SUMMARY1. The depression of synaptic transmission, which occurs during prolonged repetitive activation, was examined in the opener muscle of the crayfish walking leg.2. Excitatory post-synaptic potentials (e.p.s.p.s) initially facilitated but then declined to low amplitudes after about 4000 stimulus pulses had been delivered; this depression is presynaptic in origin.3. Axon conduction blocks occurred at points of bifurcation along the entire length of the presynaptic nerve. This resulted in failure of the nerve impulse to invade some branches of the terminal arborization.4. Nerve terminal invasion failure caused either intermittent or complete inactivation of some synaptic release sites; this was associated with depression of the post-synaptic response.5. The statistics of transmitter release during prolonged repetitive stimulation were examined by focal extracellular recording methods. Transmitter release could be described by binomial statistics, and depression involved a drop in m, n and p.6. The rate of spontaneous quantal release did not decrease, however, arguing against transmitter depletion.7. It is concluded that repetitivestimulationeventuallyleads to depolarization of the axon membrane. This causes impulse propagation failure which reduces the number of synaptic release sites that are activated and mimics a drop in the effective stimulation rate; both effects cause synaptic depression.

SUMMARY1. After blocking K+ currents with 10 mM-tetraethylammonium (TEA) or TEA plus 250 /tM-3,4-diaminopyridine (3,, motor nerve terminal Ca2+ currents were recorded using focal extracellular electrodes. Two transmitters released from the terminal, ATP and acetylcholine (ACh), were then applied, and the effects on the nerve terminal Ca2+ current were measured.2. ATP (50 /IM) reduced the Ca2+ current by 34%, but this action is prevented when hydrolysis to adenosine is blocked by a,fi-methyladenosine 5'-diphosphate (200 /tM). Thus, inhibition by ATP presumably occurs subsequent to ATP hydrolysis to adenosine.3. Adenosine (50,UM) inhibited the terminal Ca2+ current by 29%. This was mimicked by the adenosine analogue L-phenylisopropyl adenosine (L-PIA) and blocked by theophylline (100 ,LM), which antagonizes adenosine receptors at micromolar concentrations.4. ACh (100,tM) or the anticholinesterase methane sulphonyl fluoride (MSF; 1 mm) also depressed the terminal Ca2+ current. This response was mimicked by muscarine (100 ftM) and antagonized by atropine (100 1uM) or pirenzipine (4/,M), which is generally specific for M1 receptors.5. Addition of Ba2+, which blocks adenosine-mediated K+ currents, had no effect on the inhibitory effects of either adenosine or ACh; similarly, neither adenosine nor ACh in the bath affected K+ current records obtained after blocking all inward currents with 10 mM-Co2+ and focal application of tetrodotoxin.6. Incubation of the muscle for 4 h in pertussis toxin (10-5 g ml-) eliminated both adenosine-and ACh-induced inhibition of the terminal Ca2+ current. This result indicates the possible involvement of a G protein in the transduction of the feedback pathway.7. Neither cyclic AMP analogues, the adenylate cyclase activator forskolin (10 ftM), the phorbol ester phorbol 12-myristate 13-acetate (PMA; 3,M) nor the diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG; 3 /M) had any effect on adenosine-or ACh-induced depression of the terminal Ca2+ current. Therefore, * To whom reprint requests should be addressed. MS 8023B. R. HAMILTON AND D. 0. SMITH pathways involving these particular second messengers are most probably not involved.8. The effects of adenosine and ACh are non-additive. 9. These results indicate that ATP and ACh, which are released during exocytosis, may inhibit their own release through attenuation of the terminal Ca2+ current via autoreceptors coupled to a G protein.

SUMMARY1. The phenomena leading to action potential conduction block during repetitive stimulation of the excitor axon of the opener muscle in the crayfish walking leg were studied.2. Action potentials, recorded extracellularly with micro-electrodes, failed to propagate past sites of axonal bifurcation following at least 3000 impulses; reduction of the rate or brief cessation of stimulation resulted in restored conduction.3. Failure occurred initially at branch points located most peripherally and then more centrally as stimulation continued; this centripetal progression of the site of block resulted in a stepwise reduction of the number of synaptic terminals from which transmitter was released.4. Prior to conduction failure, the conduction velocity and the sodium inward current of the action potentials decreased.5. Local application of hyperpolarizing current or of physiological saline with low [K+] in the vicinity of a block can restore propagation; thus depolarization of the membrane most probably causes failure.6. Soaking the preparation for as long as 2 hr in the metabolic inhibitor 2,4-dinitrophenol had no effect on the number of stimulus impulses before initial conduction block; however, the time required for recovery from the failure was prolonged.7. The number of impulses prior to block was related directly to the temperature of the preparation; this had a Q10 calculated to be about 1*3.8. It is suggested that during repetitive activity, the K + gradient across the membrane is reduced, resulting in depolarization and eventually in conduction failure.

Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.

SUMMARY1. The levels of adenine nucleotides and adenosine which accumulate in the neuromuscular junction during nerve stimulation of the rat extensor digitorum longus (EDL) muscle were assayed biochemically. The sources were also determined by the use of different inhibitors.2. ATP and total adenine nucleotide release increased as stimulation frequency increased, consistent with previous evidence indicating ATP release from presynaptic sources.3. Adenosine levels also increased during nerve stimulation. However, accumulation decreased by 46-58 % when muscle activation was blocked by the addition of d-tubocurarine (dTC). Adenosine levels also decreased by 40-59% when adenine nucleotide hydrolysis to adenosine was blocked by the addition of 1 mM-X,fmethyladenosine 5'-diphosphate. Thus, approximately half of the extracellular adenosine is released from activated muscle while the other half is derived from adenine nucleotide hydrolysis.4. Similar quantities of adenine nucleotide and acetylcholine (ACh) accumulated during nerve stimulation. With adenine nucleotide and ACh hydrolysis blocked by a,,-methyladenosine 5'-diphosphate and eserine, respectively, the calculated amounts of adenine nucleotide and ACh released were 1-2 x 10-18 and 1 5 x 101 mol (stimulus impulse)-' endplate-1.5. AH5183 (vesamicol), which blocks ACh release, reduced extracellular ACh and adenine nucleotide accumulation by 40 and 45%, respectively. It did not affedt adenosine release from the activated muscle.6. Theophylline (100,M), which blocks adenosine receptors, caused ATP accumulation to increase by 38 %; extracellular levels of adenosine derived from adenine nucleotide hydrolysis also increased by 17 %. These results are consistent with the presence of adenosine-mediated inhibition of adenine nucleotide release.7. It is concluded that adenine nucleotides (presumably in the form of ATP) and ACh are released jointly, and that ATP is hydrolysed fairly rapidly to adenosine. Adenosine resulting from ATP hydrolysis accounts for about half of the extracellular adenosine accumulating during nerve stimulation, while the other half is released directly by the underlying muscle.