Anatomical and electrophysiological study of multitransmitter neuron R14 of Aplysia.

This study provides detailed information on the Aplysia neuron R14, including its endogenous electrical activity and extensive axonal projections to a variety of vascular and vascular-related tissues. With the aid of intracellular recording techniques, R14 was found to display in vitro variable spontaneous patterns of silent, beating, or bursting activity. Electrophysiological tracing and intracellular cobalt staining revealed the peripheral processes and target tissues of R14. The white-colored axons of R14 exit the parietovisceral ganglion in the genito-pericardial, spermathecal, branchial, and vulvar nerves. These processes extended 20 mm or more into peripheral tissues: the pericardial wall and lumen, digestive gland sheath, aortae, arteries, and veins. R14 axons also project to the right bag cell cluster. Its extensive axonal projections to tissues associated with the cardiovascular system verify physiological studies that show that R14 plays a role in cardiovascular regulation. This neuron appears to have a wide influence over several aspects of circulation in contrast to individual neurons of the R3-13 group, each of which projects to limited numbers of vascular and vascular-related tissues. R14 also uniquely innervates digestive tissues, thus suggesting that it may act as a nexus between influences on digestive and renal physiology such as ion/water regulation, in addition to modulating cardiovascular homeostasis.

Anatomy and ultrastructure of the axons and terminals of neurons R3-R14 in Aplysia.

Using light and electron microscopy and autoradiography, we have traced the axons of neurons R3-R14 in the parietovisceral ganglion (PVG) of Aplysia to terminal fields associated with vascular tissue. The axons are identified by their large size (15-30 micrometer diameter), extensive glial infolding, characteristic dense core vesicles (DCV; approximately 180 nm diameter), and specific, rapid uptake of 3H-glycine. Each neuron in this homogeneous group sends an axon via the branchial nerve to the pericardial region surrounding the junction of the efferent gill vein and the heart. R14 also sends axons to major arteries near the PVG. The R3-R14 axons branch extensively; we estimate that there are at least several hundred per cell. Branches along axons in the branchial nerve exit the nerve, subdivide, and end blindly in the sheath which is bathed by hemolymph. Similar blind endings from R3R14 occur in the sheath of the PVG (Coggeshall, '67). Axonal branches in the pericardial region and the special R14 axons in the arterial walls form both varicose endings near and terminals in contact with vasvular smooth muscle. All R3-R14 endings are free of glia, packed with DCV, show occasional omega-shaped profiles and rapidly take up 3H-glycine. R3-R14 manufacture specific low molecular weight peptides (Gainer and Wollberg, '74), and both the cell bodies (Iliffe et al., '77) and the germinals contain unusually high concentrations of glycine. The presence of peptides as putative neurohormones and sheath endings (neurohormonal release areas) are consistent with R3-R14 being neurosecretory (Coggeshall et al., '66). While glycine could not be a circulating hormone due to its high circulating levels (Iliffe et al., '77), glycine could act as a local chemical messenger between R3-R14 and smooth muscle. The terminal morphology of R3-R14 is consistent with these neurons having both synaptic-type and neurosecretory-type functions.

Anatomy and innervation of the anterior aorta of Aplysia and the ultrastructure of specialized neuromuscular junctions on vascular smooth muscle.

The fine structure of the cellular layers and innervation of smooth muscle in the anterior aorta of Aplysia were examined. The inner layer of circular muscle is not innervated but its fibers may be electrically coupled. In contrast, longitudinal fibers in the outer layer are well separated and richly innervated by highly specialized neuromuscular junctions (NMJ). Three distinct types of NMJ are present on this smooth muscle, each identifiable by a set of quantitatively described morphological features including size, degree of contact with sarcolemma, density of active zones, number of mitochondria and vesicular content. The three types of NMJ are likely to arise from the identified serotonergic (RDAAE), cholinergic (RDAAI), and glycinergic (R14) neurons that provide the major known excitatory, inhibitory, and modulatory inputs to this vessel. Each longitudinal muscle fiber is separately innervated by one or more NMJ of each type. Since there are no intercellular junctions between longitudinal fibers, coordination of contractility is clearly a function of the pattern of neural activity. This report further characterizes the rapid and fine control of the vasculature in Aplysia and demonstrates the utility of this preparation for cellular-level studies on the neural control of smooth muscle and neurochemical messengers mediating its activity.

Localization of axonally transported [3H]glycine in vesicles of identified neurons.

A specific association of axonally transported, free [3H]glycine with vesicles in the identified neurons R3-R15 of Aplysia is demonstrated by high resolution autoradiography. The association of glycine with vesicles, the first such finding in any animal for a neuroactive amino acid, adds to evidence that glycine may be utilized as a neuro-chemical messenger by R3-R14.

Peripheral axons of the parabolic burster neuron R15.

In a combined electrophysiological and anatomical study, the parabolic burster neuron R15 was found to project axons through the genito-pericardial nerve onto the pericardial wall and digestive gland sheath and, more variably, into the heart and pericardial coelom. Projection into these tissues is consistent with the hypothesis that R15 is neurosecretory and may play a role in circulation and/or ion-water regulation in Aplysia.

Specific glycine uptake by identified neurons of Aplysia californica. I. Autoradiography.

The identified giant neurons R3-R14 in the Aplysia parietovisceral ganglion (PVG) have a rapid, Na+-dependent and Hg2+-sensitive uptake system for glycine not found in neighboring neurons. In autoradiographs of PVG incubated in [3H]glycine (glutaraldehyde fix), the cytoplasms of R3-R14 have 3--4 times more silver grains (No./100 sq.micrometer) than other neurons. The glycine uptake system in R3-R14 is selective (alanine, serine, leucine, and proline are taken up equally by all neurons) and is unaffected by reserpine and anisomycin. Neurons R3-R14 contain 2 times less label when ganglia are fixed in formaldehyde than when glutaraldehyde is used as a fixative. Because formaldehyde fixes free amino acids poorly, much of the glycine taken up by R3-R14 is, therefore, not incorporated into protein. In autoradiographs of PVG incubated in [3H]glycine, silver grains are distributed randomly throughout the cytoplasm and nucleus of R3-R14; no association of the grains with the dense core granules characteristic of these neurons7 or other cellular components was found. In contrast, grains in the neurosecretory "bag cells" of the PVG were clustered in numerous discrete areas of the cytoplasm (Golgi complex areas) and the nucleus was only sparsely labeled. The existence of a rapid and selective glycine uptake system in R3-R14, together with their high endogenous glycine concentrations17, suggests that glycine may be a neurotransmitter in these neurons.

Modulation of arterial muscle contraction in Aplysia by glycine and neuron R14.

Glycine and electrical activity in neuron R14 both enhance the contractility of the anterior aorta of the gastropod Aplysia californica. Glycine and R14 do not seem to cause contraction directly, change membrane permeabilities or alter junctional potentials occurring in the muscle fibers, yet they increase the force of contractions induced by other means. Modulation of muscle contraction is a new function for glycine.

Specific glycine uptake by identified neurons of Aplysia californica. II. Biochemistry.

Glycine is taken up twice as rapidly by neurons R3-R14 as by other identified neurons in the Aplysia parietovisceral ganglion. Earlier studies had shown that R3-R14 have much higher glycine concentrations than other Aplysia neurons. Most of the glycine taken up by R3-R14 was biochemically untransformed for at least 1 h following its uptake. Glycine is actively transported into into R3-R14 and other Aplysia neurons by carrier-mediated processes. Glycine uptake by R3-R14 was markedly reduced in the absence of Na+ and in the presence of Hg2+, while these treatments had little effect on glycine uptake by other Aplysia neurons. There appears to be a special glycine uptake system present in R3-R14 and a general glycine uptake system common to all Aplysia neurons. The elevated glycine concentrations and special glycine uptake associated with R3-R14 may indicate that glycine is utilized as a neurotransmitter by those neurons.

Metastatic spread of fibrosarcoma of bone; A report on forty-nine cases, and a comparison with osteosarcoma.

Of tumours arising in otherwise normal bones, fibrosarcoma is about one-third as common as osteosarcoma and may have a very slightly better prognosis. A comparison of the aetiology and behaviour of forty-nine fibrosarcomata and 152 osteosarcomata indicates several similar features. Fibrosarcoma lacks the characteristic peak incidence in adolescence of osteosarcoma, but the age and sex distributions of both tumour types in patients of middle life--twenty-five to sixty-five years--are remarkably similar, even in their frequency. With fibrosarcoma, perhaps, lung metastases are fewer and appear later, thus contributing to the slightly better survival, but there is some increase in the proportion of extra-pulmonary secondaries. As with osteosarcoma, patients with fibrosarcoma show some increase in the length of post-metastatic survival when metastases are of later appearance. For the whole series the five-year crude survival rate was 21 per cent, better results being recorded for patients with histologically well differentiated tumours (30 per cent) and for long bone tumours when the patient was metastasis-free initially and the tumour was treated by prompt ablation (40 per cent). These are probably the best results one may expect for osseous fibrosarcoma without recourse to adjuvant antimetastatic therapy. Complete control of the primary tumour is likewise mandatory, and can be assured only by complete surgical removal when this is technically feasible.

Chondrosarcoma of the bones of the hand.

Nineteen chondrosarcomas are reported arising in proximal phalanges or metacarpal bones of the hand mainly in elderly patients, predominantly women. The usual clinical presentation was of a progressively painful large tumour, often arising in a dormant lesion near the metacarpo-phalangeal joint. Radiologically most showed some bone expansion with a poorly defined area of destruction and a considerable soft-tissue swelling. Histologically, malignancy was usually obvious, but confusion might arise from the inclusion of bland areas of chondromatous tissue that probably represented the original lesion. Four tumours, initially curetted and grafted, recurred locally and necessitated amputation of the digit or ray. Amputation was the primary treatment for fourteen other tumours and was curative except in one patient who eventually needed amputation through the forearm for a large second recurrence. One tumour was satisfactorily controlled by excision of the affected phalanx. None of these nineteen tumours is known to have metastasised. Correct treatment implies a carefully considered balance between conservation of function and complete removal of all tumour tissue.

Ewing's tumour. A study of behaviour and treatment in forty-seven cases.

Forty-seven histologically confirmed cases of Ewing's tumour are reported, with typical age, sex and skeletal distributions. The disease-free survival curves are like those for osteosarcoma of corresponding types of bone, but with higher mortality. The two-year disease-free survival rates were 24 percent for tumours of long bones and 5 per cent for those of other bones. Osseous metastases were most frequent, although the lung was the commonest single site involved. There were secondary deposits in lymph nodes in nine patients, possibly also in four more with mediastinal enlargement. Although initially radiosensitive, one-third of tumours recurred locally, almost always with the appearance of metastases. Neither whole bone irradiation nor high dosage alone ensures complete control of the primary tumour, and it is suggested that recently reported improved results of treatment should be attributed to adjuvant multi-drug chemotherapy. Inadequate control of the primary tumour diminishes the chance of survival and for some sites radical operation merits reconsideration.

Cobalt toxicity after McKee hip arthroplasty.

The significance of cobalt as a cause of symptoms after McKee hip arthroplasty is discussed. Seven patients are described in whom such arthroplasties. became unsatisfactory after periods varying from nine months to four years. Six of these patients were cobalt-positive but nickel- and chrome-negative on patch testing. Macroscopic and histological necrosis of bone, muscle and joint capsule around the prostheses was found in five patients whose hips were explored. The symptoms were progressive pain, a feeling of instability, and in two cases spontaneous dislocation. Radiological features included acetabular fracture, bone resorption, loosening and dislocation of the prosthesis. Increased cobalt concentrations (determined by atomic absorption spectrophotometry) in the urine of four patients and in a variety of tissues in one patient are presented. Patch testing is recommended in the investigation of patients with troublesome McKee hip arthroplasties

Osteosarcoma in children. A study of 125 cases.

A study is presented of the aetiology and results of treatment in a group of 125 proven osteosarcomas present in children under fifteen years of age. These cases have been collected from the records of one English and six European treatment centres. There is a slight male preponderance, but the striking aetiological feature is the very high proportion of tumours of the long bones of the limbs (96 per cent). The two and a half and five year disease-free survival rates were respectively 15 and 12 per cent, with a further 9 per cent still living, but under observation for less than two and a half years. Evidence of metastasis after two and a half years is very unusual, but no child with a tumour of an axial or girdle bone lived this length of time. Although the differences in the results of the different methods of treatment employed are not statistically valid, the largest number of long survivors had been treated by early amputation, which method also provided the lowest rate of local tumour recurrences. Reasons are discussed which indicate that prompt ablation is the treatment of choice, perhaps with certain advantages in the light of recent advances in adjuvant treatment. The past situation in connection with childhood osteosarcoma certainly provides strong support for immediate carefully designed clinical trials of the new adjuvant methods cited.

Influences of glycine and neuron R14 on contraction of the anterior aorta of Aplysia.

Firing neuron R14 in the Aplysia parietovisceral ganglion enhances the force of stimulated contractions and causes rhythmic contractions rather than individual contractions in response to a single stimulation of the anterior aorta of Aplysia. Bath application of 1.0 mM glycine to the artery causes a small depolarization, rhythmic contractions and enhances the force of individual, neurally induced contractions of the anterior aorta. These observations suggest that the physiological role of the innervation of the anterior aorta by R14 is to convert the mode of contraction of the anterior aorta from direct neural control to a myogenic mode. R14 activity does not produce classical junction potentials and inotophoretically applied glycine does not produce detectable potential changes in the anterior aorta muscle fibers. Glycine increases the force and frequency of Aplysia heart beat, and blood pressure. Glycine causes release of preloaded Ca2+ from the ventricle of the Aplysia heart, implying that glycine causes intracellular release of Ca2+. The similarity of the actions of glycine and electrical activity in R14 extend previous evidence that R14 may utilize glycine to modulate the contractility of Aplysia circulatory muscle.