M1 and M2 mAChRs activate PDK1 and regulate PKC ßI and ε and the exocytotic apparatus at the NMJ.

Neuromuscular junctions (NMJ) regulate cholinergic exocytosis through the M1 and M2 muscarinic acetylcholine autoreceptors (mAChR), involving the crosstalk between receptors and downstream pathways. Protein kinase C (PKC) regulates neurotransmission but how it associates with the mAChRs remains unknown. Here, we investigate whether mAChRs recruit the classical PKCßI and the novel PKCε isoforms and modulate their priming by PDK1, translocation and activity on neurosecretion targets. We show that each M1 and M2 mAChR activates the master kinase PDK1 and promotes a particular priming of the presynaptic PKCßI and ε isoforms. M1 recruits both primed-PKCs to the membrane and promotes Munc18-1, SNAP-25, and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and PKC phosphorylation. In summary, our results discover a co-dependent balance between muscarinic autoreceptors which orchestrates the presynaptic PKC and their action on ACh release SNARE-SM mechanism. Altogether, this molecular signaling explains previous functional studies at the NMJ and guide toward potential therapeutic targets.

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with µ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway.

Neurotrophin-4 couples to locally modulated ACh release at the end of neuromuscular synapse maturation.

We use immunocytochemistry to show that neurotrophin-4 (NT-4) and its receptor proteins (p75(NTR) and tropomyosin-related tyrosine kinase B) are present in neonatal neuromuscular junctions (NMJ) colocalized with several synaptic markers. NT-4 incubation (1h, in the range 2-12 nM) does not change the size of the endplate potential between P6 and P45. However, extended exposure (3h) to a relatively low dose of NT-4 (2 nM) potentiates ACh release (approx. 70%) in adult but not in neonatal muscles. The present results suggest that the developmental mechanism of axonal competition and neonatal elimination of redundant synapses cannot be modulated by added NT-4. However, this neurotrophin was able to modulate synaptic transmission locally in the adult NMJ.

Involvement of brain-derived neurotrophic factor (BDNF) in the functional elimination of synaptic contacts at polyinnervated neuromuscular synapses during development.

We use immunohistochemistry to describe the localization of brain-derived neurotrophic factor (BDNF) and its receptors trkB and p75(NTR) in the neuromuscular synapses of postnatal rats (P6-P7) during the synapse elimination period. The receptor protein p75(NTR) is present in the nerve terminal, muscle cell and glial Schwann cell whereas BDNF and trkB proteins can be detected mainly in the pre- and postsynaptic elements. Exogenously applied BDNF (10 nM for 3 hr or 50 nM for 1 hr) increases ACh release from singly and dually innervated synapses. This effect may be specific for BDNF because the neurotrophin NT-4 (2-8 nM) does not modulate release at P6-P7. Blocking the receptors trkB and p75(NTR) (with K-252a and anti-p75-192-IgG, respectively) completely abolishes the potentiating effect of exogenous BDNF. In addition, exogenous BDNF transiently recruits functionally depressed silent terminals, and this effect seems to be mediated by trkB. Calcium ions, the L-type voltage-dependent calcium channels and protein kinase C are involved in BDNF-mediated nerve ending recruitment. Blocking experiments suggest that endogenous BDNF could operate through p75(NTR) receptors coupled to potentiate ACh release in all nerve terminals because the anti-p75-192-IgG reduces release. However, blocking the trkB receptor (K-252a) or neutralizing endogenous BDNF with the trkB-IgG fusion protein reveals a trkB-mediated release inhibition on almost mature strong endings in dual junctions. Taken together these results suggest that a BDNF-induced p75(NTR)-mediated ACh release potentiating mechanism and a BDNF-induced trkB-mediated release inhibitory mechanism may contribute to developmental synapse disconnection.

Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

Coupling of presynaptic muscarinic autoreceptors to serine kinases in low and high release conditions on the rat motor nerve terminal.

We used intracellular recording to investigate how muscarinic acetylcholine receptors and the serine kinase signal transduction cascade are involved in regulating transmitter release in the neuromuscular synapses of the levator auris longus muscle from adult rats. Experiments with M1 and M2 selective blockers show that these subtypes of muscarinic receptors were involved in enhancing and inhibiting acetylcholine (ACh) release, respectively. Because the unselective muscarinic blocker atropine considerably increased release, the overall presynaptic muscarinic mechanism seemed to moderate ACh secretion in normal conditions. This muscarinic function did not change when more ACh was released (high external Ca2+) or when there was more ACh in the cleft (fasciculin II). However, when release was low (high external Mg2+ or low external Ca2+) or when there was less ACh in the cleft (when acetylcholinesterase was added, AChE), the response of M1 and M2 receptors to endogenously released ACh shifted to optimize release, thus producing a net potentiation of the Mg2+-depressed level. Protein kinase A (PKA) (but not protein kinase C, PKC) has a constitutive role in promoting a component of normal release because when it is inhibited with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2 HCl, release diminishes. The imbalance of the muscarinic acetylcholine receptors (mAChRs) (with the selective block of M1 or M2) inverts the kinase function. PKC can then tonically stimulate transmitter release, whereas PKA is uncoupled. The muscarinic function can be explained by an increased M1-mediated PKC activity-dependent release and a decreased M2-mediated PKA activity-dependent release. In the presence of high external Mg2+ or low Ca2+, or when AChE is added, both mAChRs may potentiate release through an M2-mediated PKC mechanism and an M1-mediated mechanism downstream of the PKC.

Protein kinase C activity affects neurotransmitter release at polyinnervated neuromuscular synapses.

By using intracellular recording, we studied how protein kinase C (PKC) activity affected transmitter release in singly and dually innervated endplates of the Levator auris longus muscle of 5-6-day-old rats during axonal competition in the postnatal synaptic elimination period. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when the stimulation intensity is increased. The nerve terminals that generate the lowest and the highest EPP amplitudes are designated "small-EPP generating ending" (SEGE) and "large-EPP generating ending" (LEGE), respectively. Blocking PKC with calphostin C, staurosporine, or chelerythrine results in an increased release from SEGE ( approximately 80%), whereas release from LEGE and from endings generating only one EPP (OEGE) is not significantly affected. Blocking PKC also leads to the recruitment of silent synapses (acetylcholine cannot be released before PKC inhibition). The mean number of functional axon terminals per synapse increases by approximately 47%, and these are now designated the "recruited-EPP generating endings" (REGE). This suggests that axonal PKC can modulate postnatal synaptic elimination by favoring the nerve terminal disconnection of certain weak axonal endings (REGE and SEGE). We conclude that a PKC-mediated mechanism should occupy a pivotal place in neonatal synapse elimination, because functional axonal withdrawal can indeed be turned back by PKC block.

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat.

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.

Calcium inflow-dependent protein kinase C activity is involved in the modulation of transmitter release in the neuromuscular junction of the adult rat.

Using intracellular recording, we studied how protein kinase C activity affected miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in the neuromuscular junctions of the levator auris longus muscle from adult rats. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA, 10 nM) increased the quantal content by approximately 150% (P<0.05). On the other hand, the quantal content decreased by approximately 40% (P<0.05) for all the protein kinase C inhibitors tested (Calphostin-C, 10 microM; Chelerythrine, 1 microM; Staurosporine, 200 nM). These changes in acetylcholine release were maintained at plateau for 1 to 7 h. Moreover, none of the protein kinase C activators or inhibitors used could modify the spontaneous MEPP mean size (P>0.05). We reduced the calcium influx in nerve terminals using the P/Q-type channel blocker omega-Aga-IVA(100 nM) or with 5 mM magnesium in physiological solution. In neither situation was the quantal content modified by PMA or by CaC. However, when high Ca2+ (5 mM) was added to a preparation that was previously blocked with omega-Aga-IVA, PMA and CaC had their full effect. We conclude that under physiological conditions PKC is dependent on the calcium inflow through the P/Q-type voltage-dependent calcium channels during evoked activity and works near the maximum rate at normal external calcium concentration.

Muscarinic autoreceptors related with calcium channels in the strong and weak inputs at polyinnervated developing rat neuromuscular junctions.

Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials in singly and dually innervated endplates of the levator auris longus muscle from 3 to 6-day-old rats. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when we increase the stimulation intensity. The lowest and highest EPP amplitudes are designated "small-EPP" and "large-EPP," respectively. In singly innervated endplates and large-EPP, we found an inhibition of acetylcholine release by M1-receptor antagonists pirenzepine and MT-7 (more than 30%) and M2-receptor antagonists methoctramine and AF-DX 116 (more than 40%). The small-EPP was also inhibited by both M2-receptor antagonists methoctramine (approximately 70%) and AF-DX 116 (approximately 40%). However, the small-EPP was enhanced by M1-receptor antagonists pirenzepine (approximately 90%) and MT-7 (approximately 50%). The M4-receptor selective antagonists tropicamide and MT-3 can also increase the small-EPP amplitude (75% and 120%, respectively). We observed a graded change from a multichannel involvement (P/Q- N- and L-type voltage-dependent calcium channels) of all muscarinic responses (M1-, M2- and M4-mediated) in the small-EPP to the single channel (P/Q-type) involvement of the M1 and M2 responses in the singly innervated endplates. This indicates the existence of a progressive calcium channels shutoff in parallel with the specialization of the adult type P/Q channel. In conclusion, muscarinic autoreceptors can directly modulate large-EPP generating ending potentiation, and small-EPP generating ending depression through their association with the calcium channels during development.

Decreased calcium influx into the neonatal rat motor nerve terminals can recruit additional neuromuscular junctions during the synapse elimination period.

Individual skeletal muscle fibers in newborn vertebrates are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in an activity-dependent process of synaptic elimination by axonal competition. Because synaptic activity depends strongly on the influx of calcium from the external media via presynaptic voltage-dependent calcium channels, we investigate the relationship between calcium channels, synaptic activity and developmental axonal elimination. We studied how several calcium channel blockers affect (after 1 h of incubation) the total number of functional axons per muscle fiber (poly-innervation index) of the Levator auris longus muscle of 6-day-old rats. We determined the poly-innervation index by gradually raising the stimulus amplitude and recorded the recruitment of one or more axons that produced a stepwise increment of the endplate potential.The L-type channel blocker nitrendipine (1 microM) increased the mean poly-innervation index (35.79% +/- 3.91; P<0.05). This effect was not washed out with normal Ringer, although the poly-innervation index returned to the control value when high-calcium Ringer (5 mM) was used. The P-type channel blocker omega-agatoxin-IVA (100 nM) also increased the number of recruitable endplate potentials (27.49% +/- 1.78; P<0.05), whereas N-type channel blocker omega-conotoxin-GVIA (1 microM) was ineffective (P>0.05). However, neither nitrendipine nor omega-agatoxin-IVA modified the poly-innervation index on high-calcium Ringer (P>0.05 in both cases). A more intense inhibition of calcium influx (by the sequential use of two calcium channel blockers) did not recruit any additional silent synapses. Moderately increasing the magnesium ions (by 500 microM) in the physiological solution produces a synaptic recruitment (36.78% +/- 2.1; P<0.05) similar to that with L- and P-type calcium channel blockers incubation. This magnesium effect was not washed with normal Ringer but a Ringer that is high in calcium can reverse it. The recruited endings were identified by selective activity-dependent loading with styryl dyes. Rhodaminated alpha-bungarotoxin-labeled acetylcholine receptors were present in the postsynaptic counterpart. Based on these findings we suggest that, before their complete retraction, functionally silent nerve terminals can be manifested or recovered if calcium influx is reduced by a calcium channel blocker or if external magnesium is increased. The normal activation of this calcium-dependent silencing mechanism during development may be related to the final loss of the supernumerary axons.

Calcium channels coupled to neurotransmitter release at dually innervated neuromuscular junctions in the newborn rat.

We studied the effect of several calcium channel blockers (omega-Conotoxin-GVIA, 1 and 3microM; omega-Agatoxin-IVA, 100nM; Nitrendipine, 1 and 10microM) on evoked transmitter release at singly and dually innervated endplates of the levator auris longus muscle from three- to six-day-old rats. In dually innervated fibers, a second endplate potential may appear after the first one when we increase the stimulation intensity. The lowest and highest endplate potential amplitudes are designated "small endplate potential" and "large endplate potential", respectively. The percentage of doubly innervated junctions remains almost constant throughout the age range examined. Nevertheless, the percentage of junctions innervated by three or more terminal axons drops, whereas the singly innervated junctions increase. Therefore, between postnatal days 3 and 6, roughly half the neuromuscular junctions may experience the final process of axonal elimination. The synaptic efficacy of the large endplate potential in dual junctions, measured as the mean amplitude of the synaptic potential and mean quantal content, was the same as in the junctions that had become recently mono-innervated in the same postnatal period. In singly innervated fibers, the endplate potential size was strongly reduced by both the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA (79.17+/-4.02%; P < 0.05) and the N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA (56.31+/-7.80%; P < 0.05), whereas endplate potential amplitude was not significantly changed by the L-type voltage-dependent calcium channel blocker Nitrendipine. In dually innervated fibers, the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA and L-type voltage-dependent calcium channel blocker Nitrendipine increased the size of the small endplate potential (161.29+/-47.87% and 109.32+/-11.03%, respectively; P < 0.05 in both cases) and reduced the large endplate potential (74.42+/-15.32% and 70.91+/-10.04%, respectively; P < 0.05 in both cases). The N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA significantly increased the small endplate potential in the first few minutes after toxin application (at 10min: 90.23+/-17.38%; P < 0.05). This increase was not maintained, while the large endplate potential was strongly inhibited (69.25+/-7.5%; P < 0.05). In conclusion, in the dually innervated endplates of the newborn rat, presynaptic calcium channel types can have different roles in transmitter release from each of the two inputs, which suggests that nerve terminal voltage-dependent calcium channels are involved in neonatal synaptic maturation.

Pertussis toxin-sensitive G-protein and protein kinase C activity are involved in normal synapse elimination in the neonatal rat muscle.

Individual skeletal muscle fibers in most new-born rodents are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in a process of synaptic elimination. Previous studies showed that the naturally occurring serine-protease thrombin mediates the activity-dependent synapse reduction at the neuromuscular junction (NMJ) in vitro and that thrombin-receptor activation may modulate nerve terminal consolidation through a protein kinase mechanism. To test whether these mechanisms may be operating in vivo, we applied external thrombin and its inhibitor hirudin, and several substances affecting the G protein-protein kinase C system (GP-PKC) directly over the external surface of the neonatal rat Levator auris longus muscle. Muscles were processed for immunocytochemistry to simultaneously detect acetylcholine receptors (AChRs) and axons for counting the percentage of polyinnervated NMJ. We found that exogenous thrombin accelerated synapse loss and hirudin blocked axonal removal. Phorbol-12-myristate-13-acetate, a potent PKC activator, had a similar effect as thrombin, whereas the PKC inhibitors, calphostin C and staurosporine, prevented axonal removal. Pertussis toxin, an effective blocker of GP function, blocked synapse elimination. These findings suggest that the normal synapse elimination in the neonatal rat muscle may be modulated, at least in part, by the pertussis-sensitive G-protein and PKC activity and that thrombin could play a role in the postnatal synaptic maturation in vivo.

Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)-mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time- and dose-related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite-associated receptor aggregates in nerve-muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity-dependent synapse reduction and does so, at least in part, by altering AChR stability.

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-bungarotoxin-induced sprouting.

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48-169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedure permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxin-induced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization.

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals.

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; DL50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 microM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker omega-conotoxin-GVIA (1 microM) inhibited neurotransmitter release by 50-70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker omega-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and omega-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.

Physiological activity-dependent ultrastructural plasticity in normal adult rat neuromuscular junctions.

The major finding of the present study is that the ultrastructural organization of the neuromuscular synapse can be modified by a small, 4-week-long, physiological increase in the locomotor activity of the extensor digitorum longus muscle of normal adult rats trained to walk. This study measures these plastic adaptations using several synaptic morphological parameters. The observed changes in neuromuscular junctions affect both pre- and postsynaptic membranes. In particular, the presynaptic membrane densities in the active zones and the postsynaptic adaxonal membrane densities become larger, which shows that in the normal adult mammal neuromuscular junction, there is an activity-dependent modulation of the neurotransmission-related structures in response to slight physiologic functional demands. The nature and magnitude of these changes are discussed.

Activity-dependent plastic changes in the motor nerve terminals of the adult rat.

Small and short-lasting physiologic variations in the locomotor activity of normal adult rats can induce remodelling in the motor nerve endings of the fast extensor digitorum longus muscle. The specificity and relative importance of the different plastic adaptations occurring in the presynaptic axonal tree have been studied, in silver impregnated nerve endings, by using an automatic image analysis treatment of the nerve terminals' geometric properties and a discriminant analysis of the morphometric parameters. Changes observed, like selective length variations in certain terminal segments and positional rearrangements, agree with a mechanism of neural connectivity regulation in the adult that arises as a consequence of normal neuromuscular activity.