Presence of soluble amyloid beta-peptide precedes amyloid plaque formation in Down's syndrome.

Abnormal and excessive accumulation of the amyloid beta-peptide (A beta) in the brain is a major and common characteristic of all Alzheimer's disease (AD) forms irrespective of their genetic background. Insoluble aggregates of A beta are identified as amyloid plaques. These deposits are thought to form when the amount of A beta is increased in the brain parenchyma as a result of either overexpression or altered processing of the amyloid precursor protein (APP). Soluble A beta ending at carboxyl-terminal residue 40 (A beta 40) and, in lesser amount, the form ending at residue 42 (A beta 42), are normal products of the APP metabolism in cell cultures. Increased secretion of soluble A beta 42 has been observed in cells transfected with constructs modeling APP gene mutations of familial forms of AD (refs 4, 5). On the basis of these in vitro data it has been hypothesized that the presence of soluble A beta 42 plays a role in the formation of amyloid plaques. Subjects affected by Down's syndrome (DS) have an increased APP gene dosage and overexpress APP. Apparently because of this overexpression, they almost invariably develop amyloid deposits after the age of 30 years, although they are free of them at earlier ages. Moreover, it has been observed that A beta 42 precedes A beta 40 in the course of amyloid deposition in DS brain. Thus, DS subjects provide the opportunity to investigate in the human brain the metabolic conditions that precede the formation of the amyloid deposits. Here we report that soluble A beta 42 is present in the brains of DS-affected subjects aged from 21 gestational weeks to 61 years but it is undetectable in age-matched controls. It is argued that overexpression of APP leads specifically to A beta 42 increase and that the presence of the soluble A beta 42 is causally related to plaque formation in DS and, likely, in AD brains.

Establishment and characterization of a new human cloned myelomonocytic cell line (ZC-1.6).

A human cell line, ZC, derived from the blood of a patient with acute myelomonocytic leukemia, was established and cloned. One of the clones, ZC-1.6, was subsequently characterized. As for its morphology and cytochemistry, ZC-1.6 clone shares a number of features with immature cells of the monocytic or myelocytic lineages. Surface marker analysis shows positivity for 4F2 (100% of the cells), and OKM1 (38%) monoclonal antibodies, and presence of surface HLA-D/DR antigens (100%) and Fc (11%) and complement (C3b) receptors (100%). Functional capabilities of ZC-1.6 cells include adherence, spreading, and phagocytosis of latex and opsonized zymosan particles. Despite its morphological immaturity, the ZC-1.6 clone produces relevant amounts of O2- in the presence of different stimuli (phorbol myristate acetate, opsonized zymosan, or latex particles). The production of O2- by ZC-1.6 cells is the first evidence that reactive oxygen intermediate production may represent an early feature of cells of the myelomonocytic lineage.

Lipoperoxidation is selectively involved in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by extensive neurofibrillary tangle (NFT) formation and neuronal loss in selective neuronal populations. Currently, no clues to the biological events underlying the pathological process have emerged. In Alzheimer disease (AD), which shares with PSP the occurrence of NFTs, advanced glycation end products (AGEs) as well as oxidation adducts have been found to be increased in association with neurofibrillary pathology. The presence and the amount of lipid and protein oxidation markers, as well as of pyrraline and pentosidine. 2 major AGEs, was assessed by biochemical, immunochemical, and immunocytochemical analysis in midbrain tissue from 5 PSP cases, 6 sporadic AD cases, and 6 age-matched control cases. The levels of 4-hydroxynonenal (HNE) and thiobarbituric acid reactive substances (TBARS), 2 major products of lipid peroxidation, were significantly increased by 1.6-fold (p < 0.04) and 3.9-fold (p < 0.01), respectively, in PSP compared with control tissues, whereas in AD only TBARS were significantly increased. In PSP tissue the intensity of neuronal HNE immunoreactivity was proportional to the extent of abnormal aggregated tau protein. The amount of protein oxidation products and AGEs was instead similar in PSP and control tissues. In AD, a higher but not significant level of pyrraline and pentosidine was measured, whereas the level of carbonyl groups was doubled. These findings indicate that in PSP, unlike in AD, lipid peroxidation is selectively associated with NFT formation. The intraneuronal accumulation of toxic aldehydes may contribute to hamper tau degradation, leading to its aggregation in the PSP specific abnormal filaments.

Density and pattern of dopamine D2-like receptors in the cerebellar cortex of aged rats.

The pharmacological properties and the anatomical localization of dopamine (DA) D2-like receptors were studied in the cerebellum of 3 months, 12 months, and 24 months male Wistar rats using combined radioligand binding and autoradiographic techniques with [3H]-spiroperidol as a ligand. The binding was consistent with the labelling of the DA D3 receptor subtype. The affinity for DA D2-like receptors was similar in the cerebellar cortex of the three animal groups investigated, whereas the density of binding sites (Bmax value) assessed using conventional radioligand binding techniques was reduced as a function of aging. Light microscope autoradiography revealed the localization of binding sites primarily in the molecular layer and to a lesser amount in Purkinje neurons layer. A loss of binding sites was noticeable with aging in the grey matter of the cerebellar cortex. It affected primarily the molecular layer. Analysis of radioligand binding data and light microscope autoradiography suggests that age-related changes of DA D2-like receptors depend in part by structural alterations of cerebellar cortex and in part by modifications in receptor expression.

Neurotransmitters, neuroreceptors and aging.

At first glance, it is satisfying to see the progress which has been made in the study of neurotransmitters. We have learned a great deal in the last number of years. First, we have been able to identify previously unknown compounds which affect the nervous system or associated peripheral organs. We now know a great deal about the metabolism of these molecules including their synthesis and catabolism. We have learned to identify and to classify their receptors. We have learned that alterations in the effects of neurotransmitters may be responsible for certain pathologies or may be a function of normal aging. Yet, we still have far to go in our research. There are neurotransmitters still to be discovered. We need to continue our efforts because there is still a large amount of confusion in the literature, for example, far too many contradictory reports concerning the effects of age confuse rather than clarify. Possibly order may return to the literature if investigators can agree on some basic tenets. For example, we need a basic definition of old. Some research groups consider 12-month-old rats as old while other groups consider them to be young individuals. We need to have standardization of methodology so that the conclusions can have validity. Once again certain investigators use whole brain homogenates while others use only discrete portions. We need to consider whether the effect we see in our experiment is primary or secondary to aging. We can be certain that due to the aging population, the importance of basic research of age-dependent changes in neurotransmitters and neuroreceptors will increase in the future.

Muscarinic cholinergic receptors in the hippocampus of aged rats: influence of choline alphoscerate treatment.

The present study was designed to investigate age-dependent changes of muscarcinic M1 and M2 cholinergic receptors in the rat hippocampus using radioreceptor assay and autoradiographic techniques with [3H]pirenzepine and [3H]AF-DX 116 as ligands. The analysis was performed on 2-, 12- and 27-month-old male Wistar rats, considered young, adult and old, respectively. Moreover, the influence of a 6-month treatment with choline alphoscerate on the density and pattern of M1 and M2 cholinergic receptors was assessed. Choline alphoscerate (L-alpha-glyceryl phosphorylcholine) is a precursor in the biosynthesis of several brain phospholipids which increases the availability of acetylcholine in various tissues. Muscarinic M1 cholinergic receptors were significantly decreased with increasing age whereas M2 cholinergic receptors did not show changes. Choline alphoscerate treatment countered, in part, the loss of muscarinic M1 receptor sites in old rats. Light microscope autoradiography revealed a loss of silver grains developed after exposure of sections of hippocampus to [3H]pirenzepine in the stratum oriens of CA1 and CA3 fields in rats of 12 and 27 months in comparison with young animals. Choline alphoscerate restored, in part, the decrease of silver grains noted in old rats. Quantitative analysis of the density of silver grains developed in the cell body of pyramidal neurons of CA1 and CA3 fields processed for the demonstration of muscarinic M1 receptor sites revealed a decrease of these grains in rats of 27 months in comparison with younger cohorts. These findings suggest that the reduction in muscarinic M1 sites noticeable between 2- and 12-month rats is probably dependent on the loss of nerve cells and/or terminals in these hippocampal fields rather than to a reduction of their density per neuron. Treatment with choline alphoscerate increased the expression of muscarinic M1 cholinergic receptors within the cell body of pyramidal neurons of CA1 and CA3 fields compared to age-matched control old rats. Consistent with radioreceptor assay data, no changes in the density of muscarinic M2 cholinergic receptors in the animal groups examined were demonstrated by light microscope autoradiography. The possible pharmacological relevance of the increased expression of muscarinic M1 cholinergic receptors elicited by choline alphoscerate in the hippocampus of aged rats is discussed.

Choline acetyltransferase and acetylcholinesterase in the hippocampus of aged rats: sensitivity to choline alphoscerate treatment.

The influence of aging on the acetylcholine synthesising and the degrading enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was studied in the hippocampus of male Wstar rats at 2 months (young), 12 months (adult) and 27 months (old) of age using biochemical, immunocytochemical and histochemical techniques. The influence of treatment for 6 months with a daily dose of 100 mg/kg of choline alphoscerate (L-alpha-glycerylphosphorylcholine) on the parameters examined was also investigated in old rats. Biochemical analysis of ChAT and AChE revealed the highest of the enzymatic activities in the hippocampus of adult rats and no significant differences between young and old animals. Immunocytochemical analysis of ChAT immunoreactivity revealed the highest immunostaining in adult rats followed in descending order by young then old animals. Histochemical evaluation of AChE reactivity revealed the highest expression in adult rats followed in descending order by old then young animals. Biochemical analysis of the effects of choline alphoscerate did not reveal any effect on ChAT activity and in increased expression of AChE activity. Moreover, the compound restored, in part, ChAT immunoreactivity in the hippocampus of old rats and increased the expression of AChE reactivity primarily in the CA3 sub field in old rats. The above results suggest that appropriate quantitative immunocytochemical and histochemical techniques may represent a useful tool for assessing age-dependent changes in cholinergic neurotransmission markers. The functional and pharmacological significance of the effects of choline alphoscerate on the expression of ChAT and AChE in the hippocampus of aged rats should be clarified in future studies.

Age-related changes of glial fibrillary acidic protein immunoreactive astrocytes in the rat cerebellar cortex.

Age-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity were investigated in the cerebellar cortex of young (3 months), adult (12 months) and old (24 months) rats using immunohistochemical techniques associated with image analysis. In young rats, cell bodies of GFAP-immunoreactive astrocytes were found in the white matter and in the granular layer of cerebellar cortex. Radially-oriented branches of astrocytes which are sited in the granular layer were also observed in the molecular layer. The number of GFAP-immunoreactivity astrocytes of white matter was decreased in adult and old rats in comparison with young cohorts, whereas their size increased progressively from 3 to 24 months old. The number and the size of GFAP-immunoreactive astrocytes of the granular layer was similar in young and adult rats. An increased number and size of GFAP-immunoreactive astrocytes was noticeable in old rats in comparison with younger cohorts. The number of radially oriented branches of the molecular layer was the same in the three age groups investigated. The above results indicate that GFAP-immunoreactive astrocytes of rat cerebellar cortex undergo age-related changes. The not homogeneous sensitivity to aging of cerebellar astrocytes suggests that evaluation of changes of different cell populations of cerebellar cortex should represent an important step of research on aging cerebellum.

Sensitivity to ageing of the limbic dopaminergic system: a review.

The limbic system includes the complex of brain centres, nuclei and connections that provide the anatomical substrate for emotions. Although the presence of small amounts of dopamine (DA) in several limbic structures has been recognized for a long time, for many years it was thought that limbic DA represented a precursor of noradrenaline in the biosynthetic pathway of catecholamines. More recent evidence has shown that limbic centres and nuclei are supplied with a dopaminergic innervation arising from the ventral tegmental area (field A10) and in smaller amounts from the mesencephalic A9 field. The dopaminergic limbic system is sensitive to ageing. Parameters of dopaminergic neurotransmission (DA levels, biosynthetic and catabolic markers and DA receptors) undergo age-related changes which depend on the structure and species investigated and are characterized mainly by a decline of different parameters examined. In this paper, the influence of ageing on DA biosynthesis, levels, metabolism and receptors are reviewed in laboratory rodents, monkeys and humans as well as in cases of Alzheimer's disease and Parkinson's disease. The possibility that changes of dopaminergic neurotransmission markers in the limbic system are associated with cognitive impairment and psychotic symptoms affecting the elderly is discussed. Better knowledge of dopaminergic neurotransmission mechanisms in the so-called physiological ageing and in senile dementia may provide new insights in the treatment of behavioural alterations frequently occurring in old age.

Decreased density of beta-adrenergic and muscarinic cholinergic receptor sites in the vasa nervorum of aged rats.

The pharmacological profile and the anatomical localization of beta-adrenergic and muscarinic cholinergic receptors of the vasa nervorum were studied in sections of sciatic nerve using radioreceptor binding and light microscope autoradiography techniques. Sprague-Dawley rats of 4 and 24 months of age were used. [3H]Dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) were used to label beta-adrenergic and muscarinic cholinergic receptors, respectively. The ligands were bound to sections of rat sciatic nerve in a manner consistent with the labelling of beta-adrenergic or muscarinic cholinergic receptors in the 2 age groups investigated. The dissociation constant (Kd) values (about 1.37 nM for [3H]DHA and 0.75 nM for [3H]QNB) did not significantly change between 4- and 24-month-old rats. The maximum concentration of binding sites (Bmax) for [3H]DHA was decreased by about 35% in 24 in comparison with 4-month-old rats. The Bmax value for [3H]QNB was reduced by about 50% in the aged rats. Light microscope autoradiography revealed the development of specific silver grains in the medial layer of epineurial and perineurial arteries in sections of sciatic nerve exposed either to [3H]DHA or [3H]QNB. The number of silver grains developed in epineurial and perineurial arteries of rats of 24 months is significantly lower than in animals of 4 months. The above results suggest the occurrence of an age-dependent loss in the density of beta-adrenergic and muscarinic cholinergic receptors of vasa nervorum. Vasa nervorum are the blood vessels which supply peripheral nerve trunks. They are constituted by outer (epineurial) and inner (perineurial) arteries and veins as well as by a capillary (endoneurial) plexus. Vasa nervorum are innervated by both sympathetic and non-sympathetic nerves which probably play a role in the pathogenesis of some neuropathies. Several different neurotransmitter containing nerve fibres have been identified in the vasa nervorum perivascular plexuses. However, no information is so far available concerning the neurotransmitter receptors of vasa nervorum. Moreover, although the occurrence of age-dependent changes in peripheral nerve morphology and function is well documented, very few reports were centered on the age-dependent changes of the vasa nervorum. The aims of the present study were to characterize pharmacologically and to localize anatomically the beta-adrenergic and muscarinic cholinergic receptors in the rat vasa nervorum. Moreover, the effect of ageing on the density and pattern of these receptors was investigated.

Age-related structural changes in the rat cerebellar cortex: effect of choline alfoscerate treatment.

The influence of ageing and of 3 months choline alfoscerate treatment on age-related microstructural changes in cerebellar cortex was studied in 3-, 12- and 24-month-old male Sprague-Dawley rats. The number of Purkinje and granule neurons, the density of Nissl bodies in the cytoplasm of Purkinje and granule neurons and the density of silver-gold impregnated fibres within molecular and granule cells layers were assessed by neurohistological and neurohistochemical techniques associated with microdensitometry and quantitative image analysis. The number of Purkinje and granule neurons was approximately the same in rats of 3 and 12 months and significantly decreased in 24-month-old animals. The density of Nissl bodies and of fine processes of silver-gold impregnated fibres were greatest in the cerebellar cortex of rats of 12 months of age, followed in descending order by 3- and 24-month-old rats. Both the density of Nissl bodies and of silver-gold impregnated fibres were significantly lower in the cerebellar cortex of the oldest age group considered in comparison with the young and middle age groups. Treatment with choline alfoscerate, a precursor in the biosynthesis of brain phospholipids which increases bioavailability of choline in the nervous tissue, noticeably reduced the loss of Purkinje and granule neurons in rats of 24 months. Moreover, it restored the density of Nissl bodies in the cytoplasm of Purkinje and granule neurons as well as the density of silver-gold stained fibres in the molecular and in the granule cells layers to values not significantly different from those found in rats of 3 months. These findings suggest that choline alfoscerate treatment may be effective in counteracting the age-dependent disarrangement of rat cerebellar cortex. The possible mechanisms of action of the compound on the microstructural changes of cerebellar cortex occurring with age are discussed.

Effect of long-term treatment with the dihydropyridine-type calcium channel blocker darodipine (PY 108-068) on the cerebral capillary network in aged rats.

The effects of treatment with the dihydropyridine Ca+2 antagonist darodipine (PY 108-068) on age-related changes in the cerebral capillary network was studied using alkaline phosphatase histochemistry with quantitative image analysis. The investigation was performed on male Wistar rats of 12 months (adult reference group) and 27 months. The 27-month-old rats consisted of two groups, the first of control untreated animals and the second of rats receiving an oral dose of 5 mg/kg/day of darodipine from the 21st to the 27th month. The cerebral areas examined included the frontal cortex, the occipital cortex, Ammon's horn of the hippocampus, and the dentate gyrus. The number and the average length of alkaline phosphatase-positive capillaries were decreased in old rats, when compared with adult rats. The intercapillary distance, which is considered as a sensitive parameter for capillary density was increased in aged rats in comparison to adult rats. The capillary diameter was increased slightly or unchanged in old rats. The Ammon's horn and the frontal cortex were the cerebral areas most affected by age-dependent changes of the capillary network. Treatment with darodipine increased the number and the average length of alkaline phosphatase-reactive capillaries and reduced the intercapillary distance and the diameter of cerebral capillaries in old rats. The pericapillary microenvironment of the Ammon's horn was the most sensitive to treatment with darodipine. The above results showed that treatment with darodipine is capable of counteracting some microvascular changes occurring in the brain of aged rats. This suggests that the blockade of dihydropyridine-type Ca2+ channels has a positive effect on the brain microvascular system and may counteract the impairment of pericapillary microenvironment occurring with aging.

Effect of treatment with the dihydropyridine-type calcium antagonist darodipine (PY 108-068) on the expression of calbindin D-28K immunoreactivity in the cerebellar cortex of aged rats.

The influence of long term treatment with the dihydropyridine-type Ca2+ antagonist darodipine (PY 108-068) on age-dependent changes in calbindin D-28K immunoreactivity in the cerebellar cortex of male Wistar rats was assessed. In 12-month-old rats used as an adult reference group, specific calbindin D-28K immunoreactivity was found within the cytoplasm of Purkinje neurons and their dendritic processes. The number of Purkinje neurons displaying calbindin D-28K immunoreactivity was decreased in the cerebellar cortex of aged in comparison with adult rats. The pattern of calbindin D-28K immunoreactivity was similar in the cerebellar cortex of 24-month-old rats (aged), although a significant decrease in the intensity of immunoreactivity was noticeable. Treatment of aged rats with darodipine for 6 months increased the percentage of immunoreactive Purkinje neurons and the intensity of calbindin D-28K immunoreactivity in the cytoplasm of Purkinje neurons. Calbindin D-28K is a Ca2+ binding protein probably involved in the modulation of Ca2+ homeostasis. The observation of a positive effect of darodipine treatment on calbindin D-28K immunoreactivity in the cerebellar cortex suggests that manipulation of dihydropyridine-type Ca2+ channels may contribute to counter age-dependent changes of Ca2+ homeostasis.

HLA-DR Schwann cell reactivity in peripheral neuropathies of different origins.

HLA-DR antigens have been found on Schwann cells in peripheral neuropathies of different origins but not in normal control cases. Class II antigen reactivity was more intense in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and hereditary motor and sensory neuropathy type 1 (HMSN), but was also observed in toxic or metabolic neuropathies. The expression of HLA-DR antigen on Schwann cells does not appear to be related to the inflammatory or autoimmune origin of the disease.

Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.

BACKGROUND: Progressive supranuclear palsy (PSP) is characterized by a pure neurofibrillary tau pathology involving mainly basal ganglia and brainstem nuclei. In addition to a haplotype of the tau gene potentially favoring tau aggregation, lipoperoxidation has been shown to be associated with PSP tau pathology. OBJECTIVE: To analyze cdk5/p35 complex, a kinase that regulates neurite outgrowth, as a potential cellular mechanism underlying tau phosphorylation in brain tissues from PSP and control cases and comparatively in cerebral cortex from subjects with AD. METHODS: Cdk5/p35 protein levels and distribution were evaluated by immunoblotting and immunocytochemistry in brain regions from seven PSP, six AD, and seven control cases, with similar postmortem intervals. RESULTS: Total cdk5 protein levels were significantly increased by more than threefold in PSP tissue and were augmented in PSP neurons, codistributed with tau immunoreactivity. P35, the regulatory subunit of cdk5, was degraded by postmortem proteolysis to the same extent in PSP, AD, and control tissues. CONCLUSIONS: The proteolysis in vivo of p35, the regulatory subunit of the kinase, is not ascertainable because it is masked by its postmortem degradation. The study, however, indicates that in PSP, the alteration of cdk5 is different from that described in AD and suggests that the absence of amyloid beta protein deposition may account for the different pathways responsible for the same kinase activation.

Dopamine D1-like receptors in the thymus of aged rats: a radioligand binding and autoradiographic study.

Age-dependent changes in the density and pattern of dopamine D1-like receptors were studied in the thymus of young (3 months), adult (12 months) and aged (24 months) male Wistar rats using combined radioligand binding and autoradiographic techniques. [3H]SCH 23390, which was used as a ligand, was specifically bound to sections of the thymus in a manner consistent with the labelling of dopamine D5 receptor. The dissociation constant value was similar in the thymus of the three animal groups examined. The maximal density of binding sites, evaluated with conventional radioligand binding techniques, was significantly reduced in the thymus of adult in comparison with young rats and further reduced in aged animals. Silver grains which correspond to [3H]SCH 23390 binding sites were revealed by light microscope autoradiography primarily in the cortex of the thymus and in lesser amounts within thymic corpuscles. A progressive decrease in the density of silver grains more pronounced in the cortex than in thymic corpuscles was observed in the thymus of adult and old in comparison with young rats. The loss of silver grains revealed with autoradiography is more moderate than the decrease in the density of binding sites shown by radioligand binding. Silver grains developed per single cells (probably lymphocytes) of the thymic cortex were reduced between young and adult rats and further decreased in old rats. The above findings suggest that the age-related decline in the density of dopamine D5 receptor assayed in the thymus is due in part to the reduced thymic mass with aging.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent changes in the expression of dopamine receptor subtypes in human peripheral blood lymphocytes.

The pharmacological profile and the density of dopamine D3 and D5 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 D3 receptor was assayed with [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]7-OH-DPAT) as a ligand. Dopamine D5 receptor was assayed using [3HIR]-(+)-(-chloro-2,3,4,5, tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([3H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [3H]7-OH-DPAT and [3H]SCH 23390 at dopamine D3 and D5 receptor, respectively, were similar in subjects of different ages. The density of dopamine D3 receptor binding sites was slightly decreased in subjects of 30-39 years in comparison with younger individuals. A remarkable loss of dopamine D3 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 [3H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D5 receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D3 but not D5 receptor between adult and mature subjects. The possibility that dopamine D3 receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.

Dopamine D1-like receptor subtypes in human peripheral blood lymphocytes.

Molecular biology studies have shown that human peripheral blood lymphocytes express a dopamine D5 receptor, whereas no information is available on dopamine D receptor, the other dopamine D1-like receptor subtype. Radioligand binding assay investigations with the nonsubtype selective dopamine D1-like receptor antagonist [3H]SCH 23390 as radioligand have suggested the presence of a dopamine D5 receptor in human peripheral blood lymphocytes. However, so far no evidence was provided as whether or not human peripheral blood lymphocytes express a dopamine D1 receptor. In this study, we have investigated dopamine D1 and D5 receptor mRNA and the influence of antibodies against dopamine D1 and D5 receptors on [3H]SCH 23390 binding to intact human peripheral blood lymphocytes. The two receptors were also analyzed by immunocytochemistry. Dopamine D5 receptor, but not D1 mRNA, was detected in human peripheral blood lymphocytes. Anti-dopamine D5 receptor antibodies, but not anti-dopamine D1 receptor antibodies, significantly decreased [3H]SCH 23390 binding to human peripheral blood lymphocytes. A dark-brown immunoreactivity was visualized in cytospin centrifuged human peripheral blood lymphocytes exposed to anti-dopamine D5, but not to anti-dopamine D1 receptor antibodies. These data collectively indicate that dopamine D5 receptor is the only dopamine D1-like receptor subtype expressed by human peripheral blood lymphocytes.

Expression of common acute lymphoblastic leukemia antigen (CD 10) by myelinated fibers of the peripheral nervous system.

The common acute lymphoblastic leukemia antigen (CALLA), CD10, is a 100-kDa surface glycoprotein endowed with neutral endopeptidase activity, shared by a number of hemopoietic and non-hemopoietic cells. In this report, immunohistochemical and Western blot techniques, using different anti-CD10 monoclonal antibodies, were utilized to demonstrate that CD10 is also expressed by myelin sheaths of the human peripheral nervous system (PNS), but not of the central nervous system. CD10-positive immunoreactivity appeared to be localized in the outer and inner borders of myelinated fibers, in nodes of Ranvier and in the Schmidt-Lantermann clefts, thus showing a distribution pattern very similar to that of myelin-associated glycoprotein (MAG). The above findings suggest that CD10 antigen, through its enzymatic activity, may have a functional role in the assembly and maintenance of PNS myelin. In addition, it is not known whether CD10, similarly to MAG, may be a target antigen in some PNS immune-mediated disorders.

NADPH-diaphorase activity in brain macrophages during postnatal development in the rat.

NADPH-diaphorase histochemistry, that allows the visualization of cells producing the gaseous intercellular messenger nitric oxide, was used in the study of the forebrain during the first three postnatal weeks in the rat. Subpopulations of NADPH-diaphorase positive neurons were observed at all ages studied. In addition, non-neuronal NADPH-diaphorase-stained cells were detected in the subcortical white matter, and were very numerous in the supraventricular portion of the corpus callosum, and in the internal and external capsules. These cells were present during the first two postnatal weeks, and were especially prominent at the end of the first postnatal week. They were round-shaped and morphologically similar to the brain macrophages, whose phagocytic activity has been shown in previous studies to play a role in naturally occurring cell death and elimination of exhuberant axons. Series of sections adjacent to those stained with NADPH-diaphorase were processed with immunohistochemistry, using two different antibodies (OX-42 and ED-1) that detect macrophagic and microglial markers, and antibodies that recognize the neuronal form of nitric oxide synthase. Furthermore, brain sections from rats at postnatal day 7 were sequentially processed for either OX-42 or nitric oxide synthase immunohistochemistry followed by NADPH-diaphorase histochemistry. The morphological features and distribution of the non-neuronal NADPH-diaphorase-positive cells were superimposable to those obtained with OX-42 and ED-1 immunohistochemistry. In addition, these cells did not display nitric oxide synthase immunoreactivity. Double-labelled NADPH-diaphorase-positive and OX-42-immunoreactive cells were detected at postnatal day 7. The present results show that brain macrophages express NADPH-diaphorase activity during the early stages of the normal postnatal maturation and suggest that nitric oxide produced by brain macrophages could be involved in the development reshaping of the central nervous system.