Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons.

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.

Synaptic vesicle dynamics in rat fast and slow motor nerve terminals.

We have investigated whether rat motor nerve terminals with different in vivo activity patterns also have different vesicle trafficking characteristics. To do this, we monitored, using combined optical and electrical techniques, the rate of exocytosis (during different frequencies and patterns of activity), the releasable pool size, and the recycle time of synaptic vesicles in terminals on soleus (slow-twitch) and extensor digitorum longus [(EDL); fast-twitch] muscle fibers. EDL terminals had a higher initial quantal content (QC) than soleus, but during tonic or phasic stimulation at 20-80 Hz, EDL QC ran down to a greater extent than soleus QC. By recording loss of fluorescence from exocytosing vesicles labeled with the dye FM1-43, EDL terminals were found to destain faster than those in soleus. Simultaneous intracellular recording of end plate potentials, to count the number of vesicles released, permitted estimation of the total vesicle pool (VP) size and the recycle time by combining the optical and electrophysiological data. Soleus vesicle pool was larger than EDL, but recycle time was not significantly different. These terminals, therefore, are adapted to their in vivo activity patterns by alterations in QC and VP size but not recycle time.

Utrophin abundance is reduced at neuromuscular junctions of patients with both inherited and acquired acetylcholine receptor deficiencies.

Congenital myasthenic syndromes are a heterogeneous group of conditions in which muscle weakness resulting from impaired neuromuscular transmission is often present from infancy. One form of congenital myasthenic syndrome is due to a reduction of the number of acetylcholine receptors (AChRs) at the neuromuscular junction. We describe four new cases of AChR deficiency, characterized by a reduction in both miniature endplate potential amplitude and AChR abundance accompanied by elongation of the neuromuscular junction and some decrease in postsynaptic folding. A number of cytoplasmic proteins are normally associated with the postsynaptic membrane and may contribute to the clustering of AChRs at the neuromuscular junction. We therefore investigated the expression of several of these proteins in these AChR-deficiency patients. In each patient, immunolabelling of the neuromuscular junction for rapsyn, dystrophin, beta-dystroglycan and a form of beta-spectrin was strong but that for utrophin was markedly reduced or absent. This suggested that a defect in utrophin expression might underlie the congenital AChR deficiency. However, a reduction in utrophin labelling was also seen in three patients with adult acquired autoimmune myasthenia gravis in whom AChR loss results directly from the extracellular binding of autoantibodies. We conclude that the loss of AChRs in AChR deficiency does not result from the absence of rapsyn or beta-dystroglycan and that reduction of utrophin is probably secondary to the loss of AChRs. The possible role of AChRs and/or utrophin in determining the extent of postsynaptic folding is discussed.

Spatial relationships of utrophin, dystrophin, beta-dystroglycan and beta-spectrin to acetylcholine receptor clusters during postnatal maturation of the rat neuromuscular junction.

At the adult mammalian neuromuscular junction, acetylcholine receptors are concentrated at the tops of the postsynaptic folds and voltage-gated sodium channels are concentrated in their depths. It is likely that this arrangement involves linkage of the ion channels to components of the underlying membrane cytoskeleton. In rats, the mature distribution of acetylcholine receptors arises as part of the developmental remodelling of the junctional region during the first few weeks after birth. We have followed the changes during this period in the distribution of four proteins associated with the postsynaptic cytoskeleton at mature neuromuscular junctions (utrophin, dystrophin, beta-dystroglycan and beta-spectrin) to see if any of them co-localizes with acetylcholine receptors during the remodelling process, as would be required if it serves to link acetylcholine receptors to the cytoskeleton. Each protein was visualized with specific monoclonal antibodies and its distribution at various stages was compared with that of the acetylcholine receptors, labelled with alpha-bungarotoxin. We also related the changes in distribution of these postsynaptic proteins to the main stages in fold formation and, in the Discussion, to reported observations of the accumulation of voltage gated sodium channels during development. Our results show that utrophin labelling is closely co-localized with that of acetylcholine receptors throughout postnatal maturation. beta-dystroglycan labelling is present at most sites of high acetylcholine receptors density throughout maturation although it often extends beyond the region of highest acetylcholine receptors labelling density. By contrast, dystrophin and beta-spectrin labelling is not consistently concentrated at most neuromuscular junctions until after P7 and P14 respectively.

Illumination partly reverses the postsynaptic blockade of the frog neuromuscular junction by the styryl pyridinium dye RH414.

The styryl pyridinium dye RH414, which we have used recently for optical monitoring of synaptic vesicle membrane trafficking in frog motor nerve terminals, reversibly inhibited contractions in the frog nerve-muscle preparations used for those studies. We report here the results of experiments into the basis of this inhibition and how the blocking effects can be modulated by incident light, by using conventional intracellular recording and iontophoretic techniques. Bath application of 5-42.5 microM RH414 blocked nerve-evoked twitches in frog cutaneus pectoris muscles, although subthreshold endplate potentials and miniature endplate potentials could still be recorded. In the presence of the dye, illumination of the recording area with light from a mercury arc lamp over a wide range of wavelengths (340-560 nm) potentiated the amplitude of endplate potentials, miniature endplate potentials, and depolarizations resulting from iontophoretic application of acetylcholine. The magnitude of both the light-induced disinhibition of endplate and iontophoretic potentials increased with the intensity of the illumination and developed exponentially with a time constant of several hundred milliseconds. Both the dye-induced inhibition and the light-induced disinhibition disappeared after washing in dye-free physiological saline. Muscle fibre resting membrane potential, input conductance and miniature endplate potential frequency, however, were not affected by these manipulations. These data are consistent with a specific, curare-like interaction of the dye with acetylcholine receptors which can be modulated by light.

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle.

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. Neuro Report 1992; 3: 857-860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by approximately 0.3 microns, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.

Restoration of motor unit properties and fiber type distribution in reinnervated axolotl skeletal muscle.

The contractile properties of functionally isolated motor units and the muscle fiber type distribution of reinnervated iliotibialis posterior muscles were examined in axolotls (Ambystoma mexicanum) 7 to 12 months after complete transection of the hind limb nerve trunks. Motor units were continuously distributed with respect to size and contractile speeds and there was a positive correlation between motor unit size and twitch:tetanus ratio. The degree of overlap between motor units was positively correlated with motor unit size and inversely correlated with similarity of contractile speeds. In addition, the muscle fiber type distribution within the reinnervated muscles, as revealed by immunocytochemical localization of tonic, slow, and fast myosin isoforms, was indistinguishable from that in the contralateral unoperated muscles, with no evidence of fiber type grouping. Thus, the motor organization after reinnervation was very similar to that of normal muscles examined previously. The only difference found was that a small number of motor units contracted unusually rapidly in reinnervated muscles. The absence of muscle fiber type redistribution and the essentially normal motor unit characteristics suggest that the original pattern of synaptic connections was reestablished following reinnervation. These observations are consistent with the returning motor neurons selectively reinnervating their original muscle fiber types.

Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction.

1. Frog cutaneous pectoris motor nerve terminals were loaded with the fluorescent dye FM1-43, which produced a series of discrete spots along the length of terminals, each spot evidently marking a cluster of synaptic vesicles. Terminals were imaged for 2-10 min as they destained during repetitive nerve stimulation. Endplate potentials (EPPs) were recorded simultaneously from the muscle fibres innervated by these terminals; their summed amplitudes provided a measure of cumulative transmitter release. 2. Individual fluorescent spots in any one terminal varied in initial brightness but destained at similar fractional rates. 3. The rates of cumulative transmitter release and destaining increased with stimulus frequency in the range 2-30 Hz. At 40 Hz, however, both transmitter release and destaining were slower than at 30 Hz. 4. In twenty-six experiments, rates of dye loss and transmitter release were compared quantitatively. When the time course of summed EPPs was scaled to fit the time course of dye loss during the first 30-60 s of destaining, the two curves usually diverged at later times, the dye loss curve falling below the summed EPP curve. Thus, assuming that dye loss and transmitter release are proportional at early times, at later times the rate of dye loss decreases relative to the rate of transmitter release. 5. At stimulus frequencies from 2 to 30 Hz, the results could be fitted by a simple model in which vesicles lose their dye during exocytosis and, after a fixed recycle 'dead time', they re-enter the vesicle pool, mixing randomly with other vesicles. 6. Unlike stimulation at lower frequencies, at 40 Hz dye loss and summed EPP amplitude curves did not significantly diverge. Stimulation periods lasted up to about 2 min. Interpreted according to the model of vesicle recycling, this suggests that vesicle recycling is inhibited at 40 Hz. 7. The model led to predictions about the relative number, N, of vesicles (labelled and unlabelled) in the terminal at any time during stimulation. The calculated value of N decreased at times less than the recycle 'dead time', and then increased, reflecting the appearance of recycled vesicles in the vesicle pool. 8. From estimates of N and recorded EPP amplitudes, the fraction of vesicles released per shock, F, could be calculated during the entire stimulation period. At low stimulus frequencies (2-5 Hz), after an initial rapid fall, F decreased slowly and monotonically by about 50% in 6 min. At higher stimulus frequencies, a different process was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of FM1-43 staining patterns and electrophysiological measures of transmitter release at the frog neuromuscular junction.

Frog motor nerve terminals were stained with the activity-dependent dye FM1-43, which appears to stain recycled synaptic vesicles. Superficial end plates which could be visualized in their entirety were imaged and end plate potentials (EPPs) evoked by low frequency nerve stimulation were recorded from the muscle fibers which were innervated by the imaged terminals. Curare was present to block muscle contractions. The amplitude of the EPPs correlated reasonably well with the number of fluorescent spots counted in the terminals (r = 0.68). Each fluorescent spot probably represents a cluster of synaptic vesicles localized at an active zone. Several other morphological and electrophysiological values were measured and calculated. The results are consistent with the ideas that FM1-43 stains recycled synaptic vesicles, and that the number of vesicle clusters in a terminal is a good predictor of synaptic efficacy.

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.

Different distributions of dystrophin and related proteins at nerve-muscle junctions.

The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.

Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.

Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.

The fluorescent dyes FM1-43 and RH414 label motor nerve terminals in an activity-dependent fashion that involves dye uptake by synaptic vesicles that are recycling. This allows optical monitoring of vesicle recycling in living nerve terminals to determine how recycled vesicles reenter the vesicle pool. The results suggest that recycled vesicles mix with the pool morphologically and functionally. One complete cycle of release of transmitter, recycling of a vesicle, and rerelease of transmitter appears to take about 1 minute.

Characteristics of end-plates formed in mouse skeletal muscles reinnervated by their own or by foreign nerves.

Morphological and electrophysiological techniques were used to study the neuromuscular junctions of soleus and extensor digitorum longus muscles in normal mice and 6 months after reinnervation by either their original or foreign nerves. In muscles reinnervated by foreign nerves, there were increased incidences of morphological abnormalities, including ultra-terminal axonal sprouting, multiaxonal innervation of end-plates, and ectopic synapse formation, as compared with both normal muscles and muscles reinnervated by their correct nerves. In spite of the morphological abnormalities, however, there was no evidence that the effectiveness of synapses (as estimated from the mean quantal content, m, of end-plate potentials) formed between nerves and inappropriate muscles was impaired. As had also been found in normally innervated muscles, the value of m was again higher in extensor digitorum longus than in soleus following reinnervation by inappropriate nerves. These results suggest that in mammals, when muscles are reinnervated by foreign nerves, mechanisms exist to maintain the efficacy of neuromuscular transmission, in contrast to the situation in amphibians (Sayers and Tonge, 1982: J. Physiol. (Lond.), 330:57-68). The abnormalities observed in this study following reinnervation may be a morphological manifestation of these corrective mechanisms.

Organization of motor units in the axolotl: a continuously growing animal.

The characteristics of motor units in the iliotibialis posterior muscle of the axolotl hindlimb are described. Tension recording and intracellular electrophysiological methods demonstrate that the physiological properties of the population of motor units are continuously distributed rather than grouped into a series of discrete types. Overlap between motor units occurs and this is positively correlated with motor unit size but negatively correlated with differences in time to peak tension. Immunocytochemical staining with antimyosin antibodies combined with histochemical demonstration of actomyosin ATPase activity revealed at least four types of muscle fibre which were distributed asymmetrically within iliotibialis posterior. The results are discussed in terms of the continuous growth of the muscle and the interactions between muscle and nerve in the formation of the axolotl motor system.