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.     

Rescue of Aβ1–42-induced memory impairment in day-old chick by facilitation of astrocytic oxidative metabolism: implications for Alzheimer's disease

Administration of small oligomeric β-amyloid (Aβ)_1–42 45 min before one-trial bead discrimination learning in day-old chicks abolishes consolidation of learning 30 min post-training (Gibbs et al. Neurobiol. Aging , in press). Administration of the β₃-adrenergic agonist CL316243, which specifically stimulates astrocytic but not neuronal glucose uptake, rescues Aβ impaired memory. Weakly reinforced training can be consolidated by various metabolic substrates and we have demonstrated neuronal dependence on oxidative metabolism of glucose soon after training versus astrocytic glucose dependence 20 min later. Based on these findings we examined whether different metabolic substrates were able to counteract memory inhibition by Aβ_1–42. Although lactate, the medium-chain fatty acid octanoate, and the ketone body β-hydroxybutyrate consolidated weakly reinforced training when injected close to learning, none of them were able to salvage Aβ-impaired memory; at this early time. All three metabolites and the astrocytic-specific acetate consolidated weak learning and rescued Aβ-impaired memory when injected 10–20 min post-training. However, neither glucose nor insulin rescued memory when injected at 20 min. Rescue of memory by providing astrocytes with alternative substrates for oxidative metabolism suggests that Aβ_1–42 exerts its amnestic effects specifically by impairing astrocytic glycolysis.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Apoptotic Cell Death Induced by β-Amyloid1–42 Peptide Is Cell Type Dependent

Abstract: β-Amyloid peptide (Aβ), a proteolytic fragment of the β-amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ_1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB₂a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ-induced cell death (necrosis or apoptosis) may depend on the cell type.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

A traditional medicinal herb Paeonia suffruticosa and its active constituent 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose have potent anti-aggregation effects on Alzheimer's amyloid β proteins in vitro and in vivo

The deposition of amyloid β (Aβ) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains; therefore, inhibition of Aβ fibril formation and destabilization of pre-formed Aβ fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa , a traditional medicinal herb, not only inhibited fibril formation of both Aβ_1–40 and Aβ_1–42 but it also destabilized pre-formed Aβ fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Aβ burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Aβ in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta- O -galloyl-β- d -glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa , PGG alone inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils in vitro and in vivo . Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Aβ fibrils in vitro and in vivo . PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD.     

Pathologic Amyloid β-Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells

Abstract: Cerebrovascular amyloid β-protein (Aβ) deposition is a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Aβ_1–40 containing the E22Q HCHWA-D mutation, but not wild-type Aβ_1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µ M neither HCHWA-D Aβ_1–40 nor wild-type Aβ_1–40 appreciably assembles into β-pleated sheet-containing fibrils in solution over a 6-day incubation period. In contrast, at the same concentrations, HCHWA-D Aβ_1–40, but not wild-type Aβ_1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA-D Aβ_1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA-D Aβ_1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.     

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.     

β-Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late-onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to β-amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ_1–42, Aβ_1–40, and Aβ_25–35, increase significantly apoE-liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ_25–35, was further shown to increase significantly the internalization of both apoE3- and apoE4-liposomes, without affecting apoE degradation. Conversely, Aβ_1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE-liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE 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.     

Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella

Abstract: The β₄ and β₁₀ thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β₄ is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β₁₀ is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β-thymosins were initially expressed in granule cells, although expression, especially of thymosin β₄, was truncated compared with the in vivo time course. As in vivo, thymosin β₄ was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β₁₀. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.     

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β-amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α₂-macroglobulin (α₂M^⋆), a physiological ligand for the low-density lipoprotein receptor-related protein (LRP) that is abundantly expressed in the CNS. These α₂M^⋆/¹²⁵I-Aβ complexes are immunoreactive with both anti-Aβ and anti-α₂M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α₂M^⋆/¹²⁵I-Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor-associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free ¹²⁵I-Aβ is not inhibited by RAP and thus must be mediated via an LRP-independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H₂O₂ and free radical-mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H₂O₂-scavenging enzyme catalase protects neurons in culture against Aβ-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H₂O₂. Aβ-mediated elevation in intracellular H₂O₂ production is suppressed by addition of a potent H₂O₂ scavenger without any significant neuroprotection. Three intracellular biochemical markers of H₂O₂-mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to Aβ. Our results challenge a pivotal role for H₂O₂ generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.     

Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam₃CSK₄) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre-treatment of the cells with 10 μg/mL of a TLR2-specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam₃CSK₄ response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam₃CSK₄ response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ-induced tumor necrosis factor alpha production in a human monocytic cell line.     

In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1- and β3-Subunit mRNAs

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABA_A receptor α₁- and β₃-subunit mRNA was conducted using synthetic 3'- end ³⁵S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α₁-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β₃-subunit mRNA were not affected. Dramatically increased levels of GABA_A receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α₁ subunit and in cerebral cortex only for the β₃-subunit. These data provide further support to the structural and pharmacological GABA_A receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.