Cell contacts and smooth muscle bundle formation in tissue transplants into the anterior eye chamber

Summary Segments of the taenia coli from guinea-pig were transplanted into the anterior chamber of the eye. Depending on such factors as the total volume of the transplant and the presence or absence of ganglion cells degeneration was either very extensive (90% or more of the total number of muscle cells) or localized (alternating regions of degenerating and normal structure). During days 1–2 muscle cells lost their plasma membranes so that their cytoplasmic contents were dispersed into the intercellular spaces. Many cells produced numerous small processes which were pinched off and dispersed in a similar manner. Following a period of intense mitotic activity (3–8 days) numerous cells with the characteristics of embryonic smooth muscle cells were evident. Within 10–14 days these differentiating cells produced bulbous protrusions and assumed more irregular outlines than at 3–8 days. The protrusions formed close contacts (50–100Å intercellular space) and tight junctions between adjacent muscle cells. Aggregation of muscle cells into bundles was under way between 14–28 days. At approximately 4–6 weeks these developing muscle groups were invaded by nerve fiber bundles. The pattern of the innervation and the form and size of the muscle bundles simulated the normal. These findings are discussed in relation to the possible functions of the intercellular contacts and cellular protrusions which characterise various periods of regeneration.This work was supported by the Australian Research Grants Committee. The transplants were carried out by Dr. T. Malmfors assistant, Miss Ulla Enberg.     

Phosphatidylinositol-cleaving activity in smooth muscle from rat vas deferens

• 1.1. Phosphatidylinositol-cleaving activity was studied in subcellular fractions from smooth muscle of rat vas deferens.
• 2.2. In the presence of calcium ions and deoxycholate most of the endogenous phosphatidylinositol was broken-down in 60 min, whilst the other phospholipids were stable.
• 3.3. The enzymatic activity responsible for this breakdown catalyses a phospholipase C-type cleavage of the glycerol-phosphate bond, the water soluble products from exogenous [³²P]-labelled phosphatidylinositol being d-myoinositol 1:2-cyclic phosphate (702-80%) and d-myoinositol 1-phosphate (202-30%).
• 4.4. Activity was abolished by 1 mM ethanedioxybis(ethylamine)tetra-acetate (EGTA) and in the presence of deoxycholate both the soluble and total particulate fractions showed maximum activity at pH 6.52-6.8. The soluble fraction showed a second peak of activity at pH 5.52-5.8 that was independent of deoxycholate; this was not observed in the particulate fraction.
• 5.5. About two-thirds of the activity was soluble. The remaining activity was particulate, with a preferential concentration in the microsomal fraction.

     

Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens

Summary Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 31/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescence microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.Supported by the Swedish Medical Research Council (04X-3185), and by grants from Magnus Bergvalls Stiftelse, Tidningen Expressens Prenatalforskningsfond and Karolinska Institutets fonder. We thank miss Ingrid Strömberg for skilful technical assistance. Nialamide^® was generously donated by Swedish Pfizer AB.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Knowing the ontogenesis of the central monoamine neurons of the rat it is possible to obtain, by free-hand dissection from embryos and newly born animals, pieces containing dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) neurons that are small enough to permit homologous transplantation to the anterior chamber of the eye of adult animals. With this technique it was established that all three types of immature monoamine neurons are able to survive in the anterior chamber. Fluorescence histochemical analysis of whole mount preparations of the sympathetically denervated host irides revealed that both the catecholamine- and the 5-HT-neurons are able to partly reinnervate the irides, forming networks of varicose nerve terminals similar to the normally present sympathetic adrenergic ground plexus.Monoamine nerve cell bodies are attached to the irides but the majority of fluorescent nerve cell bodies is located within the transplants. Serial sectioning of these transplants showed rather well organized brain tissue, containing groups of fluorescent and non-fluorescent cell bodies, many areas being innervated by monoamine nerve terminals. When brain tissue was transplanted before the normal appearance of fluorescent neuroblasts (embryos with a crown-rump length less than 8 mm) monoamine neurons developed and matured within the eye.The amount of newly formed nerves of central origin recovered on the irides increased with time between the 2nd and 4th postoperative week and persisted after 2 months. The yield of new fibers was better using transplants from embryos with a crown-rump length between 15 and 30 mm than using transplants from larger embryos and newly born animals.If embryonic brain tissue known to be devoid of monoamine nerve cell bodies but containing monoamine nerve terminals in the adult state (cortex cerebri and cerebelli, spinal cord) was transplanted to sympathetically non-denervated eyes, the sympathetic adrenergic fibers seemed to be able to innervate the transplants.This work was supported by grants from the Swedish Medical Research Council (14×–3185), Karolinska Institutets fonder, and Magnus Bergvalls Stiftelse. We thank Miss Monica Eliasson, Mrs. Ulla Flyger, Mrs. Barbro Norstedt and Miss Ingrid Strömberg for skilful technical assistance. The generous gifts of Nialamide, Pfizer, and Pargyline, Abbott are gratefully acknowledged.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Small pieces of fetal rat brain selected to contain a high number of noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) neuroblasts were transplanted to the anterior chamber of the eye of adult rats. The sympathetic ground plexus of the host iris was removed by superior cervical ganglionectomy so that transmitter mechanisms of the different central monoamine fibers innervating the iris could be selectively studied after intraocular maturation. Such irides, containing NA, DA, or 5-HT nerve terminals were incubated with radiolabelled transmitters and then stimulated by an electrical field while superfused, to investigate the spontaneous and stimulation-induced release of amine, both in drug-free buffer and buffer containing drugs acting on monoamine receptors.The central monoamine neurons of all three types were able to take up exogenous amines and release them upon stimulation by an electrical field, in much the same way as corresponding nerves in situ in slices of cerebral cortex (NA, 5-HT) or olfactory tubercle (DA).The -adrenergic receptor blocking agent phentolamine increased the stimulation-induced release of ³H-NA from central NA fibers on the iris significantly. The dopamine receptor stimulating agent apomorphine decreased the stimulation-induced release of ³H-DA from central DA fibers on the iris. Pimozide, a DA receptor blocking drug tended to increase the ³H-DA release. The 5-HT receptor stimulating agent ergocornine tended to reduce the stimulation-induced release of ³H-5-HT from central 5-HT fibers on the iris. It was concluded that all three types of central monoamine nerve fibers develop essentially normal transmitter storage and release mechanisms also in an environment completely devoid of normal postsynaptic receptors. The drug experiments add strong support to the view that there are presynaptic monoamine receptors (autoreceptors) able to modulate transmitter release present on the monoamine nerve terminals.Supported by the Swedish Medical Research Council (04X-3185 and 04X-2330) and by grants from Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder, we thank Miss Ingrid Strömberg and Miss Ulla Enberg for skilful technical assistance.     

Terminal axons ensheathed in smooth muscle cells of the vas deferens

Summary The ultrastructure of axon profiles which were completely ensheathed in smooth muscle cells has been described in the guinea pig, mouse and rat vas deferens. The axon profiles contained both small (500 Å) and large (1,000 Å) vesicles, neurotubules and mitochondria. Adrenergic axons were clearly identified within smooth muscle cells after treatment of the tissue with 5-or 6-hydroxydopamine, drugs which cause specific ultrastructural changes in adrenergic axons. The ensheathed axons were separated from the surrounding muscle cells by narrow, regular gaps, usually about 100–300 Å wide. Schwann cells seldom accompanied the ensheathed axons. Axons often penetrated the muscle cells in the nuclear region and profiles were sometimes observed among the perinuclear organelles.     

Reinnervation of regenerating smooth muscle cells in minced vas deferens of the guinea-pig

Summary In adult guinea-pigs, a portion of the wall of the vas deferens was removed, minced and replaced. This caused muscle cells to dedifferentiate, divide and redifferentiate. Reinnervation of redifferentiating cells was followed using electron microscopy and histochemistry. Adrenergic nerves were first observed to re-enter the regenerating area 5 days after operation, and close contacts (within 20 nm) with muscle cells were first seen at 10 days. The total number of adrenergic nerves per 100 muscle cells reached control values by 5 weeks, and by 15 weeks was higher than control levels. Cholinergic nerves first appeared in the regenerating area about 3–4 weeks after the operation. The total number of cholinergic nerves present had not reached control values even at 15 weeks, and no nerve muscle contacts within 20 nm were observed. The ratio of adrenergic to cholinergic nerves in the regenerating area was higher at 15 weeks than in control tissue.This work was supported by grants from the Wellcome Trust and the Medial Research Council     

Dense-cored membranous structures in smooth muscle cells of the vas deferens of the rat

Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior.     

The nervous environment of individual smooth muscle cells of the guinea pig vas deferens

Smooth muscle cells of the external longitudinal coat of the guinea pig vas deferens were followed for 480 mu at 4.5-mu intervals. Muscle bundles and fibers interwove, facilitating intermuscular and neuromuscular contacts. The ribbon- or rodlike muscle cells were about 450 mu long, 3,000 mu3 in volume, and 4,500 mu2 in area. The thickened nuclear zone day anywhere along the middle one-third of the cell. Intercellular distances were 500-800 A. Intrusions were rare, and tight-junctions absent. At any level in a field of 80 muscle fibers there were 10-15 nerve bundles, each containing several varicose axons. Bundles and axons divided. Axons, en passage, were frequently within 500-1,000 A of a muscle fiber. En passage close contacts were rate. Axon terminations were bare, and bare axons invariably terminated. Bare terminations had scattered vesicle-laden varicosities and were from 10-60 mu in length, and all ended within 500 A of muscle fibers. Some made close contact with muscle fibers. Less than half of the muscle cells received this close contact, but some cells were approached by more than one termination. Most terminations involved more than one cell. Some cells had little or no innervation. Some groups of cells had a rich innervation. There was very little evidence of sensory innervation. These conclusions are not valid for other smooth muscles.     

Facilitated diffusion of monosaccharides in smooth muscle of rat vas deferens in vitro

The suitability of rat vas deferens for investigating sugar transport in smooth muscle was determined in vitro, with the nonmetabolized glucose analog 3-O-methyl-D-glucose as test sugar. Vas deferens smooth muscle contains a facilitated diffusion system for monosaccharides, as shown by saturation of the transport sites and by competition between 3-O-methyl-D-glucose and D-glucose. The activity of the facilitated diffusion system could be enhanced by hyperosmolarity and by contractile activity, but frequency dependency could not be established. A high concentration of insulin (100 mU/mL) was required to stimulate sugar transport. As smooth muscle is not a primary tissue for the storage of energy reserves, it does not require large numbers of insulin receptors.     

Unit activity of the septum and hippocampus transplanted alone or together into the anterior chamber of the rat eye

Unit activity of grafts of the septum and hippocampus, developing for 3–6 months in the anterior chamber of the eye was investigated in acute experiments on curarized orcerveau isolé rats. Whereas neurons in the transplanted septum had spontaneous activity of irregular, regular, or rhythmic bursting type, activity was absent in hippocampal grafts or consisted of very infrequent synchronized population sites. If grafts of the septum and hippocampus developed together and contact was established between them, the same types of activity developed in the hippocampus as in the septum. In many paired grafts spontaneous epileptic phenomena were observed; they were easily provoked also by electrical stimulation of one of the grafts. Superfusion with medium with a high Mg⁺⁺ concentration and low Ca⁺⁺ concentration abolished spontaneous activity in most neurons of hippocampal but not septal grafts, and also suppressed some of the epileptic phenomena, evidence of the leading role of the septum in the organization of spontaneous hippocampal unit activity.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 61–69, January–February, 1985.     

Development of mammalian nervous tissue transplanted into the brain and anterior chamber of the eye: problems and prospects

Some theoretical problems arising in connection with nervous tissue grafting in mammals are discussed. The survival of grafts in the brain and anterior eye chamber is provided by a complex of factors, including peculiarities of immunological reaction, blood-brain barrier and certain characteristics of embryonic nervous tissue. Organotypic development of ectopic grafts suggests a significant autonomy of inner genetic programmes in self-organization of brain structures. Development of the graft-host brain nervous connections is, to a great extent, determined by the factors of topographic closeness and the presence of free postsynaptic structures, without prominent specificity of the graft-brain relationships. Complex neurotrophic interactions, mainly provided by the glial cells, are also found between the graft and damaged host brain. A study of electric activity of the grafted neurons has shown a varying degree of dependence of the functional organization of the brain structures on the environmental afferent influences. The grafts can serve as a chronic endogenous source of neurotransmitters and neurohormones, and, possibly, restore interrupted structural connections, thus providing the compensation of some complex brain functions.     

Involvement of calcium in calcium-current inactivation in smooth muscle cells from rat vas deferens

Summary Ca-channel currents were recorded in Cs-loaded single smooth muscle cells from rat vas deferens to define the dependence of the inactivation time course on Ca concentration. The decay of Ca-channel current obtained in a Ba²⁺- or Sr²⁺-containing external solution during long voltage-clamp pulses was much slower than that in a Ca-containing solution. The difference was not due to a change in the surface potential of the membrane as judged from the steady-state activation and inactivation curves. When Ca was the charge carrier, increasing external Ca concentration slightly accelerated the rate of inactivation. In addition, the rate of inactivation of Ca-channel current in 10.8mm Ba was also accelerated by adding Ca to the external solution in a concentration-dependent manner. The time course of Ca-current inactivation was slowed when the cells were dialyzed with a high concentration of citrate, a Ca-chelating agent. From these results, we concluded that a mechanism regulated by intracellular Ca activity plays a role in the inactivation of Ca channels in smooth muscle. The Ca-dependent process may protect against Ca overload by regulating Ca entry in smooth muscle cells.     

An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig

Summary Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3–5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.This work was supported by grants from the Wellcome Trust and the Medical Research Council     

Electron microscopical study of synaptic glomeruli in cerebellum transplanted to the anterior eye chamber

Fetal cerebellar anlage from rat fetuses of 15-16 operational days were grafted into the anterior chamber of the eye of adult female albino rat recipients. Survival time of the transplants--containing both cerebellar cortex and cerebellar nuclei--was 2 to 2 1/2 months. Electron microscopical (EM) studies of the thin, under-developed granular layer of the laminated cerebellar cortex revealed the presence of well differentiated cerebellar glomeruli, surrounded by granule cell perikarya. As in the normal cerebellar cortex, the central profile of the glomerular complex was the large mossy terminal, containing spheroid synaptic vesicles, and forming synaptic contacts with dendrites and dendritic digits of the granule cells. Golgi cell axonal varicosities, containing ovoid or pleomorphic synaptic vesicles were found also on the periphery of the glomeruli. In addition, in several synaptic glomeruli, a third neuronal element was also observed, containing flat, discoidal vesicles and receiving synaptic contacts from mossy and Golgi axons, but being also presynaptic to granule cell dendrites. It is suggested that all mossy terminals in the cerebellar transplant originate from the cerebellar nucleus. Morphological evidence is also provided that the presynaptic dendrite-like processes--never found in normal cerebellar cortex--are also processes of nuclear neurons.