Types of nerve terminals in fetal and neonatal rabbit myocardium

Summary With the use of electron microscopy 4 types of axonal profiles were observed in the developing myocardium of rabbits: 1) adrenergic axons which contained mainly small dense-core vesicles and which presumably can store 5-hydroxydopamine; 2) cholinergic axons which contained small clear synaptic vesicles and which were acetylcholinesterase-positive; 3) axons which contained large vesicles filled with moderately electron-dense material and which resembled purinergic axons; and 4) profiles filled with mitochondria, vesicles of various sizes, lysosome-like bodies, and microtubules and which resembled sensory terminals.Supported in part by the Kentucky Heart Association and in part by the Human Development Studies Program, University of Kentucky. Excellent technical assistance of Mrs Merle Wekstein is gratefully acknowledged.     

Cholinergic and adrenergic innervation of the penis artery of the bull: transmitter concentrations and synaptic vesicles.

The penile artery of the bull contained significant amounts of acetylcholine, noradrenaline and dopamine, and its axon profiles contained either numerous small granular or agranular vesicles, as well as some large granular vesicles. In the dorsal metatarsal artery, only noradrenaline and dopamine were detectable, and the axon profiles observed contained numerous small granular vesicles. In the penile artery, the axons with small agranular vesicles, probably cholinergic axons, were in close contact with axons containing small granular vesicles. It is suggested that, in the penile artery of the bull, one of the functions of the cholinergic nerves is suppression of excitatory adrenergic neurotransmission.     

Cholinergic and adrenergic innervation of the penis artery of the bull: Transmitter concentrations and synaptic vesicles

Summary The penile artery of the bull contained significant amounts of acetylcholine, noradrenaline and dopamine, and its axon profiles contained either numerous small granular or agranular vesicles, as well as some large granular vesicles. In the dorsal metatarsal artery, only noradrenaline and dopamine were detectable, and the axon profiles observed contained numerous small granular vesicles. In the penile artery, the axons with small agranular vesicles, probably cholinergic axons, were in close contact with axons containing small granular vesicles. It is suggested that, in the penile artery of the bull, one of the functions of the cholinergic nerves is suppression of excitatory adrenergic neurotransmission.This study has been supported by grants from the Sigrid Jusélius Foundation (to O.E.), from the Medical Research Council of the Academy of Finland (to E.K.) and from the Magnus Bergvall Memorial Fund (to N.O.S.). Excellent technical assistance by Mrs Paula Hasenson and Miss Seija Ovaskainen is gratefully acknowledged.     

Different types of synaptic vesicles in axons of the retractor penis muscle of the bull.

Three types of axon profiles were observed in the smooth muscle of the retractor penis and the penile artery of the bull: 1. profiles containing small granular vesicles, presumably representing adrenergic axons; 2. profiles containing small agranular vesicles, presumably representing cholinergic axons; 3. profiles containing numerous large and small granular vesicles. The third type of profile was not found in the vas deferens or the metatarsal artery. It is therefore possible that this type of profile represents the non-adrenergic, non-cholinergic inhibitory nerves, the presence of which has previously been pharmacologically indicated in these tissues.     

Different types of synaptic vesicles in axons of the retractor penis muscle of the bull

Summary Three types of axon profiles were observed in the smooth muscle of the retractor penis and the penile artery of the bull: 1. profiles containing small granular vesicles, presumably representing adrenergic axons; 2. profiles containing small agranular vesicles, presumably representing cholinergic axons; 3. profiles containing numerous large and small granular vesicles. The third type of profile was not found in the vas deferens or the metatarsal artery. It is therefore possible that this type of profile represents the non-adrenergic, non-cholinergic inhibitory nerves, the presence of which has previously been pharmacologically indicated in these tissues.This study has been supported by grants from the Sigrid Jusélius Foundation (to O. E.), from the Medical Research Council of the Academy of Finland (to E. K.) and from the Magnus Bergvall Memorial Fund (to N. O. S.). Excellent technical assistance by Mrs.Paula Hasenson is gratefully acknowledged.     

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) “pearling” of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, “podiasomes” and PLT “ghosts”. Interestingly, a flow cytometry showed a population of TOM20⁺LC3⁺ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.     

Sequential ultrastructural study of mucosal innervation following parietal cell vagotomy and antrectomy

Rats having undergone parietal cell vagotomy (PCV) or PCV with antrectomy were sacrificed and gastric mucosal samples studies by electron microscopy. Degeneration of axons was followed by the appearance of small, neurotubule-rich axons which increased in size and number with increasing postoperative interval. The source of these regenerating fibers is unknown but may have come from the fundus.     

Sequential ultrastructural study of mucosal innervation following parietal cell vagotomy and antrectomy

Summary Rats having undergone parietal cell vagotomy (PCV) or PCV with antrectomy were sacrificed and gastric mucosal samples studies by electron microscopy. Degeneration of axons was followed by the appearance of small, neurotubule-rich axons which increased in size and number with increasing postoperative interval. The source of these regenerating fibers is unknown but may have come from the fundus.     

The synapsins: beyond the regulation of neurotransmitter release

The synapsins are a family of five closely related neuron-specific phosphoproteins associated with the membranes of synaptic vesicles. The synapsins have been implicated in the regulation of neurotransmitter release. They tether synaptic vesicles to actin filaments in a phosphorylation-dependent manner, controlling the number of vesicles available for release at the nerve terminus. A growing body of evidence suggests that the synapsins play a broad role during neuronal development. They participate in the formation and maintenance of synaptic contacts among central neurons. In addition, each synapsin has a specific role during the elongation of undifferentiated processes and their posterior differentiation into axons and dendrites. In this review, we focus on these novel roles of synapsins during the early stages of development. Received 26 September 2001; received after revision 8 November 2001; accepted 9 November 2001     

Evidence that the establishment of pregnancy requires activation of lipoxygenase and phospholipase-A2

Summary The present work investigates the possibility that lipoxygenase products are involved in the biochemical mechanisms of blastocyst implantation by utilizing nordihydroguaiaretic acid (NDGA) and caffeic acid (CA), inhibitors of lipoxygenase enzymes, and quinacrine (QU), an inhibitor of phospholipase-A₂. It has been shown previously that inhibition of cyclooxygenase results in blockade of implantation. The inhibitors were dissolved in a standard medium and 5 l of the solutions were micro-injected into the uterine horns of day-4 pregnant mice. The contralateral horns acted as controls and received only vehicle. A sham-operated group provided normal controls. In 14 NDGA-treated mice, the control horns contained 40 implantations while the treated horns contained only 6 small implantations and 8 resorbing sites. These control horns were comparable to the sham controls. In 14 CA-treated mice, treated horns contained 17 small implantations plus 4 resorptions, whereas the control horns contained 26 small implantations and 4 resorptions. Twelve QU-treated mice exhibited 7 small implantations and 4 resorptions in the treated horns, plus 24 small sites and no resorptions in the control horns. Fourteen sham-operated mice had 95 implantation sites and no resorptions in their 28 horns. The results provide evidence for the involvement of the lipoxygenase enzymes and phospholipase-A₂ in the initial implantation process and in the subsequent development of early pregnancy.     

Evidence that the establishment of pregnancy requires activation of lipoxygenase and phospholipase-A2

The present work investigates the possibility that lipoxygenase products are involved in the biochemical mechanisms of blastocyst implantation by utilizing nordihydroguaiaretic acid (NDGA) and caffeic acid (CA), inhibitors of lipoxygenase enzymes, and quinacrine (QU), an inhibitor of phospholipase-A2. It has been shown previously that inhibition of cyclooxygenase results in blockade of implantation. The inhibitors were dissolved in a standard medium and 5 microliter of the solutions were micro-injected into the uterine horns of day-4 pregnant mice. The contralateral horns acted as controls and received only vehicle. A sham-operated group provided normal controls. In 14 NDGA-treated mice, the control horns contained 40 implantations while the treated horns contained only 6 small implantations and 8 resorbing sites. These control horns were comparable to the sham controls. In 14 CA-treated mice, treated horns contained 17 small implantations plus 4 resorptions, whereas the control horns contained 26 small implantations and 4 resorptions. Twelve QU-treated mice exhibited 7 small implantations and 4 resorptions in the treated horns, plus 24 small sites and no resorptions in the control horns. Fourteen sham-operated mice had 95 implantation sites and no resorptions in their 28 horns. The results provide evidence for the involvement of the lipoxygenase enzymes and phospholipase-A2 in the initial implantation process and in the subsequent development of early pregnancy.     

Cholera toxin B-subunit incorporation into synaptic vesicles of the neuromuscular junction of the rat

The B-subunit of cholera toxin, a nontoxic macromolecule which binds specifically to GM1 ganglioside, was conjugated to colloidal gold and injected into skeletal muscle of the rat. It was taken up rapidly in vesicles in the terminal axons at the neuromuscular junctions. Injection of albumin-colloidal gold conjugates resulted in an insignificant uptake. The results indicate that uptake of extracellular macromolecules into the terminal axon of the neuromuscular junction may be greatly enhanced by binding to gangliosides at the presynaptic membrane, and that it may occur without association with vesicular recycling related to transmitter release.     

Cholera toxin B-subunit incorporation into synaptic vesicles of the neuromuscular junction of the rat

Summary The B-subunit of cholera toxin, a nontoxic macromolecule which binds specifically to GM1 ganglioside, was conjugated to colloidal gold and injected into skeletal muscle of the rat. It was taken up rapidly in vesicles in the terminal axons at the neuromuscular junctions. Injection of albumin-colloidal gold conjugates resulted in an insignificant uptake. The results indicate that uptake of extracellular macromolecules into the terminal axon of the neuromuscular junction may be greatly enhanced by binding to gangliosides at the presynaptic membrane, and that it may occur without association with vesicular recycling related to transmitter release.The study was supported by grants from the Swedish Medical Research Council (proj. No. 07122).—Part of this study was performed at INSERM U-153, Paris, headed by Dr M. Fardeau.     

Turnover of 5-hydroxydopamine in adrenergic nerves

Summary In adrenergic nerve endings of the guinea-pig vas deferens the population of small granular vesicles increases from 19% in control animals to 80–90% 1–3 h after the administration of 5-hydroxydopamine, and gradually declines to control values in 10 days. Large granular vesicles were also loaded but the loss of enhanced granulation was more rapid than in the small granular vesicles.M. R. C. Scholar. I am greatly indebted to Prof. G. Burnstock, Prof. G. Gray and Dr G. Gabella for helpful advice and criticism. The technical assistance of J. Barron and G. Barrett is gratefully acknowledged.     

Stimulation induced depletion of the synaptic vesicles in excitatory motor nerve terminals of the locust,Locusta migratoria L

Summary Neural stimulation at high frequency induced gross changes in the ultrastructural properties and depleted the synaptic vesicles in fast axon terminals of locust extensor tibiae muscles. After a 1-h-rest the synaptic vesicle populations recovered and the ultrastructural properties of these recovering neuromuscular junctions closely resembled those of the controls.Acknowledgment. The authors wish to thank Mr M. J. Parker for invaluable help with the statistical analyses. R. P. Botham gratefully acknowledges the SRC for financial assistance.     

Non-synaptic chemical neurotransmission.

Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained on their vesicles must act at non-synaptic membranes.     

Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth

Axons of primary olfactory neurons are intimately associated with olfactory ensheathing cells (OECs) from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, little is understood about the nature and role of interactions between OECs and axons during development of the olfactory nerve pathway. We have used high resolution time-lapse microscopy to examine the growth and interactions of olfactory axons and OECs in vitro. Transgenic mice expressing fluorescent reporters in primary olfactory axons (OMP-ZsGreen) and ensheathing cells (S100ß-DsRed) enabled us to selectively analyse these cell types in explants of olfactory epithelium. We reveal here that rather than providing only a permissive substrate for axon growth, OECs play an active role in modulating the growth of pioneer olfactory axons. We show that the interactions between OECs and axons were dependent on lamellipodial waves on the shaft of OEC processes. The motility of OECs was mediated by GDNF, which stimulated cell migration and increased the apparent motility of the axons, whereas loss of OECs via laser ablation of the cells inhibited olfactory axon outgrowth. These results demonstrate that the migration of OECs strongly regulates the motility of axons and that stimulation of OEC motility enhances axon extension and growth cone activity.     

Comparison of protein synthesis profiles in chronic lymphocytic leukaemia cells and B-lymphocytes from peripheral blood,cord blood and tonsil

2D-gel electrophoresis was used to investigate protein synthesis in leukaemic cells from a series of 15 chronic lymphocytic leukaemia (CLL) patients, and in non-malignant B-cell populations from different sources. The protein synthesis profiles of CD5+ B-cells from umbilical cord blood and from tonsil were determined, and the levels of expression of their proteins were observed to be similar to the CLL cells. The CD5-cells from cord blood resembled peripheral blood B-lymphocytes, and the protein synthesis profile of CD5-cells from tonsils was very complex. One protein was also identified which consistently appeared to be synthesised at a low level in CD5+ B-cells from tonsil but which was always more prominant in CLL cells and other non-malignant B-lymphocytes. On the basis of these data it is possible that the closest non-malignant counterpart to CLL is the CD5+ B-lymphocyte from cord blood.     

Distribution of H3-dimetacrine in rat cerebral cortex by electron microscopic autoradiography

Summary The cellular distribution of H³-dimetacrine in rat cerebral cortex was studied by electron microscopic autoradiography. A considerable proportion of autoradiographic grains (40.6%) was located over the synaptic areas, while the other structures (i.e., dendrites, axons, glial and neuronal cells) contained less autoradiographic activity.Acknowledgments. The authors wish to thank Siegfried AG., Switzerland, for the generous supply of 3-bromo-dimetacrine. Dimetacrine was kindly donated by Nippon Chemiphar Co., Ltd., Tokyo, Japan.     

Non-synaptic chemical neurotransmission

Summary Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained in their vesicles must act at non-synaptic membranes.Supported by grant MA4183 of the Medical Research Council of Canada.