Ex vivo expansion of cord blood.

A marked increase in the utilization of umbilical cord blood (UCB) transplantation has been observed in recent years; however, the use of UCB as a hematopoietic stem cell (HSC) source is limited primarily by the number of progenitor cells contained in the graft. Graft failure, delayed engraftment and profound delay in immune reconstitution lead to significant morbidity and mortality in adults. The lack of cells available for post transplant therapies, such as donor lymphocyte infusions, has also been considered to be a disadvantage of UCB. To improve outcomes and extend applicability of UCB transplantation, one potential solution is ex vivo expansion of UCB. Investigators have used several methods, including liquid suspension culture with various cytokines and expansion factors, co-culture with stromal elements and continuous perfusion systems. Techniques combining ex vivo expanded and unmanipulated UCB are being explored to optimize the initial engraftment kinetics as well as the long-term durability. The optimal expansion conditions are still not known; however, recent studies suggest that expanded UCB is safe. It is hoped that by ex vivo expansion of UCB, a resulting decrease in the morbidity and mortality of UCB transplantation will be observed, and that the availability of additional cells may allow adoptive immunotherapy or gene transfer therapies in the UCB setting.

Peripheral placement of apheresis catheters in children: feasibility, safety, and efficacy in the collection of blood stem cells--initial experience.

An 8-F 24-cm-long apheresis catheter was placed in the basilic vein with imaging-guided percutaneous technique in 15 children undergoing leukapheresis for collection of autologous peripheral blood stem cells. There were no immediate or long-term complications. This is a low-morbidity procedure requiring minimal sedation that results in successful collection of peripheral blood stem cells and allows flow rates comparable to those with surgically placed central catheters.

A comparison of the electrophysiological effects of two organophosphates, mipafox and ecothiopate, on mouse limb muscles.

Adult male albino mice were given single subcutaneous injections of either mipafox (110 mumol/kg) or ecothiopate (0.5 mumol/kg), two organophosphorus compounds (OPs). Acetylcholinesterase activity was measured in the soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles. At 7 and 28 days after dosing, in vitro electrophysiological measurements were carried out in the soleus and EDL. Action potentials and end-plate potentials were evoked at 30 Hz and recorded intracellularly from single muscle fibers. The amplitudes, time course, and latencies of these potentials were measured and the variability (jitter) of latencies was calculated. Recordings after mipafox were also made with 3-Hz stimulation. Acetylcholinesterase activity was inhibited by mipafox (65% in the soleus; 76% in the EDL) and ecothiopate (59% in the soleus; 42% in the EDL). Mipafox and ecothiopate both increased postjunctional (muscle action potential) jitter in the soleus and EDL at 7 days after dosing. Organophosphates caused an increase in end-plate potential amplitudes in the soleus. Mipafox caused an increase in prejunctional (end-plate potential) jitter at 28 days after dosing in both muscles. A single dose of ecothiopate also caused an increase in prejunctional jitter at 28 days in the soleus. The OP-induced increase in jitter was different at different frequencies of stimulation. The results show that there are electrophysiological changes in both muscles after administration of organophosphorus compounds. The slow-twitch soleus appears more sensitive to prejunctional changes caused by OPs than the fast-twitch EDL.

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning.

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Determination of diarrheic shellfish toxins in mussels by microliquid chromatography-tandem mass spectrometry.

A fast, sensitive, and specific procedure for determining toxins that cause diarrheic shellfish poisoning (DSP) using microliquid chromatography coupled with tandem mass spectrometry (micro-LC-MS-MS) is reported. The lipophylic polyether acidic toxins okadaic acid (OA), its isomer dinophysistoxin-2 (DTX-2), the 35-methylokadaic acid dinophysistoxin-1 (DTX-1), and the novel toxin dinophysistoxin-1B (DTX-2B; recently isolated from Irish mussels) were extracted from shellfish tissues with acetone and chromatographed by isocratic elution at 10 microL/min with CH3 CN-H2O, 80 + 20 (v/v), containing 0.1% trifluoroacetic acid, through a C18 reversed-phase column (1.0 mm id). The chromatograph is coupled via an ion spray interface to an atmospheric pressure ionization source. Collision-induced-dissociation (CID) ion mass spectra of the protonated molecule, [M + H]+, at m/z 805 for OA, DTX-2, and DTX-2B and at m/z 819 for DTX-1, were obtained in MS-MS experiments to identify 2 diagnostic fragment ions for each analyte that could be used for selected-reaction-monitoring (SRM) micro-LC-MS-MS analysis. The CID spectrum of DTX-2B confirmed it to be a new OA isomer, like DTX-2. Standard curves obtained by SRM micro-LC-MS-MS were linear (r2 > or = 0.9992) over the range 0.05-1.00 micrograms/mL (i.e., 0.10-2.00 micrograms toxin/g hepatopancreas), and a detection limit of 15 pg/injection was obtained for each DSP toxin. Average recoveries ranged from 95 to 101%, and coefficients of variation ranged from 1.8 to 3.4%. This novel SRM micro-LC-MS-MS method was used to confirm acidic DSP toxins in Irish and Italian toxic mussels. It offers a high degree of specificity because analyte confirmation is based on retention time, molecular weight, structural information obtained from the presence of 2 diagnostic fragments for each analyte, and ion ratios. OA was found in both Irish (< or = 0.7 micrograms/g hepatopancreas) and Italian (< or = 1.5 micrograms/g hepatopancreas) mussels. DTX-1 was found only in Italian mussels (< or = 0.3 micrograms/g hepatopancreas). DTX-2 (< or = 6.1 micrograms/g hepatopancreas) and DTX-2B (< or = 0.08 micrograms/hepatopancreas) were unique to Irish shellfish.

Identification of a new diarrhoetic toxin in shellfish using liquid chromatography with fluorimetric and mass spectrometric detection.

A new toxin, dinophysistoxin-2B (DTX-2B) was isolated from Irish mussels using silica chromatography, gel permeation, octadecylsilane solid-phase extraction and repeated preparative high-performance liquid chromatography (HPLC). Dinophysistoxin-2 (DTX-2) was also isolated from shellfish using the same procedures. The separation of these toxins in chromatographic fractions was monitored using fluorimetric HPLC following derivatization with 9-anthrylmethyldiazomethane or 1-bromoacetylpyrene. Flow-injection analysis-mass spectrometry (FIA-MS) with an atmospheric pressure ionization (API) and an ionspray (ISP) interface showed a mass spectrum dominated by the protonated molecule, [M+H]+, at m/z 805 for DTX-2B, thus indicating that this new toxin has the same mol.wt as okadaic acid and DTX-2. The low-energy fragment ion spectrum, as produced in FIA-MS experiments by up-front collision-induced dissociation of the protonated molecule of DTX-2B, showed fragment ions corresponding to successive losses of water molecules from the [M+H]+ ion. This low collision energy fragmentation pattern is typical of marine polyether toxins such as okadaic acid, DTX-2 and DTX-1. These results provide strong evidence that DTX-2B is another okadaic acid isomer.

The effects of multiple low doses of organophosphates on target enzymes in brain and diaphragm in the mouse.

1. Multiple low doses of the direct acting organophosphates, ecothiopate, paraoxon and mipafox produced persistent and additive inhibition of diaphragm acetylcholinesterase. Paraoxon and mipafox had similar effects on brain acetylcholinesterase. There was greater recovery from inhibition between doses for paraoxon and ecothiopate than for mipafox. 2. Ecothiopate did not inhibit brain acetylcholinesterase but there was a small increase in activity. 3. Mipafox also had a cumulative inhibitory effect on brain neuropathy target esterase. 4. These results have particular implication for the use of multiple low doses of organophosphates occupationally by man.

Effects of multiple doses of organophosphates on evoked potentials in mouse diaphragm.

1. Male albino mice were injected s.c. with an organophosphate (mipafox, ecothiopate or paraoxon). Treatments were either a single injection or multiple daily injections with lower doses for 5 or 8 days. At 3 h after injection the activity of brain and diaphragm acetylcholinesterase and of brain neuropathy target esterase (NTE) was measured. Also measured in the diaphragm at 3 h post dose was the duration of spontaneous miniature endplate potentials (eMEPPs), recorded extracellularly. 2. At 7 and 28 days after dosing action potentials and evoked endplate potentials, produced by stimulating the phrenic nerve at 30 Hz, were recorded in diaphragm muscle. The amplitudes, time-course and latencies of these potentials were measured and the variability of latencies (jitter) was calculated. 3. Single doses of mipafox (20 mg/kg), ecothiopate (0.192 mg/kg) or paraoxon (0.415 mg/kg) in the mouse produced ca. 70% inhibition of diaphragm acetylcholinesterase at 3 h after dosing. All three OPs produced a prolongation of the half-decay times of eMEPPs. 4. All three OPs in the above single doses produced increased muscle action potential (postjunctional) jitter but only mipafox produced an increase in endplate potential (prejunctional) jitter. Mipafox in a slightly reduced single dose (17.5 mg/kg) had no effect on prejunctional or postjunctional jitter. 5. Multiple dosing with mipafox (8 mg/kg daily for 5 days) increased both postjunctional and prejunctional jitter at both 7 and 28 days after the end of dosing. After multiple dosing with mipafox (5 mg/kg daily for 5 days) postjunctional (but not prejunctional) jitter was increased. Multiple doses of paraoxon (0.166 mg/kg daily for 5 days) or ecothiopate (0.76 mg/kg daily for 5 days) increased prejunctional and postjunctional jitter. 6. Depending on the dosing regime, all three OPs tested were capable of increasing both prejunctional and postjunctional jitter. Neither ecothiopate nor paraoxon inhibited NTE, so this prejunctional effect is not likely to be related to 'classical' OP-induced delayed neuropathy. The prejunctional effects may be related to long-term inhibition of acetylcholinesterase and the triggering mechanism for increase in prejunctional jitter may involve a relationship between the inhibition of acetylcholinesterase and the time for which it is inhibited. The differences between the time-courses of increases in prejunctional and postjunctional jitter and the differential effects of the different multiple dosing regimes indicate that it is likely that the triggering relationship between enzyme inhibition and time is different for prejunctional and postjunctional effects.

Isolation of dinophysistoxin-2 and the high-performance liquid chromatographic analysis of diarrhetic shellfish toxins using derivatisation with 1-bromoacetylpyrene.

The rare diarrhetic shellfish toxin, dinophysistoxin-2 (DTX-2), was isolated from the digestive glands of mussels (Mytilus edulis). This was achieved by chromatography on silica and Sephadex LH-20 followed by reversed-phase solid phase extraction and semi-preparative high-performance liquid chromatography (HPLC) with an Ultremex C18 column. Using 1-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, the diarrhetic shellfish toxins, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and DTX-2, were determined by HPLC with fluorimetric detection. Derivatisation using BAP was compared with 9-anthryldiazomethane (ADAM) and, although the latter exhibited a four-fold better sensitivity, the BAP method gave fewer artefact peaks from reagent decomposition. The limits of detection of OA and DTX-2 were 0.4 ng on-column using BAP, which permits this method to be used for the regulatory control of these toxins in shellfish.

Dinophysistoxin-2: the predominant diarrhoetic shellfish toxin in Ireland.

Diarrhoetic shellfish poisoning (DSP) in Europe is due mainly to the presence of the dinoflagellate toxin, okadaic acid (OA). However, analysis of cultivated mussels (Mytilus edulis) from southwest Ireland revealed that an isomer of OA, dinophysistoxin-2, was the major toxin present during DSP episodes. Using fluorimetric HPLC, following derivatisation with 9-anthryldiazomethane, both OA and DTX-2 were found in shellfish during a prolonged toxic episode in 1991. However, examination of similar mussel cultivation locations in 1994 showed that DTX-2 was even more predominant. During this DSP period, OA levels were less than 0.7 microgram/g, whereas maximum DTX-2 levels of 6.3 micrograms/g hepatopancreas were recorded. This toxicity in shellfish occurred soon after high cell counts of Dinophysis acuta were observed. As well as large seasonal variability in toxin levels in rope cultured mussels, substantial variations were also observed, both horizontally and vertically, within the water column.

Comparative studies of two organophosphorus compounds in the mouse.

A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 mumol/kg, s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 mumol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE.

The origin of the effects of an anticholinesterase on the latencies of action potentials in mouse skeletal muscles.

1. Subcutaneous injection in mice of a single dose of an organophosphorous anticholinesterase, ecothiopate (0.5 mumol kg-1), produced increased variability in the latency (jitter) of indirectly-elicited action potentials in diaphragm muscles 5 days after treatment, but there was no effect on the variability of latencies of endplate potentials. This study was designed to elucidate the mechanism(s) of the increase in action potential jitter. 2. Action potentials evoked directly by electrical stimulation at one end of muscle fibres and recording near the other end had less jitter than indirectly-evoked action potentials and ecothiopate had no effect on directly-evoked action potentials. 3. In preparations with uncut fibres, pretreatment with ecothiopate reduced by about 20% both muscle fibre input resistance and the amplitude of spontaneous miniature endplate potentials. Ecothiopate had no effect on muscle fibre resting membrane potential or on the threshold potential for excitation. 4. In untreated preparations, indirectly-evoked action potentials recorded at the endplate had similar jitter to action potentials recorded at the tendon when latencies were measured at 10% of peak amplitude. However, when latencies were measured at peak, there was greater jitter of action potentials at the endplate. Ecothiopate increased jitter of action potentials recorded at the endplate at 10% of peak but did not significantly increase jitter of action potentials recorded at the endplate when measured at the peak. 5. In cut-fibre preparations, the first endplate potential of trains was significantly increased after ecothiopate but there was no effect of ecothiopate on the amplitude of plateau endplate potentials later in the train. Analysis of plateau endplate potentials showed that 5 days after administration, ecothiopateproduced an increase in the variance of endplate potential amplitudes and changes in the binomial parameters n and p.6. It was concluded that the increased jitter produced by ecothiopate is not a generalized effect on the plasma membrane and that none of the above observations could explain the increased jitter. The possibility is discussed that increased jitter is produced by variability in times to threshold of endplate potentials and/or by variability in the locus of generation of the action potential in the perijunctional area.

Electrophysiological and biochemical effects following single doses of organophosphates in the mouse.

Single doses of organophosphates (mipafox or ecothiopate) were given subcutaneously to mice. At intervals up to 77 days after dosing animals were killed and muscle action potentials and endplate potentials were recorded intracellularly in mouse phrenic-nerve/hemidiaphragm preparations. Activities of acetylcholinesterase and neuropathy target esterase in brain and acetylcholinesterase in diaphragm were also measured. Mipafox (0.11 mmol/kg), a neurotoxic organophosphate, produced an increase in prejunctional jitter (i.e. the variabilities of the latencies) of endplate potentials. This increase began 14-21 days after administration and lasted more than 23 days. No clinical signs of neuropathy were observed during this study. Mipafox also produced an increase in postjunctional (muscle action potential) jitter. Mipafox inhibited brain and diaphragm acetylcholinesterase and brain neuropathy target esterase. By comparison, a non-neurotoxic organophosphate, ecothiopate (0.5 mumol/kg), was a potent inhibitor of diaphragm acetylcholinesterase and produced a large increase in postjunctional jitter but ecothiopate did not inhibit brain neuropathy target esterase and had no effect on prejunctional jitter. Doses were chosen so that the inhibition of diaphragm acetylcholinesterase by each of the two organophosphates was similar. It is concluded that the neurotoxic organophosphate, mipafox, produced measurable changes in nerve function. These long-term changes may represent a new phenomenon, unrelated to the classical organophosphate induced delayed neuropathy. Alternatively, they may represent a neuropathic process which precedes or is below the threshold for clinical signs.

Protection against the effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles.

1. Adult male albino mice were injected subcutaneously with an organophosphorous anticholinesterase to initiate excessive variability in the latency of indirectly elicited muscle action potentials (jitter) when assessed 5 days later. 2. Pretreatment of the mice with a single dose of pyridostigmine prevented the development of jitter after subsequent dosing with an organophosphate. 3. Treatment with one dose of pralidoxime (2PAM) prevented the development of jitter if given less than 1 h after treatment with ecothiopate, a reactivatable inhibitor of cholinesterase. Similar treatment with 2PAM after a non-reactivatable inhibitor did not prevent the development of jitter. The repeated administration of 2PAM over 12 h did ameliorate jitter. 4. Pretreatment of mice orally with alpha-tocopherol and N-acetylcysteine, known to prevent ecothiopate-induced myopathy, did not prevent the development of jitter after ecothiopate. 5. It is concluded that the development of jitter was a consequence of the inhibition of acetylcholinesterase, and although jitter did not develop acutely, the potential for the full development of jitter was achieved about 1 h after intoxication with ecothiopate. The development of jitter did not involve the generation of free radicals. Reduction of the early effects of intoxication with anticholinesterases by pyridostigmine or 2PAM prevented the development of jitter.

Effects of suramin on the concentration--response relationship of alpha, beta-methylene ATP on the mouse vas deferens.

1. The effect of suramin on the concentration-effect curve for the contractile response of the isolated mouse vas deferens to alpha, beta-methylene ATP (alpha, beta-meATP) was investigated. 2. The concentration-response curve to alpha, beta-meATP had a consistent discontinuity at about 3 x 10(-6) M, giving it a biphasic appearance. 3. Suramin in a dose-dependent, reversible manner both shifted the curve to the right and at the same time elevated the maximum response. 4. The P2y inhibitor Reactive Blue 2, on the other hand, both shifted the curve to the left and elevated the maximum response. 5. These results show that alpha, beta-meATP in this preparation is an agonist both at excitatory, presumably P2y receptors and inhibitory P2y receptors, and that suramin antagonizes both effects.

The effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles.

1. The purpose of this investigation was to determine the long-term effects of a single dose of persistent anticholinesterases on muscle action potentials evoked by nerve stimulation. 2. Action potentials (APs), elicited by stimulation of the phrenic nerve, were recorded intracellularly in muscle fibres of mouse diaphragm. The time between stimulus and AP was measured and the variability of this latency was calculated during trains of APs. At the beginning of trains of APs there was an increase in latency, and this delay was also measured. 3. Within 3 h of subcutaneous injection, a single dose (500 nmol kg-1) of the anticholinesterase, ecothiopate produced about 90% reduction in the acetylcholinesterase activity of homogenates of mouse diaphragm muscle, but five days after injection, this activity was not different from values in untreated animals. The initial delay of APs and the variability of latencies were increased four fold and two fold respectively, remained at these maxima from the 1st to the 5th day after ecothiopate, and returned to the values in untreated animals between 15 and 27 days after ecothiopate. 4. These effects of ecothiopate on AP latency were dose-dependent and were also seen in extensor digitorum longus and soleus muscles. 5. Other anticholinesterases used were BOS (pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate), a quaternary compound, and diisopropyl fluorophosphate, a tertiary compound, which had effects similar to those of ecothiopate; the greater duration of the effects of this compound may be related to the greater duration of reduction in cholinesterase activity. 6. Ecothiopate had no effect on the delay or variability of latencies of endplate potentials which were recorded in cut-fibre preparations 5 days later. 7. It is concluded that the effects of ecothiopate on the latencies of indirectly-evoked muscle APs are postjunctional, may not be related to the degree of reduction in cholinesterase activity at the time of recording, and are not directly linked to necrosis.

The nature of the presynaptic effects of (+)-tubocurarine at the mouse neuromuscular junction.

1. The effects of (+)-tubocurarine (TC) on tetanic run-down and quantum content of end-plate potentials (EPPs) were investigated in cut-fibre preparations of mouse diaphragm. 2. (+)-Tubocurarine, 0.15 microM, halved the amplitude of spontaneous miniature EPPs (MEPPs) and steepened the tetanic run-down of EPPs evoked at 10 Hz by increasing the quantum content of the first EPP of the train while having no effect on quantum content of plateau EPPs. With stimulation at 1 Hz, there was little run-down and the quantum content of all EPPs was increased by TC. 3. The use of binomial statistics to analyse release indicated that after TC the increase in the quantum content of the first EPP in the train at 10 Hz was due to an increase in n and that during the run-down there was a decrease in p so that plateau EPP quantum content at 10 Hz was not different from control. 4. To elucidate a possible role of cholinoreceptors in the presynaptic effects of TC, studies were made on the effects of pancuronium or of alpha-bungarotoxin (BTX), with concentrations and exposure times where they had postsynaptic effects equal to 0.15 microM-TC. The run-down of EPPs was unaffected by BTX, while pancuronium steepened it to a lesser extent than TC. 5. The anticholinesterase, ecothiopate, decreased the quantum content of plateau EPPs only at high frequencies of stimulation (50 Hz) and did not affect the presynaptic effects of TC at 10 Hz. 6. At concentrations which reduced MEPP amplitude, atropine (10 microM) or hexamethonium (50 microM) had no effect on EPP run-down. 7. These results indicate that TC could have presynaptic effects via a presynaptic acetylcholine receptor, but that such a receptor may not have the same binding specificities as the postsynaptic receptor.

Statistics of neuromuscular transmitter release in young and old mouse muscle.

1. It was reported previously that in limb muscles of old (27-30 months) CBF-1 mice, quantal content (m) of evoked transmitter release was increased compared to that in young (9-12 months) mice. In diaphragm muscles there was no change with age. The object of the present study was to determine whether the age-related increase in transmitter release was due to increase in the binomial parameter n or the parameter p. The analysis also involved consideration of goodness-of-fit between observed and expected binomial distribution of the data. 2. Spontaneous miniature end-plate potentials (m.e.p.p.s) and evoked end-plate potentials (e.p.p.s) were recorded with intracellular techniques from soleus and diaphragm muscles bathed in low-Ca high-Mg medium. The goodness-of-fit between the observed e.p.p amplitude distribution and that expected from a binomial distribution was evaluated by chi 2 test. 3. In different muscles and at different ages, the percentage of fibres with binomial e.p.p. distributions varied from 17 to 44%, even though in all fibres there was a similar proportionality between direct quantal content and the reciprocal of the square of the coefficient of variation of e.p.p. amplitudes. In addition, apparent graphical agreement between observed and theoretical binomial e.p.p. distributions was often not substantiated by the chi 2 criterion. 4. In soleus muscles from young mice, lowering the stimulus frequency from 10 to 0.5 Hz and shortening the train length from 250 to 100 pulses increased the prevalence of binomial e.p.p. distributions, but the same result was not obtained in diaphragm or soleus muscles from old mice. If the mean amplitude of groups of 10 e.p.p.s in any train showed any drift (greater than 10%) then that train was excluded from the results. Thus, in order to make valid age comparisons, only fibres with binomial e.p.p. distributions were analysed further. 5. There was no change with age in m, n or p in diaphragm muscles, but in soleus muscles from old animals a nearly 2-fold increase in n entirely accounted for the increase in m. 6. If, as proposed by others, n represents the number of release sites, then the ageing soleus neuromuscular junction may have increased numbers or length of active zones or associated membrane components.

Sustained transmitter output by increased transmitter turnover in limb muscles of old mice.

The ability of neuromuscular junctions in old animals to maintain tetanic output was tested in phasic and tonic limb muscles and the physiologic mechanism of maintenance was elucidated by analysis of the turnover of a false transmitter during prolonged tetani. Transmitter release during and after tetani was compared in limb muscles of young (8-9 month) and old (28-30 month) male CBF-1 mice. Amplitudes of end-plate potentials (epp's) in curarized preparations and of spontaneous miniature end-plate potentials (mepp's) were measured in vitro at 30 degrees C in soleus and extensor digitorum longus (edl) muscles. In both young and old soleus muscles, epp amplitude was maintained at about 45% of resting level during the latter part of trains of 1,200 stimuli at 10 Hz but recovered to about 90% control within a few seconds after stimulation ceased. In edl muscles of young mice, epp amplitudes during a 20 Hz train of 1,200 impulses steadily declined to about 20% of control and gradually recovered over 2 min after the tetanus. In old edl muscles, tetanic decay of the epp's was greater and recovery slower than in young muscles, but absolute epp amplitudes were invariably greater. During trains of 6,000 impulses at 10 Hz, plateau epp amplitude decayed to 40-50% in young soleus muscle and 30-40% control in old muscle, but recovery was similar and absolute epp amplitudes were greater in old soleus muscle. A false transmitter precursor, homocholine (HoCh), was used to investigate the mechanism of this prolonged output, and, therefore, the use of HoCh in this system was first validated.(ABSTRACT TRUNCATED AT 250 WORDS)