β-Carbolines characterized as benzodiazepine receptor agonists and inverse agonists produce bi-directional changes in palatable food consumption

Drugs which bind to specific benzodiazepine recognition sites fall into three categories: agonists, antagonists and inverse agonists. A set of biochemical parameters is available which distinguishes between the three. In addition, actions of the drugs result in physiological and behavioural effects which are distinguishable. β-Carboline derivatives provide a group of compounds which show high affinity for the benzodiazepine sites and which contains examples belonging to each of the three categories. Evidence is reviewed which shows that β-carboline benzodiazepine receptor agonists (ZK 93423, ZK 91296) produce increases in the consumption of a palatable diet by non-deprived rats, that β-carboline inverse agonists (FG 7142, DMCM) produce an anorectic effect and that the β-carboline ZK 93426 acts as a benzodiazepine receptor antagonist. The resuks support the proposal of bi-directional control of feeding responses through the action of drugs at a common benzodiazepine receptor. Furthermore, benzodiazepine receptor inverse agonists provide a novel class of anorectic agents. Evidence is also reviewed which is suggestive of the modulation of food-related reward by drug actions at benzodiazepine receptors.     

The anxiogenic β-carboline FG-7142 increases in vivo and in vitro tyrosine hydroxylation in the prefrontal cortex

Systemic administration of the anxiogenic β-carboline FG-7142, a benzodiazepine inverse agonist, results in a regionally selective increase in dopamine (DA) utilization in the anteromedial prefrontal cortex (PFC). We have examined both in vivo and in vitro tyrosine hydroxylation in the PFC and other mesotelencephalic DA system terminal fields in order to determine if FG-7142 effects changes in DA synthesis, and to determine if the β-carboline biochemically activates certain DA neurons through an action occurring at the cell body level (impulse-dependent regulation) or at the terminal field level (presynaptic regulation). FG-7142 increased in vivo tyrosine hydroxylation in the PFC and in the ventral tegmental area, midbrain source of the DA innervation of the PFC; no changes were observed in mesolimbic or nigrostriatal regions. The β-carboline also increased in vitro tyrosine hydroxylation in the PFC, but decreased tyrosine hydroxylation in striatal slices. The effects of FG-7142 were blocked by the benzodiazepine antagonist RO 15-1788. Another β-carboline inverse agonist, methyl-β-carboline-carboxylate, also increased in vitro tyrosine hydroxylation in the PFC. GABA exerted opposite effects to those of the β-carbolines, decreasing in vitro tyrosine hydroxylation increasing DA synthesis in the CP. These data indicate that the benzodiazepine inverse agonists increase both in vivo and in vitro tyrosine hydroxylation in the PFC, and that the β-carboline may act to increase DA synthesis at both the terminal field and the cell body level.     

Ethyl β-carboline-3-car☐ylate antagonizes the action of GABA and benzodiazepines in the hippocampus

In urethane-anaesthetized rats, the β-carboline derivative βCCE (0.3–1.0 mg/kg i.v.) excited hippocampal pyramidal cells which were inhibited by GABA (applied iontophoretically) and benzodiazepines (applied iontophoretically or intravenously). While benzodiazepines facilitated the action of GABA, the effects of GABA and benzodiazepines were antagonized by βCCE. This electrophysiological study supports the behavioural observations that βCCE is a benzodiazepine receptor antagonist.     

β-Carboline kindling of the benzodiazepine receptor

β-Carboline (norharman), an antagonist of the benzodiazepine receptor, given daily at a dose of 20 mg/kg (i.p.), produces kindled seizures. The mechanism of this epileptogenesis is unknown; however, other ligands of the benzodiazepine receptor, i.e. diazepam, R015-1788 and CL218-872 block the expression of kindled seizures in a dose-dependent manner. The results suggest that β-carboline kindling is mediated through the benzodiazepine receptor.     

The anorectic effect of FG 7142, a partial inverse agonist at benzodiazepine recognition sites, is reversed by CGS 8216 and clonazepam but not by food deprivation

The benzodiazepine partial inverse agonist N′-methyl-β-carboline-3-carboxamide (FG 7142; 5.0 and 10.0 mg/kg, i.p.) produced a dose-dependent reduction in the consumption of a familiar, highly palatable diet by non-food-deprived male rats. At dose levels which exhibited no significant intrinsic effects, the benzodiazepine receptor antagonist 2-phenylpyrazolo-[4,3-c]-quinoline-3(5H)-one (CGS 8216; 1.25–5.0 mg/kg, i.p.) reversed the anorectic effect of FG 7142. When clonazepam and FG 7142 were given in combination, mutual cancelling of their opposite effects occurred. These results are consistent with an action of FG 7142 at benzodiazepine recognition sites to reduce the level of palatable food consumption, and imply that a bidirectional control of food intake via benzodiazepine recognition sites can be achieved. The anorectic effect of FG 7142 was not reversed by 24-h food deprivation, indicating a possible separation from the effects of hunger mechanisms.     

Reduction of predator odor-induced anxiety in mice by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to an aversive, anxiety-inducing, predator (cat) odor were examined in an odor preference test. Control untreated mice displayed an anxiogenic response to the cat odor, spending a minimal amount of time in a Y-maze in the vicinity of the cat odor. Intracerebroventricular (i.c.v.) administrations of 3αHP had an anxiolytic action, resulting in significant dose-related (0.01–1.0 μg) increases in the amount of time spent in the proximity of the cat odor. These anxiolytic effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP) having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the response to cat odor indicating that the anxiolytic actions of 3αHP were unlikely to be related to any analgesic effects. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results indicate that the allylic neurosteroid 3αHP has anxiolytic actions involving interactions with the GABA_A receptor.     

β-Carboline inhibition of benzodiazepine receptor binding in vivo

The in vivo potencies of 4 β-carboline derivatives as inhibitors of benzodiazepine receptor binding were investigated. For all 4 derivatives maximal inhibition was seen within 2 min after i.v. application but decreased continuously for the next 20 min and after this time accounted for less than one-third of maximal inhibition. The in vivo potencies of the β-carbolines as inhibitors of benzodiazepine receptor binding are much smaller than one would expect from their affinities measured in vitro. Thus, it is assumed than only a small portion of the i.v. dose reaches the brain.     

Mice selected for differences in sensitivity to a benzodiazepine receptor inverse agonist vary in intermale aggression

Brain γ-aminobutyric acid (GABA) levels are involved in intermale aggression in mice. It was therefore expected that animals genetically selected for their sensitivity to the convulsive effects of methyl β-carboline-3-carboxylate (β-CCM; BS, β-CCM sensitive, and BR, β-CCM resistant), a benzodiazepine (BZ) inverse agonist that specifically binds to the BZ site on the GABA-A receptor complex, would differ in their levels of aggressive behavior. Using two different aggression tests, in two independent experiments, we showed that BS mice are more aggressive than BR animals. The precise mechanisms underlying the observed line differences in β-CCM sensitivity and aggression remain to be determined. Received: 18 January 1999 / Accepted: 24 June 1999 / Published online: September 1 1999     

Bidirectional changes in the consumption of food produced by β-carbolines

β-Carboline derivatives provide examples of benzodiazepine receptor ligands which span the range: full agonist-partial agonist-antagonist-partial inverse agonist-full inverse agonist. Taken together, the effects of these compounds illustrate two important principles: firstly, the bidirectionality of effects which can be achieved using benzodiazepine receptor ligands; secondly, the selectivity of effects which are produced by partial agonists. Applied to the study of feecling processes, these principles imply that both hyperphagic and anorectic effects can be generated by actions of selected ligands at benzodiazepine receptors. Furthermore, they suggest that a hyperphagic effect may occur in the absence of side-effects (e.g., sedation, muscle-relaxation), which are characteristic of classical benzodiazepines. Experimental data in support of these predictions are presented. A microstructural approach to feecling behaviour indicated that a benzodiazepine receptor agonist and an inverse agonist extend and abbreviate, respectively, the duration of individual bouts of eating. Preference for a saccharin solution was attenuated by the β-carboline inverse agonist, FG 7142, but rejection of a quinine solution was not increased. Adrenalectomy had no effect on the anorectic effect of inverse agonists.     

Male preference for the odors of estrous female mice is enhanced by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to the odors of estrous female mice were examined in an odor preference test. Control untreated mice displayed a significant preference for the odors of an estrous female, spending more time in a Y-maze in the vicinity of the odors of an estrous than a non-estrous female. Intracerebroventricular (i.c.v.) administrations of 3αHP enhanced male preference for the odors of estrous females, causing a significant dose-related (0.01–1.0 μg) increase in the amount of time spent in the proximity of the odors of the estrous female, while having no significant effect on the responses to the non-estrous female odors. These effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP), having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the responses to female odors suggesting that the enhanced preference for estrous female odors were unlikely to be directly due to any analgesic effects of 3αHP. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results suggest that the neurosteroid 3αHP has facilitatory effects on olfactory mediated male sexual interest or motivation that involve interactions with the GABA_A receptor.     

Convulsions Induced by Methyl β-Carboline-3-Carboxylate in Mice: Effects of Preceding Saline Injections

The convulsant effects of a high (5 mg/kg intraperitoneally, i.p.) dose of benzodiazepine (BZD) receptor ligand methyl β-carboline-3-carboxylate (β-CCM), whether or not preceded by administration of two lower doses of β-CCM (0.5 and 1 mg/kg i.p.) or of saline were studied in nine inbred mouse strains. In five of the strains (A/J, BALB/cBy, C3H/HeJ, CBA/H, and DBA/2J), neither saline nor preceding injections of β-CCM had any effect on subsequent reactivity to the subsequent convulsant dose. In the other 4 strains, such injections induced either tolerance (CPB-K, NZB), or sensitization (C57BL/6J, XLII), whatever the compound subsequently administered (β-CCM or saline). In these strains, the data rule out any tolerance or sensitization effect due to β-CCM, but suggest that such effects could be due to injection itself.     

Sex with knockout models: behavioral studies of estrogen receptor α

Estrogens are an important class of steroid hormones, having multiple targets, in the body and brain, and exerting ubiquitous effects on behavior. At present, two estrogen receptors (ERα and β) have been cloned and sequenced in mammals. In the brain these receptors are regionally specific, but both have widespread distributions, which are largely non-overlapping. Given the newly emerging complexities of estrogen's mechanisms of action it is important to distinguish which pathways are involved in modifying which behaviors. We use a knockout mouse, lacking functional copies of the estrogen receptor α (ERα) gene, to study the mechanisms by which estrogens mediate behaviors. There are pronounced ramifications of ERα gene disruption on behavior. First, female ERα knockout (ERαKO) mice do not display normal feminine sexual behavior. Second, treatment of adult mice with androgens promotes masculine sexual behavior in both sexes. However, male-typical sexual behavior is severely compromised in male and female ERαKOs. Third, male ERαKOs do not exhibit the same social preferences for female mice as do wildtype (WT) littermates. Thus, the ERα is essential for normal expression of sexual behaviors. In addition, gonadectomized ERαKO and WT mice rapidly learn to escape from the Morris water maze. Exogenous estrogen treatment prevents WT females from learning this task, yet, has no effect in ERαKO mice, suggesting that estrogens effects on learning in adult females involves the ERα. Based on these data we hypothesize that ERα mediates many of the effects of estrogen on sexual behavior, learning, and memory.     

Treatment with Anti-interferon-γ Monoclonal Antibodies Modifies Experimental Autoimmune Encephalomyelitis in Interferon-γ Receptor Knockout Mice

The role of interferon-γ (IFN-γ) in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) is still controversial. We have studied the function of IFN-γ and its receptor in the EAE model using two different IFN-γ receptor knockout (IFN-γ R^−/−) mouse types: C57Bl/6×129Sv, with a disruption of the IFN-γ receptor cytoplasmic domain, and 129Sv, homozygous for a disrupted IFN-γ receptor gene. Mice were immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. A subgroup of mice was treated with anti-IFN-γ monoclonal antibodies (mAb) on day 8 postimmunization. Clinical scoring and both histological and immunohistochemical studies were undertaken for all groups. We hereby show that treatment with anti-IFN-γ mAb worsened the disease course of 129Sv wild-type mice. However, it decreased the mean daily score in IFN-γ R^−/− 129Sv and the incidence of the disease down to 50% in C57Bl/6×129Sv IFN-γ R^−/− mice. Moreover, after anti-IFN-γ mAb treatment, oxidative stress levels, metallothionein I and II antioxidant protein expression, and apoptoticneuronal death were increased in wild-type mice while decreased in IFN-γ R^−/− mice. These results suggest a putative alternative mechanism of action of this cytokine that works independent of its receptor.     

The effects of des-enkephalin-γ-endorphin and des-Tyr-α-endorphin on regional serotonin metabolism in rat brain

Des-enkephalin-γ-endorphin (β-endorphin_6–17; DEγE) causes a significant reduction of the serotonin concentration of the raphe area of the mesencephalon and of the dorsal hippocampus of the rat. DEγE does not, however, alter the pargyline-induced accumulation of serotonin in either of these two brain regions or in the mediobasal hypothalamus. Des-Tyr¹-α-endorphin (β-endorphin_2–16; DTαE), on the other hand, inhibits the accumulation of serotonin following MAO-inhibition by pargyline in all 3 brain regions, while, in addition to causing a transient reduction in the serotonin concentration of the raphe area, it decreases the serotonin concentration of the mediobasal hypothalamus. These results indicate that DEγE and DTαE, which have been shown to exert opposite effects on behavior in various behavioral tests, both affect regional serotonin metabolism in rat brain and that they do so differently.     

Modulation of humoral immune response by central administration of leucine-enkephalin: Effects of μ, δ and κ opioid receptor antagonists

The effect of leucine-enkephalin (Leu-Enk) on primary humoral immune response was investigated following intracerebroventricular (i.c.v.) administration of the peptide in the rat. Leu-Enk stimulated plaque-forming cell (PFC) response in rats i.c.v. injected with 0.1 and 1 μg/kg, whereas doses of 20 and 50 μg/kg exerted immunosuppressive effects. I.c.v. treatment of rats with δ opioid receptor antagonist ICI 174864 and κ opioid receptor antagonist nor-binaltorphimine (nor-BNI) blocked stimulation and suppression of PFC response induced by Leu-Enk, respectively. The μ opioid receptor antagonist β-funaltrexamine (β-FNA) reversed both immunomodulatory effects produced by Leu-Enk. Since β-FNA alone had no effect on PFC response (unlike ICI 174 864 and nor-BNI), these data showed that central effects of Leu-Enk on PFC response were mediated by brain μ opioid receptors, and suggested a possible involvement of δ and κ opioid receptors.     

Purification of γ-aminobutyric acid (GABA) receptor from rat brain by affinity column chromatography using a new benzodiazepine, 1012-S, as an immobilized ligand

The purification of γ-aminobutyric acid (GABA) and benzodiazepine receptors from the rat brain was employed by the affinity column using a new benzodiazepine, 1012-S, as immobilized ligand. The 1012-S has a aliphatic primary amino group and exhibited an extremely high potency for displacing [³H]flunitrazepam binding to solubilized benzodiazepine receptor preparation (IC₅₀ = 6.0 × 10⁻¹¹M). This benzodiazepine affinity gel retained almost all of the solubilized GABA receptors from synaptic membranes applied to the column, and 25.6% of the receptor was eluted bio-specifically following the application of 1 mM 1012-S. The highest purification fold thus obtained was 4576 (specific activity: 0.99 nmol/mg protein). Furthermore, the successive application of 1–2 M NaSCN also resulted the elution of a highly enriched GABA receptor (specific activity: 0.41 nmol/mg protein; purification fold: 1889). SDS-polyacrylamide gel electrophoretic profiles of the bio-specifically eluted fraction with 1012-S showed the existence of two major bands having the molecular weights of approximately 48,500 and 54,500 in which the former band was selectively photoaffinity-labelled with [³H]flunitrazepam. On the other hand, it was found that the non-specifically eluted fraction with NaSCN contained 4 additional minor bands having molecular weights of 41,000 to 51,000. These results indicate that GABA receptor of the rat brain is coupled, at least in part, with benzodiazepine receptor and is readily purified by the use of highly specific benzodiazepine affinity gel, 1012-S-acetamide adipic hydrazide Sepharose 4B. The present results also suggest that the purified GABA/benzodiazepine receptor complex may contain two different kinds of subunits having the molecular weights of 48,000 and 54,500, in which the former subunit may possess benzodiazepine binding sites.     

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1β or an IL-1β fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ. Cells were also tested under identical conditions with an IL-1β synthetic peptide fragment (IL-1β_208–240) previously shown to possess biological activity. IL-1β, TNFα and IFNγ potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1β_208–240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1β, rabbit IL-1β_208–240 and human IL-1β_208–240 was blocked by the IL-1ra, while TNFα- and IFNγ-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1β and human IL-1β_208–240 was also blocked by the sIL-1R. Our findings suggest that IL1β_208–240 acts as an IL-1β agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.     

Effects of β-endorphin and its fragments on inhibitory avoidance behavior in rats

The effects on retrieval of a one-trial learning inhibitory avoidance response of β-endorphin, α-endorphin, and γ-endorphin, given prior to test have been studied in rats. β-Endorphin (β-LPH_61–91) in a relatively low dose (1.5 μg sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 μg sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of γ-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of β-endorphin (β-LPH_78–91) was ineffective. α-Endorphin facilitated inhibitory avoidance behavior in a dose-dependent manner. Naltrexone pretreatment antagonized the bimodal effect of β-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same.It is suggested that β-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate receptors as well as biotransformation into α- and γ-endorphin may contribute to the multiple behavioral effects of this neuropeptide.     

The role of neurosteroids and non-genomic effects of progestins and androgens in mediating sexual receptivity of rodents

Progestins and androgens modulate sexual receptivity in rodents, in part through mechanisms independent of traditional intracellular steroid receptors. Progesterone (PROG) in the ventromedial hypothalamus (VMH) and ventral tegmental (VTA) facilitates lordosis but has different actions in these brain areas. Primarily using lordosis in rodents as an in vivo experimental model, we have examined the effects that progestins exert in the midbrain and hypothalamus. Localization and blocker studies indicate that PROG’s actions in the VMH require intracellular progestin receptors (PRs) but in the VTA they do not. Progestins that have rapid, membrane effects, and/or are devoid of affinity for PRs, facilitate lordosis when applied to the VTA. Manipulation of GABA and/or GABA_A/benzodiazepine receptor complexes (GBRs) in the VTA alters lordosis, which suggests that progestins may interact with GBRs to facilitate receptivity by enhancing the function of GABAergic neurons. Interfering with PROG’s metabolism to, or the biosynthesis of, 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG or allopregnanolone), the most effective endogenous GBR agonist, in the VTA attenuates female sexual behavior in rodents. Stimulation of mitochondrial benzodiazepine receptors (MBRs), which enhances neurosteroid production, by infusions of an MBR agonist to the VTA enhances lordosis. 3α,5α-TH PROG is increased in the midbrain of mated>proestrous>diestrous rodents. These data suggest that in the VTA, PROG may facilitate lordosis following metabolism to and/or biosynthesis of 3α,5α-TH PROG, which may have subsequent actions at GBRs and/or MBRs to acutely modulate female sexual behavior in rodents. The 3α-hydroxysteroid oxidoreduced metabolite of dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-androstanediol), is important for termination of sexual receptivity in rodents and has these effects in the absence of functional intracellular androgens receptors. As well, altering GBR function in the hypothalamus can influence 3α-androstanediol’s inhibition of sexual receptivity. Through actions in the hypothalamus that are independent of intracellular androgen receptors but involving GBRs, 3α-androstanediol inhibits lordosis. These findings suggest that the PROG metabolite and pregnane neurosteroid, 3α,5α-TH PROG, and the testosterone metabolite and androstane neurosteroid, 3α-androstanediol, can have proximate influences on lordosis that is via nonclassical actions at intracellular steroid receptors.