Alpha2-containing GABA(A) receptors are involved in mediating stimulant effects of cocaine.

alpha2 subunit-containing GABA(A) receptors are involved in incentive learning associated with cocaine, and in cocaine addiction. Deletion of alpha2-containing receptors abolishes cocaine-induced behavioural sensitisation (BS), while selective activation of alpha2 receptors, achieved using Ro 15-4513's agonist properties in alpha2(H101R) mice, induced BS. Here, we investigate further the mechanisms underlying Ro 15-4513-induced behavioural sensitisation in alpha2(H101R) mice. alpha2(H101R) mice sensitised to Ro 15-4513 (10 mg/kg) showed an enhanced stimulant response to cocaine (10 mg/kg). In contrast, cocaine (10 mg/kg)-sensitised alpha2(H101R) mice did not show enhanced sensitivity to the stimulant effects of Ro 15-4513 (1, 3 and 10 mg/kg), suggesting that the neural adaptations underlying Ro 15-4513 induced BS are related to, but not identical with those associated with cocaine-induced plasticity. Secondly, we investigated whether alpha2-containing receptors are involved in mediating the ability of BZs to facilitate cocaine-induced activity. The non-selective (i.e., alpha1, alpha2, alpha3 and alpha5 subtype) benzodiazepine GABA(A) receptor agonist midazolam (10 and 30 mg/kg) potentiated cocaine (10 mg/kg) hyperactivity in wildtype mice, but not in alpha2(H101R) mice, in which alpha2-containing receptors are insensitive to benzodiazepines. To determine where alpha2 receptors are localised we compared BZ-insensitive sites between wildtype (alpha4 and alpha6) and alpha2(H101R) (alpha2, alpha4 and alpha6) mice, using quantitative autoradiography to estimate [(3)H]Ro 15-4513 binding in the presence of 10 muM diazepam. alpha2 receptors were found in projection areas of the mesolimbic dopamine pathway including accumbens, central amygdala, and basolateral amygdala as well as CA1 and CA3 areas of the hippocampus. The involvement of the alpha2-containing receptor in mediating BZ's potentiating effect on cocaine hyperactivity suggests that the locomotor stimulant effects of BZs and psychostimulants may be mediated by a common neural system, but the lack of cross sensitisation to Ro 15-4513 in cocaine-sensitised alpha2(H101R) mice, suggests that this form of BS may occur downstream of plastic events underlying cocaine sensitisation.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain.

BACKGROUND AND PURPOSE: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. EXPERIMENTAL APPROACH: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. KEY RESULTS: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. CONCLUSIONS AND IMPLICATIONS: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.

Inositol monophosphatase--a putative target for Li+ in the treatment of bipolar disorder.

Attenuation of the phosphatidylinositol (PI) signal transduction pathway as a consequence of inhibition of inositol monophosphatase (IMPase) has been proposed as the mechanism for the efficacy of Li+ in the treatment of bipolar disorder. Nevertheless, Li+ also affects other aspects of PI signal transduction, and it is therefore not clear whether modulation of PI responses by Li+ can be attributed solely to inhibition of IMPase. However, inhibitors of IMPase mimic the effects of Li+ on some aspects of PI cell signalling, thus highlighting the potential of IMPase as a target for the treatment of bipolar disorder. The recent description of the three-dimensional structure of IMPase in conjunction with site-directed mutagenesis and kinetic studies has led to the elucidation of the enzyme mechanism. These structural and mechanistic data should prove useful in the development of novel inhibitors of IMPase that might ultimately prove useful clinically.

An inverse agonist selective for alpha5 subunit-containing GABAA receptors improves encoding and recall but not consolidation in the Morris water maze.

RATIONALE: Compounds selective for the GABAA receptors containing an alpha5 subunit have been reported to enhance performance in the hippocampally mediated delayed-matching-to-position version of the Morris water maze, in which reduction in the time required to find a hidden platform relative to an initial trial is used as an index of learning and memory. OBJECTIVE: In the present study, we have used one such compound, alpha5IA-II, to examine whether these effects occur during the encoding, consolidation or recall phases of this paradigm. METHODS: alpha5IA-II was administered in the absence or presence of the benzodiazepine site antagonist flumazenil, so as to limit its action to periods associated with encoding, consolidation and recall. Drug doses and timings of administrations were defined using occupancy data derived from an in vivo [3H]flumazenil binding assay. Similar experiments were carried out to study the memory-disruptive properties of chlordiazepoxide (CDP). RESULTS: The trial 1 to trial 2 difference was increased when alpha5IA-II was given before either trial 1 or trial 2, indicating an effect on the encoding and recall phases, respectively, of learning and memory. Conversely, alpha5IA-II had no effect on performance when given immediately after trial 1, suggesting that it had no effect on the consolidation phase. In contrast to the facilitation of performance produced by the alpha5-selective inverse agonist alpha5IA-II given during the encoding and recall but not the consolidation phase, the non-selective agonist CDP impaired performance when given during the encoding and recall phases, whilst having no effect on the consolidation phase. CONCLUSIONS: These data further highlight the cognition-enhancing properties of GABAA alpha5-selective inverse agonists and define the functional specificity of these effects in terms of encoding and recall processes in the Morris water maze.

Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice.

The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

Cerebrospinal fluid inositol monophosphatase: elevated activity in depression and neuroleptic-treated schizophrenia.

BACKGROUND: Inositol monophosphatase (IMPase) is a key enzyme in the regulation of the activity of the phosphatidyl inositol (PI) signaling pathway. This enzyme is also found in the cerebrospinal fluid (CSF), where it may prove useful as a marker of dysfunctional PI signal transduction. METHODS: IMPase activity was measured in lumbar CSF of depressed and neuroleptic-treated schizophrenic patients. In addition, and to gain an insight into the factors that influence the levels of CSF IMPase, enzyme activity was measured in subgroups of schizophrenic patients treated for 3-7 days with lithium or 7 days with inositol. RESULTS: CSF IMPase activity was significantly increased by 88% in depressed and by 172% in schizophrenic patients relative to control subjects. Lithium produced a marked increase in CSF IMPase activity in the group as a whole, and this group effect could be more specifically attributed to 3 of the 8 individuals in whom enzyme activity increased by over 300%. On the other hand, inositol had no effect on CSF IMPase activity. CONCLUSIONS: In the absence of a clear relationship between CSF IMPase activity and neuronal PI signaling pathways it is not possible to correlate these changes with altered neuronal function. Nevertheless, increased CSF IMPase activity in depression and schizophrenia may be a marker of the pathophysiological processes underlying these disorders. Moreover, the large lithium-induced increase in IMPase activity seen in a subgroup of schizophrenic subjects suggests a differential regulation of CSF enzyme activity in these patients.

Cerebrospinal fluid production is normal in Down syndrome.

The rate of production of cerebrospinal fluid (CSF) and the caudorostral gradients of total CSF protein were measured in seven subjects with Down syndrome (DS) and compared to age-matched healthy normal volunteers. The CSF production rate in DS subjects (0.35 +/- 0.02 mL/min.) did not differ significantly from normal subjects (0.37 +/- 0.09 mL/min.). In addition, the caudorostral gradient of total protein was similar in DS and normal subjects, with more caudal fractions of lumbar CSF having higher total protein levels than more rostral fractions. These data suggest that there is no gross disturbance in CSF dynamics in DS.

CSF and serum concentrations of albumin and IgG in Alzheimer's disease.

Cerebrospinal fluid (CSF) and serum concentrations of albumin and immunoglobulin G (IgG) were measured in 31 patients with presumptive Alzheimer's disease (AD) and in 14 healthy control subjects. The albumin and IgG quotients, and IgG index were calculated to evaluate the permeability of the blood-brain barrier and the intrathecal production of immunoglobulins. X-ray computerized tomography (CT) of the head was performed to investigate the relation between cerebral atrophy and CSF protein concentrations. The albumin and IgG quotients, and the IgG index did not differ significantly between the AD and control groups. Cerebral atrophy, as measured by CSF volume, was not related to CSF protein concentrations in either group. The results do not support the hypothesized roles of blood-brain barrier disruption or of immunologically-mediated injury of the central nervous system in the pathogenesis of AD.

Structure and mechanism of inositol monophosphatase.

Since lithium inhibits IMPase and modulates phosphatidylinositol (PtdIns) cell signalling at therapeutically relevant concentrations (0.5-1.0 mM), IMPase has attracted attention as a putative molecular target for lithium in the treatment of manic depression. IMPase is a homodimer, with each subunit organised in an alpha beta alpha beta alpha arrangement of alpha-helices and beta-sheets, and this type of structure seems crucial to the two-metal catalysed mechanism in which an activated water molecule serves as a nucleophile. Lithium appears to inhibit the enzyme following substrate hydrolysis by occupying the second metal binding site before the phosphate group can dissociate from its interaction with the site 1 metal. The understanding of IMPase structure and the mechanism of substrate hydrolysis and lithium inhibition should be useful in the development of novel inhibitors which may prove clinically useful in the treatment of manic depression.

Carbamate analogues of (-)-physostigmine: in vitro inhibition of acetyl- and butyrylcholinesterase.

Reaction of (-)-eseroline (1) with alkyl, aryl and aralkylisocyanates afforded a series of carbamate analogues of (-)-physostigmine (2) which were assayed for inhibition of acetyl- and butyrylcholinesterase (AChE and BChE, respectively) in vitro. Included in this study were two N-alkyl-substituted carbamates 9 and 14 obtained from (-)-eseroline (1) with dialkylcarbamoyl chlorides, and allophanates 12 and 13 obtained as by-products in the reaction of 1 and benzylcarbamoyl eseroline (8) with benzyl isocyanate. Whereas none of the analogues studied was more potent than 2 against electric eel AChE, and carbamates 6, 7 and 8 were all more than 3 times more potent against human plasma BChE than 2.

Lumbar cerebrospinal fluid choline in healthy aging and in Down's syndrome.

Choline concentrations were measured in lumbar cerebrospinal fluid (CSF) and plasma of 37 healthy normal subjects and 13 young (age range, 21 to 34 years) and 6 older (age, greater than or equal to 45 years) healthy adults with Down's syndrome (DS). All subjects with DS had a trisomy 21 karyotype, and 3 of the 6 older subjects were demented as judged from a history of mental deterioration, disorientation, and memory loss. In healthy normal subjects, there was a significant correlation between age and CSF choline concentrations. Compared with age-matched controls, in young subjects with DS, CSF, but not plasma, choline concentrations were elevated (by 49%), whereas in older subjects with DS, CSF and plasma choline concentrations were similar to control values. The CSF choline concentrations were unrelated to body height in the DS and control groups, and rostrocaudal CSF choline gradients did not differ between either the control and DS groups or the young and old subjects with DS, suggesting that the elevation in the CSF choline concentration in young subjects with DS was not related to their shorter stature. Since increased CSF choline concentrations in young adult subjects with DS were accompanied by normal plasma choline concentrations, these results suggest that young adults with DS have either an increased release of choline from the brain or a reduced rate of clearance of choline from CSF.

Cerebrospinal fluid somatostatin and neuropeptide Y. Concentrations in aging and in dementia of the Alzheimer type with and without extrapyramidal signs.

Cerebrospinal fluid somatostatin and neuropeptide Y concentrations were measured in 26 healthy normal subjects, 27 patients with dementia of the Alzheimer type (DAT), and seven patients with DAT with extrapyramidal signs (EDAT). In healthy normal subjects, there was no significant correlation between age and either somatostatin or neuropeptide Y concentration. However, the concentrations of both peptides correlated significantly with each other. In patients with DAT and EDAT, the concentrations of somatostatin (17.5 +/- 5.0 and 16.4 +/- 5.0 pg/mL, respectively) were significantly reduced relative to age-matched control subjects (23.1 +/- 8.2 pg/mL) but were unrelated to dementia severity and did not change significantly during the progression of the disease. Neuropeptide Y concentrations did not differ significantly between the age-matched control, DAT, and EDAT groups (38.2 +/- 12.8, 37.0 +/- 12.3, and 30.3 +/- 7.8 pg/mL, respectively). These results suggest that in DAT, dysfunction of cortical somatostatin but not neuropeptide Y transmitter systems is reflected by reduced cerebrospinal fluid concentrations.

Reductions in [3H]nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study.

In Alzheimer's disease (AD) and Parkinson's disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic [3H]acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers.

Cerebrospinal fluid production is reduced in healthy aging.

In order to study age-related differences in cerebrospinal fluid (CSF) production in humans, we measured the rate of CSF production in 7 young (age 21 to 36 years) and 7 elderly (age 67 to 84 years) healthy volunteers, using a modified Masserman method. In addition, we evaluated CSF protein gradients by collecting CSF in serial fractions up to the 30th ml and assaying for total protein concentration. The mean rate of CSF production was significantly less in the elderly than in the young subjects. Mean CSF total protein concentrations were higher in the elderly than in the young, and significant rostrocaudal protein gradients with similar slopes were present in both groups. However, there was no correlation between CSF production and CSF total protein concentrations or protein gradient slopes. Age-related reductions in CSF production, together with the ventricular dilatation that occurs with aging, should presumably result in reduced CSF turnover and therefore influence measured concentrations of lumbar CSF constituents.

Cerebrospinal fluid monoamine markers are decreased in dementia of the Alzheimer type with extrapyramidal features.

We measured monoamine metabolites and biopterin in the CSF of 37 patients with dementia of the Alzheimer type (DAT), with or without extrapyramidal signs, and in 14 age-matched healthy controls. Compared with concentrations in DAT and controls, the concentrations of homovanillic acid (HVA) and biopterin were significantly decreased in DAT with extrapyramidal signs (EDAT). CSF 3-methoxy-4-hydroxy-phenethyleneglycol and 5-hydroxyindoleacetic acid did not differ significantly among these groups. Age at onset of dementia was positively correlated with CSF HVA (r = 0.49, p less than 0.05). The two dementia groups did not differ significantly in the extent of ventricular dilation as measured by quantitative CT, but EDAT patients had lower Mini-Mental State Examination scores than did DAT patients. When patients were matched for age and dementia severity, CSF HVA and biopterin concentrations remained significantly lower in EDAT than in DAT patients. These results indicate that EDAT patients form a distinct subgroup of DAT with evidence of central monoamine dysfunction.

Regional differences in the inhibition of mouse in vivo [3H]Ro 15-1788 binding reflect selectivity for alpha 1 versus alpha 2 and alpha 3 subunit-containing GABAA receptors.

The benzodiazepines flunitrazepam, diazepam, and Ro 15-1788 and the beta-carboline DMCM bind with equivalent affinity to the benzodiazepine binding site of GABAA receptors containing different alpha subunits (i.e., alpha 1, alpha 2, alpha 3, or alpha 5); whereas, the triazolopyridazine CL 218,872 and imidazopyridine zolpidem have higher affinity for alpha 1 subunit-containing GABAA receptors. In the present study, the in vivo binding of [3H]Ro 15-1788 in mouse cerebellum and spinal cord was used to establish the occupancy of the benzodiazepine binding site of GABAA receptors containing primarily alpha 1 and alpha 2/alpha 3 subunits, respectively. Thus, the nonselective compounds flunitrazepam, diazepam, and DMCM all produced a similar inhibition of binding in cerebellum and spinal cord (respective ID50 values of 0.2 to 0.3 mg/kg, 2 mg/kg, and 10 mg/kg i.p.); whereas, the alpha 1 selective compounds CL 218,872 and zolpidem were more potent at inhibiting [3H]Ro 15-1788 binding in the cerebellum (ID50 values 4.5 mg/kg and 10 mg/kg i.p.) compared to the spinal cord (ID50 values 12 mg/kg and > 30 mg/kg i.p.). Thus, the reduction of in vivo f[3H]Ro 15-1788 binding in tissues containing alpha 1 and alpha 2/alpha 3 receptor populations reflects the in vitro affinities of subtype selective compounds and should help to interpret the behavioral profile of such compounds.

GABA(A) alpha 1 subunit knock-out mice do not show a hyperlocomotor response following amphetamine or cocaine treatment.

The GABA(A) receptor system provides the major inhibitory control in the CNS, with the alpha 1 beta 2 gamma 2 subunit combination being the most abundant and widely distributed form of the receptor. The alpha1 subunit knock-out (alpha1 KO) mice had a surprisingly mild overt phenotype, despite having lost approximately 60% of all GABA(A) receptors. The alpha1 KO mice had normal spontaneous locomotor activity, but were more sensitive to the sedating/ataxic effects of diazepam than wildtype (WT) mice. Pharmacological modulation of dopamine and N-methyl-D-aspartate (NMDA) receptors also produced altered responses in alpha1 KO mice compared with WT mice. As expected, the NMDA receptor antagonist MK801, amphetamine and cocaine increased locomotor activity in WT mice. Although MK801 increased locomotor activity in alpha1 KO mice, amphetamine and cocaine induced stereotypy not hyperlocomotion. Binding studies showed no gross changes in the total number of D1, D2 or NMDA receptors. Furthermore, pre-pulse inhibition of acoustic startle and the effects of cocaine in conditioned place preference were similar in both alpha1 KO and WT mice, indicating selective rather that global changes in response to dopaminergic agents. These data demonstrate subtle changes in behaviours mediated by neurotransmitters other than GABA in alpha1 KO mice and suggest that compensation may have occurred beyond the GABAergic system.

Synthesis and anticholinesterase activity of (-)-N1-norphysostigmine, (-)-eseramine, and other N(1)-substituted analogues of (-)-physostigmine.

(-)-N1-Benzylnorphysostigmine (4), prepared from synthetic (-)-O-methyl-N1-noreseroline (1) by N-benzylation, ether cleavage, and reaction of (-)-N1-benzylnoreseroline (3) with methyl isocyanate, was the intermediate used to prepare the title compounds. Catalytic debenzylation of 4 afforded (-)-N1-norphysostigmine (5), and (-)-eseramine (6) was obtained by reaction of 5 with methyl isocyanate. Reductive N-methylation of 5 gave (-)-physostigmine (9) while reaction of 5 with allyl bromide and phenethyl bromide afforded carbamates 7 and 8, respectively. Data on the in vitro potencies (IC50) and activities of certain of these compounds (4-8) as inhibitors of electric eel acetyl cholinesterase are reported. (-)-N1-Norphysostigmine (5) was found to be similarly potent against AChE as (-)-physostigmine (9).

Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells.

Cell surface expression of the NR1a subunit has been examined in mouse L cell lines permanently transfected with the complementary DNA for human NR1a or with the complementary DNAs for NR1a and NR2A. The expression of the subunits was under the control of the murine mammary tumour virus promoter and following induction of expression by dexamethazone both cell lines expressed high levels of the NR1a subunit as determined by immunofluorescence using permeabilized cells and immunoblotting of cell membranes with subunit specific antibodies. However, cell surface expression of the NR1a subunit was found only in the cells expressing both the NR1a and NR2A subunits. This was confirmed by cell surface biotinylation of the two cell lines and affinity isolation of the receptor subunits. To determine if this result was solely due to the use of a particular cell line and or the choice of expression vector, Cos-7 cells were transiently transfected with either NR1a or NR1a plus NR2A. Here too cell surface expression was only found following co-transfection of both subunits.