neuronal nicotinic ACh receptor (nAChR) channels. Activation of nAChR channels in outside-out membrane patches by either ACh (3 microM) or cytisine (3 microM) indicates the presence of at least three distinct conductances (20, 35, and 47 pS) in rat intracardiac neurons. In the presence of 3 microM SP, the large conductance nAChR channels are preferentially inhibited. The open probabilities of the large conductance classes activated by either ACh or cytisine were reversibly decreased by 10- to 30-fold in the presence of SP. The single-channel conductances were unchanged, and mean apparent channel open times for the large conductance nAChR channels only were slightly decreased by SP. Given that individual parasympathetic neurons of rat intracardiac ganglia express a heterogeneous population of nAChR subunits represented by the different conductance levels, SP appears to preferentially inhibit those combinations of nAChR subunits that form the large conductance nAChR channels. Since ACh is the principal neurotransmitter of extrinsic (vagal) innervation of the mammalian heart, SP may play an important role in modulating autonomic control of the heart.     

Safety factor at the neuromuscular junction

Reliable transmission of activity from nerve to muscle is necessary for the normal function of the body. The term 'safety factor' refers to the ability of neuromuscular transmission to remain effective under various physiological conditions and stresses. This is a result of the amount of transmitter released per nerve impulse being greater than that required to trigger an action potential in the muscle fibre. The safety factor is a measure of this excess of released transmitter. In this review we discuss the practical difficulties involved in estimating the safety factor in vitro. We then consider the factors that influence the safety factor in vivo. While presynaptic transmitter release may be modulated on a moment to moment basis, the postsynaptic features that determine the effect of released transmitter are not so readily altered to meet changing demands. Different strategies are used by different species to ensure reliable neuromuscular transmission. Some, like frogs, rely on releasing a large amount of transmitter while others, like man, rely on elaborate postsynaptic specialisations to enhance the response to transmitter. In normal adult mammals, the safety factor is generally 3-5. Both pre- and postsynaptic components change during development and may show plasticity in response to injury or disease. Thus, both acquired autoimmune and inherited congenital diseases of the neuromuscular junction (NMJ) can significantly reduce, or even transiently increase, safety factor.     

Different effects of types A and B botulinum toxin on transmitter release at the rat neuromuscular junction

Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either crystalline type A botulinum toxin or purified type B botulinum neurotoxin. At 1, 3, 5 and 7 days after injection, the extensor digitorum longus nerve-muscle preparation was excised and analyzed in vitro for alterations in spontaneous and nerve stimulus-evoked quantal transmitter release. Muscles receiving type A toxin were paralyzed up to and including 7 days after injection. Muscles treated with type B toxin, although completely paralyzed at 1 and 3 days, twitched in response to nerve stimulation at 5 and 7 days after injection. Both toxins induced a marked decrease in the frequency of miniature endplate potentials but type A did so to a greater extent. The remaining population of miniature endplate potentials contained a greater frequency of potentials with small or large amplitudes and prolonged rise times compared to normal muscle. These changes were more pronounced with type A toxin than with type B toxin. In the presence of alpha-dinitrophenol (1 mM), high frequency, fast-rising miniature endplate potentials of uniform size reappeared. High K+ (20 mM) was less effective in this respect. At 3 days after toxin injection nerve impulse evoked transmitter release was reduced more for type A treated muscles than for type B. However, 3,4-diaminopyridine, an agent which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type B-treated muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prejunctional effects of anticholinesterase drugs at the endplate

Anticholinesterase drugs induce antidromic firing of motor axons in mammalian nerve-muscle preparations. Antidromic firing is observed in response to a conditioning stimulus applied to the nerve and hence is known as back-firing; the present investigation intends to clarify the mechanisms underlying this phenomenon. Acetylcholinesterase (AChE) was inhibited with neostigmine. Antidromic action potentials were recorded extracellularly from the nerve trunk. When the muscle was cut on one side of the main endplate region 0.1–0.2 cm away from the outermost myelinated nerve branches, back-firing gradually disappeared. This indicated that postsynaptic factors at least contribute to the generation of back-firing. One can conceive of a plausible mechanism whereby postsynaptic events influence presynaptic excitability: potassium ions leaking from extensively depolarized muscle fibers (block of AchE) might cause depolarization of nerve endings at some endplates (potassium hypothesis). Postsynaptic potassium currents were calculated according to a simple mathematical model of the muscle membrane. These simulations predict high postsynaptic potassium currents under the following experimental conditions: (1) low concentration of extracellular potassium, (2) exchange of extracellular chloride with nitrate, (3) small fiber diameter. Back-firing was found to increase under such conditions. The notion that postsynaptic potassium efflux may influence presynaptic membrane polarization was further substantiated: when carbamoylcholine chloride (Carbachol; 10 mol · 1^–1) was added to the bathing fluid, miniature endplate current frequency could be increased at some voltage clamped endplates by depolarizing the muscle fiber well below the potassium equilibrium potential.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38, project N     

Voltage dependence of agonist effectiveness at the frog neuromuscular junction: resolution of a paradox.

1. End-plate currents produced by nerve-released acetylcholine and iontophoretically applied acetylcholine and carbachol have been recorded from voltage-clamped frog cutaneous pectoris neuromuscular junctions made visible with Nomarski differential interference contrast optics. 2. The effectiveness of agonists - that is, the end-plate conductance change produced by a given dose-has been determined as a function of post-junctional membrane potential. 3. As the post-junctional membrane potential is made more negative, nerve-released acetylcholine becomes less effective whereas iontophoretically-applied agonists become more effective. 4. This voltage dependence of agonist effectiveness is mediated neither by end-plate current iontophoresis of agonist into the cleft nor through electric field effects on the esterase. 5. Influences of membrane potential on the opening and closing of end-plate channel gates can account quantitatively for the voltage-dependent effectiveness of both nerve-released and iontophoretically applied agonist.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

Actions of polypeptides at the neuromuscular junction

The effects of several polypeptides, e.g. angiotensin II, substance P, oxytocin and vasopressin, on the isolated frog gastrocnemius, chick biventer cervicis and rat hemodiaphragm preparations were studied using electrophysiological and neurochemical techniques. The effects of angiotensin II, substance P, oxytocin and vasopressin on neuromuscular transmission and muscle contraction were investigated by studying the following parameters:

1. the directly and indirectly-elicited twitch and tetanic contractions,
2. nerve compound action potential,
3. uptake of³H-methylcholine into nerve-muscle preparations,
4. the contractures produced by depolarizing drugs, e.g. ACh or TEA.

The results showed that angiotensin II (10^?10–10^?6 M) and substance P (10^?7–10^?6 M) enhanced neuromuscular transmission and muscle contraction by increasing the amplitudes of the indirectly-elicited twitch and tetanic contractions. Oxytocin and vasopressin (1–100 mU/ml^?1) both depressed neuromuscular transmission by reducing the contractile and electrical response in the frog, chick and rat skeletal muscle. It was concluded that, like their effects on ganglionic transmission, the peptides can modify neuromuscular transmission. The mechanism by which these peptides produce their effects may be dependent on external calcium concentration. These peptides may affect both pre- and postjunctional mechanisms; prejunctionally by increasing/decreasing the release of ACh, and postjunctionally by affecting the sensitivity of the postjunctional membrane to depolarizing drugs and/or producing a contracture in the skeletal muscle.     

Inhibition of acetylcholinesterase accelerates axon terminal withdrawal at the developing rat neuromuscular junction

Summary In developing skeletal muscles, the rate at which superfluous innervation is lost from the endplates depends on the general level of neuromuscular activity. Whether it is activity of the presynaptic or postsynaptic structures (or both) that is critical is not well established. In this work, we transitorily inhibited the AChE of soleus muscle in postnatal rats, in order to increase postsynaptic activity, without directly altering activity of the nerve terminals. We then followed the time course of disappearance of axon terminals from the endplates of treated and normal muscles, using electron-microscope techniques. Three hours after inhibition of AChE, the muscle fibres exhibited local supercontracture and ultrastructural damage in the region of the endplate, consistent with local elevation of Ca²⁺ levels. At the same time, small electron-opaque vesicles, apparently of muscular origin, appeared in the synaptic cleft. The nerve terminals, however, were entirely normal in number and appearance. One day after treatment, endplates of esteraseinhibited muscles showed accelerated loss of nerve terminals, compared to endplates of normally developing muscles. No further loss of nerve terminals occurred, once AChE activity returned at the endplate. These results suggest that the rate at which superfluous nerve terminals retract from the developing neuromuscular junction is regulated by the level of activation of the muscle. It seems likely that activity of postsynaptic sites may similarly regulate changes in innervation patterns, in other developing or adapting neuro-neuronal or neuro-effector systems.