Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers.

The freeze-fracture technique was used to study differences in membrane structure which could explain differences in the number of quanta released from axon terminals on twitch and tonic muscle fibers in Anolis intercostal muscles. The protoplasmic leaflets of axon terminals facing lizard twitch muscle fibers have intramembrane particle specializations characterized by two parallel linear particle arrays each composed of two particle rows which lie perpendicular to the axis of shallow ridges in the axolemma. During K+ depolarization, vesicles open between the arrays, confirming that these structures are the active zones for synaptic vesicle opening. Active zones at axon terminals on tonic fibers are defined by one linear particle array composed of two parallel particle rows oriented along the axis of a shallow presynaptic ridge; vesicles open beside these arrays. Thus, there are more particles near active zone vesicles in terminals on twitch fibers. Even though terminals on twitch and tonic muscle fibers seem to have similar numbers of synaptic vesicles associated with their active zones, a presynaptic action potential is reported to release at least 10 times more quanta from terminals on twitch fibers. We postulate that the differences in quantal output are related to the observed differences in the number of active zone particles flanking synaptic vesicles at the active zone. Indeed, the correlation between the distribution of these particles and the level of transmitter release provides additional support for the idea that they are the calcium channels which couple transmitter release to the action potential.

Excitatory and inhibitory activity in the dorsal musculature of the nematode Ascaris evoked by single dorsal excitatory motonerons.

A physiological preparation, in which identified motoneurons of the nematode Ascaris lumbricoides can be individually stimulated, was used to map the response evoked by single dorsal excitatory (DE) motoneurons in muscle cells innervated along the length of the dorsal nerve cord. As previously reported (Walrond, J. P., I. S. Kaas, A. O. W. Stretton, and J. E. Donmoyer (1985) J. Neurosci. 5: 1-8), stimulation of a DE cell produces excitatory responses in muscle cells which it directly innervates. Excitatory activity propagates along the most strongly activated region of muscle at a velocity of approximately 28 cm/sec, then relaxes into a slower velocity of approximately 12 cm/sec. When either the DE1 or DE3 neurons were stimulated, excitatory responses were also observed in muscle cells not directly innervated by the neuron. These signals propagate in the opposite direction from the fast-propagating activity at a velocity of approximately 13 cm/sec. Injection of hyperpolarizing current into muscle cells blocks this slower propagation but fails to block the faster conduction. We conclude that the fast-conducting responses result from signals propagating in the motor axon, whereas the slow responses are conducted through gap junctions which connect Ascaris muscle cells. Stimulating a single DE motoneuron also evokes hyperpolarizing muscle responses in regions adjacent to the zones of fast and slow excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal inhibition in the motor nervous system of the nematode Ascaris: direct control of ventral inhibitory motoneurons by dorsal excitatory motoneurons.

In previous physiological experiments (Stretton, A. O. W., R. M. Fishpool, E. Southgate, J. E. Donmoyer, J. P. Walrond, J. E. R. Moses, and I. S. Kass (1978) Proc. Natl. Acad. Sci. U. S. A. 75: 3493-3497), we have shown that the dorsal cord of the nematode Ascaris lumbricoides includes the processes of three types of dorsal excitatory (DE) motoneurons and one type of ventral inhibitory (VI) motoneuron. Ultrastructural studies have revealed that the axons of the DE motoneurons make monosynaptic contacts with the dorsal processes of VI motoneurons. In this paper, we describe a physiological preparation with which to investigate the properties of these synapses. We show that activation of a DE neuron can excite a VI neuron producing inhibition in ventral muscle cells shortly after dorsal muscle cells are excited, thus mediating reciprocity between dorsal and ventral muscles. Each VI dendrite receives input from four or five DE neurons; activation of any one of these DE neurons is sufficient to activate the VI neuron.

Structural plasticity at crustacean neuromuscular synapses.

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.

Submicrovillar tubules in distal segments of squid photoreceptors detected by rapid freezing.

Invertebrate phototransduction is believed to involve an inositol trisphosphate (InsP3)-mediated release of calcium from intracellular storage compartments. Although light-induced production of InsP3 has been demonstrated for squid retinas, morphological evidence for the presence of internal calcium stores has been lacking. Because squid retinas are about 1 mm thick and composed of densely packed receptor cells, conventional aldehyde fixatives may not penetrate rapidly enough to preserve subcellular organelles. To reduce the time for fixative penetration, receptor cells were isolated from intact retinas before fixation, but these techniques provided little improvement in the preservation of membrane-bound compartments. Alternatively, the distal ends of the receptors were ultra-rapidly frozen by dropping 1 mm2 pieces of intact retina against a liquid helium-cooled copper block. Electron micrographs of thick sections from rapidly frozen and freeze-substituted retinas showed elongated saccules oriented parallel to the long axis of the receptor cell and located about 40 nm from the microvillar openings. Freeze-fracture and etch views of rapidly frozen cells showed that the saccules are 130 nm diameter tubules and extend for at least several micrometers along the length of the receptor cell. We call these organelles submicrovillar tubules (SMT). The gap between the SMT and the plasma membrane contains a network of filaments that appear to be actin. Freeze-fracture and etch views of the rhabdomeres also indicate that adjacent microvilli are separated by a 6-8-nm-wide extracellular space along most of their length. This space is spanned by extracellular connections linking adjacent microvilli. The position and orientation of the SMT suggest that these organelles may serve the same function as the more voluminous and highly convoluted submicrovillar cisternae found in other invertebrates. The SMT is likely to be the intracellular compartment that stores and releases calcium as part of the InsP3-mediated light response.

Two structural adaptations for regulating transmitter release at lobster neuromuscular synapses.

The distal accessory flexor muscle (DAFM) in the lobster (Homarus americanus) walking leg consists of 5 muscle fiber bundles. All five bundles, one proximal, one distal, and 3 medial, are innervated by one excitatory and one inhibitory motor neuron. Both neurons release more transmitter on the distal bundle than on the proximal bundle. The aim of our studies was to investigate the structural basis of this differentiation. Thin sections cut at 50 microns intervals showed a similar number of excitatory synapses on the two bundles. Freeze-fracture views of excitatory synapses showed that synapse size, active zone number per synapse, and intramembrane particle density in the postsynaptic membrane are similar proximally and distally. Active zones at synapses on the distal bundle are larger and contain about 50% more large intramembrane particles, which are thought to include the voltage-gated Ca2+ channels that couple the action potential to transmitter release, than their counterparts on the most proximal bundle. This difference in channel number appears to produce a disproportionate increase in the probability of transmitter release sufficient to account for most of the proximal-distal disparity in the amplitude of the excitatory postsynaptic potential. In contrast, staining the inhibitor for antibodies to the inhibitory neurotransmitter, GABA, showed that it forms more varicosities on the distal bundle than on the proximal bundle. Because most of the synapses are located in the varicosities, differences in synapse number likely regulate the proximal-distal disparity in the amount of inhibitory transmitter released. Therefore, the regional differentiation in the amount of transmitter released in the DAFM appears to be based on two distinct mechanisms. In the inhibitor, transmitter release appears to be regulated differentially by differences in synapse number. In the excitor, transmitter release appears to be regulated differentially from a similar number of synapses by differences in active zone structure.

Inhibitory innervation of a lobster muscle.

Inhibitory neuromuscular synapses formed by the common inhibitor (CI) neuron on the distal accessory flexor muscle (DAFM) in the lobster, Homarus americanus, were studied with electrophysiological and electron-microscopic (thin-section and freeze-fracture) techniques. Postsynaptic inhibition as indicated by inhibitory junctional potentials was several-fold stronger on distal compared to proximal muscle fibers. This difference correlated with the results of serial thin-section studies, which showed more inhibitory synapses on distal fibers than on their proximal counterparts. Effects of postsynaptic inhibition on excitatory junctional potentials via current shunting had a morphological correlate in the spatial relationship between inhibitory and excitatory synapses on the distal fibers. Inhibitory synapses were larger than their excitatory counterparts and had fewer glial processes. In freeze-fracture views, inhibitory synapses did not appear as raised plateaus in the P-face as do excitatory synapses, and their active zones were more widely scattered. The intramembrane particles in the inhibitory postsynaptic membrane - representing neurotransmitter receptors - are arranged in parallel rows in the sarcolemmal P-face and have complementary furrows in the sarcolemmal E-face. Altogether, our findings help to describe a population of inhibitory neuromuscular synapses formed by the CI neuron in lobster muscle.

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction.

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called "active zones." Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.

Avermectin B1a, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris.

Avermectin B1a (AVM) is an antiparasitic agent that paralyzes nematodes without causing hypercontraction or flaccid paralysis. Using selective stimulation techniques, we have shown that AVM blocks transmission between interneuron(s) and excitatory motorneurons in the ventral nerve cord of Ascaris. It also inhibits transmissin between inhibitory motoneurons and muscle but has little effect on excitatory neuromuscular transmission. Picrotoxin can reverse the AVM-induced block of interneuron-excitatory motoneuron transmission but has no effect on the inhibitory motoneuronal synapse in either the presence or absence of AVM. Our results provide an explanation of how AVM may cause paralysis of nematodes.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Effects of Delphinium alkaloids on neuromuscular transmission.

The Delphinium alkaloids methyllycaconitine (MLA), nudicauline, 14-deacetylnudicauline (14-DN), barbinine, and deltaline were investigated for their effects on neuromuscular transmission in lizards. The substituent at C14 provides the only structural difference among the alkaloids MLA, nudicauline, 14-DN, and barbinine. Deltaline lacks the N-(methylsuccinyl)anthranilic acid at C18 common to the other four alkaloids. Each alkaloid reversibly reduced extracellularly recorded compound muscle action potential (CMAP) amplitudes in a concentration-dependent manner. The IC(50) values for CMAP blockade were between 0.32 and 13.2 microM for the N-(methylsuccinimido)anthranoyllycacotonine-type alkaloids and varied with the C14 moiety; the IC(50) value for deltaline was 156 microM. The slopes of the concentration-response curves for CMAP blockade were similar for each alkaloid except barbinine, whose shallower curve suggested alternative or additional mechanisms of action. Each alkaloid reversibly reduced intracellularly recorded spontaneous, miniature end-plate potential (MEPP) amplitudes. Alkaloid concentrations producing similar reductions in MEPP amplitude were 0.05 microM for 14-DN, 0.10 microM for MLA, 0.50 microM for barbinine, and 20 microM for deltaline. Only barbinine altered the time constant for MEPP decay, further suggesting additional or alternative effects for this alkaloid. MLA and 14-DN blocked muscle contractions induced by exogenously added acetylcholine. All five alkaloids are likely nicotinic receptor antagonists that reduce synaptic efficacy and block neuromuscular transmission. The substituent at C14 determines the potency and possibly the mechanism of nicotinic acetylcholine receptor blockade for MLA, nudicauline, 14-DN, and barbinine at neuromuscular synapses. The lower potency of deltaline indicates that the N-(methylsuccinyl)anthranilic acid at C18 affects alkaloid interactions with nicotinic acetylcholine receptors at neuromuscular junctions.

Identification of excitatory and inhibitory motoneurons in the nematode Ascaris by electrophysiological techniques.

A physiological preparation in which it is possible to record responses in muscle to stimulation of single motoneurons of the nematode Ascaris lumbricoides is described. With this preparation we have determined the physiological sign (E or I; excitatory or inhibitory) of the neuromuscular synapses of 21 identified motoneurons--12 are excitatory and 9 inhibitory. Ascaris motoneurons had previously been classified by morphological criteria into seven classes (Stretton, A. O. W., R. M. Fishpool, E. Southgate, J. E. Donmoyer, J. P. Walrond, J. E. R. Moses, and I. S. Kass (1978) Proc. Natl. Acad. Sci. U. S. A. 75: 3493-3497). Physiological studies were performed on members of five of these classes. Three classes of neurons (DE1, DE2, and DE3) are excitatory to dorsal muscle cells. Two classes (DI and VI) are inhibitory neurons which innervate the dorsal and ventral muscle cells, respectively. The motoneurons in Caenorhabditis elegans (White, J. E., E. Southgate, J. N. Thomson, and S. Brenner (1976) Philos. Trans. R. Soc. Lond. (Biol.) 275: 327-348) can be divided into seven morphological classes which are very similar to those in Ascaris. Based upon the structure-function correlation in Ascaris, we have predicted which motoneurons are excitatory and which are inhibitory in C. elegans.

Structure and physiological activity of the motoneurons of the nematode Ascaris.

The nervous system of the nematode worm Ascaris contains about 250 nerve cells; of these, the motoneurons consist of five segmental sets, each containing 11 cells. Morphologically, the motoneurons can be divided into seven different types. Their geometry is simple: some are unbranched, others have one branch point, and the most complex have two. There is no neuropil in the nerve cords; synapses are made by axo-axonal contact or onto short spines. These features enable us to study the anatomy and physiology of the system with a degree of completeness that would be difficult in other systems. The physiological activity of five of the motoneurons has been investigated, three being excitatory and two inhibitory. The excitatory motoneurons receive input from intersegmental interneurons. The inhibitory motoneurons do not receive input from the interneurons; instead they receive their input from the excitatory motoneurons in a circuit that can mediate reciprocal inhibition between the dorsal and the ventral musculature.