Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region

The properties and connections of supernumerary sensory and motor nerve cells in the central nervous system of an abnormal leech

The characteristic features of individual neurons in leech ganglia are highly consistent from animal to animal. Hy chance a leech was dis‐ covered that had more cells to be seen than usual in many of its ganglia. In some segmental ganglia of the abnormal animal there were four or even five sensory cells responding independently to touch (T cells) instead of the usual complement of three on each side. Ganglia also contained three or four pressure sensitive cells (P cells) and three or four nociceptive cells (N cells) instead of two of each. The membrane properties, as well as the shapes, sizes, and positions, of all these cells were normal. Their axons reached the skin by the appropriate nerve bundles. Two sensory neurons of one modality, often independently innervated, overlay regions of skin that normally would be supplied by one cell. On the other hand, the areas innervated by some supernumerary cells were abnormal in their shape and position. In some ganglia instead of one motor neuron that raised the skin into bumps (the annulus erector, or AE cell) there were two, each of which could independently erect annuli. The regions innervated by the extra AE cells appeared normal and overlapped extensively. Normal synaptic interactions were found between supernumerary sensory cells and motor cells. The results with this animal indicate that two or more nerve cells can form effective connections with other neurons and with skin that normally would be innervated by a single cell

Inducing Ulnar Nerve Function while Eliminating Claw Hand and Reducing Chronic Neuropathic Pain

Ulnar nerve injury induces chronic neuropathic pain and is frequently devastating due to loss of cupping the hand around objects (finger clawing) and diminished grip strength. There is little chance of restoring good function, eliminating finger clawing, or reducing the pain. A novel technique was tested for its efficacy in promoting ulnar nerve function and reducing finger clawing and chronic neuropathic pain. A 25-year-old subject presented 5.7 months after a wrist gunshot that created three nerve gaps proximal to the deep ulnar nerve branch. He sought restoration of function due to developing ulnar nerve injury-induced claw hand and increasingly severe chronic neuropathic pain. After resection of the scarred nerve tissue, each gap was 10 cm long. The gaps were bridged with two nonreversed sural nerve grafts within a PRP-filled NeuroMend collagen tube (Collagen Matrix, Oakland, N.J.). Some axons regenerated entirely across all three 10-cm-long repaired nerve gaps, restoring excellent topographically correct sensitivity of S4, including two-point discrimination of 4 mm, good M4 motor function, and full ROM. The ulnar nerve injury-induced finger clawing was eliminated, and the chronic neuropathic pain of 7 was reduced to 0 on a 0–10 validated scale and did not return over the following 3.75 years. Thus, this novel technique induces good sensory and motor function, despite repairing three 10-cm-long nerve gaps while eliminating ulnar nerve injury-induced hand clawing and chronic neuropathic pain. Further studies are required to determine whether the effects were due to PRP