Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

Innervation and membrane specialization at neuromuscular junctions in submaxillaris muscle of the frog

The submaxillaris muscle of the frog after zinc iodide-osmium staining reveals the presence of polyneural innervation. Cholinesterase staining shows that the longer terminals have postsynaptic folds whereas the smaller terminals (up to 5 micron) lack them. Thin-section electron microscopy shows that muscle fibers with or without an M line have terminals with and without postsynaptic folds. The terminals with postsynaptic folds have presynaptic membrane outpocketings above folds. These outpocketings are rudimentary or absent in the terminals without postsynaptic folds. In longer junctions, the P face of the presynaptic membrane has double rows of paired particles on active zone ridges perpendicular to the axis of the muscle. In smaller junctions active zone ridges are rudimentary or absent and double rows of particles form various patterns. Postsynaptic active zones in longer junctions consist of clusters of particles leaving gaps in between, whereas in the smaller junctions they lack gaps. The polyneural innervation and different deployment of membrane particles at neuromuscluar junctions could be a factor responsible for different physiological properties of this muscle.     

Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.     

Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze- fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.     

Exocytosis and its control at the synapse.

Several new approaches have given fresh insight into the mechanism and control of exocytosis. Electrophysiological and morphological studies show that many or all of the intramembrane particles at presynaptic active zones are voltage-gated Ca2+ channels. The sensitivity and time resolution of voltammetry allow the time course with which a single vesicle releases transmitter to be studied. Membrane proteins of the cell surface and synaptic vesicles have been shown to interact, and may join to form the fusion-pore complex.     

Structural changes after transmitter release at the frog neuromuscular junction

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.     

A freeze-fracture study of membrane events during neurohypophysial secretion

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.     

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.     

Microdomain arrangement of the SERCA-type Ca2+ pump (Ca2+-ATPase) in subplasmalemmal calcium stores of paramecium cells.

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of approximately 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( approximately 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.     

Sodium-calcium exchange affects local calcium signal decay and the rate of exocytotic secretion in single chromaffin cells

The effects of Na+ deprivation on local calcium signal decay and the rate of exocytotic secretion were measured in single bovine chromaffin cells to determine whether Na-Ca exchange influences the local cytosolic Ca2+ signal for neurohormone release. Na+ replacement with N-methylglucamine caused a marked slowing of the decay of the local Ca2+ signal near points of its initiation, as measured by high-resolution fluorescent Ca2+ imaging in the confocal laser scanning microscope. Na+ replacement also resulted in a doubling of the rate and magnitude of exocytotic secretion measured in single cells by high-resolution microamperometry. Release rates provide an independent measure of local active zone Ca2+. Five repetitive stimulations of the same cell in Na+-free, but not in Na+-containing, medium resulted in a progressively increasing rate of catecholamine release, suggesting an increasing level of active zone Ca2+ and a role of Na-Ca exchange activity in Ca2+ clearance between stimulations. As secretory activity and its triggering Ca2+ signals are known to be co-localized in active zones along the plasma membrane, the results suggest that Na-Ca exchange may influence the decay of the local Ca2+ signal for exocytotic secretion. This would be consistent with a contribution to local Ca2+ clearance by a novel mechanism utilizing the insertion of secretory vesicle Na-Ca exchangers into the plasma membrane during exocytosis.     

Structural organization of the "zipper line" in Drosophila species with giant spermatozoa

The "zipper line" of Drosophila melanogaster and of Drosophila species characterized by giant spermatozoa (D. hydei, D. kanekoi and D. bifurca) was studied by electron microscopy using conventional thin-sections, lectin labeling and freeze-fracture replicas. In cross sections the membrane specializations are located either at the level of the short cistern close to the large mitochondrial derivative where a small tuft of glycocalyx is visible or, in species characterized by long spermatozoa, along a cistern beneath the plasma membrane. In correspondence of such cistern, the plasma membrane exhibits a thick and extended glycocalyx. At this level, as well as at the short tuft of D. melanogaster, alpha-mannose residues were detected. The "zipper" of D. melanogaster consists of rows of intramembrane particles longitudinally disposed along the sperm tail and associated with the external face of the plasma membrane. On the protoplasmatic face a narrow ribbon of transversal grooves is visible. Freeze-fracture replicas have revealed, in the region characterized by extended glycocalyx, the presence of a large ribbon of intramembrane particles disposed in parallel transversal rows, associated with the protoplasmatic membrane face. On the complementary external face a ribbon of parallel transversal grooves was observed. It is suggested that membrane specializations are mechanical devices to protect spermatozoa from torsion and bending in the seminal vesicles and then in the female storage organ.     

Fine structure of the palisade zone of the rat median eminence as revealed by freeze-fracturing

Summary Nerve terminals in the palisade zone of the rat median eminence were investigated with freeze-fracture electron microscopy. Fracture face P of the specific terminals showed two populations of intramembranous particles (IMP): a large and a small variety. The large IMP-s often formed small irregular clusters. In nerve terminals the total number of both populations of IMP-s was considerably less than that found on P membrane faces of ependymal feet. On fracture face E of the nerve terminals, the number of IMP-s was about a quarter of that seen on fracture face P.On both fracture faces of most terminals a few small round impressions (or elevations respectively) were found which may be interpreted as broken necks of either exo- or endocytotic vesicles. Neither gap nor tight junctions occurred at lateral membranes of the specific axon terminals. Similarly, no membrane specializations were observed with freeze-fracturing on membrane areas adjacent to the basement membrane. The findings are discussed in relation to a possible exocytosis mechanism of the hypothalamic releasing and inhibiting hormones.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

The Action of Black Widow Spider Venom on Cholinergic Mechanisms in Synaptosomes

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high-affinity choline uptake into the preparation. Both activities occurred in the absence of [Ca²⁺]₀. When Na⁺ in Krebs-Ringer was replaced isotonically by sucrose, BWSV did not cause any release of [³H]ACh. On the other hand, BWSV was still effective if Na⁺ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10^?5 M) failed to prevent the ACh-releasing action of the venom. The uptake of [³H]norepinephrine and [³H]tyrosine into the P₂ fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of [³H]deoxyglucose was slight. In addition, the venom-induced release of [³H]norepinephrine was much higher than that of [³H]deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage-sensitive fluorescence probe, 3, 3′-dipentyl-2, 2′-oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca²⁺ -free or K⁺ -free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P₂ fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein-cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P₂ preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.     

Exocytosis and membrane recycling

Exocytosis implies the fusion of the membrane of secretion granules with, and the insertion into, the plasmalemma. In non-growing systems such an insertion is temporary in that the inserted membrane is eventually removed. Turnover results indicate that the removed membrane is not destroyed but recycled within the cell and reused. In some systems exocytosis occurs over the entire plasmalemma, while in others it is restricted to discrete regions, characterized by peculiar morphology and composition. Thus the fusion of the two membranes is probably preceded by a recognition step. Structural specializations were detected in interacting granule and plasma membranes by freeze-fracture and surface labelling techniques: arrays of intramembrane particles in protozoans and nerve terminals; clearing of particles and surface antigens in other systems. Direct evidence, obtained in some secretory systems, indicates that after exocytosis the granules and plasma membranes do not intermix, but remain segregated. The subsequent recapture of membrane patches of the granule type (in many systems by means of coated pits and vesicles) could then account for the striking specificity of the recycling process, documented by both composition and structural studies. In different systems the recycling of granule membranes is carried out at greatly different rates. Recent results in the parotid gland and neuromuscular junction indicate that this process is Ca2+-dependent.     

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.     

Presynaptic active zones at neuromuscular junctions of larval frogs

Freeze-fracturing presynaptic membranes at tadpole neuromuscular junctions display small clusters of large P-face particles, including short double linear arrays. Short pairs of double particle rows are randomly oriented at some junctions. At others, presynaptic membranes are crossed at regular intervals by long pairs of double rows indistinguishable from those characterizing the active zones of adult amphibian neuromuscular junctions. Formation of double particles rows, pairing of the double rows, and transverse alignments of the pairs are shown to be independent processes.     

Pre- and post-synaptic structures in insect CNS: intramembranous features and sites of alpha-bungarotoxin binding

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous articles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated alpha-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that alpha-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.     

Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.