Parental Social Isolation during Adolescence Alters Gut Microbiome in Rat Male Offspring.

Previous work from our laboratory demonstrated that parental stress, induced by social isolation starting at puberty, leads to behavioral, endocrine, and biochemical changes in the male, but not female, offspring (ISO-O) of Sprague-Dawley rats. Here, we report alterations in the gut microbiota composition of ISO-O vs. grouped-housed offspring (GH-O), although all animals received the same diet and were housed in the same conditions. Analysis of bacterial communities by next-generation sequencing (NGS) of 16S rRNA gene revealed alterations at family and order levels within the main phyla of Bacteroides, Proteobacteria, and Firmicutes, including an almost total deficit in Limosilactobacillus reuteri (formerly Lactobacillus reuteri) and a significant increase in Ligilactobacillus murinus (formerly Lactobacillus murinus). In addition, we found an increase in the relative abundance of Rhodospirillales and Clostridiales in the families of Lachnospiraceae and Ruminococcaceae, and Bacteroidales in the family of Prevotellaceae. Furthermore, we examined plasma levels of the proinflammatory cytokines interleukin-1-beta and tumor necrosis factor-alpha, which did not differ between the two groups, while corticosterone concentrations were significantly increased in ISO-O rats. Our findings suggest that adverse environmental conditions experienced by parents may have an impact on the likelihood of disease development in the subsequent generations.

Chronic treatment with hormonal contraceptives alters hippocampal BDNF and histone H3 post-translational modifications but not learning and memory in female rats.

Hormonal contraceptives prevent ovulation with subsequent reduction in endogenous levels of estradiol, progesterone and its neuroactive metabolite allopregnanolone. These neurosteroids modulate several brain functions, including neuronal plasticity, cognition and memory. We hypothesized that hormonal contraceptives might affect synaptic plasticity, learning and memory, as a consequence of suppressed endogenous hormones levels. Female rats were orally treated with a combination of ethinyl estradiol (EE, 0.020 mg) and levonorgestrel (LNG, 0.060 mg) once daily for four weeks. Decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and altered histone H3 post-translational modifications (PTMs) were observed 14 days after discontinuation from chronic EE-LNG treatment. These effects were not accompanied by alterations in long-term plasticity at glutamatergic synapses, recognition memory in the novel object and novel place location tests, or spatial learning, memory, and behavioral flexibility in the Morris water maze test. Thus, decreased BDNF content does not affect synaptic plasticity and cognitive performance; rather it might be relevant for the occurrence of certain psychiatric symptoms, reported by some women using hormonal contraceptives. These results provide the first evidence of hippocampal epigenetic changes induced by hormonal contraceptives and complement previous studies on the neurobiological actions of hormonal contraceptives; the finding that effects of chronic EE-LNG treatment on BDNF content and histone PTMs are observed 14 days after drug discontinuation warrants further investigation to better understand the implications of such long-term consequences for women's health.

Increased Voluntary Alcohol Consumption in Mice Lacking GABAB(1) Is Associated With Functional Changes in Hippocampal GABAA Receptors.

Gamma-aminobutyric acid type B receptor (GABABR) has been extensively involved in alcohol use disorders; however, the mechanisms by which this receptor modulates alcohol drinking behavior remain murky. In this study, we investigate alcohol consumption and preference in mice lacking functional GABABR using the 2-bottle choice paradigm. We found that GABAB(1), knockout (KO), and heterozygous (HZ) mice drank higher amounts of an alcoholic solution, preferred alcohol to water, and reached higher blood alcohol concentrations (BACs) compared to wild-type (WT) littermates. The GABABR agonist GHB significantly reduced alcohol consumption in the GABAB(1) HZ and WT but not in the KO mice. Next, because of a functional crosstalk between GABABR and δ-containing GABAA receptor (δ-GABA A R), we profiled δ subunit mRNA expression levels in brain regions in which the crosstalk was characterized. We found a loss of the alcohol-sensitive GABAAR δ subunit in the hippocampus of the GABAB(1) KO alcohol-naïve mice that was associated with increased É£2 subunit abundance. Electrophysiological recordings revealed that these molecular changes were associated with increased phasic inhibition, suggesting a potential gain of synaptic GABAAR responsiveness to alcohol that has been previously described in an animal model of excessive alcohol drinking. Interestingly, voluntary alcohol consumption did not revert the dramatic loss of hippocampal δ-GABAAR occurring in the GABAB(1) KO mice but rather exacerbated this condition. Finally, we profiled hippocampal neuroactive steroids levels following acute alcohols administration in the GABAB(1) KO and WT mice because of previous involvement of GABABR in the regulation of cerebral levels of these compounds. We found that systemic administration of alcohol (1.5 g/kg) did not produce alcohol-induced neurosteroid response in the GABAB(1) KO mice but elicited an expected increase in the hippocampal level of progesterone and 3α,5α-THP in the WT controls. In conclusion, we show that genetic ablation of the GABAB(1) subunit results in increased alcohol consumption and preference that were associated with functional changes in hippocampal GABAAR, suggesting a potential mechanism by which preference for alcohol consumption is maintained in the GABAB(1) KO mice. In addition, we documented that GABAB(1) deficiency results in lack of alcohol-induced neurosteroids, and we discussed the potential implications of this finding in the context of alcohol drinking and dependence.

Are preconceptional stressful experiences crucial elements for the aetiology of autism spectrum disorder? Insights from an animal model.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by changes in social interactions, impaired language and communication, fear responses and presence of repetitive behaviours. Although the genetic bases of ASD are well documented, the recent increase in clinical cases of idiopathic ASD indicates that several environmental risk factors could play a role in ASD aetiology. Among these, maternal exposure to psychosocial stressors during pregnancy has been hypothesized to affect the risk for ASD in offspring. Here, we tested the hypothesis that preconceptional stressful experiences might also represent crucial elements in the aetiology of ASD. We previously showed that social isolation stress during adolescence results in a marked decrease in the brain and plasma concentrations of progesterone and in the quality of maternal care that these female rats later provide to their young. Here we report that male offspring of socially isolated parents showed decreased agonistic behaviour and social transmission of flavour preference, impairment in reversal learning, increased seizure susceptibility, reduced plasma oxytocin levels, and increased plasma and brain levels of BDNF, all features resembling an ASD-like phenotype. These alterations came with no change in spatial learning, aggression, anxiety and testosterone plasma levels, and were sex-dependent. Altogether, the results suggest that preconceptional stressful experiences should be considered as crucial elements for the aetiology of ASD, and indicate that male offspring of socially isolated parents may be a useful animal model to further study the neurobiological bases of ASD, avoiding the adaptations that may occur in other genetic or pharmacologic experimental models of these disorders.

Differential modulation of GABA(A) receptor function by aryl pyrazoles.

Several aryl pyrazoles characterized by a different molecular structure (flexible vs constrained), but chemically related to rimonabant and AM251, were tested for their ability to modulate the function of recombinant α1ß2γ2L GABAA receptors expressed in Xenopus laevis oocytes. The effects of 6Bio-R, 14Bio-R, NESS 0327, GP1a and GP2a (0.3-30 µM) were evaluated using a two-electrode voltage-clamp technique. 6Bio-R and 14Bio-R potentiated GABA-evoked Cl(-) currents. NESS 0327, GP1a and GP2a did not affect the GABAA receptor function, but they acted as antagonists of 6Bio-R. Moreover, NESS 0327 inhibited the potentiation of the GABAA receptor function induced by rimonabant. The benzodiazepine site seems to participate in the action of these compounds. In fact, flumazenil antagonized the potentiation of the GABAA receptor induced by 6Bio-R, and NESS 0327 reduced the action of lorazepam and zolpidem. On the contrary, NESS 0327 did not antagonize the action of "classic" GABAergic modulators (propanol, anesthetics, barbiturates or steroids). In α1ß2 receptors 6Bio-R potentiated the GABAergic function, but flumazenil was still able to antagonize the potentiation induced by 6Bio-R. Aryl pyrazole derivatives activity at the GABAA receptor depends on their molecular structure. These compounds bind to both an αßγ binding site, and to an α/ß site which do not require the γ subunit and that may provide structural leads for drugs with potential anticonvulsant effects.

Effects of voluntary ethanol consumption on emotional state and stress responsiveness in socially isolated rats.

Social isolation of rats immediately after weaning is thought to represent an animal model of anxiety-like disorders. This mildly stressful condition reduces the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) as well as increases the sensitivity of rats to the effects of acute ethanol administration on the concentrations of this neuroactive steroid. We further investigated the effects of voluntary consumption of ethanol at concentrations increasing from 2.5 to 10% over 4 weeks of isolation. Isolated rats showed a reduced ethanol preference compared with group-housed animals. Ethanol consumption did not affect the isolation-induced down-regulation of BDNF or Arc, but it attenuated the increase in the cerebrocortical concentration of 3α,5α-TH PROG induced by foot-shock stress in both isolated and group-housed animals as well as increased the percentage of number of entries made by socially isolated rats into the open arms in the elevated plus-maze test. Ethanol consumption did not affect expression of the α4 subunit of the GABA(A) receptor in the hippocampus of group-housed or isolated rats, whereas it up-regulated the δ subunit throughout the hippocampus under both conditions. The results suggest that low consumption of ethanol may ameliorate some negative effects of social isolation on stress sensitivity and behavior.

Changes in expression and function of extrasynaptic GABAA receptors in the rat hippocampus during pregnancy and after delivery.

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABA(A)-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABA(A)-Rs, and a tonic component mediated by extrasynaptic GABA(A)-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3alpha,5alpha-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the delta subunit of the GABA(A)-R and a concomitant decrease in that of the gamma(2) subunit in the hippocampus at P19. Expression of the alpha(4) subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5alpha-reductase inhibitor finasteride prevented the changes in tonic current and in delta and gamma(2) subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABA(A)-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy.

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.

Synthesis and structure-activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem.

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published procedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmacophoric (delta1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary allylammonium cation 14. The structure-affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and suggests a common mode of interaction at the PBR binding site.

Thiocolchicoside inhibits the activity of various subtypes of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes.

Thiocolchicoside is a myorelaxant drug with anti-inflammatory and analgesic properties as well as pronounced convulsant activity. To characterize the mechanisms of action of this drug at the molecular level, we examined its effects on the function of various recombinant neurotransmitter receptors expressed in Xenopus oocytes. Electrophysiological recordings from recombinant human gamma-aminobutyric acid type A (GABA(A)) receptors consisting of alpha1beta1gamma2L, alpha1beta2gamma2L, or alpha2beta2gamma2L subunit combinations revealed that thiocolchicoside inhibited GABA-evoked Cl(-) currents with similar potencies (median inhibitory concentrations of 0.13 to 0.2 microM) and in a competitive manner. Consistent with previous observations, thiocolchicoside also inhibited the binding of GABA to rat cerebral cortical membranes. Thiocolchicoside inhibited the function of recombinant human strychnine-sensitive glycine receptors composed of the alpha1 subunit with a potency (median inhibitory concentration of 47 microM) lower than that apparent with recombinant GABA(A) receptors. It also inhibited the function of human nicotinic acetylcholine receptors composed of the alpha4 and beta2 subunits, but this effect was only partial and apparent at high concentrations. In contrast, thiocolchicoside had no effect on the function of 5-HT(3A) serotonin receptors. Our results thus provide molecular evidence that the epileptogenic activity of thiocolchicoside might be due to inhibition of the function of inhibitory receptors in the central nervous system, especially that of GABA(A) receptors.

Changes in expression of the delta subunit of the GABA (A) receptor and in receptor function induced by progesterone exposure and withdrawal.

Type A receptors for GABA (GABA(A) receptors) that contain the delta subunit are located predominantly at extrasynaptic sites and are implicated in modulation of neuronal excitability through tonic inhibition. We have examined the effects of chronic exposure to and subsequent withdrawal of progesterone or the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THPROG) on expression of the delta subunit of GABA(A) receptors and on receptor function in cultured rat hippocampal neurons. Progesterone treatment for 1 day increased the amounts of both delta subunit mRNA and protein, whereas such treatment for 6 days induced marked decreases in the abundance of both the mRNA and protein. Subsequent progesterone withdrawal up-regulated expression of the delta subunit, which was significantly increased at 9-12 h after withdrawal. These effects of progesterone were mimicked by 3alpha,5alpha-THPROG and blocked by the 5alpha-reductase inhibitor finasteride. They were also accompanied by parallel changes in the function of GABA(A) receptors in hippocampal neurons. These results show that chronic exposure to and withdrawal of progesterone induce differential effects on both expression of the delta subunit of GABA(A) receptors and receptor function that are mediated by 3alpha,5alpha-THPROG. They are consistent with the notion that this progesterone metabolite plays a key physiological role in modulation of GABAergic synapses.

Anticipation and consumption of food each increase the concentration of neuroactive steroids in rat brain and plasma.

Stressful stimuli and anxiogenic drugs increase the plasma and brain concentrations of neuroactive steroids. Moreover, in rats trained to consume their daily meal during a fixed period, the anticipation of food is associated with changes in the function of various neurotransmitter systems. We have now evaluated the effects of anticipation and consumption of food in such trained rats on the plasma and brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH DOC), two potent endogenous positive modulators of type A receptors for gamma-aminobutyric acid (GABA). The abundance of these neuroactive steroids was increased in both the cerebral cortex and plasma of the rats during both food anticipation and consumption. In contrast, the concentration of their precursor, progesterone, was increased in the brain only during food consumption, whereas it was increased in plasma only during food anticipation. Intraperitoneal administration of the selective agonist abecarnil (0.1 mg/kg) 40 min before food presentation prevented the increase in the brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC during food anticipation but not that associated with consumption. The change in emotional state associated with food anticipation may thus result in an increase in the plasma and brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC in a manner sensitive to the activation of GABA(A) receptor-mediated neurotransmission. A different mechanism, insensitive to activation of such transmission, may underlie the changes in the concentrations of these neuroactive steroids during food consumption.

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function.

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.

Low tolerance and dependence liabilities of etizolam: molecular, functional, and pharmacological correlates.

The effects of prolonged exposure to and subsequent withdrawal of the thienotriazolobenzodiazepine etizolam on gamma-aminobutyric acid (GABA) type A receptor gene expression and function were compared with those of the benzodiazepine lorazepam. Exposure of rat hippocampal neurons in culture to 10 microM etizolam for 5 days reduced the amounts of alpha5 and gamma2S receptor subunit mRNAs, whereas etizolam withdrawal was associated with a persistent reduction in gamma2S mRNA and an increase in alpha2 and alpha3 mRNAs. Neither chronic exposure to nor withdrawal of etizolam affected the acute modulatory effects of etizolam or lorazepam on GABA-evoked Cl- current. Treatment with 10 microM lorazepam for 5 days reduced the amounts of alpha1 and gamma2S subunit mRNAs and increased that of alpha3 mRNA, whereas lorazepam withdrawal was associated with persistence of the changes in alpha3 and gamma2S mRNAs and an increase in alpha2 and alpha4 mRNAs. Parallel changes in the abundance of alpha1 and alpha4 subunit proteins induced by chronic exposure to and withdrawal of lorazepam, but not etizolam, were detected by immunocytofluorescence analysis. Chronic lorazepam treatment resulted in a reversible reduction in the modulatory efficacy of this drug and conferred on flumazenil the ability to potentiate GABA-evoked Cl- current. The anticonvulsant action of etizolam was not altered in mice chronically treated with this drug, whereas lorazepam-treated animals became tolerant to the acute anticonvulsant effect of this benzodiazepine. These data suggest that etizolam is endowed with a reduced liability to induce tolerance and dependence compared with classical benzodiazepines.

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate is a novel positive modulator of GABAA receptors.

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate (TG41) enhanced the binding both of gamma-aminobutyric acid (GABA) and of flunitrazepam to rat cerebral cortical membranes. Electrophysiological recordings from Xenopus oocytes expressing various recombinant GABA(A) receptor subtypes revealed that TG41 enhanced the function of all receptor subunit combinations tested. The potency of TG41 at receptors containing alpha1, beta2, and gamma2L subunits was greater than that of alphaxalone, etomidate, propofol, or pentobarbital. The potency of TG41 was also greater at receptors containing alpha1 or alpha2 subunits than at those containing alpha4 and it was markedly higher at receptors containing beta2 or beta3 subunits than at those containing beta1. This drug induced a reversible loss of the righting reflex in Xenopus tadpoles and it elicited hypnosis (5 mg/kg) after intravenous administration in rats. These results indicate that the pharmacological profile of TG41 is similar to that of general anesthetics which potentiate the activity of GABA(A) receptors containing the beta2 or beta3 subunit.

Novel anellated pyrazoloquinolin-3-ones: synthesis and in vitro BZR activity.

A series of pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-one derivatives 6, 7a-c, 8a,b, 9a,b and 10-12 were synthesized as modified pyrazoloquinolinone analogs (PQs) and evaluated for their ability to inhibit radioligand to central and peripheral benzodiazepine receptors (BZRs) and their effect on GABA(A) alpha1beta2gamma2L receptors expressed in Xenopus laevis oocytes. Multistep synthesis starting from 5-nitroindole, via the Gould-Jacobs reaction to the quinoline nucleus, yielded key intermediates 9-chloro-3H-pyrrolo[3,2-f]quinoline-8-carboxylates. The reaction of the latter with methyl-hydrazine and various phenyl-hydrazines furnished the final compounds. In order to confirm the expected tetracyclic 2-substituted-2H-pyrazolopyrroloquinolin-3-one structure, IR spectrophotometric, mono-1H and 13C and bi-dimensional spectrometric and HRMS analyses were carried out: all compounds were found to be 2-substituted 3-keto tautomers; compound 6 only differed because it turned out to be 1-methyl-2H-pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-olo. The results of this work are consistent with those previously reported for PQs: 7-9 show high potency in displacing specific [3H]flunitrazepam from its receptor site; no compound was active in inhibiting the binding of [3H]PK 11195. They all act as antagonists at central BZR.

Synthesis, central and peripheral benzodiazepine receptor affinity of pyrazole and pyrazole-containing polycyclic derivatives.

A series of new pyrazole-condensed 6,5,5 tricyclic compounds were synthesized and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Some 1-aryl-5-phenylpyrazole derivatives were also prepared and tested for comparison with their corresponding rigid tricyclic analogs. Among the newly synthesized 1-aryl-1,4-dihydro-indeno[1,2-c]pyrazoles bearing both an ethoxycarbonyl group at position 3 and a carbonyl function at the position 4, compound 4b emerged as a new potent (IC(50) = 26.4 nM) and selective CBR ligand. The 4-oxo-1-aryl-1,4-dihydro-indeno[1,2-c]pyrazole diethylamide derivative 14a was instead identified as a relatively potent (IC(50) = 124 nM) but highly selective PBR ligand.

High affinity central benzodiazepine receptor ligands. Part 3: insights into the pharmacophore and pattern recognition study of intrinsic activities of pyrazolo[4,3-c]quinolin-3-ones.

Novel 2-phenyl-2,5-dihydropyrazolo[4,3-c]quinolin-3-(3H)-ones (PQs) endowed with high affinity for central benzodiazepine receptor (BzR) were synthesized. In particular, 9-fluoro-2-(2-fluorophenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (2(2)) showed binding affinity in the subnanomolar concentration range and proved to be in vitro a potent antagonist. This finding allowed the nature of the hydrogen bonding receptor site H(2) to be established, as located between the N-1 nitrogen of the PQ nucleus and the ortho position of the N-2-aryl group. [35S]tert-Butylbicyclophosphorothionate ([35S]TBPS) binding assays and electrophysiological measurements of the effects on GABA-evoked Cl(-) currents at recombinant human alpha(1)beta(2)gamma(2)(L) GABA(A) receptors, expressed in Xenopus laevis oocytes, were used to assess the intrinsic activities of a large series of PQs. With the aim of extracting discriminant information and distinguishing BzR ligands with different profiles of efficacy, 51 PQ derivatives, including full and partial agonists, antagonists, and inverse agonists, were analyzed in a multidimensional chemical descriptor space, defined by the lipophilicity parameter CLOG P and 3-D molecular WHIM descriptors, by means of principal component analysis, k-nearest neighbors (k-NN) method, and linear discriminant analysis (LDA). The classification methods were applied to subsets of pairs of efficacy classes, and lipophilicity and 3-D size descriptors were detected as the discriminant variables by a stepwise linear discriminant analysis. LDA proved to be superior to k-NN, especially in classifying PQ ligands (60-84% of success in prediction ability) into categories of efficacies which were contiguous and quite overlapped in the hyperspace of variables.

Structure-activity relationships in carboxamide derivatives based on the targeted delivery of radionuclides and boron atoms by means of peripheral benzodiazepine receptor ligands.

The structure-activity relationship studies on 2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands have been refined with the aim of using these ligands as carriers of radionuclides and boron atoms. Some new ligands show enhanced affinity and steroidogenic activity with respect to reference compound 1 and are interesting candidates for radiolabeling and PET studies. Moreover, carborane derivative 3q, representing the first example of PBR ligand bearing a carborane cage, can be useful to explore an alternative mechanism in BNCT.

Synthesis and biological evaluation of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines as new ligands of central and peripheral benzodiazepine receptors.

A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d, with those of highly active and selective PBR ligands, taken as reference, was performed.