Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

Preimplantation genetic diagnosis for BRCA1/2--a novel clinical experience.

OBJECTIVE: To describe our 2-year experience with preimplantation genetic diagnosis (PGD) for carriers of mutations in the genes BRCA1 and BRCA2, the dilemmas incurred and the lessons learned. METHODS: We collected data on those carriers of BRCA1/2 mutations who applied for PGD counseling and who decided to proceed. We describe the PGD procedures that were conducted and their outcome. RESULTS: Ten carriers of BRCA1/2 mutations applied for PGD counseling, seven were healthy, and three were BC survivors. Eight women needed in vitro fertilization (IVF) because of coexisting infertility. After counseling, six opted for the procedure and five of them underwent PGD for the BRCA mutation. In one of these PGD, fluorescence in situ hybridization (FISH) analysis for chromosomes 21, X and Y was also performed. Three women conceived, each in the first treatment attempt. One of them gave birth to twins, the second to a singleton and the third is currently pregnant. During the pregnancies, dilemmas concerning PGD confirmation were discussed. CONCLUSIONS: PGD is an acceptable reproductive option for BRCA mutation carriers, especially for those who require IVF due to fertility problems. Discussion of this option should be carried out with sensitivity, taking into account the age of the woman, her health, fertility status and emotional state. Confirmatory prenatal diagnosis may not always be encouraged.

Allosteric modulation of the NMDA receptor by dihydrolipoic and lipoic acid in rat cortical neurons in vitro.

The mitochondrial cofactor dihydrolipoic acid (DHLA) was observed to potentiate N-methyl-D-aspartate (NMDA), but not non-NMDA, receptor-mediated whole-cell responses in cultured neurons. This potentiation was readily reversed by the oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB). DHLA was unable to increase NMDA responses previously potentiated by dithiothreitol, nor did it have an effect on NMDA receptors alkylated with N-ethylmaleimide. Single-channel recordings revealed that DHLA produced an increase in NMDA channel open frequency, with no change in single-channel conductance or open time. In contrast, lipoic acid reversed the potentiation of NMDA-evoked responses produced by dithiothreitol and had no effect on NMDA receptors previously oxidized by DTNB. DHLA and lipoic acid are pervasively found substances that readily permeate cellular membranes and thus may influence NMDA receptor activity in vivo by modifying its redox site.

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site.

A novel modulatory site on the N-methyl-D-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.

Selective modulation of NMDA responses by reduction and oxidation.

Electrophysiological responses to the glutamate analog N-methyl-D-aspartate (NMDA) measured in three different central neuronal preparations are subject to a novel modulatory mechanism: they are substantially potentiated after exposure to the disulfide reducing agent dithiothreitol, while oxidation with 5-5-dithiobis-2-nitrobenzoic acid decreases the magnitude of the response. Modification of the NMDA response by either oxidation or reduction does not appear to affect the pharmacological properties of the receptor-channel complex. Since we observe that the redox state of the native receptor-channel complex varies widely among neurons, an in vivo mechanism that can strongly regulate NMDA-activated functions by either reduction or oxidation may exist. In addition, these results suggest that it may be possible to design specific redox agents for characterizing the NMDA receptor-channel complex.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Apoptotic surface delivery of K+ channels.

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.

A vital role for voltage-dependent potassium channels in dopamine transporter-mediated 6-hydroxydopamine neurotoxicity.

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.

NMDA and glutamate evoke excitotoxicity at distinct cellular locations in rat cortical neurons in vitro.

The development of cortical neurons in vivo and in vitro is accompanied by alterations in NMDA receptor subunit expression and concomitant modifications in the pharmacological profile of NMDA-activated ionic currents. For example, we observed that with decreasing NR2B/NR2A subunit expression ratio, the block of NMDA receptor-mediated whole-cell responses by the NR2B-selective antagonist haloperidol was also decreased. In mature cultures (>22 d in vitro), however, NMDA responses obtained from excised nucleated macropatches, which comprised a large portion of the soma, remained strongly antagonized by haloperidol. These results suggest that in more mature neurons NR1/NR2B receptors appear to be preferentially expressed in the cell body. As predicted from the whole-cell recording pharmacological profile, NMDA-induced toxicity was largely unaffected by haloperidol in mature cultures. However, haloperidol effectively blocked glutamate toxicity in the same cultures, suggesting that the neurotoxic actions of this amino acid were mostly due to the activation of somatic NMDA receptors. In experiments in which the potency of glutamate toxicity was increased by the transport inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid, the neuroprotective effects of haloperidol were significantly diminished. This was likely because of the fact that glutamate, now toxic at much lower concentrations, was able to reach and activate dendritic receptors under these conditions. These results strongly argue that exogenous glutamate and NMDA normally induce excitotoxicity at distinct cellular locations in mature mixed neuronal cultures and that NR1/NR2B receptors remain an important component in the expression of glutamate, but not NMDA-induced excitotoxicity.

p38 activation is required upstream of potassium current enhancement and caspase cleavage in thiol oxidant-induced neuronal apoptosis.

Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2'-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 microm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.

Novel role for the NMDA receptor redox modulatory site in the pathophysiology of seizures.

Redox-active compounds modulate NMDA receptors (NMDARs) such that reduction of NMDAR redox sites increases, and oxidation decreases, NMDAR-mediated activity. Because NMDARs contribute to the pathophysiology of seizures, redox-active compounds also may modulate seizure activity. We report that the oxidant 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and the redox cofactor pyrroloquinoline quinone (PQQ) suppressed low Mg(2+)-induced hippocampal epileptiform activity in vitro. Additionally, in slices exposed to 4-7 microM bicuculline, DTNB and PQQ reversed the potentiation of evoked epileptiform responses by the reductants dithiothreitol and Tris(2-carboxyethyl)phosphine (TCEP). NMDA-evoked whole-cell currents in CA1 neurons in slices were increased by TCEP and subsequently decreased by DTNB or PQQ at the same concentrations that modulated epileptiform activity. However, DTNB and PQQ had little effect on baseline NMDA-evoked currents in control medium, and PQQ did not alter NMDAR-dependent long-term potentiation. In contrast, in slices returned to control medium after low Mg(2+)-induced ictal activity, DTNB significantly inhibited NMDAR-mediated currents, indicating endogenous reduction of NMDAR redox sites under this epileptogenic condition. These data suggested that PQQ and DTNB suppressed spontaneous ictal activity by reversing pathological NMDAR redox potentiation without inhibiting physiological NMDAR function. In vivo, PQQ decreased the duration of chemoconvulsant-induced seizures in rat pups with no effect on baseline behavior. Our results reveal endogenous potentiation of NMDAR function via mass reduction of redox sites as a novel mechanism that may enhance epileptogenesis and facilitate the transition to status epilepticus. The results further suggest that redox-active compounds may have therapeutic use by reversing NMDAR-mediated pathophysiology without blocking physiological NMDAR function.

The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic brain injury.

Pyrroloquinoline quinone is a ubiquitous redox cofactor and putative essential nutrient in mammals. Pyrroloquinoline quinone has recently been demonstrated to depress N-methyl-D-asparate induced electrical responses and is neuroprotective in vitro. In addition, pyrroloquinoline quinone has been demonstrated to act as a free radical scavenger in mammalian tissues. In this study, we demonstrate a neuroprotective effect of pyrroloquinoline quinone in an in vivo cerebral hypoxia/ischemia model in the rodent. Significant reduction in infarct size resulted from pyrroloquinoline quinone pretreatment and also when pyrroloquinoline quinone was administered following induction of hypoxia/ischemia. The neuroprotective effect was not dependent on change in core or cranial temperatures, as there was no difference between temperature measurements in pyrroloquinoline quinone-treated and vehicle-treated controls. No changes in electroencephalographic activity were observed at neuroprotective doses. These findings suggest that pyrroloquinoline quinone may represent a novel class of quinoid reagents of potential use in the treatment of neurological disorders that involve excitotoxicity. This study demonstrates a protective effect of the novel essential nutrient pyrroloquinoline quinone on brain injury in a rodent model of cerebral hypoxia/ischemia. Pyrroloquinoline quinone was neuroprotective when administered before and even after the insult, and did not appear to have significant neurobehavioral side effects. Pyrroloquinoline quinone represents a new class of agents with potential use in the therapy of stroke.

Lack of interaction between nitric oxide and the redox modulatory site of the NMDA receptor.

1. The inhibitory effects of nitric oxide (NO) on N-methyl-D-aspartate (NMDA) receptor function have been proposed to be mediated via the interaction of this gas with a redox-sensitive thiol moiety on the receptor. Here, we evaluated this suggested mechanism by examining the actions of various NO donors on native neuronal receptors as well as in wild-type and cysteine-mutated recombinant NMDA receptors expressed in Chinese hamster ovary (CHO) cells. 2. The NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydraxino)ethanamine (NOC-12; 100 microM) produced a rapid and readily reversible inhibition of whole-cell currents induced by NMDA (30 microM) in cultured cortical neurons. The inhibition was apparent at all holding potentials, though a more pronounced block was observed at negative voltages. The effects of NOC-12 disappeared when the donor was allowed to expire. A similar receptor block was observed with another NO-releasing agent, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). 3. The blocking effects of NO released by SNAP, 3-morpholinosydnonimine (SIN-1; 1 mM), and 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamin e (NOC-5; 100 microM) on currents mediated by recombinant NRI/NR2B receptors were virtually indistinguishable from those observed on native receptors. Furthermore, mutating cysteines 744 and 798 of NR1, which constitute the principal redox modulatory site of the NR1/NR2B receptor configuration, did not affect the inhibition produced by NO. 4. The NR2A subunit may contribute its own redox-sensitive site. However, the effects of NO on NR1/NR2A receptors were very similar to those seen for all other receptor configurations evaluated. Hence, we conclude that NO does not exert its inhibition of NMDA-induced responses via a modification of any of the previously described redox-sensitive sites on the receptor.

Reduction of NMDA receptors with dithiothreitol increases [3H]-MK-801 binding and NMDA-induced Ca2+ fluxes.

1. We have investigated the modulation of N-methyl-D-aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). 2. Increases in [3H]-MK-801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2mM) and diminished by DTNB (0.5 mM). 3. The inhibition of [3H]-MK-801 binding by CGS 19755 and 7-chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT. 4. NMDA-induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB-reversible fashion. In addition to augmenting the magnitude of NMDA-induced increase in intracellular free Ca2+, 10 mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl. 5. These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.

The action of CGS-19755 on the redox enhancement of NMDA toxicity in rat cortical neurons in vitro.

The effects of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) were studied in cultures of rat cerebral cortex under normal and altered redox conditions. CGS-19755 was effective in preventing delayed neuronal death produced by an acute exposure to either glutamate (500 microM) or NMDA (200 microM), but was ineffective in protecting neurons against the toxicity induced by a prolonged exposure to kainate (500 microM). We observed that the reducing agent dithiothreitol (DTT, 500 microM), could dramatically enhance toxicity and electrophysiological responses produced by 50 microM NMDA. CGS-19755 (100 microM) could effectively block both of these effects of DTT. Any toxicity produced by DTT alone was also antagonized by CGS-19755. In contrast, oxidized DTT did not enhance NMDA toxicity nor was it toxic when added alone. These results indicate that CGS-19755 is an effective and specific neuroprotectant acting at the NMDA receptor in vitro, and that the enhancement in NMDA toxicity induced by DTT is mediated by an increase in activity at this receptor complex.

Blockade of nicotinic responses in rat retinal ganglion cells by neuronal bungarotoxin.

The effects of neuronal bungarotoxin (NBT) on nicotinic acetylcholine responses recorded from rat retinal ganglion cells in culture were studied with patch electrodes. We observed that the concentration of this toxin needed to induce a total blockade of nicotinic currents varied according to the method of toxin application utilized. Rapid addition of 20 microM NBT by pressure ejection from micropipettes produced total blockade of 50-microM acetylcholine-induced currents. In contrast, when added slowly via the physiological solution which continuously superfused the cells, NBT was able to produce a complete blockade of the response at 200 nM. The IC50 determined for NBT by the superfusion method was 55 nM. Recovery from block was slow and incomplete with both drug application methods, although some differences were found. The results are discussed with reference to a scheme in which NBT binds with both low and high affinity to functional nicotinic receptors in rat retinal ganglion cells in culture.

Effects of nicotinic agonists on the NMDA receptor.

The cholinergic nicotinic agonists (-)nicotine and lobeline were observed to partially inhibit whole-cell N-methyl-D-aspartate (NMDA)-induced responses in rat cortical neurons in culture. In addition we found that (-)nicotine, (+)nicotine, and lobeline, but not the nicotine metabolite (-)cotinine nor acetylcholine, were able to displace [3H]dizocilpine ([3H]MK 801) binding in well-washed membranes obtained from rat brain. These results show that certain nicotinic agonists can interact with the NMDA receptor and block its function.

Intrinsic redox properties of N-methyl-D-aspartate receptor can determine the developmental expression of excitotoxicity in rat cortical neurons in vitro.

The sensitivity of central neurons in culture to N-methyl-D-aspartate (NMDA) receptor-mediated cell death increases with development. In this study, we show that this phenomenon in vitro may be due, at least in part, to changes in the redox properties of the NMDA receptor itself. With increasing days in culture, NMDA-induced electrical responses in rat cortical neurons are less sensitive to dithiothreitol-induced potentiation and spontaneously oxidize less readily than in younger cells. These results imply that at earlier developmental ages NMDA receptors prefer a more oxidized state. Hence, in the presence of a reducing agent, NMDA-induced neurotoxicity was produced in normally resistant younger neurons. The observed changes in NMDA receptor properties with development could not be attributed to long-range diffusible redox endogenous factors. An oxidized NMDA receptor thus confers maturing neurons a protective mechanism against glutamate toxicity during development.

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons.

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.

Axonal transport of alpha-bungarotoxin binding sites in rat sciatic nerve.

[125I]alpha-Bungarotoxin (alpha-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [125I]alpha-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [125I]alpha-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature. D-Tubocurarine, nicotine, decamethonium and atropine displaced [125]alpha-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [125I]alpha-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.