Proteasomal abnormalities in cortical Lewy body disease and the impact of proteasomal inhibition within cortical and cholinergic systems.

Dementia with Lewy bodies (DLB) accounts for 15-20% of the millions of people worldwide with dementia. In the current work we investigate the association between proteasome dysfunction and the development of cortical Lewy body pathology. Analysis of post-mortem cortical tissue indicated levels of the alpha-subunit of the 20S proteasome were significantly reduced in DLB cortex, but not Alzheimer's, in comparison to control and this reduction correlated with both the severity and duration of dementia. Application of proteasome inhibitors to rodent cortical primary neurones in vitro and by direct injection onto rodent cholinergic forebrain neurons in vivo gave rise to dose dependent neuronal death and in rodent cortex -- marked cholinergic deficits accompanied by the accumulation of inclusions that stained positive for alpha-synuclein and ubiquitin. These findings suggest that proteasomal abnormalities are present within cortical Lewy body disease and the experimental inhibition of proteasomal function mirrors the neuropathological changes seen within the disorder.

Group III metabotropic glutamate receptors act as hetero-receptors modulating evoked GABA release in the globus pallidus in vivo.

In vitro studies suggest that group III metabotropic glutamate (mGlu) receptors function as hetero-receptors to modulate GABA release within the globus pallidus. In the present study we examined this hypothesis in vivo, using microdialysis to assess the ability of locally infused group III mGlu receptor agonists to modulate KCl-evoked GABA release in the globus pallidus of anaesthetised rats. Extra-cellular levels of GABA in dialysate samples were assayed using High Pressure Liquid Chromatography (HPLC) coupled to electrochemical detection. Infusion of KCl (50-100 mM) evoked a dose-dependent increase in GABA levels in the globus pallidus. Addition of the group III mGlu receptor agonists l-AP4 (30 and 300 microM) and l-SOP (3-300 microM) significantly reduced the extra-synaptic levels of GABA that resulted after 100 mM KCl challenge. The effect of L-SOP (30 microM) was almost totally abolished by co-infusion with M-SOP (30 microM). These data confirm that activation of group III mGlu receptors inhibits GABA release in the globus pallidus, thereby supporting their hetero-receptor role in vivo.

Autoradiographic localisation of [3H]2-BFI imidazoline I2 binding sites in mouse brain.

Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain.

Locomotor effects of imidazoline I2-site-specific ligands and monoamine oxidase inhibitors in rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway.

The present study examined the ability of the selective imidazoline I(2)-site ligands 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) and 2-[4,5-dihydroimidaz-2-yl]-quinoline (BU224) and selected monoamine oxidase (MAO) inhibitors to evoke locomotor activity in rats bearing a lesion of the nigrostriatal pathway. Male Sprague-Dawley rats were injected with 12.5 microg 6-hydroxydopamine (6-OHDA) into the right median forebrain bundle to induce a unilateral lesion of the nigrostriatal tract. After 6 weeks, test drugs were administered either alone or in combination with L-DOPA (l-3,4-dihydroxyphenylamine) and the circling behaviour of animals was monitored as an index of anti-Parkinsonian activity. Intraperitoneal (i.p.) administration of the irreversible MAO-B inhibitor deprenyl (20 mg kg(-1)) or the imidazoline I(2)-site ligands BU224 (14 mg kg(-1)) and 2-BFI (7 and 14 mg kg(-1)) produced significant increases in ipsiversive rotations compared to vehicle controls totaling, at the highest respective doses tested, 521 +/-120, 131 +/- 37 and 92.5 +/- 16.3 net contraversive rotations in 30 (deprenyl) or 60 (BU224 and 2-BFI) min. In contrast, the reversible MAO-A inhibitor moclobemide (2.5-10 mg kg(-1)) and the reversible MAO-B inhibitor lazabemide (2.5-10 mg kg(-1)) failed to instigate significant rotational behaviour compared to vehicle. Coadministration of lazabemide (10 mg kg(-1)), moclobemide (10 mg kg(-1)) or 2-BFI (14 mg kg(-1)) with L-DOPA (20 mg kg(-1)) significantly increased either the duration or total number of contraversive rotations emitted over the testing period in comparison to L-DOPA alone. These data suggest that I(2)-specific ligands have dual effects in the 6-OHDA-lesioned rat model of Parkinson's disease; a first effect associated with an increase in activity in the intact hemisphere, probably via an increase in striatal dopamine content, and a secondary action which, through the previously documented inhibition of MAO-A and/or MAO-B, increases the availability of dopamine produced by L-DOPA.

Chronic administration of 2-(2-benzofuranyl)-2-imidazoline (2-BFI) induces region-specific increases in [3H]2-BFI binding to rat central imidazoline I2 sites.

Chronic administration of I(2) ligands increases the density of central I(2) sites as measured in brain homogenates. Here, we have used autoradiography to examine whether the increase in I(2) site density induced by chronic administration of 2-(2-benzofuranyl)-2-imidazoline (2-BFI) is uniform across brain regions. We dosed rats with 2-BFI 7 mg/kg or with saline vehicle i.p. over 96 days. Compared with vehicle-treated rats, this treatment significantly increased specific [(3)H]2-BFI binding only in the arcuate nucleus and area postrema, by 63% and 67% respectively. There were no significant effects in the pineal gland or interpeduncular nucleus which, like the arcuate nucleus and area postrema, are rich in I(2) sites. These data indicate that chronic administration of 2-BFI selectively alters radioligand binding in two I(2) rich brain ideas, namely the arcuate nucleus and area postrema, suggesting there may be more than one population of I(2) sites in the rat brain.

Activation of group III metabotropic glutamate receptors in selected regions of the basal ganglia alleviates akinesia in the reserpine-treated rat.

1. This study examined whether group III metabotropic glutamate (mGlu) receptor agonists injected into the globus pallidus (GP), substantia nigra pars reticulata (SNr) or intracerebroventricularly (i.c.v.) could reverse reserpine-induced akinesia in the rat. 2. Male Sprague-Dawley rats, cannulated above the GP, SNr or third ventricle, were rendered akinetic with reserpine (5 mg kg(-1) s.c.). 18 h later, behavioural effects of the group III mGlu receptor agonists L-serine-O-phosphate (L-SOP) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) were examined. 3. In reserpine-treated rats, unilateral injection of L-SOP (2000 and 2500 nmol in 2.5 microl) into the GP produced a significant increase in net contraversive rotations compared to vehicle, reaching a maximum of 83+/-21 rotations 120 min(-1) (n=8). Pretreatment with the group III mGlu receptor antagonist methyl-serine-O-phosphate (M-SOP; 250 nmol in 2.5 microl) inhibited the response to L-SOP (2000 nmol) by 77%. Unilateral injection of L-SOP (250-1000 nmol in 2.5 microl) into the SNr of reserpine-treated rats produced a dose-dependent increase in net contraversive rotations, reaching a maximum of 47+/-6 rotations 30 min(-1) (n=6). M-SOP (50 nmol in 2.5 microl) inhibited the response to L-SOP (500 nmol) by 78%. 4. Following i.c.v. injection, L-SOP (2000-2500 nmol in 2.5 microl) or L-AP4 (0.5-100 nmol in 2 microl) produced a dose-dependent reversal of akinesia, attaining a maximum of 45+/-17 (n=8) and 72+/-3 (n=9) arbitrary locomotor units 30 min(-1), respectively. 6. These studies indicate that injection of group III mGlu receptor agonists into the GP, SNr or cerebral ventricles reverses reserpine-induced akinesia, the mechanism for which remains to be established.

Potential serotonergic and noradrenergic involvement in the discriminative stimulus effects of the selective imidazoline I2-site ligand 2-BFI.

The functional significance of imidazoline I2 binding sites is unknown but microdialysis studies have indicated that the administration of I2-site ligands leads to an increase in extracellular levels of monoamines. The specific I2-site ligand 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) generates a cue in drug discrimination, thereby indicating functional consequences of I2-site ligand binding. In the present work, we explored the ability of selective noradrenergic and serotonergic ligands to substitute for 2-BFI. Hooded Lister rats were trained in two-lever operant chambers with condensed milk reward to distinguish 2-BFI (7 mg/kg) from saline vehicle, by pressing the correct lever to a predetermined success criterion. Training sessions were then interspersed with sessions in which animals were administered test substances and the proportion of lever presses on the 2-BFI-associated lever (substitution) recorded. Several agents exhibited significant partial substitution for 2-BFI: The monoamine-releasing agents D-amphetamine and fenfluramine dose-dependently substituted for 2-BFI, while norepinephrine (desipramine, reboxetine) and serotonin (clomipramine, citalopram) reuptake inhibitors substituted at one or more doses. Further investigation using specific receptor agonists and antagonists indicated a possible role for activation of alpha1-adrenoceptors but failed to support involvement of alpha2-adenoceptor, beta-adrenoceptor or 5-HT1A receptor activation. These results support the concept that the 2-BFI cue may contain both noradrenergic and serotonergic components.

Characterization of the discriminable stimulus produced by 2-BFI: effects of imidazoline I(2)-site ligands, MAOIs, beta-carbolines, agmatine and ibogaine.

1. The molecular nature and functions of the I(2) subtype of imidazoline binding sites are unknown but evidence suggests an association with monoamine oxidase (MAO). Rats can distinguish the selective imidazoline I(2)-site ligand 2-BFI from vehicle in drug discrimination, indicating functional consequences of occupation of these sites. We have used drug discrimination to investigate the nature of the discriminable stimulus, especially in relation to MAO inhibition. 2. Following training to distinguish 2-BFI 7 mg kg(-1) i.p. from saline vehicle in two-lever operant-chambers, male Hooded Lister rats underwent sessions where test substances were given instead and the proportion of lever presses on the 2-BFI-associated lever (substitution) recorded. 3. 2-BFI; its cogeners BU216, BU224, BU226 and LSL60101; the reversible MAO-A inhibitors moclobemide and RO41-1049; the beta-carbolines harmane, norharmane and harmaline which also reversibly inhibit MAO-A, and the anti-addictive substance ibogaine exhibited potent, dose-dependent substitution for 2-BFI. 4. Agmatine, and LSL60125 substituted at one dose only. The reversible MAO-B inhibitors lazabemide and RO16-1649; the sigma(2)-site ligand SKF10,047 and the I(2A)-site ligand, amiloride, failed to substitute. The irreversible inhibitor of MAO, deprenyl, substituted for 2-BFI while clorgyline did not. 5. These results suggest imidazoline I(2) site ligands produce a common discriminable stimulus that appears associated with reversible inhibition of MAO-A rather than MAO-B, possibly through increases in extracellular concentration of one or more monoamines. Ibogaine exhibits a commonality in its subjective effects with those of I(2)-site ligands.