Biological bases of anxiety.

Anxiety is a complex psychological and behavioural trait, related to behavioural responses as different as seizures, memory, impulsivity or aggression. The biological bases analysed in the present article involve neurotransmitter systems, specific regions in the brain and genetic factors. The genetic approach is given special emphasis as it offers, in rodents, a promising field for acquiring knowledge on biological factors modulating anxiety.

3-Amino-beta-carboline derivatives and the benzodiazepine receptor. Synthesis of a selective antagonist of the sedative action of diazepam.

Seven 3-N-substituted derivatives of 3-amino-beta-carboline were synthesized and their affinities for the benzodiazepine receptor were assessed in vitro. Two compounds, 3-(ethylamino)-beta-carboline and 3-[(methoxycarbonyl)amino]-beta-carboline (beta-CMC), showing IC50 values of 460 and 71 nM, respectively, were selected for in vivo studies. The former compound showed long-lasting proconvulsant activity in Papio papio baboons while beta-CMC was shown in mice to selectively antagonize the sedative effects of diazepam without exhibiting convulsant, proconvulsant, or anxiogenic activity by itself.

Synthesis and benzodiazepine receptor affinities of rigid analogues of 3-carboxy-beta-carbolines: demonstration that the benzodiazepine receptor recognizes preferentially the s-cis conformation of the 3-carboxy group.

1H-Indolo[3',2':4,5]pyrido[3,2-b]-2-penten-5-olide (6) and 1H,5H-indolo[3',2'-c]-6,7-dihydro-2-pyridone (7), rigid analogues of methyl 4-ethyl-beta-carboline-3-carboxylate (8) and N-methyl-4-ethyl-beta-carboline-3-carboxamide (9), respectively, were synthesized and their in vitro binding affinities to the central type benzodiazepine receptors were compared. The IC50 values of 6 and 8 were approximately equivalent (42 and 27 nM, respectively). The amide derivative 9, for which theoretical energy calculations indicate that the s-trans carbonyl conformation is the preferred one, displayed very low affinity (IC50 greater than 10(4) nM). However, when the carbonyl group of 9 was forced to adopt the s-cis conformation as in lactam 7, binding to the benzodiazepine receptor was largely restored (IC50 = 150 nM), indicating that the s-cis carboxy conformation at C-3 of beta-carbolines is preferentially recognized by this receptor. In vivo, compound 6 showed neither convulsant, proconvulsant, nor anticonvulsant activity in mice. Moreover, 6 did not antagonize methyl beta-carboline-3-carboxylate induced convulsions in mice. This lack of activity of 6 was attributed to its inability to cross the blood-brain barrier since no significant displacement of [3H]Ro 15-1788 from mouse brain benzodiazepine receptors by 6 could be observed in vivo.

Mouse chromosome 9 involvement in beta-CCM-induced seizures.

Analysis of beta-CCM induced seizures in three inbred strains of mice, ABP/Le, C57BL/6J and C57BL/6ByJ, and their F1s and F2s progeny, allowed identification of a putative seizure susceptibility controlling locus on chromosome 9 near the short-ear locus. The involvement of a gene in the medial segment of this chromosome in both seizure activity and GABA-controlled behaviour is discussed.

Balance control and posture in anxious mice improved by SSRI treatment.

A task requiring dynamic postural stabilisation during locomotion in a conflicting visual vestibular environment (rotating beam), has been devised to assess anxiety-related balance impairments and postural changes in mice. The model, already validated with acutely administered diazepam, was used to assess the action of two chronically administered selective serotonin reuptake inhibitors (SSRIs), fluoxetine and paroxetine. On three behavioural measures (imbalance, elevation of trunk and angle of tail), observed in anxious BALB/cByJ mice, both compounds had the same diazepam-like effects: reduction in number of imbalances, higher elevation of trunk and increase in tail angle. These data suggest, for the first time, that SSRIs should be useful in the treatment of anxiety-induced balance impairments.

Chromosomes 4 and 13 in beta-carboline-induced seizures in mice: benzodiazepine binding.

Methyl beta-carboline-3-carboxylate (beta-CCM) is a ligand for the benzodiazepine (BZD) binding site of the GABA-A receptors with convulsive properties. We provided evidence for the involvement of a fragment of mouse chromosomes 4 and 13 in beta-CCM-induced seizures in a previous paper. Here, we analyzed, through [3H]-flumazenil binding, whether central BZD binding sites could be involved in the physiological processes underlying these differences of genetic sensitivities. In the JE/Le strain, where the effects of the chromosome 4 fragment can be analyzed, we found associations between [3H]-flumazenil binding and the convulsive action of beta-CCM. On the contrary, this no longer holds true in C3XtEso strain, where the effects of the chromosome 13 fragment were observed.

Convulsive effects of a benzodiazepine receptor inverse agonist: are they related to anxiogenic processes?

The linkage-testing strain of ABP/Le mice carries six mutations which express in easily identifiable phenotypes. By crossing this strain with a traditional inbred strain (C57BL/6ByJ) which is the 'wild type' for the mutated ABP/Le loci, we produced Mendelian populations, intercrosses and backcrosses so as to estimate whether the sensitivity to methyl beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine receptor inverse agonist, and anxiety-related behaviour could be related to a common genetically determined substrate. We have shown that one locus on chromosome 9 is associated with beta-CCM-induced seizures and three loci on chromosomes 4, 7 and 9 are associated with anxiogenic processes. Analysis of [3H]flumazenil binding suggested a possible involvement of a Bmax decrease in both beta-CCM-induced seizures and anxiogenic processes. The putative common genetic regulation of both mechanisms is discussed.

Olfactory memory in rats, cholinergic agents and benzodiazepine receptor ligands.

Drugs and their effects on olfactory learning processes in rats were tested using a modified version of the runway apparatus developed by Ades. Rats were first exposed to a conspecific urine sample and 24 h later were exposed to the same stimulus in the runway. Observations recorded the time spent investigating the urine and the number of sniffs at the site, these being considered to be indices of memory. Diazepam-treated rats (4 or 6 mg/kg) and scopolamine-treated rats (0.5 or 1 mg/kg) showed increases for both parameters. When both drugs were administered simultaneously, the impairing effect was potentiated. However, no changes in learning responses were observed in rats treated with physostigmine (0.125, 0.25, 0.5 mg/kg) or methyl beta-carboline-3-carboxylate (0.3, 0.5, 1 mg/kg), although the administration of physostigmine or methyl beta-carboline-3-carboxylate was shown to antagonize the impairing effect of diazepam or scopolamine respectively. These observations support the hypothesis of interactions existing between cholinergic agents and benzodiazepine receptor ligands and of such interactions affecting olfactory acquisition processes. The runway apparatus appears to be a valid candidate model to be used for the assessment of pharmacological influences on olfactory learning in rats.

Effects of Ginkgo biloba extract (EGb 761) on learning and possible actions on aging.

A study of the effect of Ginkgo biloba extract (EGb 761) has shown enhancing effects on training in adult and aged Swiss mice. An analysis of inbred mice has confirmed this sensitivity to EGb 761, but depending on the strains, with different effects at different ages. The most interesting results are related to improvements in performances observed with aged mice of the DBA/2J strain. The results obtained with inbred strains in the study of the mossy fibers of the hippocampus make it possible to suggest a link between the improvements in training and the histological structure of the hippocampus. This possibility, which can be confirmed by further studies, is presented here.

Intermale aggression, GAD activity in the olfactory bulbs and Y chromosome effect in seven inbred mouse strains.

The capacity to attack a passive standard opponent in a resident-intruder test and the GAD activity in the olfactory bulbs were measured in 140 male mice from seven different inbred mouse strains. The effect of the non-pseudo autosomal region of the Y-chromosome (YNPAR) on these two phenotypes has also been investigated using a quartet of reciprocal strains congenic for the YNPAR. A strong negative correlation was found between the two variables but the YNPAR is not involved. This result suggests that males of more attacking strains have a lower olfactory threshold, making the olfactory discrimination of the opponent easier and its identification as a stranger more efficient.

Intermale aggression and dark/light preference in ten inbred mouse strains.

The capacity of males to attack a passive standard opponent in a resident-intruder test and the preferences in a dark/light choice situation were measured in 200 male mice from 10 different inbred mouse strains. Large strain differences were found for all variables recorded, i.e., the proportion of attacking males, the time spent in the brightly lit box, and the number of transitions between the lit and the dark boxes. A strong negative correlation was found between the first two variables. This result suggests that males of more attacking strains have a higher level of anxiety but do not differ for their level of activity. An involvement of GABA as mediating factor is suggested.

Unexpected absence of correlation between the genetic mechanisms regulating beta-carboline-induced seizures and anxiety manifested in an elevated plus-maze test.

Among the ligands of the benzodiazepine site, one can mention the benzodiazepines as agonists and some beta-carbolines (e.g. methyl-beta-carboline-3-carboxylate, abbreviated hereafter beta-CCM) as inverse agonists. Most benzodiazepines and beta-carbolines act on processes involved in memory, anxiety, and convulsions with opposite physiological effects. Since these molecules have influences on both anxiety and convulsions, we predicted that there would exist a genetic correlation between anxiety evaluated in an elevated plus-maze and susceptibility to beta-CCM-induced seizures. Using inbred strains of mice, the genetic correlation was estimated with the Hegmann and Possidente model. An absence of genetic correlation was found, showing that the mechanisms responsible for basal anxiety measured with the elevated plus-maze test and those leading to susceptibility to beta-CCM-induced seizures do not share the same genetic pathways.

Involvement of regions of the 4th and 7th chromosomes in the open-field activity of mice.

Reactivity to a new environment was studied in mice, using an open-field procedure in two strains, C57BL/6By and ABP/Le, the F1 populations and the intercrosses F2 and backcross segregating populations. The analysis of the behavioral traits: peripheral and central activities, leaning, rearing and defecation in the parental strains made it possible to show that the ABP/Le strain was more reactive than C57BL/6By. In addition, the study of segregating, for four phenotypic markers, in F2 and backcross populations strongly suggested that two autosomal regions were involved in the control of open-field behavior: one in chromosomal region comprising the b locus on chromosome 4 and one in chromosomal region comprising the p locus on chromosome 7.

Effects of adrenal medulla graft on recovery of GABAergic and dopaminergic neuron deficits in mice: behavioural, pharmacological and immunohistochemical study.

We studied the capacity of adrenal medullary transplant to restore the deficits of GABAergic and dopaminergic neurons in mice injected with quinolinic acid (QA), using an open field test as well as pharmacological and immunohistochemical techniques. We analysed behavioural traits-total locomotor activity, peripheral and central activities, grooming, leaning and rearing in the QA-lesioned mice and mice that had undergone adrenal medulla (AM) transplantation. We found that the adrenal transplant recovered a loss of GABAergic neurons. It reduced QA-induced hyperactivity in locomotion and improved emotional indices. In addition, immunohistochemical studies of catecholaminergic markers-tyrosine hydroxylase (TH), dopamine (DA) and neuronal vesicular monoamine transporter type 2- and a single post-trial injection of tetrabenazine (TBZ; 5 mg/kg) indicated that catecholamines-synthesising chromaffin cells in the AM grafts were also involved in the beneficial effects. A likely interpretation of this behavioural pattern of results is that adrenal medullary transplants set into play an interaction between GABAergic and DAergic factors. Our results may contribute to the clarification of the beneficial effects of AM transplants in striatal function.

Involvement of adrenal medulla grafts in the open field behavior.

Immunohistochemical and behavioral techniques were used to study the effects of adrenal medulla grafts, implanted in striatum after bilateral kainic acid (KA) lesions of this structure, on the open field behavior of mice. KA-induced behavioral changes in leaning, grooming and locomotor activity of the open field test were significantly improved after grafting of the adrenal medulla, and in some respects, fully restored. Immunohistochemical identification showed that grafts contained neuron-like cells with a tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and enkephalin-like immunostainings. A likely interpretation of this complex pattern of results is that adrenal medullary grafts may restore the deficits of GABAergic neurons which in turn reverse the abnormalities in emotionality and locomotion. Neurobiologically, these behavioral improvements probably involve GABAergic and catecholaminergic factors of adrenal medulla grafts, although other neuroactive substances, such as acetylcholine and enkephalins, cannot be excluded.

Balance control and posture differences in the anxious BALB/cByJ mice compared to the non anxious C57BL/6J mice.

A relation between anxiety disorders and balance control dysfunctions has been observed in many studies in humans. A mismatch in the integration of sensory inputs could trigger these disturbances. Very few experimental animal procedures have been designed to study the functional link between anxiety and balance control. A task was therefore developed, challenging the visual, vestibular and somesthesic sensory systems in mice. The test, called the 'rotating beam', gave an accurate assessment of balance control and the posture, using sensitive measures (number of falls and imbalances, position of tail and trunk). Striking differences were observed between the two inbred strains of mice known to have radically different anxiety-related behaviour. The highly anxious strain, BALB/cByJ, performed poorly compared to the non anxious strain, C57BL/6J. Balance control and postural abilities of anxious mice were improved by acute anxiolytic diazepam treatment. Lower behavioural performance level was registered in non anxious mice given anxiogenic beta-CCM treatment. The findings account for a strong relationship between anxiety and balance control in mice. Finally, the highly sensitive procedure proved to be well suited to the study of functional links between anxiety and sensorimotor processes.

Genetic difference in sensitivity to beta-carboline: evidence for the involvement of brain benzodiazepine receptors.

The convulsive effects of methyl beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine receptor ligand, are different in two inbred strains of mice: BALB/cBy mice are more sensitive to beta-CCM than C57BL/6J mice. In the present article, we report the effects of [3H]flunitrazepam binding in these two strains, which suggest a possible explanation of the differences in their sensitivity to beta-CCM by the involvement of brain benzodiazepine receptors.

Genetic factors regulate processes related to anxiety in mice.

The propensity for anxiety-related behavior has been studied by comparing two highly inbred strains of mice, ABP/Le and C57BL/6ByJ, in two behavioral procedures, open-field and light-dark preference. Their Mendelian F2 population allowed us to evaluate the putative involvement of four easily identifiable loci in anxiogenic processes. In fact, chromosomal regions containing the brown, pink-eyed dilution and short-ear loci on the 4th, 7th and 9th chromosomes respectively are associated with anxiety-related behavior patterns. In addition, binding of [3H]flumazenil to brain GABA(A) receptors was measured as a biochemical index that may be associated with observed behavior patterns.

Cognitive enhancing properties of beta-CCM infused into the nucleus basalis magnocellularis of the rat.

Peripheral administration of various benzodiazepine derivatives or beta-carbolines (inverse agonists at benzodiazepine receptors), has been shown to affect memory. In this study, the effect of local infusion of a beta-carboline-methyl beta carboline-3-carboxylate (beta-CCM) into the nucleus basalis magnocellularis (NBM) of rats was examined in a two-trial recognition task. The results show that beta-CMM (3 micrograms/0.5 microliter) enhances recognition performance when injected both before or immediately after the acquisition trial. These effects appear to be mediated by a benzodiazepine (BZD) receptor since they were blocked by pretreatment with Ro 15-1788, a BZD receptor antagonist. This study supports the involvement of the NBM in cognitive processes, and demonstrates that these processes can be influenced by alteration of GABAergic neurotransmission.

Genetic selection of mouse lines differing in sensitivity to a benzodiazepine receptor inverse agonist.

Mice were selectively bred according to their sensitivity or their resistance to the convulsive effects of a 4-mg/kg dose of methyl beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine (BZ) receptor inverse agonist. The selection proved to be easy, with a clear separation of the two lines, convulsing with short latencies or resistant, already at the first generation of selection. Selection of a third line of animals convulsing with long latencies did not succeed. 3H-Ro 15-1788 binding analysis provided evidence for a strong decrease in Bmax in the resistant line.