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Biomedical subjects

C P Ko

Publications and source records attributed to C P Ko.

29 records · Page 2Linked to original sources
PubMed1986

Absence of sterol-specific complexes at active zones of degenerating and regenerating frog neuromuscular junctions.

Freeze-fracture combined with filipin treatment has been used as a cytochemical probe for membrane cholesterol. As previously shown at the frog neuromuscular junction, distinctive sterol-specific complexes were formed on the presynaptic membrane after filipin treatment, except at active zones. The absence of sterol-specific complexes from active zones was confirmed using two other cytochemical agents--digitonin and saponin. We also studied the maintenance and differentiation of the presynaptic membrane heterogeneity revealed by membrane cholesterol probes at degenerating and regenerating neuromuscular junctions. During degeneration, active zones in frog nerve terminals were disorganized, but still lacked sterol-specific complexes. After engulfing the degenerating nerve terminals, Schwann cells occupied the synaptic gutters and displayed a uniform distribution of sterol-specific complexes. Schwann cell ridges opposite the postjunctional folds also had prominent sterol-specific complexes in regions formerly occupied by active zones. By 2 weeks after nerve crush, nerve terminals reinvaded the endplate region and active zones began to regenerate. While the intramembrane particles of the early regenerating active zones were not arranged in the normal double-rowed organization, filipin-sterol complexes were nevertheless excluded from these primitive active zones. Areas of nerve terminal membrane opposite to junctional folds but lacking active zones were covered with filipin-sterol complexes. These results show that the normal double-rowed organization is not required for the expression of the membrane heterogeneity associated with the active zone. In addition, the absence of sterol-specific complexes is closely associated with the active zone particles and not simply the membrane regions opposite to the postjunctional folds. The membrane heterogeneity does not seem to be directly linked with the functional state of the active zone since it is still associated with degenerating active zones after transmission failure has occurred.

Animals↗
PubMed1986

A comparison of active zone structure in frog neuromuscular junctions from two fast muscles with different synaptic efficacy.

To search for ultrastructural correlates of differences in synaptic safety factor and neurotransmitter release, neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi muscles of the grass frog Rana pipiens were freeze fractured. Synaptic efficacy in these muscles was determined by the extent to which isometric twitch tension could be blocked by lowering [Ca2+] in the bathing solution. We found that junctions in the cutaneous pectoris were significantly more effective than those of the cutaneous dorsi. Morphometric analysis of 16 junctions from each type of muscle showed significant differences in some aspects of active zone structure. Cutaneous pectoris terminals had longer active zone segments and active zones spaced more closely together. This resulted in 20% more active zone length per unit terminal length in the cutaneous pectoris. Cutaneous dorsi terminals had active zones that were more often segmented into two or more sections at a single junctional fold. Mean active zone length per junctional fold and the number of active zone particles per micrometre of active zone length were not significantly different. As a result of the somewhat larger terminal width in the cutaneous dorsi, the percentage of terminal width occupied by active zone was greater in the cutaneous pectoris. As an attempt to indirectly estimate active zone spacing with the light microscope, we applied rhodamine-conjugated alpha bungarotoxin to neuromuscular junctions from the cutaneous pectoris and cutaneous dorsi. No significant difference in the spacing of fluorescently labelled acetylcholine receptor bands was found between the two types of junctions. Our results indicated that the greater active zone length per unit terminal length in the cutaneous pectoris was associated with its increased synaptic efficacy. In addition the continuity and particle organization of active zones may have contributed to the observed differences in synaptic safety factor at frog neuromuscular junctions.

Animals↗
PubMed1985

Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs.

Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.

Age Factors↗
PubMed1984

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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

Reinnervation of the amphibian cardiac ganglion after complete or partial denervation.

The interactions between regenerating and sprouted nerve terminals during reinnervation of neurones were tested in the parasympathetic cardiac ganglion in frogs. 1. After partial (unilateral) vagotomy, remaining intact preganglionic vagal axons rapidly sprouted and innervated the entire ganglion. At later intervals after nerve damage, regenerating vagal axons were able to reinnervate ganglion cells despite the presence of synapses from sprouted nerve terminals. 2. When vagal reinnervation took place after unilateral vagotomy, synaptic input from the sprouted vagus nerve declined. 3. The presence of synapses from intact and sprouted nerve terminals in the ganglion after partial denervation measurably delayed the rate of vagal reinnervation. 4. After complete denervation (bilateral vagotomy), ganglionic reinnervation was rapid and complete. However, cells initially received an excessive number of preganglionic inputs and an abnormal distribution of left/right vagal innervation in the ganglion. 5. At long intervals (up to 85 weeks) after ganglionic reinnervation, some reduction of excess vagal inputs took place, indicating there was a slow re-organization of ganglionic synapses. 6. The number of boutons per cell body as revealed by zinc iodide-osmium staining remained constant after vagal reinnervation, despite an initial excessive synaptic reinnervation and subsequent synaptic remodelling.

Action Potentials↗
PubMed1981

Electrophysiological and freeze-fracture studies of changes following denervation at frog neuromuscular junctions.

1. Changes which occur at frog neuromuscular junctions following denervation have been studied by combining intracellular recording and freeze-fracture electron microscopy. 2. Shortly after nerve section, both neuromuscular transmission and intramembrane structures of neuromuscular junctions remain normal. 3. Later, neuromuscular transmission fails, beginning with the disappearance of end-plate potentials (e.p.p.s) and followed by the disappearance of miniature end-plate potentials (m.e.p.p.s). The frequency of m.e.p.p.s which persist after cessation of e.p.p.s is not increased dramatically in K+-rich or hypertonic solutions. 4. Concomitant with the changes of transmission are changes in intramembrane structures. The first sign of these changes in disruption of active zones, which become disorganized, fragmented or vanish. Nerve terminals then disintegrate and eventually are engulfed by Schwann cells. 5. When neuromuscular transmission has failed completely, former sites of the neuromuscular junction are occupied by Schwann cells. These cells develop transverse ridges which lie opposed to junctional folds, just like active zones of nerve terminals. However, the ridges on Schwann cells do not contain organized rows of particles or clusters of any synaptic organelles, even at later stages when Schwann cell m.e.p.p.s commence. 6. It is suggested that the failure of e.p.p.s involves at least an impairment of the transmitter release mechanism at the nerve terminal, which is probably associated with the disruption of active zones. The cessation of m.e.p.p.s is thought to be caused by the engulfment of terminals by Schwann cells.

Animals↗
PubMed1978

Impulse blockade in frog cardiac ganglion does not resemble partial denervation in changing synaptic organization.

Partial denervation of parasympathetic neurons in the frog heart by surgical section of one vagus nerve results in a marked reorganization of functional synaptic connections made by the remaining vagus nerve. These changes are not simply due to a lack of impulse activity per se in the sectioned nerve because blockage of impulses in one vagus with tetrodotoxin-impregnated cuffs did not cause similar changes in the innervation pattern of the ganglion. Furthermore, tetrodotoxin-blocked vagal fibers retain their ability to sprout and can form new synapses on denervated neurons.

Action Potentials↗
PubMed1976

Synaptic transmission between rat spinal cord explants and dissociated superior cervical ganglion neurons in tissue culture.

Physiological properties of the synapses formed between explants of spinal cord and dissociated autonomic ganglion neurons in tissue culture were studied using intracellular and extracellular stimulation and recording techniques (as well as iontophoresis) with a culture perfusion system allowing continuous microscopic observation during repeated changes of the bathing medium. The principal neurons of the superior cervical ganglion (SCGN) were dissociated from perinatal rats and the spinal cord explants were obtained from 15-day rat fetuses; these were allowed to mature for 3-10 weeks in co-culture. Recordings from over 1000 SCGN established that: (a) spontaneous small depolarizations and action potentials occurred in 20% of the SCGN studied, (b) the EPSPs observed in SCGN after spinal cord stimulation were sensitive to decreased Ca2+ and increased Mg2+, as well as to D-tubocurare, hexamethonium and mecamylamine, but not to atropine (at 10(-6) M concentration) or to the alpha-adrenergic blocking agents phentolamine or phenoxybenzamine; no potentiation of the EPSPs was seen with neostigmate or eserine, (c) acetylcholine directly applied to the SCGN was seen to mimic the responses seen after spinal cord stimulation; tetrodotoxin blocked both direct and iontophoretically fired action potentials, with only a suprathreshold acetylcholine potential remaining. These synapses were not sensitive to alpha-bungarotoxin. It is concluded that the synapses formed by spinal cord neurites on principal SCGN in tissue culture are nicotinic cholinergic, and that the evoked EPSPs recorded in this study are thus similar to the orthodromic fast EPSPs observed in vivo. No slow synaptic responses were observed and no demonstrable effects were noted that could be attributed to adrenergic transmission.

Acetylcholine↗
PubMed1976

Synaptic transmission between rat superior cervical ganglion neurons in dissociated cell cultures.

The principal neurons of the rat superior cervical ganglion (SCGN) when established as dissociated cells in tissue culture form synapses among themselves. In the present study we have examined this synaptic interaction when these neurons are co-cultured with several other types of tissues. Dissociated SCGN were prepared from perinatal rats and studied, after 3-4 weeks maturation, with intracellular recording techniques. Synaptic interactions between sympathetic neurons were demonstrated when these cells were: (a) grown with explants from newborn rat thoracic spinal cord, (b) when the SCGN had survived for several weeks subsequent to removal of the spinal cord explants, and (c) when the SCGN were grown in the presence of an adrenergic target (interscapular brown fat cells). Unidirectional, reciprocal, recurrent and complex chemical synaptic networks, consisting of convergence and divergence, characterized connections between SCGN. All synaptic responses were cholinergic since they were reversibly blocked by hexamethonium or mecamylamine but were not sensitive to 10(-5) M phenoxybenzamine. Removal of the spinal cord explants did not significantly alter the proportion of chemical synaptic interactions between SCGN (more than 25%) from matched cultures. Anatomical observations established that in cultures with brown fat, innervating neurites appeared on the fat cells; these neurites frequently expanded to form varicosities that resembled the adrenergic terminals normally seen on brown fat in the animal. Synaptic profiles also occurred on the neurons in these cultures and some of these were shown to be cholinergic. The proportion of neuronal interactions in the combined SCGN + fat cultures was low, however, suggesting that co-culture with target tissue might influence the frequency of interconnections developed between SCGN in culture. Other factors, such as the presence of non-neuronal cells, degree of dissociation, cellular density, culture age and the survival of certain types of SCGN in culture are discussed as variables related to the formation of synapses between SCGN. Non-rectified electrical coupling between SCGN was also observed in 17 out of 679 pairs (2.5%) of neurons. Attenuation factor for electrically coupled action potentials ranged between 1 and 43.5.

Adipose Tissue, Brown↗