Olavi 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.

Improved localization of phosphorylase by the use of polyvinyl pyrrolidone and high substrate concentration.

585 Truks’uschui romoob 1itsmiroki (.1. Hislochem. (]ylochem. 3: 153, 1955) s!s’ns’mihues! a sois’thod for histochenouical ohesoionsslmrotions of phosphom)’lase. This moset-hod is thus’oretictohby notososh amusl ovorks ins practice. Howeven, ooit-hu tinsussri less roctive, for instance, than liven titus! susumncle, t hue suet hos! I)movs’51 somuuewhat ctui)mis’isuuos in siuom hunumotlri asis! toe decisled to investigate the effect of riotous’ factors involved ins the ns’ros’tioss ins thus’ hoops’ to imOupmovs’ the nsiethooh. The suunsiss resuslts time enlists’s! us Table I. The main diffs’rens’es his’tween the above proceslurs’ and that given by Taks’uchi and Kumiaki rums’ the presence of I)ol)’visuyh pyrrohidone tons! I-he higher concs’mstmabiomo of glucose-I-phosphate ins the substrate nuixtusme. Both of these chtomugs’s significantly iniprovesl the sensitivity ansI ths’ localizing power of the reaction. The advamutageoums effect of l)olyvinYl pymmohidone has bes’ss ohsemves! earlier in dehys!mogemsrose techniquss’s ansi it os-as ascnil)s’d to osmsuobic protection (Scarpelhi rosusi

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.

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.

CHOLINESTERASES AND ESERINE‐RESISTANT CARBOXYLIC ESTERASES IN DEGENERATING AND REGENERATING MOTOR END PLATES OF THE RAT

KUPFER (1951) was the first to demonstrate histochemically the persistence of cholinestrase activity for about a month in degenerating motor end plates of the rat. Since then numerous publications have appeared on this subject and cholinesterase activity has been reported to persist in the motor end plate long after the degeneration of the motor nerve (BAUER, BLUMBERG and ZACKS, 1962; BERGNER, 1957; BRZIN and MAJCEN-TKACEV, 1963; BRZIN and ZAJICEK, 1958; CSILLIK and SAVAY, 1958; GEREBTZOFF and VANDERMISSEN, 1956; GUTH and ZALEWSKI, 1963; HINES, 1960; SNELL and MCINTYRE, 1956 and WASSER and HADORN, 1961). In all the above-cited studies the total cholinesterase or esterase activity was demonstrated, often assuming acetylcholinesterase (AChE) responsible. However, overwhelming evidence is available of the multiplicity of esterases involved in producing the histochemical reactions with substrates such as cr-naphthyl acetate (see ERANKO, HARKONEN, KOKKO and RAISANEN, 1964; HARKONEN 1964; KOKKO 1965). Acetylthiocholine and butyrylthiocholiiie are more specific substrates in being, as a rule, hydrolysed only by cholinesterases, the former by both acetylcholinesterase (AChE) and nonspecific or butyrylcholinesterase (BuChE) the latter by BuChE only. Discrimination between these two types of cholinesterase activity is readily possible in histochemical systems with the aid of selective inhibitors (see KOELLE, 1963; ERANKO et al., 1964; ERANKO and TERAVAINEN, 1967). Though admittedly simplified, division of histochemically demonstrable esterase activities into a non-specific type, which can be demonstrated in the presence of potent inhibitors of cholinesterases such as esterine, and either AChE or BuChE has proved valuable for practical purposes (see ERXNKO et al., 1964). Although likely guesses of the proper classification (see DIXON and WEBB, 1964) of the enzymes involved can be made (in the present case AChE = E.C. 3.1.1.7., BuChE = E.C 3.1.1 .S., ‘non-specific esterases’ = E.C. 3.1 . l . 1. + E.C. 3.1.1.2. + E.C. 3.1.1.6. + others ?), it is preferred to use here the less precise terms in describing the histochemical reactions employed. As demonstrated by many authors, the motor end plate of the striated muscle of the rat contains not only AChE but also BuChE and non-specific esterase (see ERANKO and TERAVAINEN, 1967). While the multiplicity of esterases is well documented in the normal end plate, adequate substrate-inhibitor combinations have been used by amazingly few investigators to discriminate between these enzymes in studying

Electron microscopic demonstration of cholinesterases in nervous tissue

SummaryAcetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rats spinal cord using the Karnovsky-Roots modification of Koelles thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.

Light and electron microscopic histochemical evidence of granular and non-granular storage of catecholamines in the sympathetic ganglion of the rat

SynopsisSuperior cervical ganglia of adult rats were studied for light microscopy with the formaldehyde-induced catecholamine fluorescence technique and by electron microscopy after fixation in 3% potassium permanganate in phosphate-buffered Krebs-Ringer solution.In ganglia frozen-dried for a week at −45°C or below, the nerve cells showed a diffuse blue catecholamine fluorescence throughout the perikaryon and a more intense fluorescence in granular structures predominantly in the periphery of the cytoplasm. All the nerve cells exhibited the same blue fluorescence, although there was a great variation in the fluorescence intensity between individual cells. Clusters of small cells exhibited a very intense catecholamine fluorescence. In specimens frozen-dried at temperatures higher than −35°C or for less than a week, no granular fluorescence was observed in the perikarya or nerve cell processes.Clusters of small granular vesicles, 30–50 nm in diameter, were observed by electron microscopy in the peripheral cytoplasm of most nerve cells. Some nerve cells showed small granular vesicles scattered throughout the perikaryon and others were entirely devoid of such vesicles. A few granular vesicles were seen in the Golgi region of most cells. Large granular vesicles, about 100 nm in diameter, were seen in many cells but their number was much smaller than that of the small granular vesicles. Small cells containing numerous granular vesicles of even larger size, about 150 nm in diameter, were also observed. They corresponded to the small very intensely fluorescent cells seen by fluorescence microscopy.It is concluded, firstly, that the fluorescent granules correspond to the clusters of small granular vesicles demonstrable by electron microscopy. Since most nerve cells showed small granular vesicles only in the periphery of the cytoplasm but a diffuse fluorescence throughout the cytoplasm, it is concluded, secondly, that a large proportion of the cytoplasmic catecholamines is stored in a non-granular pool, presumably in the endoplasmic reticulum.