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Age-related changes of the metabolic characteristics of large, medium and small brances of the rat superior mesenteric artery were investigated in young (4 month) and old (25 month) male Wistar rats, using enzyme histochemical techniques. The following enzymatic activities were examined: lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH); NADPH(2)-tetrazolium reductase (NADPHD) and NADH(2)-tetrazolium reductase (NADHD). The intensity of enzymatic reactions within the tunica media of the large, medium and small branches and within the tunica intima of the large and medium branches of the artery was assessed microphotometrically. In young rats the reactivity of enzymes investigated is similar in both the tunica media and the intima with the exception of SDH which reacts stronger in the media than in the intima. LDH and NADHD displayed the most intense reaction whereas SDH reaction was less intense. In old rats LDH and NADPHD reactivity decreased, SDH reactivity increased in the intima of large and medium branches; and SDH increased in the media of large branches. The possibility is discussed that an impaired endothelial function may represent one of the most consistent phenomena of aging of the superior mesenteric artery.

The functional relationships between sympathetic nerve fibres and meningeal mast cells were studied in whole mounts of rat dura mater and pia-arachnoid membrane using formaldehyde histofluorescence technique. Mast cells displaying a yellow fluorescence indicative of their 5-hydroxytryptamine content were found in the dura mater, primarily perivascularly and in lesser amounts in the meningeal tissues proper without any apparent relationship with blood vessels. Bilateral removal of the superior cervical ganglion produced the degranulation of part of dura mater mast cells as well as a remarkable reduction of the intensity of their 5-hydroxytryptamine cytoplasmic fluorescence. On the contrary, the electrical stimulation of the superior cervical ganglion increased the intensity of cytoplasmic fluorescence of this kind of cells.Taken together the above data seem to be strongly suggestive of a functional link between meningeal mast cells and sympathetic nerve fibres originating from superior cervical ganglion.

Summary The pharmacological profile and the anatomical localization of a putative dopamine D4 receptor were assessed in sections of rat and human atria and ventricles using combined radioligand binding and autoradiographic techniques with [3H]-spiperone as a ligand. [3H]-Spiperone was bound specifically to sections of human and rat atria and ventricles. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high affinity sites. In atria, the dissociation constant value (Kd) was 0.45 nM in rats and 0.32 nM in humans, and the maximum density of binding sites (Bmax) was 31.6 ± 2.9 fmol mg−1 tissue in rats and 18.8 ± 0.7 fmol mg−1 tissue in humans. In ventricles, Kd was 0.38 nM in rats and 0.39 nM in humans, and the Bmax was 43.5 ± 3.0 fmol mg−1 tissue in rats and 56.4 ± 3.2 fmol mg−1 tissue in humans. The pharmacological profile of [3H]-spiperone binding to sections of both rat and human atria and ventricles was consistent with the labelling of dopamine D2-like receptors. [3H]-Spiperone binding was more sensitive to displacement by the neuroleptic clozapine in sections of atria than of ventricles, suggesting the expression of a dopamine D4 receptor in atrial tissue. Moreover, preincubation of some section with a dopamine D4 receptor antibody and subsequent exposure to [3H]-spiperone caused a remarkable decrease of radioligand binding to sections of atria, but only a slight reduction of binding to section of ventricles. Light microscope autoradiography revealed the accumulation of silver grains over atrial tissue within atrial myocardiocytes. A higher density of silver grains was developed in rat than in human atria. In ventricles, silver grains were accumulated primarily in cluster areas both in rats and in humans. The above findings suggest the expression of a dopamine D4 receptor in rat atria, but not in ventricles. A similar site was identified in human atria. The possible relevance of a dopamine D4 receptor in the heart is discussed.

The pharmacological profile and the density of dopamine D 3 and D 5 receptor subtypes expressed by human peripheral blood lymphocytes of subjects of different ages (ranging from 20 to 75 years) were assessed using radioligand binding techniques. Dopamine D 3 receptor was assayed with [ 3 H]7-hydroxy-N,N-di- n -propyl-2-aminotetraline ([ 3 H]7-OH-DPAT) as a ligand. Dopamine D 5 receptor was assayed using [ 3 H][R]-(+)-(-chloro-2,3,4,5,tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([ 3 H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [ 3 H]7-OH-DPAT and [ 3 H]SCH 23390 at dopamine D 3 and D 5 receptor, respectively, were similar in subjects of different ages. The density of dopamine D 3 receptor binding sites was slightly decreased in subjects of 30–39 years in comparison with younger individuals. A remarkable loss of dopamine D 3 receptor was then found between 40 and 49 years of age in comparison with younger subjects. A further slight decrease was noticeable between 50 and 59 years of age. The number of [ 3 H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D 5 receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D 3 but not D 5 receptor between adult and mature subjects. The possibility that dopamine D 3 receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.

Ca2+ channels of the L-type were characterised in intact human peripheral blood lymphocytes using a radioligand binding technique and the dihydropyridine-type Ca2+ channel antagonist [3H](+)-PN 200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridine carboxylate) as a ligand. [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity. The dissociation constant (Kd) value was 0.4 ± 0.02 nM and the maximum binding capacity (Bmax) was 33.5 ± 1.6 fmol/106 cells. Pharmacological analysis of [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was consistent with the labelling of a Ca2+ channel of the L-type. In fact, dihydropyridine derivatives were the most potent competitors of [3H](+)-PN 200-110 binding, whereas phenylalkylamine and benzothiazepine compounds or non-selective Ca2+ channel modulators were weak or ineffective displacers. These findings are the first observation that human peripheral blood lymphocytes express Ca2+ channels of the L-type. The possibility that Ca2+ channel antagonists may interfere with immune system function is discussed.

Combined radioligand binding and light microscope autoradiography techniques were used for investigating the pharmacological profile and the microanatomical localisation of dopamine receptor subtypes in the cardiovascular system and in the kidney. In superior mesenteric and renal arteries the predominant dopamine D1-like receptor belongs to the D5 (or D1b) subtype. This site is located within smooth muscle of the tunica media. The same receptor subtype predominates in the kidney, where it has a vascular and tubular localisation. The dopamine D2-like receptor subtype expressed by systemic arteries belongs to the D2 receptor subtype. It has a prejunctional and endothelial localisation. In the kidney the predominating dopamine D2-like receptor belongs to the dopamine D3 subtype. Atria but not ventricles express dopamine D2-like receptors belonging to the D4 receptor subtype. The above results suggest that in spite of the emerging complexity of the dopamine receptor profile demonstrated by molecular biology techniques, radioligand binding and autoradiographic techniques, if performed with appropriate radioligands and/or in the presence of compounds active on specific receptor subtypes, may represent a useful tool for better understanding the biological significance of peripheral dopamine receptors. (Hypertens Res 1995; 18 Suppl. I: S23-S27)

1. 1. The density of nerve cells and of silver-gold impregnated fibres were evaluated in the hippocampus and in the cerebellar cortex in adult (12-month-old) and old (24-month-old) Sprague-Dawley rats. 2. 2. The effects of long-term choline alfoscerate (GFC) treatment (100 mg/Kg/day for 6 months) on the above parameters were investigated in old rats. 3. 3. The number of nerve cell profiles and the area occupied by silvergold impregnated fibres were decreased both in the hippocampus and in the cerebellar cortex in old in comparison with adult rats. 4. 4. GFC treatment countered the age-dependent reduction of nerve cells and silver-gold impregnated fibres. The hippocampus was more sensitive than the cerebellar cortex to the activity of GFC. 5. 5. These results suggest that GFC treatment is effective in slowing down the expression of structural changes occurring in aging brain.

Treatment with l -deprenyl increases mean and maximum life span in the rat and reverses memory and learning deficits associated with old age. Since only sparse information is available concerning the influence of l -deprenyl administration on the aging brain microanatomy, we have investigated the effect of long-term treatment with l -deprenyl on the structure of the cerebellar cortex in the aged rat. The cerebellar cortex was used since it represents a useful model for assessing age-related changes in nervous system anatomy and function. Male Sprague-Dawley rats were treated from the 19th to the 24th month of age with a daily oral dose of 1.25 mg/kg or 5 mg/kg l -deprenyl. Age-matched rats were left untreated and used as a control group. Eleven-month-old untreated rats were used as an adult reference group. The density of Purkinje and granule neuron profiles as well as the intensity of Nissl's staining within the cytoplasm of Purkinje neurons were reduced in 24-month in comparison with 11-month rats. Moreover, an increased accumulation of lipofuscin was noticeable in the cytoplasm of Purkinje neurons of old rats as well as an increase in MAO-B activity in the molecular layer of the cerebellar cortex. The two doses of l -deprenyl increased the density of both Purkinje and granule neuron profiles and the intensity of Nissl's staining in the cytoplasm of Purkinje neurons and reduced lipofuscin deposition within Purkinje neurons. The lower dose of l -deprenyl caused only a slight decrease in MAO-B activity, whereas the 5-mg/kg/day dose remarkably reduced it. These results suggest that long-term treatment with l -deprenyl counters the expression of some age-related microanatomical changes in the rat cerebellar cortex. The possible independence of the effects of the compound on age-related microanatomical changes of the cerebellar cortex and on MAO-B inhibitory activity is discussed.

We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [3H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M1 and M2 receptors in the right coronary artery. Autoradiography showed the localization of M1 sites primarily in the medial layer of the right coronary artery. M2 sites were located primarily in the adventitia. No [3H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.

The influence of ageing on the noradrenergic innervation of the ovary was studied in female Wistar rats using high pressure liquid chromatography (HPLC) with electrochemical detection and catecholamine histofluorescence techniques. Old age was accompanied by a significant decrease in ovarian noradrenaline levels. In young animals (3-month-old) noradrenergic nerve fibres were distributed primarily to blood vessels and in lesser amounts to the interstitial glands. In aged animals (24-month-old) perivascular noradrenergic fibres were reduced by more than 40%; interstitial gland nerve fibres were reduced by approximately 20%. The possibility that impaired noradrenergic ovarian innervation occurring in old age may be in some way related with age-dependent failure in reproductive activity is discussed.

By the use of combined radioreceptor binding and autoradiographic techniques we attempted to analyse the biochemical characteristics and the anatomical localization of GABA-B receptors in sections of rat circle of Willis and pialarachnoid araeries. 3 H-GABA in the presence of 40 μm isoguvacine and 2·5 m m CaCl 2 was used to label GABA-B receptor sites. Sections of rat cerebellar cortex were also processed as a reference tissue. No specific 3 H-GABA binding was detectable either in radioreceptor binding or autoradiographic experiments. In contrast, the ligand was bound to sections of cerebellar cortex in a manner consistent with the labelling of GABA-B receptor sites. These findings indicate that cerebrovascular physiological responses to GABA are not linked, in the rat cerebrovascular tree, to the activation of GABA-B receptors.

The density and pattern of the sympathetic noradrenergic innervation of the extramedullary and intramedullary blood vessels of the spinal cord was studied in 3-, 12- and 25-month-old male Wistar rats using combined catecholamine histofluorescence and quantitative image analysis techniques. The study of innervation of intramedullary vessels was accomplished in spinal cord-transected rats to avoid the interference of descending spinal monoamine fibres in the observations. No age-related changes in the density of noradrenergic innervation of the anterior spinal artery or of sympathetic fibres associated with spinal cord blood vessels occurred. These results suggest that unlike perivascular noradrenergic nerves supplying the cerebrovascular tree, the sympathetic innervation of spinal cord blood vessels does not undergo age-dependent changes. It cannot be excluded that the lesser vulnerability of the spinal compared to the cerebral vascular tree to certain kinds of age-related diseases, may depend on the unchanged sympathetic trophic regulation of spinal vessels with age.

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.

The distribution of acetylcholine esterase (AChE) in the ovary of normal as well as of sympathectomized guinea pigs was studied. In normal animals AChE-positive nerve fibres were found organized in a perivascular plexus. Some nerve fibres reach the corpus luteum and the follicular wall. Chemical sympathectomy performed with the neurotoxin 6-hydroxydopamine caused an almost complete disappearance of AChE positive nerve fibres suggesting that most of the AChE activity of ovarian nerves is localized in adrenergic nerve fibres. Enzyme activity was also present in smooth muscle cells of the vascular tree.

By using combined radioreceptor binding and autoradiographic techniques, we were able to localize the GABA ‘A’ receptor agonist 3 H-muscimol in the rat choroid plexus. Within sections of rat choroid plexus, 3 H-muscimol was bound specifically with a K D of 37 nM and a B max of 253 pmol/mg tissue. These values are consistent with the labelling of GABA ‘A’ receptor sites. The autoradiographic studies demonstrated that 3 H-muscimol was attached to the epithelium of the choroid plexus. The blood vessels of the plexus did not exhibit specific labelling. Examination of these data suggests the existence of GABAergic mechanisms which control cerebrospinal fluid production or flow.

Summary In the present study the distribution of specific binding sites for the GABA-A receptor agonist, 3 H-muscimol, was studied in the rat uterus using an autoradiographic technique. Specific binding sites were present in both myometrium and endometrium suggesting a dual role for GABA in this organ.

Summary Cholinergic innervation of the blood vessels of the female reproductive system was investigated in human by the cholinesterase method. The following results were obtained: 1. The uterine and vaginal arteries are provided with a rich cholinergic innervation. 2. The ovarian, tubaric and ovarian-uterine artery are poorly innervated. 3. The ovarian and uterine vein are not provided with a cholinergic innervation.

Vasoactive intestinal polypeptide (VIP) levels and distribution were studied in the penis of young-adult (3-month-old) and old (30-month-old) Wistar rats by radioimmunoassay and immunofluorescence. No significant changes in tissue VIP concentrations or distribution occurred in the penis of old rats compared to young-adult rats. The present data indicate that the age-related impairment of male sexual function is not dependent on modifications of the VIP-ergic innervation of penile tissue.

The cholinergic innervation of the aorta has been studied in some mammalian species. Cholinergic nerve fibers (CNF) are organized in the aorta of the rabbit, kid, lamb, and guinea pig in 2 plexuses. The plexuses of CNF consist of a superficial plexus localized in the adventitial layer and a deep one localized in the adventitial-medial transitional zone. Some CNF reach the outer layers of the media. In the aorta of the dog, mouse and rat CNF are organized in a single plexus localized in the adventitial layer. This nerve plexus is formed of CNF that for the most part are distributed to the vasa vasorum. The chemical sympathectomy don't alters the pattern of the cholinergic innervation of the aorta, indicating that stained CNF are parasympathetic in nature. The meaning of a cholinergic innervation of the aortic wall is discussed.

The influence of ageing on the metabolic profile of lateral ventricle choroid plexus epithelial cells from young (3-month-old) and aged (26-month-old) male Wistar rats was studied using enzyme histochemical techniques. The following enzymatic activities related to energy transduction were examined: lactate- (LDH) and succinate- (SDH) dehydrogenases; NADH 2 -tetrazolium reductase (NADHD) and α-glycerophosphate-dehydrogenase (GPDH). The intensity of enzymatic staining within single choroid plexus epithelial cells from young and old animals was assessed microphotometrically. In the choroid plexus epithelial cells of young rats NADHD was the enzymatic activity more heavily stained; cell levels of LHD and GPDH were approximately the same and SDH reactivity was less intense. In old age LDH was reduced by 9.3%, SDH was reduced by 26.1%, NADHD was reduced by 8.6% and GPDH was reduced by 3.6%. The possibility that impaired energy transduction mechanisms at the level of choroid plexus epithelium in old age may influence functional activity of the choroid plexus is discussed.

The influence of aging on the metabolic profile of cerebellar cortex was studied in young (3-month-old), adult (12-month-old) and aged (26-month-old) male Sprague—Dawley rats using enzyme histochemical techniques. The following enzymatic activities related to energy transduction were examined: lactate-(LDH) and succinate-(SDH) dehydrogenases; NADH 2 -tetrazolium reductase (NADHD) and alpha-glycerophosphate-dehydrogenase (GPDH). The intensity of enzymatic staining within the neuropil of molecular and granular layers as well as within the cytoplasm of Purkinje neurons of young, adult and aged animals was assessed microphotometrically. In the molecular layer LSH, SDH and NADHD levels were reduced in old rats; GPDH was decreased both in adult and old animals. In Purkinje neurons no age-related changes of the enzymatic activities under study were observed. In the granular layer LDH and GPDH showed an age-dependent loss; SDH and NADHD were unchanged. The possibility that age-related changes of the enzymatic activities under study may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs resulting from the documented age-dependent loss of synapses in the molecular or in the granular layers of cerebellar cortex is discussed.

The effects of ageing and of 6 months of Hydergine treatment on lipofuscin deposition within the cytoplasma of pyramidal neurons of rat prefrontal cortex, hippocampus (fields CA1 and CA3) and of Purkinje neurons were assessed microfluorimetrically. No lipofuscin autofluorescence was detected in the nerve cell populations of 3-month-old rats, but lipopigment had accumulated in nerve cell bodies of 16-month-old animals and increased significantly thereafter in rats of 22 months of age. In 22-month-old rats, Hydergine administration (0.6 and 1 mg/kg p.o.) started at 16 months caused a significant dose-related decrease in lipofuscin accumulation within the cytoplasm of the various kinds of nerve cells examined.

The direct immunohistochemical detection of the potent beta-blocker (−)-alprenolol was accomplished in rat cerebellum. (−)-Alprenolol binding sites were found in Purkinje cells and in portions of the molecular and granular layers lying close to the Purkinje cells.

The existence of a double catecholaminergic and cholinergic innervation was demonstrated in the human greater saphenous vein. Catecholamine-containing nerve fibres are organized in a network-like plexus localized at the adventitial-medial border. Acetylcholinesterase-containing nerve fibres are arranged in a plexus found at the adventitial-medial border as well. Catecholamine and acetylcholinesterase-containing nerve fibres, while localized in close apposition since they occupy the same portion of the vein, represent two distinct and independent populations of nerve fibres coming likely from the sympathetic and parasympathetic sections of the autonomic nervous system respectively. Our findings demonstrating a close relationship between catecholaminergic and cholinergic nerve fibres within the wall of the human greater saphenous vein offer morphological support to physiological and pharmacological results reported in the literature of a presynaptic control exerted by cholinergic nerves on norepinephrine release at the level of the saphenous vein.