Olfactory bulbectomy alters NMDA receptor levels in the rat prefrontal cortex.

Olfactory bulbectomized (OBX) rats show a variety of behavioral and biochemical deficits that parallel human depression. We investigated the expression of glutamate receptor subtypes in cortical and subcortical brain regions following bilateral olfactory bulbectomy in adult rats. Quantitative receptor autoradiography using [(125)I]MK-801 (NMDA receptor), [(3)H]AMPA (AMPA receptor), and [(3)H]kainate (kainate receptor) was performed on brain sections at 1-5 weeks following olfactory bulbectomy. Our results show an elevation of NMDA receptors in the medial prefrontal cortex within 1 week following bulbectomy, which persisted up to at least 5 weeks post-bulbectomy. Neither kainate nor AMPA receptors were altered in any brain region examined. The potential significance of these results is discussed in light of experimental findings supporting a role for NMDA receptors in the mechanism of action of antidepressant drugs and the pathophysiology of major depression.

Pronounced impact of Th1/E47cs mutation compared with -491 AT mutation on neural APOE gene expression and risk of developing Alzheimer's disease.

Possession of the apolipoprotein E (APOE) epsilon4 allele is the most frequently associated genetic susceptibility factor for Alzheimer's disease (AD). Recently, new polymorphisms in the regulatory region of the APOE gene have been described. We analysed the effects of three of these mutations (-491 AT, -427 CT and Th1/E47cs) on disease risk in a large case-control study, and tested their impacts on APOE allelic expression in brain tissues. The Th1/E47cs T allele was associated with an increased risk of occurrence of AD, while the -491 T allele was associated with a decreased risk, independently of the APOE epsilon2/epsilon3/epsilon4 polymorphism effect. However, the impact of the Th1/E47cs mutation was the strongest. The -427 CT polymorphism was not associated with the disease. In AD subjects heterozygous for the epsilon4 allele, analysis of allelic expression showed that the relative expression levels of the epsilon4 allele were higher than those of the corresponding controls. Consistent with epidemiological data, the relative level of expression of the epsilon4 allele was modified accordingly to the presence or absence of the two main promoter polymorphisms, indicating, in vivo, the deleterious effect of the Th1/E47cs T allele and the protective effect of the -491 T allele in population. These data indicate that in addition to the qualitative effect of the APOE epsilon2/epsilon3/epsilon4 polymorphisms on the AD occurrence, the quantitative variation of expression of these alleles due to functional APOE promoter mutations, is a key determinant of AD development.

Ionotropic glutamate receptor subtypes in the aged memory-impaired and unimpaired Long-Evans rat.

The comparative quantitative autoradiographic distribution of ionotropic glutamate receptor subtypes were investigated in young adults (six months) and aged (24-25 months) cognitively impaired and unimpaired male Long-Evans rats. Aged rats were behaviorally characterized as either cognitively impaired or unimpaired based upon their performances in the Morris water maze task compared to the young adult controls. The status of the N-methyl-D-aspartate, [125I]dizocilpine maleate, [3H]kainate and amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA, [3H]AMPA) receptor binding sites were then established in these three subgroups of animals as a function of their cognitive performance in the Morris water maze task. The apparent densities of both N-methyl-D-aspartate/[125I]dizocilpine maleate and kainate binding sites were significantly decreased in various regions of the aged rat brain. Marked losses in [125I]dizocilpine maleate binding sites were observed in outer laminae of the frontal, parietal and temporal cortices, and the stratum radiatum of the CA3 subfield of the hippocampus. Interestingly, losses in [125I]dizocilpine maleate binding sites were generally most evident in the cognitively unimpaired aged subgroup, suggesting a possible inverse relationship between losses of this receptor subtype and cognitive performances in the Morris water maze task. The levels of [3H]kainate binding were most significantly diminished in various cortical and hippocampal areas as well as the striatum and septal nuclei of both groups of aged rats. In contrast, the apparent density of [3H]AMPA binding was increased in most hippocampal subfields and the superficial laminae of the occipital cortex of the cognitively impaired vs young adult rats. Changes in [3H]AMPA labeling failed to reach significance in the unimpaired cohort. Taken together, these results show that while losses in [3H]kainate binding were similar in both subgroups of aged rats, differences were seen with respect to cognitive status for both [125I]dizocilpine maleate/N-methyl-D-aspartate and [3H]AMPA binding sites. Decreases in [125I]dizocilpine maleate binding sites were mostly restricted to cortical areas of cognitively unimpaired rats, while increases in the AMPA binding subtype were seen in the memory-impaired subgroup. It would thus appear that changes in N-methyl-D-aspartate and AMPA receptor subtypes may be more critical than alterations in kainate binding sites for the emergence of the functional deficits seen in the aged cognitively impaired rat.

Comparative ontogenic profile of cholinergic markers, including nicotinic and muscarinic receptors, in the rat brain.

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [3H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [3H]N-methylcarbamylcholine (alpha(4)beta(2) nicotinic receptor sites), [3H]hemicholinium-3 (high-affinity choline uptake sites), [3H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [3H]4-DAMP (muscarinic M1/M3 receptor sites), [3H]pirenzepine (muscarinic M1 receptor sites), and [3H]AF-DX 116 and [3H]AF-DX 384 (muscarinic M2 receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M1/M3, M1, and possibly M2 receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M1/M3 and M1 sites were at least as high as in the adult, whereas M2 and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M1/M3 and M1 sites and, finally, by M2 and high-affinity choline uptake sites.

The distribution of somatostatin binding sites in the brain of gymnotiform fish, Apteronotus leptorhynchus.

The neuropeptide somatostatin (SS) and its binding sites display a wide distribution in the central nervous system of vertebrates. By employing semi-quantitative autoradiography, we identified such binding sites in the brain of the weakly electric fish Apteronotus leptorhynchus (Gymnotiformes, Teleostei). Whereas (SS1) binding sites for the octapeptide analogue Tyr3-SMS-201-995 appear to be absent in the gymnotiform brain, (SS2) binding sites for the analogue [Tyr0-D-Trp8]-somatostatin-14 were found in many brain regions and showed a similar distribution to that observed by other authors in the amphibian and mammalian central nervous system. Telencephalon While binding in the ventral telencephalon was typically low, all cell groups of the dorsal portion displayed a high degree of binding. The highest density of binding sites was found in the dorsal and caudal subdivision 2 of the dorsomedial telencephalon. Diencephalon Many cell groups of the diencephalon showed a medium to high degree of binding density. The highest level was seen in the habenula. Mesencephalon All layers of the optic tectum contained a medium number of binding sites, except the stratum marginale. In the torus semicircularis, the different layers displayed distinct binding density. While laminae 7-8 showed the highest degree of binding, the lowest density was found in lamina 6. Rhombencephalon Binding was generally low or absent in the tegmentum. Low levels of binding density were observed in the electrosensory lateral line lobe. Cerebellum Extremely high levels of binding were found in the eminentia granularis medialis and the eminentia granularis posterior. Throughout most regions of the brain, the relative density of binding sites and the relative amount of somatostatin immunoreactivity in fibres, as determined in previous studies, were in good agreement.

Levomepromazine receptor binding profile in human brain--implications for treatment-resistant schizophrenia.

The receptor binding profile of levomepromazine (LMP) in human brain was compared with that of clozapine (CLOZ) and chlorpromazine (CPZ). LMP showed significantly greater binding affinity for both alpha-1 and serotonin-2 binding sites than either CLOZ or CPZ, and significantly greater binding to alpha-2 sites than CPZ. A potent pharmacological effect at these receptor sites may explain the beneficial effect of LMP on psychotic symptoms and akathisia in treatment-resistant schizophrenia recently described in 2 open studies. LMP requires further appraisal as a potentially useful neuroleptic in the management of treatment-resistant schizophrenia.

Characterization and autoradiographic distribution of [3H]AF-DX 384 binding to putative muscarinic M2 receptors in the rat brain.

The novel radioligand [3H]AF-DX 384 binds specifically and saturably to putative muscarinic M2 receptor sites in homogenates of rat cerebral cortex. In saturation studies, [3H]AF-DX 384 appears to bind to two subpopulations of sites/states, one of high affinity (Kd1 = 0.28 +/- 0.08 nM) and another of low affinity (Kd2 = 28.0 +/- 5.0 nM). The maximal binding capacity (Bmax) of [3H]AF-DX 384 binding sites represented 9.7 +/- 2.3 fmol/mg protein (Bmax1) and 1993 +/- 551 fmol/mg protein (Bmax2) for the high and low affinity sites/states, respectively. The ligand selectivity profile of [3H]AF-DX 384 (at 2 nM) revealed that (-)-quinuclidinyl benzylate = atropine greater than 4-diphenylacetoxy-N-methylpiperidine methobromide greater than AQ-RA 741 greater than AF-DX 384 greater than UH-AH 371 much greater than methoctramine greater than oxotremorine-M greater than hexahydro-sila-defenidol much greater than pirenzepine greater than carbamylcholine much much greater than nicotine. This suggests that under our assay conditions [3H]AF-DX 384 binds mostly to M2-like muscarinic receptors in the rat central nervous system. This is further supported by the clear M2-like pattern of distribution observed using quantitative receptor autoradiography. High densities of specific labelling were seen in areas such as the hypoglossal nucleus, the pontine nucleus, the superior colliculus, the motor trigeminal nucleus, various thalamic nuclei and certain cortical laminae. Compared to [3H]AF-DX 116, the percentage of specific binding detected with [3H]AF-DX 384 was much higher. This is likely to be related to the greater chemical stability and affinity of [3H]AF-EX 384. In addition, autoradiograms obtained with [3H]AF-DX 384 (2 nM) are of better quality with film exposure periods five shorter than those needed for [3H]AF-DX 116 (10 nM). Therefore, [3H]AF-DX 384 displays a good selectivity for muscarinic M2 sites and offers major advantages, including higher affinity and greater stability, over previously used ligands.

Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases.

We have recently reported on the differential alterations of various cholinergic markers in cortical and subcortical regions in Alzheimer's disease (AD). The main purpose of the present study was to determine if cholinergic deficits observed in patients with AD are unique to this disorder or can be generalized to others such as idiopathic Parkinson's disease (PD) and PD with Alzheimer-type dementia (PD/AD). Muscarinic M1, M2, and nicotinic receptor binding parameters (KD and Bmax) were determined in various cortical and subcortical areas using selective radioligands ([3H]pirenzepine, [3H]AF-DX 116, and N[3H]methylcarbamylcholine). Choline acetyltransferase activity was also determined as a marker of the integrity of cholinergic innervation. Alterations of cholinergic markers are comparable in cortical areas in AD, PD, and PD/AD brains. In frontal and temporal cortices, as well as in the hippocampus, choline acetyltransferase activity and binding capacities of M2 and nicotinic binding sites are similarly decreased in these three disorders compared with age-matched control values. M1 receptor binding parameters are not significantly modified in cortical areas in patients with these disorders. In contrast, important differences between AD and PD brain tissues are found in subcortical areas such as the striatum and the thalamus. The density of M1 sites is significantly increased in striatal areas only in patients with AD, whereas densities of nicotinic sites are decreased in thalamus and striatum in PD and PD/AD, but not AD, brain tissues. The binding capacity of M2 sites is apparently unchanged in subcortical areas in all three disorders, although tendencies toward reductions are observed in the striatum of PD and PD/AD patients. Thus, although comparable alterations of various cholinergic markers are observed in cortical areas in the three neurological disorders investigated in the present study, important differences are seen in subcortical areas. This may be relevant to the respective etiological and clinical profiles of AD and PD.

Characterization of 5-HT1D receptor binding sites in post-mortem human brain cortex.

The present study provides further evidence for the presence of serotonin1D (5-HT1D) receptors in post-mortem human brain. Receptor binding parameters in temporal cortex homogenates were assessed using [3H]5-HT in the presence of 100 nM 8-OH-DPAT, 1 microM propranolol and 1 microM mesulergine to prevent labelling of the 5-HT1A, 5-HT1B and 5-HT1C sites, respectively. Under these conditions, [3H]5-HT apparently bound to a class of high affinity (Kd = 5.0 +/- 1.0 nM) low capacity (Bmax = 96 +/- 23 fmol/mg protein) sites. In competition experiments, 5-HT and 5-carboxyamidotryptamine (5-CT), as well as ergotamine, lysergic acid, sumatriptan and RU-24969 exhibited high affinity for these sites. This pharmacological profile is concordant with the ligand selectivity pattern reported for 5-HT1D receptors in other species and thus provides further evidence for its existence in human temporal cortex. In addition, the competition profile of some ligands, particularly of unlabelled 5-HT, 5-CT and ergotamine, revealed the existence of a lower affinity binding site. The latter suggests receptor heterogeneity or the presence of a lower affinity state of 5-HT1D receptors.

Presence of sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla.

Receptor binding studies with [3H]-(+)SKF-10047 were carried out to characterize the putative sigma (sigma) and phencyclidine (PCP) receptors in membrane preparations of bovine adrenal medulla. Specific binding of the radiolabelled compound was observed after incubation with the membrane preparation at 37 degrees, the equilibrium being reached at 20 min and the maximal binding being observed with 0.6 mg/ml protein. Saturation binding studies were performed at equilibrium (30 min at 37 degrees with 0.5 mg/ml of membrane protein) in the presence of haloperidol (1 microM) or 1-[1-(2-thienyl) cyclohexyl] piperidine (TCP; 0.2 microM) to block sigma or PCP receptors, respectively. The binding of [3H]-(+)SKF-10047 was characterized by two distinct components. A high affinity binding site (haloperidol sensitive) had an apparent KD of 8.3 nM and a Bmax of 67 pmol/g protein. A lower affinity binding site (TCP sensitive) had an apparent KD of 32.7 nM and a Bmax of 83 pmol/g protein. The drug specificity of the high affinity binding site resembled that of the putative sigma receptor, being potently inhibited by haloperidol and pentazocine. The binding pharmacology of the low affinity site resembled that of the phencyclidine receptor, being potently displaced by TCP and PCP. The binding of [3H]-(+)SKF-10047 to both receptors showed marked stereoselectivity for the dextrorotatory (+) isomer of SKF-10047 and was insensitive to the receptor specific opioid ligands DAGO (mu), DSLET (delta) and U-69593 (kappa). These data indicate that bovine adrenal medulla contains sigma and PCP-like receptors.

Structural identification, subcellular localization and secretion of bovine adrenomedullary neuromedin C [GRP-(18-27)].

Bombesin-like immunoreactivity (BLI) was purified from acid (HCl) extracts of bovine adrenal medulla. High performance liquid chromatography (HPLC) on a mu-Bondapak C18 column revealed the presence of five molecular forms of BLI, one coeluting with synthetic gastrin releasing peptide (GRP), the mammalian counterpart of amphibian bombesin, one coeluting with neuromedin C, one coeluting with neuromedin B and the two other ones coeluting with the oxidized forms of neuromedins B and C. The material corresponding to neuromedin C was purified to homogeneity and its amino acid composition and sequence corresponded to those expected for neuromedin C. HPLC analysis on an analytical SP-5PW column of subcellular extracts of bovine adrenal medulla indicated that neuromedin C is almost exclusively localized in secretory granules. The neuropeptide function of neuromedin C and/or other BLI peptides at this level was supported by the stimulatory effect of carbamylcholine (500 microM) on the release of BLI (4.5-fold increase over the basal release of 19 fmol/5 min) from perfused bovine adrenal glands.