De-Maw Chuang

PET imaging with [11C]PBR28 can localize and quantify upregulated peripheral benzodiazepine receptors associated with cerebral ischemia in rat

Peripheral benzodiazepine receptors (PBRs) are upregulated on activated microglia. We recently developed a promising positron emission tomography (PET) ligand, [11C]PBR28, with high affinity and excellent ratio of specific to nonspecific binding. We assessed the ability of [11C]PBR28 PET to localize PBRs in a rat permanent middle cerebral artery occlusion (MCAO) model of neuroinflammation. [11C]PBR28 was intravenously administered to rats at 4 and 7 days after permanent MCAO. In all experiments, arterial blood was sampled for compartmental modeling of regional distribution volumes, and rat brains were sampled after imaging for in vitro [3H]PK 11195 autoradiography and histological evaluation. [11C]PBR28 PET and [3H]PK 11195 autoradiography showed similar areas of increased PBRs, especially in the peri-ischemic core. Results from these in vivo and in vitro methods were strongly correlated. In this first study to demonstrate neuroinflammation in vivo with small animal PET, [11C]PBR28 had adequate sensitivity to localize and quantify the associated increase in PBRs.

Glyceraldehyde-3-phosphate dehydrogenase is over-expressed during apoptotic death of neuronal cultures and is recognized by a monoclonal antibody against amyloid plaques from Alzheimer's brain

The age-induced apoptotic death of cerebellar neurons in culture is associated with over-expression of a 38-kDa particulate protein identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both the age-induced apoptosis and the 38-kDa protein overexpression were effectively suppressed by the presence of tetrahydroaminoacridine, an antidementia drug, or aurintricarboxylic acid. This over-expressed 38-kDa protein and purified GAPDH were found to react with a monoclonal antibody (mAb), Am-3, which was raised against amyloid plaques from Alzheimers brain, but not with mAb, AmT-1, which was produced using synthetic amyloid beta peptide. These results raise the possibility that GAPDH is also involved in the neurodegeneration during the development of Alzheimers disease.

ONO-1603, a potential antidementia drug, shows neuroprotective effects and increases m3-muscarinic receptor mRNA levels in differentiating rat cerebellar granule neurons

We have reported that the antidementia drug tetrahydroaminoacridine (THA; 30 microM) is neuroprotective and neurotrophic and selectively increases m3-muscarinic acetylcholine receptor (mAChR) mRNA levels in differentiating cerebellar granule cells. Here, we examined whether novel prolyl endopeptidase inhibitor ONO-1603, a potential antidementia drug, induces similar effects in these cerebellar neurons. Supplement of ONO-1603 (0.03 microM) to cultures grown in 15 mM KCl-containing media was found to markedly promote neuronal survival and neurite outgrowth and enhance [3H]N-methylscopolamine binding to mAChRs. Moreover, ONO-1603 increased the level of m3-mAChR mRNA and stimulated mAChR-mediated phosphoinositide turnover. The common actions of ONO-1603 and THA suggest that these properties could be related to their putative antidementia activities and that this model system may be used to screen for drugs effective in the treatment for Alzheimers disease.

Chronic haloperidol treatment attenuates receptor-mediated phosphoinositide turnover in rat brain slices

The long-term effects of haloperidol on phosphoinositide turnover in rat brain slices were investigated. Continuous treatment with haloperidol decanoate (21 mg/kg I.M. biweekly for 6 weeks) significantly attenuated carbachol- and norepinephrine (NE)-induced inositol phosphate accumulation in rat frontal cortex and hippocampus. In the striatum, the haloperidol treatment also significantly decreased carbachol-stimulated inositol phosphate level but did not significantly affect NE-sensitive phosphoinositide turnover. These effects were not observed in rats treated with a single dose of haloperidol (1.5 mg/kg). Basel levels of inositol phosphate in these 3 brain regions did not change following continuous or single haloperidol doses.

Effects of chronic nicotine and haloperidol administration on muscarinic receptor-mediated phosphoinositide turnover in rat brain slices

Muscarinic receptor-mediated phosphoinositide (PI) turnover in rat brain slices was assessed after chronic administration of nicotine (12 mg/kg/day) or haloperidol decanoate (1.5 mg/kg/day), either alone or in combination, for 6 weeks. Nicotine alone did not significantly alter carbachol-induced inositol monophosphate (IP1) accumulation in the frontal cortex, but did result in a significant increase in the hippocampus, and in a decrease in the striatum. Haloperidol alone attenuated carbachol-stimulated IP1 accumulation in all three brain regions. Chronic treatment with combined nicotine and haloperidol resulted in no significant change in carbachol-sensitive IP1 accumulation in either the frontal cortex or hippocampus but did result in a decrease in the striatum. The results suggest significant cross-talk between cholinergic and dopaminergic systems in affecting PI metabolism.

Tetrahydroaminoacridine increases m3-, but not m2-, muscarinic acetylcholine receptor mRNA levels in differentiating cerebellar granule cells

We used Northern blot hybridization to determine whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a potential antidementia drug, selectively altered the levels of muscarinic acetylcholine receptor (mAChR) mRNA in differentiating cerebellar granule cells. Granule cells were cultured for 8 days in media containing 15 mM K+, 25 mM K+ or 15 mM K+ plus 30 microM THA. High K+ markedly increased the levels of m2- and m3-mAChR mRNA in the surviving cells. In contrast, THA increased the levels of m3-mAChR mRNA, but had little or no effect on m2-mAChR mRNA levels. These results suggest that THA selectively up-regulates the synthesis of m3-mAChR mRNA.

Autoradiographic demonstration of an increase in muscarinic cholinergic receptors in cerebellar granule cells treated with tetrahydroaminoacridine

The neurotrophic and neurosurviving effects of 9-amino-1,2,3,4-tetrahydroacridine (THA), a putative antidementia agent, were studied in cultured granule cells using biochemical and morphological methods. The addition of 30 microM THA to cultures grown in 15 mM K(+)-containing media markedly increased cell survival and enhanced [3H]N-methylscopolamine binding to muscarinic cholinergic receptors (mAChRs). Furthermore, receptor autoradiographic studies revealed that neuronal cells were labelled over both cell bodies and fibers by the [3H]receptor ligand. These observations provide direct evidence that THA promotes the expression of mAChR binding sites in differentiating cerebellar granule cells.

ETHANOL INDUCES SUBTYPE-SPECIFIC UP-REGULATION OF MUSCARINIC ACETYLCHOLINE RECEPTOR mRNA IN NEUROHYBRID CELL LINES

Effects of sub-chronic ethanol treatment on the mRNA levels of muscarinc acetylcholine receptor (mAChR) subtypes were studied in two neurohybrid cell lines, NG108-15 and NCB-20. The former expresses only m4-mAChRs, while the latter expresses m1- and m4-mAChRs. Exposure to 200 mM ethanol for 1 to 3 days induced a time-dependent increase in the levels of m4-mAChR mRNA in NG108-15 and NCB-20 cells. In contrast, the levels of ml-mAChR mRNA and mAChR-mediated phosphoinositide hydrolysis in NCB-20 cells were unchanged. The ethanol-induced up-regulation of m4-mAChR mRNA was associated with a parallel increase in the levels of mAChR binding sites assessed by using [3H]quinuclidinyl benzilate. The mRNA up-regulation was closely correlated with a decrease in intracellular cyclic AMP concentration and the ethanol up-regulation was blocked by 8-bromo-cyclic AMP and forskolin, suggesting that m4-mAChR mRNA is under negative regulation by cyclic AMP. Thus, neurally-related cell lines are useful models for studying molecular mechanisms underlying ethanol-induced alteration of mAChR expression and function in the nervous system.

Effect of chronic haloperidol treatment on dopamine-induced inositol phosphate formation in rat brain slices

The effects of chronic haloperidol administration on the accumulation of inositol phosphates were examined in rat brain slices pre-labeled with [3H]myo-inositol and incubated with various dopaminergic drugs. Rats were treated with haloperidol-decanoate or its vehicle (sesame oil) for two, four or six weeks. Dopamine and the selective D1 agonist, SKF38393, induced a significant increase in lithium-dependent accumulation of [3H]inositol monophosphate (IP1) in the frontal cortex, hippocampus and striatum of vehicle-treated animals, while the selective D2 agonist quinpirole did not show any effect on IP1 accumulation. The actions of dopamine and SKF38393 were blocked by the D1 antagonist, SCH23390, but not by the D2 antagonist, spiperone, in all three brain regions. Haloperidol treatment did not affect basal phosphoinositide turnover in the three brain regions. Four or six weeks of haloperidol treatment significantly decreased dopamine-induced IP1 accumulation in the striatum (by 30% and 25%, respectively), but not in the frontal cortex and the hippocampus. Four weeks of treatment with haloperidol significantly decreased IP1 levels in the striatal slices when measured in the presence of quinpirole. However, the accumulation of IP1 measured in the presence of SKF38393 was not significantly altered after haloperidol treatment. The loss of dopamine-sensitive IP accumulation was not observed in the presence of spiperone after haloperidol treatment. The number, but not the affinity, of [3H]sulpiride binding sites in the striatum was significantly increased (by 34–46%) after chronic haloperidol treatment. A timecourse study suggests that the inhibition by chronic haloperidol treatment of dopamine-induced phosphoinositide hydrolysis may involve an effect secondary to an increase in the number of dopamine D2 receptors in the striatum.

Differential effects of butyrate and dibutyryl cAMP on mRNA levels of muscarinic acetylcholine receptor subtypes expressed in neurohybrid cell lines

NCB-20 cells expressed m1- and m4-muscarinic acetylcholine receptor (mAChR) mRNAs, while NG108-15 cells expressed only m4-mAChR mRNA. Butyrate induced a time-dependent increase in the level of m1-mAChR mRNA with no change in the m4-mAChR mRNA level in NCB-20 cells. Similarly, butyrate did not affect the m4-mAChR mRNA level in NG108-15 cells. In contrast, dibutyryl cAMP caused a significant time-dependent decrease in the level of m4-mAChR mRNA in NCB-20 and NG108-15 cells as well as m1-mAChR mRNA in NCB-20 cells. Our results suggest that these two differentiating agents are important physiological regulators of the transcription and/or stability of the mRNA of certain mAChR subtypes expressed in these two neurohybrid cell lines.