Liisa Eränkö

Small, Intensely Fluorescent Granule-containing Cells in the Sympathetic Ganglion of the Rat

Publisher Summary In a study, among typical ganglion cells, occasional small cells were observed to exhibit an extremely bright yellow fluorescence. It was later shown that the fluorescence of these cells, called “small intensely fluorescent cells” (SIF cells) remained essentially unchanged after division of pre- or postganglionic nerves close to the ganglion although the latter caused an almost complete disappearance of histochemically demonstrable catecholamines from the cytoplasm of the ordinary ganglion cells. The small cells were, therefore, considered a new variety of non-chromaffin amine-storing cells, and because the color of the fluorescence was yellow, a monoamine, perhaps 5-hydroxytryptamine, was thought to be stored in the secretory granules of the SIF cells in the same manner as catecholamines in the adrenal medulla. The study presented in this chapter was undertaken to study the constancy, number, and distribution of the SIF cells, as yet almost unexamined, as well as the problem of the amine responsible for their intense formaldehyde-induced fluorescence. Sometimes the SIF cell processes surrounded ganglion cells in intimate contact with them, and their beadings were impinging on the ganglion cell cytoplasm. Clusters of SIF cells were also observed among nerve tracts inside the ganglion in preganglionic or postganglionic nerve trunks near the ganglion and sometimes directly on ganglion cell bodies.

Immunoreactivity for substance P in the gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye

SummaryThe distribution of substance P (SP) immunofluorescence was investigated in the Gasserian ganglion, ophthalmic nerve and in the anterior segment of the rabbit eye. About one third of the nerve cell bodies in the Gasserian ganglion exhibited SP immunofluorescence, which was also observed in some nerve fibres of the ophthalmic nerve. In the cornea, some SP-positive iris contained numerous nerve fibres with SP immunofluorescence. In the sphincter area such fibres were circular, while the orientation of the SP fibres was radial in the dilator muscle. Both in the iris and in the ciliary body, the largest vessels were surrounded by nerves exhibiting SP immunofluorescence. A few nerve fibres also appeared in the stroma of the ciliary processes.

Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

SynopsisPineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.

Ultrastructure of the developing sympathetic nerve cell and the storage of catecholamines

Abstract Postnatal development of the superior cervical ganglion of the rat was studied by electron microscopy after fixation in potassium permanganate. The ultrastructural appearances were correlated with fluorescence microscopic observations on the distribution of catecholamines. Small cells with numerous granular vesicles (90–150 nm) were observed even in the newborn rat ganglion; they corresponded to small intensely fluorescent (SIF) cells. Most sympathetic cells at this stage were weakly fluorescent and contained little cytoplasm with few cytoplasmic organelles. These cells were considered early sympathicoblasts; they did not contain any granular vesicles, but multivesicular bodies were common. Outer cell membranes of neighbouring sympathicoblasts were often in direct contact, although slender satellite cell processes were sometimes seen between them. Nerve terminals with numerous empty vesicles, presumably cholinergic preganglionic synapses, were observed on the perikaryon of some early sympathicoblasts. Cells with well developed Golgi apparatus, endoplasmic reticulum, mitochondria and multivesicular bodies appeared a few days after birth. Such cells, considered late sympathicoblasts, were predominant in the ganglia of 1-week-old rats. Although an even catecholamine fluorescence was seen throughout the cytoplasm of such cells, these usually contained only a few large granular vesicles (60–110 nm) near the outer cell membrane and near the Golgi apparatus, most of the cytoplasm being devoid of them. However, occasional late sympathicoblasts showed large granular vesicles (60–110 nm) scattered throughout the cytoplasm, while other were entirely free of them. Direct cell membrane contacts between late sympathicoblasts were common. Further differentiation resulted in the formation of young nerve cells, whose diameter was about twice of that of the early sympathicoblasts, whose cytoplasm was densely filled with cell organelles, and which were completely surrounded by a layer of satellite cell cytoplasm. Young nerve cells exhibited an even catecholamine fluorescence thoughout the cytoplasm, but many cells also showed intensely fluorescent granules near the outer cell membrane. Numerous small granular vesicles (30–60 nm) were seen in the cell periphery of most young nerve cells, while the cytoplasm was devoid of them elsewhere; some granular vesicles were seen in the Golgi region. Occasional young nerve cells showed numerous small granular vesicles (30–60 nm) throughout the cytoplasm, and others were without granular vesicles altogether. It is concluded that: (1) catecholamines are stored in developing sympathetic cells mainly outside granular vesicles, although granular storage progressively increases with the cell maturation; (2) granular vesicles are large (60–110 nm) in sympathicoblasts but become small (30–60 nm) in nerve cells; (3) the 3 types of sympathicoblasts and nerve cells observed, those with some granular vesicles in the periphery, those with granular vesicles throughout the cytoplasm and those without any granular vesicles, presumably represent different functional stages of the same cell; and (4) the small granule-containing (SIF) cells are distincly different from sympathicoblasts or sympathetic nerve cells.

Loss of histochemically demonstrable catecholamines and acetylcholinesterase from sympathetic nerve fibres of the pineal body of the rat after chemical sympathectomy with 6-hydroxydopamine.

SynopsisNewborn albino rats were injected daily for 8 days with 50 μg/g of 6-hydroxydopamine. They were killed 3 weeks after the last injection together with untreated litter mate controls. Monoamines were demonstrated histochemically in the pineal body, in the iris and in the superior cervical ganglion with the formaldehyde-induced fluorescence method. Acetylcholinesterase was demonstrated in the pineal using acetylcholine as substrate and tetraisopropy-pyrophosphoramide (iso-OMPA) to inhibit non-specific cholinesterases.Treatment with 6-hydroxydopamine caused a complete disappearance of amine-containing fibres from the pineal, whereas some fluorescent ganglion cells remained in the superior cervical ganglion and in some rats a few amine-containing fibres in the iris. Acetylcholinesterase activity, located in fine nerve fibres of the pineal body, disappeared completely after treatment with 6-hydroxydopamine.Since 6-hydroxydopamine causes a selective destruction of the aminergic sympathetic fibres, it is concluded that the disappearance of the acetylcholinesterase activity indicates that in the pineal body this enzyme activity is located exclusively in truly aminergic nerve fibres.

Histochemical evidence of chemical sympathectomy by guanethidine in newborn rats

SynopsisGuanethidine is known to cause a loss of catecholamines from sympathetically innervated tissues and sympathetic ganglia in adult animals but its effect on newborn animals has not been examined.Newborn rats were injected daily with guanethidine (20 mg/kg body weight) for 8 days. They were killed when 1 month-old along with untreated litter mate controls. Catecholamines were demonstrated in the iris, in the pineal body and in sympathetic ganglia, using the formaldehyde-induced fluorescence method.In the guanethidine-treated rats there was a complete loss of fluorescent nerve fibres from the pineal body and an almost complete loss of similar fibres from the iris. The sympathetic ganglia were reduced to less than 10% of the control ganglia, and the number of nerve cell bodies per unit area was decreased in the ganglion remnants.It is concluded that guanethidine causes, in newborn rats, an irreversible destruction of most sympathetic neurons, i.e. a chemical sympathectomy closely resembling that obtainable in newborn animals by injections of 6-hydroxydopamine or antiserum to nerve growth factor.

Effect of sensory and sympathetic denervation on substance P immunoreactivity in nerve fibres of the rabbit eye

Abstract Substance P (SP) immunoreactivity was demonstrated in ocular tissues of rabbit. SP was found in nerve fibres of cornea iris, ciliary body, choroid and in the inner plexiform layer of the retina. In order to verify the origin of these nerves the animals were subjected to two different denervation procedures: intracranial combined maxillary and ophthalmic neurotomy or superior cervical ganglionectomy. The former operation destroyed all SP immunoreactive nerves of the ipsilateral eye except for the retina, whereas the latter had no effect. It is concluded that the ocular SP immunoreactive nerves are sensory trigeminal fibres. SP immunoreactivity in the retina is not due to sensory nerves but probably to amacrine cells.

Substance P-like immunoreactivity in the rabbit inner ear

The auditory and vestibular sense-organs of the rabbit were examined for the presence of substance P(SP) by an immunohistochemical technique using a monoclonal SP-antibody. Between 30 and 40 per cent of the cells of the vestibular ganglia and about 50 per cent of the spiral ganglion cells innervating the extreme basal part of the cochlea showed SP-like immunoreactivity. The neural elements in the vestibular sensory epithelia, notably the calyx-formed nerve terminals of type I sensory cells of the maculae, also showed strong SP-like immunoreactivity. The findings suggest the possibility of a modulator or transmitter role for SP in the inner ear function.

Postnatal development of histochemically demonstrable catecholamines in the superior cervical ganglion of the rat

SynopsisCatecholamines were demonstrated histochemically in the superior cervical ganglion of albino rats aged o (newborn), 3, 6, 12, 24 or 90 days (adult). After freezing in propane at −190°C, the ganglia were driedin vacuo at −50°C for a week and embedded in epoxy resin. Fluorescence microscopy was carried out with maximum excitation at about 400 nm and emission above 460 nm, avoiding fading due to over-exposure to u.v. light. Most sympathicoblasts in the ganglia of newborn rats exhibited a weak, blue fluorescence which was evenly distributed throughout the cytoplasm. Most cells were still weakly fluorescent in rats 3 and 6 days old, although the number of more intensely fluorescent cells increased with age. The size and the fluorescence intensity of the nerve cells increased with development until the adult stage. The cytoplasmic fluorescent granular aggregates were first seen in the peripheral cytoplasm of nerve cells of 12-day-old rats, and in the ganglia of 24-day-old rats the peripheral granules showed the adult picture. Fluorescent processes were seen to emerge from nerve cells of 3-day-old rats, but fluorescent nerve fibres were first seen between the nerve cell bodies in the ganglia of 6-day-old rats, and intensely fluorescent granular formations appeared in the intercellular nerve cell processes in 24-day-old rats. Small intensely fluorescent cells were already numerous at birth and at all stages of development were easily discriminated from sympathicoblasts and the developing nerve cells by virtue of their much smaller size.It is concluded that the nerve cell perikaryon matures when rats are 3 weeks old, which coincides with the maturation of the peripheral nerve net in peripheral target organs. This maturation is characterized by the appearance of a granular pool of catecholamines in the perikaryon and the terminal nerve fibres, corresponding to clusters of small granular vesicles at the ultrastructural level.

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

SummaryNewborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.