Acetylcholine Release in the Rat Prefrontal Cortex In Vivo: Modulation by α2-Adrenoceptor Agonists and Antagonists

Abstract: We have previously shown that the release of acetylcholine (ACh) in the medial prefrontal cortex of the conscious rat, as measured by microdialysis, is increased following intraperitoneal injection of the selective α₂-adrenoceptor antagonist (+)-efaroxan. To characterize further the receptor pharmacology of this response, the effects of other selective α₂-adrenoceptor ligands were examined. The α₂-adrenoceptor antagonists idazoxan (2.5 and 20 mg/kg), atipamezole (2.5 mg/kg), and fluparoxan (10 mg/kg) increased ACh outflow by up to 250–325% of basal levels over a 3-h period following intraperitoneal injection. The α₂-adrenoceptor agonists UK-14304 (2.5 mg/kg) and guanabenz (2.5 mg/kg) reduced ACh outflow by 80 and 60%, respectively. Clonidine (0.00063–0.16 mg/kg) had no significant depressant effect and at 2.5 mg/kg increased ACh outflow to 233% of basal levels. These results indicate a modulatory role for α₂-adrenoceptors on the release of ACh in the rat prefrontal cortex in vivo. Based on the facilitatory effects produced by the antagonists alone, this α₂-adrenoceptor modulation appears to be tonic and inhibitory. The ability of α₂-adrenoceptor antagonists to enhance ACh outflow suggests a therapeutic usefulness in disorders where cortical ACh release deficits have been implicated.     

Laminar Alterations in γ-Aminobutyric AcidA, Muscarinic, and β Adrenoceptors and Neuron Degeneration in Cingulate Cortex in Alzheimer's Disease

The laminar distribution of binding to a number of postsynaptic neurotransmitter receptors was assessed autoradiographically in postmortem samples of area 23a in posterior cingulate cortex from 13 Alzheimer and nine age-matched control cases. Specific binding in all Alzheimer cases was compared to that in control cases, and the following alterations were observed: reduced muscimol binding in most layers; no changes in pirenzepine binding; and elevated cyanopindolol binding in layers Ic, IIIc, and IV. The Alzheimer cases were classified further on the basis of neuronal degeneration: class 1, no neuron loss; class 2, greatest losses in layer II or III; class 3, greatest losses in layer IV; and class 4, greatest losses in layer V or VI. This classification uncovered further alterations in ligand binding patterns. First, muscimol binding was reduced in layers II and III only in class 2 cases and in layers V and VI only in class 4 cases. Second, pirenzepine binding was reduced in layers Ic, IIIa-b, and VI of class 1 cases and layers Va and VI of class 4 cases. In spite of neuron degeneration in classes 2 and 3, there was no change in pirenzepine binding in these classes. Third, elevated cyanopindolol binding occurred in classes 3 and 4, whereas classes 1 and 2 had normal levels of binding. These results suggest that cases of Alzheimer's disease express heterogeneities in neocortical pathology which are reflected in the laminar patterns of binding to postsynaptic receptors. Reductions in muscimol binding to the gamma-aminobutyric acidA receptor had the closest relationship with neuron degeneration, whereas pirenzepine binding appeared to reflect a compensation in muscarinic receptors for changes in neuron densities.     

Down-Regulation of β-Adrenergic Receptors by Pindolol in Gsα-Transfected S49 cyc− Murine Lymphoma Cells

The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (GS alpha) in the down-regulation of beta-adrenergic receptors by pindolol was studied in S49 cyc- cells (normally GS alpha-deficient) transfected to express functional recombinant rat GS alpha. An inducible cell line (S49 GS alpha IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of GS alpha under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. GS alpha was not detectable in S49 GS alpha IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, GS alpha was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-32P]ADP-ribosylation. Membranes prepared from GS alpha IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGS alpha +, contained the coding sequence of GS alpha under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 GS alpha CST cells, expressed GS alpha at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GS alpha CST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of beta-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc- or GS alpha IND cells. When GS alpha CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that GS alpha is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.     

β2-Adrenergic Receptors on Peripheral Nerves

We report that peripheral nerves have a functional adenylate cyclase-coupled beta-adrenergic receptor. The pharmacological specificity of this receptor is shown to be of the beta 2 subtype. Two peripheral nerves, the sciatic from the frog and rat and the vagus from the rat, responded to beta 2-agonists with 10-50-fold increases in intracellular cyclic AMP level. This increase was inhibited by the beta-adrenergic antagonist propranolol. In contrast, a central nerve tract, the corpus callosum, responded to isoproterenol with only a minimal one- to twofold increase in cyclic AMP level. These studies demonstrate that peripheral nerves have beta 2-adrenergic receptors that are responsive to exogenously applied catecholamines and suggest a role for these ligands in the previously described modulation of axonal conduction.     

α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

Antidepressant Treatments, Including Sibutramine Hydrochloride and Electroconvulsive Shock, Decrease β1 but Not β2-Adrenoceptors in Rat Cortex

The beta 1- and beta 2-adrenoceptor populations in rat cortex were individually quantified by labelling all of the receptors with [3H]dihydroalprenolol and displacing with isoprenaline (200 microM) or CGP 20712A (1-(2-[(3-carbamoyl-4-hydroxy)phenoxy]ethylamino)-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol methanesulphonate; 100 nM) to define total beta-adrenoceptors and beta 1-adrenoceptors, respectively. Binding parameters for beta 2-adrenoceptors were calculated by the difference. Oral administration of the monoamine reuptake inhibitors sibutramine HCl (3 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg), or zimeldine (10 mg/kg) for 10 days decreased the total number of beta-adrenoceptors present in rat cortex. This effect was entirely due to a reduction in the number of beta 1-adrenoceptors. Similarly, 10 days of treatment with the monoamine oxidase inhibitor tranylcypromine (10 mg/kg p.o.) or five electroconvulsive shocks (ECSs; 200 V, 2 s) spread over this period also down-regulated beta-adrenoceptors by reducing the content of the beta 1-subtype. By contrast, treatment with clenbuterol (5 mg/kg p.o.) for 10 days reduced the number of cortical beta-adrenoceptors by an effect on the beta 2-adrenoceptor population. The effects of short-term treatment with these drugs were also investigated, and, using the doses shown above, the results of 3 days of administration or a single ECS were determined. Sibutramine HCl and desipramine were alone in producing a reduction in number of beta-adrenoceptors after 3 days. Once again, this was exclusively due to a loss of beta 1-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Decreased Density of Presynaptic α2-Adrenoceptors in Postmortem Brains of Patients with Alzheimer's Disease

The full agonist [3H]bromoxidine (UK 14304) was used to quantitate alpha 2-adrenoceptors in postmortem brains of patients with Alzheimer's disease. The effects of aging and human serum Cohn fraction IV on [3H]bromoxidine binding were also assessed. In patients with Alzheimer's disease, the binding capacity (Bmax) of [3H]bromoxidine was lower in the frontal cortex (37%), hypothalamus (33%), and cerebellum (52%) than in matched controls. In the hippocampus, amygdala, and head of caudate, the binding capacities (Bmax) were unchanged. Quantitative autoradiographic analyses with [3H]bromoxidine confirmed the existence of a marked reduction (55-60%) in alpha 2A-adrenoceptor density in the frontal cortex (layers I and III). In patients with dementia who did not meet neuropathological criteria for Alzheimer's disease, the density of alpha 2-adrenoceptors was unchanged. In control subjects, the density of alpha 2A-adrenoceptors in the frontal cortex showed a significant negative correlation with age at death. The inhibitory effect of human serum Cohn fraction IV on [3H]bromoxidine was very similar in control subjects and patients with Alzheimer's disease. The observed decrease in the density of brain alpha 2-adrenoceptors in Alzheimer's disease may represent direct biochemical evidence of a presynaptic location of this receptor on noradrenergic nerve terminals in the human CNS.     

Characterization of l-[3H]Nicotine Binding in Human Cerebral Cortex: Comparison Between Alzheimer's Disease and the Normal

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 degrees 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.     

Contribution of Noradrenergic Neurons to the Regulation of Dopaminergic (D1) Receptor Denervation Supersensitivity in Rat Prefrontal Cortex

3,4-Dihydroxyphenylethylamine (DA, dopamine) levels in the rat prefrontal cortex were selectively decreased by 52%, leaving noradrenaline (NA) levels unaffected, 4 weeks following restricted bilateral electrolytic lesions of the ventral mesencephalic tegmentum (VMT). These lesions also induced a significant increase in DA-sensitive, but not isoproterenol-sensitive, adenylate cyclase activity in tissue homogenates (+38%). We had shown previously that chemical (6-hydroxydopamine, 6-OHDA) lesions of the VMT destroy both ascending DA and NA fibers but do not alter the D1-receptor density in the prefrontal cortex. In this study, electrolytic lesions of the VMT were combined with bilateral injections of 6-OHDA made laterally in the pedunculus cerebellaris superior to assess the role of NA fibers in the development of D1-receptor supersensitivity. This combined treatment produces a large decrease of cortical NA levels (-95%), an increase in beta-adrenergic-sensitive adenylate cyclase activity (+110%), and a decrease in DA levels (-60%), but does not alter D1-receptor density in the prefrontal cortex. These results indicate that the development of D1-receptor supersensitivity in the prefrontal cortex following electrolytic lesion of the VMT depends on the presence of an intact NA innervation.     

Stress Preferentially Increases Extraneuronal Levels of Excitatory Amino Acids in the Prefrontal Cortex: Comparison to Hippocampus and Basal Ganglia

Abstract: The technique of intracerebral microdialysis was used to assess the effect of stress on the extracellular concentrations of excitatory amino acids, glutamate and aspartate, in the rat medial prefrontal cortex, hippocampus, striatum, and nucleus accumbens. A 20-min restraint procedure led to an increase in extracellular glutamate in all regions tested. The increase in glutamate levels was significantly higher in the prefrontal cortex than that observed in other regions. With the exception of the striatum, extracellular levels of aspartate were increased in all regions. Furthermore, the increase in aspartate levels was significantly higher in prefrontal cortex compared to hippocampus and nucleus accumbens. Local perfusion of tetrodotoxin during the restraint procedure significantly decreased the stress-induced increase in extracellular excitatory amino acids. In order to ensure that the above results were not an artifact of restraint not associated with stress (e.g., decreased mobility), we also examined the effect of swimming stress on the extracellular levels of excitatory amino acids in selected regions, i.e., striatum and medial prefrontal cortex. Both regions displayed a significant increase in extracellular levels of aspartate and glutamate following 20 min of swimming in room temperature water. This study provides direct evidence that stress increases the neuronal release of excitatory amino acids in a regionally selective manner. The implications of the present findings for stress-induced catecholamine release and/or hippocampal degeneration are discussed.     

Molecular determinants for the action of general anesthetics at recombinant α2β3γ2γ-aminobutyric acidA receptors

General anesthetics modulate the activity of ligand-gated ion channels including the GABA(A) receptor. Mutational studies mainly on the benzodiazepine-insensitive alpha(2)beta(1(M286W)) and alpha(6)beta(3(N289M))gamma(2) GABA(A) receptors revealed that a serine in transmembrane domain 2 and a methionine in transmembrane domain 3 are essential for the action of most general anesthetics. We investigated whether these residues would similarly be relevant for their action at the benzodiazepine-sensitive GABA receptor subtype, alpha(2)beta(3)gamma(2). We found that not only the N265M but also the M286W mutation nearly abolished the modulatory effect of etomidate. However, the anti-convulsant loreclezole, a structural homologue of etomidate, was inactive on the N265M mutant, but displayed normal modulatory activity on the M286W mutant. Both mutations did not affect the modulatory action of the neurosteroid alphaxalone. The direct action of alphaxalone, however, was dramatically increased in the M286W mutant to about twice the maximal GABA current but not significantly affected in the N265M mutant. These data demonstrate that the structural requirements for modulatory and direct actions of various general anesthetics are distinct. The molecular switches induced by these mutations can be exploited to identify the molecular determinants for the action of general anesthetics.     

α2-Macroglobulin Enhances the Clearance of Endogenous Soluble β-Amyloid Peptide via Low-Density Lipoprotein Receptor-Related Protein in Cortical Neuron

Apolipoprotein E and alpha2-macroglobulin (alpha2M) are genetic risk factors for late-onset Alzheimer's disease, and both bind a cell surface receptor, the low-density lipoprotein receptor-related protein (LRP). To investigate the role of LRP on preventing the accumulation of beta-amyloid peptide (A beta), we examined the effects of alpha2M on the clearance of endogenous A beta. Studies were performed in primary Tg2576 transgenic mouse cortical neuronal cultures expressing human mutant amyloid precursor protein (APP) 695. This system allowed us to follow endogenous A beta using immunoblots to detect monomeric forms of the peptide. A beta and APP levels were measured in conditioned media. We found that activated alpha2M (alpha2M*) substantially decreased soluble A beta levels and had no effect on secreted or full-length APP levels. Native alpha2M, which is not a ligand for LRP, did not affect A beta levels. The receptor-associated protein, which inhibits interaction of all ligands with LRP in vitro, prevented alpha2M*-induced decreases of soluble A beta levels. These data suggest that alpha2M* affects soluble A beta clearance rather than A beta production. Further studies showed that similar A beta clearance via an LRP-mediated pathway was observed after treatment with another LRP ligand, lactoferrin. Taken together, these data demonstrate that alpha2M* enhances the clearance of soluble A beta via LRP in cortical neurons.     

α2-Adrenergic Receptor Blockade Markedly Potentiates Duloxetine- and Fluoxetine-Induced Increases in Noradrenaline, Dopamine, and Serotonin Levels in the Frontal Cortex of Freely Moving Rats

Abstract: Evidence exists that a reinforcement in monoaminergic transmission in the frontal cortex (FCX) is associated with antidepressant (AD) properties. Herein, we examined whether blockade of α₂-adrenergic receptors modified the influence of monoamine reuptake inhibitors on FCX levels of serotonin (5-HT), noradrenaline (NAD), and dopamine (DA). The selective α₂-adrenergic receptor agonist S 18616 (0.16 mg/kg, s.c.) suppressed extracellular levels of NAD, DA, and 5-HT (by 100, 51, and 63%, respectively) in single dialysates of FCX of freely moving rats. In contrast, the selective α₂-adrenergic receptor antagonists atipamezole (0.16 mg/kg, s.c.) and 1-(2-pyrimidinyl)piperazine (1-PP; 2.5 mg/kg, s.c.) increased levels of NAD (by 180 and 185%, respectively) and DA (by 130 and 90%, respectively), without affecting 5-HT levels. Duloxetine (5.0 mg/kg, s.c.), a mixed inhibitor of 5-HT and NAD reuptake, and fluoxetine (10.0 mg/kg, s.c.), a selective 5-HT reuptake inhibitor, both increased levels of 5-HT (by 150 and 120%, respectively), NAD (by 400 and 100%, respectively), and DA (by 115 and 55%, respectively). Atipamezole (0.16 mg/kg, s.c.) markedly potentiated the influence of duloxetine and fluoxetine on levels of 5-HT (by 250 and 330%, respectively), NAD (by 1,030 and 215%, respectively), and DA (by 370 and 170%, respectively). 1-PP similarly potentiated the influence of duloxetine on 5-HT, NAD, and DA levels (by 290, 1,320, and 600%, respectively). These data demonstrate that α₂-adrenergic receptors tonically inhibit NAD and DA and phasically inhibit 5-HT release in the FCX and that blockade of α₂-adrenergic receptors strikingly potentiates the increase in FCX levels of 5-HT, NAD, and DA elicited by reuptake inhibitors. Concomitant α₂-adrenergic receptor antagonism and inhibition of monoamine uptake may thus provide a mechanism allowing for a marked increase in the efficacy of AD agents.     

Involvement of Dopamine Neurons in the Regulation of β-Adrenergic Receptor Sensitivity in Rat Prefrontal Cortex

The contribution of dopamine (DA) afferents to the regulation of beta-adrenergic receptor sensitivity (isoproterenol-stimulated adenylate cyclase activity) in the rat prefrontal cortex was investigated by comparing the effects of lesions affecting either both DA and noradrenaline (NA) or NA fibers alone. Bilateral 6-hydroxydopamine (6-OHDA) lesions made in the ventral tegmental area destroyed ascending DA and to a variable extent ascending NA fibers innervating the prefrontal cortex. Two opposite effects were observed depending on the extent of cortical NA denervation: (a) When NA denervation was complete (less than 4% of controls), a marked increase in the isoproterenol-sensitive adenylate cyclase activity (+78%) was found. The amplitude of this denervation supersensitivity was similar to that occurring following complete and selective destruction of NA innervation induced by bilateral 6-OHDA injections made into the pedunculus cerebellaris superior. (b) When 6-OHDA injections into the ventral tegmental area led to a partial destruction of cortical NA afferents (10-40% of control values), a hyposensitivity of the isoproterenol-induced adenylate cyclase activity (-30%) was observed. This effect contrasted with the moderate supersensitivity seen in rats with partial, but selective, destruction of NA innervation (pedunculus cerebellaris superior lesions). The hyposensitivity of beta-adrenergic receptors obtained in rats with partial lesions of cortical NA fibers, but devoid of cortical DA innervation, suggests that DA neurons may regulate, under certain conditions, the denervation supersensitivity of beta-adrenergic receptors.     

Electroconvulsive Shock and Reserpine: Effects on β-Adrenergic Receptors in Rat Brain

Abstract Electroconvulsive shock (ECS) administered once daily for up to 14 days decreases β-adrenergic receptor binding in the cortex and hippocampus in a time-dependent manner. The decrease in binding in the cortex lasts at least 1 week after the last shock. In the striatum, hypothalamus, or cerebellum, 14 days of ECS did not produce significant changes in β-adrenergic receptor binding. The brain regional pattern of β-adrenergic receptor changes suggests that repeated ECS affects β-adrenergic receptors in brain regions that receive a noradrenergic innervation activated by ECS. The effects of ECS on neurotransmitter receptor binding appear to be highly selective. Of five receptors in the cortex and three receptors in the hippocampus measured, only β-adrenergic receptor binding is decreased. Chronic footshock stress does not alter β-adrenergic receptor binding sites in the cortex, indicating that the effects of ECS are not due to stress alone. The effects of ECS on reserpine-induced alterations in β-adrenergic receptor binding sites were also examined. Ten days of ECS following chronic reserpine injections reverses the increased binding of β-adrenergic receptors     

Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.