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Notes: The neuropeptide galanin (GAL) is widely distributed in the mammalian CNS. Several lines of evidence suggest that GAL may play a critical role in cognitive processes such as memory and attention through an inhibitory modulation of cholinergic basal forebrain activity. Furthermore, GAL fibers hyperinnervate remaining cholinergic basal forebrain neurons in Alzheimer's disease (AD). This suggests that GAL activity impacts cholinergic dysfunction in advanced AD. Pharmacological and in vitro autoradiographic studies indicate the presence of heterogeneous populations of GAL receptor (GALR) sites in the basal forebrain which bind GAL with both high and low affinity. Interestingly, we have recently observed that GALR binding sites increase in the anterior basal forebrain in late-stage AD. Three G protein-coupled GALRs have been identified to date that signal through a diverse array of effector pathways in vitro, including adenylyl cyclase inhibition and phospholipase C activation. The repertoire and distribution of GALR expression in the basal forebrain remains unknown, as does the nature of GAL and GALR plasticity in the AD basal forebrain. Recently, GAL knockout and overexpressing transgenic mice have been generated to facilitate our understanding of GAL activity in basal forebrain function. GAL knockout mice result in fewer cholinergic basal forebrain neurons and memory deficits. On the other hand, mice overexpressing GAL display hyperinnervation of basal forebrain and memory deficits. These data highlight the need to explore further the putative mechanisms by which GAL signaling might be beneficial or deleterious for cholinergic cell survival and activity within basal forebrain. This information will be critical to understanding whether pharmacological manipulation of GALRs would be effective for the amelioration of cognitive deficits in AD.

Notes: Abstract: 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.

Notes: Abstract: U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin l-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 μM. Nonspecific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of γ-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 μM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP+-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.

Notes: Abstract: 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°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 ≥ 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 ≥ mazindol ≥ 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. Moderately elevated densities of [125I]RTI-55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [125I]RTI-55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [125I]RTI-55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [125I]RTI-55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [125I]RTI-55 labels multiple recognition sites on dopamine and serotonin transporters.

Notes: Abstract: Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.

Notes: Abstract: Transferrin receptors were characterized with 125Iferrotransferrin on membrane fractions prepared from the rodent forebrain. The distribution of transferrin receptors in the rat brain was investigated further by in vitro autoradiography. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 2 nM and a binding site density of 15 pmol/g. The Hill coefficient derived from these data was 1.05. indicating the absence of cooperativity and that 125I-ferrotransferrin binds to a single class of sites. Estimates of the kinetically determined Kd for forebrain membranes were within the 2–4 nM range, in agreement with the equilibrium measurements. Apotransferrin and ferrotransferrin competitively displaced the binding of 125I-ferrotransferrin, while ferritin, albumin, and cytochrome c failed to compete for the binding site. Ceruloplasmin, the copper transport protein, was a weak inhibitor of 125I-ferrotransferrin binding. Autoradiographic localization studies demonstrate a heterogeneous distribution of transferrin receptors in the rat brain. Transferrin receptor densities were markedly elevated over the cerebral cortex and the hippocampus. Moderate to high 125I-ferrotransferrin binding was also apparent throughout areas involved in motor functions, including the caudate-putamen, the nucleus accumbens, the substantia nigra, the red nucleus, and the cerebellum.

Notes: Abstract: Putative nicotine receptors in the human cerebral cortex were characterized with l-[3H]nicotine. l-[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25°C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimer's disease were comparable to age-matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimer's disease had a 50% reduction in the number of l-[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimer's disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimer's disease.

Notes: Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT-PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap 〈 0.0001). In hippocampus this was localized in CA1–3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell-types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.

Notes: Chronic cocaine and withdrawal induce significant alterations in nucleus accumbens (NAc) glutamatergic function in humans and rodent models of cocaine addiction. Dysregulation of glutamatergic function of the prefrontal cortical–NAc pathway has been proposed as a critical substrate for unmanageable drug seeking. Previously, we demonstrated significant up-regulation of NMDA, (±)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor subunit mRNAs and protein levels in the ventral tegmental area (VTA), but not the substantia nigra, of cocaine overdose victims (COD). The present study was undertaken to examine the extent of altered ionotropic glutamate receptor (iGluR) subunit expression in the NAc and the putamen in cocaine overdose victims. Results revealed statistically significant increases in the NAc, but not in the putamen, of NMDA receptor subunit (NR)1 and glutamate receptor subunit (GluR)2/3 wit trends in GluR1 and GluR5 in COD. These results extend our previous finding and indicate pathway-specific alterations in iGluRs in COD. In order to determine that changes were related to cocaine intake and not to other factors in the COD victims, we examined the effects of cocaine intravenous self-administration in rhesus monkeys for 18 months (unit dose of 0.1 mg/kg/injection and daily drug intake of 0.5 mg/kg/session). Total drug intake for the group of four monkeys was 37.9 ± 4.6 mg/kg. Statistically significant elevations were observed for NR1, GluR1, GluR2/3 and GluR5 (p 〈 0.05) and a trend towards increased NR1 phosphorylated at serine 896 (p = 0.07) in the NAc but not putamen of monkeys self-administering cocaine compared with controls. These results extend previous results by demonstrating an up-regulation of NR1, GluR2/3 and GluR5 in the NAc and suggest these alterations are pathway specific. Furthermore, these changes may mediate persistent drug intake and craving in the human cocaine abuser.

Notes: The presence of neuronal elements that are indicative of dopaminergic neurotransmission in cerebellum suggest that this brain region may contribute to the motor symptoms or dyskinesia seen in Parkinson's disease. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of markers for dopaminergic neurotransmission in the cerebellum from postmortem brain tissue obtained from normal subjects and patients dying with Parkinson's disease who were receiving treatment with dopaminergic drugs. Dopamine D1−3 receptors, tyrosine hydroxylase and dopamine transporter mRNA was detected in the uvula and nodulus (lobules 9 and 10, respectively) of the vermis of cerebellum from normal individuals. In Parkinson's disease, the level of dopamine D1 and D3 receptor mRNA was significantly reduced in lobule 9 and the level of tyrosine hydroxylase mRNA was significanty reduced in lobule 10. No alteration in the level of dopamine D2 receptor or dopamine transporter mRNA was found in either lobule in patients with Parkinson's disease. These results show that mRNA expression for the functional components of dopaminergic neurotransmission is present in human cerebellum. The discrete changes in the levels of dopamine D1 and D3 receptors and tyrosine hydroxylase mRNA in cerebellum from l-DOPA treated Parkinson's disease patients suggests that this brain area has a role in the symptoms of Parkinson's disease and/or the beneficial/side-effects of treatment.