Search

Objective: To evaluate the performance of INTERGROWTH-21st and World Health Organization (WHO) fetal growth charts to identify small-for-gestational-age (SGA) and fetal growth restriction (FGR) neonates as well as their specific risks for adverse neonatal outcomes. Design: Multicenter cross-sectional study. Setting: Ten maternity units across four Latin American countries, 2016-2018. Population: 67,968 singleton live births. Methods: According to each standard, the neonates were classified as SGA and FGR (birthweight

International audience; Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimer's disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by MRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.

SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide) is the lead compound of an original pyridazinoindole series of potent and highly selective TSPO (peripheral benzodiazepine receptor) ligands. Isotopic labeling of SSR180575 with the short-lived positron-emitter carbon-11 (T1/2: 20.38 min) at its 5-methylpyridazino[4,5-b]indole moiety as well as at its N,N-dimethylacetamide function by methylation of the corresponding nor-analogues was investigated. Best results in terms of radiochemical yields and purities were obtained for the preparation of [indole-N-methyl-11C]SSR180575, where routine production batches of 4.5–5.0 GBq of radiochemically pure (>99%) i.v. injectable solutions (specific radioactivities: 50–90 GBq/µmol) could be prepared within a total synthesis time of 25 min (HPLC purification included) starting from a 55 GBq [11C]CO2 cyclotron production batch (non-decay-corrected overall radiochemical yields: 8–9%). The process comprises (1) trapping at −10°C of [11C]methyl triflate in DMF (300 µl) containing 0.2–0.3 mg of the indole precursor for labeling and 4 mg of K2CO3 (excess); (2) heating at 120°C for 3 min; (3) dilution of the residue with 0.5 ml of the HPLC mobile phase and (4) purification using semi-preparative reversed-phase HPLC (Zorbax® SB-C-18). In vivo pharmacological properties of [indole-N-methyl-11C]SSR180575 as a candidate for imaging neuroinflammation with positron emission tomography are currently evaluated. Copyright © 2010 John Wiley & Sons, Ltd.

Recent epidemiological, biological and genetic data indicate a relationship between cholesterol and Alzheimer's disease (AD) including the association of polymorphisms of ABCA1 (a gene that is known to participate in cholesterol and phospholipid transport) with AD prevalence. Based on these data, we postulated that genetic variation in the related and brain-specific ABCA2 gene leads to increase risk of AD. A large case-control study was conducted where the sample was randomly divided into a hypothesis-testing sample (230 cases/286 controls) and a validation sample (210 cases/233 controls). Among the 45 SNPs we tested, one synonymous SNP (rs908832) was found significantly associated with AD in both samples. Additional analyses performed on the whole sample showed a very strong association between this marker and early-onset AD (OR = 3.82, 95% C.I. = [2.00 - 7.30], P = 5 x 10(-5)). Further research is needed to understand the functional role of this polymorphism. However, together with the reported associations of AD with APOE, CYP46A1 and ABCA1, the present result adds a very significant support for the role of cholesterol and phospholipid homeostasis in AD and a rationale for testing novel cholesterol homeostasis-related therapeutic strategies in AD.

Alzheimer's disease (AD) is characterized by a substantial degeneration of pyramidal neurons and the appearance of neuritic plaques and neurofibrillary tangles. Here we present a novel transgenic mouse model, APP(SL)PS1KI that closely mimics the development of AD-related neuropathological features including a significant hippocampal neuronal loss. This transgenic mouse model carries M233T/L235P knocked-in mutations in presenilin-1 and overexpresses mutated human beta-amyloid (Abeta) precursor protein. Abeta(x-42) is the major form of Abeta species present in this model with progressive development of a complex pattern of N-truncated variants and dimers, similar to those observed in AD brain. At 10 months of age, an extensive neuronal loss (>50%) is present in the CA1/2 hippocampal pyramidal cell layer that correlates with strong accumulation of intraneuronal Abeta and thioflavine-S-positive intracellular material but not with extracellular Abeta deposits. A strong reactive astrogliosis develops together with the neuronal loss. This loss is already detectable at 6 months of age and is PS1KI gene dosage-dependent. Thus, APP(SL)PS1KI mice further confirm the critical role of intraneuronal Abeta(42) in neuronal loss and provide an excellent tool to investigate therapeutic strategies designed to prevent AD neurodegeneration.

Transgenic mice over-expressing a mutated form of the human amyloid precursor protein (APP, 695 isoform) bearing a mutation associated with Alzheimer's disease (V642I, so-called London mutation, hereafter APPLd2) and wild-type controls were studied at age periods (3 and 10 months) prior to the overt development of neuritic amyloid plaques. Both 3- and 10-month-old APPLd2 mice had reflex eyelid responses like those of controls, but only younger mice were able to acquire a classical conditioning of eyelid responses in a trace paradigm. In vitro studies on hippocampal slices showed that 10-month-old APPLd2 mice also presented deficits in paired-pulse facilitation and long-term potentiation, but presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals. It is proposed that definite functional changes may appear well in advance of noticeable structural alterations in this animal model of Alzheimer's disease, and that specific learning tasks could have a relevant diagnostic value.

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits (alpha6(*) nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human alpha6beta2, alpha6beta4, alpha6beta2beta3, alpha6beta4beta3, or alpha6beta4beta3alpha5 nAChR. Cells expressing subunits needed to form alpha6beta4beta3alpha5 nAChR exhibited saturable [(3)H]epibatidine binding (K(d) = 95.9 +/- 8.3 pM and B(max) = 84.5 +/- 1.6 fmol/mg of protein). The rank order of binding competition potency (K(i)) for prototypical nicotinic compounds was alpha-conotoxin MII (6 nM)nicotine (156 nM) approximately methyllycaconitine (200 nM)alpha-bungarotoxin (10 microM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that beta3 and alpha5 subunits are likely partners of the alpha6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that alpha6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human alpha5 subunit makes a critical contribution to alpha6beta4beta3alpha5 nAChR assembly into a ligand-binding form with native alpha6(*)-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

Nicotine activates specific receptors that are cation-permeable ionic channels located in the central and autonomous nervous systems, as well as at the neuromuscular junction. Administration of nicotine to animals and humans has been shown to enhance cognitive processes. However, side effects linked to the activation of peripheral nicotinic receptors limit the usefulness of nicotine for the treatment of cognitive disorders such as Alzheimer's disease (AD) or mild cognitive impairments (MCI). The synthesis and properties of TC-1827, a novel metanicotine derivative that activates brain α4β2 nicotinic receptors is described. TC-1827 has high affinity for nicotine-labeled receptors in the cortex (Ki=34 nM), full-agonist intrinsic activity in α4β2-mediated neurotransmitter release studies in synaptosomes, and has no functional activity at nicotinic receptors in ganglionic or muscular cell lines. The compound enhances long-term potentiation in hippocampal slices, a form of synaptic plasticity thought to be involved in information storage at the cellular level. In vivo studies demonstrate that TC-1827 dose-dependently occupies thalamic nicotinic receptors labeled with [3H]-cytisine, increases cortical extracellular acetylcholine levels following oral administration, and enhances cognitive performance in rat and mice behavioral procedures of learning and memory. Pharmacokinetic studies in mice, rats, and monkeys indicated that TC-1827 has good oral absorption with a first pass effect resulting in bioavailabilities of 13–65% across dose/species. Cardiovascular safety studies indicate good cardiovascular tolerability for this compound. The present data demonstrate that TC-1827 is a selective and potent activator of brain α4β2 nicotinic receptors and is a prototypical member of a new class of compounds with potential utility in the symptomatic treatment of cognitive disorders including AD and MCI. Drug Dev. Res. 62:26–40, 2004. © 2004 Wiley-Liss, Inc.

Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism (EO-AR). Here we show that inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection.

Alzheimer's disease (AD) is a genetically complex neurodegenerative disorder and the leading cause of dementia of the elderly. Recently, Hu et al. suggested that a trinucleotide deletion in intron 13 of the APBB1 gene was a factor protecting against late-onset AD. We report here the results of a case/control study aimed at replicating this association. Our study included 461 AD patients and 397 matched controls. We compared the allele and genotype frequencies of the polymorphism between the two groups but did not find any statistically significant difference ( P =0.08 and P =0.09, respectively). By contrast, adjusting for age and sex, we found a slight risk associated with the deletion (odds ratio=1.47, 95% confidence interval=1.05–2.04). Stratification by age showed that the risk effect associated with the deletion concerned subjects aged less than 65 years.

In astrocytes, thrombin and thrombin receptor-activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [3H]-thymidine incorporation into DNA (EC50 = 1 nM and +650% at 100 nM for thrombin; EC50 = 3 microM and +600% at 100 microM for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 nM thrombin or 100 microM TRAP-14). The [3H] thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as Gi or G(o), is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.

Progesterone is synthesized in the nervous system by neurons and glial cells. Because of their simple structure, plasticity and capacity of regeneration, peripheral nerves are particularly well suited for studying the biosynthesis, mechanisms of action and effects of the hormone. Schwann cells, the myelinating glial cells in the peripheral nervous system, synthesize progesterone in response to a diffusible neuronal signal. In peripheral nerves, the local synthesis of progesterone plays an important role in the formation of myelin sheaths. This has been shown in vivo, after cryolesion of the mouse sciatic nerve, and in vitro, in cocultures of Schwann cells and sensory neurons. Schwann cells also express an intracellular receptor for progesterone, which thus functions as an autocrine signalling molecule. Progesterone may promote myelination by activating the expression of genes coding for transcription factors (Krox-20) and/or for myelin proteins (P0, PMP22). Recently, it has been proposed that progesterone may indirectly regulate myelin formation by influencing gene expression in neurons. Steroid hormones also influence the proliferation of Schwann cells: estradiol becomes a potent mitogen for Schwann cells when levels of cAMP are elevated and glucocorticosteroids have been shown to increase the mitogenic effects of peptide growth factors.

The aim of this study was to investigate the facilitatory effects of subanalgesic or low doses of different drugs (acetylsalicylic acid, ibuprofen and morphine) on the antinociceptive responses induced by the endogenous opioid peptides, enkephalins, protected from their catabolism by the dual enkephalin-degrading enzymes inhibitor RB101. According to the analgesic profile of the three studied compounds different antinociceptive assays were used: the hot plate and formalin tests in mice, and the tail flick and paw pressure tests on inflamed paws in rats and polyarthritic rats. Facilitatory effects of subanalgesic doses of acetylsalicylic acid and ibuprofen on RB101-induced antinociceptive responses were observed in the early and late phases of the formalin test, respectively. In the hot plate, tail flick and paw pressure tests, the dose-dependent analgesic effects of RB101 were strongly potentiated by subanalgesic doses of morphine (0.5 mg/kg), while in these tests, acetylsalicylic acid and ibuprofen were unable to modify the RB101-induced antinociceptive responses. The synergism in antinociceptive effects observed with the combination of RB101 and morphine supported by isobolographic analysis, may have interesting clinical implications, considering both the lack of opiate drawbacks observed with RB101 and the high potentiation of its antinociceptive effects with very low doses of morphine.

We analyzed the expression of native GABA(A) receptors in choline acetyltransferase and glutamic acid decarboxilase positive cells, from lamina IX of the lumbar region of rat spinal cord. More than one isoform of each subunit was detected within a single cell. The alpha3, alpha5, alpha1, beta3 and gamma2 subunit was the most frequent combination in both cell populations. However, the total number of subunit expressed by each cell type was different, being the ChAT positive cells the simplest. Interestingly, the ChAT and GAD positive cells also displayed a different pattern of distribution of both spliced isoforms of the gamma2 subunit. These results indicate that several GABA(A) receptors, with different molecular composition, are expressed in a single cell and that different cell types can express different GABA(A) receptors.

Among the chemokine family, fractalkine shows unusual properties: it exists as a membrane-bound and soluble protein, and both fractalkine and its receptor CX(3)CR1 are expressed predominantly in the central nervous system. In rat cell culture models, the chemokine fractalkine was expressed in neurons and microglia, but not in astrocytes and its receptor exclusively localized to microglial cells, where its expression was downregulated by treatment with the bacterial endotoxin (LPS). In microglial cultures, LPS (10 ng/ml) induced a marked increase in the release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The effects of LPS on TNF-alpha secretion were partially blocked (30%) by fractalkine and the effects of fractalkine were reversed by a polyclonal anti-fractalkine antibody. When microglial-associated fractalkine was neutralized by anti-fractalkine antibody, the LPS response was increased by 80%, suggesting tonic activation of microglial fractalkine receptors by endogenous fractalkine. The effects of the antibody were antagonized by the addition of fractalkine. LPS-activated microglia were neurotoxic when added to neuronal hippocampal culture, producing 20% neuronal death, as measured by NeuN-positive cell counting. An anti-fractalkine antibody produced neurotoxic effects of similar magnitude in this co-culture system and also markedly potentiated the neurotoxic effects of LPS-activated microglia (40% neuronal death). These results suggest that endogenous fractalkine might act tonically as an anti-inflammatory chemokine in cerebral tissue through its ability to control and suppress certain aspects of microglial activation. These data may have relevance to degenerative conditions such as multiple sclerosis, in which cerebral inflammatory processes may be activated.

Peripheral benzodiazepine receptors (PBR) and their endogenous ligands, the diazepam-binding inhibitor derived-peptides, are present in Schwann cells in the peripheral nervous system. The aim of this study was to determine the influence of reversible (freeze-injury) and permanent (transection and ligature) nerve lesion on PBR density and on the levels of their endogenous ligands, by autoradiography (using [ 3 H ]PK11195) and radioimmunoassay (using antisera directed against the octadecaneuropeptide (ODN), a diazepam-binding inhibitor fragment). The potential role of PBR on peripheral nerve steroidogenesis, was studied by investigating the effect of specific PBR agonists and antagonists on pregnenolone levels in the sciatic nerve. Sixteen to 30 days after nerve lesion, PBR density and ODN–LI level were highly increased. Their expression returned to normal level when regeneration was completed 60 days after freeze-injury, but remained elevated when regeneration did not occur in transected distal stumps. Reverse-phase HPLC analysis of ODN–LI showed that in control nerve extracts, the major immunoreactive peak co-elutes with triakontatetraneuropeptide (TTN). After freeze-injury, intermediate molecular forms eluting between ODN and TTN were predominant and remained elevated at day 60. The greater accumulation of intermediate forms when regeneration is allowed to occur may indicate a particular role of these forms in axonal elongation and myelination. Ro5–4864, a high affinity PBR agonist increased pregnenolone concentration in the sciatic nerve. This effect was antagonised by PK11195, a high affinity PBR antagonist, which had no effect on pregnenolone basal level, indicating a specific action of PBR in neurosteroid production. These results suggest a role for PBR and their endogenous ligands in peripheral nerve regeneration. A trophic effect could be exerted via stimulation of steroid synthesis.

In this study we have investigated the expression of the peripheral benzodiazepine receptor (PBR) and its endogenous ligand octadecaneuropeptide (ODN) in the sciatic nerve of the adult rat by immunohistochemistry. We have also determined the effect of nerve freezing lesion or chronic denervation on PBR and ODN expression. In the sciatic nerve of control rats PBR- and ODN-immunoreactivities (IR) were associated to Schwann cells. Ten days after nerve injury, PBR- and ODN-IR increased significantly in the distal stump. PBR-IR was associated to Schwann cells and macrophages, whereas ODN-IR was only detected in Schwann cells. Immunoreactivities returned to normal levels when axons were allowed to regenerate for 2 months after nerve freezing-injury. Under chronic denervation conditions, PBR- and ODN-IR remained elevated in the distal stump, at least for this period of time. These results suggest that PBR and ODN are regulated by signals from regenerating axons and that PBR and its endogenous ligand may play a role in peripheral nerve regeneration.

We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical alpha1-alpha3 gamma-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of alpha1 and alpha3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-alpha1- or anti-alpha3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the alpha1-alpha3 GABAA receptors represented 54.7 +/- 5.0 and 23.6 +/- 3.3% of the alpha3 and alpha1 populations, respectively. Therefore, alpha1 and alpha3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these alpha1-alpha3 GABAA receptors from the cortex constitute a large proportion of the total alpha3 population and a relatively minor component of the alpha1 population. The pharmacological analysis of the alpha1- or alpha3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both alpha1 and alpha3 subunits, with alpha1 and alpha3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-alpha1-anti-alpha3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of alpha1 and alpha3 subunits, associated with beta2/3, and gamma2 subunits. The pharmacological analysis of alpha1-alpha3 GABAA receptors demonstrated that, despite the fact that each alpha subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the alpha1 subunit is pharmacologically predominant over the alpha3 subunit. These results indicate the existence of active and nonactive alpha subunits in the native alpha1-alpha3 GABAA receptors from rat cortex.

International audience; Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is characterized by the invariant cerebral accumulation of ␤-amyloid peptide. This event occurs early in the disease process. In humans, [18F]-fluoro-2-deoxy-D-glucose ([18F]-FDG) positron emission tomography (PET) is largely used to follow-up in vivo cerebral glucose utilization (CGU) and brain metabolism modifications associated with the Alzheimer's disease pathology. Here, [18F]-FDG positron emission tomography was used to study age-related changes of cerebral glucose utilization under resting conditions in 3-, 6-, and 12-month-old APP SweLon /PS1 M146L , a mouse model of amyloidosis. We showed an age-dependent increase of glucose uptake in several brain regions of APP/PS1 mice but not in control animals and a higher [18F]-FDG uptake in the cortex and the hippocampus of 12-month-old APP/PS1 mice as compared with age-matched control mice. We then developed a method of 3-D microscopic autoradiography to evaluate glucose uptake at the level of amyloid plaques and showed an increased glucose uptake close to the plaques rather than in amyloid-free cerebral tissues. These data suggest a macroscopic and microscopic reorganization of glucose uptake in relation to cerebral amyloidosis.

The effects of fluid percussion trauma on brain interleukin (IL)-6, IL-1 and tumor necrosis factor-alpha (TNF-alpha) levels have been studied. In the cortex and hippocampus of control and sham-operated rats, the levels of these cytokines were very low (below 4 units/mg protein) and constant. IL-6 and IL-1 levels in the ipsilateral cortex increased rapidly following trauma to reach a maximum of 350 and 16 units/mg protein, respectively, 8 h after the lesion, remained elevated until 18 h and decreased thereafter to basal values. TNF-alpha levels were maximally elevated (12 units/mg protein) at 3 h and 8 h and returned to basal values by 18 h. Qualitatively similar changes, but with 25-80-fold smaller amplitude, were seen in the contralateral cortex and in the ipsi- and contralateral hippocampus. The levels of IL-6 in the plasma of sham-operated and lesioned rats were only slightly elevated, whereas IL-1 and TNF-alpha were undetectable. Histological studies of brain tissue at early stages after trauma demonstrated an acute hemorrhage associated with neutrophil invasion. The administration of Ro5 4864 (0.5 mg/kg i.p.), a specific ligand of p (peripheral-type benzodiazepine) binding sites, did not result in any significant effect on the levels of IL-6, IL-1 or TNF-alpha in the brain of control or sham-operated animals. However, when administered 24 h before or 15 min after trauma, this benzodiazepine enhanced the increase of these cytokines by 2-4-fold in the ipsilateral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the ω1 and ω2 populations of central ω benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with μM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20–40 μM (zolpidem) and 5–15 μM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 μM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 μM zolpidem or 1 μM alpidem, but were displaced by 100 μM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 μM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central ω site subtypes which display high (IC50 = 31 and 6.1 nM, respectively; ω1 sites), intermediate (IC50 = 480 and 122 nM, respectively; ω2I) and low (IC50 = 20100 and 2300 nM, respectively; ω2L) affinity for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between ω1 and ω2 sites but not between two ω2 receptor subpopulations. ω1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior collicus. ω2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, ω2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures. In conclusion, the present results indicate that in adult and neonatal rat CNS sections zolpidem and alpidem can discriminate between 3 populations of [3H]flumazenil binding sites that may be akin to ω binding sites associated with GABAA receptors containing α1-subunits (ω1), α2- or α3-subunits (ω2I) and α5-subunits (ω2L, respectively.

The potential heterogeneity in the allosteric coupling between GABA and omega 1 binding sites within the native GABAA receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [3H]zolpidem binding to omega 1 site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 +/- 47%) in cortical membranes than in cerebellar (165 +/- 48%) or in hippocampal (118 +/- 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than omega 1 since: (1) the pharmacological properties of the [3H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of omega 1 sites in each structure; and (3) GABA did not increase the Bmax for [3H]zolpidem. Regional differences in the effect of 100 microM GABA on [3H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3-4-fold) effects of GABA in [3H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [3H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [3H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroinflammation is involved in neurological disorders through the activation of microglial cells. Imaging of neuroinflammation with radioligands for the translocator protein (18 kDa) (TSPO) could prove to be an attractive biomarker for disease diagnosis and therapeutic evaluation. The indoleacetamide-derived 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide, SSR180575, is a selective high-affinity TSPO ligand in human and rodents with neuroprotective effects.Here we report the radiolabelling of SSR180575 with (11)C and in vitro and in vivo imaging in an acute model of neuroinflammation in rats.The image contrast and the binding of [(11)C]SSR180575 are higher than that obtained with the isoquinoline-based TSPO radioligand, [(11)C]PK11195. Competition studies demonstrate that [(11)C]SSR180575 has high specific binding for the TSPO.[(11)C]SSR180575 is the first PET radioligand for the TSPO based on an indoleacetamide scaffold designed for imaging neuroinflammation in animal models and in the clinic.

The potential neuroprotective effects of IL-6 against the excitotoxic neuronal loss induced by N- methyl- d -aspartate (NMDA) have been studied. Infusion into the rat striatum of excitotoxic amounts (250 nmol) of NMDA resulted in a 45% decrease in striatal choline acetyl transferase activity (ChAT; a marker of cholinergic neurons) and glutamate decarboxylase (GAD, a marker of GABAergic neurons) at 2 days post-injection. Co-infusion of 10 U of IL-6 reduced the loss of ChAT activity to 21% but failed to prevent the loss of GAD activity. IL-6 per se, up to the dose of 500 U, failed to affect ChAT or GAD activities. The in vivo effects of IL-6 are not mediated by a direct antagonism of NMDA toxicity, since IL-6 (up to a concentration of 500 and 5000 U/ml, respectively) did not antagonize either the increase in cyclic GMP levels resulting from NMDA receptor activation in cerebellar slices or the glutamate-induced release of lactate dehydrogenase, an index of neurotoxicity, by cultured cortical neurons. These results suggest that the increase in IL-6 levels observed in experimental brain lesions may play a role in the protection and regeneration of cholinergic neurons.

The activation of phosphoinositide turnover in rat cerebral cortex and choroid plexus is triggered by the stimulation of 5-HT2 and 5-HTIC receptors, respectively. To characterize the 5-HT receptor subtype mediating the activation of phosphoinositide turnover in the hippocampus, the potency of several 5-HT agonists and antagonists on total [3H]inositol phosphate formation has been compared in the hippocampus, cerebral cortex and choroid plexus of immature rats. 5-HT, α-methyl-5-HT, quipazine, MK-212, mCPP (m-chlorophenylpiperazine) and TFMPP (m-trifluoromethylphenylpiperazine) are less potent and efficient in stimulating phosphoinositide turnover in the hippocampus and cerebral cortex than in the choroid plexus. However, for a number of 5-HT receptor antagonists (ketanserin, spiperone, ritanserin, pizotifen, cyproheptadine, mesulergine, mianserin, methiothepin, methysergide) there is a good correlation (r = 0.82) between their antagonistic potency in the hippocampus and choroid plexus while such correlation is not observed for the hippocampus and cerebral cortex. The specific 5-HT2 receptor antagonist spiperone only partially antagonizes (37% inhibition at 1 μM) the stimulation by 5-HT of phosphoinositide turnover in the hippocampus. These results suggest that in the immature rat hippocampus the activation of phosphoinositide turnover by 5-HT is mainly mediated by the 5-HTIC receptor subtype.

The anatomical localization of the increase in ω 3 (peripheral type benzodiazepine) binding site densities (an index of glial reaction) following intraperitoneal injection of convulsant doses of kainic acid has been studied by autoradiography in the rat brain. Consistent increase in ω 3 site densities were observed in the olfactory, piriform and entorhinal cortices, amygdaloid nucleus, hippocampal formation and thalamus. In the hippocampal formation, the most pronounced increases were seen in the stratum lucidum of the CA3 field and in the stratum oriens and pyramidale of the CA1 and CA2 fields. This pattern of changes in ω 3 site densities closely paralleled the pattern of neuropathological alterations (assessed by histological methods) observed in these brain regions. Thus, ω 3 site autoradiography may provide a sensitive and reliable index of the neuronal damage resulting from kainic acid administration.

Saturation studies with [ 3 H]ifenprodil (in the presence of 3 μ M (+)-3-PPP and 10 μM GBR 12909) demonstrated the presence of a high-affinity ( K d = 0.45 μ M) population of binding sites in sagittal rat brain sections. This binding was inhibited by spermine (IC 50 = 69 μ M) and spermidine (IC 50 = 623 μ M) but not by putrescine (1 mM). Ifenprodil displaced this binding in a biphasic fashion with a high affinity component (IC 50 = 0.992 μ M) accounting for approximately 50% of the spermine-displaceable [ 3 H]ifenprodil binding. The polyamine-sensitive [ 3 H]ifenprodil binding sites were heterogenously distributed in the rat brain, the highest binding densities being found in the hippocampus and in the nucleus accumbens. The anatomical distribution of [ 3 H]ifenprodil binding sites closely matches that previously reported for the N- methyl- d -aspartate (NMDA) receptor.

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 μM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) > ipsapirone (20) > gepirone (120) > RU 24969 (140) > buspirone (560) > 1-(m-trifluoromethylphenyl)piperazine (1,500) > methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r= 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 μM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 μM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 μM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 uM) and arachidonic acid (100 μM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 μM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 μM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a γ-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor. The latter possibility is supported by the fact that incubation of immature rat hippocampal slices with 8-OH-DPAT (10 μM) caused a down-regulation (-16%) of [3H]quinuclidinyl benzylate binding.

The possible involvement of lymphokines in the glial reaction/proliferation that follows brain injury has been investigated by measuring the density of omega 3 (peripheral type benzodiazepine) binding sites associated to glial cells and macrophages after local injection of lymphokines in the rat cerebral cortex and striatum. omega 3 Site densities were measured either by quantitative autoradiography in brain sections or by conventional binding in membrane using [3H]PK 14105 or [3H]PK 11195 as ligands. Intracortical or intrastriatal infusion of interleukin-1 (10 and 20 units) caused a marked increase in the density of omega 3 sites (+83% and +80%, respectively, when compared to saline-infused animals) around the injection site at 7 days postinjection. There was a good spatial correspondence between the autoradiographic distribution of omega 3 sites and the distribution of reactive astrocytes (as assessed by GFAP immunostaining) or acid phosphatase rich cells (phagocytes). Significant increases in omega 3 site densities were also observed in striatal homogenates 1 week after local administration of tumor necrosis factor-alpha (TNF-alpha). The maximal increase (+80%) was observed after the administration of 3 units, higher and lower doses resulting in smaller increases. Intrastriatal injection of E. coli lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate interleukin-1 and TNF-alpha production by microglial cells in culture, also resulted in significant increases in omega 3 site densities in striatal homogenates (maximal increase, +170% 1 week after the injection of 200 ng).(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the nature and origin of the serotonergic innervation of two distinct anatomical cerebrovascular compartments, namely, small pial vessels and major cerebral arteries, in the rat. To this end, the levels of serotonin [5-hydroxytryptamine (5-HT)] and 5-hydroxyindoleacetic acid (5-HIAA) were measured by HPLC in both cerebrovas-cular compartments after either bilateral sympathectomy or destruction of the ascending serotonergic pathways, which originate from the raphe nuclei. We first showed that the small pial vessel samples were not contaminated by underlying cortical tissues through the use of an immunohistochemical approach that revealed the glia limitans, the most superficial cortical layer. Superior cervical ganglionectomy caused a marked decrease in noradrenaline concentrations in major cerebral arteries (−77%), although the reduction was less pronounced (−34%) in small pial vessels. Sympathectomy decreased by 33% 5-HT concentrations in the major cerebral arteries but was without effect on 5-HT levels in the small pial vessels. Destruction of the ascending serotonergic pathways (via local administration of 5,7-dihydroxytryptamine into the ventral tegmental area) produced a dramatic fall in 5-HT and 5-HIAA concentrations in both vascular compartments. To establish the authenticity of the serotonergic innervation, the synthesis of 5-HT [as assessed by measuring the accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition] was measured in the two vascular beds under control conditions and after destruction of the ascending serotonergic pathways. The rate of accumulation of 5-HTP was higher in the small pial vessels than in major cerebral arteries, an observation that indicates an important de novo synthesis of 5-HT in small pial vessels. The neurotoxic lesion of the ascending serotonergic fibers caused a marked reduction of 5-HTP accumulation in small pial vessels (−67%) and a slight but significant decrease (−24%) in major cerebral arteries. These results collectively point to the existence of a true serotonergic innervation of small pial vessels that originates in the rostral raphe nuclei; in contrast, most of the 5-HT present in major cerebral arteries seems to originate from sources other than these nuclei.

The microscopic distribution of ω 3 (peripheral type benzodiazepine) binding sites in sections from normal and ischaemic rat brain has been determined by emulsion autoradiography with the photoaffinity ligand [ 3 H]PK 14105. In the normal rat brain, high densities of ω 3 sites were present in the ependymal cells, the apical pole of the secretory cells in the choroid plexus and astrocyte-like cells in the outer cellular layer of the olfactory bulb. In ischaemic brain lesions caused by middle cerebral artery occlusion in spontaneously hypertensive rats or in spontaneous ischaemic and haemorrhagic lesions in spontaneously hypertensive stroke-prone rats, the proliferating ω 3 sites were associated with reactive glial cells and macrophages.

In recent years, there has been considerable effort to design and synthesize radiotracers suitable for use in Positron Emission Tomography (PET) imaging of the α4β2 neuronal nicotinic acetylcholine receptor (nAChR) subtype. A new fluoropyridinyl derivative of (−)-cytisine ( 1 ), namely (−)-9-(2-fluoropyridinyl)cytisine ( 3 , K i values of 24 and 3462 nM for the α4β2 and α7 nAChRs subtypes, respectively) has been synthesized in four chemical steps from (−)-cytisine and labelled with fluorine-18 ( T 1/2 : 119.8 min) using an efficient two-step radiochemical process [(a) nucleophilic heteroaromatic ortho -radiofluorination using the corresponding N -Boc-protected nitro-derivative, (b) TFA removal of the Boc protective group]. Typically, 20–45 mCi (0.74–1.67 GBq) of (−)-9-(2-[ 18 F]fluoropyridinyl)cytisine ([ 18 F]- 3 , 2–3 Ci/μmol or 74–111 GBq/μmol) were easily obtained in 70–75 min starting from a 100 mCi (3.7 GBq) aliquot of a cyclotron-produced [ 18 F]fluoride production batch (20–45% non decay-corrected yield based on the starting [ 18 F]fluoride). The in vivo pharmacological profile of (−)-9-(2-[ 18 F]fluoropyridinyl)cytisine ([ 18 F]- 3 ) was evaluated in rats with biodistribution studies and brain radioactivity monitoring using intracerebral radiosensitive β-microprobes. The observed in vivo distribution of the radiotracer in brain was rather uniform, and did not match with the known regional densities of nAChRs. It was also significantly different from that of the parent compound (−)-[ 3 H]cytisine. Moreover, competition studies with (−)-nicotine (5 mg/kg, 5 min before the radiotracer injection) did not reduce brain uptake of the radiotracer. These experiments clearly indicate that (−)-9-(2-[ 18 F]fluoropyridinyl)cytisine ([ 18 F]- 3 ) does not have the required properties for imaging nAChRs using PET.

Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms.

In the present study, we have investigated the potential neuroprotective effects of a novel peripheral benzodiazepine binding site (PBR) ligand, 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575), in models of central and peripheral neurodegeneration in vivo and its effect on steroid concentrations in plasma and nervous tissue. SSR180575 shows high affinity (IC(50), 2.5-3.5 nM) and selectivity for the rat and human PBR and potently inhibits the in vivo binding of [(3)H]alpidem to PBR in the rat brain and spleen after oral or i.p. administration (ID(50), 0.1-0.3 mg/kg). In an experimental model of motoneuron degeneration induced by facial nerve axotomy in the immature rat, SSR180575 given i.p. or orally for 8 days rescued facial motoneurons, increasing their survival by 40 to 72% at 6 and 10 mg/kg p.o. b.i.d. Moreover, in this model, SSR180575 (10 mg/kg p.o. b.i.d.) increased by 87% the number of motoneurons immunoreactive to peripherin, a type III intermediate filament, whose expression is up-regulated during nerve regeneration. SSR180575 also improved functional recovery in acrylamide-induced neuropathy in the rat when given therapeutically at 2.5 to 10 mg/kg/day p.o. Furthermore, SSR180575 (3 mg/kg i.p. b.i.d.) accelerated functional recovery of the blink reflex after local injury of the facial nerve in the rat. SSR180575 increased pregnenolone accumulation in the brain and sciatic nerve (+100% at 3 mg/kg i.p.), suggesting that its neuroprotective effects are steroid-mediated. These results indicate that PBR ligands (e.g., SSR180575) promote neuronal survival and repair in axotomy and neuropathy models and have potential for the treatment of neurodegenerative diseases (e.g., peripheral neuropathies or amyotrophic lateral sclerosis).

N-Methyl-o-aspartate (NMD A) receptor activation selectively releases the poly amines spermine and spermidine from the rat striatum in vivo. The intrastriatal injection of spermine or spermidine is neurotoxicbut this toxicity is not blocked by MK-801 and unlikely to be mediated via the NMDA receptor. The neurotoxic effects of intrastriatally injected NMDA can, however; be reduced by polyamine synthesis inhibition with difluoromethylornithine. Alterations in polyamine metabolism in the ischaemic brain, although perhaps induced by NMDA receptor activation\, may contribute to ischaemic cell loss via NMDA-independent mechanisms, possibly related to the diverse effects of polyamines on calcium homoeostasis and channel function.

The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC(50) values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of three-dimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and co-transported ions.

Changes in gene expression within the hippocampus induced by denervation after electrolytic fimbria-fornix lesion in rat were compared to morphological and biochemical alterations. Fimbria-fornix lesion results in degeneration of hippocampal cholinergic terminals as evidenced by a sustained (2 days to1 month) decrease in cholineacetyltransferase (ChAT) activity (50%). These changes were accompanied by a decrease in growth associated protein 43 (GAP-43) immunoreactivity in all hippocampal layers 4 days after lesion followed by a subsequent increase and return to normal levels by 20 days postinjury. This increase in GAP-43 expression in the hippocampus between 7 to 20 days after lesion may reflect heterotypic sprouting. TUNEL-positive cells were revealed by in situ assay within the hippocampus at 10 days, but not at 3 days, after lesion. Two subtracted cDNA libraries from the dorsal hippocampus of control and injured rats (at 3 and 10 days postlesion) were constructed in order to search for new genes potentially implicated in degeneration/regeneration phenomena. We analysed 1,536 clones from each library by differential screening and found a total of 46 up-regulated genes. Among the 15 known genes, 6 coded for proteins involved in signal transduction pathways. The up-regulation of growth arrest DNA damage induced gene (GADD153), brain-specific RING finger protein, JNK interacting protein (JIP-1), protein kinase A (PKA), and Na+K+ ATPase was studied by quantitative polymerase chain reaction (PCR). Two of these genes, GADD153 and JIP-1, have been previously shown to participate in cell modifications induced by stress and apoptosis. J. Neurosci. Res. 59:209–217, 2000 © 2000 Wiley-Liss, Inc.

The concentration of peripheral type benzodiazepine binding sites (PTBS) in the brain parenchyma is greatly increased following brain lesions, reflecting the glial reaction and/or presence of hematogenous cells. Thus, PTBS density is a sensitive and reliable marker of brain injury in a large number of experimental models (ischemia, trauma, excitotoxic lesions, brain tumors) and equivalent human neuropathological conditions. PTBS density can be measured using specific radioligands and a conventional binding technique, or by quantitative autoradiography in tissue sections.

In the present study, we have assessed the efficacy of eliprodil, a neuroprotective agent which blocks both the modulatory polyamine site of the NMDA receptor and neuronal voltage-sensitive calcium channels, alone or in combination with the thrombolytic agent, rt-PA, in a rat embolic stroke model using a neurological score and the volume of the infarct as endpoints. Embolization was induced by intracarotid injection of an arterial blood clot. Eliprodil, administered at the dose of 1 mg/kg, iv. 10 min and 2 h 30 after embolization, reduced the neurological deficit by 54% (P < 0.01) and the total volume of the brain lesion by 49%. Thrombolysis with rt-PA (2.5 mg/kg, as a 30 min iv infusion beginning 1 h after embolization) decreased the neurological deficit by 48% (P < 0.05) and the size of the total infarct by 55% (P < 0.05). Combined therapy greatly improved the degree of neuroprotection as assessed by neurological and histological outcomes (70% (P < 0.001) and 89% (P < 0.01) neuroprotection, respectively). These results demonstrate that the administration of a neuroprotective drug (eliprodil) or a thrombolytic agent (rt-PA) similarly reduce the volume of brain damage and the neurological deficit in a rat embolic stroke model. Combined cytoprotective therapy and thrombolysis markedly improved the degree of neuroprotection and may, thus, represent a valuable approach for the treatment of stroke in humans.

Amisulpride, a benzamide derivative with an atypical neuroleptic profile relieves the negative symptoms of schizophrenia when administered at low doses (50–150 mg). In an attempt to define the anatomical substrates involved in this action we have studied the effects of amisulpride on regional cerebral glucose utilisation (RCGU) in the awake lightly restrained rat, by quantitative autoradiography using [14C]2-deoxyglucose ([14C]2-DG). Amisulpride was administered 1 h before [14C]2DG i.v. injection, at a dose of 5 mg/kg which resulted in a striatal D2 receptor occupancy of 10% similar to that induced by doses of this compound used for the treatment of negative symptoms of schizophrenia. Amisulpride induced significant RCGU increases in cortical areas, in visual relays, in auditory structures and in several limbic structures. The pattern of changes in RCGU seen with amisulpride clearly differs from that of haloperidol, given at a dose resulting in a similar occupancy of striatal D2 receptors (0.01 mg/kg), which was mostly ineffective. The amisulpride-induced activation of RCGU in specific brain areas involved in the control of cognitive functions and motivational and emotional behavior, may at least in part, explain the efficacy of this compound in the treatment of negative symptoms of schizophrenia.

The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [3H]thymidine incorporation and reversal of stellation induced by Bt2cAMP as end-points. Thrombin increased [3H]thymidine incorporation into DNA with an EC50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [3H]thymidine incorporation were mimicked by TRAP-14 (EC50=3 μM) and a peptide containing non-natural amino acids Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (A6Y; EC50=0.8 μM). Similarly, these two peptides reversed Bt2cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [3H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg- d -Cys-Gly-Lys-His-Ser-Arg; IC50=180 μM against thrombin and TRAP-14 and 800 μM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets. © 1997 Elsevier Science B.V. All rights reserved.

Drugs acting upon the benzodiazepine binding sites of the GABAA receptor complex constitute one of the most widely used class of psychotropic agents. These drugs increase the sensitivity of the GABAA receptor to its natural agonist, γ-aminobutyric acid (GABA). This allosteric facilitation of GABAergic neurotransmission has found clinical application in the treatment of a large number of disorders [1–3]. These applications are based on the sedative, anxiolytic, anticonvulsant and myorelaxant activities of benzodiazepine receptor agonists. Moreover, these drugs also display untoward effects such as memory disruption, and after chronic treatment, induction of tolerance and dependence. These pleitropic actions constitute one of the most important drawback of benzodiazepines. For this reason, much research has tried to dissociate the effects responsible for the therapeutic activity from those considered as secondary. For example, to dissociate anxiolytic from sedative effects, or hypnotic effects from memory impairment or myorelaxant activities, and also to avoid the induction of tolerance or dependence.

Using the in vivo microdialysis technique, we have studied the effect of the systemic administration of several alpha 1-adrenoceptor antagonists on the extracellular levels of serotonin (5-HT) in the rat hippocampus. Prazosin, and to a lesser extent, terazosin, decreased these levels by 50-65% for 0.03-0.4 mg/kg, i.v. and by 30-40% for 0.1-0.4 mg/kg, i.v., respectively. In contrast, alfuzosin, an alpha 1-adrenoceptor antagonist with poor brain penetration, did not significantly affect these levels even at the high dose of 0.4 mg/kg, i.v. When perfused into the hippocampus through the dialysis probe, prazosin (1-10 microM) induced a more limited (20-30%) and delayed decrease in 5-HT outflow. These results support the existence of a central noradrenergic facilitatory influence, mediated by alpha 1-adrenoceptors, on serotonergic neurons projecting to the hippocampus. In the striatum prazosin (0.4 mg/kg, i.v.) decreased 5-HT levels to a smaller extent (-35%) than in the hippocampus (-65%), suggesting the existence of differences in the degree of noradrenergic influence on median and dorsal raphe nuclei, which preferentially project to the hippocampus and striatum, respectively.

The extracellular concentration of inositol 1,4,5-trisphosphate (IP3) has been monitored in the ventral hippocampus of the anesthetized rat by using a microdialysis technique coupled to a radioreceptor assay. Three hours after the implantation of the cannula, basal extracellular concentration of IP3 (corrected for a 9% recovery) was 71 nM (0.39 pmol/60-microliters fraction) and remained stable for at least 5 h. Local infusion of carbachol for 60 min caused a significant concentration-related increase in extracellular IP3 levels (0, 24, and 57% at 1, 50, and 100 microM, respectively). Acetylcholine (100 microM) and muscarine (100 microM) increased IP3 outflow by 40 and 42%, respectively. The effect of carbachol was fully prevented by coinfusion of 10 microM pirenzepine and reduced by 1 microM tetrodotoxin indicating that the carbachol response is mediated by neuronal muscarinic receptors. These data demonstrate the feasibility of using microdialysis and a radioreceptor assay to measure IP3 in the extracellular space. This approach could prove useful for the study of the in vivo operation of muscarinic and, by extension, a number of receptors coupled to phosphoinositide turnover.

Our search to understand the molecular links between immune and nervous systems has led us to propose that central benzodiazepine receptors (CBR) and peripheral benzodiazepine receptors (PBR) could represent an integrative network whereby endogenous ligands could modulate anxiety as well as immune functions11.

The neuroprotective potential of ifenprodil and SL 82.0715, two non-competitive NMDA antagonists, the mechanism of which is related to a blockade of the polyamine regulatory site, has been evaluated in experimental paradigms of focal cerebral ischaemia in the rat, cat and mouse. When first administered 30 min after middle cerebral artery occlusion in the rat, SL 82.0715 (1 and 10 mg/kg p.o.) reduced by 34 and 48%, respectively the consolidated volume of infarction, as assessed histologically. Ifenprodil and SL 82.0715 (0.3–3 mg/kg i.v.) administered as a perfusion over 3 h after occlusion of the feline middle cerebral artery reduced the volume of infarcted tissue (measured 4 days after occlusion) in a dose-related manner by 25 to 42%. The administration of SL 82.0715 (10 mg/kg i.p.), 5 min, 6 h, 18 h after middle cerebral artery occlusion in the mouse and then twice a day until sacrifice evoked a decrease of similar magnitude (ca. 60–70%) in the volume of the infarction and in the proliferation of ω3 (peripheral type benzodiazepine) binding site levels (an index of reactive gliosis). A significant diminution of cortical ω3 sites was still noted when the first administration was delayed until 3 h post-occlusion. The overall consistency of these results in animal models of stroke strongly suggest that ifenprodil and SL 82.0715 are of potential value in the treatment of ischaemic cerebrovascular diseases.