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Metabolic syndrome (MetS) is a disorder characterized primarily by the development of insulin resistance. Insulin resistance and subsequent hyperinsulinemia, originating from abdominal obesity, increases the risk of cerebrovascular and cardiovascular disease and all-cause mortality. Obesity is probably a risk factor for Alzheimer's disease and vascular dementia and is associated with impaired cognitive function. The obese Zucker rat (OZR) represents a model of type 2 diabetes exhibiting a moderate degree of arterial hypertension and of increased oxidative stress. To clarify the possible relationships between MetS and brain damage, the present study has investigated brain microanatomy in OZRs compared with their littermate controls lean Zucker rats (LZRs). Male OZRs and LZRs of 12 weeks of age were used. Their brain was processed for immunochemical and immunohistochemical analysis of glial fibrillary acidic protein (GFAP). In frontal and parietal cortex of OZRs a significant increase in the number of GFAP immunoreactive astrocytes was observed. Similar findings were found in the hippocampus, where an increased number of GFAP immunoreactive astrocytes were detected in the CA1 and CA3 subfields and dentate gyrus of OZRs compared to the LZRs. These findings indicating the occurrence of brain injury accompanied by astrogliosis in OZRs suggest that these rats, developed as an animal model of type 2 diabetes, may also represent a model for assessing the influence of MetS on brain. The identification of neurodegenerative changes in OZRs may represent the first step for better characterizing neuronal involvement in this model of MetS and possible treatment for countering it.

The expression and microanatomical localization of adenosine A1 and A3 receptor subtypes were investigated in rat pial and intracerebral arteries by immunoblotting, immunohistochemistry and in situ hybridization techniques. Pial artery membranes develop immune bands of approximately 79 and 52 kDa when exposed to anti-A1 and anti-A3 receptor protein antibodies respectively. Sympathectomy performed by bilateral superior cervical ganglionectomy did not change the pattern of adenosine A1 or A3 receptor immunochemistry. Sections of pial and intracerebral arteries processed for A1 or A3 receptor protein immunohistochemistry developed immune reaction in the tunica media, within arterial smooth muscle. A1 receptor immunoreactivity was more pronounced in large-sized compared to medium- and small-sized pial arteries and was stronger in small than in medium- or large-sized intracerebral arteries. A3 receptor immunoreactivity was more pronounced in smaller sized pial arteries compared to larger pial arteries, whereas no differences in the intensity of immune staining were noticeable between different sized intracerebral arteries. In situ hybridization histochemistry revealed a strong signal for A1 receptor and a moderate signal for A3 receptor in the tunica media of pial arteries, within smooth muscle. The present study indicates that rat pial and intracerebral arteries besides to the well characterized A2a and A2b receptors, express also A1 and A3 receptor subtypes. The identification of cerebrovascular A1 and A3 adenosine receptor subtypes may stimulate further research for detailing the mechanism(s) of regulation of cerebral circulation by adenosine.

The volume and the morphology of brain white matter as well as the number and the size of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were investigated in 6-month-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar–Kyoto (WKY) rats. The volume of frontal and occipital cortex and of hippocampus was decreased in SHR in comparison with normotensive rats, whereas the volume of neostriatum was unchanged. A remarkable decrease of the volume of internal capsule and striosomes, a moderate reduction of that of corpus callosum and no changes of the volume of external capsule and of white matter of hippocampus were also observed in SHR. In SHR the number of astrocytes was higher in the frontal and occipital cortex and in the white matter of the CA 1 and CA 3 subfields of the hippocampus, but not in the corpus callosum or in the grey matter of the CA 1 and CA 3 subfields. Staining for myelin did not reveal alterations in single fibre sheath morphology. These findings indicate the occurrence of changes of forebrain white matter in SHR, consisting in the reduction of it without qualitative modifications of myelinated fibres. The development of gliosis apparently not related with changes of volume of white matter was also found.

The expression of dopamine D4 receptor was investigated in human peripheral blood lymphocytes with a radioligand binding assay technique, using [3H]clozapine as radioligand. [3H]Clozapine was specifically bound to human peripheral blood lymphocytes. The binding was time-, temperature-, and concentration-dependent and of high affinity, with a dissociation constant (K(d)) value of 0.34 +/- 0.02 nM and a maximum density of binding sites (B(max)) value of 27 +/- 1.4 fmol/10(6) cells. The pharmacological profile of [3H]clozapine binding to human peripheral blood lymphocytes was similar to that found in Chinese hamster ovary (CHO) cells transfected with the D4 clone (D4.2 variant). The above results are consistent with molecular biology studies demonstrating the expression of a dopamine D4 receptor in immune cells and in human peripheral blood lymphocytes. The availability of a rapid and sensitive radioligand binding assay technique for the dopamine D4 receptor in human peripheral blood lymphocytes may contribute to better define the role of this dopamine receptor subtype in neurological and psychiatric disorders.

The presence of a cholinergic system in the placenta is suggested by several data, but no information is available concerning cholinergic receptor expression by placenta. The present study was designed to investigate muscarinic cholinergic receptors in sections of human placenta using a radioligand binding techniques with [ 3 H] N -methyl scopolamine ([ 3 H]NMS) as a ligand. [ 3 H]NMS was bound to sections of human placenta in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant ( K d ) value was 0.1±0.03 nM and the maximum density of binding site ( B max ) value was 10.82±0.09 fmol/mg of tissue. The binding was time-, temperature- and concentration-dependent, belonging to one class of high affinity sites. Analysis of [ 3 H]NMS displacement curves by compounds acting on the different subtypes of muscarinic cholinergic receptor subtypes suggests that human placenta expresses the four subtypes (M 1 –M 4 ) of muscarinic cholinergic receptor assayable with radioligand binding assay techniques. The demonstration of muscarinic cholinergic recognition sites in human placenta may contribute to define the possible significance of placental cholinergic system. Moreover, human placenta can be used as an easily obtainable human source of M 1 –M 4 muscarinic cholinergic receptor subtypes.

The expression of dopamine D5 receptor was investigated in peripheral blood lymphocytes of 11 migraine patients and of ten healthy control subjects using a radioligand binding technique with [3H]SCH 23390 as a ligand. [3H]SCH 23390 is a benzazepine derivative with potent antagonist properties at the dopamine D1-like receptors. [3H]SCH 23390 was specifically bound to peripheral blood lymphocytes of migraineurs and control subjects in a manner consistent with the labelling of a dopamine D5 receptor. In migraineurs a statistically significant higher density of lymphocyte dopamine D5 receptor compared with controls was noticeable, whereas the affinity of the radioligand was unchanged. The increased density of dopamine D5 receptor in peripheral blood lymphocytes may reflect the dopaminergic hypersensitivity displayed by migraineurs and may represent a relatively simple and reliable peripheral marker of altered dopaminergic function.

The present study assesses the effect of unilateral lesions of the nucleus basalis magnocellularis (NBM) and of treatment with L-alpha-glyceryl phosphorylcholine (GFC, choline alfoscerate) on the acetylcholine-synthesizing (choline acetyltransferase (ChAT)), and acetylcholine-degradating (acetylcholinesterase (AChE)) enzymes in the rat fronto-parietal cortex ipsilateral to the lesion. Ibotenic acid injections in the right NBM area caused a significant decrease of both ChAT and AChE activities as well as of histochemically reactive stores of AChE in the right fronto-parietal cortex. Treatment with GFC restored in part the loss of ChAT and AChE activities. Moreover, AChE reactivity is restored in the fronto-parietal cortex of NBM-lesioned rats treated with GFC. GFC is a precursor in the biosynthesis of brain phospholipids which increases the bioavailability of acetylcholine in the nervous tissue. The possible relevance of the restoration of the marker enzymes of cholinergic neurotransmission by GFC in an animal model of cholinergic hypofunction is considered.

Circulation to the brain is affected by hypertension. Hypertension-dependent cerebrovascular changes were documented primarily in brain pial arteries, whereas no information is so far available concerning changes of peripheral nerve vascularization in hypertension. This study was designed to assess the occurrence of structural changes of interfascicular and intrafascicular arteries supplying peripheral nerves (the so called vasa nervorum) in spontaneously hypertensive rats (SHR). The investigation was performed in 8-month-old SHR, by using standard microanatomical techniques associated with quantitative image analysis. In SHR a significant increase of systolic pressure values accompanied by thickening of the arterial wall, narrowing of the lumen and increase of the wall-to-lumen ratio were observed in comparison with age-matched normotensive Wistar-Kyoto rats. Hypertension-related structural changes involved primarily interfascicular arteries and to a lesser extent intrafascicular arteries. These findings indicate that similarly as documented for cerebral arteries, the vascular supply to peripheral nerves is impaired in hypertension. Structural changes of interfascicular and intrafascicular arteries of SHR could lead to ischemia of peripheral nerves. Further work is in progress to evaluate the functional relevance of hypertensive changes to peripheral nerve vasculature.

The subtypes of muscarinic cholinergic receptors were studied in human peripheral blood lymphocytes with radioligand binding techniques and the non-selective muscarinic cholinergic receptor antagonist [3H]quinuclidinyl benzylate (QNB) as a ligand. [3H]QNB was bound to human peripheral lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.60 +/- 0.08 nM and the maximum density of binding sites (Bmax) was 2.33 +/- 0.03 fmol/2.2 x 10(6) cells. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high affinity sites. Analysis of the pharmacological profile of [3H]QNB binding in the presence of compounds specific for the different muscarinic receptor subtypes suggests that human peripheral blood lymphocytes express mainly muscarinic cholinergic M2 and M3 receptor subtypes and to a lesser extent muscarinic M4 receptors. The characterization of the subtypes of muscarinic cholinergic recognition sites expressed by human peripheral blood lymphocytes may represent a tool for investigating the possible relationships between immune and cholinergic systems in normal and pathologic conditions.

The pharmacological properties and the anatomical localisation of dopamine D 3 receptor were assessed in the rat cerebellar cortex using radioligand binding techniques associated with light microscope autoradiography and 7-[ 3 H]hydroxy- N,N -di-n-propyl-2-aminotetralin (7-[ 3 H]OH-DPAT) as a ligand. 7-[ 3 H]OH-DPAT was specifically bound to sections of rat cerebellar cortex with a dissociation constant ( K d ) of 0.5 nM and a maximum density of binding sites ( B max ) of 97 ± 4 fmol/mg tissue. The rank order of potency of competitors of 7-[ 3 H]OH-DPAT binding and the observation that guanosine triphosphate did not affect radioligand binding suggest the labelling of a dopamine D 3 receptor. 7-[ 3 H]OH-DPAT binding sites are located mainly in the molecular layer and in lesser amounts in the Purkinje neuron layer, primarily within the cell body of Purkinje neurons. No specific accumulation of silver grains was observed in the granule neuron layer or in the white matter of the cerebellar cortex. The localisation of a putative dopamine D 3 receptor within Purkinje neurons suggests that this site may have functional relevance in the cerebellar cortex.

The effect of unilateral or bilateral lesions of the nucleus basalis magnocellularis (NBM) on the dentate gyrus of the hippocampus were assessed using microanatomical and electrophysiological techniques. NBM is the main cholinergic basal forebrain nucleus that supplies the fronto-parietal cortex. Lesions were induced using the neurotoxin ibotenic acid or a radio-frequency system and did not affect glutamic acid decarboxylase activity both in the frontal cortex and in the hippocampus. At 4 weeks after lesioning, a loss of choline acetyltransferase (ChAT) activity and of ChAT-immunoreactive fibres was observed in the frontal cortex but not in the hippocampus and no changes in the density of granule neurons of the dentate gyrus or in the hippocampal long-term potentiation (LTP) were noticeable. At 8 weeks after lesioning the loss of both ChAT activity and of ChAT-immunoreactive fibres persisted in the frontal cortex of NBM-lesioned rats. Moreover, at this time a significant decrease in the density of granule neurons in the dentate gyrus accompanied by a reduced probability of dentate LTP induction were observed in both ibotenic acid- and radio-frequency-lesioned rats. These findings have shown that although NBM does not send direct cholinergic projections to the hippocampus, lesions of this cholinergic nucleus are accompanied by delayed neurodegenerative changes involving the dentate gyrus. This suggests the occurrence of indirect connections between NBM and the hippocampus, the functional relevance of which should be explored.

The presence and micro-anatomical localization of muscarinic cholinergic receptors were assessed in dorsal root ganglia of chick embryo during development using radioligand binding and immunocytochemical techniques, respectively. The non-selective muscarinic cholinergic receptor radioligand [3H]quinuclidinyl benzilate was specifically bound to sections of chick dorsal root ganglia with a dissociation constant value (Kd) of 0.75 +/- 0.02 nM and a maximum density of binding sites (Bmax) of 7.2 +/- 0.5 fmol/mg tissue. [3H]Quinuclidinyl benzilate binding was partially sensitive to pirenzepine displacement. This suggests that muscarinic cholinergic receptors expressed by dorsal root ganglia of chick embryo at least in part belong to the M1 muscarinic receptor subtype. Immunocytochemical analysis confirmed the presence of muscarinic receptors in the ganglia. These findings suggest that neurons of dorsal root ganglia, which are known to express cholinergic markers such as choline acetyltransferase, acetylcholinesterase and high affinity choline uptake, are also cholinoceptive.

The present study was designed to analyze age-dependent changes in the expression of calbindin D-28k immunoreactivity in the cerebellar cortex of male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old). Calbindin D-28k immunoreactivity was localized primarily in the cytoplasm of Purkinje neurons and in the basal portion of their dendritic processes entering in the molecular layer. The expression of calbindin D-28k immunoreactivity was highest in Purkinje neurons of adult rats and lowest in Purkinje neurons of old rats. Moreover, the number of Purkinje neurons displaying calbindin D-28k immunoreactivity was decreased in aged rats. These results demonstrate the occurrence of age-related-changes in the expression of immunoreactivity of the Ca2+ binding protein calbindin D-28k in the rat cerebellar cortex. The possibility that impairment in the expression of Ca2+ binding proteins may be involved in changes of intracellular Ca2+ homeostasis reported in aging and in some neuro-degenerative disorders is discussed.

The expression of gp75LNGFR (low-affinity receptor of the nerve growth factor (NGF) family of neurotrophins) immunoreactivity was studied in the cerebellar cortex of male Wistar rats of 3 months (young), 12 months (adult) and 24 months of age using immunohistochemical techniques and a monoclonal antibody against rat-gp75LNGFR. The percentage of Purkinje neurons displaying gp75LNGFR immunoreactivity (IR), and the intensity of gp75LNGFR IR in the cytoplasm of Purkinje and granule neurons, and in the neuropil of molecular layer of the cerebellar cortex were determined by quantitative image analysis and microdensitometry. The number of Purkinje neurons displaying gp75LNGFR IR and the intensity of gp75LNGFRIR in the cytoplasm of Purkinje neurons was significantly higher in rats of 12 months of age in comparison with 24- and 3-month-old rats. No significant changes were observed in the intensity of gp75LNGFRIR in the granule neuron layer of young, adult and old rats. In the molecular layer, the highest gp75LNGFRIR was found in young animals. It was reduced as a function of age. The present results show that changes in gp75LNGFRIR observed as a function of age affect to a different extent the three layers of the rat cerebellar cortex.

The effect of dopamine on the 3',5'-cyclic adenosine monophosphate (cAMP) generating system of membrane particles of rat prostate gland was studied. Dopamine increased the concentration of cAMP in a dose-dependent manner. The selective D1 receptor inhibitor SCH 23390 caused a decrease in dopamine-elicited cAMP levels. Any effect of dopamine on prostatic cAMP concentration was abolished by the simultaneous addition to the incubation medium of SCH 23390 and of the D2 receptor blocking agent (-)-sulpiride. Also the D2 receptor agonist bromocriptine decreased cAMP levels. The present data indicate the existence, in the rat prostate gland, of two types of dopamine receptors mediating, respectively, the activation (D1 effect) and the inhibition (D2 effect) of adenylate cyclase activity.

The occurrence and distribution of 4-aminobutyrate:2-oxoglutarate transaminase (GABA-T) activity were examined in the guinea pig lung using biochemical and enzymehistochemical methods. Specific GABA-T reactivity was confined primarily to the arteries and to a lesser extent to the veins. No activity could be observed in association with bronchi, alveoli and nerve fibers. Our findings indicate that the GABA-T activity in the lung is specifically located in blood vessels. This study is the first to demonstrate GABA-T activity in peripheral blood vessels.

Using a fluorescence histochemical method we found that the precursor in the synthesis of serotonin, 5-hydroxytryptophan (5-HTP), is taken up by a population of nerve fiber-like structures, presumably indoleaminergic in nature, within the wall of cerebral arteries. These findings may indicate that the therapeutic action of 5-HTP in the management of some types of headache is determined by a direct peripheral action on cerebral blood vessel.

The presence of a cholinergic innervation of the main cerebral blood vessels has been studied in the dog. Cholinergic nerve fibers are found in all examined arteries and veins, organized in two or in a single nerve plexus. The cerebral veins appear to be less innervated than arteries. The meaning of a cholinergic innervation in the cerebral circulation is discussed.

The effect of dopamine (DA) on 3′,5′-cyclic adenosine monophosphate (cAMP) generation in the rabbit portal vein was investigated. Dopamine added to homogenates of rabbit portal vein increased the concentration of cAMP. DA-elicited cAMP increase was exclusively inhibited by DA antagonists fluphenazine and haloperidol suggesting the existence of DA receptors (of the DA1 subtype) in the portal vein.

Using a fluorescence histochemical technique we found that the precursor of serotonin, 5-hydroxytryptophan (5-HTP), is taken up by a population of nerve fiber-like structures, indoleaminergic in nature since selectively destroyed by the neurotoxin 6-hydroxytryptamine, within rat choroid plexus. Nerve fiber-like structures are localized within the wall of choroid blood vessels. More infrequently nerve fibers end in the stroma or in close relation to epithelial cells of the plexus. The electrolytic lesioning of raphe nuclei causes the disappearance of 5-HTP fluorescence, suggesting that these nerve fibers originate from raphe nuclei. These results suggest a direct influence of indoleaminergic pathways originating from the brainstem on the blood flow through choroid plexus as well as on the production of cerebrospinal fluid.

The effect of dopamine on 3', 5'-cyclic adenosine monophosphate (cAMP) generation in the rabbit renal artery was studied. Dopamine added to homogenates of rabbit renal artery increased the concentration of cAMP. The stimulation of cAMP levels elicited by dopamine was remarkably reduced by the dopamine receptor antagonists fluphenazine and haloperidol, but it was unaffected by the ergot-like dopaminergic agonist bromocriptine. These findings appear to indicate that the effects of dopamine on rabbit renal artery are mediated through a D 1 receptor.

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.