Biomedical subjects

D C Mash

Publications and source records attributed to D C Mash.

At least 73 records · Page 4Linked to original sources

PubMed1996

Quantitation of N,N-dimethyltryptamine and harmala alkaloids in human plasma after oral dosing with ayahuasca.

Harmine, harmaline, tetrahydroharmine (THH), and N,N-dimethyltryptamine (DMT) were quantitated in plasma from 15 healthy male volunteers after the ingestion of ayahuasca, a beverage that has been used for religious purposes in Brazil since pre-Columbian times. A growing awareness of the interest in this ancient shamanistic practice in modern urban cultures and the widespread popular dissemination of the inebriant effects and type and sources of the plant admixtures used to prepare the beverage have provided additional impetus for this study. The three harmala alkaloids were quantitated from protein-precipitated plasma by high-performance liquid chromatography using fluorescence detection. Recovery from blank human plasma was quantitative, and the limit of quantitation (LOQ) was below 2 ng/mL of plasma for each of the harmala alkaloids. Standard concentrations ranged from 10 to 250 ng/mL for harmine and THH and from 1.0 to 25.0 ng/mL for harmaline, respectively. Linearity was observed for harmine, harmaline, and THH within these respective ranges. The highest concentrations of harmala alkaloids in human plasma were found to be 222.3 ng/mL for harmine, 134.5 ng/mL for THH, and 9.4 ng/mL for harmaline. DMT was quantitated by gas chromatography using nitrogen-phosphorus detection after liquid-liquid extraction with diphenhydramine as an internal standard. DMT recovery was quantitative, and the limit of detection and LOQ were 0.5 and 5 ng/mL, respectively. Linearity for DMT was observed from 5 to 1000 ng/mL. The one-step extraction method for DMT and the protein precipitation method for the three harmala alkaloids afford rapid, sensitive, and quantitative analyses of these alkaloids with minimal analyte loss. The analytical methods also may be applicable to other matrices, including whole blood and urine samples and homogenized tissue specimens. These are the first reported observations of DMT and harmala alkaloids in plasma after ritual ingestion of ayahuasca.

Administration, Oral↗

PubMed1995

The serotonin agonist m-chlorophenylpiperazine (mCPP) binds to serotonin transporter sites in human brain.

The serotonin (5-HT) agonist m-chlorophenylpiperazine (mCPP) is commonly used as a neuroendocrine challenge agent in humans, and it is well accepted that its mechanism of action involves direct stimulation of postsynaptic 5-HT receptors. In the present work, we demonstrate that mCPP also displays appreciable affinity (IC50 = 230 nM) for 5-HT transporters labeled by [125I]3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester ([125I]RTI-55) in human occipital cortex. These data agree with reports that show mCPP increases extracellular 5-HT in rat brain via a mechanism involving 5-HT transporters. Our results indicate that mCPP may exhibit a presynaptic 5-HT action in man, and studies utilizing mCPP to assess 5-HT receptor sensitivity should be interpreted with caution.

Adult↗

PubMed1995

Properties of ibogaine and its principal metabolite (12-hydroxyibogamine) at the MK-801 binding site of the NMDA receptor complex.

The putative anti-addiction alkaloid ibogaine and its principal metabolite 12-hydroxyibogamine appear to act at the (+)-5 methyl-10,11,dihydro-5H- dibenzo[a,d]cycloheten-5-10-imine maleate (MK-801) binding site in the N-methyl-D-aspartate (NMDA)-receptor cation channel. This conclusion is based on findings that both compounds competitively displaced specific [3H]MK-801 binding to membranes from postmortem human caudate and cerebellum and from frog spinal cord. Ibogaine was 4-6-fold more potent than its metabolite and both compounds were less potent (50-1000-fold) than MK-801 binding to the NMDA receptor. In addition, ibogaine (100 microM) and 12-hydroxyibogamine (1 mM) blocked (85-90% of control) the ability of NMDA (100 microM, 5 s) to depolarize frog motoneurons in the isolated frog spinal cord. The prevention of NMDA-depolarizations in frog motoneurons showed use-dependency and was very similar to the block produced by MK-801. In view of the abilities of MK-801 to affect the responses to addictive substances in pre-clinical investigations, our results are compatible with the idea that the ability of ibogaine and 12-hydroxyibogamine to interrupt drug-seeking behavior may, in part, result from their actions at the MK-801 binding site.

Adult↗

PubMed1995

Mapping dopamine transporters in the human brain with novel selective cocaine analog [125I]RTI-121.

The novel cocaine analog RTI-121 [3 beta-(4-iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester] was evaluated as a probe for the in vitro labeling and localization of the dopamine transporter in the human brain. Saturation binding experiments conducted in sucrose-phosphate buffer (10 mM sodium phosphate, pH 7.4, 0.32 M sucrose) revealed high- and low-affinity binding components with affinity values (KD) of 0.25 +/- 0.04 and 4.9 +/- 1.6 nM (mean +/- SE) and densities (Bmax) of 56.8 +/- 13.8 and 147.7 +/- 23.4 pmol/g tissue, respectively. In contrast, when saturation binding experiments were performed in phosphate-buffered saline (10 mM Na2HPO4, 1.8 mM KH2PO4, 136 mM NaCl, 2.8 mM KCl, 10 mM NaI, pH 7.4), a 9-fold decrease in the density of the low-affinity component was noted, suggesting that the low-affinity RTI-121 binding site is associated with the region of the transporter involved in the ionic dependence of substrate recognition and/or uptake. The rank order of potency for inhibition of [125I]RTI-121 binding to human caudate membranes demonstrates that the radioligand selectively labels the dopamine transporter (GBR 12909 > RTI-121 > mazindol > nomifensine > (-) cocaine > desipramine > citalopram). Autoradiographic mapping of [125I]RTI-121 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderate densities were also observed over the substantia nigra and the ventral tegmental area. Low-to-background labeling of [125]RTI-121 was seen throughout the cerebral cortex, amygdaloid nuclei, globus pallidus, and thalamus. In comparison with the autoradiographic distribution of the cocaine analogs [3H]WIN 35,428 (or CFT) and [125I]RTI-55 (or beta-CIT), the labeling pattern for [125I]RTI-121 was more restricted. These studies demonstrate that [125I]RTI-121 labels dopamine-rich brain regions with greater selectivity than other currently available cocaine analogs, which makes it a potentially superior imaging probe for mapping the dopamine transporter in the human brain.

Adult↗

PubMed1995

Attenuation of muscarinic receptor-G-protein interaction in Alzheimer disease.

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [3H]PIP2 hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease. Whether this defect reflects an alteration in the M1 muscarinic receptor, its respective guanine nucleotide binding (G) protein or both is not known. This study compares the number and both basal and muscarinic receptor-mediated function of G-proteins in synaptosomal membranes from cerebral cortical samples of age-matched control subjects and Alzheimer disease patients. Immunoblotting with anti-G alpha q/11 and anti-G beta antibodies demonstrated no alteration in the number of these G-protein subunits in Alzheimer disease. Basal [35S]GTP gamma S binding and hydrolysis of [gamma-32P]GTP by high-affinity GTPase also were not significantly altered in Alzheimer disease compared to control membrane samples. However, muscarinic agonist-stimulated GTP gamma S binding and GTP hydrolysis were significantly reduced (80-100%) in Alzheimer disease cortical samples. Diminished agonist-stimulated GTP gamma S binding and GTP hydrolysis correlated with the loss of guanine nucleotide-sensitive, high-affinity agonist binding (KL/KH) ratio) to the M1 receptor subtype. These data provide further evidence for the loss of muscarinic receptor-G protein coupling in Alzheimer disease and support the hypothesis that muscarinic receptor-mediated cortical activation may be compromised in Alzheimer disease.

Aged↗

PubMed1995

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies.

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.

Alzheimer Disease↗

PubMed1995

Identification of a primary metabolite of ibogaine that targets serotonin transporters and elevates serotonin.

Ibogaine is a hallucinogenic indole with putative efficacy for the treatment of cocaine, stimulant and opiate abuse. The purported efficacy of ibogaine following single dose administrations has led to the suggestion that a long-acting metabolite of ibogaine may explain in part how the drug reduces craving for psychostimulants and opiates. We report here that 12-hydroxyibogamine, a primary metabolite of ibogaine, displays high affinity for the 5-HT transporter and elevates extracellular 5-HT. In radioligand binding assays, 12-hydroxyibogamine was 50-fold more potent at displacing radioligand binding at the 5-HT transporter than at the DA transporter. Ibogaine and 12-hydroxyibogamine were equipotent at the dopamine transporter. In vivo microdialysis was used to evaluate the acute actions of ibogaine and 12-hydroxyibogamine on the levels of DA and 5-HT. Administration of 12-hydroxyibogamine produced a marked dose-related elevation of extracellular 5-HT. Ibogaine and 12-hydroxyibogamine failed to elevate DA levels in the nucleus accumbens over the dose range tested. The elevation in synaptic levels of 5-HT by 12-hydroxyibogamine may heighten mood and attenuate drug craving. The effects of the active metabolite on 5-HT transmission may account in part for the potential of ibogaine to interrupt drug-seeking behavior in humans.

Adult↗

PubMed1995

Differential regulation of molecular subtypes of muscarinic receptors in Alzheimer's disease.

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1-m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up-regulated significantly and may offer a target for new memory-enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.

Aged↗

PubMed1995

Identification and quantitation of ibogaine and an o-demethylated metabolite in brain and biological fluids using gas chromatography-mass spectrometry.

This report describes a sensitive method for quantitating ibogaine and a single major metabolite in biological fluids and brain tissue. We identified the metabolite as 12-hydroxy-ibogamine (12-OH-ibogamine or noribogaine) by full-scan, electron-impact gas chromatography-mass spectrometry (GC-MS). Ibogaine, 12-OH-ibogamine, and o-(methyl)-ibogaine-d3 (ibogaine-d3) internal standard were isolated by solvent extraction under basic conditions. The resulting organic extract was evaporated to dryness, and the residue was derivatized at room temperature with ethyl iodide in the presence of trimethyl anilinium hydroxide in dimethyl sulfoxide. The reaction was terminated by acidification and washed with organic solvents to remove impurities. The aqueous phase was then alkalinized and reextracted. The organic extract was concentrated and analyzed by GC-MS. Quantitation was based upon the ratios of the molecular ions at m/z 310 for ibogaine, m/z 313 for ibogaine-d3, and m/z 324 for 12-OH-ibogamine ethyl ether. The limit of detection was 5 ng/mL for both ibogaine and derivatized 12-OH-ibogamine, and limits of quantitation were between 5 and 10 ng/mL for all matrices tested. Calibration curves were linear in the range of 3-1000 ng/mL or ng/g for both analytes.

Administration, Oral↗

PubMed1994

Frequency and type of electrocardiographic abnormalities in cocaine abusers (electrocardiogram in cocaine abuse).

Electrocardiographic abnormalities of 200 asymptomatic, chronic cocaine abusers (aged < or = 45 years, 69% black) admitted for rehabilitation (group 1) were compared with 38 cocaine abusers treated in the emergency room (group 2), 21 cocaine abusers who died suddenly (group 3), and 425 control subjects from the general population. In group 1, 39% of electrocardiograms were abnormal: Increased QRS voltage was noted in 27%, ST elevation in 22%, ST-T changes in 17%, and prior myocardial infarction in 3%. Increased QRS voltage (35% vs 10%, p = 0.00007) and ST elevation (26% vs 13%, p = 0.0278) were more prevalent in blacks than in whites. With use of Minnesota coding, electrocardiograms in group 1 were compared with those of 141 black and 284 white men (aged < 40 years) from the general population. ST elevation was more prevalent in both black (22% vs 8%, p = 0.00073) and white (15% vs 1%, p < 0.00001) cocaine abusers than in the general population. Compared with group 1, group 2 had higher prevalence of sinus tachycardia (16% vs 1%, p = 0.0002), supraventricular tachycardia (5% vs 0%, p = 0.024), ST-T changes (34% vs 17%, p = 0.0164), and QTc > 440 ms (26% vs 4%, p = 0.00003); mean QTc was also greater among group 2 subjects (427 +/- 38 vs 404 +/- 19 ms, p < 0.0001). In group 3, QTc was > 440 ms in 6 of 8 subjects (75%) with 12-lead electrocardiograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Localization of muscarinic receptor subtypes in brain stem areas regulating sleep.

Muscarinic cholinergic receptors (mAChRs) within the pontine brain stem play a key role in generating rapid eye movement (REM) sleep. Using an in vitro autoradiographic technique that permits selective labeling of mAChR subtypes by radioligand binding, this study provides the first quantitative map of mAChR subtypes in cat brain stem areas important for REM sleep generation. M1, M2 and M3 mAChR subtypes were distributed heterogeneously throughout the brain stem. For all 3 mAChR subtypes, the greatest levels of binding were found in the dorsal raphe and locus coeruleus, and the least amount of binding was in the reticular formation. These findings are consistent with data from in vivo studies showing that multiple mAChR subtypes are involved in REM sleep generation.

Animals↗

PubMed1994

[3H]-(+)-pentazocine binding to sigma recognition sites in human cerebellum.

The binding characteristics of the sigma-1 selective benzomorphan [3H]-(+)-pentazocine were determined in human cerebellar membranes. Saturation binding analysis revealed two affinity sites with a KDH of 1.4 +/- 0.7 nM and a KDL of 33.6 +/- 11.9 nM. Kinetic studies performed at 25 degrees C demonstrated reversible binding with association and dissociation rate constants determined for two classes of sites. In saturation binding studies, the addition of (+)-SKF 10,047 occluded binding of [3H]-(+)-pentazocine to high affinity sigma binding sites. The affinity profile of ligands displacing [3H]-(+)-pentazocine was consistent with the labeling of sigma-1 recognition sites with haloperidol > (+)-pentazocine > (+)-SKF 10,047 > (+)-3-PPP > DTG > (-)-pentazocine > (-)-SKF 10,047. The potency of the putative D3 receptor-selective ligand (+)-7-OH-DPAT was close to that measured for (+)-pentazocine in displacement experiments. These data suggest that [3H]-(+)-pentazocine labels sigma-1 sites in human cerebellum under appropriate assay conditions.

Binding Sites↗

PubMed1994

Visualizing dopamine and serotonin transporters in the human brain with the potent cocaine analogue [125I]RTI-55: in vitro binding and autoradiographic characterization.

The cocaine analogue RTI-55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [125I]RTI-55 at 25 degrees C to putamen membranes was monophasic. In contrast, dissociation of [125I]RTI-55 occurred in two phases with t1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [125I]RTI-55 binding demonstrated two binding sites in the human putamen with KD values of 0.10 +/- 0.02 and 1.81 +/- 0.46 nM. The binding of [125I]RTI-55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine > or = fluoxetine > citalopram). In the human occipital cortex, [125I]RTI-55 recognized multiple binding sites with KD values of 0.02 +/- 0.01 and 4.18 +/- 0.46 nM. The rank order of potency for inhibition of [125I]RTI-55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 > or = mazindol > or = nisoxetine > benztropine) suggests that [125I]RTI-55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [125I]RTI-55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Selective labeling and localization of the M4 (m4) muscarinic receptor subtype.

We report here a novel strategy for the selective labeling and localization of the M4 (m4) muscarinic receptor subtype, based on the distinct kinetics of the muscarinic antagonists dexetimide and N-methylscopolamine (NMS) and on the selectivity profile of guanylpirenzepine and AF-DX 116 for the m1-m5 muscarinic receptor subtypes expressed in CHO-K1 cells. Incubation with 10 nM dexetimide, a nonselective antagonist, resulted in > 90% occupancy of each of the m1-m5 receptor subtypes. The relatively rapid rates of dexetimide dissociation from the m1, m2, and m4 receptor subtypes (t1/2 values of < 12.5 min) and the slower rates of dexetimide dissociation from the m3 and m5 receptor subtypes (t1/2 values of 65 and 75 min, respectively) favored the labeling of the m1, m2, and m4 receptor subtypes with short incubations with [3H]NMS. Inclusion of 200 nM guanylpirenzepine and 250 nM AF-DX 116 prevented the binding of [3H]NMS to the majority of the m1 and m2 receptor subtypes, respectively, resulting in primary labeling of the m4 receptor subtype. Brief dissociation of the radioligand in the presence of 1 microM atropine improved the ratio of m4 to m2 labeling by selectively removing [3H]NMS from the m2 subtype. Under these conditions, the ratio of [3H]NMS binding to the m4 versus m1, m2, m3, and m5 receptor subtypes was 4:1. In vitro autoradiography combined with these m4-selective labeling conditions demonstrated that the M4 (m4) receptor subtype was localized to the primary visual area (V1, area 17, occipital cortex) and the basal ganglia, a distribution distinct from that demonstrated for the M1 (m1), M2 (m2), and M3 (m3) receptor subtypes. These results demonstrate that a combination of the distinct kinetics of dexetimide and NMS and the receptor subtype selectivity of guanylpirenzepine and AF-DX 116 provides a valuable labeling strategy to examine the distribution and localization of the M4 (m4) muscarinic receptor subtype in brain, peripheral tissues, and cell lines.

Animals↗

PubMed1994

High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims.

Cocaine mediates its powerful reinforcement by binding to recognition sites on the dopamine (DA) transporter. The pharmacological identity of cocaine recognition sites and their relevance to dopamine transport function has remained unclear. Ligand binding studies with transport inhibitors and cocaine congeners have provided evidence for multiple sites or "states" of the DA transporter. The potent cocaine congener [3H]WIN 35,428 ((CFT), 2B-carbomethoxy-3 beta-(4-fluorophenyl)-tropane) has been shown to recognize high and low affinity binding sites on the DA transporter. We have used [3H]WIN 35,428 to map and quantify the high affinity cocaine recognition site on the DA transporter in victims of fatal cocaine overdose. Region-of-interest densitometric analysis of the autoradiograms demonstrated a 2- to 3-fold elevation in the apparent density of [3H]WIN 35,428 binding in particular sectors of the striatum from victims of cocaine overdose as compared to age-matched and drug-free control subjects. The most marked increase in [3H]WIN 35,428 binding was seen in the nucleus accumbens. The apparent increase in the density of high affinity sites was confirmed by saturation binding analysis of [3H]WIN 35,428 to putamen membranes. Saturation analysis revealed high and low affinity binding components with affinities (KD values) of 4.3 +/- 1.2 and 84.7 +/- 19.7 nM (mean +/- S.E.) and densities of 9.9 +/- 4.0 and 193.0 +/- 28.6 pmol/g of tissue, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Preliminary laboratory studies of inactivation of HIV-1 in needles and syringes containing infected blood using undiluted household bleach.

To evaluate the effectiveness of bleach disinfection of injection equipment, we tested HIV-1 inactivation by household bleach in needles and syringes. We obtained blood from HIV-1 infected injecting drug users (IDUs), placed small aliquots in needles and syringes. Blood with and without anticoagulant was incubated at room temperature for 3, 6, 18, and 24 h, and some needles and syringes from each condition were exposed to undiluted bleach for 15 and 30 s. The needles and syringes were then rinsed and the rinses were used to inoculate peripheral blood mononuclear cells (PBMNCs). HIV-1 replication was monitored using p24 enzyme linked immunosorbent assay (ELISA). We describe results that HIV-1 is inactivated in clotted and unclotted blood allowed to stand at room temperature for 3, 6, 18, and 24 h in needles and syringes using undiluted household bleach at 30 s of exposure time. These results are consistent with earlier findings that micropellets of HIV-1 were inactivated by bleach under similar conditions of exposure to bleach; 10% bleach was not effective at an exposure time of 30 s and undiluted bleach was not effective at an exposure time of 15 s to inactivate HIV-1 in clotted blood. Bleach concentration and exposure time are critical and HIV disinfection may not occur with inadequate exposure to bleach HIV.

Disinfection↗

PubMed1993

Cholinergic innervation of the amygdaloid complex in the human brain and its alterations in old age and Alzheimer's disease.

The cholinergic innervation of the human amygdaloid complex was studied immunohistochemically with a choline acetyltransferase (ChAT) antibody in eight brains: five control and three with Alzheimer's disease (AD). All amygdaloid nuclei displayed ChAT-immunopositive axons and varicosities. The density of these axons reached levels that were higher than in any other part of the forebrain except for the striatum. The highest level of ChAT-immunopositive profiles was seen in the basolateral nucleus and the second highest in the lateral part of the central nucleus. The basomedial, accessory basal, and cortical nuclei, the amygdalohippocampal and cortico-amygdaloid transition areas, as well as the anterior amygdaloid area, showed a moderate density of ChAT-positive varicosities and fibers. The lateral nucleus displayed a relatively low density of cholinergic innervation, and there were only rare ChAT-positive fibers in the medial nucleus. Although the level of cholinergic innervation in the lateral nucleus was relatively lower than in many of the other amygdaloid nuclei, it was approximately equivalent to that of entorhinal cortex, a region that receives one of the heaviest cholinergic inputs in the cerebral cortex. The distribution of the cholinergic fibers as studied by ChAT immunohistochemistry was nearly identical to that observed with AChE histochemistry. Quantitative densitometry in control specimens showed that there was no decline of amygdaloid cholinergic input when middle-aged subjects were compared with senescent subjects. In AD there was a severe and regionally selective depletion of this innervation in the amygdaloid complex. The cortical, accessory basal, and lateral nuclei displayed the most severe loss of ChAT-positive profiles, whereas the basolateral, and especially the central, nuclei displayed relatively little change. There was no consistent relationship between the loss of cholinergic fibers and the density of amyloid plaques and neurofibrillary tangles in amygdaloid nuclei.

Aged↗

PubMed1993

Distinct kinetic binding properties of N-[3H]-methylscopolamine afford differential labeling and localization of M1, M2, and M3 muscarinic receptor subtypes in primate brain.

Three classes of muscarinic receptors in mammalian brain have been postulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstration of the localization of putative M1, M2, and M3 receptor subtypes in the brain because of the overlapping affinities of virtually all muscarinic antagonists. In the present study, the conditions for selective occupancy of the M1, M2, and M3 receptor subtypes in the brain of the rhesus monkey were based on the distinct kinetic and equilibrium binding properties of N-[3H]-methylscopolamine (NMS) at cloned m1-m4 muscarinic receptor subtypes expressed in A9L transfected cells. Quantitative autoradiography of the M1, M2, and M3 muscarinic receptor subtypes in the primate brain was performed according to the following strategy. The M1 (m1) receptor subtype was labeled directly with a non-saturating concentration of [3H]-pirenzepine. The M2 (m2) subtype was labeled by incubations consisting of short, two minute pulses of [3H]-NMS after a preincubation with 0.3 microM pirenzepine to occlude m1, m3, and m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occlude the m1, m2, and m4 sites, equilibrium with 0.5 nM [3H]-NMS, followed by a 60 minute tracer dissociation in the presence of 1 microM atropine. In vitro autoradiography demonstrated that the M1 receptor subtype was confined to forebrain structures. M1 receptors were prevalent throughout the cerebral cortical mantle, amygdala, hippocampus, and the striatum. Low to background levels of the M1 receptor subtype were measured over the thalamus, hypothalamus, and brainstem. The M2 subtype was widely distributed with elevated densities of binding sites seen over all primary sensory cortical areas, and within discrete thalamic, hypothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the M1 sites labeled by non-saturating concentrations of [3H]-pirenzepine with some notable exceptions. Within the cerebral cortical mantle, the M3 receptor exhibited an elevated gradient over the orbitofrontal gyrus and the temporal lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the M1 receptors were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus pallidus and within the substantia nigra pars reticulata. In contrast to the pattern of the M1 receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗