David T. Masuoka

An autoradiographic method of mapping the distribution and density of monoamine neurons in mouse brain

A combined in vitro uptake and autoradiographic procedure as an important complement to the histochemical fluorescence method is described. Slabs of fresh mouse brain were incubated with 14C-NE, 14C-DA or 14C-5-HT, freeze-dried, and placed against X-ray film for autoradiography, Catecholamine nerve terminals were labeled by in vitro incubation with 14C-NE or 14C-DA. Dopaminergic terminals were labeled by 14C-NE incubation preceded by desipramine (to block uptake into NE terminals). With 14C-5-HT incubation, the uptake pattern indicated the possibility that 5-HT nerve terminals were being labeled. Advantages of this method are that it allows the visualization of overall density and distribution of selected monoamine nerve terminals or uptake sites of other putative neurotransmitters in whole coronal or sagittal sections, so that data are obtained from many areas of brain or spinal cord rather than in only those areas preselected for microscopic viewing.

Aging and unusual catecholamine-containing structures in the mouse brain.

Brains of C57BL/6J mice, aged 4, 8 and 20--29 months, were examined by the Falck-Hillarp histochemical fluorescence technique. Numerous large, intensely fluorescent green to yellow-green spots (LIFS) were observed in the brains of senescent mice. LIFS were generally round to ovoid in shape and ranged in size from about 10 micrometer to about 30 micrometer. Histochemical and pharmacological procedures and spectral analysis indicated that the formaldehyde-induced fluorescence of the LIFS was due to the presence of catecholamines (CA) rather than aging pigment. Their distribution in the brain suggests an association with nerve axons or terminals rather than cell bodies. The number of LIFS in the hypothalamus increased progressively during aging. It is proposed that LIFS may represent age-related, unusual CA accumulation in enlargements proximal to axonal or terminal portions undergoing spontaneous degeneration.

HISTOCHEMICAL DEMONSTRATION OF FLUORESCENT CATECHOLAMINE TERMINALS IN CRYOSTAT SECTIONS OF BRAIN TISSUE

of cells. St rong react iomms were foummd in sectiomis of rat liver amid ear amid tracheal cartilages, espe(ially iii young amsimals (Fig. 1). Less intense amid consistent reactions were seems in the skits, lstmsg, spleen, kidney amid intestine (Fig. 2). Similar react iomss were present its rabbit, guinea pig amid twmsise tissues. Sect motis atsd smears of 48-hr susbcssltures of three strains of fl-xylosidase commtainiimg fsimmgi3 were also reactive (Figs. 3 atsd 4). Staimsing appeared to l)e localized along cell walls of the mycelia, amid spore forms were comssistetstly mmegative. Ilydrolysis and coupling occurred between phI 4.4 atmd 5.4, amid the reactiomm appeared to be optimal at p11 5.0 its both tissues amid fumigi. The staimsitig immtensity greatly increased with fixat iotm its either glutteraldehyde or formalims, bust there was otmly limited improvensetmt imm the reInlively poor cyt ologic localizat ion of the react ion product. Staitsitmg was occasiotmally presetmt at the edges and folds of the sections that had beets fixed. Greatly decreased st ai msimsg occurred without spertssiditse its the iticssbat ion solsit ioms, amid the reaction product was not visualized either in sect iomss or as a precipitate in the solustioms without ferri-ferrocyatmide. Oxidat ioms with hydrogen peroxide, ceriutm animomsisim sulfate amid cystine in plmuce of the iroms complex was attempted withoust success. The reaction was mmot seems its heated sections evetm after imscubatiomm its the presemsce of reactive t isssse. Likewise, NaF, et hylenediamimmetetracetic acid blocked the reaction. Although o-xylose is a 5-carbon sugar, it forms a pyratioside its solution, and so is a strtmctusral analogue of n-glucose. Inhi.

[3H]Dopamine release by d-amphetamine from striatal synaptosomes of reserpinized rats.

The injection of reserpine, 5 mg/kg i.p. (ipRes), the regimen employed by a majority of investigators, results in synaptosomal and vesicular preparations which are incompletely reserpinized as determined by [3H]dopamine ([3H]DA) accumulation. Reserpine administered by the subcutaneous route, 5 mg/kg (scRes), appears to produce complete reserpinization. Release of [3H]DA by d-amphetamine (Amph) was observed from striatal synaptosomes prepared both from normal rats and those pretreated with reserpine intraperitoneally but not from those injected subcutaneously. In the more completely reserpinized scRes synaptosomes, so little [3H]DA had accumulated that release by Amph was not measurable, indicating that if a labile, reserpine-resistant, extravesicular DA storage pool releasable by Amph is present under these conditions, it must be extremely small. In scRes monoamine oxidase (MAO)-inhibited preparations, Amph released preloaded [3H]DA located in the cytosol in the absence of functional vesicles. Although chromatographic analysis of the superfusate from ipRes striatal synaptosomes showed that significant amounts of preloaded [3H]DA were released by Amph, the level of dihydroxyphenylacetic acid was not increased over controls, indicating that Amph releases only DA and not its metabolite and is also acting as a MAO inhibitor. No [3H]DA could be released by Amph from superfused hyposmotically shocked normal or ipRes synaptosomes, suggesting that an intact membrane is required for Amph-induced release.

6-Hydroxydopamine: effect on mouse brain monoamines and 14C-noradrenaline uptake.

Abstract Intraventricular (i.vent.) injections of 6-hydroxydopamine (6-OH-DA) were made by a convenient technique in mice. Dose and time studies on the effect of 6-OH-DA on brain monoamines were conducted. When in vitro uptake of 14 C-noradrenaline by brain tissue from control and 6-OH-DA-treated mice was studied by autoradiography, there was a corresponding decrease of radioactive distribution in most catecholamine (CA) nerve terminal areas of treated mice. With histochemical fluorescence microscopy, corresponding changes in the distribution of CA nerve terminals and accumulation of CA fluorescence in the medial forebrain bundle were observed in the brain of 6-OH-DA-pretreated mice. These results confirm in mice evidence obtained in other species that selective doses of i.vent. injected 6-OH-DA cause selective degeneration of CA nerve terminals in the brain. Decreased 14 C-noradrenaline uptake and parallel changes in endogenous CA measured chemically and by fluorescence histochemistry, further supports the view that exogenous noradrenaline is specifically taken up by CA neurons.

Distribution of [14C]amphetamine in mouse brain: An autoradiographic study

Summary The localization and distribution of [ 14 C]amphetamine in mouse brain has been studied by an autoradiographic technique and by thin-layer chromatography. Thin-layer chromatography revealed that radioactivity in the brain is chiefly due to unchanged amphetamine. The highest content was found in the cerebral cortex, hippocampus, thalamus, septum and basal ganglia. The possible relationship of this localization to the site of action of amphetamine and the distribution of catecholamine nerve terminals is discussed.

A combined procedure for the histochemical fluorescence demonstration of monoamines and microautoradiography of water-soluble drugs.

A procedure for combining microautoradiography and the histochemical fluorescence method for aromatic monoamines on the same tissue section is described. The use of aqueous solutions is completely avoided, thus eliminating artifacts due to diffusion and dislocation of the labeled water-soluble drug and endogenous amines. The combination of these two techniques provides a means for obtaining data hitherto unavailable to correlate the site of binding and/or storage of water-soluble drugs with monoaminergic nerve cells and terminals.

Uptake of 3H-norepinephrine by fluorescent nerves of the heart.

The localization of uptake 5 minutes after the injection of a low dose of 3H-norepinephrine by adrenergic nerve fibers in the heart has been observed by combining autoradiography with the formaldehyde-induced histochemical fluorescence method for aromatic monoamines in the same tissue section. The distribution of 3H-label corresponds exactly to that of the fluorescent nerves. Furthermore, uptake studies in animals depleted of catecholamines by the tyrosine hydroxylase inhibitor (H 44/68), which does not block uptake, indicate that the dosage of 3H-norepinephrine used, under the conditions of the present experiment, was not in itself sufficient to produce the intense formaldehyde-induced fluorescence in nerves. It may be concluded that 3H-norepinephrine was taken up only by those nervous structures in the heart which show a specific fluorescence reaction due to the presence of endogenous catecholamine.

Histochemical Fluorescence Microscopy and Micro-autoradiography Techniques Combined for Localization Studies

Publisher Summary This chapter deals with the procedures in which the histochemical fluorescence technique is combined with autoradiography in the complete absence of aqueous solutions. This criterion must be met to eliminate possible artifacts due to diffusion and dislocation of labeled water-soluble drugs and endogenous amines. Thus, liquid tissue fixation and dehydration through aqueous alcohol solutions before exposure to photographic emulsion and the use of liquid or wet photographic emulsions over the tissue sections are the steps that must be avoided when water-soluble compounds are employed. With the development of a specific formaldehyde induced histochemical fluorescence method for aromatic monoamines, the interesting possibility of combining this technique with that of autoradiography presents itself. Such studies might be of value in the localization of binding and storage of putative transmitters. The tissue distribution of a variety of drugs, for example sympathomimetics, adrenergic blockers, enzyme inhibitors, and monoamine releasers, may be correlated with monoamine structures. In addition to histochemical fluorescence and autoradiography, it is possible to demonstrate acetylcholinesterase activity in adjacent thin cryostat sections.